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• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • 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
  • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
• • 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
  • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D213/81—Amides; Imides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D213/81—Amides; Imides
  • C07D213/82—Amides; Imides in position 3
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/08—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing alicyclic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems

Definitions

• TBK1 phosphorylates interferon regulatory transcription factor 3 (IRF3) which drives the production of type I interferons and supports the induction of an adaptive immune response (Zhu, Y. et al. Mol. Cancer.2019, 18: 152).
• IRF3 interferon regulatory transcription factor 3
• PARP inhibitor-induced STING pathway activation and anti-tumour immune responses have been demonstrated in multiple tumour models, providing rationale for exploiting combinations of PARP inhibitors with immunotherapies for improved therapeutic efficacy (Sen, T. et al. Cancer Discov. 2019; 9: 646–661).
• EBV has also been shown to be a causative factor in multiple sclerosis (MS) whereby EBV infection greatly increases the risk of subsequent MS (Bjornevik, K. et al. Science (2021); 375: 296-301).
• First-generation PARP inhibitors generally demonstrate non-selective activity at PARP1 and 2.
• Haematological toxicities such as anaemia, neutropenia and thrombocytopenia are associated with clinical use of these molecules which restricts their use in combination with cytotoxic chemotherapies and other targeted agents due to dose-limiting cytopenias (LaFargue, CJ. et al. Lancet Oncol. 2019, 20, e15 ⁇ e28).
• AZD5305 was described as a potent PARP1 inhibitor and trapper with 500-fold selectivity over PARP2 and less off-target activity against secondary pharmacology targets than first-generation PARP inhibitors (Johannes, JW. et al. J. Med. Chem. 2021; 64: 14498- 14512). Importantly, significantly less haematotoxicity was observed for AZD5305 in rodent models than with first-generation PARP inhibitors, confirming the reported pathogenic role of PARP2 in haematologic toxicity (Illuzzi, G. et al. Clin. Cancer Res.2022; CCR-22-0301).
• PARP1 inhibitors and in particular PARP1 inhibitors for use in medicine. It is a further aim to provide pharmaceutical compositions comprising such inhibitors, and in particular to provide compounds and pharmaceutical compositions for treating a cancer. It is also an aim to provide methods of synthesis of the compounds. Summary In one aspect, there is provided a PARP1 inhibitor compound for use in medicine.
• the PARP1 inhibitor compound has the following structure: wherein: R 1 is selected from H and a substituted or unsubstituted organic group; R 2 is absent or selected from H and a substituted or unsubstituted organic group; R 3 is absent or selected from H and a substituted or unsubstituted organic group; R 4 is selected from H and a substituted or unsubstituted organic group; Z 1 and Z 2 are each independently selected from C and N; and L is a group having the following structure: wherein: each X 1 is independently selected from C and N; each X 2 is independently selected from C, N, O and S; n is a number selected from 0, 1, 2, 3, 4, 5 and 6; and m is a number selected from 0, 1, 2, 3, 4, 5 and 6; with the proviso that m + n is a number selected from 1, 2, 3, 4, 5, and 6; p is a number selected from 0, 1, 2, 3, 4, 5 and 6; and q is a number selected from 0, 1, 2, 3, 4, 5 and 6; with
• p + q is a number selected from 2, 3, 4, 5, and 6.
• p + q is a number selected from 2, 3, 4, 5, and 6
• m + n is a number selected from 2, 3, 4, 5 and 6.
• Another aspect provides a pharmaceutical composition comprising a PARP1 inhibitor compound as defined herein.
• a further aspect provides a pharmaceutical kit for treating a cancer.
• the kit comprises a PARP1 inhibitor compound as defined herein, and a further agent for treating cancer.
• the compound and the further agent are suitable for administration simultaneously, sequentially or separately.
• Another aspect provides a method of treating a disease and/or a condition and/or a disorder, which method comprises administering to a patient a compound, a composition or a kit as provided herein.
• Still another aspect provides a compound selected from: Another aspect provides a method of synthesis of a PARP1 inhibitor compound as provided herein. The method comprises conducting a reaction between a first reactant comprising ring E bearing a first portion of group L and a second reactant comprising a remainder of group L, to form the PARP1 inhibitor compound.
• a compound is considered to be a PARP1 inhibitor if its presence is capable of preventing or reducing the ability of immobilised PARP1 to undergo auto-poly-ADP ribosylation (AutoPARylation) following incubation with biotinylated-NAD+ as compared to the same process in its absence.
• the compound is considered to be a PARP1 inhibitor if it has an IC50 ⁇ 10 ⁇ M in a suitable assay.
• the cis and trans suffixes may refer to pairs of diastereomers having the indicated configuration of the A ring.
• Nuclear Overhauser Effect nuclear magnetic resonance spectroscopy (“NOE NMR”) may be used to determine the stereochemistry of compounds as described herein.
• the suffix ‘a’ in a compound number denotes an enantiomer eluted as a first fraction when a mixture of two enantiomers is separated by supercritical fluid chromatography (“SFC”) using a chiral column.
• the suffix ‘b’ in a compound number denotes an enantiomer eluted as a second fraction when a mixture of two enantiomers is separated by supercritical fluid chromatography (“SFC”) using a chiral column.
• SFC supercritical fluid chromatography
• Some structural formulae presented herein illustrate stereochemistry assigned by the applicant. In the event of any discrepancy between order of elution (as represented by compound numbering) and assigned stereochemistry, the order of elution takes precedence.
• Example 3 hereinbelow describes the synthesis of compound 6.
• Compound 6 is obtained as a mixture of diastereomers.
• the diastereomers are separated into two fractions by prep-HPLC.
• the first fraction to be eluted comprises a pair of enantiomers 6cis-a and 6cis-b.
• the second fraction comprises a pair of enantiomers 6trans-a and 6trans-b.
• the mixture of 6cis-a and 6cis-b is passed through a Regis (R,R)-Whelk-O chiral chromatography column under the conditions indicated above.
• the first fraction to be eluted in the second separation step comprises compound 6cis-a, and the second fraction comprises compound 6cis-b.
• the improved pharmacokinetics and improved CNS penetration may at least in part be due to improved physicochemical properties of the molecules compared to those having bicyclic head groups, as well as modifying the conformation of the molecule: the head groups of bicyclic head groups tend to be flatter and more lipophilic.
• R 5 group refers generally to groups R 5A , R 5B , and R 5C .
• An “R 5A ” group is an R 5 group which is attached to ring A, and so on.
• Some of the formulae presented herein use more specific identifiers for R 5 groups. For example, “R 5A1 ” identifies a subset of R 5A groups.
• the groups comprising heteroatoms described above, as well as any of the other groups defined above, may comprise one or more heteroatoms from any of groups IIIA, IVA, VA, VIA or VIIA of the Periodic Table, such as a B, Si, N, P, O, or S atom or a halogen atom (e.g. F, Cl, Br or I).
• groups IIIA, IVA, VA, VIA or VIIA of the Periodic Table such as a B, Si, N, P, O, or S atom or a halogen atom (e.g. F, Cl, Br or I).
• R5 groups may in particular be absent or selected from: H, deuterium, a halogen (such as –F, -Cl, -Br, and –I; preferably F or Cl), a nitrile group, a substituted or unsubstituted C 1 -C 6 alkyl group, a substituted or unsubstituted linear or branched C 1 -C 6 halogenated alkyl group (preferably CF 3 or CHF 2 ), a cyclopropyl group, an -OH group, a substituted or unsubstituted linear or branched C 1 -C 6 alcohol group, a substituted or unsubstituted linear or branched C 1 -C 7 amino carbonyl group (such as -NH-CO-Me), an -NH 2 group,
• Qb When ring A is a 4-membered ring, Qb may be -CH 2 - or a bond.
• Qc is a bond or -CH 2 -, and preferably Qc is a bond.
• Qc when ring B is a 5-, 6-, or 7-membered ring, Qc may be a bond.
• Qc When ring B is a 3- or 4-membered ring, Qc may be selected from -CH 2 -; -O-; and -NR 8 -.
• R 8 is H or a substituted or unsubstituted organic group, optionally H or a C1 to C3 alkyl group.
• both X 1 atoms of ring A may have an R configuration or both X 1 atoms of ring A may have an S configuration.
• the A ring may have an (R,R) configuration or an (S,S) configuration.
• the (S,S) enantiomer may be more active than the (R,R) enantiomer.
• ring A is an aromatic ring. When ring A is aromatic, the PARP1 inhibitor compound is neither A ring cis nor A ring trans.
• Example 3 Synthesis of 6cis-a, 6cis-b, 6trans-a, 6trans-b Preparation of 3-(5-ethyl-6-methoxypyridin-2-yl)cyclopent-2-en-1-one (1203) To a solution of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclopent-2-en-1-one 1201 (200 mg, 0.96 mmol) and 6-chloro-3-ethyl-2-methoxypyridine 1202 (197 mg, 1.15 mmol) in dioxane (5 mL) and H 2 O (0.5 mL) was added Pd(dppf)Cl 2 (70 mg, 0.10 mmol) and sodium carbonate (255 mg, 2.40 mmol) at room temperature.
• Pd(dppf)Cl 2 70 mg, 0.10 mmol
• sodium carbonate 255 mg, 2.40 mmol
• the reaction mixture was concentrated under reduced pressure.
• the residue was purified by prep-HPLC (Gemini 5 ⁇ m C18150*21.2 mm, mobile phase: ACN - H 2 O (0.1 % FA), gradient: 2 % - 95 %) to afford the first fraction as 6cis-a / 6cis-b racemic mixture (cis stereochemistry assumed across cyclopentane based on NOE experiments) (10 mg, 95 % purity, white solid) and the second fraction as 6trans-a / 6trans-b racemic mixture (trans stereochemistry assumed across cyclopentane based on NOE experiments) (3 mg, 95 % purity, white solid).
• NaBH (OAc ) 3 (433 mg, 2.04 mmol) was added to the reaction mixture and stirred at 60 °C for 1 h.
• NaBH 3 CN (534 mg, 8.5 mmol) was added to the reaction mixture and stirred at 60 °C for 2 days. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure.
• the reaction mixture was concentrated under reduced pressure.
• the residue was purified by prep-HPLC (Gemini 5 ⁇ m C18 150*21.2mm, mobile phase: ACN - H 2 O (0.05 % NH 3 .H 2 O), gradient: 25 % - 95 %) to afford the first fraction as a racemic mixture of 7trans-a and 7trans-b (23 mg, white solid) and the second fraction as a racemic mixture of 7cis-a and 7cis-b (33 mg, white solid).
• 7trans-a / 7trans-b LCMS (ESI) calcd for C 23 H 3 FN 5 O 2 [M + H] + m/z 428.25, found 428.20.
• Example 5 Synthesis of 12cis-a / 12cis-b / 12trans-a / 12trans-b Preparation of methyl 5-bromo-6-methoxypicolinate (1402) To the solution of 5-bromo-6-methoxypicolinic acid 1401 (5.00 g, 21.65 mmol) in MeOH (200 mL) was added dropwise SOCl 2 (7.67 g, 64.47 mmol). The resulting mixture was stirred for 2 h at 0 °C. The reaction solution was concentrated under reduced pressure, then adjusted to pH 9 with an aqueous solution of Na 2 CO 3 , the aqueous layer was extracted with EtOAc (300mL x 3). The combined organic layer was concentrated under reduced pressure.
• the residue was purified by prep-HPLC (column: Gemini 5 ⁇ m C18150 ⁇ 21.2 mm, mobile phase: ACN - H 2 O (0.1 % FA), gradient: 10 - 25) to give 6-(3-(4-(4- chlorophenyl)piperazin-1-yl)cyclopentyl)-3-ethylpyridin-2(1H)-one 28cis racemic mixture (60 mg, 99 % purity, 11 % yield) and 6-(3-(4-(4-chlorophenyl)piperazin-1-yl)cyclopentyl)-3- ethylpyridin-2(1H)-one 28trans racemic mixture (40 mg, 99 % purity, 7 % yield) as white solid.
• Example 9 Synthesis of 79cis-a / 79cis-b / 79trans-a / 79trans-b
• a and B were prepared: A: a solution of NaNO 2 (3.4 g, 0.049 mol) in H 2 O (100 mL).
• A was added to B dropwise at 0 °C, the reaction mixture was stirred at 0 °C for 20 min.
• reaction mixture was stirred at 100 °C for 16 h under N 2 atmosphere.
• the reaction solution was cooled to rt and poured into water, then extracted with EtOAc (30 mL ⁇ 3). The combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated under reduced pressure.
• the residue was purified by prep-HPLC (column: Gemini - C18150 ⁇ 21.2 mm, 5 ⁇ m; mobile phase: ACN - H 2 O (0.05 % NH 3 ); gradient: 20 - 60) to obtain 5-(4-(4-(5-ethyl-6-oxo-1,6-dihydropyrimidin-2-yl)bicyclo[2.1.1]hexan-2- yl)piperazin-1-yl)-6-fluoro-N-methylpicolinamide 82 racemate (2.7 mg, 97.60 % purity, 15 % yield) as a white solid.
• Example 12 Exemplary compounds of the invention were prepared and tested to determine their effect as PARP1 and PARP2 inhibitors. Typical assays are described below.
• Example 12A PARP1 biochemical dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA assay) Optiplate HB 384-well plates were coated with anti-FLAG antibody, supplied as a 4 mg/ml solution, using a Na 2 CO 3 /HCO 3 coating buffer at pH 9.6, overnight at 4 °C, in order to achieve a final immobilisation per well of 0.3 Pg.
• DELFIA assay biochemical dissociation-enhanced lanthanide fluorescence immunoassay
• a Cy5-labelled binding probe is shown below and described in Papeo, G. et al. J. Biomol. Screen. 2014; 19:1212-1219.
• Cy5 probe structure NanoBRET cellular target occupancy assay NanoBRET assays were employed to demonstrate cellular target engagement and selectivity at PARP1 and PARP2. These assays are based on bioluminescence resonance energy transfer (BRET) between a Nano-luc-tagged protein (e.g.
• PARP1 or PARP2 and a fluorescent group on a high affinity NAD + competitive binding probe.
• Such cellular probe displacement assays can be utilised to measure inhibitor affinities and selectivity ratios at PARP1 and 2.
• Frozen HEK293 cells transiently transfected with either PARP1-NanoLuc® fusion or PARP2- NanoLuc® fusion constructs (Promega) were thawed and dispensed as a suspension in 384- well microplates each at a density of 1750 cells per well. NanoBRET TM TE PARP Tracer-01 was then added to final concentrations of 11 and 2 nM for PARP1 and PARP2 assays, respectively.
• Probe Displacement HTRF and NanoBRET assay categories - indicates IC 50 or K d value above 10 PM + indicates IC 50 or K d value above 1 PM up to 10 PM ++ indicates IC 50 or K d value above 100 nM up to 1 PM +++ indicates IC 50 or K d value above 10 nM up to 100 nM ++++ indicates IC 50 or K d value of 10 nM or less
• the selectivity values relate to the selectivity preference of PARP1 over PARP2.

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