WO2026018015A2 - Nouveaux procédés et utilisations - Google Patents

Nouveaux procédés et utilisations

Info

Publication number
WO2026018015A2
WO2026018015A2 PCT/GB2025/051597 GB2025051597W WO2026018015A2 WO 2026018015 A2 WO2026018015 A2 WO 2026018015A2 GB 2025051597 W GB2025051597 W GB 2025051597W WO 2026018015 A2 WO2026018015 A2 WO 2026018015A2
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
nlrx1
4alkyl
substance
halo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/GB2025/051597
Other languages
English (en)
Other versions
WO2026018015A3 (fr
Inventor
Neil Miller
Anthony Richard RUTTER
Ovadia LAZARI
Tammy LADDUWAHETTY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nrg Therapeutics Ltd
Original Assignee
Nrg Therapeutics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nrg Therapeutics Ltd filed Critical Nrg Therapeutics Ltd
Publication of WO2026018015A2 publication Critical patent/WO2026018015A2/fr
Publication of WO2026018015A3 publication Critical patent/WO2026018015A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4192—1,2,3-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/423—Oxazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4245—Oxadiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/443—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/465—Nicotine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • the invention relates inter alia to methods for identifying a substance useful for the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity, and uses of said substances.
  • the mitochondria permeability transition pore is a high conductance channel residing on the inner mitochondrial membrane that is activated under certain conditions of cellular stress, in particular excessive Ca 2+ loading and oxidative stress. Due to the role of the mPTP in regulating oxidative phosphorylation and in mitochondria-related apoptosis or necrosis, it has been implicated in a number of degenerative and metabolic diseases.
  • genetic knockout of Ppif was shown to be protective in various preclinical in vivo transgenic models of neurodegenerative disease including Alzheimer’s disease, Parkinson’s disease and motor neuron disease, also known as Amyotrophic Lateral Sclerosis (ALS), demonstrating the therapeutic potential of mPTP inhibition.
  • ALS Amyotrophic Lateral Sclerosis
  • the present inventors have previously identified novel compounds which inhibit mPTP activity. For example, see WO2022/049376, WO2022/049377, and WO2023/166303.
  • NLRX1 is a member of the NOD-like receptor (NLR) family of cytoplasmic pattern recognition receptors (PRRs), and so is understood to have an important role in innate immunity (Liu et al.2022).
  • NLRX1 is at present the only NLR known to be located in mitochondria which therefore allows it to co-ordinate the link between mitochondrial function and innate immunity (Chu et al. 2019).
  • NLRX1 has been shown to have an important role in maintaining mitochondrial homeostasis and metabolic balance, as well as apoptosis and autophagy.
  • NLRX1 is a known regulator of inflammation, although its precise role is somewhat unclear.
  • NLRX1 has been reported to weaken the innate immune response via (i) reduction in activation of the RIG-I-MAVS pathway in response to viral infection, for example via direct binding to the mitochondrial antiviral signal protein (MAVS) (Nagai-Singer et al. 2019) or (ii) competition with the retinoic acid-inducible gene I (RIG-I) protein resulting in reduced formation of the immunostimulatory interferon regulatory factor 3 (IRF3) homodimer (Allen et al.2011).
  • MAVS mitochondrial antiviral signal protein
  • RIG-I retinoic acid-inducible gene I
  • NLRX1 may inhibit the inflammatory response which is activated upon infliction of damage to the mitochondria (Chu et al. 2019).
  • NLRX1 has also been reported to be pro-inflammatory in alternative contexts.
  • genetic knock-out of NLRX1 has been shown to dampen pro-apoptotic signalling and limit resultant cell death in mice suffering from hyperoxic acute lung injury (Kim et al. 2023).
  • a deficiency in NLRX1 has been demonstrated to result in an increase in the number of reparative macrophages, which are able to regulate the immune response and heal damaged tissue, in the context of ischaemic injury (Li et al.
  • NLRX1 has been reported to have a pro-inflammatory role in the development of diet-induced type 2 diabetes mellitus (Costford et al.2018). In view of the known role of NLRX1 in regulating inflammation, it has been implicated in a number of diseases. In particular, NLRX1 has been implicated in an experimental autoimmune encephalomyelitis (EAE) model (Eitas et al. 2014) and, consequently, multiple sclerosis (MS) (Gharagozloo et al. 2019), which is itself an autoimmune disease characterised by mitochondrial dysfunction.
  • EAE experimental autoimmune encephalomyelitis
  • MS multiple sclerosis
  • NLRX1 has been linked to Alzheimer’s disease and Parkinson’s disease since each of these conditions may be caused by a glutamate imbalance, whilst NLRX1 is known to regulate the glutamate cycle (Leber et al.2018).
  • the above neurological degenerative diseases are also associated with the mPTP.
  • novel compounds developed previously by the present inventors, which inhibit the mPTP have been indicated for use in the treatment of inter alia Alzheimer’s disease, Parkinson’s disease, ALS, and multiple sclerosis. The discovery by the present inventors that said mPTP inhibitors actually bind to NLRX1 explains this overlap in disease association.
  • NLRX1 is associated with a myriad of other diseases and conditions, which are not necessarily associated with the mPTP.
  • NLRX1 autoimmune diseases
  • SLE systemic lupus erythematosus
  • IBD inflammatory bowel disease
  • RA rheumatoid arthritis
  • AD atopic dermatitis
  • NLRX1 has been purported to have a role in cancer, for example hepatocellular carcinoma, breast cancer, head and neck squamous cell carcinoma, gastric cancer, and histiocytic sarcoma (Casta ⁇ o-Rodr ⁇ guez et al. 2015; Wang et al. 2017). NLRX1 has also been implicated in metabolic diseases such as type 2 diabetes mellitus (Zeng et al. 2017), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH) (Kors et al. 2018).
  • metabolic diseases such as type 2 diabetes mellitus (Zeng et al. 2017), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH) (Kors et al. 2018).
  • NLRX1 may clearly have a role in pathogen infection, for example wherein the pathogen is Helicobacter pylori (Casta ⁇ o-Rodr ⁇ guez et al. 2015) or influenza (Varga and Palese, 2011).
  • the pathogen is Helicobacter pylori (Casta ⁇ o-Rodr ⁇ guez et al. 2015) or influenza (Varga and Palese, 2011).
  • the present inventors have consequently discovered that said existing compounds may have utility in the treatment of a range of additional diseases or disorders associated with altered NLRX1 activity.
  • the present invention provides a method for identifying a substance useful for the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity , which method comprises determining whether said substance (i) binds to NLRX1 and (ii) inhibits activity of the mPTP and, if it does, identifying said substance as a substance useful for the prevention or treatment of said disease, disorder, or condition associated with altered NLRX1 activity.
  • the present invention also provides a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting activity of the mPTP, for use in the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity .
  • the present provides known inhibitors of mPTP activity, in particular a substance of formula (I), (II), (III), (IV), (V), (VA), (VI), or (VIB), as described below, for use in the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity, in particular wherein said disease, disorder, or condition is an Other NLRX1-Related Disease, Disorder or Condition (as defined herein).
  • a disease, disorder, or condition associated with altered NLRX1 activity in particular wherein said disease, disorder, or condition is an Other NLRX1-Related Disease, Disorder or Condition (as defined herein).
  • Figures 1 and 2 Show the proteins/peptides identified, and in particular NLRX1, as potential binding partners of known inhibitors of the mPTP via affinity-based chemoproteomics experiments (see Background Example 1).
  • Figure 3 Shows the effect of genetic deletion of NLRX1 on activity of the mPTP, as detected by fluorescence in a mitochondrial calcium retention assay. Moreover, shows the effect of treatment with cyclosporin A and a known inhibitor of the mPTP on the activity of the mPTP in wild-type and NLRX1 knock-out (KO) cells (see Background Example 2).
  • Figure 4 Shows the effect of a known inhibitor of the mPTP, Test Substance 1, on the Tm (i.e. midpoint value of melting temperature) of NLRX1 in a cellular thermal shift assay (CETSA; see NLRX1 Binding Example 1).
  • Figure 5 Shows the ability of a range of Test Substances to bind and stabilise NLRX1 to varying degrees in a cellular thermal shift assay (CETSA; see NLRX1 Binding Example 1).
  • Figures 6 and 7 Show the ability of two different Probe Substances to specifically bind to NLRX1-expressing membranes to be used in a filtration binding assay according to the present invention. Moreover, show the lack of any specific binding of said Probe Substances to membranes which lack NLRX1 (see NLRX1 Binding Example 2).
  • Figures 8 and 9 Show the ability of two different Test Substances to bind to NLRX1, quantified by a reduction in bioluminescence resonance energy transfer (BRET) signal, in a nano-BRET assay (see NLRX1 Binding Example 3).
  • Figure 10 Shows the effect of an mPTP Inhibitor, upon treatment at a dose of 50 mg/kg or 150 mg/kg, on the levels of the neurofilament-light (NFL) protein, in both the plasma and cerebrospinal fluid (CSF), in the ⁇ NLS TDP43 mouse model of amyotrophic lateral sclerosis (ALS) (see Biological Example 1).
  • NNLS TDP43 cerebrospinal fluid
  • Figures 11 and 12 Show the effect of an mPTP Inhibitor, upon treatment at a dose of 10 mg/kg, 50 mg/kg, or 150 mg/kg, on the levels of the astrocyte activation marker, GFAP, in the plasma in the ⁇ NLS TDP43 mouse model of amyotrophic lateral sclerosis (ALS) (see Biological Example 1).
  • Figure 13 Shows the effect of an mPTP Inhibitor, upon treatment at a dose of 50 mg/kg, on the level of the pro-inflammatory cytokine IL-1 ⁇ , in the brain cortices in the ⁇ NLS TDP43 mouse model of amyotrophic lateral sclerosis (ALS) (see Biological Example 1).
  • Figure 14 Shows the effect of an mPTP Inhibitor, upon treatment at a dose of 50 mg/kg, on the expression of specific mRNA transcripts in the brain cortices, which are differentially expressed between ⁇ NLS TDP43 and wild-type mice, as assessed via RNAseq (see Biological Example 1).
  • Figure 15 Shows a table reporting the effect of an mPTP Inhibitor, upon treatment at a dose of 50 mg/kg, on the expression of specific mRNA transcripts in the brain cortices, which are differentially expressed between ⁇ NLS TDP43 and wild-type mice, as assessed via RNAseq and qPCR analyses (see Biological Example 1).
  • Figure 16 Shows the effect of an mPTP Inhibitor, upon treatment at a dose of 10 mg/kg or 50 mg/kg, on the expression of the Isg15 and GFAP mRNA transcripts, which are differentially expressed between ⁇ NLS TDP43 and wild-type mice (see Biological Example 1).
  • Figure 17 Shows the effect of an mPTP Inhibitor, upon treatment at a dose of 50 mg/kg or 150 mg/kg, on the expression of pro-inflammatory cytokine mRNA transcripts, namely TNF ⁇ , IL-6, IL-1 ⁇ , and CCL2, in the brain cortices of rats treated with pilocarpine, and thus induced into a seizure, as assessed via qPCR (see Biological Example 2).
  • Figure 18 Shows the effect of an mPTP Inhibitor, upon treatment at a dose of 10 mg/kg, 50 mg/kg or 150 mg/kg, on the levels of the neurofilament-light (NFL) protein, in both the plasma and cerebrospinal fluid (CSF), in the pilocarpine-induced rat seizure model (see Biological Example 2).
  • Figure 19 Shows the ability of a radiolabelled Test Substance to bind to wild-type and NLRX1-knockout out coronal brain sections, without specific binding of said radiolabelled Test Substance to brain sections which lack NLRX1 (see NLRX1 Binding Example 4).
  • Figure 20 Shows the saturation curve for binding of a radiolabelled Test Substance to wild- type coronal brain sections (see NLRX1 Binding Example 4).
  • the present invention provides a method for identifying a substance useful for the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity, which method comprises determining whether said substance (i) binds to NLRX1 and (ii) inhibits activity of the mPTP and, if it does, identifying said substance as a substance useful for the prevention or treatment of said disease, disorder, or condition associated with altered NLRX1 activity.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP in particular there is provided a substance which (i) binds NLRX1 and (ii) inhibits the activity of the mPTP.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP which is identified by a method according to the present invention, for example a method described under the sub-heading ‘Methods for Identifying Substances for use according to the present invention’ below. That is, there is provided a substance which (i) binds NLRX1 and (ii) inhibits the activity of the mPTP, which is identified by a method according to the present invention, for example a method described under the sub-heading ‘Methods for Identifying Substances for use according to the present invention’ below.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, for use in the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity there is provided a substance which (i) binds NLRX1 and (ii) inhibits the activity of the mPTP, for use in the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP which is identified by a method according to the present invention, for example a method described under the sub-heading ‘Methods for Identifying Substances for use according to the present invention’ below, for use in the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity.
  • a substance which (i) binds NLRX1 and (ii) inhibits the activity of the mPTP which is identified by a method according to the present invention, for example a method described under the sub-heading ‘Methods for Identifying Substances for use according to the present invention’ below, for use in the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity.
  • the terms ‘therapeutic effect’, ‘treatment’ or ‘treating’ include the control, mitigation, reduction or modulation of the state of the disease, disorder, or condition, or the symptoms of the disease, disorder, or condition.
  • a disease, disorder, or condition associated with altered NLRX1 activity may be defined as any disease, disorder, or condition in which the activity of NLRX1 is thought or is known to contribute to the establishment, progression, maintenance, or symptoms of the disease, disorder, or condition, for example those diseases, disorders, and conditions mentioned herein below.
  • the disease, disorder, or condition associated with altered NLRX1 activity is associated with excessive or undesired activity of NLRX1, which may be defined as any disease, disorder, or condition in which the activity of NLRX1 at a level which exceeds the activity of NLRX1 within a normal range, or its dysregulation, in particular in a healthy individual, is thought or is known to contribute to the establishment, progression, maintenance, or symptoms of the disease, disorder, or condition, for example those diseases, disorders, and conditions mentioned herein below.
  • the mitochondria permeability transition pore mPTP
  • mPTP mitochondria permeability transition pore
  • the binding to NLRX1 is believed to lead to regulatory modulation of NLRX1, for example, activation or inhibition of NLRX1 activity (such as NLRX1 signalling activity), particularly inhibition of NLRX1 activity. Therefore, in any of the above-described aspects of the invention, the substance which binds to NLRX1 is suitably capable of inhibiting the activity of NLRX1, i.e. the substance which binds to NLRX1 is an inhibitor of NLRX1.
  • the present invention provides methods for identifying inhibitors of NLRX1 which are useful for the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity, and further provides inhibitors of NLRX1 activity which have been identified by methods according to the present invention. Therefore, diseases, disorders, or conditions associated with altered NLRX1 activity include (i) diseases, disorders, or conditions associated with activation of the mPTP, and (ii) other diseases, disorders, or conditions associated with altered NLRX1 activity , i.e. but which are not defined herein as diseases, disorders, or conditions associated with activation of the mPTP (herein referred to as “Other NLRX1-Related Diseases, Disorders or Conditions”).
  • a disease, disorder, or condition associated with activation of the mitochondria permeability transition pore may be defined as any disease, disorder, or condition in which the activity of the mPTP at a level which exceeds the activity of the mPTP within a normal range, in particular in a healthy individual, is thought or is known to contribute to the establishment, progression, maintenance, or symptoms of the disease, disorder, or condition, for example those diseases, disorders, and conditions mentioned herein below.
  • the disease, disorder, or condition associated with activation of the mPTP is associated with excessive activity of the mPTP, which may be defined as any disease, disorder, or condition in which the activity of the mPTP at a level which exceeds the activity of the mPTP within a normal range, in particular in a healthy individual, is thought or is known to contribute to the establishment, progression, maintenance, or symptoms of the disease, disorder, or condition, for example those diseases, disorders, and conditions mentioned herein below.
  • the disease, disorder, or condition, which is associated with altered NLRX1 activity is an autoimmune, an acute or chronic inflammatory, an allergic, a degenerative such as a neurodegenerative, a mitochondrial, a respiratory or pulmonary, a cardiovascular, a central nervous system i.e. neurological, a metabolic, an infectious or pathogenic, a hyperproliferative (e.g. cancer), a renal, a hepatic, or a skin, disease, disorder, or condition.
  • a degenerative such as a neurodegenerative, a mitochondrial, a respiratory or pulmonary, a cardiovascular, a central nervous system i.e. neurological, a metabolic, an infectious or pathogenic, a hyperproliferative (e.g. cancer), a renal, a hepatic, or a skin, disease, disorder, or condition.
  • a single disease, disorder, or condition, which is associated with altered NLRX1 activity, and which is associated with activation of the mPTP, or which is an Other NLRX1-Related Disease, Disorder or Condition may fall into one of more of the above categories of disease, disorder, or condition.
  • a disease, disorder, or condition, which is associated with altered NLRX1 activity may simultaneously be an acute or chronic inflammatory disease, disorder, or condition and a respiratory or pulmonary disease, disorder, or condition, e.g. asthma.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is a degenerative such as a neurodegenerative, or a central nervous system i.e.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is a degenerative disease, disorder, or condition such as a neurodegenerative disease, disorder, or condition.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is selected from Parkinson’s disease, dementia with Lewy bodies, Alzheimer’s disease, amyotrophic lateral sclerosis, multiple sclerosis, frontal temporal dementia, chemotherapy induced neuropathy, Huntington’s disease, epilepsy, Charcot-Marie-Tooth disease, multiple system atrophy, spinocerebellar ataxias, progressive supranuclear palsy, hereditary spastic paraplegia, Duchenne muscular dystrophy, congenital muscular dystrophy, inclusion body myositis, traumatic brain injury and Friedreich’s ataxia.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is a degenerative disease, disorder, or condition, which is not a degenerative disease, disorder, or condition, of the central nervous system i.e. neurological disease, disorder, or condition.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is selected from hereditary spastic paraplegia, Duchenne muscular dystrophy, inclusion body myositis, and congenital muscular dystrophy.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is a central nervous system disease, disorder, or condition i.e. is a neurological disease, disorder, or condition.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is a degenerative disease, disorder, or condition, such as a neurodegenerative disease, disorder, or condition of the central nervous system.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is selected from Parkinson’s disease, dementia with Lewy bodies, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, frontal temporal dementia, chemotherapy induced neuropathy, Huntington’s disease, epilepsy, Charcot-Marie-Tooth disease, multiple system atrophy, spinocerebellar ataxias, progressive supranuclear palsy, traumatic brain injury and Friedreich’s ataxia.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is Parkinson’s disease.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is amyotrophic lateral sclerosis.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is a mitochondrial disease, disorder, or condition.
  • the disease, disorder, or condition, which is associated with activation of the mPTP is selected from Reye syndrome, Leber’s hereditary optic neuropathy and associated disorders and disorders, such as those diseases and disorders disclosed in CA2884607A1 (Stealth Peptides International Inc.), which is herein incorporated by reference in its entirety.
  • the disease, disorder, or condition, which is an Other NLRX1-Related Disease, Disorder or Condition is an autoimmune, an acute or chronic inflammatory, an allergic, a respiratory or pulmonary, a cardiovascular, an infectious or pathogenic, a hyperproliferative (e.g. cancer), a renal, a hepatic, or a skin, disease, disorder, or condition.
  • the disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition is an autoimmune disease, disorder, or condition.
  • the disease, disorder, or condition is selected from inflammatory bowel disease (IBD) (e.g., Crohn’s disease and ulcerative colitis), irritable bowel syndrome (IBS), lupus, systemic lupus erythematosus, rheumatoid arthritis, Sjogren’s syndrome, systemic scleroderma, type 1 diabetes mellitus, psoriasis (including psoriatic arthritis), autoimmune encephalitis, sarcoidosis, Guillain-Barre syndrome, Grave’s disease, antiphospholipid syndrome and cancer-immunotherapy-induced autoimmune diseases.
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • lupus systemic lupus erythematosus
  • rheumatoid arthritis Sjogren’s syndrome
  • the disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition is an acute or chronic inflammatory, or an allergic, disease, disorder, or condition.
  • the disease, disorder, or condition is selected from asthma, cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, chronic granulomatous disease, graft versus host disease, tumor necrosis factor receptor associated periodic syndrome, hay fever (seasonal allergies), sinusitis, eczema (atopic dermatitis), contact dermatitis, hives (urticaria), and eosinophilic gastrointestinal disorder.
  • the disease, disorder, or condition, which is an Other NLRX1-Related Disease, Disorder or Condition is a respiratory or pulmonary disease, disorder, or condition.
  • the disease, disorder, or condition is selected from asthma, bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, idiopathic pulmonary fibrosis (IPF), and emphysema.
  • the disease, disorder, or condition, which is an Other NLRX1-Related Disease, Disorder or Condition is a metabolic disease, disorder, or condition.
  • the disease, disorder, or condition is selected from type 1 diabetes mellitus, type 2 diabetes mellitus, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, diabetic ketoacidosis, obesity, and pre-diabetes.
  • the disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition, is an infectious or pathogenic disease, disorder, or condition.
  • the disease, disorder, or condition is selected from a bacterial infection, a viral infection and a fungal infection, and any disease, disorder, or condition resulting from said bacterial, viral or fungal infection e.g.
  • Non-limiting examples of bacterial infection include infections of Bacillus anthracis, Bacillus cereus, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis Campylobacter jejuni Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes,
  • Non-limiting examples of viral infection include infections of viruses in the family adenoviridae; viruses in the family herpesviridae, such as herpes simplex type 1 (HSV1), herpes simplex type 2 (HSV2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus (HHV); viruses in the family papillomaviridae, such as human papillomaviruses; viruses in the family polyomaviridae; viruses in the family poxviridae; viruses in the family hepadnaviridae such as hepatitis B virus (HBV); viruses in the family parvoviridae; viruses in the family astroviridae; viruses in the family caliciviridae; viruses in the family picomaviridae such as coxsackievirus, hepatitis A virus, poliovirus, and rhinovirus; viruses in the family coronavi
  • Non-limiting examples of fungal infections include infection of fungi of the genus Aspergillus, which cause aspergillosis; fungi of the genus Blastomyces, which cause blastomycosis; fungi of the genus Candida, such as Candida albicans, which cause candidiasis; fungi of the genus Coccidioides; fungi of the genus Cryptococcus, such as Cryptococcus neoformans and Cryptococcus gattii, which cause cryptococcosis; dermatophytes fungi, which cause ringworm; fungi of the genus Histoplasma, such as Histoplasma capsulatum, which cause histoplasmosis; fungi of the order Mucorales, which cause mucormycosis; fungi of the genus Saccharomyces, such as Saccharomyces cerevisiae; fungi of the genus Pneumocystis, such as P
  • the disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition is an hyperproliferative disease, disorder, or condition, e.g. cancer.
  • the disease, disorder, or condition is selected from breast cancer, lung cancer, liver cancer e.g. hepatocellular carcinoma, skin cancer e.g.
  • the disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition is a skin disease, disorder, or condition.
  • the disease, disorder, or condition is selected from psoriasis, cutaneous lupus erythematosus, dermatomyositis, pemphigoid, pemphigus, scleroderma, vasculitis, epidermolysis bullosa acquisita, vitiligo, lichen planus, scleritis, dermatitis, erythema nodosum, pyoderma gangrenosum, skin fissures, acne, enterocutaneous fistula, skin tags, canker sores, acrodermatitis enteropathica, pyoderma vegetans, leukocytoclastic vasculitis, anal fissures, Sweet’s syndrome, rosacea, alopecia, keratoderma blennorrhagica, rosacea, cold sores, urticaria, actinic keratosis, carbuncle, cellulitis,
  • a method for identifying a substance useful for the prevention or treatment of a disease, disorder, or condition associated with activation of the mPTP comprises determining whether said substance (i) binds to NLRX1 and (ii) inhibits activity of the mPTP and, if it does, identifying said substance as a substance useful for the prevention or treatment of said disease, disorder, or condition associated with activation of the mPTP.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP which is identified by a method according to the present invention, for example a method described under the sub-heading ‘Methods for Identifying Substances for use according to the present invention’ below, for use in the prevention or treatment of a disease, disorder, or condition associated with activation of the mPTP.
  • a substance which (i) binds NLRX1 and (ii) inhibits the activity of the mPTP which is identified by a method according to the present invention, for example a method described under the sub-heading ‘Methods for Identifying Substances for use according to the present invention’ below, for use in the prevention or treatment of a disease, disorder, or condition associated with activation of the mPTP.
  • the present invention provides a method for identifying a substance useful for the prevention or treatment of a disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition, which method comprises determining whether said substance (i) binds to NLRX1 and (ii) inhibits activity of the mPTP and, if it does, identifying said substance as a substance useful for the prevention or treatment of said disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, for use in the prevention or treatment of a disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition there is provided a substance which (i) binds NLRX1 and (ii) inhibits the activity of the mPTP, for use in the prevention or treatment of a disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP which is identified by a method according to the present invention, for example a method described under the sub- heading ‘Methods for Identifying Substances for use according to the present invention’ below, for use in the prevention or treatment of a disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition.
  • a substance which (i) binds NLRX1 and (ii) inhibits the activity of the mPTP which is identified by a method according to the present invention, for example a method described under the sub- heading ‘Methods for Identifying Substances for use according to the present invention’ below, for use in the prevention or treatment of a disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, for use in the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity is useful for the prevention or treatment of such a disease, disorder, or condition in a mammal, in particular a human. Therefore, a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, for use in the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity, is suitably administered to a mammal, in particular a human.
  • a method for preventing or treating a disease, disorder, or condition associated with altered NLRX1 activity comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, for example wherein the substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP is identified by a method according to the present invention, for example a method described under the sub-heading ‘Methods for Identifying Substances for use according to the present invention’ below.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP for example wherein the substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP is identified by a method according to the present invention, for example a method described under the sub-heading ‘Methods for Identifying Substances for use according to the present invention’ below, in the manufacture of a medicament for preventing or treating a disease, disorder, or condition associated with altered NLRX1 activity.
  • substances which are capable of binding to NLRX1 suitably are substances capable of regulatory modulation of NLRX1, for example activation or inhibition of NLRX1 activity (such as NLRX1 signalling activity), particularly inhibition of NLRX1 activity.
  • the present invention suitably provides substances which are (i) capable of binding to and inhibiting the activity of NLRX1, i.e.
  • the present invention relates to methods for identifying a substance useful for the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity, which method comprises determining whether said substance (i) binds to NLRX1 and (ii) inhibits activity of the mPTP and, if it does, identifying said substance as a substance useful for the prevention or treatment of said disease, disorder, or condition associated with altered NLRX1 activity. That is, the method of the present invention comprises step (i) of determining whether a substance binds to NLRX1, and step (ii) of determining whether a substance inhibits the activity of the mPTP.
  • the method of the present invention is an in vitro method. In an alternative, less preferred, aspect, the method of the present invention is an in vivo method. In one aspect, the method of the present invention comprises step (i) of determining whether a substance binds to NLRX1, wherein the determination of whether a substance binds to NLRX1 is performed by means comprising a thermal shift assay of NLRX1 binding.
  • the thermal shift assay is a cellular thermal shift assay (CETSA). Therefore, in one aspect, the present invention provides an assay method for a substance that binds to NRLX1 which comprises means comprising a thermal shift assay of NLRX1 binding.
  • the present invention provides an assay method for a substance that binds to NRLX1 which comprises means comprising a cellular thermal shift assay (CETSA) of NLRX1 binding.
  • a thermal shift assay measures changes in the thermal denaturation temperature and hence stability of a protein under varying conditions, in particular drug/ligand concentration.
  • the binding of a drug/ligand may increase the thermal stability of a target protein, as described in Koshland, 1958.
  • Measuring the thermal shift of a protein may be achieved via a number of techniques including differential scanning fluorimetry or thermofluor.
  • a cellular thermal shift assay CETSA
  • protein thermal shift/melting curves are generated in intact cells.
  • denatured proteins including a target protein
  • Stable proteins in the resultant supernatant can be identified via a number of techniques including Western Blotting, enzyme-linked immunosorbent assay (ELISA), or mass spectrometry.
  • ELISA enzyme-linked immunosorbent assay
  • the cellular thermal shift assay (CETSA) comprises: (i) contacting a test substance with cells expressing NLRX1 to produce a test sample; (ii) exposing the test sample to a stepwise heat challenge; (iii) calculating the Tm value of the test sample; and (iv) comparing the Tm value of the test sample to the Tm value of a control sample; wherein binding to NLRX1 is indicated by an increased Tm value of the test sample relative to the Tm value of a control sample.
  • step (ii) of exposing the test sample to a stepwise heat challenge comprises exposing samples to a 3 step, for example a 4 step, for example a 5 step, for example a 6 step, for example an 8 step, for example a 10 step, for example a 12 step heat challenge.
  • a stepwise heat challenge is a process by which a test sample, typically via separation into aliquots, is exposed to a series of specific temperatures within a defined range.
  • a 3 step heat challenge involves exposure of a test sample to 3 different temperatures within a defined range.
  • the heat challenge of step (ii) is performed between 40 and 100 o C, such as between 45 and 85 o C, such as between 50 and 75 o C.
  • Step (iii) of calculating the Tm value i.e. the temperature at which 50% of melting is achieved, of test samples is performed firstly by quantifying the amount of melted NLRX1 in the test sample at each step of the stepwise heat challenge, plotting said amounts against temperature, and calculating the temperature at which 50% of melting is achieved.
  • step (iii) of calculating the Tm value of the test sample further comprises a process of Western Blotting, in particular wherein NLRX1 (e.g. melted NLRX1) is targeted with an anti-NLRX1 antibody and said anti-NLRX1 antibody is targeted with a secondary antibody conjugated to horseradish peroxidase.
  • the activity of the horseradish peroxidase which represents the amount of NLRX1 (e.g. melted NLRX1) in a test sample, is measured via chemiluminescence.
  • NLRX1 e.g. melted NLRX1
  • said anti-NLRX1 antibody is targeted with a secondary antibody conjugated to a dye, such as a fluorescent dye, in particular an infra-red or near-infra-red dye.
  • a dye such as a fluorescent dye, in particular an infra-red or near-infra-red dye.
  • the fluorescence of the dye such as the infra-red or near-infra-red dye, which represents the amount of NLRX1 (e.g.
  • step (iv) of comparing the Tm value of the test sample to the Tm value of a control sample comprises comparison to a control sample to which no test substance was added and/or comparison to a control sample to which vehicle, or an alternative known inactive test substance was added.
  • the Tm value of test samples is increased, relative to the control sample, by 0.5 o C or more, such as by 0.75 o C or more, such as by 1 o C or more, such as by 1.2 o C or more, such as by 1.5 o C or more.
  • the method of the present invention comprises step (i) of determining whether a substance binds to NLRX1, wherein the determination of whether a substance binds to NLRX1 is performed by means comprising a filtration binding assay of NLRX1 binding.
  • the present invention provides an assay method for a substance that binds to NRLX1 which comprises means comprising a filtration binding assay of NLRX1 binding.
  • a filtration binding assay measures the binding affinity between two molecules by means of a method involving a filtration step which separates bound from unbound molecules.
  • a first molecule typically a protein (and in this case NLRX1)
  • a second molecule typically a ligand or DNA.
  • the amount of ligand bound to the protein can then be quantified by filtration-based separation of the protein from the unbound ligand.
  • the first molecule, i.e. protein is retained on the filter following filtration and only the second molecule, i.e. ligand, bound to the first molecule, i.e. protein, is retained, whilst unbound second molecule, i.e.
  • second molecule i.e. ligand
  • first molecule i.e. protein
  • second molecule i.e. ligand
  • this method may involve use of a radioisotope labelled second molecule, i.e. ligand (radioligand), with detection achieved via liquid scintillation.
  • the second molecule i.e. ligand
  • the second molecule may be fluorescently labelled and detection of bound ligand may be achieved via chemiluminescence.
  • the binding affinity of the second molecule, i.e. ligand, for the first molecule, i.e. protein can be determined.
  • a first molecule typically a protein (and in this case NLRX1)
  • the second test molecule typically a ligand or DNA
  • the filter is only retained by the filter upon specific binding to the first molecule. This specific binding may be ascertained via a number of different techniques, such as mass spectrometry or fluorescent labelling or radioisotope labelling and measurement via liquid scintillation.
  • the filtration binding assay according to the present invention comprises: (i) contacting a test substance with NLRX1, to produce a test sample; (ii) filtering the test sample through a filter, on which NLRX1 is retained; (iii) detecting, and optionally quantifying, the amount of test substance which is retained on the filter; wherein binding to NLRX1 is indicated by detection, and optional quantification, of test substance retained on the filter.
  • the filtration binding assay according to the present invention comprises: (i) contacting a probe substance, which binds to NLRX1, with membranes on which NLRX1 is present; (ii) filtering test substances through a filter, in the presence of said membranes on which NLRX1 is present; and (iii) detecting, and optionally quantifying, the amount of test substance binding to membranes on which NLRX1 is present, or detecting, and optionally quantifying, the amount of probe substance displaced from binding to membranes on which NLRX1 is present; wherein binding to NLRX1 is indicated by detection, and optional quantification, of test substance binding to membranes on which NLRX1 is present or detection, and optional quantification, of probe substance displaced from binding to membranes on which NLRX1 is present.
  • membranes on which NLRX1 is present are membranes expressing NLRX1, for example which have been produced from cells expressing NLRX1.
  • membranes on which NLRX1 is present are membranes onto which NLRX1 is attached.
  • step (ii) of filtering the test sample through a filter, on which NLRX1 is retained, or filtering test substances through said membranes on which NLRX1 is present comprises filtration via centrifugation.
  • step (ii) of filtering the test sample through a filter, on which NLRX1 is retained or filtering test substances through said membranes on which NLRX1 is present comprises vacuum-based aspiration.
  • step (iii) of detecting, and optionally, quantifying the amount of test substance which is retained on the filter is achieved via mass spectrometry, for example using a process of liquid chromatography, for example, ultra high-performance liquid chromatography, in particular using a C18 column; Poroshell 120 E-C18, Agilent, coupled to triple quadrupole mass spectrometer.
  • the filter used in the aforementioned filtration binding assays may comprise glass fibre paper or nitrocellulose paper, in particular glass fibre paper.
  • step (iii) of detecting, and optionally, quantifying the amount of test substance binding to membranes on which NLRX1 is present, or detecting, and optionally quantifying, the amount of probe substance displaced from binding to membranes on which NLRX1 is present comprises detecting, and optionally quantifying, the amount of probe substance displaced from binding to membranes on which NLRX1 is present.
  • step (iii) comprises detecting, and optionally quantifying, the amount of probe substance displaced from binding to membranes on which NLRX1 is present
  • said step comprises a process of liquid chromatography, for example, ultra high-performance liquid chromatography, in particular using a C18 column; Poroshell 120 E-C18, Agilent, coupled to triple quadrupole mass spectrometer.
  • binding to NLRX1 is indicated by detection, and optional quantification, of test substance retained on the filter on which NLRX1 is retained. More suitably, binding to NLRX1 is indicated by detection, and optional quantification, of an increased amount of test substance retained on the filter on which NLRX1 is retained relative to a control sample.
  • a control sample used in the filtration binding assay described above is a sample to which no test substance was added or to which vehicle, or an alternative known inactive test substance was added.
  • binding to NLRX1 is detected, either via detection of an increased amount of test substance retained on the filter on which NLRX1 is retained at an amount that is in accordance with one or more of the suitable EC50, IC50, pIC50, and Ki values described below.
  • binding to NLRX1 is indicated by detection, and optional quantification, of test substance binding to membranes on which NLRX1 is retained or is present.
  • binding to NLRX1 is indicated by detection, and optional quantification, of probe substance displaced from binding to membranes on which NLRX1 is present.
  • binding to NLRX1 is indicated by detection, and optional quantification, of an increased amount of test substance binding to membranes on which NLRX1 is present or detection, and optional quantification, of an increased amount of probe substance displaced from binding to membranes on which NLRX1 is present, relative to a control sample.
  • a control sample used in the filtration binding assay described above is a sample to which no test substance was added or to which vehicle, or an alternative known inactive test substance was added.
  • binding to NLRX1 is detected either via detection of an increased amount of test substance binding to membranes on which NLRX1 is present or detection of an increased amount of probe substance displaced from binding to membranes on which NLRX1 is present, is specific binding to NLRX1.
  • said specific binding is identified via exposure of test substances to NLRX1 expressing membranes in the presence of an excess concentration of a competing compound, or via exposure of test substances to NLRX1 knock-out cell membranes, or via exposure of said test substances to alternative proteins to which they do not bind.
  • binding to NLRX1 is detected, either via detection of an increased amount of test substance binding to membranes on which NLRX1 is present or detection of an increased amount of probe substance displaced from binding to membranes on which NLRX1 is present, at an amount that is in accordance with one or more of the suitable EC50, IC50, pIC50, and Ki values described below.
  • the method of the present invention comprises step (i) of determining whether a substance binds to NLRX1, wherein the determination of whether a substance binds to NLRX1 is performed by means comprising a proximity assay of NLRX1 binding.
  • the proximity assay is a bioluminescence resonance energy transfer (BRET) assay, and in particular is a nano-BRET assay, of NLRX1 binding.
  • the present invention provides an assay method for a substance that binds to NRLX1 which comprises means comprising a proximity assay of NLRX1 binding.
  • the present invention provides an assay method for a substance that binds to NRLX1 which comprises means comprising a bioluminescence resonance energy transfer (BRET) assay, and in particular a nano-BRET assay, of NLRX1 binding.
  • BRET bioluminescence resonance energy transfer
  • a proximity assay directly detects interaction of proteins, extracellular vesicles, and other cellular components.
  • two different primary antibodies are typically raised, in different animal species, against two proteins or cellular components of interest.
  • secondary antibodies are raised which target the constant region of said primary antibodies.
  • Each of the secondary antibodies is typically conjugated to a short, sequence-specific DNA molecule.
  • the DNA strands conjugated to the secondary antibodies can participate in rolling circle DNA synthesis upon subsequent addition of appropriate substrates and enzymes.
  • fluorescently labelled complementary oligonucleotides can be added, which binds to amplified DNA, and omit levels of fluorescence which may be detected. It will be understood that proximity assays may be adapted to use alternative mechanisms for generating fluorescent signals upon two proteins or cellular components of interest coming into close proximity.
  • one secondary antibody may be conjugated to a fluorophore donor, and the other secondary antibody may be conjugated to a fluorophore acceptor.
  • a BRET assay is a specific example of a proximity assay which takes advantage of the natural phenomenon of dipole-dipole energy transfer from a luminescence donor to a fluorophore acceptor.
  • a first protein or cellular component of interest is conjugated to a luminescence donor, typically an enzyme such as luciferase
  • a second protein or cellular component of interest is conjugated to a fluorophore acceptor, such as a fluorescent protein such as green-fluorescent protein (GFP), or yellow-fluorescent protein (YFP).
  • GFP green-fluorescent protein
  • YFP yellow-fluorescent protein
  • a nano-BRET assay is a further limitation to the BRET assay in which a smaller, brighter, and more stable luciferase (nano-luciferase) is used resulting in a high fluorescence emission intensity and improved spectral resolution, which in turn improves the sensitivity and range of existing BRET assays.
  • the BRET or nano-BRET assay according to the present invention comprises: (i) contacting a test substance with cells expressing an NLRX1-luciferase construct to produce a test sample; (ii) contacting a tracer molecule, which comprises a substance known to bind to NLRX1 and a substance which is required for luciferase activity, with the test sample; and (iii) contacting luciferase substrate with the test sample; and (iv) quantifying the luciferase signal of the test sample; wherein binding to NLRX1 is indicated by a decrease in the luciferase signal relative to a control sample.
  • the BRET or nano-BRET assay according to the present invention comprises: (i) contacting a test substance with cells expressing an NLRX1-luciferase construct to produce a test sample; (ii) contacting a tracer molecule, which comprises a substance known to bind to NLRX1 and a substance which is required for luciferase activity, with the test sample; and (iii) contacting luciferase substrate with the test sample; and (iv) quantifying the light emission signal of the NLRX1-luciferase test sample and the fluorescence of the tracer molecule; wherein binding to NLRX1 is indicated by a change in the BRET ratio relative to a control sample.
  • a control sample used in the BRET or nano-BRET assay described above is a sample to which no test substance was added or to which vehicle, or an alternative known inactive test substance was added.
  • cells expressing an NLRX1-luciferase construct are provided via transfection of cells with an NLRX1-luciferase construct comprising NLRX1 conjugated to luciferase, such as a nano-luciferase, at the C-terminus.
  • the tracer molecule of step (ii) comprises a substance known to bind to NLRX1 conjugated via click chemistry to NanoBRETTM 590-Azide-C3 (Promega).
  • NanoBRETTM 590-Azide-C3 reagent uses thereof, and assays using said reagents, are further described, in addition to further suitable reagents, in US Patent No. 10,024,862, US Patent No. 10,067,149, and EP2932267, each of which is herein incorporated by reference in its entirety.
  • a BRET or nano-BRET assay according to the present invention in particular wherein said assay utilises the NanoBRETTM 590-Azide-C3 (Promega) reagent, is performed in accordance with the assays described in one or more of US Patent No.10,024,862, US Patent No.10,067,149, and EP293226.
  • step (iv) of quantifying the light emission of the luciferase signal of the test sample and/or the fluorescence of the tracer molecule is performed using a CLARIOstar reader.
  • step (iv) of quantifying the light emission of the luciferase signal of test samples and/or the fluorescence of the tracer molecule is performed via detection at a wavelength of about 470 nm and about 615 nm in order to calculate the BRET ratio (acceptor ca 615 nm emission/donor ca 470 nm emission).
  • BRET ratio acceptor ca 615 nm emission/donor ca 470 nm emission
  • the method of the present invention comprises step (ii) of determining whether a substance inhibits the activity of the mPTP, wherein the determination of whether a substance inhibits the activity of the mPTP is performed by means comprising a mitochondrial calcium retention assay.
  • pharmacological inhibition or modulation of the mPTP can be measured in such a mitochondrial calcium retention assay since, in vitro, isolated mitochondria rapidly sequester exogenous calcium (Ca2+) until the intramitochondrial Ca2+ concentration reaches the threshold for mPTP activation.
  • the mitochondrial calcium retention assay is a rat liver mitochondrial calcium retention assay.
  • the mitochondrial calcium retention assay is a rat brain mitochondrial calcium retention assay.
  • the mitochondrial calcium retention assay is human platelet mitochondrial calcium retention assay.
  • mitochondrial calcium retention assay is a rat liver mitochondrial calcium retention assay
  • mitochondria are suitably isolated from female rats, such as female Sprague Dawley rats, using the following method: Cervical dislocation is performed on the female rat, prior to perfusing the liver of said female rat, in situ, with between 20 and 60 mL, such as between 30 and 50 mL, such as about 40 mL of a saline solution, and preferably Dulbecco’s Phosphate Buffered Saline (DPBS).
  • DPBS Dulbecco’s Phosphate Buffered Saline
  • the liver is subsequently dissected and transferred into between 10 and 50 mL, such as between 20 and 40 mL, such as about 30 mL, of an isolation buffer, suitable examples of which are known to the skilled person.
  • a particularly suitable isolation buffer comprises 250 mM sucrose, 10 mM potassium chloride, 1 mM ethylene glycol-bis( ⁇ -aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM ethylene diaminetetraacetic acid (EDTA), and 25 mM HEPES, wherein the pH of said isolation buffer is adjusted to 7.5 using 1 M sodium hydroxide.
  • the liver is then removed from the buffer and minced into pieces of between 1 and 10 mm, in particular about 5 mm, prior to transfer into a homogenization tube, such as a 50 mL Potterton dounce homogenization tube, comprising between 10 and 50 mL, such as about 30 mL homogenization/centrifugation buffer, suitable examples of which are known to the skilled person.
  • a homogenization tube such as a 50 mL Potterton dounce homogenization tube, comprising between 10 and 50 mL, such as about 30 mL homogenization/centrifugation buffer, suitable examples of which are known to the skilled person.
  • a particularly suitable homogenization/centrifugation buffer comprises 300 mM trehalose, 25 mM HEPES, 1 mM EGTA, 1 mM EDTA, and 10 mM potassium chloride, wherein the pH of said homogenization/centrifugation buffer is adjusted to 7.5 using 1 M sodium hydroxide, and wherein said homogenization/centrifugation buffer is supplemented with 0.1% bovine serum albumin (BSA) and complete protease inhibitor cocktail (one tablet per 50 mL buffer). Homogenization is suitably carried out using a Teflon® pestle at between 1500 and 2500 rpm, such as 1600 and 2000 rpm, such as at about 1800 rpm.
  • BSA bovine serum albumin
  • Teflon® pestle at between 1500 and 2500 rpm, such as 1600 and 2000 rpm, such as at about 1800 rpm.
  • the homogenization product is centrifuged at between 500 and 1500 g, such as between 600 and 100 g, such as at about 800 g, for between 1 and 20 minutes, such as between 2 and 16 minutes, such as between 5 and 15 minutes, such as for about 10 minutes, at a temperature of 1-10oC, such as 2-8oC, such as 2-5oC, such as at about 4oC.
  • the resulting supernatant was centrifuged at between 5000 and 20000 g, such as between 6000 and 16000 g, such as between 8000 and 12000 g, such as at about 10000 g, for between 1 and 20 minutes, such as between 2 and 16 minutes, such as between 5 and 15 minutes, such as for about 10 minutes, at the same temperature as above.
  • the resultant pellet was washed once with an assay buffer, suitably FLIPR assay buffer comprising 75 mM mannitol, 25 mM sucrose, 5 mM potassium phosphate monobasic, 20 mM Tris, 100 mM potassium choride, and 0.1% BSA, adjusted to pH 7.4 using hydrochloric acid, before being centrifuged again and subsequently resuspended in said assay buffer, in particular FLIPR assay buffer, to a target protein concentration of between 5 and 15 mg/mL, such as 6 and 12 mg/mL, such as 7.5 to 10 mg/mL, and most preferably of about 8.8. mg/mL.
  • an assay buffer suitably FLIPR assay buffer comprising 75 mM mannitol, 25 mM sucrose, 5 mM potassium phosphate monobasic, 20 mM Tris, 100 mM potassium choride, and 0.1% BSA, adjusted to pH 7.4 using hydrochloric acid, before being centrifuge
  • mitochondria are suitably isolated from rats, such as female rates, such as female Sprague Dawley rats, using the following method: Anaesthetized rats, which may be performed via methods known in the art, are suitably perfused, in situ, with between 20 and 60 mL, such as between 30 and 50 mL, such as about 40 mL of a saline solution, and preferably Dulbecco’s Phosphate Buffered Saline (DPBS). The brain is subsequently dissected and transferred into between 10 and 50 mL, such as between 20 and 40 mL, such as about 30 mL, of an isolation buffer, suitable examples of which are known to the skilled person.
  • DPBS Dulbecco’s Phosphate Buffered Saline
  • a particularly suitable isolation buffer comprises 225 mM mannitol, 75 mM sucrose, and 1 mM ethylene glycol-bis( ⁇ -aminoethyl ether)-N,N,N′,N′- tetraacetic acid (EGTA), wherein the pH of said isolation buffer is adjusted to 7.4 using 1 M sodium hydroxide.
  • the brain is then removed from the buffer and minced into pieces of between 1 and 10 mm, in particular about 5 mm, prior to transfer into a homogenization tube, such as a 50 mL Potterton dounce homogenization tube, comprising between 5 and 25 mL, such as about 10 mL homogenization/centrifugation buffer, suitable examples of which ware known to the skilled person.
  • a homogenization tube such as a 50 mL Potterton dounce homogenization tube, comprising between 5 and 25 mL, such as about 10 mL homogenization/centrifugation buffer, suitable examples of which ware known to
  • a particularly suitable homogenization/centrifugation buffer comprises 300 mM trehalose, 25 mM HEPES, 1 mM EGTA, 1 mM EDTA, and 10 mM potassium chloride, wherein the pH of said homogenization/centrifugation buffer is adjusted to 7.5 using 1 M sodium hydroxide, and wherein said homogenization/centrifugation buffer is supplemented with 0.1% bovine serum albumin (BSA) and complete protease inhibitor cocktail (one tablet per 50 mL buffer). Homogenization is suitably carried out using a Teflon® pestle at between 1500 and 2500 rpm, such as 1600 and 2000 rpm, such as at about 1800 rpm.
  • BSA bovine serum albumin
  • Teflon® pestle at between 1500 and 2500 rpm, such as 1600 and 2000 rpm, such as at about 1800 rpm.
  • the homogenization product is centrifuged at between 500 and 5000 g, such as between 1000 and 2500 g, such as at about 2000 g, for between 1 and 20 minutes, such as between 2 and 16 minutes, such as between 5 and 15 minutes, such as for about 10 minutes, at a temperature of 1-10 o C, such as 2-8 o C, such as 2-5 o C, such as at about 4 o C.
  • the resulting supernatant was centrifuged at between 5000 and 20000 g, such as between 6000 and 16000 g, such as between 8000 and 14000 g, such as at about 12000 g, for between 1 and 20 minutes, such as between 2 and 16 minutes, such as between 5 and 15 minutes, such as for about 9 minutes, at the same temperature as above.
  • the resultant pellet is suitably resuspended in an isolation buffer, for example as described above, but with the further addition of about 0.02% digitonin, prior to centrifugation at between 5000 and 20000 g, such as between 6000 and 16000 g, such as between 8000 and 14000 g, such as at about 12000 g, for between 1 and 20 minutes, such as between 2 and 16 minutes, such as between 5 and 15 minutes, such as for about 11 minutes, at the same temperature as above, followed by subsequent resuspension in 1-10 mL, such as 2-8 mL, such as about 5 mL, isolation buffer, preferably wherein the isolation buffer has the composition described above but wherein the EGTA concentration is reduced to about 0.1 mM.
  • test substances are prepared, typically from stocks in 10 mM dimethyl sulfoxide (DMSO), via serial dilution in DMSO in half- log steps to produce a desire number of test concentrations, suitably in the range 30 ⁇ M to 1 nM. Simultaneously, an intermediate dilution of 1-10 ⁇ L, such as about 5 ⁇ L, of DMSO samples in 50-500 ⁇ L, such as 100-400 ⁇ L, such as 200-300 ⁇ L, or about 250 ⁇ L, assay buffer.
  • DMSO dimethyl sulfoxide
  • Suitable assay buffers are known to the skilled person and may include FLIPR assay buffer, which has the composition described above.5 ⁇ L samples were then added into duplicated wells of a multi-well plate, preferably a 384-well plate. Control wells may suitably contain 0.5% (v/v) DMSO and 5 ⁇ M cyclosporin A. Next, a stock assay solution was prepared via addition of 0.1-2 mL, such as 0.5 -1.5 mL, such as about 1 mL, of the mitochondrial sample solution (derived from liver or brain) to 2-10 mL, such as 4-8 mL, such as 5-6 mL, and in particular about 5.6 mL, assay buffer.
  • the assay buffer is FLIPR assay buffer with the composition described above supplemented with 10 mM succinate disodium salt, 1 ⁇ M rotenone and 2 ⁇ M Fluo5N pentapotassium salt (Invitrogen).
  • the assay buffer is comprises 120 mM mannitol, 40 mM MOPS, 5 mM monopotassium phosphate, 60 mM potassium chloride, 10 mM pyruvate, 2 mM malate, 2 mM magnesium chloride, 20 ⁇ M ADP, 1.26 ⁇ M oligomycin A, supplemented with 2 ⁇ M Fluo5N pentapotassium salt (Invitrogen).
  • a 5-25 ⁇ L, such as 15 ⁇ L, sample is then transferred into the wells of the multi-well plate (which also contain the test substances), prior to incubation for between 1 and 20 minutes, such as between 5 and 15 minutes, such as for about 10 minutes, at room temperature.
  • Multi-well plates are then transferred to a plate reader, a particularly suitable example of which is a FLIPR® Tetra Plate Reader.
  • Dye fluorescence is suitably read every 1 to 5 seconds, such as 3 seconds, for between 1 and 20 minutes, such as between 5 and 15 minutes, such as for about 10 minutes.
  • a 1-5 ⁇ L, such as 2.5 ⁇ L, bolus of calcium chloride (75 ⁇ M) was added from a source comprising calcium chloride, in particular at a concentration of 675 ⁇ M, in assay buffer, preferably FLIPR assay buffer.
  • assay buffer preferably FLIPR assay buffer.
  • the assay is instead performed using a mitochondrial membrane potential flow cytometry assay in stimulated human platelets, since stimulation of platelets results in the rapid influx of Ca 2+ across the platelet membrane.
  • human platelets are suitably harvested and isolated using the following method: A volume of human blood, typically between 5-50 mL, such as 10-40 mL, such as 15-25 mL, such as about 20 mL, is collected from a consenting donor, and added to a solution of sodium citrate, in particular 3.2% sodium citrate. The sample is centrifuged at between 10 and 500 g, such as between 50 and 400 g, such as between 100 and 300 g, such as about 200 g, for between 5 and 30 minutes, such as between 10 and 25 minutes, such as for about 20 minutes, at room temperature. The platelet rich plasma layer is then transferred to a new, i.e. clean, vessel.
  • Prostaglandin 12 is added to the platelet sample at a concentration of 1-40 ng/mL, such as 5-30 ng/mL, such as 10-25 ng/mL, such as about 20 ng/mL.
  • the sample is centrifuged at between 100 and 1000 g, such as between 200 and 800 g, such as between 500 and 750 g, such as at about 640 g, for between 1 and 20 minutes, for example 5 to 15 minutes, for example for about 10 minutes.
  • the resultant pellet is resuspended in 1-10 mL, such as 2-5 mL, such as about 4 mL, of an assay buffer, and preferably HEPES buffer which comprises 137 mM sodium chloride, 2.7 mM potassium chloride, 11.9 mM sodium bicarbonate, 0.42 mM monosodium phosphate, 1 mM magnesium chloride, 5.5 mM glucose, 0.1% BSA, and 10 mM HEPES, adjusted to pH 7.4, before storage on ice.
  • HEPES buffer which comprises 137 mM sodium chloride, 2.7 mM potassium chloride, 11.9 mM sodium bicarbonate, 0.42 mM monosodium phosphate, 1 mM magnesium chloride, 5.5 mM glucose, 0.1% BSA, and 10 mM HEPES, adjusted to pH 7.4, before storage on ice.
  • mitochondrial calcium retention assay is a human platelet mitochondrial calcium retention assay test substances are prepared, typically from stocks in 10 mM DMSO, via serial dilution into an assay buffer, examples of which are known to the skilled person, suitably comprising 0.1-0.5%, such as about 0.4%, DMSO, prior to addition of 10-50 ⁇ L, such as 20- 30 ⁇ L, such as about 25 ⁇ L samples to wells of a multi-well plate, preferably a 96-well plate.
  • an assay buffer examples of which are known to the skilled person, suitably comprising 0.1-0.5%, such as about 0.4%, DMSO, prior to addition of 10-50 ⁇ L, such as 20- 30 ⁇ L, such as about 25 ⁇ L samples to wells of a multi-well plate, preferably a 96-well plate.
  • nM mitochondrial membrane potential dye DiOC6(3) (3,3’-dihexyloxacarbocyanine Iodide; Invitrogen)
  • DiOC6(3) 3,3’-dihexyloxacarbocyanine Iodide; Invitrogen
  • nM mitochondrial membrane potential dye
  • 20-400 nM such as 50-300 nM, such as 100-250 nM, such as about 200 nM, for between 5 and 60 minutes, for example between 15 and 45 minutes, for example for about 30 minutes
  • 5-250 ⁇ L such as 10-100 ⁇ L, such as 20-80 ⁇ L, such as 40-60 ⁇ L, such as about 50 ⁇ L, of the platelet sample to each well of a multi-well plate, preferably a 96-well plate.
  • Control wells may suitably contain 0.1% DMSO only, or 5 ⁇ M cyclosporin A.
  • the wells were incubated for between 5 and 30 minutes, such as between 10 and 20 minutes, such as for about 15 minutes.
  • platelets were stimulated upon the addition of an assay buffer, suitable examples of which are known by the skilled person, further comprising calcium chloride, alpha thrombin, and convulxin to achieve final concentrations of 1-5 nM, e.g. 2 nM calcium chloride; 0.01 to 0.03, e.g. about 0.017 U/mL alpha thrombin; and 0.1 to 0.3, e.g. about 0.167 ⁇ g/mL convulxin.
  • the samples were incubated for between 1 and 20 minutes, such as between 5 and 18 minutes, such as between 10 and 15 minutes, for example for about 14 minutes before the reaction was then stopped upon addition of 5-50 ⁇ L, such as 10-40 ⁇ L, such as 20-30 ⁇ L, such as about 25 ⁇ L, EDTA, suitably at a concentration of 5-25 mM, such as 10-20 mM, such as about 15 mM.
  • the mitochondrial membrane potential across the population of platelets is suitably quantified via flow cytometry, for example using a Guava® easyCyte 5 Benchtop Flow Cytometer, with 1000 to 5000, such as 2000 to 4000, such as about 3000 events per well.
  • the method of the present invention comprises step (i) of determining whether a substance binds to NLRX1, wherein the determination of whether a substance binds to NLRX1 is performed by means comprising a thermal shift assay of NLRX1 binding, and step (ii) of determining whether a substance inhibits the activity of the mPTP, wherein the determination of whether a substance inhibits the activity of the mPTP is performed by means comprising a mitochondrial calcium retention assay.
  • the method of the present invention comprises step (i) of determining whether a substance binds to NLRX1, wherein the determination of whether a substance binds to NLRX1 is performed by means comprising a thermal shift assay of NLRX1 binding, wherein the thermal shift assay is a cellular thermal shift assay (CETSA), and step (ii) of determining whether a substance inhibits the activity of the mPTP, wherein the determination of whether a substance inhibits the activity of the mPTP is performed by means comprising a mitochondrial calcium retention assay.
  • CETSA cellular thermal shift assay
  • the method of the present invention comprises step (i) of determining whether a substance binds to NLRX1, wherein the determination of whether a substance binds to NLRX1 is performed by means comprising a filtration binding assay of NLRX1 binding, and step (ii) of determining whether a substance inhibits the activity of the mPTP, wherein the determination of whether a substance inhibits the activity of the mPTP is performed by means comprising a mitochondrial calcium retention assay.
  • the method of the present invention comprises step (i) of determining whether a substance binds to NLRX1, wherein the determination of whether a substance binds to NLRX1 is performed by means comprising a proximity assay of NLRX1 binding, and step (ii) of determining whether a substance inhibits the activity of the mPTP, wherein the determination of whether a substance inhibits the activity of the mPTP is performed by means comprising a mitochondrial calcium retention assay.
  • the method of the present invention comprises step (i) of determining whether a substance binds to NLRX1, wherein the determination of whether a substance binds to NLRX1 is performed by means comprising a proximity assay of NLRX1 binding, wherein the proximity assay is a bioluminescence resonance energy transfer (BRET) assay, and step (ii) of determining whether a substance inhibits the activity of the mPTP, wherein the determination of whether a substance inhibits the activity of the mPTP is performed by means comprising a mitochondrial calcium retention assay.
  • BRET bioluminescence resonance energy transfer
  • the method of the present invention comprises step (i) of determining whether a substance binds to NLRX1, wherein the determination of whether a substance binds to NLRX1 is performed by means comprising a proximity assay of NLRX1 binding, wherein the proximity assay is a nano-BRET assay, and step (ii) of determining whether a substance inhibits the activity of the mPTP, wherein the determination of whether a substance inhibits the activity of the mPTP is performed by means comprising a mitochondrial calcium retention assay.
  • the mitochondrial calcium retention assay of any of the above aspects is a rat liver mitochondrial calcium retention assay, or a rat brain mitochondrial calcium retention assay, or a human platelet mitochondrial calcium retention assay.
  • Characteristics of Substances for use according to the present invention provides for substances which are (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, for use in the treatment of disease, disorder, or condition associated with altered NLRX1 activity, and methods for identifying said substances.
  • a substance which inhibits mPTP activity may be defined as any molecules which are able to directly inhibit the activity of the mPTP via any direct interaction with the mPTP.
  • These substances also include substances which directly inhibit the activity of cellular components which function upstream or downstream of the mPTP, consequently inhibiting mPTP activity.
  • Inhibitors of mPTP activity include substances which directly or indirectly inhibit the activity or function, or the expression of the mPTP or any upstream or downstream components.
  • inhibitors, of the mPTP may directly or indirectly inhibit the activity or function of the mPTP.
  • the substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP is a modulator of NLRX1, for example an activator or an inhibitor of NLRX1 activity (such as NLRX1 signalling activity), in particular an inhibitor of NLRX1 activity.
  • a substance which modulates, activates, or inhibits nucleotide- binding oligomerization domain, leucine rich repeat containing X1 protein (NLRX1) activity, a substance with NLRX1 modulatory, activatory, or inhibitory activity, or an NLRX1 modulator, activator, or inhibitor, or variations thereof, may be defined as any molecules which are able to directly modulate, activate, or inhibit the activity of NLRX1 through interaction with NLRX1. These molecules are to be differentiated from indirect modulators, activators, or inhibitors of NLRX1, that is molecules which directly modulates, activate, or inhibit the activity of cellular components which function upstream or downstream of NLRX1, consequently modulating, activating, or inhibiting NLRX1 activity.
  • Modulators, activators, or inhibitors of NLRX1 activity include molecules which directly modulate, activate, or inhibit the activity or function, or the expression of NLRX1.
  • modulators, activators, or inhibitors, of NLRX1 directly modulate, activate, or inhibit the activity or function of NLRX1.
  • Modulators, activators, or inhibitors of NLRX1 activity will thus bind NLRX1, typically non-covalently.
  • the substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, for example which is suitably identified by a method according to the present invention is a small molecule.
  • small molecule defines a molecule with a molecular weight of about 1500 g/mol or less, for example about 1200 g/mol or less, for example about 1000 g/mol or less, such as about 900 g/mol or less, such as about 800 g/mol or less, such as about 750 g/mol or less.
  • a small molecule has a molecular weight of about 50 g/mol or more, for example about 100 g/mol or more, for example about 150 g/mol or more, for example about 200 g/mol or more, such as about 250 g/mol or more, such as about 300 g/mol or more.
  • a small molecule may have a molecular weight of about 50-1500 g/mol, for example about 100-1500 g/mol, for example about 200-1200 g/mol, for example about 250-1000 g/mol.
  • the half maximal effective concentration EC50 is a measure of the concentration of a drug required to induce a particular biological or biochemical response. Specifically, EC50 is a quantitative measure that indicates how much of a substance is needed to obtain 50% of a given biological response, for example NLRX1 binding.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP binds to NLRX1 with an EC50 value of less than about 100 ⁇ M, for example less than about 75 ⁇ M, for example less than about 50 ⁇ M, for example less than about 25 ⁇ M, for example less than about 15 ⁇ M, or less than about 10 ⁇ M.
  • the substance suitably binds to NLRX1 with an EC50 value of 0.000001-100 ⁇ M, such as 0.000001-75 ⁇ M, such as 0.000001-50 ⁇ M, such as 0.00001-50 ⁇ M, 0.0001-50 ⁇ M, 0.001-50 ⁇ M, 0.01-50 ⁇ M, 0.1-50 ⁇ M or 1-50 ⁇ M.
  • the half maximal inhibitory concentration (IC50) is a measure of the potency of a substance in inhibiting a biological or biochemical function. Specifically, IC50 is a quantitative measure that indicates how much of a substance is needed to inhibit, in vitro, a given biological process or biological component, for example mPTP activity, by 50%.
  • the IC50 value is the concentration of competing substance that is required to displace 50% of the specific binding of a probe molecule, such as a radioligand, which may be measured via, for example, liquid scintillation.
  • the pIC50 value is the negative log of the IC50 value of an inhibitor, and this logarithmic measure may be used since it more accurately reflects the logarithmic nature of potency values/concentrations. For example, an IC50 value of 1 ⁇ M corresponds to a pIC50 of 6.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP binds to NLRX1 with a pIC50 value of greater than about 4.0, for example greater than about 5.0, in particular greater than about 6.0, for example greater than about 7.0.
  • the substance suitably binds to NLRX1 with a pIC50 value between 4.0 and 9.0, such as between 5.0 and 9.0, such as between 5.5 and 9.0, such as between 5.5 and 8.5, such as between 6.0 and 8.0.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP inhibits the activity of the mPTP with a pIC50 value of greater than about 4.0, for example greater than about 5.0, in particular greater than about 6.0, for example greater than about 7.0.
  • the substance suitably inhibits the activity of the mPTP with a pIC50 value between 4.0 and 9.0, such as between 5.0 and 9.0, such as between 5.5 and 9.0, such as between 5.5 and 8.5, such as between 6.0 and 8.0.
  • K i is an absolute value specifying the concentration of competing substance in a competition binding assay which would occupy 50% of a binding target, e.g. NLRX1, if no other binding partners were present.
  • the substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP binds to NLRX1 with a K i value of less than about 500 nM, for example less than about 250 nM, for example less than about 200 nM, for example less than about 100 nM, for example less than about 75 nM, for example less than about 50 nM, less than about 25 nM, or less than about 10 nM.
  • the substance suitably binds to NLRX1 with a K i value of 0.000001-500 nM, such as 0.000001-250 nM, such as 0.000001-100 nM, such as 0.00001-100 nM, 0.0001-100 nM, 0.001-100 nM, 0.01-100 nM, 0.1-100 nM or 1-100 nM.
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP may suitably have one or more of any of the following advantageous properties: - low inhibition of cytochrome P450 enzymes, such as CYP3A4 and/or CYP2D6; - adequate solubility and/or low intrinsic clearance (CLint) and/or low efflux by multi-drug resistance protein 1 (MDR1) resulting in e.g. high oral bioavailability and/or high systemic exposure and/or high brain penetration.
  • cytochrome P450 enzymes such as CYP3A4 and/or CYP2D6
  • CLint low intrinsic clearance
  • MDR1 multi-drug resistance protein 1
  • a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, to be useful for the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity is administered to a patient in an amount such that the dose of the substance is from about 0.1 mg to about 10000 mg per day, for example from about 0.2 mg to about 8000 mg per day, for example from about 0.5 mg to about 5000 mg per day.
  • the dose of the substance is suitably from about 1 mg to about 5000 mg per day, for example from about 5 mg to about 5000 mg per day, for example from about 10 mg to about 5000 mg per day.
  • the present invention also provides known inhibitors of mPTP activity, for use in the prevention or treatment of a disease, disorder, or condition associated with altered NLRX1 activity.
  • the present invention provides known inhibitors of mPTP activity, for use in the prevention or treatment of a disease, disorder, or condition associated which is an Other NLRX1-Related Disease, Disorder or Condition. Suitable diseases, disorders or conditions which are Other NLRX1-Related Diseases, Disorders or Conditions are described above, but are not limited to those specified, under the heading “Conditions to be Treated”.
  • alkyl as used herein, such as in C 1-3 alkyl, is a straight or branched fully saturated hydrocarbon chain containing the specified number of carbon atoms.
  • C1-3alkyl groups include methyl, ethyl, n-propyl and iso-propyl.
  • Me means methyl.
  • Et means ethyl.
  • alkylthio refers to an alkyl group (e.g. a C1- 3alkyl group) as defined above singularly bonded to a sulphur atom e.g. -S-C1-3alkyl.
  • Examples of C1-3alkylthio include methylthio, ethylthio and propylthio.
  • alkylene as used herein, such as C1-4alkylene, whether alone or forming part of a larger group e.g.
  • Examples of C1-4alkylene groups include methylene (i.e.
  • alkynyl as used herein, such as in C 2-3 alkynyl, is a straight or branched divalent hydrocarbon chain with a least one carbon-carbon triple bond. Examples of C 2-3 alkynyl include ethynyl, 1-propynyl and 2-propynyl.
  • alkenyl refers to a straight or branched hydrocarbon group containing the specified number of carbon atoms and at least one carbon- carbon double bond, such as one or two double bonds.
  • haloalkenyl as used herein, such as in C 2-6 haloalkenyl is a straight or a branched alkenyl chain containing the specified number of carbon atoms and at least one halogen atom, such as fluoro or chloro, for example fluoro.
  • alkoxy as used herein, such as in C1-3alkoxy, refers to an alkyl group (e.g. a C1- 3alkyl group) as defined above, singularly bonded to an oxygen atom.
  • C1-3alkoxy groups include methoxy, ethoxy, 1-propoxy and 2-propoxy, especially methoxy.
  • cycloalkyl as used herein, such as in C3-5cycloalkyl or C3-6cycloalkyl, is a fully saturated hydrocarbon ring containing the specified number of carbon atoms, such as 3 to 5 or 3 to 6.
  • Examples of C3-6cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, in particular cyclopropyl.
  • the cycloalkyl may be substituted as defined herein.
  • heterocycloalkyl as used herein, such as in 4-10 membered heterocycloalkyl (e.g. 4-7 membered heterocycloalkyl), whether alone or forming part of a larger group such as C1- 4alkylene(4-10 membered heterocycloalkyl) and C1-4alkyleneO(4-10 membered heterocycloalkyl), is a fully saturated hydrocarbon ring containing the specified number of carbon atoms, wherein at least one of the carbon atoms is replaced by a heteroatom such as N, S or O.
  • the heterocycloalkyl may be substituted as defined herein.
  • a heterocycloalkyl group may be monocyclic.
  • a heterocycloalkyl group may be polycyclic such as a fused bicyclic or a bridged bicyclic ring system.
  • a heterocycloalkyl group may incorporate a fused ring.
  • a heterocycloalkyl group may incorporate a bridged ring.
  • a bicyclic heterocycloalkyl group is spirocyclic i.e. a bicyclic cycloalkyl group wherein the two rings are connected through just one atom. The rings can be different or identical.
  • 4-10 membered heterocycloalkyl groups include those comprising one heteroatom such as containing one heteroatom (e.g.
  • heteroatoms such as two heteroatoms e.g. two nitrogen atoms or one nitrogen atom and one oxygen atom.
  • heteroatoms such as two heteroatoms e.g. two nitrogen atoms or one nitrogen atom and one oxygen atom.
  • 4-10 membered heterocycloalkyl groups containing one nitrogen atom include azetidinyl, pyrrolidinyl, piperidinyl and azepanyl.
  • 4-10 membered heterocycloalkyl groups containing two nitrogen atoms include diazetidinyl, imidazolidinyl, pyrazolidinyl, diazinanyl, and diazepanyl.
  • 4-10 membered heterocycloalkyl groups include oxetanyl, thietanyl, dioxetanyl, dithietanyl, tetrahydrofuranyl, tetrahydrothiophenyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydropyranyl, thianyl, morpholinyl, thiomorpholinyl, dioxanyl, dithianyl, triazinanyl, trioxanyl, trithianyl, oxepanyl, and thiepanyl.
  • heterocycle means a non-aromatic cyclic group of carbon atoms wherein from one to four of the carbon atoms is/are replaced by one or more heteroatoms independently selected from nitrogen (N), oxygen (O) or sulfur (S).
  • a heterocycle group may, for example, be monocyclic or bicyclic. In a bicyclic heterocycle group there may be one or more heteroatoms in each ring, or only in one of the rings. Where a heterocycle group contains more than one ring, not all rings must contain a heteroatom.
  • a heteroatom may be S, O or N, and is suitably O or N.
  • a heterocycle group contains 5-10 ring members.
  • a heterocycle may contain one heteroatom selected from N, S and O, for example N and O, especially N.
  • a heterocycle may contain two heteroatoms selected from N, S and O.
  • a heterocycle may contain three heteroatoms selected from N, S and O, for example N and O.
  • the heterocycle is monocyclic, such as a 5- or 6- membered heterocycle ring. Examples of heterocycles include morpholinyl, tetrahydrofuran, and tetrahydropyran.
  • C5-11spirocarbocyclyl means a cyclic ring system containing 5 to 11 carbon atoms, wherein R4 and R5 are part of the sprirocarbocyclyl with the carbon that they are attached to.
  • An example of a C5-11spirocarbocyclyl is spiropentane.
  • aryl as used herein in relation to substances of formula (I), (II), and (III), whether alone or forming part of a larger group e.g. C 1-4 alkylene(aryl), OC 1-4 alkylene(aryl) or C 1- 4 alkyleneO(aryl), refers to a phenyl ring.
  • aryl may be substituted as defined herein.
  • aryl refers to a mono (i.e. phenyl) or polycyclic ring system (e.g. comprising one or two, such as one additional ring) containing at least one phenyl ring, suitably an aryl group contains 6-10 ring members. Additional rings in a polycylic ring system may be saturated (e.g forming indane or tetralin) or partially unsaturated (e.g. forming indene) or fully unsaturated (e.g.
  • aryl refers to a mono (i.e. phenyl) or a bicyclic ring system containing at least one phenyl ring (and no heteroaryl rings).
  • heteroaryl refers to mono or polycyclic ring system (e.g bicyclic) with at least one ring having aromatic character and containing at least one heteroatom selected from N, O and S, for example N.
  • a heteroaryl group contains 5-10 ring members.
  • heteroaryl is monocyclic, such as a 5- or 6- membered heteroaryl ring (e.g. containing one or two heteroatom selected from N, S and O).
  • heteroaryl is bicyclic, such as a 5,5-, 5,6- or 6,6- bicyclic system (e.g. containing one, two or three heteroatoms selected from N, S and O).
  • a heteroaryl may contain one heteroatom selected from N, S and O, for example N and O, especially N.
  • a heteroaryl may contain two heteroatoms selected from N, S and O.
  • a heteroaryl may contain three heteroatoms selected from N, S and O, for example N and O.
  • 6-membered heteroaryls include one nitrogen atom (pyridinyl), two nitrogen atoms (pyridazinyl, pyrimidinyl or pyrazinyl) and three nitrogen atoms (triazinyl).
  • heteroaryl include triazolyl, indolyl, indazolyl, benzofuranyl, benzimidazolyl, benzoxazolinyl, quinolinyl, isoquinolinyl and quinazolinyl.
  • haloalkyl as used herein, such as in C1-4haloalkyl, whether alone or forming part of a larger group such as OC1-4haloalkyl, is a straight or branched alkyl group containing the specified number of carbon atoms, substituted by one or more halo atoms, for example fluoromethyl (CH2F), di-fluoromethyl (CHF2), tri-fluoromethyl (CF3), 1-fluoroethyl (CH2FCH2) and 2-fluoroethyl (CH2CH2F).
  • haloalkoxy as used herein, such as in C 1-4 haloalkoxy, refers to an haloalkyl group (e.g.
  • fluoroalkyl as used herein, such as in C 1-3 fluoroalkyl, is a straight or branched alkyl group containing the specified number of carbon atoms, substituted by one or more fluoro atoms, for example fluoromethyl (CH 2 F), di-fluoromethyl (CHF 2 ), tri-fluoromethyl (CF 3 ), 1-fluoroethyl (CH 2 FCH 2 ) and 2-fluoroethyl (CH 2 CH 2 F).
  • fluoroalkoxy as used herein, such as in C 1-3 fluoroalkoxy, is a linear or branched, saturated, monovalent C 1-3 alkoxy group, as defined above, in which one or more of the hydrogen atoms is replaced, identically or differently, with a halogen atom for example -OCF 3 , -OCHF 2 , -OCH 2 F, -OCF 2 CF 3 and -OCH 2 CF 3 .
  • halo or ‘halogen’ as used herein, refers to fluorine, chlorine, bromine or iodine. Particular examples of halo are bromine, fluorine and chlorine, especially fluorine.
  • the optional substituent may be attached to an available carbon atom, which means a carbon atom which is attached to a hydrogen atom i.e. a C-H group or the optional substituent may be attached to an available nitrogen atom, which means a nitrogen atom which is attached to a hydrogen atom i.e. a N-H group.
  • the optional substituent replaces the hydrogen atom attached to the carbon atom or the hydrogen atom attached to the nitrogen atom.
  • the present invention provides a substance of formula (I): wherein: R 1a is H or methyl; R 1b is H or F; A is group (Aa), (Ab), (Ac) or (Ad): wherein group (Aa) is: wherein: R2 is H, C1-4alkyl, C1-4alkylene(aryl), C1-4alkylene(OH), C1-4alkylene(C3-6cycloalkyl), C1- 4alkylene(4-7 membered heterocycloalkyl), C1-4alkoxy, OC1-4alkylene(aryl), C1-4alkyleneOC1- 4alkyl, C1-4alkyleneOC3-6cycloalkyl, C1-4alkyleneO(4-7 membered heterocycloalkyl), C1- 4alkyleneO(aryl), C3-6alkynyl or C1-4alkenylO(C3-6alkynyl); wherein said aryl, heterocycloalkyl and cycloalkyl are optionally substituted by 1,
  • Suitable substances of formula (I), and compositions containing them, and processes for preparing them, are further described in WO2022/049376, which is herein incorporated by reference in its entirety. It will be understood that suitable and/or preferred substances of formula (I) may include any of the substances (generic or specific) disclosed in WO2022/049376.
  • the present invention provides a substance of formula (I), for use according to the present invention, wherein A is group (Aa):
  • a of the substance of formula (I), for use according to the present invention is group (Aa):
  • R2 is C1-4alkyl, C1-4alkylene(aryl), C1-4alkylene(OH), C1-4alkyleneOC1-4alkyl, C1- 4alkyleneOC3-6cycloalkyl, C1-4alkyleneO(aryl), C1-4alkylene(4-7 membered heterocycloalkyl), C 1-4 alkyleneO(4-7 membered heterocycloalkyl) or C 1-4 alkyleneO(C 3-6 alkynyl); wherein said aryl, heterocycloalkyl and cycloalkyl may be optionally substituted by up to 3 substituents each independently selected from C 1-4 alkyl, C 3-6 cycloalkyl, C 1-4 alkoxy, C 1-4 haloalkyl, halo and
  • the aryl is unsubstituted. In another aspect of the invention, the aryl is substituted by 1, 2 or 3 substituents independently selected from methyl, chloro and fluoro.
  • the heterocycloalkyl is unsubstituted. In another aspect of the invention, the heterocycloalkyl is substituted by 1, 2 or 3 substituents independently selected from CH 2 CH 2 F and fluoro.
  • each R3 is independently fluoro or methyl.
  • m is suitably 1 or 2.
  • the present invention provides a substance of formula (I), for use according to the present invention, wherein A is group (Ab):
  • a of the substance of formula (I), for use according to the present invention is group (Ab)
  • R4 is H, methyl or benzyl.
  • R5 is suitably H.
  • each R6 is independently fluoro or methyl.
  • n is 1.
  • the present invention provides a substance of formula (I), for use according to the present invention, wherein A is group (Ac):
  • a of the substance of formula (I), for use according to the present invention is group (Ac), R7 is methyl, CH2OH or CH2OMe.
  • a of the substance of formula (I), for use according to the present invention is group (Ac), o is suitably 2.
  • the present invention provides a substance of formula (I), for use according to the present invention, wherein A is group (Ad):
  • a of the substance of formula (I), for use according to the present invention is group (Ad), X is a bond or O.
  • each R 8 is independently OCH 2 - cyclopropyl, OCH 2 -oxetanyl, OCH 2 CH 2 F, methyl, OMe, oEt or fluoro, for example methyl, OMe or fluoro
  • each R 8 is independently OCH 2 -cyclopropyl, OCH 2 -oxetanyl, OCH2CH2F or oEt
  • each R8 is independently OCH2CH2F, OMe or oEt, especially OMe.
  • a of the substance of formula (I), for use according to the present invention is group (Ad), suitably p is 0 or 1.
  • each R9 is independently fluoro.
  • a of the substance of formula (I), for use according to the present invention is group (Ad), suitably q is 1 or 2.
  • the present invention provides a substance of formula (I), for use according to the present invention, wherein group B is (Ba):
  • group B of the substance of formula (I), for use according to the present invention is group (Ba)
  • Y is C(R11)(R12) or N(R13), wherein, suitably, (i) R11 is H or methyl and R12 is H, or (ii) R11 and R12 are both H, or (iii) R11 and R12 together to the carbon atom to which they are attached form a cyclopropyl ring
  • R 13 is suitably C 1-4 alkyl, such as methyl, ethyl, propyl or butyl, especially methyl; or C 3-6 cycloalkyl, such as cyclopropyl.
  • B of the substance of formula (I), for use according to the present invention is group (Ba)
  • Y is O or S.
  • each R 10 is independently fluoro, chloro or methyl.
  • r is suitably 0 or 1.
  • the present invention provides a substance of formula (I), for use according to the present invention, wherein group B is (Bb):
  • each R14 is independently fluoro or methyl.
  • the present invention provides a substance of formula (I), for use according to the present invention, wherein group B is (Bc):
  • group B is group (Bc):
  • R15 is methyl, ethyl, cyclopropyl, CF3, CN, OMe, chloro or fluoro e.g.
  • R15 is OMe, chloro or fluoro or R15 is methyl, CN, chloro or fluoro.
  • B of the substance of formula (I), for use according to the present invention is group (Bc), D, E and F are C(R16), or; D is N, and E and F are C(R16) or; E is N, and D and F are C(R16) or; F is N, and D and E are C(R16); wherein each R16 is independently H, fluoro, chloro or methyl.
  • the present invention provides a substance of formula (I), for use according to the present invention, wherein the substance of formula (I) is selected from the group consisting of: (E)-3-(1H-benzo[d][1,2,3]triazol-6-yl)-N-(3-fluoro-2-methylphenyl)acrylamide; (E)-N-(3-fluoro-2-methylphenyl)-3-(2-oxo-2,3-dihydrobenzo[d]thiazol-5-yl)acrylamide; (E)-3-(3,3-dimethyl-2-oxoindolin-6-yl)-N-(3-fluoro-2-methylphenyl)acrylamide; (E)-N-(3-fluoro-2-methylphenyl)-3-(2'-oxospiro[cyclopropane-1,3'-indolin]-6'-yl)acrylamide; (E)-N-(3-fluoro-2-methyl
  • the present invention provides a substance of formula (II): wherein: R 1a is H or methyl; R 1b is H or fluoro; A is group (Aa), (Ab), (Ac) or (Ad): wherein group (Aa) is: wherein: R 2 is H, C 1-4 alkyl, C 1-4 alkylene(aryl), C 1-4 alkylene(OH), C 1-4 alkylene(C 3-6 cycloalkyl), C 1- 4 alkylene(4-7 membered heterocycloalkyl), C 1-4 alkoxy, OC 1-4 alkylene(aryl), C 1- 4 alkyleneOC 1-4 alkyl, C 1-4 alkyleneOC 3-6 cycloalkyl, C 1-4 alkyleneO(4-7 membered heterocycloalkyl), C 1-4 alkyleneO(aryl), C 3-6 alkynyl and C 1-4 alkyleneO(C 3-6 alkynyl); wherein said aryl, heterocycloalky
  • Suitable substances of formula (II), and compositions containing them, and processes for preparing them, are further described in WO2022/049377, which is herein incorporated by reference in its entirety. It will be understood that suitable and/or preferred substances of formula (II) may include any of the substances (generic or specific) disclosed in WO2022/049377.
  • the present invention provides a substance of formula (II), for use according to the present invention, wherein A is group (Aa):
  • a of the substance of formula (II), for use according to the present invention is group (Aa):
  • R2 is C1-4alkyl, C1-4alkylene(aryl), C1-4alkylene(OH), C1-4alkyleneOC1-4alkyl, C1- 4alkyleneOC3-6cycloalkyl, C1-4alkyleneO(aryl), C1-4alkylene(4-7 membered heterocycloalkyl), C1-4alkyleneO(4-7 membered heterocycloalkyl) and C1-4alkyleneO(C3-6alkynyl); wherein said aryl, heterocycloalkyl or cycloalkyl may be optionally substituted by up to 3 substituents each independently selected from C1-4alkyl, C3-6cycloalkyl; C1-4alkoxy, C1-4haloalkyl, halo and CN.
  • the aryl is unsubstituted. In another aspect of the invention, the aryl is substituted by 1, 2 or 3 substituents independently selected from methyl, chloro and fluoro. In one aspect of the invention, the heterocycloalkyl is unsubstituted. In another aspect of the invention, the heterocycloalkyl is substituted by 1, 2 or 3 substituents independently selected from CH2CH2F and fluoro.
  • each R 3 is independently fluoro or methyl.
  • m is suitable 1 or 2.
  • the present invention provides a substance of formula (II), for use according to the present invention, wherein A is group (Ab):
  • a of the substance of formula (II), for use according to the present invention is group (Ab)
  • R4 is H, methyl or benzyl.
  • R5 is suitably H.
  • each R6 is independently fluoro or methyl.
  • n is suitably 0.
  • the present invention provides a substance of formula (II), for use according to the present invention, wherein A is group (Ac):
  • a of the substance of formula (II), for use according to the present invention is group (Ac), R7 is methyl, CH2OH or CH2OMe.
  • a of the substance of formula (II), for use according to the present invention is group (Ac), o is suitably 2.
  • the present invention provides a substance of formula (II), for use according to the present invention, wherein A is group (Ad):
  • a of the substance of formula (II), for use according to the present invention is group (Ad)
  • X is a bond or O.
  • each R8 is independently methyl, OMe or fluoro.
  • p is suitably 0 or 1.
  • each R9 is independently fluoro.
  • q is suitably 1 or 2.
  • D, E and F are C(R10) or; D is N, and E and F are C(R10) or; E is N, and D and F are C(R10) or; F is N, and D and E are C(R10); wherein each R10 is independently H, fluoro, chloro or methyl.
  • the present invention provides a substance of formula (II), for use according to the present invention, wherein the substance of formula (II) is selected from the group consisting of: (E)-N-(3-fluoro-2-methylphenyl)-3-(7-methyl-1H-indazol-6-yl)acrylamide; (E)-N-(2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-6-yl)acrylamide; (R,E)-N-(2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-6-yl)acrylamide; (E)-3-(1H-indazol-6-yl)-N-((1R,2R)-2-methylcyclohexyl) acrylamide (racemic mixture); (E)-3-(1H-indazol-6-yl)-N-((1R,2R)-2-methylcyclohexyl) acryl
  • the present invention provides a substance of formula (III): wherein: R1a is H or methyl; R1b is H or F; A is group (Aa), (Ab), (Ac), (Ad) or (Ae): wherein group (Aa) is: wherein: R 2 is H, halo, C 1-4 alkyl, C 1-4 haloalkyl, C 1-4 alkylene(aryl), C 1-4 alkylene(OH), C 1- 4 alkylene(C 3-6 cycloalkyl), C 1- 4alkylene(4-10 membered heterocycloalkyl), 4-10 membered heterocycloalkyl, C 1-4 alkoxy, OC 1-4 alkylene(aryl), C 1-4 alkyleneOC 1-4 alkyl, C1-4alkyleneOC3-6cycloalkyl, C1-4alkyleneO(4-10 membered heterocycloalkyl), C1- 4alkyleneO(aryl), C 3-6 alkynyl, C 1-4 alken
  • Suitable substances of formula (III), and compositions containing them, and processes for preparing them, are further described in WO2023/166303, which is herein incorporated by reference in its entirety. It will be understood that suitable and/or preferred substances of formula (III) may include any of the substances (generic or specific) disclosed in WO2023/166303.
  • the present invention provides a substance of formula (III), for use according to the present invention, wherein A is group (Aa): .
  • R 2 is halo, C 1-4 haloalkyl, C 1- 4alkylene(8-10 membered heterocycloalkyl), C 1- 4 alkyleneO(8-10 membered heterocycloalkyl), 4-10 membered heterocycloalkyl such as 1- methyl piperazinyl, C 1-4 alkylene(CO 2 H), such as CH 2 CH 2 (CO 2 H), OC 1-4 alkylene(CO 2 H), C 1- 4 alkyleneOC 1-4 alkylene(CO 2 H), N(R 2aa )C 1-4 alkylene(CO 2 H), C 1-4 alkylene(NR 2ab R 2ac ), OC 1- 4 alkylene(NR 2ab R 2ac ) or N(R 2aa )C 1-4 alkylene(NR 2ab R 2ac ), in particular wherein R 2 is 4-10 membered heterocycloalkyl
  • R2x is C1-4alkylene(4-10 membered heterocycloalkyl), 4-10 membered heterocycloalkyl, such as 1-methyl piperazinyl, C 1-4 alkyleneO(4-10 membered heterocycloalkyl), C 1-4 alkylene(CO 2 H), such as CH 2 CH 2 (CO 2 H), OC 1-4 alkylene(CO 2 H), C 1- 4 alkyleneOC 1-4 alkylene(CO 2 H), N(R 2xaa )C 1-4 alkylene(CO 2 H), C 1-4 alkylene(NR 2xab R 2xac ), OC 1- 4alkylene(NR2xabR2xac) or N(R2xaa)C1-4alkylene(NR2xabR2xac), in particular wherein R2x is 4-10 membered heterocycloalkyl, such as 1-methyl piperazinyl, C 1-4 alkyleneO(4-10 membered heterocycloalkyl), C 1-4 alky
  • a of the substance of formula (III), for use according to the present invention is group (Aa), suitably, m is 2 and each R3 is independently fluoro and methyl.
  • the present invention provides a substance of formula (III), for use according to the present invention, wherein A is group (Ae):
  • a of the substance of formula (III), for use according to the present invention is group (Ae)
  • R 17 is C 1-4 alkyl, such as methyl, ethyl or propyl, especially methyl
  • R 18 is C 1-4 alkyl, such as methyl, ethyl or propyl, especially methyl
  • R 20 is C 1-4 alkoxy, such as OMe or OEt, especially OM
  • R 21 is H.
  • the present invention provides a substance of formula (III), for use according to the present invention, wherein B is group (Bc):
  • B of the substance of formula (III), for use according to the present invention is group (Bc)
  • R15 is methyl, ethyl, cyclopropyl, CF3, CN, OMe, chloro or fluoro e.g. methyl, ethyl, cyclopropyl, CF3 or CN.
  • D, E and F are C(R16) and each R16 is independently H, fluoro, chloro or methyl.
  • the present invention provides a substance of formula (III), for use according to the present invention, wherein the substance of formula (III) is selected from the group consisting of: (E)-3-(3-fluoro-4-methyl-5-(3-(3-methyl-1H-indazol-6-yl)acrylamido)phenyl)propanoic acid; (E)-3-(3-(3-(3-chloro-1H-indazol-6-yl)acrylamido)-5-fluoro-4-methylphenyl)propanoic acid; (E)-3-(3-fluoro-5-(3-(3-fluoro-1H-indazol-6-yl)acrylamido)-4-methylphenyl)propanoic acid; (E)-3-(3-(3-(3-cyano-1H-indazol-6-yl)acrylamido)-5-fluoro-4-methylphenyl)propanoic acid; (E)-3-(3-(3-
  • the present invention provides a substance of formula (III), for use according to the present invention, wherein the substance of formula (III) is selected from the group consisting of: (2E)-N-(6-methoxy-2,4-dimethylpyridin-3-yl)-3-(3-methyl-1H-indazol-6-yl)prop-2-enamide; (2E)-3-(3-fluoro-1H-indazol-6-yl)-N-(6-methoxy-2,4-dimethylpyridin-3-yl) prop-2-enamide ; (2E)-3-(3-cyano-1H-indazol-6-yl)-N-(6-methoxy-2,4-dimethylpyridin-3-yl)prop-2-enamide; (2E)-3-(5-fluoro-3-methyl-1H-indazol-6-yl)-N-(6-methoxy-2,4-dimethylpyridin-3-yl)prop-2-enamide;
  • the present invention provides a substance of formula (IV): wherein: R1 is H or F; wherein: R2 is C1-3alkyl, -CH2OC1-3alkyl, C1-3fluoroalkyl, C1-3alkoxy, -CN, halo or C3-5cycloalkyl; R3 is H, C1-3alkyl, C1-3fluoroalkyl, C1-3alkoxy, -CN, halo or C3-5cycloalkyl; R 4 is H, C 1-3 alkyl, -CH 2 OC 1-3 alkyl, C 1-3 fluoroalkyl, C 1-3 alkoxy, C 1-3 fluoroalkoxy, -CN, halo or C3-5cycloalkyl; and R5 is H, C1-3alkyl, -CH2OC1-3alkyl, C1-3fluoroalkyl, C1-3alkoxy, -CN, halo or C3- 5cycloalkyl; provided that when R5 is H then R1 is H then R
  • the substance of formula (IV) for use according to the present invention is a substance of formula (IVA): wherein: R 2 is C 1-3 alkyl, -CH 2 OC 1-3 alkyl, C 1-3 fluoroalkyl, C 1-3 alkoxy, -CN, halo or C 3-5 cycloalkyl; R 3 is H, C 1-3 alkyl, C 1-3 fluoroalkyl, C 1-3 alkoxy, -CN, halo or C 3-5 cycloalkyl; R 4 is H, C 1-3 alkyl, -CH 2 OC 1-3 alkyl, C 1-3 fluoroalkyl, C 1-3 alkoxy, C 1-3 fluoroalkoxy, -CN, halo or C 3-5 cycloalkyl; and R 5 is H, C1-3alkyl, -CH2OC1-3alkyl, C1-3fluoroalkyl, C1-3alkoxy, -CN, halo or C3- 5
  • the present invention provides a substance of formula (IVA), or prodrug thereof, for use according to the present invention, wherein B is group (Ba1):
  • B of the substance of formula (IVA), for use according to the present invention is group (Ba1), R8 is F.
  • B of the substance of formula (IVA), for use according to the present invention is group (Ba1), R8 is H.
  • B of the substance of formula (IVA), for use according to the present invention is group (Ba1)
  • R6 is H.
  • B of the substance of formula (IVA), for use according to the present invention is group (Ba1), Y is CH2.
  • B of the substance of formula (IVA), for use according to the present invention is group (Ba1)
  • Y is NMe
  • the present invention provides a substance of formula (IVA), or prodrug thereof, for use according to the present invention, wherein B is group (Bb1):
  • B of the substance of formula (IVA), for use according to the present invention is group (Bb1)
  • D is N.
  • B of the substance of formula (IVA), for use according to the present invention is group (Bb1)
  • D is C(R 11 ) and R 11 is F, Cl, -CN, OMe, preferably wherein D is C(R11) and R11 is F.
  • B of the substance of formula (IVA), for use according to the present invention is group (Bb1)
  • D is C(R11) and R11 is H.
  • R13 is suitably H.
  • B of the substance of formula (IVA), for use according to the present invention is group (Bb1)
  • R10 is suitably H, Cl, F, Me or -CN, preferably H, Cl, Me or -CN.
  • the present invention provides a substance of formula (IVA), or prodrug thereof, for use according to the present invention, wherein R2 is Me.
  • the present invention provides a substance of formula (IVA), for use according to the present invention, wherein R5 is C1-3alkyl, -CH2OMe, C1-3fluoroalkyl, C1-3alkoxy, -CN, halo or C3-5cycloalkyl, preferably is Me, F or Cl more preferably is Me or Cl.
  • R 5 is C1-3alkyl, -CH2OMe, C1-3fluoroalkyl, C1-3alkoxy, -CN, halo or C3-5cycloalkyl, preferably is Me, F or Cl more preferably is Me or Cl.
  • a substance of formula (IVA), or prodrug thereof for use according to the present invention, wherein R 3 is Me, R 4 is H and R 5 is Me, F or Cl e.g. is Me or Cl.
  • the substance of formula (IVA) for use according to the present invention is a substance of formula (IVB): wherein: R 2 is C 1-3 alkyl, -CH 2 OC 1-3 alkyl, C 1-3 fluoroalkyl, C 1-3 alkoxy, -CN, halo or C 3-5 cycloalkyl; R3 is H, C1-3alkyl, C1-3fluoroalkyl, C1-3alkoxy, -CN, halo or C3-5cycloalkyl; R4 is H, C1-3alkyl, -CH2OC1-3alkyl, C1-3fluoroalkyl, C1-3alkoxy, C1-3fluoroalkoxy -CN, halo or C3-5cycloalkyl; and R5 is F, Cl or -CN
  • the present invention provides a substance of formula (IVB), or prodrug thereof, for use according to the present invention, wherein B is group (Ba).
  • B of the substance of formula (IVB), for use according to the present invention is group (Ba), suitably R7 is F and R6 and R8 are each H.
  • B of the substance of formula (IVB), for use according to the present invention is group (Ba), suitably R8 is F and R6 and R7 are each H.
  • B of the substance of formula (IVB), for use according to the present invention is group (Ba), suitably Y is N(Me).
  • the present invention provides a substance of formula (IVB), or prodrug thereof, for use according to the present invention, wherein B is group (Bb).
  • B of the substance of formula (IVB), for use according to the present invention is group (Bb)
  • D is C(R11) and R11 is F.
  • B of the substance of formula (IVB), for use according to the present invention is group (Bb), suitably, D is C(R 11 ) and R 11 is H.
  • R12 and R13 are suitably H.
  • R 10 is suitably selected from H, Cl, Me and -CN.
  • R 2 is suitably Me, -(CH 2 )OMe or - CN.
  • a substance of formula (IVB), or prodrug thereof, for use according to the present invention suitably wherein R 2 is Me, R 3 is H, R4 is H and R5 is Cl.
  • a substance of formula (IVB), or prodrug thereof, for use according to the present invention suitably wherein R 2 is Me, R 3 is H, R 4 is H and R5 is F; or R2 is -(CH2)OMe, R3 is H, R4 is H and R5 is F; or R2 is -(CH2)OMe, R3 is H, R4 is H and R5 is Cl; or R2 is Me, R3 is H, R4 is H and R5 is -CN; or R2 is -CN, R3 is H, R4 is H and R5 is Cl; or R2 is Me, R3 is H, R4 is H and R5 is Me.
  • the present invention provides a substance of formula (IV), for use according to the present invention, wherein the substance of formula (IV) is selected from the group consisting of: (2Z)-2-fluoro-3-(7-fluoro-1H-indazol-6-yl)-N-(5-fluoro-2,4-dimethylpyridin-3-yl)prop-2- enamide; (2Z)-2-fluoro-3-(3-fluoro-1H-indazol-6-yl)-N-(5-fluoro-2,4-dimethylpyridin-3-yl) prop-2- enamide; (2Z)-N-(2,5-dimethylpyridin-3-yl)-2-fluoro-3-(3-methyl-1H-indazol-6-yl)prop-2-enamide; (2Z)-N-(5-chloro-2-methylpyridin-3-yl)-2-fluoro-3-(3-methyl-1H-ind
  • the present invention provides a substance of formula (V): wherein: R 1 is H or C1-4alkyl; R 2 is H, halo, C1-4alkyl, C1-4haloalkyl; C1-4alkoxy, C1-4haloalkoxy or C0-4alkylene(OH); R 3 is H, halo or C1-4alkyl; R 4 is H, halo or C1-4alkyl; R 5 is halo, C1-6alkyl, C1-6haloalkyl, C1-6alkoxy, C1-6haloalkoxy, C2-6alkenyl, C2- 6haloalkenyl, C0-6alkylene(C3-6cycloalkyl), C0-6alkylene(OH); or R 4 and R 5 together with the carbon atom to which they are attached form aC5-11 spiro carbocyclyl, 4 to 7 m embered heterocycle or C3-6cycloalkyl wherein said spiro carbocyclyl, heterocycloalkyl or
  • Suitable substances of formula (V), and compositions containing them, and processes for preparing them, are further described in PCT/GB2024/050146, which is herein incorporated by reference in its entirety and in the Examples section below.
  • mPTP Inhibitor 1 further described in the Examples section, is an example of this genus. It will be understood that suitable and/or preferred substances of formula (V) may include any of the substances (generic or specific) disclosed in PCT/GB2024/050146.
  • the present invention provides a substance of formula (VA) for use according to the present invention: wherein: R 1 is H or C 1-4 alkyl; R 2 is H, halo, C 1-4 alkyl, C 1-4 haloalkyl; C 1-4 alkoxy, C 1-4 haloalkoxy or C 0-4 alkylene(OH); R 3 is H, halo or C 1-4 alkyl; R 4 is H or C 1-4 alkyl; R 5 is H, halo, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 2-6 alkenyl, C 2- 6 haloalkenyl, C 0-6 alkylene(C 3-6 cycloalkyl), C 0-6 alkylene(OH); or R 4 and R 5 together with the atom to which they are attached form a C 3-6 cycloalkyl wherein said cycloalkyl may be optionally substituted by one
  • R 4 and R 5 have the following stereochemistry: .
  • R 4 and R 5 may also have the following stereochemistry: .
  • Mixtures of enantiomers including racemic mixtures of compound of formula (V) and (VA) with respect to the chiral centre shown are also provided.
  • a substance of formula (V) or (VA) for use according to the present invention, suitably wherein R1, R2 and R3 are each H.
  • the present invention provides a substance of formula (V) or (VA), for use according to the present invention, wherein R4 is H or methyl, particularly H.
  • the present invention provides a substance of formula (V) or (VA), for use according to the present invention, wherein R 5 is C 1-6 alkyl, such as methyl, ethyl, n- propyl or iso-propyl, especially ethyl.
  • R 5 is C 1-6 alkyl, such as methyl, ethyl, n- propyl or iso-propyl, especially ethyl.
  • the present invention provides a substance of formula (V) or (VA), for use according to the present invention, wherein R5 is C0-6alkylene(C3-6cycloalkyl), such as C3-6cycloalkyl, for example cyclopropyl or cyclobutyl, especially cyclopropyl.
  • the present invention provides a substance of formula (V) or (VA), for use according to the present invention, wherein R4 and R5 together with the atom to which they are attached form a C3- 6 cycloalkyl, such as a cyclopropyl, cyclobutyl or cyclopentyl ring, especially a cyclobutyl ring.
  • the present invention provides a substance of formula (V) or (VA), for use according to the present invention, wherein R 6 is H and R 7 is H.
  • a substance of formula (V) or (VA) for use according to the present invention, wherein A is phenyl.
  • a substance of formula (V) or (VA), for use according to the present invention wherein A is selected from the group consisting of: wherein: A 1A is halo, such as F, Br or Cl; C1-6alkyl, such as methyl; or C1-6alkoxy, such as OMe; and A 1B is H or halo, such as F, Br or Cl, especially F.
  • a substance of formula (V) or (VA), for use according to the present invention wherein B is selected from the group consisting of: such , wherein: B 1A is H or C1-6alkyl, such as methyl; and B 2A is H or C1-6alkyl, such as methyl.
  • the present invention provides a substance of formula (V) or (VA), for use according to the present invention, wherein the substance of formula (V) or (VA) is selected from the group consisting of: N-((1-benzylcyclobutyl)methyl)-5-hydroxynicotinamide; N-((1-benzylcyclobutyl)methyl)-5-hydroxy-6-methylnicotinamide; N-((1-benzylcyclobutyl)methyl)-5-hydroxy-4-methylnicotinamide; N-((1-benzylcyclobutyl)methyl)-1H-1,2,3-triazole-5-carboxamide; N-((1-benzylcyclobutyl)methyl)-2-oxo-2,3-dihydropyrimidine-4-carboxamide; N-((1-benzylcyclobutyl)methyl)-6-oxo-1,6-dihydropyrimidine-2-carboxamide; N-(((1-benzylcyclobut
  • the present invention provides a substance of formula (VI): wherein: R 1a is H or C1-4alkyl; R 1b is H or C1-4alkyl; R 2a is H, halo, C1-4alkyl or C1-4haloalkyl; R 3a is H, halo or C1-4alkyl; R 4a is H or C1-4alkyl; R 5a is H, C1-6alkyl, C1-6haloalkyl, C1-6alkoxy, C1-6haloalkoxy, C2-6alkenyl, C2- 6haloalkenyl, C 0-6 alkylene(C 3-6 cycloalkyl), C 0-6 alkylene(OH); or R 4a and R 5a together with the carbon atom to which they are attached form a C 3-6 cycloalkyl wherein said cycloalkyl may be optionally substituted by one or more groups selected from C 1- 4 alkyl, C 1-4 haloalkyl and halo; R 6a is H
  • Suitable substances of formula (VI), and compositions containing them, and processes for preparing them, are further described in PCT/GB2024/050145, which is herein incorporated by reference in its entirety. It will be understood that suitable and/or preferred substances of formula (VI) may include any of the substances (generic or specific) disclosed in PCT/GB2024/050145.
  • the present invention provides a substance of formula (VIB) for use according to the present invention: wherein: R 1a is H or C1-4alkyl; R 1b is H or C 1-4 alkyl; R 2a is H, halo, C 1-4 alkyl or C 1-4 haloalkyl; R 3a is H, halo or C 1-4 alkyl; R 4a is H or C 1-4 alkyl; R 5a is H, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 2-6 alkenyl, C 2- 6 haloalkenyl, C 0-6 alkylene(C 3-6 cycloalkyl), C 0-6 alkylene(OH); or R 4a and R 5a together with the atom to which they are attached form a C 3-6 cycloalkyl wherein said cycloalkyl may be optionally substituted by one or more groups selected from C 1-4 alkyl
  • a substance of formula (VI) or (VIB), for use according to the present invention wherein R 1a and R 1b are each H.
  • the present invention provides a substance of formula (VI) or (VIB), for use according to the present invention, wherein R 2a and R 3a are each H.
  • the present invention provides a substance of formula (VI) or (VIB), for use according to the present invention, wherein R 4a and R 5a together with the atom to which they are attached form a C3-6cycloalkyl, such as cyclobutyl.
  • the present invention provides a substance of formula (VI) or (VIB), for use according to the present invention, wherein R 4a and R 5a are each methyl.
  • a substance of formula (VI) or (VIB) wherein, suitably, R 6a is H.
  • a substance of formula (VI) or (VIB) for use according to the present invention, wherein x is 1.
  • the present invention provides a substance of formula (VI) or (VIB), for use according to the present invention, wherein AA is phenyl.
  • the present invention provides a substance of formula (VI) or (VIB), for use according to the present invention, wherein AA is substituted by one or more AA1, and wherein at least one AA1 is halo.
  • the present invention provides a substance of formula (VI) or (VIB), for use according to the present invention, wherein BA is selected from the group consisting of: wherein: B 1B is H or C1-6alkyl, such as methyl; B 2B is H or C 1-6 alkyl, such as methyl; and B 2B is C0-6alkylene(OH), such as OH.
  • the present invention provides a substance of formula (VI) or (VIB), for use according to the present invention, wherein the substance of formula (VI) or (VIB) is selected from the group consisting of: (6-hydroxypyrazin-2-yl)(8-phenyl-6-azaspiro[3.4]octan-6-yl)methanone; (4-hydroxypyridin-2-yl)(8-phenyl-6-azaspiro[3.4]octan-6-yl)methanone; (3,3-dimethyl-4-phenylpyrrolidin-1-yl)(5-hydroxypyridin-3-yl)methanone; 2-methyl-5-(8-phenyl-6-azaspiro[3.4]octane-6-carbonyl)-2,4-dihydro-3H-1,2,4-triazol-3-one; (6-hydroxypyrazin-2-yl)(8-phenyl-6-azaspiro[3.4]octane-6-carbonyl)-2,
  • a method for preventing or treating a disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a known inhibitor of mPTP activity.
  • the known inhibitor of mPTP activity is a substance of formula (I), (II), (III), (IV), (V), (VA), (VI), or (VIB), as described above.
  • the known inhibitor of mPTP activity is a substance of formula (I), (II), (III), (IV), (V), (VA), (VI), or (VIB), as described above.
  • compositions and Administration Routes Suitably a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, and in particular (i) binds NLRX1 and (ii) inhibits the activity of the mPTP, and which is suitably identified by a method according to the present invention, for example a method described under the sub-heading ‘Methods for Identifying Substances for use according to the present invention’, for use in the treatment of a disease, disorder or condition associated with altered NLRX1 activity, is formulated as part of a pharmaceutical composition.
  • a known inhibitor of mPTP activity in particular a substance of formula (I), (II), (III), (IV), (V), (VA), (VI) or (VIB) for use in the treatment of a disease, disorder or condition which is an Other NLRX1-Related Disease, Disorder or Condition, is formulated as part of a pharmaceutical composition. Therefore, in one aspect the present invention provides a pharmaceutical composition comprising a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, and in particular (i) binds NLRX1 and (ii) inhibits the activity of the mPTP, and one or more pharmaceutically acceptable excipients.
  • Said pharmaceutical composition is suitably for use in the treatment of a disease, disorder or condition associated with altered NLRX1 activity.
  • the present invention provides a pharmaceutical composition comprising a substance of formula (I), (II), (III), (IV), (V), (VA), (VI), or (VIB), and one or more pharmaceutically acceptable excipients.
  • Said pharmaceutical composition is suitably for use in the treatment of a disease, disorder or condition which is an Other NLRX1-Related Disease, Disorder or Condition.
  • the present invention also provides a method for preventing or treating a disease, disorder, or condition associated with altered NLRX1 activity, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a pharmaceutical composition comprising a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, and in particular (i) binds NLRX1 and (ii) inhibits the activity of the mPTP, and one or more pharmaceutically acceptable excipients.
  • the present invention provides use of a pharmaceutical composition
  • a pharmaceutical composition comprising a substance which is (i) capable of binding to NLRX1 and (ii) capable of inhibiting the activity of the mPTP, and in particular (i) binds NLRX1 and (ii) inhibits the activity of the mPTP, and one or more pharmaceutically acceptable excipients, in the manufacture of a medicament for preventing or treating a disease, disorder, or condition associated with altered NLRX1 activity.
  • the present invention also provides a method for preventing or treating a disease, disorder or condition which is an Other NLRX1-Related Disease, Disorder or Condition, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a pharmaceutical composition comprising a substance of formula (I), (II), (III), (IV), (V), (VA), (VI) or (VIB), and one or more pharmaceutically acceptable excipients.
  • the present invention provides use of a pharmaceutical composition
  • a pharmaceutical composition comprising a substance of formula (I), (II), (III), (IV), (V), (VA), (VI), or (VIB), and one or more pharmaceutically acceptable excipients, in the manufacture of a medicament for preventing or treating a disease, disorder, or condition which is an Other NLRX1-Related Disease, Disorder or Condition.
  • the pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients selected from the group consisting of adjuvants, antioxidants, bacteriostats, binders, detergents, diluents, disintegrants, emulsifiers, extenders, lubricants, salts, solvents, solutes, stabilisers, sugars, suspending agents, surfactants, such as non-ionic surfactants, oils, preservatives, thickeners, tonicity adjusting (osmotic) agents, buffers, viscosity enhancers, and water.
  • pharmaceutically acceptable excipients selected from the group consisting of adjuvants, antioxidants, bacteriostats, binders, detergents, diluents, disintegrants, emulsifiers, extenders, lubricants, salts, solvents, solutes, stabilisers, sugars, suspending agents, surfactants, such as non-ionic surfactants, oils, preservatives, thickeners, tonicity
  • the substances of the invention may be provided in the form of a pharmaceutically acceptable salt and/or solvate thereof.
  • said substance may be provided in the form of a pharmaceutically acceptable salt and/or solvate, such as a pharmaceutically acceptable salt.
  • the salts of the substances of the present invention including substances of formula (I), (II), (III), (IV), (V), (VA), (VI), and (VIB), should be pharmaceutically acceptable.
  • Non-pharmaceutically acceptable salts of the said substances may be of use in other contexts such as during preparation of the substances of the invention.
  • Suitable pharmaceutically acceptable salts will be apparent to those skilled in the art.
  • Pharmaceutically acceptable salts include those described by Berge et al. (1977). Such pharmaceutically acceptable salts include acid and base addition salts.
  • Pharmaceutically acceptable acid additional salts may be formed with inorganic acids e.g. hydrochloric, hydrobromic, sulphuric, nitric or phosphoric acid and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid.
  • Other salts e.g.
  • oxalates or formates may be used, for example in the isolation of substances of the invention and are included within the scope of this invention.
  • Certain substances of the invention including substances of formula (I), (II), (III), (IV), (V), (VA), (VI), and (VIB), may form acid or base addition salts with one or more equivalents of the acid or base.
  • the present invention includes within its scope all possible stoichiometric and non-stoichiometric forms.
  • the substances of the present invention including substances of formula (I), (II), (III), (IV), (V), (VA), (VI), and (VIB), may be prepared in crystalline or non-crystalline form and, if crystalline, may optionally be solvated, e.g. as the hydrate.
  • This invention includes within its scope stoichiometric solvates (e.g. hydrates) as well as substances containing variable amounts of solvent (e.g. water).
  • the present disclosure includes all isotopic forms of the substances of the present invention, including substances of formula (I), (II), (III), (IV), (V), (VA), (VI), and (VIB), whether in a form (i) wherein all atoms of a given atomic number have a mass number (or mixture of mass numbers) which predominates in nature (referred to herein as the “natural isotopic form”) or (ii) wherein one or more atoms are replaced by atoms having the same atomic number, but a mass number different from the mass number of atoms which predominates in nature (referred to herein as an “unnatural variant isotopic form”).
  • unnatural variant isotopic form also includes embodiments in which the proportion of an atom of given atomic number having a mass number found less commonly in nature (referred to herein as an “uncommon isotope”) has been increased relative to that which is naturally occurring e.g. to the level of >20%, >50%, >75%, >90%, >95% or >99% by number of the atoms of that atomic number (the latter embodiment referred to as an "isotopically enriched variant form").
  • the term “unnatural variant isotopic form” also includes embodiments in which the proportion of an uncommon isotope has been reduced relative to that which is naturally occurring.
  • Isotopic forms may include radioactive forms (i.e. they incorporate radioisotopes) and non-radioactive forms. Radioactive forms will typically be isotopically enriched variant forms.
  • An unnatural variant isotopic form of a compound may thus contain one or more artificial or uncommon isotopes such as deuterium ( 2 H or D), carbon-11 (11C), carbon-13 (13C), carbon-14 (14C), nitrogen-13 (13N), nitrogen-15 (15N), oxygen-15 ( 15 O), oxygen-17 ( 17 O), oxygen-18 ( 18 O), phosphorus-32 ( 32 P), sulphur-35 ( 35 S), chlorine-36 ( 36 Cl), chlorine-37 ( 37 Cl), fluorine-18 ( 18 F) iodine-123 ( 123 I), iodine-125 ( 125 I) in one or more atoms or may contain an increased proportion of said isotopes as compared with the proportion that predominates in nature in one or more atoms.
  • Unnatural variant isotopic forms comprising radioisotopes may, for example, be used for drug and/or substrate tissue distribution studies.
  • the radioactive isotopes tritium, i.e. 3 H, and carbon-14, i.e. 14 C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
  • Unnatural variant isotopic forms which incorporate deuterium i.e. 2 H or D may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
  • unnatural variant isotopic forms may be prepared which incorporate positron emitting isotopes, such as 11 C, 18 F, 15 O and 13 N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
  • the substances of the present invention including substances of formula (I), (II), (III), (IV), (V), (VA), (VI), and (VIB), are provided in a natural isotopic form.
  • the substances of the present invention including substances of formula (I), (II), (III), (IV), (V), (VA), (VI), and (VIB), are provided in an unnatural variant isotopic form.
  • the unnatural variant isotopic form is a form in which deuterium (i.e. 2 H or D) is incorporated where hydrogen is specified in the chemical structure in one or more atoms of a substance of the invention.
  • the atoms of the substances of the invention are in an isotopic form which is not radioactive.
  • one or more atoms of the substances of the invention are in an isotopic form which is radioactive.
  • radioactive isotopes are stable isotopes.
  • the unnatural variant isotopic form is a pharmaceutically acceptable form.
  • substances of the present invention including substances of formula (I), (II), (III), (IV), (V), (VA), (VI), and (VIB), are provided whereby a single atom of the compound exists in an unnatural variant isotopic form.
  • substances of the present invention including substances of formula (I), (II), (III), (IV), (V), (VA), (VI), and (VIB), two or more atoms exist in an unnatural variant isotopic form.
  • Unnatural isotopic variant forms can generally be prepared by conventional techniques known to those skilled in the art or by processes described herein e.g. processes analogous to those described in the accompanying Examples for preparing natural isotopic forms.
  • unnatural isotopic variant forms could be prepared by using appropriate isotopically variant (or labelled) reagents in place of the normal reagents employed in the Examples.
  • the substances of the invention are intended for use in pharmaceutical compositions it will readily be understood that they are each preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are on a weight for weight basis). Impure preparations of the substances of the invention may be used for preparing the more pure forms used in the pharmaceutical compositions.
  • the amount of active ingredient which is required to achieve a prophylactic or therapeutic effect will, of course, vary with the particular substance, the route of administration, the subject under treatment or prophylaxis, including the type, species, age, weight, sex, and medical condition of the subject and the renal and hepatic function of the subject; and the particular disease, disorder, or condition being treated or prevented, as well as its severity.
  • An ordinarily skilled physician, veterinarian or clinician can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the disease, disorder, or condition.
  • compositions according to the present invention include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous [bolus or infusion], and intraarticular), intranasal (also known as nasal administration), inhalation (including fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators) insufflation, rectal, intraperitoneal, topical (including dermal, buccal, sublingual, and intraocular) and intrathecal administration, although the most suitable route may depend upon, for example, the disease, disorder, or condition of the subject.
  • suitable pharmaceutical compositions according to the present invention are those suitable for oral, intrathecal and parenteral administration; and more suitably are those suitable for oral or intrathecal administration.
  • pharmaceutical compositions of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • Oral dosages of the present invention, when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 100 mg/kg/day, suitably 0.01 mg per kg of body weight per day (mg/kg/day) to 10 mg/kg/day, and most suitably 0.1 to 5.0 mg/kg/day, for adult humans.
  • compositions are suitably provided in the form of tablets or other forms of presentation provided in discrete units, such as capsules, cachets, pills, containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • the oral composition may be presented as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, for example as elixirs, tinctures, suspensions or syrups; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • An oral composition typically contains from about 0.01 mg to about 500 mg of the active ingredient, suitably from about 1 mg to about 100 mg of active ingredient.
  • the oral dosages of the present invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired nit.
  • a tablet may be made by compression or moulding, optionally with one or more pharmaceutically acceptable carriers.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered substance moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
  • the pharmaceutical compositions therefore may be administered in a form suitable for immediate release or extended release. Immediate release or extended release can be achieved by the use of suitable pharmaceutical compositions comprising a substance of the present invention, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.
  • the pharmaceutical compositions of the invention may also be administered liposomally.
  • compositions for oral administration include suspensions which can contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents such as those known in the art; and immediate release tablets which can contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate, calcium sulfate, sorbitol, glucose and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art.
  • Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
  • Disintegrators include without limitation starch, methylcellulose, agar, bentonite, xanthan gum and the like.
  • Pharmaceutical compositions of the invention may also be administered via the oral cavity by sublingual and/or buccal administration.
  • compositions for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerine or sucrose and acacia.
  • exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene). Molded tablets, compressed tablets or freeze-dried tablets are exemplary forms which may be used.
  • Exemplary pharmaceutical compositions include those formulating a substance of the present invention with fast dissolving diluents such as mannitol, lactose, sucrose and/or cyclodextrins.
  • compositions may be high molecular weight excipients such as celluloses (avicel) or polyethylene glycols (PEG).
  • Such compositions can also include an excipient to aid mucosal adhesion such as hydroxy propyl cellulose (HPC), hydroxy propyl methyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleic anhydride copolymer (e.g., Gantrez), and agents to control release such as polyacrylic copolymer (e.g. Carbopol 934).
  • HPC hydroxy propyl cellulose
  • HPMC hydroxy propyl methyl cellulose
  • SCMC sodium carboxy methyl cellulose
  • Gantrez sodium carboxy methyl cellulose
  • agents to control release such as polyacrylic copolymer (e.g. Carbopol 934).
  • Lubricants, glidants, flavors, coloring agents and stabilizers may also be added for ease of fabrication and use.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • Intrathecal administration involves injection of the pharmaceutical composition of the invention into the spinal canal, or into the subarachnoid space so that it reaches the cerebrospinal fluid. This is advantageous for the administration of substances which may not be able to pass the blood brain barrier via other routes of administration, such as oral administration.
  • compositions of the present invention may be administered intrathecally by continuous infusion such as with a catheter, or a pump, or intrathecally by a single bolus injection or by intermittent bolus injection.
  • Pharmaceutical compositions of the invention which are administered intrathecally and continuously are suitably administered via implantable delivery devices, such as an implantable pump. Examples of such delivery devices include devices which can be implanted subcutaneously in the body or in the cranium, and provides an access port through which the pharmaceutical compositions may be delivered to the nerves or brain.
  • the pharmaceutical composition may also be administered intermittently.
  • the intermittent administration may be, for example, every thirty minutes, every hour, every several hours, every 24 hours, every couple of days (for example every 48 or 72 hours) or any combination thereof.
  • Intrathecal dosages of the present invention when used for the indicated effects, will typically be less than 1 mg, such as less than 500 ⁇ g, for example less than 250 ⁇ g per kg of body weight when administered in a single dose or intermittently for adult humans.
  • the intrathecal dosages of the present invention When administered continuously, will typically be less than 250 ⁇ g per kg body weight per hour, such as less than 125 ⁇ g per kg body weight per hour for adult humans.
  • compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the pharmaceutical compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use.
  • compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer’s solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • suitable non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer’s solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • suitable doses will range from about 0.1 to about 10 mg/kg/minute during a constant rate infusion.
  • a pharmaceutical composition of the invention is administered by intranasal, inhalation (including fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators) or insufflation administration.
  • intranasal, inhalation including fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators
  • insufflation administration Such a method of administration allows for low doses of the pharmaceutical composition of the invention to be administered, which can lead to a reduction in side-effects.
  • Exemplary pharmaceutical compositions for intranasal, aerosol or inhalation administration include solutions in saline, which can contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.
  • compositions of the invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • Pharmaceutical compositions for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter, synthetic glyceride esters or polyethylene glycol.
  • Such carriers are typically solid at ordinary temperatures but liquefy and/or dissolve in the rectal cavity to release the drug.
  • Examples General Methods The substances used in the Examples below, including the Background Probes and Test Substances, are disclosed in, or may suitably be prepared according to the methods disclosed in WO2022/049376, and/or WO2022/049377, and/or WO2023/166303, and/or WO2024/153945, and/or WO2024/153946, and/or WO2025/052129.
  • Assay for inhibition of mPTP activity Rat liver mitochondria assay Pharmacological inhibition or modulation of the mPTP can be measured in well characterised ‘Ca 2+ retention’ assays performed in isolated mitochondria.
  • isolated mitochondria rapidly sequester exogenous Ca 2+ until the intramitochondrial Ca 2+ concentration reaches the threshold for mPTP activation. Once the pore is activated, mitochondrial integrity is compromised and the stored Ca2+ is released.
  • the distribution of Ca2+ between extra- and intra-mitochondrial compartments can be measured in real time with the use of membrane- impermeant Ca 2+ sensitive fluorescent dyes.
  • inhibition or modulation of the mPTP either delays the opening of the pore or increases the concentration of Ca 2+ required to induce mPTP opening.
  • mPTP activity is measured in mitochondria freshly isolated from female Sprague Dawley (250 to 300 gram) rat livers using the folllowing method.
  • Cervical dislocation is performed on the rat.
  • the liver is then perfused in-situ with ⁇ 40 ml cold Dulbecco’s Phosphate Buffered Saline (DPBS) prior to dissection and transfered into 30 ml Isolation Buffer (250mM Sucrose, 10mM KCl, 1mM EGTA, 1mM EDTA, 25mM HEPES, adjusted to pH 7.5 with 1M NaOH).
  • DPBS Phosphate Buffered Saline
  • Each lobe of the liver is then removed from the buffer, minced using tweezers and a scalpel into ⁇ 5mm pieces then transferred into a 50 ml Potterton dounce homogenization tube on ice containing 30 ml ice-cold centrifugation buffer (300mM Trehalose, 25mM HEPES, 1mM EGTA, 1mM EDTA, 10mM KCl, adjusted to pH 7.5 with 1M NaOH and supplemented with 0.1% bovine serum albumin (BSA) and complete protease inhibitor cocktail (one tablet of inhibitor per 50mls of buffer). Homogenisation is carried out using a teflon pestle at 1800 rpm.
  • BSA bovine serum albumin
  • the slurry is centrifuged at 800 g for 10 min at 4 o C, then the supernatant centrifuged at 10,000 g for 10 min.
  • the pellet is washed once with FLIPR assay buffer (75mM Mannitol, 25mM Sucrose, 5mM Potassium Phosphate Monobasic, 20mM Tris base, 100mM KCl, 0.1 % BSA adjusted to pH 7.4 with 5M HCl) centrifuged again, then resuspended in FLIPR assay buffer to a concentration of 8.8 mg/ml protein.
  • FLIPR assay buffer 75mM Mannitol, 25mM Sucrose, 5mM Potassium Phosphate Monobasic, 20mM Tris base, 100mM KCl, 0.1 % BSA adjusted to pH 7.4 with 5M HCl
  • Tested compounds (10 mM stock in DMSO) are serially diluted in DMSO in half log steps to generate 10 test concentrations (final concentrations in assay 30 ⁇ M to 1 nM).
  • An intermediate dilution of 5 ⁇ l DMSO samples into 247 ⁇ l FLIPR assay buffer is carried out prior to transfer of 5 ⁇ l into duplicate wells of a 384 well polypropylene assay plate.
  • Control wells are 0.5 % (v/v) DMSO and 5 ⁇ M cyclosporin A.
  • a stock mitochondria/Fluo5N assay solution is prepared in 5.6 ml FLIPR assay buffer (at RT) supplemented with succinate disodium salt (10mM), rotenone (1 ⁇ M), Fluo5N pentapotassium salt (2 ⁇ M) and 1 ml mitochondria suspension, then transferred (15 ⁇ l) into the assay plate containing test compounds and incubated for 10 min at RT.
  • Assay plates are processed for fluorescence detection on either a FLIPR Tetra (Molecular Devices) or CLARIOstar (BMG) plate reader. For kinetic fluorescence detection on the FLIPR, dye fluorescence is measured every 3 sec for a total of 10 min.
  • a 2.5 ⁇ l bolus of CaCl 2 (75 ⁇ M) is added from a source plate containing 675 ⁇ M CaCl 2 in FLIPR assay buffer.
  • the 2.5 ⁇ l addition of CaCl 2 is performed using a Viaflow 384, plates areincubated at RT for 10 min, and dye fluorescence measured after 10 min on the CLARIOstar plate reader.
  • pIC50 values for tested compounds are calculated using the fluorescence value collected at the 10 min timepoint on either plate reader, with % inhibition calculated using the DMSO control and cyclosporin A values as 0 and 100 % respectively.
  • mPTP activity is measured in brain mitochondria freshly isolated from female Sprague Dawley (250 to 300 gram) rats. Anaesthetised rats were perfused in-situ with ⁇ 40 ml cold Dulbecco’s Phosphate Buffered Saline (DPBS), then brains dissected and transferred into 30 ml Isolation Buffer (225mM mannitol, 75 mM sucrose, 1mM EGTA, adjusted to pH 7.4 with 1M NaOH).
  • DPBS Phosphate Buffered Saline
  • the brain is minced using tweezers and a scalpel into ⁇ 5mm pieces then transferred into a 50 ml Potterton Dounce homogenization tube on ice containing 10 ml ice-cold isolation buffer (as above with addition of Complete Protease inhibitor; 1 tablet per 50 ml buffer). Homogenisation is carried out using a teflon pestle at 1800 rpm. The slurry is centrifuged at 2000 g for 10 min at 4 o C, then the supernatant centrifuged at 12,000 g for 9 min.
  • Test compounds are prepared in 384 well polypropylene assay plates as described above for the liver mitochondria assay.
  • a stock mitochondria/Fluo5N assay solution is prepared in 5.6 ml assay buffer (120 mM mannitol, 40 mM MOPS, 5 mM KH2PO4, 60 mM KCl, 10 mM pyruvate, 2 mM malate, 2 mM MgCl2, 20 ⁇ M ADP, 1.26 ⁇ M oligomycin A, adjusted to pH 7.4) supplemented with Fluo5N pentapotassium salt (2 ⁇ M) and 1 ml mitochondria suspension, then transferred (15 ⁇ l) into the assay plate containing test compounds and incubated for 10 min at RT. Assay plates are then transferred to a FLIPR Tetra plate reader (Molecular Devices).
  • Dye fluorescence is then measured every 3 sec for a total of 10 min. After 12 sec, a 2.5 ⁇ l bolus of Ca2+ (75 ⁇ M) is added from a source plate containing 675 ⁇ M CaCl2 in FLIPR assay buffer. pIC50 values for test compounds are calculated using the fluorescence value collected at the 10 min timepoint with % inhibition calculated using the DMSO control and cyclosporin A values at 100 % and 0 % respectively.
  • Background Example 1 Identification of NLRX1 as a binding partner of inhibitors of the mPTP Potential binding partners of known inhibitors of the mPTP were identified by a process of affinity-based chemoproteomics, using Background Probe 1 below: which is known to be a potent inhibitor of the mPTP. Moreover, Background Probe 1 is suitable for immobilization on azide-activated affinity resin. Conjugation of Background Probe 1 to azide-agarose beads were carried out by click chemistry using two different coupling densities, specifically 0.6 mM and 1.2 mM, to yield low- density (LD) and high-density (HD) beads respectively.
  • LD low- density
  • HD high-density
  • Affinity-based purification of target proteins to be screened was performed in clarified detergent cell lysates, specifically containing 1 mg/mL protein in 0.5% Triton X-100 extract, prepared from HEK293 cells.
  • control beads i.e. empty azide-agarose beads
  • LD beads LD beads
  • HD beads were incubated in duplicate with the clarified detergent cell lysates referred to above, and in the presence of DMSO control, or Background Probe 2 below: at a concentration of 1 ⁇ M or 30 ⁇ M, each in 0.3% DMSO, at 4 o C.
  • Background Probe 2 is another known inhibitor of the mPTP, and in particular is known to be a far more potent inhibitor of the mPTP than Background Probe 1 above.
  • proteins identified in accordance with the criteria above are plotted according to the % displacement value, which is itself the mean of duplicate values, of each of the LD and HD beads by Background Probe 2.
  • the results show that only two proteins, PTDSS1 and NLRX1, are common, strong, hits between LD and HD beads. Of these two proteins, only NLRX1 is a mitochondrial protein.
  • NLRX1 was the protein maximally displaced by Background Probe 2, relative to all other proteins identified as potential binding partners of known inhibitors of the mPTP. These findings implicate NLRX1 as a binding partner of known inhibitors of the mPTP.
  • a repeat chemoproteomic screen was performed, with the addition of a further negative control.
  • NLRX1 is enriched by more than 5-fold in Background Probe 1 bead samples relative to control (empty) bead samples and displaced by greater than 60% in both the first and second experiments. In fact, NLRX1 had a mean displacement of 93% across all samples. These results provide strong evidence to suggest that NLRX1 is a binding partner of known inhibitors of the mPTP.
  • Background Example 2 Evaluation of the role of NLRX1 in interaction with, and modulation of, the mPTP The activity of the mPTP in both wild-type and NLRX1 knock-out (KO) cells was evaluated using methods known in the art and described in published protocols (see Kuznetsov et al. 2008).
  • wild-type and NLRX1 KO HeLa cells were purchased from a commercial provider (Abcam). Subsequently, both cell types were resuspended in digitonin lysis buffer (130 mM potassium chloride, 10 mM MOPS-Tris, 1mM Trizma-Pi, 1 mM EGTA, 100 ⁇ M digitonin) and incubated on ice for 5 minutes to ensure permeabilization of the plasma membrane). Following two centrifugation and wash steps (i.e.
  • the cells were added to wells of a 96-well plate at a density of 1 x 10 6 cells per well, in 200 ⁇ L assay buffer (130 mM potassium chloride, 10 mM MOPS-Tris, 1mM Trizma-Pi, 10 ⁇ M EGTA-Tris, 5 mM glutamate, 2.5 mM malate, 0.5 ⁇ M Calcium Green 5N, pH 7.4), with wells containing either test substance or 0.1% DMSO control.
  • the 96-well plate was then immediately transferred to a FLIPR® Tetra Plate Reader for kinetic fluorescence detection.
  • NLRX1 Binding Examples
  • NLRX1 is therefore a novel target against which substances may be screened for binding ability.
  • the present inventors have therefore developed novel methods and assays which are suitable for identifying substances which are capable of binding to NLRX1.
  • NLRX1 Binding Example 1 Screening new substances for NLRX1 binding using a cellular thermal shift assay (CETSA) New substances may be screened for binding to NLRX1 using a cellular thermal shift assay (CETSA), the protocol for which has been developed and optimised by the present inventors. Specifically, HEK293T cells were suspended in Hanks’ balanced salt solution (HBSS; Gibco) at a concentration of 40 million cells/mL, prior to mixing in equal parts with test substances (suspended in HBSS supplemented with 0.4% DMSO) to yield a final cell density of 20 million cells/mL in 0.2 % DMSO.
  • HBSS Hanks’ balanced salt solution
  • test substances sustained in HBSS supplemented with 0.4% DMSO
  • chemiluminescent Western Blotting detection was achieved using a horseradish peroxidase (HRP) conjugated anti-mouse secondary antibody (W4021; Promega), at a dilution of 1:20000, by incubation with the anti-NLRX1 antibody treated Westen Blot sample for 1 hour at room temperature, and subsequent incubation with Clarity Western ECL substrates (Bio- Rad), according to the manufacturer’s instructions.
  • HRP horseradish peroxidase
  • W4021 horseradish peroxidase secondary antibody
  • Clarity Western ECL substrates Bio- Rad
  • Test Substance 1 The results of testing a known potent inhibitor of the mPTP, herein referred to as Test Substance 1, via the above-described CETSA are shown in Figure 4.
  • Tm 61.0 o C
  • Test Substance 1 is the same as Background Probe 2 referred to above.
  • Test Substance 3 is the same as Background Probe 1 referred to above.
  • the results of Figure 5 show that the above-described CETSA protocol is a suitable method for the identification of substances which bind to NLRX1. In particular, Test Substances 1, 3, and 4, were demonstrated to bind to NLRX1 with different affinities.
  • Test Substances 2 and 5 failed to demonstrate significant specific NLRX1 binding.
  • EC50 values (of NLRX1 binding) were calculated for each of Test Substances 1, 3, and 4 and are provided in Table 2 below: Table 2: T est Substance No. Calculated EC50 Value ( ⁇ M)* Test Substance 1 6 Test Substance 3 34 Test Substance 4 2 *mean of 3 or more replicate experiments NLRX1 Binding Example 2: Screening new substances for NLRX1 binding using a filtration binding assay New substances may be screened for binding to NLRX1 using a filtration binding assay, the protocol for which has been developed and optimised by the present inventors.
  • NLRX1-expressing membranes were prepared from a HeLa cell line stably transfected with the full-length human NLRX1 gene.
  • cultured cells were detached and suspended with Buffer A (50 mM Tris HCl pH 7.4, 5 mM EDTA/Tris pH 7.4, 20 mM NaCl, 5 mM KCl, 5 mM MgCl2, 10 ⁇ g/ml trypsin inhibitor, 1 ⁇ g/ml of leupeptin, and 75 ⁇ g/ml PMSF) at 4°C and at a density of 500 x 106 cells per 10 mL. Cells were then lysed using an ultrasound probe for 1 minute.
  • Buffer A 50 mM Tris HCl pH 7.4, 5 mM EDTA/Tris pH 7.4, 20 mM NaCl, 5 mM KCl, 5 mM MgCl2, 10 ⁇ g/ml trypsin inhibitor, 1 ⁇ g/ml of leupeptin,
  • the homogenate obtained was adjusted to 40 mL Buffer A and centrifugated at 50000 g.
  • the pellet was removed using a 23G gauge needle then resuspended by agitation with a 25G gauge needle in Buffer B (Buffer A with 10% glycerol). Protein quantification was determined according to the Bradford method and aliquots were stored at -80°C.
  • NLRX1-expressing membranes are suspended in incubation buffer (50 mM Tris/HCl, 5 mM EDTA/Tris (pH 7.4), 150 mM NaCl, 5 mM KCl and 2 mM MgCl2) to achieve a protein concentration of 150 ⁇ g/mL, in a 200 ⁇ L sample volume Probe Substances alone, or in combination with Test Substances, were then added to the samples and the sample incubated for 15 minutes (Probe Substance 1) or 180 minutes (Probe Substance 2) at 4oC.
  • the structure of Probe Substances 1 and 2 are shown in Table 3 below: Table 3: P robe Substance No.
  • Probe Substance 1 Probe Substance 2 Next, the samples were added to AcroPrepTM GF/B filter plates, which have previously been wetted with filter buffer (50 mM Tris/HCl (pH 7.4), and 0.001% N-dodecyl- ⁇ -maltoside), for 60 minutes at room temperature. Excess filter buffer was then removed via centrifugation for 1 minute at 1000 rpm. Samples were then filtered via centrifugation for 1 minute at 1000 rpm, and subsequently washed twice with filter buffer.
  • filter buffer 50 mM Tris/HCl (pH 7.4), and 0.001% N-dodecyl- ⁇ -maltoside
  • chromatographic separation was optimised to define the gradient parameters necessary to elute Probe Substances 1 and 2 with optimal peak shape and resolution.
  • MRM multiple reaction monitoring
  • the limit of detection (LoD) and limit of qualification (LoQ) values were 0.15 nM and 0.6 nM respectively for Probe Substance 1 and 1.5 nM and 3 nM respectively for Probe Substance 2.
  • Non-specific binding was defined and accounted for in subsequent experiments via addition of 10 ⁇ M of either Substance A, used for Probe Substance 1, or Substance B, used for Probe Substance 2, the structures of which are shown in Table 4 below: Table 4: S ubstance ID Structure Substance A Substance B Prior to screening a series of Test Substances using the filtration binding assay protocol described above, binding of Probe Substances 1 and 2 to NLRX1-expressing membranes was confirmed and similarly compared to binding of said Probe Substances to membranes derived from cells which have not been transfected with NLRX1. For this experiment, Probe Substance 1 was added to samples at a concentration of 1 nM and Probe Substance 2 was added to samples at a concentration of 5 nM.
  • Probe Substance 1 was added to samples at a concentration of 10 nM and Probe Substance 2 was added to samples at a concentration of 2 nM.
  • Test Substances were added to samples at concentrations ranging between 10 ⁇ M and 1 nM. Specific binding of Test Substances to NLRX1-expressing membranes was assessed via calculation of K i values.
  • Table 5 A summary of the Test Substances screened using the filtration binding assay protocol of the invention, and the Ki values derived from their screening (and the Probe Substance said Test Substance was used to elute), are summarised in Table 5 below: Table 5: T est Substance No.
  • Test Substance Structure Ki 95.0 (Probe Test Substance 6 Substance 1) 13.0 (Probe Test Substance 7 Substance 2) 69.0 (Probe Test Substance 8 Substance 1) 2.8 (Probe Test Substance 9 Substance 1) 8.7 (Probe Test Substance 10 Substance 2) 23.0 (Probe Test Substance 11 Substance 1) 19.0 (Probe Test Substance 12 Substance 1) 5.4 (Probe Test Substance 13 Substance 2)
  • NLRX1 Binding Example 3 Screening new substances for NLRX1 binding using a nano- BRET assay New substances may be screened for binding to NLRX1 using a nano-BRET assay, the protocol for which has been developed and optimised by the present inventors. Specifically, a vector comprising full length human NLRX1 (Uniprot ID Q86UT6) fused to a NanoLuc® tag (Promega) at the C-terminus in a pFC32 NanoLuc® Protein Fusion Flexi® Vector (Promega) was produced by DNA synthesis and molecular cloning.
  • a vector comprising full length human NLRX1 (Uniprot ID Q86UT6) fused to a NanoLuc® tag (Promega) at the C-terminus in a pFC32 NanoLuc® Protein Fusion Flexi® Vector (Promega) was produced by DNA synthesis and molecular cloning.
  • HEK293T cells ECAAC; 2 x 10 5 cells/mL
  • ECAAC EAAAC
  • HEK293T cells ECAAC
  • 2 x 10 5 cells/mL EAAAC
  • OptiMEM Invitrogen
  • Control samples were prepared similarly but with replacement of the NLRX1- NanoLuc® vector with an equivalent volume of OptiMEM.
  • Cell samples were incubated for 20 minutes at room temperature prior to mixing with lipid mixtures and subsequent incubation for 24 hours at 37 o C, 5% CO 2 , in a humidified incubator.
  • test Substances were then added using the Multidrop Pico 8 dispenser device (ThermoFisher Scientific), at a top concentration of 10 ⁇ M, and as a ten-point, 1:3, serial dilution in duplicate wells. A tracer molecule was then added at a final concentration of 1 ⁇ M to all wells of the 96- well plate. Samples were then incubated for 2 hours at 37 o C, 5% CO2, in a humidified incubator.
  • the tracer molecule was produced via click chemistry to conjugate the NanoBRET TM 590- Azide-C3 (Promega) to the alkyne moiety of the below compound: Following incubation, 50 ⁇ L of a development solution of Nano-Glo® substrate (Promega) diluted 1 in 166 and supplemented with a 1 in 500 dilution of extracellular inhibitor in OptiMEM, was added to each well of the 96-well plate. The plate contents were then mixed by shaking and incubation for 10 minutes at room temperature. Assay signals (luciferase signal) were obtained using a CLARIOstar reader (top optic: Em: 470/480; Em F: 615-18).
  • the BRET ratio (acceptor 618 nm emission/donor 470 nm emission) for each concentration was obtained, multiplied by 1000 and then plotted.
  • the results for this analysis for two Test Substances, 2 and 6, are shown in Figures 8 and 9.
  • the BRET ratio decreased approximately two-fold, indicative of specific NLRX1 binding, whilst no such decrease was seen in the presence of Test Substance 2.
  • Table 6 Table 6: T est Substance No.
  • a radiolabelled Test Substance was produced via synthesis of tritium, [3H], labelled Test Substance 15, the structure of which is illustrated below: , which radiolabelled Test Substance has a radioactive concentration of 1 mCi/mL and a specific activity of 28 Ci/mmol.
  • binding of said Test Substance to brain slices from wild-type and NLRX1-knockout mice was evaluated by ex vivo autoradiography.
  • brains from an adult wild-type or NLRX1-knockout mice were dissected and fresh frozen at between -25 and -30 oC, using 2-methyl-butane to regulate the freezing process, prior to storage at -80 oC before use.
  • brains were sectioned coronally, from anterior to posterior (to include the cortical region as well as both dorsal and ventral hippocampus), on a cryostat to provide brain slices of about 20 ⁇ m thickness, which slices are then placed onto 1 % gelatin-coated slides using the ‘freeze thaw’ method. Excess water was removed from the sections by incubating with CaSO4 for 1 hour at 4 oC before storing those sections at -80 oC until use.
  • the coronal brain sections were incubated with 3.3 nM radiolabelled Test Substance 15 for 120 minutes at 4 o C, in the presence of an intracellular buffer comprising 50 mM Tris, 10 mM NaCl, 0.5 mM MgCl2, 140 mM KCl, and having pH 7.0.
  • the specific binding component was determined by incubating adjacent coronal brain sections with the radiolabelled Test Substance 15 (as above) in the presence of a saturating concentration (10 ⁇ M) of unlabelled Test Substance 15, which yields a measure of non-specific binding.
  • Triplicate 5 ⁇ L aliquots of each stock were counted on a beta counter to confirm the concentration of each radioligand dilution.
  • coronal brain sections were washed twice (each time for 1 minute) in ice cold buffer (comprising 50 mM Tris and 1.4 mM MgCl2, having pH 7.4 at 4 °C) before being briefly submerged in water to remove excess salts.
  • the coronal brain sections were then dried immediately in a continuous cool air stream overnight, prior to apposition.
  • Total binding of 3.3 nM radiolabelled Test Substance 15 to wild-type brain slices was 22 ⁇ 4 fmol/mg protein, whereas binding to NLRX1-knockout brain slices was below the limit of quantification consistent with specific binding of the radioligand to NLRX1 (see Figure 19).
  • the homogenate was centrifuged at 32000 g at 4 oC for 20 minutes.
  • the resultant pellet was washed in the same buffer and centrifuged again at 32000 g at 4 oC for 20 minutes.
  • the pellet was then washed a further three times to obtain a P2 membrane homogenate.
  • This final pellet was resuspended in wash buffer, i.e.50 mM Tris, of pH 7.4 at 4°C.
  • the protein concentration was determined by BCA assay (Pierce BCA kit) and the homogenate stored at 80 0C.
  • rat brain homogenates were thawed on ice and made up to an assay volume of 0.25 mL per well, with 55 ⁇ g protein per well, using intracellular buffer (comprising 50 mM Tris, 10 mM NaCl, 0.5 mM MgCl2, and 140 mM KCl, and having pH 7.0) at 4°C. Homogenate suspensions were then incubated at 4 oC for 120 minutes with 0.1 to 150 nM radiolabelled Test Substance 15. As above, the specific binding component was determined by incubating the membrane suspension with radioligand in the presence of a saturating concentration, 10 ⁇ M, of unlabelled Test Substance 15.
  • Assays were terminated by filtration through Whatman GF/B filters which were pre-soaked in 0.001% dodecyl maltopyranoside, before washing said filters four times with ice cold wash buffer comprising 50 mM Tris of pH 7.4 at 4°C. Known volumes of stock radioligand were counted to produce a standard curve. Using this method, the Kd for the binding of Test Substance 15 to NLRX1 was determined to be 7.3 nM, which value was the mean of triplicate experiments (see Figure 20). Next, competition binding assays were performed using the above-described filtration binding assay protocol, to determine binding of Test Substances to NLRX1-expressing coronal brain sections.
  • Test Substance 15 was added to coronal brain sections at a concentration of 10 nM.
  • Test Substances were added to samples at concentrations ranging between 10 ⁇ M and 0.01 nM.
  • Specific binding of Test Substances to NLRX1-expressing membranes was assessed via calculation of K i values.
  • Table 7 A summary of the Test Substances screened using the filtration binding assay protocol of the invention, and the Ki values derived from their screening are summarised in Table 7 below: Table 7: T est Substance No.
  • Example 1 Investigating the mPTP inhibitory activity of substances that bind to NLRX1 using an mPTP activity assay in isolated rat liver mitochondria
  • pharmacological inhibition of the mPTP can be measured in ‘Ca 2+ retention’ assays performed in isolated mitochondria.
  • activity of the mPTP was measured in mitochondria freshly isolated from female Sprague Dawley (250 to 300 gram) rat livers. Firstly, cervical dislocation was performed on the rat prior to perfusion of the liver in-situ with about 40 mL cold Dulbecco’s Phosphate Buffered Saline (DPBS).
  • DPBS Phosphate Buffered Saline
  • the liver was then dissected and transfered into 30 mL isolation buffer (250 mM sucrose, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 25 mM HEPES, adjusted to pH 7.5 with 1 M NaOH).
  • isolation buffer 250 mM sucrose, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 25 mM HEPES, adjusted to pH 7.5 with 1 M NaOH).
  • Each lobe of the liver was then removed from the buffer, minced using tweezers and a scalpel into ⁇ 5 mm pieces then transferred into a 50 mL Potterton dounce homogenization tube on ice containing 30 mL ice-cold centrifugation buffer (300 mM trehalose, 25 mM HEPES, 1 mM EGTA, 1 mM EDTA, 10 mM KCl, adjusted to pH 7.5 with 1 M NaOH and supplemented with 0.1% bovine serum albumin (BSA) and complete protease inhibitor cocktail (one tablet of inhibitor per 50 mL buffer). Homogenisation was carried out using a teflon pestle at 1800 rpm.
  • BSA bovine serum albumin
  • the slurry was centrifuged at 800 g for 10 minutes at 4 oC, then the supernatant centrifuged at 10,000 g for 10 minutes.
  • the pellet was washed once with FLIPR assay buffer (75 mM mannitol, 25 mM sucrose, 5 mM potassium phosphate (monobasic), 20 mM Tris base, 100 mM KCl, 0.1 % BSA adjusted to pH 7.4 with 5 M HCl) centrifuged again, then resuspended in FLIPR assay buffer to a concentration of 8.8 mg/mL protein.
  • FLIPR assay buffer 75 mM mannitol, 25 mM sucrose, 5 mM potassium phosphate (monobasic), 20 mM Tris base, 100 mM KCl, 0.1 % BSA adjusted to pH 7.4 with 5 M HCl
  • Test Substances (stored in 10 mM stock in DMSO) were serially diluted in DMSO in half log steps to generate 10 test concentrations (final concentrations in assay 30 ⁇ M to 1 nM).
  • An intermediate dilution of 5 ⁇ l DMSO samples into 247 ⁇ l FLIPR assay buffer was carried out prior to transfer of 5 ⁇ l into duplicate wells of a 384 well polypropylene assay plate. Control wells were 0.5 % (v/v) DMSO and 5 ⁇ M cyclosporin A.
  • a stock mitochondria/Fluo5N assay solution was prepared in 5.6 mL FLIPR assay buffer (at room temperature) supplemented with succinate disodium salt (10 mM), rotenone (1 ⁇ M), Fluo5N pentapotassium salt (2 ⁇ M) and 1 mL mitochondria suspension, prior to being transferred (15 ⁇ L) into the assay plate containing Test Substances and incubated for 10 minutes at room temperature. Assay plates were then transferred to a FLIPR® Tetra Plate Reader (Molecular Devices). Dye fluorescence was then measured every 3 seconds for a total of 10 minutes.
  • Test Substance 1 7.9 Test Substance 2 ⁇ 4.52 Test Substance 3 6.7 Test Substance 4 6.9 Test Substance 5 5.5 Test Substance 6 7.5 Test Substance 7 6.4 Test Substance 8 7.3 Test Substance 9 8.4 Test Substance 10 6.5 Test Substance 11 8.0 Test Substance 12 7.7 Test Substance 13 6.7 Test Substance 14 7.7 *mean of 3 or more replicate experiments
  • the rank order of Test Substances in NLRX1 binding affinity correlates well with the rank order of said Test Substances in mPTP inhibitory activity.
  • Test Substances 2 and 5 which fail to demonstrate any specific NLRX1 binding activity, similarly fail to inhibit the activity of the mPTP with any significant potency.
  • the present inventors consider that any weak mPTP inhibitory activity of Test Substances 2 and 5 is observed merely due to inclusion of said Test Substances at very high concentrations. This further supports the conclusion that inhibition of mPTP activity is mediated via binding to and consequential regulatory modulation of NLRX1.
  • the reaction mixture was cooled to 0 °C and LiAlH4 (1.0M in THF, 1.10 mL, 1.10 mmol) added dropwise at 0 °C.
  • the reaction mixture was stirred at RT for 2 hrs.
  • the reaction mixture was cooled to 0 °C, quenched with aqueous Rochelle's salt solution (5 mL) and extracted with EtOAc (30 mL).
  • the organic phase was washed with sat. brine solution (30 mL), passed through a phase separator cartridge (Biotage) and the filtrate evaporated in vacuo.
  • the crude product was purified by catch release cartridge (Biotage SCX-2), washing with MeOH and eluted with 1M NH 3 -MeOH.
  • Biological Example 1 Investigating the effect of mPTP Inhibitor 1 in a ⁇ NLS TDP43 mouse model of amyotrophic lateral sclerosis (ALS)
  • ALS amyotrophic lateral sclerosis
  • human TDP43 protein is expressed, via a transgene, with mutations introduced into the nuclear localisation sequence (NLS), such that the human TDP43 protein is not imported in the nucleus but mis-localised to the cell cytoplasm and other organelles e.g. mitochondria.
  • Mis-localisation of TDP43 in this mouse model is thought to resemble that observed in tissue samples/cells from amyotrophic lateral sclerosis (ALS) patients (Walker et al. 2015).
  • the human ⁇ NLS TDP43 transgene is under the control of the neurofilament (NF) gene promoter and is regulated by a doxycycline transactivation element.
  • NF neurofilament
  • ⁇ NLS TDP43 mice were generated by breeding NEFH-tTA (Jax stock 025397) with tetO-hTDP-43 ⁇ NLS bigenic mice (Jax stock 014650). Animals were maintained in 12/12 light /dark cycles at a room temperature between 20-26°C and at a relative humidity around 50%. Food and water were provided ad libitum.
  • mice and females with offspring at pre-weaning age were provided with chow containing doxycycline (200 mg/kg) to repress the hTDP43 transgene in the pups during development.
  • Pups were weaned at approximately 4 weeks of age, genotyped and kept on doxycycline (200 mg/kg)-containing chow until 7 weeks of age.
  • Mice on study were placed on regular chow.
  • Mice were group housed in OPTI-MICE ventilated cages (Animal Care Systems, CO). Animals were housed in same-genotype groups of control or transgenic mice. Mice in the same cage received the same treatment.
  • FIG. 10 illustrates that a significant increase in NFL expression was observed in both the plasma and CSF of ⁇ NLS TDP43 mice relative to wild-type mice.
  • treatment of ⁇ NLS TDP43 mice with mPTP Inhibitor 1 at either 50 mg/kg or 150 mg/kg (for 14 days) resulted in a significant reduction in NFL expression in both the plasma (64% for 50 mg/kg, 53% for 150 mg/kg) and the CSF (20% for 50 mg/kg, 62% for 150 mg/kg).
  • mPTP Inhibitor 1 in the ⁇ NLS TDP43 mouse model of amyotrophic lateral sclerosis (ALS), the level of expression of the astrocyte activation marker, GFAP, in the plasma, and the level of expression of the pro- inflammatory cytokine IL-1 ⁇ , in the brain cortices, were quantified.
  • GFAP levels were determined via Quanterix assay, using the GFAP SR-X kit.
  • IL-1 ⁇ levels were determined via lysis of individual mice brain cortices in assay kit lysis buffer (MSD, V-Plex Plus Mouse IL-1 ⁇ ) and subsequent assaying according to the manufacturers’ instructions.
  • FIG. 13 illustrates that a significant increase in IL- 1 ⁇ expression is observed in the brain cortices of ⁇ NLS TDP43 mice relative to wild-type mice. This observation is consistent with induction of neuroinflammatory pathways.
  • treatment of ⁇ NLS TDP43 mice with mPTP Inhibitor 1 at 50 mg/kg (for 14 days) resulted in a significant reduction in IL-1 ⁇ expression, which is representative of dampened neuroinflammation.
  • the effect of mPTP Inhibitor 1 on inflammation in the brain cortices of ⁇ NLS TDP43 mice was investigated by quantifying the expression levels of an array of mRNA transcripts in the brain tissue. Specifically, transcriptional expression levels were assessed via RNAseq.
  • samples were lysed, by mechanical disruption, using a TissueLyser (Qiagen) with 5 mm stainless steel beads in 750 ⁇ l QIAzol lysis buffer.
  • Isolated RNA was purified using RNeasy96 Universal Tissue Kit (Qiagen) and stored at -80 o C prior to use.
  • Directional mRNA libraries were prepared by PolyA enrichment, and samples were sequenced using Illumina PE150 Sequencing with 8 G of raw data per sample (Novogene). Differential Expressed Gene (DEG) analysis was performed by Fios Genomics.
  • DEG Differential Expressed Gene
  • 7694 mRNA transcripts (p.adj. ⁇ 0.05) were altered in ⁇ NLS TDP43 mice relative to wild-type mice.
  • Treatment of ⁇ NLS TDP43 mice with mPTP Inhibitor 1 resulted in a significant change in the expression of 143 mRNA transcripts, 74 of which were also differentially expressed between ⁇ NLS TDP43 and wild-type mice.
  • the effect of treatment with mPTP Inhibitor 1 was to normalise expression of 73 of the aforementioned 74 mRNA transcripts back in the direction towards that observed in wild-type mice.
  • ⁇ NLS TDP43 mice treatment of ⁇ NLS TDP43 mice with mPTP Inhibitor 1 results in normalisation of a subset of the transcriptome to the wild-type, i.e. disease-free phenotype.
  • mPTP Inhibitor 1 Upon further analysis, it was identified that a number of mRNA transcripts within the 74 of which were differentially expressed between both ⁇ NLS TDP43 mice and wild-type mice, and ⁇ NLS TDP43 mice treated with vehicle and ⁇ NLS TDP43 mice treated with mPTP Inhibitor 1, are associated with inflammation, and in particular are regulated by production of the pro- inflammatory cytokines, interferons.
  • 12 specific genes/mRNA transcripts were selected for further validation via qPCR, in an independent experiment.
  • mPTP Inhibitor 1 was orally administered, at a dose of either 10 mg/kg or 50 mg/kg, to ⁇ NLS TDP43 mice 28 days, and the expression of said 12 specific genes in the brain cortical cell lysates was quantified and assessed via qPCR.
  • qPCR analysis was performed by reverse transcribing total RNA (1 ⁇ g of RNA in 20 ⁇ L volume) into cDNA using the SuperScriptTM IV VILOTM Master Mix with ezDNaseTM enzyme kit.
  • FIG. 17 illustrates that a significant increase in the expression of pro-inflammatory cytokine transcripts, namely TNF ⁇ , IL-6, IL-1 ⁇ , and CCL2, was observed in rats treated with pilocarpine, and thus induced into a seizure, relative to rats treated with saline only, 24 hours post-treatment.
  • oral treatment of pilocarpine- treated mice with mPTP Inhibitor 1, at a dose of either 50 mg/kg or 150 mg/kg resulted in a significant decrease in the level of expression of each of these pro-inflammatory cytokine transcripts.
  • mPTP Inhibitor 1 elicited a more significant reduction in the expression of said pro-inflammatory cytokine transcripts than the 50 mg/kg dose.
  • mPTP Inhibitor 1 has an anti-inflammatory effect in the pilocarpine induced rat seizure model.
  • neurofilament- light (NFL) protein levels were quantified in plasma and CSF after 24 hours post-treatment. Specifically, NFL expression was quantified using the Uman Diagnostic NF-Light kit, used according to the manufacturer’s instructions.
  • FIG. 18 The results of Figure 18 illustrate that a significant increase in NFL expression was observed in both the plasma and CSF of rats treated with pilocarpine, and thus induced into a seizure, relative to rats treated with saline only, 24 hours post-treatment.
  • treatment of pilocarpine-treated mice with mPTP Inhibitor 1 at a dose of 10 mg/kg, 50 mg/kg, or 150 mg/kg, resulted in a significant reduction in expression of NFL in both the plasma and CSF.
  • the reduction in NFL expression observed following treatment of pilocarpine-treated mice with mPTP Inhibitor 1 appeared to be dose-dependent.
  • treatment with mPTP Inhibitor 1 has a neuroprotective effect in the pilocarpine induced rat seizure model.
  • treatment with mPTP Inhibitor 1 has a potent anti-inflammatory effect, as represented by a reduction in the expression of pro-inflammatory cytokine transcripts, and has a significant neuroprotective effect (i.e. protects against neurodegeneration), as represented by a reduction in the expression of NFL, which is a marker of neurodegeneration.
  • mPTP inhibitors including new substances identified by the methods described herein, are expected to have utility in the treatment of diseases, disorders, or conditions associated with altered NLRX1 activity and, in particular, in the treatment of diseases, disorders, or conditions associated with activation of the mPTP. Miscellaneous All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.
  • NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I- MAVS and TRAF6-NF- ⁇ B signaling pathways. Immunity.2011 Jun 24;34(6):854-65. doi: 10.1016/j.immuni.2011.03.026. PMID: 21703540; PMCID: PMC3166771. Casta ⁇ o-Rodr ⁇ guez N, Kaakoush NO, Goh KL, Fock KM, Mitchell HM.
  • NLRX1 The nucleotide-binding leucine-rich repeat (NLR) family member NLRX1 mediates protection against experimental autoimmune encephalomyelitis and represses macrophage/microglia- induced inflammation. J Biol Chem.2014 Feb 14;289(7):4173-9. doi: 10.1074/jbc.M113.533034. Epub 2013 Dec 23. PMID: 24366868; PMCID: PMC3924282.
  • Single-cell RNA-sequencing identifies a novel subset of CD9+ reparative macrophages modulated by CCN2 derived from Nlrx1 deficient cardiomyocytes in the infarcted heart.
  • bioRxiv 2023.04.06.535957; doi: https://doi.org/10.1101/2023.04.06.535957 Liu M, Liu K, Cheng D, Zheng B, Li S, Mo Z. The regulatory role of NLRX1 in innate immunity and human disease. Cytokine.2022 Dec;160:156055. doi: 10.1016/j.cyto.2022.156055. Epub 2022 Oct 1. PMID: 36194971.
  • NLRX1 Enhances Glutamate Uptake and Inhibits Glutamate Release by Astrocytes. Cells.2019 Apr 30;8(5):400. doi: 10.3390/cells8050400. PMID: 31052241; PMCID: PMC6562695. Nagai-Singer MA, Morrison HA, Allen IC. NLRX1 Is a Multifaceted and Enigmatic Regulator of Immune System Function. Front Immunol.2019 Oct 11;10:2419. doi: 10.3389/fimmu.2019.02419. PMID: 31681307; PMCID: PMC6797603.
  • Triantafilou K Enigmatic inflammasomes. Immunology.2021 Mar;162(3):249-251. doi: 10.1111/imm.13309. PMID: 33590488; PMCID: PMC7884641. Varga ZT, Palese P. The influenza A virus protein PB1-F2: killing two birds with one stone? Virulence.2011 Nov-Dec;2(6):542-6. doi: 10.4161/viru.2.6.17812. Epub 2011 Nov 1. PMID: 21971186; PMCID: PMC3260547. Walker AK, Spiller KJ, Ge G, Zheng A, Xu Y, Zhou M, Tripathy K, Kwong LK, Trojanowski JQ, Lee VM.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Neurology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Neurosurgery (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Psychiatry (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)

Abstract

La présente invention concerne, entre autres, des procédés d'identification d'une substance utile pour la prévention ou le traitement d'une maladie, d'un trouble ou d'un état associé à une activité de NLRX1 modifiée, et des utilisations desdites substances dans la prévention ou le traitement d'une maladie, d'un trouble ou d'un état associé à une activité de NLRX1 modifiée, par exemple dans la prévention ou le traitement de la maladie de Parkinson ou de la sclérose latérale amyotrophique.
PCT/GB2025/051597 2024-07-18 2025-07-18 Nouveaux procédés et utilisations Pending WO2026018015A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB2410534.8 2024-07-18
GBGB2410534.8A GB202410534D0 (en) 2024-07-18 2024-07-18 Novel method and uses

Publications (2)

Publication Number Publication Date
WO2026018015A2 true WO2026018015A2 (fr) 2026-01-22
WO2026018015A3 WO2026018015A3 (fr) 2026-02-26

Family

ID=92542438

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2025/051597 Pending WO2026018015A2 (fr) 2024-07-18 2025-07-18 Nouveaux procédés et utilisations

Country Status (2)

Country Link
GB (1) GB202410534D0 (fr)
WO (1) WO2026018015A2 (fr)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0293226A1 (fr) 1987-05-27 1988-11-30 Toyo Garasu Kabushiki Kaisha Dispositif pour inspecter des bouteilles de verre
EP2932267A1 (fr) 2012-12-12 2015-10-21 Promega Corporation Reconnaissance de liaison d'une cible cellulaire par un agent bioactif à l'aide d'un transfert d'énergie de résonance de bioluminescence intracellulaire
CA2884607A1 (fr) 2015-03-11 2016-09-11 Stealth Peptides International, Inc. Compositions therapeutiques renfermant des composes acrylamido ou des composes de maleimide phenyle substitues et leurs utilisations en vue de traiter et prevenir les maladies et troubles mitochondriaux
US10067149B2 (en) 2012-12-12 2018-09-04 Promega Corporation Recognition of cellular target binding by a bioactive agent using intracellular bioluminescence resonance energy transfer
WO2022049376A1 (fr) 2020-09-01 2022-03-10 Nrg Therapeutics Ltd Nouveaux composés
WO2022049377A1 (fr) 2020-09-01 2022-03-10 Nrg Therapeutics Ltd Nouveaux composés
WO2023166303A1 (fr) 2022-03-02 2023-09-07 Nrg Therapeutics Ltd Composés d'acrylamide
WO2024153945A1 (fr) 2023-01-19 2024-07-25 Nrg Therapeutics Ltd Inhibiteurs de mptp
WO2025052129A1 (fr) 2023-09-05 2025-03-13 Nrg Therapeutics Ltd Dérivés de pyridine agissant en tant qu'inhibiteurs de mptp

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2179984A1 (fr) * 2008-10-27 2010-04-28 Congenia S.r.l. Dérivés d'Acrylamido utiles comme inhibiteurs de la transition mitochondrique de perméabilité
WO2016144352A2 (fr) * 2015-03-11 2016-09-15 Stealth Peptides International, Inc. Compositions thérapeutiques comprenant des composés acrylamido ou des composés maléimide à substitution phényle, variants et méthodes d'utilisation associés
US10487057B1 (en) * 2018-07-05 2019-11-26 Landos Biopharma, Inc. NLRX1 ligands
US10676436B2 (en) * 2018-07-05 2020-06-09 Landos Biopharma, Inc. NLRX1 ligands
AU2021342568B2 (en) * 2020-09-21 2025-04-03 Landos Biopharma, Inc. Nlrx1 ligands
EP4366733A4 (fr) * 2021-08-11 2025-06-25 Landos Biopharma, Inc. Analogues de tétrahydropyrazolopyridine ligands de nlrx1 et leurs utilisations

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0293226A1 (fr) 1987-05-27 1988-11-30 Toyo Garasu Kabushiki Kaisha Dispositif pour inspecter des bouteilles de verre
EP2932267A1 (fr) 2012-12-12 2015-10-21 Promega Corporation Reconnaissance de liaison d'une cible cellulaire par un agent bioactif à l'aide d'un transfert d'énergie de résonance de bioluminescence intracellulaire
US10024862B2 (en) 2012-12-12 2018-07-17 Promega Corporation Recognition of cellular target binding by a bioactive agent using intracellular bioluminescence resonance energy transfer
US10067149B2 (en) 2012-12-12 2018-09-04 Promega Corporation Recognition of cellular target binding by a bioactive agent using intracellular bioluminescence resonance energy transfer
CA2884607A1 (fr) 2015-03-11 2016-09-11 Stealth Peptides International, Inc. Compositions therapeutiques renfermant des composes acrylamido ou des composes de maleimide phenyle substitues et leurs utilisations en vue de traiter et prevenir les maladies et troubles mitochondriaux
WO2022049376A1 (fr) 2020-09-01 2022-03-10 Nrg Therapeutics Ltd Nouveaux composés
WO2022049377A1 (fr) 2020-09-01 2022-03-10 Nrg Therapeutics Ltd Nouveaux composés
WO2023166303A1 (fr) 2022-03-02 2023-09-07 Nrg Therapeutics Ltd Composés d'acrylamide
WO2024153945A1 (fr) 2023-01-19 2024-07-25 Nrg Therapeutics Ltd Inhibiteurs de mptp
WO2024153946A1 (fr) 2023-01-19 2024-07-25 Nrg Therapeutics Ltd Inhibiteurs de mptp
WO2025052129A1 (fr) 2023-09-05 2025-03-13 Nrg Therapeutics Ltd Dérivés de pyridine agissant en tant qu'inhibiteurs de mptp

Non-Patent Citations (23)

* Cited by examiner, † Cited by third party
Title
"Uniprot", Database accession no. P30405
ALLEN ICMOORE CBSCHNEIDER MLEI YDAVIS BKSCULL MAGRIS DRONEY KEZIMMERMANN AGBOWZARD JB: "NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF- B signaling pathways", IMMUNITY, vol. 34, no. 6, 24 June 2011 (2011-06-24), pages 854 - 65, XP028229980, DOI: 10.1016/j.immuni.2011.03.026
CASTAHO-RODRIGUEZ NKAAKOUSH NOGOH KLFOCK KMMITCHELL HM: "The NOD-like receptor signalling pathway in Helicobacter pylori infection and related gastric cancer: a case-control study and gene expression analyses", PLOS ONE, vol. 10, no. 1, 30 January 2015 (2015-01-30), pages 0117870
CHU XWU SRAJU R: "NLRX1 Regulation Following Acute Mitochondrial Injury", FRONT IMMUNOL, vol. 10, 24 October 2019 (2019-10-24), pages 2431
COSTFORD SRTATTOLI IDUAN FTVOLCHUK AKLIP APHILPOTT DJWOO MGIRARDIN SE: "Male Mice Lacking NLRX1 Are Partially Protected From High-Fat Diet-Induced Hyperglycemia", J ENDOCR SOC, vol. 2, no. 4, 21 February 2018 (2018-02-21), pages 336 - 347
EITAS TKCHOU WCWEN HGRIS DROBBINS GRBRICKEY JOYAMA YTING JP: "The nucleotide-binding leucine-rich repeat (NLR) family member NLRX1 mediates protection against experimental autoimmune encephalomyelitis and represses macrophage/microglia-induced inflam", J BIOL CHEM, vol. 289, no. 7, 23 December 2013 (2013-12-23), pages 4173 - 9
GHARAGOZLOO MMAHMOUD SSIMARD CYAMAMOTO KBOBBALA DILANGUMARAN SSMITH MDLAMONTAGNE AJARJOURA SDENAULT JB: "NLRX1 inhibits the early stages of CNS inflammation and prevents the onset of spontaneous autoimmunity", PLOS BIOL, vol. 17, no. 9, 16 September 2019 (2019-09-16), pages 3000451
JANG HKIM MHONG JYCHO HJKIM CHKIM YHSOHN MHKIM KW: "Mitochondrial and Nuclear Mitochondrial Variants in Allergic Diseases", ALLERGY ASTHMA IMMUNOL RES, vol. 12, no. 5, September 2020 (2020-09-01), pages 877 - 884
KIM HRKIM MNKIM EGLEEM JSBAEK SMLEE YJKIM KWKANG MJSONG TWSOHN MH: "NLRX1 knockdown attenuates pro-apoptotic signaling and cell death in pulmonary hyperoxic acute injury", SCI REP, vol. 13, no. 1, 1 March 2023 (2023-03-01), pages 3441
KORS LRAMPANELLI ESTOKMAN GBUTTER LMHELD NMCLAESSEN NLARSEN PWBVERHEIJ JZUURBIER CJLIEBISCH G: "Deletion of NLRX1 increases fatty acid metabolism and prevents diet-induced hepatic steatosis and metabolic syndrome", BIOCHIM BIOPHYS ACTA MOL BASIS DIS, vol. 1864, no. 5, 4 March 2018 (2018-03-04), pages 1883 - 1895
KOSHLAND DE: "Application of a Theory of Enzyme Specificity to Protein Synthesis", PROC NATL ACAD SCI, vol. 44, no. 2, February 1958 (1958-02-01), pages 98 - 104
KUZNETSOV AVVEKSLER VGELLERICH FNSAKS VMARGREITER RKUNZ WS: "Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells", NAT PROTOC, vol. 3, no. 6, 2008, pages 965 - 976, XP001538967, DOI: 10.1038/nprot.2008.61
LEBER AHONTECILLAS RTUBAU-JUNI NZOCCOLI-RODRIGUEZ VABEDI VBASSAGANYA-RIERA J: "NLRX1 Modulates Immunometabolic Mechanisms Controlling the Host-Gut Microbiota Interactions during Inflammatory Bowel Disease", FRONT IMMUNOL, vol. 9, 26 February 2018 (2018-02-26), pages 363
LI JHE GWANG LWANG HMA RSUN WHU JLIANG JLI RWU X: "Single-cell RNA-sequencing identifies a novel subset of CD9+ reparative macrophages modulated by CCN2 derived from NIrx1 deficient cardiomyocytes in the infarcted heart", BIORXIV, Retrieved from the Internet <URL:https://doi.org/10.1101/2023.04.06.535957>
LIU MLIU KCHENG DZHENG BLI SMO Z: "The regulatory role of NLRX1 in innate immunity and human disease", CYTOKINE, vol. 160, 1 October 2022 (2022-10-01), pages 156055, XP087194610, DOI: 10.1016/j.cyto.2022.156055
MAHMOUD SGHARAGOZLOO MSIMARD CAMRANI AGRIS D: "NLRX1 Enhances Glutamate Uptake and Inhibits Glutamate Release by Astrocytes", CELLS, vol. 8, no. 5, 30 April 2019 (2019-04-30), pages 400
NAGAI-SINGER MAMORRISON HAALLEN IC: "NLRX1 Is a Multifaceted and Enigmatic Regulator of Immune System Function", FRONT IMMUNOL, vol. 10, 11 October 2019 (2019-10-11), pages 2419
PLOS ONE, vol. 9, no. 6, 2014, pages 98899
TRIANTAFILOU K: "Enigmatic inflammasomes", IMMUNOLOGY, vol. 162, no. 3, March 2021 (2021-03-01), pages 249 - 251
VARGA ZTPALESE P, THE INFLUENZA A VIRUS PROTEIN PB1-F2: KILLING TWO BIRDS WITH ONE STONE? VIRULENCE, vol. 2, no. 6, 1 November 2011 (2011-11-01), pages 542 - 6
WALKER AKSPILLER KJGE GZHENG AXU YZHOU MTRIPATHY KKWONG LKTROJANOWSKI JQLEE VM: "Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43", ACTA NEUROPATHOL, vol. 130, no. 5, 22 July 2015 (2015-07-22), pages 643 - 60, XP035572423, DOI: 10.1007/s00401-015-1460-x
WANG XYANG CLIAO XHAN CYU THUANG KYU LQIN WZHU GSU H: "NLRC and NLRX gene family mRNA expression and prognostic value in hepatocellular carcinoma", CANCER MED, vol. 6, no. 11, 29 September 2017 (2017-09-29), pages 2660 - 2672
ZENG CZHOU ZHAN YWEN ZGUO CHUANG SXIAO DYE XOU MHUANG C: "Interactions of TRAF6 and NLRX1 gene polymorphisms with environmental factors on the susceptibility of type 2 diabetes mellitus vascular complications in a southern Han Chinese population", J DIABETES COMPLICATIONS, vol. 31, no. 12, 1 September 2017 (2017-09-01), pages 1652 - 1657, XP085258143, DOI: 10.1016/j.jdiacomp.2017.08.013

Also Published As

Publication number Publication date
GB202410534D0 (en) 2024-09-04
WO2026018015A3 (fr) 2026-02-26

Similar Documents

Publication Publication Date Title
JP6695457B2 (ja) エストロゲン受容体調節剤としての６，７−ジヒドロ−５ｈ−ベンゾ［７］アヌレン誘導体
ES2855135T3 (es) Amidas heterocíclicas como inhibidores de quinasa
ES2905755T3 (es) Derivados de piperidina
KR102587175B1 (ko) 인간 ｅｚｈ2의 억제제 및 이의 사용 방법
ES2392553T3 (es) Derivados (3-aril-piperazina-1-ilo) de 6,7-dialcoxiquinazolina, 6,7-dialcoxiftalazina y 6,7-dialcoxiisoquinolina
CN112955459A (zh) 双环肽配体和其用途
JP2020525454A (ja) 検出可能部分を持つ二環式ペプチドリガンドおよびその使用
JP6258331B2 (ja) キナーゼ阻害剤としてのアミノ−キノリン
CN105143221A (zh) 用于治疗炎性障碍的化合物及其药物组合物
JP2009507791A (ja) ナイアシン受容体アゴニスト、このような化合物を含有する組成物、および治療方法
JP2013523658A (ja) キナーゼ阻害剤としてのピラゾリル‐ピリミジン
JP2013533316A (ja) キナーゼ阻害剤としてのキノリルアミン
EA025322B1 (ru) Производные дигидробензооксазина и дигидропиридооксазина
CA2938187C (fr) Formes solides de 2-(tert-butylamino)-4-((1r,3r,4r)-3-hydroxy-4-methylcyclohexylamino)-pyrimidine-5-carboxamide, compositions les comprenant et procedes pour les utiliser
ES2647849T3 (es) Compuestos de pirido[2,3-d]pirimidin-4-ona como inhibidores de la tanquirasa
KR20230069915A (ko) 신규 화합물
TW201103924A (en) Novel(6-oxo-1,6-dihydropyrimidin-2-yl)amide derivatives, preparation thereof and pharmaceutical use thereof as inhibitors of AKT (PKB) phosphorylation
KR20170003667A (ko) 카르복스아미드 유도체
WO2023166303A1 (fr) Composés d&#39;acrylamide
JP2021522290A (ja) １−イミダゾチアジアゾロ−２ｈ−ピロール−５−オン誘導体
EP3568133B1 (fr) Composés protecteurs de cellules et leur utilisation
AU2014266867B2 (en) Pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide derivatives and use thereof
JP2010529993A (ja) ＩＴＰＫｂ阻害剤としての化合物および組成物
WO2019091440A1 (fr) Forme cristalline i d&#39;un composé de 5-aminopyrazole carboxamide comme inhibiteur de btk