WO2025040657A1 - Antagonistes sélectifs du récepteur kappa-opioïde de peptide cyclisé - Google Patents

Antagonistes sélectifs du récepteur kappa-opioïde de peptide cyclisé Download PDF

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WO2025040657A1
WO2025040657A1 PCT/EP2024/073297 EP2024073297W WO2025040657A1 WO 2025040657 A1 WO2025040657 A1 WO 2025040657A1 EP 2024073297 W EP2024073297 W EP 2024073297W WO 2025040657 A1 WO2025040657 A1 WO 2025040657A1
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dyna
cyclized
analog
amino acid
kor
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Christian Gruber
Edin MURATSPAHIC
David Craik
Andrew White
Mariana Spetea
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Medizinische Universitaet Wien
Universitaet Innsbruck
University of Queensland UQ
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Medizinische Universitaet Wien
Universitaet Innsbruck
University of Queensland UQ
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    • 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
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid

Definitions

  • KOR agonists provide promising potential to develop safer and more effective analgesics since they lack the undesired effects which are typical for agonists of the p- or 6- opioid receptor (MOR or DOR, respectively), including respiratory depression, constipation, tolerance or addiction (Darcq, Nat Rev Neurosci 19, 2018, 499-514). Nevertheless, opioid- mediated activation of KOR is often associated with sedation, dysphoria and hallucinations, which have precluded clinical development of KOR agonists (White, J Pharmacol Exp Ther 352, 2015, 98-109; House, Sci Signal 9, 2016, rall7).
  • KOR antagonists have demonstrated the potential to relieve symptoms of depression, anxiety or drug abuse (Carroll, loc. cit.).
  • the vast majority of these ligands are small molecules, and only a few peptidic KOR antagonists have been developed to date (Carroll, loc. cit.; Aldrich, Handb Exp Pharmacol 271, 2022, 197-220; Aldrich, The AAPS Journal 11(2), 2009, 312-22).
  • GPCRs G protein-coupled receptors
  • galanin and neuropeptide Y receptors Green, Bioorg Med Chem 21, 2013, 303-10
  • orexin receptors Karhu, Peptides 102, 2018, 54-60
  • oxytocin and vasopressin receptors Melanocortin 1 receptor
  • melanocortin 1 receptor White, J Med Chem 65, 2022, 12956-69.
  • cysteine stapling using cysteine crosslinking has emerged as one potential approach to induce conformational restraint of peptides (cf. Fairlie, Peptide Science 106(6), 2016, 843-52). For example, it has been reported that cysteine stapling may improve stability of some peptides (Li, loc. cit.; Golosov, loc. cit.). Hitherto, however, cysteine stapling, especially with late-stage functionalization moieties, has only sparsely been employed for opioid receptor ligands. DynAi-i? (YGGFLRRIRPKLKWDNQ) is the endogenous peptide KOR ligand that plays a pivotal role in a plethora of physiological functions.
  • dynAi-17 does not have 'drug-like' properties: (i) it is linear and flexible, and can adopt multiple conformational states, which may explain why it displays significant affinity towards MOR and DOR (O'Connor, loc.
  • dynAi-17 or its shorter variant dynAi-13 (YGGFLRRIRPKLK); both sometimes also simply termed "dynA"
  • dynA dynAi-13
  • a range of chemical strategies including cyclization, N- or C-terminal modification, incorporation of non-canonical and D- amino acids, and/or peptide backbone modifications, are required (Vlieghe, loc. cit.; Davenport, loc. cit.; Muttenthaler, Nat Rev Drug Discov 20, loc. cit.; Aldrich, The AAPS Journal 11(2), loc. cit.).
  • dynA analog zyklophin with a small intramolecular cyclic structure was shown to exhibit antagonistic activity on KOR following systemic administration in vivo (Aldrich, Proc Natl Acad Sci U S A 106, 2009, 18396-401; Patkar, J Med Chem 52(21), 2009, 6814-21; W02009/049233).
  • Zyklophin is thus one example of a stabilized peptide KOR antagonist with an advantage over small molecules: its effects have a shorter duration as compared to long- lasting small molecule KOR antagonists (Aldrich, Proc Natl Acad Sci U S A loc. cit.).
  • significant drawbacks with respect to zyklophin were reported.
  • the problem underlying the present invention is thus the provision of medically useful means and methods for a pharmaceutical intervention in KOR-related diseases, defects or disorders, in particular diseases, defects or disorders which can be treated by KOR antagonists. More specifically, the problem underlying the present invention is the provision of medically useful KOR antagonists.
  • the technical problem is solved by the provision of the embodiments characterized in the claims.
  • the present invention relates to a compound of Formula (I), below: Formula (I), wherein
  • SQ 1 consists of 5 to 8 amino acid residues and comprises
  • amino acid sequence YGGF (SEQ ID NO.l; most preferred), or the amino acid sequence YGGF having 1, 2, 3 or 4 conservative amino acid substitutions (for example, the amino acid sequence YGGF having Y1 substituted by /Va-AcTyrl, DTyrl, Phel or Phe(p-Br)l and/or G2 substituted by DAIa2); and
  • SQ 2 consists of 4 to 6 amino acid residues and comprises 1, 2, 3, 4 or 5 (preferred) (consecutive) amino acid residue(s) of the amino acid sequence RPKLK (SEQ ID NO.3; most preferred) or IRPKLK (SEQ ID NO.4), or comprises 1, 2, 3, 4 or 5 (preferred) (consecutive) amino acid residue(s) of the amino acid sequence RPKLK or IRPKLK having 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s);
  • each of L 1 , L 2 and L 3 is independently selected from -CH2-, -CH(CH 3 )-, -C(CH 3 )2-, -CH2-CH2-, -CH(CH 3 )-CH 2 -, -CH 2 -CH(CH 3 )- and -CH(CH 3 )-CH(CH 3 )-, preferably -CH 2 -; and
  • R is selected from H, methyl and ethyl, preferably selected from H and methyl, more preferably R is H.
  • the above compound is also termed cyclic peptide of the invention or cyclized dynorphin A analog (dynA analog) of the invention.
  • constrained KOR-targeting dynAi-13 analogs were developed by using diverse peptide stapling approaches.
  • stapling with diverse cysteine stapled moieties including methylene, acetone, m-xylene and tetrazine stapled motifs was applied (cf. Figure 1).
  • a stapling strategy was utilized that could be implemented at a late stage, on unprotected peptide scaffolds. This stapling strategy is amenable to diversification (late-stage functionalization; cf. Figure 1). Further, C-terminal amidation was applied and the resulting effects were tested.
  • cyclized dynA analogs as claimed and as provided/disclosed herein exhibit the desired and even surprisingly improved pharmacological properties.
  • the present invention thus provides for useful, and an even improved, means and methods for medical/pharmaceutical intervention in KOR-related diseases, defects or disorders, in particular diseases, defects or disorders which can be treated by KOR antagonists. More specifically, present invention provides for even improved KOR antagonists.
  • the pharmacological properties of the cyclized dynA analogs of the invention include high affinity to KOR, high antagonizing activity/potency, high stability, (extraordinary) high specificity to KOR (approx, three orders of magnitude; as compared to other opioid receptors, e.g. MOR and/or DOR) and/or less adverse effects (e.g. no itch/scratching).
  • the pharmacological properties of the cyclized dynA analogs of the invention (also) include central activity/capability of modulating KOR function within the CNS.
  • the cyclized dynA analogs of the invention may even be capable of crossing the blood-brain barrier (BBB)/entering the CNS.
  • the cyclized dynA analogs of the invention are improved with respect to the affinity to KOR, the KOR-antagonizing activity/potency, the stability, the specificity to KOR and/or the adverse effects (e.g. no itch/scratching).
  • the cyclized dynA analogs of the invention may (also) be improved with respect to the central activity/capability of modulating KOR function within the CNS; or even with respect to, and/or the central activity/crossing the blood-brain barrier (BBB)/entering the CNS.
  • BBB blood-brain barrier
  • these improvements apply when being compared to the known small-molecule KOR antagonists or known peptide KOR antagonists (like Zyklophin).
  • CSD-CH2(i,8)-NH2 targets the KOR as a stable (competitive) antagonist with nanomolar affinity and an up to and even more than 1000-fold enhanced selectivity for KOR over MOR and/or DOR (as compared to dynAi-13 as such).
  • the selectivity of CSD-CH2(i,8)-NH2 for KOR is shown to be about 1/1000/1000 (KOR/MOR/DOR).
  • the selectivity of zyklophin for KOR is only about l/194/>330 (KOR/MOR/DOR; cf. DiMattio loc. cit.).
  • the C-terminal amide of CSD-CH2(i,8)-NH2 may interact with the acidic residues in the extracellular loop of KOR, thereby leading to, for example, an increase in KOR affinity and specificity (e.g. as compared to other KOR ligands; like U50,488 or the other KOR ligands disclosed herein (see, for example, Figure 1)).
  • CSD-CH2(i,8)-NH2 may, upon binding to KOR, stabilize the inactive state of KOR and promote a more inactive KOR-CSD-CH2(i,8)-NH2 complex. Also shown herein is the improved stability of CSD-CH2(i,8)-NH2 towards proteolytic degradation in human serum. In addition, the ability of CSD-CH2(i,8)-NH2to retain inhibitory KOR activity in a DRG neuronal primary cell model is shown.
  • CSD-CH2(i,8)-NH2 was shown in vivo to be centrally active; i.e.to modulate KOR function within the CNS. This even provides for evidence that CSD- CH2(i,8)-NH2 is capable of crossing the BBB and of entering the CNS, respectively.
  • CSD-CH 2 (i,8)-NH 2 is shown herein to antagonize centrally-mediated effects of the KOR by the small molecule KOR agonist U50,488 in mice.
  • the in vivo KOR antagonism of CSD-CH 2 (i,8)-NH 2 in mice after s.c. administration is demonstrated herein based on its ability to reverse antinociception and sedation/motor impairment caused by U50,488.
  • CSD-CH2(i,8)-NH2 was shown not to induce scratching/itch in vivo (based on behavioural observations in mice (data not shown); e.g. in contrast to zyklophin (cf. DiMattio loc. cit.))
  • the compound of the invention i.e. the KOR antagonist provided herewith, comprises a peptide backbone (linear peptide precursor) which is derived from dynA, in particular from dynAi-13 (YGGFLRRIRPKLK; SEQ ID NO.7).
  • the compond of the invention is thus also termed herein "dynA analog”.
  • the peptide backbone of the compound of the invention is highly similar on a structural basis to the amino acid sequence of dynAi-13.
  • the peptide backbone of the compound of the invention may comprise (in consecutive order) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues that are identical to (or constitute at least conservative substitutions of) the corresponding amino acid residues of dynAi-13 at the respective position. It is preferred that the non-identical amino acid residues are conservative substitutions.
  • the amino acid residue corresponding to Y at position 1 of dynAi-13 are conservative substitutions.
  • Yl may be substituted by /Va-AcTyrl, DTyrl, Phel or Phe(p-Br)l and/or the amino acid residue corresponding to G at position 2 (and/or 3) of dynAi-13 (G2 (and/or G3)) may be substituted by DAIa2.
  • the peptide backbone of the compound of the invention may be at least 53.9%, 61.5%, 69.2%, 76.9%, 84.6% or 92.3% identical to dynAi-13.
  • a higher degree of identity, and of structural similarity, for the peptide backbone of the herein disclosed dynA analog as compared to dynAi-13 is preferred (e.g. the higher amounts of identical amino acid residues and/or the higher values of identity are preferred).
  • the compound of the invention i.e. the KOR antagonist provided herewith, further comprises an intra-molecular cyclic structure.
  • the compond is thus also termed herein "cyclic peptide” or "cyclized dynA analog".
  • the cyclic structure of the compound of the invention is preferably formed between the N-terminal amino acid residue (e.g. the Yl residue) and an (inner) amino acid residue at position 7, 8 or 9, most preferably at position 8 (i.e. corresponding to the I residue at position 8 of dynAi-13), of the peptide backbone.
  • the compound of the invention is not envisaged to constitute a head-to tail cyclized dynA analog (i.e.
  • the (inner) amino acid residue which corresponds to the amino acid residue at the respective position of dynAi-13 is a residue which is suitable for forming the intramolecular cyclic structure.
  • this residue is a Cysteine residue (most preferred) or a Homocysteine residue (e.g in lieu of the I residue at position 8 of dynAi-13).
  • SQ 1 consists of 5 to 8 amino acid residues, i.e. 5, 6, 7 or 8 amino acid residues.
  • SQ 1 consists of 6 to 8 or 5 to 7 amino acid residues.
  • SQ 1 consists of 7 amino acid residues.
  • SQ 1 comprises
  • amino acid residues at its C-terminus, 1, 2, 3 or 4 amino acid residue(s), preferably 2 to 4 amino acid residues, most preferably 3 amino acid residues, of the N-terminal part of a biologically active "address" sequence of dynAi-17 or dynAi-13 (cf. Chavkin loc. cit.; e.g. SEQ ID NO.8); e.g. the first three (most preferred) or first four N-terminal amino acid residues of a biologically active "address" sequence of dynAi-17 or dynAi-13. It is preferred that the amino acid residues are consecutive amino acid residues of the N-terminal part of a biologically active "address" sequence of dynAi-17 or dynAi-13.
  • SQ 1 consist of the following components:
  • amino acid sequence YGGF (SEQ ID NO.l; most preferred), or the amino acid sequence YGGF having 1, 2, 3 or 4 conservative amino acid substitutions (for example, the amino acid sequence YGGF having Y1 substituted by /Va-AcTyr, DTyr, Phe or Phe(p-Br) and/or G2 substituted by DAIa2); and
  • amino acid residue(s) at its C-terminus, 1, 2, 3 or 4 amino acid residue(s), preferably 2 to 4 amino acid residues, most preferably 3 amino acid residues, of the amino acid sequence LRR (preferred) or LRRI (SEQ ID NO.2), or 1, 2, 3 or 4 amino acid residue(s), preferably 2 to 4 amino acid residues, most preferably 3 amino acid residues, of the amino acid sequence LRR or LRRI having 1, 2, 3 or 4 conservative amino acid substitution(s).
  • 1, 2 or 3 (preferred) amino acid residue(s) of the amino acid sequence LRR is more preferred.
  • the amino acid sequence LRR is most preferred. It is preferred that the amino acid residues are consecutive amino acid residues of the respective reference sequence.
  • SQ 1 may consist of the amino acid sequence YGGFLRR with 1, 2, 3, 4, 5, 6 or 7 conservative amino acid substitution(s) (preferred); or of the amino acid sequence YGGFLRRI with 1, 2, 3, 4, 5, 6, 7 or 8 conservative amino acid substitution(s).
  • Preferred examples of SQ 1 are the amino acid sequences YGGFLRR (SEQ ID NO.5) (most preferred) or YGGFLRRI (SEQ ID NO.6).
  • SQ 2 consists of 4 to 6 amino acid residues, i.e. 4, 5 or 6 amino acid residues.
  • SQ 2 consists of 5 to 6 or 4 to 5 amino acid residues.
  • SQ 2 consists of 5 amino acid residues.
  • SQ 2 comprises 1, 2, 3, 4 or 5 amino acid residue(s), preferably 4 or 5 amino acid residues, most preferably 5 amino acid residues, of the C-terminal part of a biologically active "address" sequence of dynAi-13 (cf. Chavkin loc. cit. ; e.g. SEQ ID NO.8). It is most preferred that the amino acid residues are consecutive amino acid residues of the C-terminal part of a biologically active "address" sequence of dynAi-13.
  • SQ 2 may consist of 4 to 6 amino acid residues and comprises 1, 2, 3, 4 or 5 amino acid residue(s), preferably 4 or 5 amino acid residues, most preferably 5 amino acid residues, of the amino acid sequence RPKLK (SEQ ID NO.3) (preferred) or IRPKLK (SEQ ID NO.4); or may consist of 1, 2, 3, 4 or 5 amino acid residue(s), preferably 4 or 5 amino acid residues, most preferably 5 amino acid residues, of the amino acid sequence RPKLK or IRPKLK having 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s). It is preferred that the amino acid residues are consecutive amino acid residues of the respective reference sequence.
  • SQ 2 may consist of the amino acid sequence RPKLK with 1, 2, 3, 4 or 5 conservative amino acid substitution(s) (preferred) or of the amino acid sequence IRPKLK with 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s).
  • Preferred examples of SQ 2 are the amino acid sequences RPKLK (SEQ ID NO.3) (most preferred) or IRPKLK (SEQ ID NO.4).
  • peptide backbone of the cyclized dynA analog of the present invention comprises, as its N-terminal part, a biologically active "message” motif of dynAi-17 or dynAi-13 (cf. Chavkin loc. cit.; e.g. SEQ ID NO.l) and, as its C-terminal part, a biologically active "address" sequence of dynAi-13 (cf. Chavkin loc. cit.; e.g. SEQ ID NO.8).
  • a biologically active "message” motif of dynAi-17 or dynAi-13 and a biologically active "address" sequence of dynAi-13 is known in the art (cf. Chavkin loc. cit. (e.g. Fig. 1)) and is disclosed herein (see, for example, SEQ ID NOs. 1, 8 and 10).
  • the skilled person is readily able to test whether a given amino acid stretch (within (a peptide backbone of) a cyclized dynA analog of the invention) is a biologically active "message” motif or a biologically active "address” sequence.
  • Respective guidance and assays are provided herein and in the appended examples and also in the art (e.g. Chavkin loc. cit.).
  • a biologically active "message” motif results in naloxone-reversible opioid activity (e.g. Chavkin loc. cit.).
  • Examples of the peptide backbone of a cyclized dynA analog of the invention are YGGFLRRCRPKLK (SEQ ID NO.ll), YGGFLRRICPKLK (SEQ ID NO.12) and YGGFLRCIRPKLK (SEQ ID NO.13).
  • the most preferred peptide backbone of a cyclized dynA analog of the invention consists of the amino acid sequence YGGFLRRCRPKLK (SEQ ID NO.ll).
  • the present invention also encompasses (a) conservative amino acid substitution(s) within these particular peptide backbones (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 conservative amino acid substitution(s)), provided that an (inner) cycle-forming residue is maintained (e.g. the depicted C residue).
  • conservative amino acid substitution(s) herein elsewhere also applies here, mutantis mutandis.
  • the technical meaning of conservative amino acid substitution(s) is well known in the art. For example, an amino acid substitution is conservative (in the context of the invention), when the replacement amino acid(s) belong(s) to the same category of amino acids than the amino acid(s) to be replaced.
  • an acidic amino acid may be replaced by another acidic amino acid
  • a basic amino acid may be replaced by another basic amino acid
  • an aliphatic amino acid may be replaced by another aliphatic amino acid
  • a polar amino acid may be replaced by another polar amino acid.
  • the term "acidic amino acid(s)” as used herein is intended to mean an amino acid selected from the group comprising Asp, Asn, Glu, and Gin
  • the term “basic amino acid(s)” as used herein is intended to mean an amino acid selected from the group comprising Arg, Lys and His
  • the term “aliphatic amino acid(s)” as used herein is intended to mean any amino acid selected from the group comprising Gly, Ala, Ser, Thr, Vai, Leu, He, Asp, Asn, Glu, Gin, Arg, Lys, Cys and Met
  • the term "polar amino acid(s)"as used herein is intended to mean any amino acid selected from the group comprising Cys, Met, Ser, Tyr, Gin, Asn and Trp.
  • a conservative amino acid substitution in accordance with the invention may mean that F is replaced by I, L or Y; G is replaced by A, C, D, E or R; I is replaced by F, L, M, N or V; K is replaced by E, M, N, Q, R or T; L is replaced by F, H, I, M, P, Q, R, V or W; P is replaced by H, L, Q, R or S; R is replaced by C, G, H, K, L, M, P, Q, T or W; Y is replaced by C, D, F, H, N or S.
  • Particular (conservative) amino acid substitutions which may occur in accordance with the invention are the substitution of Y by /Va-AcTyr, DTyr, Phe or Phe(p-Br) and/or the substitution of G by DAIa.
  • any amino acid configuration may be present in the context of the peptide backbone (linear peptide precursor) or in the dynA analog of the invention.
  • most naturally occurring amino acids are in the L-configuration.
  • one or more of the amino acid residues of the provided cyclized dynA analog may also be in the D-configuration.
  • the respective peptide backbone (linear peptide precursor) in the cyclized dynA analog may thus have a certain pattern of D- and L-amino acid residues.
  • the resulting dynA analog must be a dynA analog in accordance with the invention and function accordingly (for example as described herein elsewhere, e.g. as in one, more or all of items (a) to (g), below).
  • the skilled person is readily in the position to test which amino acid residue(s) of a dynA analog should be in the L-configuration and which may be in the D-configuration, i.e. to figure out a suitable pattern of D-and L-amino acid residues (i.e. a pattern which results in a functional dynA analog in accordance with the invention).
  • the skilled person may, for example, rely on the tests/assays disclosed herein and in the appended examples (e.g. one or more of the (pharmacological) tests/assays for testing whether a dynA analog is a dynA analog of the invention as described with respect to items (a) to (g), below, and in appended Example 1, infra).
  • the tests/assays disclosed herein and in the appended examples e.g. one or more of the (pharmacological) tests/assays for testing whether a dynA analog is a dynA analog of the invention as described with respect to items (a) to (g), below, and in appended Example 1, infra).
  • amino acid or “amino acid residue”
  • amino acid residue may be a naturally-occurring amino acid (preferred), more preferably a naturally-occurring L-amino acid.
  • amino acid residue in context of this invention may also be a D-amino acid, or a non-naturally-occurring (i.e. a synthetic) amino acid, like, for example, norleucine, R- alanine, or selenocysteine.
  • any amino acid as used/defined herein may, in principle, also represent its modified form.
  • a Cys residue as used herein may be a modified Cys residue like a homocysteine residue. Modifications may, among others, be a methylation or acylation, or the like. In principle, however, non-modified amino acid residues are preferred.
  • a cyclized dynA analog described herein which comprises such (a) modification(s) or modified amino acid(s) is still functionally active in accordance with the invention (cf., for example, items (a) to (g), infra).
  • Respective assays for determining whether a given dynA analog fulfils this requirement are known to the one skilled in the art and are, among others, also described herein, e.g. in the example part and/or in the context of items (a) to (g), infra.
  • each of L 1 , L 2 and L 3 is independently selected from -CH2-, -CH(CHs)-, -C(CHs)2-, -CH2-CH2-, -CH(CH3)-CH2-, - CH2-CH(CH3)- and -CH(CH3)-CH(CH3)-.
  • at least one of L 1 , L 2 and L 3 is is -CH2-. More preferably, at least two of L 1 , L 2 and L 3 are -CH2-.
  • L 1 and L 2 may be -CH2-
  • L 2 and L 3 may be -CH2- or L 1 and L 3 may be -CH2-.
  • R is selected from H, methyl and ethyl.
  • R is selected from H and methyl.
  • R is H.
  • the cyclized dynA analog according to the invention is of Formula (II), below: Formula (II), wherein SQ 1 and SQ 2 are defined as described herein elsewhere. More preferably, SQ 1 is YGGFLRR (SEQ ID NO.5) or SQ 2 is RPKLK (SEQ ID NO.3). Most preferably, SQ 1 is YGGFLRR (SEQ ID NO.5) and SQ 2 is RPKLK (SEQ ID NO.3).
  • CSD- CH2(i,8)-N H2 is a compound of Formula (I), wherein
  • SQ 1 is YGGFLRR (SEQ ID NO.5);
  • SQ 2 is RPKLK (SEQ ID NO.3);
  • each of L 1 , L 2 and L 3 is -CH2-;
  • the invention also provides (the use of) derivatives of the disclosed cyclized dynA analogs, such as salts, e.g. with physiologic organic and inorganic acids like HCI, H2SO4, H3PO4, malic acid, fumaric acid, citronic acid, tatratic acid, acetic acid.
  • physiologic organic and inorganic acids like HCI, H2SO4, H3PO4, malic acid, fumaric acid, citronic acid, tatratic acid, acetic acid.
  • the cyclized dynA analog according to the invention is envisaged to exhibit (or is capable of exhibiting) at least one, more (e.g. at least two, three or four), or (preferably) all of the desired and advantageous (biological) functions as described herein, in particular the (biological) functions of CSD-CH2(i,8)-NH2.
  • the cyclized dynA analog according to the invention is a specific KOR antagonist, preferably with the ability of modulating KOR function within the CNS; or even of crossing the BBB.
  • the cyclized dynA analog according to the invention is envisaged to bind to KOR (or to be capable of binding to KOR), to be a specific KOR antagonist (in particular as compared to MOR and/or DOR), to modulate KOR function within the CNS (and even cross the BBB and enter the CNS, respectively), and(/or) to be stable (e.g. (human) serum).
  • the cyclized dynA analog according to the invention exhibits (or is capable of exhibiting) at least one, more (e.g. at least two, three or four), or (preferably) all of the following biological functions:
  • the cyclized dynA analog is an antagonist of the KOR; e.g. with a KOR antagonist potency corresponding to an EC50 of ⁇ 70 nM, ⁇ 60 nM, ⁇ 50 nM, ⁇ 40 nM or ⁇ /about 30 nM (in an adenylyl cyclase assay quantifying the reduction of cyclic AMP (cAMP); e.g. as described in https://doi.org/10.1021/im0501Q5i; Patkar, Med. Chem.
  • an EC50 of ⁇ 750 nM, ⁇ 700 nM, ⁇ 650 nM, ⁇ 600 nM or ⁇ /about 560 nM in a FMP blue assay; e.g. as described in https://doi.Org/10.1016/j.biopha.2021.112173; Zhao, Biomedicine & Pharmacotherapy, 112173, 2021, 1-11; higher potency is preferred), and/or an EC50 of ⁇ 550 nM, ⁇ 500 nM, ⁇ 450 nM, ⁇ 400 nM or ⁇ /about 340 nM (in a [ 35 S]GTPgS assay; e.g. as described in https://doi.org/10.1021/jm501827k; Joshi, J. Med. Chem. 58(22), 2015, 8783-95; higher potency is preferred);
  • the cyclized dynA analog specifically binds to KOR (but not to MOR and DOR; e.g. with a >400-fold, >500-fold, >600-fold, >700-fold, >800-fold, >900-fold or >/about 1000-fold selectivity for KOR over MOR and/or DOR; higher selectivity is preferred);
  • the cyclized dynA analog binds to KOR with an affinity corresponding to a Ki of ⁇ 10 -8 M, preferably ⁇ 8.0xl0 -9 M, ⁇ 7.0xl0 -9 M or ⁇ 6.8xl0 -9 M; and/or
  • the cyclized dynA analog is stable in (human) serum for >0.5 h (e.g. >50% (preferably >75%) of the cyclized dynA analog remains in the (human) serum after (about) 0.5 h; >25% (preferably >50%) of the cyclized dynA analog remains in the (human) serum after (about) 1.0 h; >20% (preferably >30%) of the cyclized dynA analog remains in the (human) serum after (about) 1.5 h; and/or >15% (preferably >25%) of the cyclized dynA analog remains in the (human) serum after (about) 2.0 h).
  • the cyclized dynA analogs of the invention may be particularly stable; and/or may have a high/improved retention and/or half life time (for example in a patient's body (e.g. in blood or serum; see, for example, (e), above).
  • cyclized dynA analogs are preferred to lack sites susceptible for hydrolysis or cleaving proteases, like, for example, serum proteases.
  • hydrolysis and (serum) proteases” and the structure of the respective sites are well known in the art.
  • the cyclized dynA analog according to the invention may not induce scratching/itch (for example in mice upon administration; e.g. at the administration site). This can, for example, be tested on the basis of behavioral observations and/or as in DiMattio loc. cit.
  • the cyclized dynA analog according to the invention may exhibit at least one, more (e.g. at least two, three or four), or (preferably) all of its (biological) functions rapidly upon adminisration (for example upon s.c. administration). This can, for example, be tested (e.g. in mice) as described in Examples 1 and 6 (cf. also Figure 5A).
  • a (competitive) KOR- antagonizing effect e.g. reduction in antinociception; e.g. as induced by U50,488) of the cyclized dynA analog according to the invention (and/or at least one or more of its other (biological) functions
  • mice after ⁇ lh, ⁇ 45 min, ⁇ 30 min, ⁇ 15 min or even ⁇ 10 min upon administration (e.g. s.c.); and/or before a comparable effect/function(s) of, for example, zyklophin would occur and/or would be detectable.
  • Figure 1 may be used as controls in this respect (for example, dynA as negative control and/or CSD- CH2(i,8)-NH2 as a positive control).
  • Guidance and assays for testing the (biological) functions are known in the art (see, for example, the respective scientific papers and/or patent literature cited herein) and are provided hierein (see, for example, the above described (biological) functions, like the ones as described in items (a) to (g), supra) and in the appended examples (see, for example, Example 1).
  • a (cyclized) dynA analog is capable of modulating KOR function within the CNS and/or passing the BBB and entering the CNS, respectively, can, for example, be determined/tested by (i) in vivo MS imaging using a labelled (cyclized) dynA analog of interest; (ii) label-free quantification of the (cyclized) dynA analog in CSF (and or brain homogenate, for instance via mass spectrometry; and/or (iii) systemic in vivo administration (s.c., i.p., i.v. or p.o.) followed by assessing central effects (such as analgesia via central KOR receptors).
  • Respective means and methods are known in the art (Muratspahic, loc. cit.; Thell, Proc Natl Acad Sci USA 113(15), 2016, 3960-5).
  • a (cyclized) dynA analog is an antagonist of the KOR/antagonizes the KOR can be tested as described in Muratspahic (J Med Chem 64, loc. cit.) or Duerrauer, (Sci Rep 9, 2019, 19295), and in appended Examples 1, 3, 5 and 6 (cf. also Table 1 and Figures 2, 4 and 5B).
  • a cAMP assay e.g. according to the Cisbio protocol
  • Schild regression analysis may be applied in this respect.
  • a (cyclized) dynA analog according to the invention may exhibit an anxiolytic effect This can also be tested by relying on respective tests/assays described herein and in the art.
  • the elevated plus maze (EPM) test may be used in this respect (e.g. as an animal behavioral model of anxiety/anxiety disorders or anxiety conditions). This is, for example, described in Danduga (Methods Mol Biol 2761, 2024, 93-6).
  • the KOR in particular, the human KOR (hKOR), is well known in the art and is, for example, described in Lalanne (Front Psychiatry 5, 2014, 170) and Du (Nat. Common. 7, 2016, 11120).
  • any KOR is meant, in particular a KOR of any (animal) species.
  • a KOR referenced herein may, for example, be a KOR of human, mouse, rat, rabbit, monkey or goat etc. origin (see, for example, UniProt entries: OPRK_MOUSE P33534, OPRK_RAT P34975).
  • the KOR is hKOR (see, for example, UniProt entry: OPRK_HUMAN P41145).
  • the KOR to be targeted by the cyclized dynA analog of the invention is the KOR of a patient to be treated.
  • the KOR to be targeted is preferably hKOR.
  • Antagonists of the KOR are known in the art may, for example, be used as reference compounds/controls when testing/assaying whether a dynA analog functions in accordance with the invention, i.e. exhibits the relevant/advantageous property(ies)/(biological)function(s) of the cyclic peptides/cyclized dynA analogs of the invention (e.g. acting as a (superior) KOR antagonist; cf.
  • a KOR antagonist known in the art may be used a control research tool/pharmacological probe testing whether a given dynA analog (as, for example, structurally defined herein) reverses an effect produced by a KOR agonist (e.g. U50,488) just like the known KOR antagonist; or, if CSD-CH2(i,8)-NH2 is used as a further control research tool/pharmacological probe, like CSD-CH2(i,8)-NH2 itself.
  • a KOR agonist e.g. U50,488
  • the cyclized dynA analog of the invention is envisaged to act as a (h)KOR antagonist/antagonist of (h)KOR.
  • being an "antagonist" of the KOR and exhibiting “antagonistic" function on the KOR means that the KOR activity, i.e. the relevant biological function(s) of the KOR is(are) reduced, inhibited or even entirely blocked.
  • a KOR ligand is antagonizing, if it antagonizes KOR with the same, or a similar (e.g. ⁇ 1-40%), potency and/or efficacy as a known KOR antagonist (e.g. nor-BN I ).).
  • being an "antagonist" of the KOR and exhibiting “antagonistic" function on the KOR means in the context of the invention that the response of KOR to an opioid stimulus (e.g. by a KOR agonist), e.g. the intracellular cAMP reduction, is reduced, inhibited or even entirely blocked.
  • an opioid stimulus e.g. by a KOR agonist
  • inhibiting in the context of the present invention is envisaged to mean that the initial status (for example status of KOR affinity, antagonizing potency and/or efficacy of a dynA analog, activity of a dynA analog, stability of a dynA analog etc.; and the respective KOR function) is lowered (in vitro and/or in vivo) by, for example, at least 10%, by at least 20%, by at least 30%, by at least 50%, by at least 80%, by at least 90%, at least 95%, by at least 99% or even by 100% (in principle, the higher values of percentage are preferred).
  • the initial status for example status of KOR affinity, antagonizing potency and/or efficacy of a dynA analog, activity of a dynA analog, stability of a dynA analog etc.; and the respective KOR function
  • the initial status for example status of KOR affinity, antagonizing potency and/or efficacy of a dynA
  • incrementing in the context of the present invention particularly means that the initial status (for example status of KOR affinity, antagonizing potency and/or efficacy of a dynA analog, activity of a dynA analog, stability of a dynA analog etc.; and the respective KOR function) is increased (in vitro and/or in vivo) by, for example, at least 10%, by at least 20%, by at least 30%, by at least 50%, by at least 80%, by at least 90%, at least 95%, by at least 99% or by at least 100%; or by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold (in principle, the higher values of percentage are preferred).
  • incrementing means that there is already an initial degree of activity/status (baseline) which is further “increased”.
  • inducing means that there is (substantially) no initial degree of activity/status which is then “induced”.
  • the skilled person is readily in the position to test the respective degree of “increasing”, “inducing” or “improving”.
  • the skilled person is readily in the position to determine for a given drug (e.g. dynA analog as defined (e.g. by a structure) or as disclosed herein) the IC50 for the respective "increasing", “inducing” or “improving” effect/activity.
  • the present invention further relates to a pharmaceutical composition comprising the cyclized dynA analog according to the invention.
  • the pharmaceutical composition may further comprise a pharmaceutical carrier.
  • the cyclized dynA analog of the invention, or the pharmaceutical composition comprising it may be for use in the treatment of a KOR-activity-related disease, in particular of a disease resulting from or coming along with an increased KOR activity and/or which is treatable by KOR antagonism/a decreased KOR activity.
  • Medical/pharmaceutical treatments of diseases/disorders are a predominant aspect of the invention.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the cyclized dynA analog according to the invention, and optionally a pharmaceutical carrier, for use in treating, ameliorating or preventing (i) depression (major depressive disorder (MDD)) and/or a (depression-related) neuropsychiatric disease (e.g. stress-mediated symptoms in depression and mood disorders);
  • MDD major depressive disorder
  • a neuropsychiatric disease e.g. stress-mediated symptoms in depression and mood disorders
  • stress related disorders e.g. post-traumatic stress disorder (PTSD) and/or phobias (e.g. social phobias, agoraphobias, specific phobias, social anxiety disorders)
  • phobias e.g. social phobias, agoraphobias, specific phobias, social anxiety disorders
  • anxiety/anxiety disoders e.g. panic disoders, obsessive-compulsive disorder (OCD), PTSD; social phobia/social anxiety disorders, generalized anxiety disorder (GAD)); and/or
  • drug addiction e.g. opiate, methamphetamine, alcohol, nicotine, ecstasy and/or cocaine/cocaine derivative addiction.
  • phobias which may be treated, ameliorated or prevented in accordance with the invention include arachnophobia (the fear of spiders), ophidiophobia (the fear of snakes), glossophobia (the fear of public speaking), acrophobia (the fear of heights), and social phobia (social anxiety disorder; e.g. the fear of social interactions); see also https://www.verywellmind.com/social-phobia-or-anxiety-disorder-3024447.
  • Drug “addiction” in accordance with the invention may include drug “seeking behavior” (like opiate, methamphetamine, alcohol, nicotine, ecstasy and/or cocaine/cocaine derivative seeking behavior).
  • a pathological status is to be treated predominantly in accordance with the invention (for example, a pathological status like the diseases/disorders mentioned above/elsewhere).
  • certain status'/conditions may be mild, and only slightly embarrassing, respectively.
  • Examples of such mild/slightly embarrassing (non- pathological) status'/conditions are anxiety conditions or nervousness conditions (e.g. over only brief periods), like those caused by (temporary) stressful events (e.g. examinations, competitions/tournaments, court hearings etc.).
  • Other examples are mild/slightly embarrassing (non-pathological) phobia-like conditions, like arachnophobia-, ophidiophobia-, glossophobia-, acrophobia-, and social phobia-like conditions (e.g.
  • diseases/disorders pathological status'
  • diseases/disorders like depression, (depression-related) neuropsychiatric diseases, phobias, anxiety/anxiety disoders, drug addictions etc.
  • the cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it may be used in the context of an acute medication (for example in the context of an acute medication in the treatment, amelioration or prevention/prophylaxis as defined herein above).
  • the cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it may be used in (treating or ameliorating of) an acute depressive episode.
  • the cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it may be used when a rapid efficacy (e.g. a rapid onset of a desired (biological) function like the (competitive) KOR-antagonizing effect) is desired, e.g.
  • the cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it may be used when an efficacy after ⁇ lh, ⁇ 45 min, ⁇ 30 min, ⁇ 15 min or even ⁇ 10 min upon administration (e.g. s.c.) is desired, e.g. in the context of the herein defined treatment, amelioration or prevention/prophylaxis.
  • the cyclized dynA analog according to the invention may, in principle, be shortacting, medium-acting or long-acting. Accordingly, the cyclized dynA analog according to the invention may be a short-acting, medium-acting or long-acting antagonist of the KOR.
  • the cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it, may be used accordingly, e.g. in the context of respective treatment regimens.
  • Short-, medium-, and long-acting refers to the time (e.g. half-life) the compound remains in the body and/or exhibits its (biological) functions in the body (duration/period of action).
  • short-acting may refer to a time (e.g. half-life) of about one to four hours (e.g. about 1.5h, 2. Oh, 2.5h, 3. Oh, 3.5h).
  • medium-acting may refer to a time (e.g. half-life) of >4 to 36 hours (e.g. about lOh, 12h, 15h, 24h, 30h).
  • long-acting may refer to a time (e.g. half-life) of about >36 hours to weeks or months (e.g. about 4 days, 2 weeks, 3 weeks, 1 month, 2 months, 4 months).
  • Acute medication and treatment options based on short-acting efficacy may, for example, be particularly useful for the treatment of acute disorders, like an acute depressive episode, acute anxiety.
  • Treatment options based on medium-acting efficacy may, for example, be particularly useful for the treatment of stress-mediated symptoms in depression and mood disorders.
  • Treatment options based on long-acting efficacy may, for example, be particularly useful for the treatment of chronic/long-term disorders, like depressions, neuropsychiatric diseases, phobias, drug addiction.
  • the cyclized dynA analog of the invention may be used in the context of a time-dependent medication (e.g. acute, fast, short-/medium-/long-acting manner; see above), a general (timeindependent) medical use of the cyclized dynA analog of the invention is also within the scope of the invention.
  • a time-dependent medication e.g. acute, fast, short-/medium-/long-acting manner; see above
  • a general (timeindependent) medical use of the cyclized dynA analog of the invention is also within the scope of the invention.
  • the cyclized dynA analog, and pharmaceutical composition comprising it, to be medically used in accordance with the invention provides the advantageous property of contributing to (a) less adverse effect(s), and (an) improved profile(s) of (an) adverse effect(s), respectively.
  • adverse effects in particular MOR- and/or DOR- dependent adverse effects (centrally-mediated or peripherally-mediated) may be reduced/ameliorated or avoided in accordance with the invention.
  • Adverse effects to be reduced/avoided may be, for example, (adverse effects related to) itch/scratching, cold/heat hypersensitivity, hyperalgesia.
  • one or more of such adverse effects is/are to be reduced/ameliorated or avoided.
  • the present invention further relates to a kit/kit of contents/kit of parts comprising a cyclized dynA analog or pharmaceutical composition of the invention, wherein said pharmaceutical composition and/or said cyclized dynA analog may (separately and independently from the kit) be for use in accordance with the invention (e.g. in the uses described herein elsewhere).
  • the cyclized dynA analog may be contained in a (single) container or vial, and/or (a) further (active) agent(s) may be contained in (a) further container(s) or vial(s).
  • the disclosed pharmaceutical composition or cyclized dynA analog can/will be administered in a pharmaceutically/therapeutically effective dose.
  • a pharmaceutically/therapeutically effective amount of the cyclized dynA analog (active ingredient) which is to be administered is reached.
  • a pharmaceutically/therapeutically effective dose refers to that amount of the compound administered which, for example, results in amelioration (of symptoms) of and/or curation from the disease, in an improved condition and/or in a prolongation of survival of a subject. This can be determined by the one skilled in the art by routine testing.
  • administration are doses in a range of 600 pg/kg BW to 20 mg/kg BW, preferably in a range of 800 pg/kg BW to 10 mg/kg BW. Doses in the range of up to 20 mg/kg BW may, for example, be appropriate and safe in the context of all three, p.o., i.p. and i.v. administration. This also may include even low doses (e.g. ⁇ 0.01 pg/kg BW, ⁇ 0.1 pg/kg BW, ⁇ 1 pg/kg, ⁇ 10 pg/kg).
  • the pharmaceutical composition of the invention may comprise (a) pharmaceutically acceptable carrier(s), excipient(s) or diluent(s).
  • the pharmaceutical composition or cyclized dynA analog of the invention may also be administered together with (a) pharmaceutically acceptable carrier(s), excipient(s) or diluent(s).
  • Respective carriers, excipients or diluents are well known in the art. The skilled person is readily in the position to choose carriers, excipients or diluents which are suitable to be employed in accordance with the present invention.
  • treating is, if not indicated otherwise, generally envisaged to encompass both, therapy and prevention/prophylactic treatment. Therapy may result in amelioration (of (a) symptom(s) of) or even in curing/healing the disease/disorder).
  • a "patient”/"subject” in the context of the purposes of the present invention i.e. to whom (a) pharmaceutical composition(s) or cyclized dynA analog(s) according to the present invention is to be administered and/or who suffers from the disease or disorder and/or symptom(s), and/or condition, as defined and described herein, in general includes both, humans and animals, as well as other organisms.
  • the compositions and methods of this invention are applicable to, or in connection with, both, human treatments and veterinary applications, including treating and preventing procedures and methods.
  • the patient/subject is a mammal, and most preferably, the patient/subject is a human.
  • the pharmaceutical composition, cyclized dynA analog(s)/active ingredient(s), kit/kit of contents/kit of parts etc. of the invention may be comprised in, may be a part of and/or may be a medical device, a medicinal product packaging or pharmaceutical composition packaging.
  • Such device or packaging may, for example, comprise or be (a) vial(s)/(a)container(s), (a) syringe(s) or (a) blister pack(s).
  • the active ingredient(s), and pharmaceutical composition(s), respectively, may be comprised in the device or packaging separately and independently (e.g. in different vials/containers or syringes or blisters), or they may be comprised in combination (e.g.
  • any of the pharmaceutical compositions, cyclized dynA analogs/active ingredients, kits etc. of the invention may be provided together with an instruction manual or instruction leaflet.
  • the instruction manual/leaflet may comprise guidance for the skilled person/attending physician (on how) to treat or prevent a disease, disorder or symptom as described herein in accordance with the invention, for example depression (major depressive disorder (MDD)) and/or a (depression-related) neuropsychiatric disease, a stress related disorder (e.g. post-traumatic stress disorder and/or phobia (e.g.
  • the present invention further relates to a method of producing a pharmaceutical composition, e.g. for the treatments/uses described herein.
  • Said method may comprise the step of mixing (a) cyclized dynA analog(s) as defined herein (and optionally (a) further active agent(s)) with a pharmaceutically acceptable carrier, for example a pharmaceutically acceptable carrier, excipient or diluent as defined herein elsewhere.
  • the method for producing a pharmaceutical composition may comprise the step of mixing (a) cyclized dynA analog(s) as defined herein, and a pharmaceutically acceptable carrier, for example a pharmaceutically acceptable carrier/excipient/diluent as defined herein elsewhere.
  • the present invention further relates to a method of preparing the cyclized dynA analog of the invention.
  • the method of preparing the cyclized dynA analog of the invention may comprise (e.g. as a step prior to a step of cyclization) a step of generating the (linear) peptide backbone ((linear) peptide precursor).
  • Means and methods of generating a (linear) peptide are well known in the art and are, for example, described in Cheneval (J. Org. Chem. 79, 2014, 5538-44) and herein elsewhere (see also Example 1, "Peptide synthesis").
  • the (linear) peptide backbones of the cyclic peptide(s)/cyclized dynA analog(s) to be produced may also conveniently be produced by non-recombinant (chemical) peptide synthesis techniques.
  • Several approaches of such peptide synthesis are known in the art. (e.g. Williams, Chemical Approaches to the Synthesis of Peptides, CRC-Press 1997; Benoiton: Chemistry of Peptide Synthesis. CRC-Press, 2005).
  • the (linear) peptide backbones and (cyclic) peptides disclosed herein, more particular the cysteine-stapled peptides like the particular CH2-stapled peptides may, for example, be generated/synthesized as described in Example 1, "Peptide synthesis", below).
  • the method for preparing the cyclized dynA analog of the invention may comprise at least one, preferably both of, the following steps (most preferred in this order):
  • step (ii) reacting the linear peptide (peptide backbone/linear peptide precursor) of step (i) with an alkylene dihalide, e.g. a Ci-4-diiodoalkylene (preferred) or Ci-4-dibromoalkylene (such as Br- CH2-Br) or Ci-4-dichloroalkylene (such as CI-CH2-CI), or Ci-4-iodobromoalkylene or C1-4- iodochloroalkylene or Ci-4-bromochloroalkylene, which may be represented by Ha l-L 2 -Hal, wherein each Hal is independently selected from Cl, Br and I and L 2 is defined as in claim 1, preferably l-L 2 -l, wherein L 2 is defined as in claim 1; l-L 2 -l is preferably selected from I-CH2- I, l-CH(CH 3 )-l, l-C(CH 3 ) 2 -l, I-CH2-
  • (di)halide refers to (di)fluoro ((F— )...- F), (di)chloro ((Cl-).. -Cl), (di)bromo (( Br— )...— Br), or iodo ((I-)...-I).
  • alkylene examples include methylene (-CH2-), ethylene (-CH2-CH2-) and propylene (-CH2- CH2-CH2- or -CH(CH3)-CH2-), of which methylene (-CH2-) and ethylene (-CH2-CH2-) are preferred.
  • step (i) may comprise solid phase peptide synthesis (SPPS), such as Fmoc solid phase peptide synthesis (SPPS), e.g. by using a rink amide resin, PAL Resin, or a Sieber amide resin, or Boc solid phase peptide synthesis (SPPS), e.g. by using a MBHA resin.
  • SPPS solid phase peptide synthesis
  • step (ii) may be performed in an aqueous solvent, preferably in the presence of a base and preferably in the presence of a reducing agent.
  • the aqueous solvent may be (preferably) a mixture of water and tetra hydrofuran; such as a 5:1 v/v mixture of water and tetrahydrofuran.
  • the base may (preferably) be selected from a carbonate and an amine. More preferably, the base may include a carbonate and an amine.
  • the carbonate may preferably be potassium carbonate and(/or) the amine may preferably be triethyl amine.
  • the reducing agent may (preferably) be a phosphine, more preferably tris-(2-carboxyethyl)-phosphine.
  • the amount of the aqueous solvent may preferably be 1000 to 3000 I, more preferably 1700 to 2300 I; the amount of the carbonate may preferably be 1 to 2, more preferably 1.4 to 1.6, molar equivalents; the amount of the reducing agent may preferably be 2 to 4, more preferably 2.5 to 3.5, molar equivalents; the amount of the amine may preferably be 5 to 15, more preferably 8 to 12 molar equivalents; a nd (/or) the amount of the alkylene dihalide, e.g. of the Ci-4-diiodoalkylene, may preferably be 4 to 12, more preferably 6 to 10, molar equivalents.
  • the method of preparing the cyclized dynA analog of the invention can be performed by omitting the carbonate and/or reducing agent.
  • the carbonate and/or reducing agent improve(s) the reaction yields (by preventing disulfide bond formation).
  • Non-limiting examples of protecting groups that may be used for the amino acid residues in the peptide backbone (linear peptide precursor), in SQ 1 and in SQ 2 of the cyclized dynA analog to be prepared are: Fmoc-Tyr(tBu)-OH (for Y), Fmoc-Gly-OH (for G), Fmoc-Phe-OH (for F), Fmoc- Leu-OH (for L), Fmoc-Arg(Pbf)-OH (for R), Fmoc-lle-OH (for I), Fmoc-Pro-OH (for P), Fmoc- Lys(Boc)-OH (for K), Fmoc-Cys(Trt)-OH.
  • Tyr Bn (benzyl), Deb (2,6-dichlorobenzyl), BrBn (2-bromobenzyl), Z (benzyloxycarbonyl), BrZ (2- bromobanzyloxycarbonyl), Pen (3-pentyl), Boc (tert-butyloxycarbonyl), Trt (trityl), 2-CI-Trt (2- chlorotrityl), TBDMS (tert-butyldimethylsilyl), Al (allyl), oNB (o-nitrobenzyl).
  • Arg Tos (p-toluenesulfonyl), Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl), Mts (mesityl-2- sulfonyl), Mrt (4-methoxy-2,3,6-trimethylphenylsulfonyl), Mis (l,2-dimethyllindole-3-sulfonyl), bis-Boc (bis-tert-butyloxycarbonyl), Suben (5-dibenzosuberenyl), NO2, (nitro), Alloc (bisallyloxycarbonyl).
  • Lys Z (benzyloxycarbonyl), Alloc (allyloxycarbonyl), oNBS (o-nitrobenzenesulfonyl), dNBS (2,4- dinitrobenesulfonyl), Dde (l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)-3-ethyl), Phdec (phenyldisulphanylethyloxycarbonyl), Pydec (2-pyridyldisulphanylethyloxycarbonyl), Bts (benzothiazole-2-sulfonyl), Troc (2,2,2-trichloroethyloxycarbonyl), Dts (dithiasuccinoyl), pNZ (p- nitrobenzyloxycarbonyl), Poc (ropargyloxycarbonyl), Nvoc (4-nitroveratryloxycarbonyl), Azoc (azidomethoxycarbonyl).
  • the method of preparing the cyclized dynA analog of the invention may be performed in accordance with/in analogy to the scheme as depicted in Figure 1.
  • the present invention also relates to a cyclized dynA analog as obtainable or obtained by the herein described/exemplified method(s) of preparing in accordance with the herein provided disclosure.
  • a cyclized dynA analog of the invention and its components, e.g. with respect to Formula (I), Formula (II), Formula (II'), the peptide backbone (linear peptide precursor), SQ 1 , SQ 2 , L 1 , L 2 , and/or L 3 (in particular L 1 , and L 3 ), as well as with respect to the cyctein-stapling options, applies also here, mutatis mutandis.
  • the present invention also relates to a cyclized dynA analog as defined above by reference to Formula (I), Formula (II) or Formula (IK), wherein the C-terminal -NRH group (e.g. -NH2 group is replaced by an -OH group.
  • the present invention further relates to the following items:
  • SQ 1 consists of 5 to 8 amino acid residues and comprises
  • amino acid sequence YGGF (SEQ ID NO.l; most preferred), or the amino acid sequence YGGF having 1, 2, 3 or 4 conservative amino acid substitutions (for example, the amino acid sequence YGGF having Y1 substituted by /Va-AcTyrl, DTyrl, Phel or Phe(p-Br)l and/or G2 substituted by DAIa2); and
  • SQ 2 consists of 4 to 6 amino acid residues and comprises 1, 2, 3, 4 or 5 (preferred) (consecutive) amino acid residue(s) of the amino acid sequence RPKLK (SEQ ID NO.3) (preferred) or IRPKLK (SEQ ID NO.4), or comprises 1, 2, 3, 4 or 5 (preferred) (consecutive) amino acid residue(s) of the amino acid sequence RPKLK or IRPKLK having 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s);
  • each of L 1 , L 2 and L 3 is independently selected from -CH2-, -CH(CH 3 )-, -C(CH 3 )2-, -CH2- CH 2 -, -CH(CH 3 )-CH 2 -, -CH 2 -CH(CH 3 )- and -CH(CH 3 )-CH(CH 3 )-, preferably -CH 2 -; and
  • R is selected from H, methyl and ethyl, preferably selected from H and methyl, more preferably R is H.
  • SQ 1 consists of the amino acid sequence YGGFLRR (SEQ ID NO.5) (most preferred) orYGGFLRRI (SEQ ID NO.6), or of the amino acid sequence YGGFLRR or YGGFLRRI having 1, 2, 3, 4 ,5, 6, 7 or 8 conservative amino acid substitution(s).
  • SQ 2 consists of the amino acid sequence RPKLK (most preferred) or IRPKLK, or of the amino acid sequence RPKLK or IRPKLK having 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s).
  • (a) is an antagonist of the kappa opioid receptor (KOR); e.g. with a KOR antagonist potency corresponding to an EC50 of ⁇ /about 30 nM in an adenylyl cyclase assay quantifying the reduction of cyclic AMP (cAMP) (e.g. as described in https://doi.org/10.1021/jm050105i), an EC50 of ⁇ /about 560 nM in a FMP blue assay (e.g. as described in https://doi.Org/10.1016/i.biopha.2021.112173), and/or an ECso of ⁇ /about 340 nM in a [ 35 S]GTPgS assay (e.g. as described in https://doi.org/10.1021/im501827k):
  • KOR kappa opioid receptor
  • (b) modulates KOR function within the CNS and/or crosses the blood-brain barrier (BBB) and enters the central nervous system (CNS), respectively;
  • (d) binds to KOR with an affinity corresponding to a Ki of ⁇ 10 -8 M, preferably ⁇ 8. OxlO -9 M; and/or
  • (e) is stable in (human) serum for >0.5 h (e.g. >50% (preferably >75%) of the cyclic peptide/cyclized dynA analog remains in the (human) serum after (about) 0.5 h; >25% (preferably >50%) of the cyclic peptide/cyclized dynA analog remains in the (human) serum after (about) 1.0 h; >20% (preferably >30%) of the cyclic peptide/cyclized dynA analog remains in the (human) serum after (about) 1.5 h; and/or >15% (preferably >25%) of the cyclic peptide/cyclized dynA analog remains in the (human) serum after (about) 2.0 h).
  • the cyclic peptide/cyclized dynA analog according to any one of items 1 to 6, wherein
  • SQ 1 is YGGFLRR (SEQ ID NO.5);
  • SQ 2 is RPKLK (SEQ ID NO.3);
  • each of L 1 , L 2 and L 3 is -CH2-;
  • R is H.
  • a pharmaceutical composition comprising the cyclic peptide/cyclized dynA analog according to any one of items 1 to 7, and optionally a pharmaceutical carrier.
  • a pharmaceutical composition comprising the cyclic peptide/cyclized dynA analog according to any one of items 1 to 7 for use in treating, inhibiting or preventing (i) depression (major depressive disorder (MDD)) and/or a (depression-related) neuropsychiatric disease (e.g. stress-mediated symptoms in depression and mood disorders);
  • stress related disorders e.g. post-traumatic stress disorder and/or phobias (e.g. social phobias, agoraphobias, specific phobias, social anxiety disorders));
  • phobias e.g. social phobias, agoraphobias, specific phobias, social anxiety disorders
  • drug addiction e.g. opiate, methamphetamine, alcohol, nicotine, ecstasy and/or cocaine/cocaine derivative addiction.
  • drug addiction e.g. opiate, methamphetamine, alcohol, nicotine, ecstasy and/or cocaine/cocaine derivative addiction.
  • step (ii) reacting the linear peptide of step (i) with an alkylene dihalide, e.g. a C1-4- diiodoalkylene (preferred) or Ci-4-dibromoalkylene (such as Br-CH2-Br) or C1-4- dichloroalkylene (such as CI-CH2-CI), or Ci-4-iodobromoalkylene or C1-4- iodochloroalkylene or Ci-4-bromochloroalkylene, which may be represented by Ha l-L 2 -Ha I, wherein each Hal is independently selected from Cl, Br and I and L 2 is defined as in item 1, preferably l-L 2 -l, wherein L 2 is defined as in item 1; l-L 2 -l is preferably selected from I-CH2-I, l-CH(CH 3 )-l, l-C(CH 3 ) 2 -l, I-CH2-CH2-I, l-CH(CH 3 )-
  • step (i) of said method comprises solid phase peptide synthesis (SPPS), such as Fmoc solid phase peptide synthesis (SPPS), e.g. by using a rink amide resin, PAL Resin, or a Sieber amide resin, or Boc solid phase peptide synthesis (SPPS), e.g. by using a MBHA resin.
  • SPPS solid phase peptide synthesis
  • step (ii) is performed in an aqueous solvent, preferably in the presence of a base and preferably in the presence of a reducing agent, wherein the aqueous solvent is preferably a mixture of water and tetrahydrofuran, such as a 5:1 v/v mixture of water and tetrahydrofuran;
  • the base is preferably selected from a carbonate and an amine, more preferably the base includes a carbonate and an amine, where the carbonate is preferably potassium carbonate and the amine is preferably triethyl amine;
  • the reducing agent is preferably a phosphine, more preferably tris-(2-carboxyethyl)- phosphine.
  • step (ii) is performed by dissolving the linear peptide of step (i) in the aqueous solvent and adding the optional carbonate, the optional reducing agent, the amine and the alkylene dihalide, e.g. the Ci-4-diiodoalkylene, preferably in this order.
  • step (ii) relative to 1 molar equivalent, the amount of the aqueous solvent is preferably 1000 to 3000 I, more preferably 1700 to 2300 I, the amount of the carbonate is preferably 1 to 2, more preferably 1.4 to 1.6, molar equivalents, the amount of the reducing agent is preferably 2 to 4, more preferably 2.5 to 3.5, molar equivalents, the amount of the amine is preferably 5 to 15, more preferably 8 to 12 molar equivalents, and the amount of the alkylene dihalide, e.g. of the Ci-4-diiodoalkylene is preferably 4 to 12, more preferably 6 to 10, molar equivalents.
  • the amount of the aqueous solvent is preferably 1000 to 3000 I, more preferably 1700 to 2300 I
  • the amount of the carbonate is preferably 1 to 2, more preferably 1.4 to 1.6, molar equivalents
  • the amount of the reducing agent is preferably 2 to 4, more preferably 2.5 to 3.5, molar equivalents
  • the terms “optional”, “optionally” and “may” denote that the indicated feature may be present but can also be absent.
  • the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent.
  • the expression “X is optionally substituted with Y" (or “X may be substituted with Y”) means that X is either substituted with Y or is unsubstituted.
  • SEQ ID NO.2 N-terminal part of C-terminal "address" sequence of dynAi-13 and dynAi-17
  • SEQ ID NO.5 N-terminal part of dynAi-13 and dynAi-17; most preferred example of SQ 1 ) YGGFLRR
  • SEQ ID NO.6 N-terminal part of dynAi-13 and dynAi-17; example of SQ 1 )
  • SEQ ID NO.11 (most preferred example of the peptide backbone of the cyclized dynA analog of the invention) YGGFLRRCRPKLK
  • SEQ ID NO.12 (example of the peptide backbone of the cyclized dynA analog of the invention) YGGFLRRI CPKLK
  • Tripeptide (no SEQ ID NO. assigned; N-terminal part of C-terminal "address" sequence of dynAi-13 and dynAi-17) LRR
  • Dynorphin (dyn) A1-13 amide trifluoroacetate salt was purchased from Bachem (Austria). Naloxone and ( ⁇ )-trans-U50,488 methanesulfonate salt were obtained from Sigma (Austria). [ 3 H]-diprenorphine (DPN) was ordered from Perkin Elmer (Austria). cAMP Gi kit was from Cisbio (France) and jetPRIME transfection reagent from Polyplus (Austria). Bromoacetic acid was ordered from Chem-Supply (Australia). Carvacrol and capsaicin were obtained from Sigma (Austria). U50,488 and nor-BNI were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA).
  • Peptides were assembled using automated Fmoc solid phase peptide synthesis (SPPS) using either rink amide or 2-chlorotrityl chloride resins on a 0.125 mmol scale. Amino acid couplings were performed with 2 eq. of amino acid, 2 eq. of HCTU and 4 eq. of DIPEA in DMF (4 mL) for 15 min and repeated twice for each residue. Fmoc was deprotected using 30% piperidine in DMF for 5 min, repeated twice. CSD peptides were assembled using an N-terminal 3- (tritylthio)propanoic acid, manually coupled using 4 eq. of HATU, 8 eq. DIPEA in 4 mL DMF for 30 min.
  • SPPS automated Fmoc solid phase peptide synthesis
  • Each peptide was cleaved from the resin using a cleavage cocktail of TFA/TIPS/H2O (95:2.5:2.5) for 2 h and the crude peptides were precipitated with cold Et20. The precipitated peptides were then redissolved in FhO/MeCN (1:1) and lyophilized to yield a white powder. Each peptide was purified by reversed-phase high performance liquid chromatography (RP- HPLC) on a Shimadzu Prominence HPLC system prior to oxidation and stapling.
  • RP- HPLC reversed-phase high performance liquid chromatography
  • Disulfide bonded peptides were synthesized by stirring the reduced peptide precursor (5.2 pmol) in 50 mM ammonium bicarbonate (pH 8.5) at 1 mg/mL in an open reaction vessel. After 18 h the mixture was acidified with TFA and purified by RP-HPLC.
  • Acetone (ace); sometimes also termed “dca” (dichloroacetone)) stapled peptides were prepared by dissolving the reduced peptide precursor (6.5 pmol) in 15 mL of 50 mM ammonium bicarbonate (pH 9.5) with 1 eq. of TCEP. 1,3-dichloroacetone was (2 eq.) added from a 40mM stock solution in MeCN. After stirring for 2 h the reaction was acidified with TFA and purified by RP-HPLC.
  • Xylene (mXYL) stapled analogs were prepared by dissolving the reduced peptide precursor (5.2 pmol) in 8 mL of 50 mM ammonium bicarbonate (pH 9.5) with 20% MeCN and 1 eq. of TCEP. l,3-bis(bromomethy)benzene (1.5 eq.) was added dropwise from a 40 mM stock solution in DMF and mixed for 30 min before acidification with TFA and purification by RP-HPLC.
  • Tetrazine (tet) stapled peptides were prepared by dissolving the reduced peptide precursor (6.2 pmol) in 12 mL of 50 mM ammonium bicarbonate (pH 5.5). The mixture was added to a 50 mL falcon tube containing 3 eq. of 3,6-dichloro-l,2,4,5-tetrazine in 12 mL of CHCI3 and vortexed for 5 min. The organic and aqueous layers were separated by centrifuge and the aqueous layer isolated. The organic layer was further washed with 50 mM ammonium bicarbonate and aqueous isolates were combined, acidified by TFA and purified by RP-HPLC.
  • Methylene (CH2) stapled peptides were prepared by dissolving the reduced peptide precursor (6.2 pmol) in 12 mL of H 2 O/THF (5:1, v/v) with 1.5 eq of K2CO3, 3 eq. of TCEP, 10 eq. of triethylamine and 8 eq. of diiodomethane added sequentially. The mixture was stirred for 6 h before acidification with TFA and purification by RP-HPLC.
  • Peptides were purified by RP-HPLC on preparative or semipreparative Phenomenex (Germany) Jupiter Cis columns (5 pm, 300 A, 250 x 21.2 mm or 250 x 10 mm) applying a linear gradient from 5% to 65% solvent B (90% MeCN, 10% H2O, 0.05% TFA) and flow rates of 8 mL/min or 3 mL/min, respectively.
  • Automatically collected fractions were analyzed by electrospray ionization mass spectrometry (ESI-MS) using a Shimadzu LCMS 2010 system and analytical RP- HPLC on a Phenomenex Jupiter Cis column (5 pm, 300 A, 150 x 2 mm).
  • MALDI matrix-assisted laser desorption/ionization
  • TOF time-of-flight
  • Table 2 analytical RP-HPLC using a Shimadzu UPLC system
  • PBS phosphate buffered saline
  • HEK293 cells were grown at 37°C and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 50 U/mL penicillin as well as streptomycin. Cell transfection was performed with jetPRIME transfection reagent as per the manufacturer's protocol (Polyplus, France) using 2 pg of plasmids encoding mouse KOR-EGFP and/or human
  • DMEM Dulbecco's Modified Eagle's Medium
  • HEK293 cell membranes stably expressing KOR, MOR and DOR were prepared as described in Muratspahic et al. (Muratspahic, J Med Chem 64, loc. cit.). The dissociation constant Ka values for the mouse KOR was previously determined in a saturation binding assay (Muratspahic, J Nat Prod 84, 2021, 2238-48). Non-specific binding was examined in the presence of 10 pM of naloxone.
  • Displacement binding was performed in a final volume of 300 pL containing [ 3 H]- diprenorphine (1 nM final), peptide solution (4X), membranes (3.5-7 pg/assay) and standard binding buffer (50 mM Tris-HCI (pH 7.4), 10 mM MgCl2 and 0.1% BSA). Equilibrium was achieved by incubating displacement binding reactions at 37°C for 1 h. The Skatron cell harvester device was utilized to terminate reactions by rapid filtration onto GF/C filters pre-soaked with 0.1% polyethlyenimine. Displacement binding for MOR and DOR was conducted with 50-100 pg and 20 pg of HEK293 stable cell membranes, respectively. cAMP Assay
  • Peptide ligands were assayed for cAMP inhibition in HEK293 stable mouse KOR cell line in a 384- well format and triplicates according to the Cisbio protocol. Briefly, 5 pL containing 2 000 cells per well were incubated with 5 pL of peptide solutions prepared 2X in IX stimulation buffer and forskolin (10 pM final). Following an incubation of peptide ligands at 37°C for 30 min cryptate- labeled cAMP and cAMP d2-labeled antibody (5 pL of each) were added to the mixture and further incubated for at least 1 h at room temperature.
  • cAMP levels were quantified on a Flexstation 3 (Molecular Devices, USA) using homogenous time-resolved fluorescence resonance energy transfer (HTRF) and the ratio 665/620 nm.
  • HTRF time-resolved fluorescence resonance energy transfer
  • Antagonism of CSD- CH2(i,8)-NH2 at KOR was evaluated by Schild regression analysis as previously described (Duerrauer, Sci Rep 9, 2019, 19295). Briefly, concentration-response curves of U50,488 were measured in the presence and absence of CSD-CH2(i,8)-NH2.
  • the cells were pretreated with CSD- CH2(i,8)-NH2 (0.3, 1, 3 and 10 pM) for 30 min at 37°C followed by co-incubation with U50,488 for additional 30 min at 37°C.
  • the reaction was terminated by the addition of 5 pL of cAMP-d2 antibody and 5 pL of cAMP Eu Cryptate. After 1 h of incubation at 37°C fluorescence was measured on a Flexstation 3 and quantified using the 665/620nm ratio. Samples were measured in technical triplicates.
  • Bioluminescence Resonance Energy Transfer (BRET) Assay was performed as previously described (Duerrauer, Sci Rep 9, 2019, 19295). Briefly, human p-arrestin-2-nano luciferase (NIuc) and mouse KOR-EGFP were transiently expressed in HEK293 cells following co-transfection in a 1:10 ratio. 16 h post -transfection, 50,000 cells in 100 pL of phenol red-free DMEM with 10% FBS per well were transferred into white clear bottom 96-well plates and incubated overnight. On the day of assay, cells were incubated for 1 h at 37°C in phenol red- and serum-free DMEM.
  • Furimazine (Promega, USA) and peptide ligands were diluted 4X and 1:50 in Hank's balanced salt solution (HBSS), respectively. Prior to establishing the baseline and measuring kinetic BRET furimazine was incubated for 5 min at 37°C. The ability of peptide ligands to induce p-a rresti n recruitment was assessed by measuring light emissions for EGFP (510 nm) and NIuc (460 nm) over 35 min using a Flexstation 3. The BRET ratio was calculated as emission EGFP (510 nm) / emission NIuc (460 nm). The ligand-induced BRET was calculated by subtracting the BRET ratio of the ligand from the BRET ratio of the HBSS.
  • HBSS Hank's balanced salt solution
  • Relative BRET quantification was determined by calculating the mean value of ligand-induced BRET for each peptide ligand from 311 to 2,411 sec (35 min) which was then divided by the mean value of ligand-induced BRET of dynAi-i3-NH2 set as 100.
  • U50,488, nor-BNI and CSD-CH2(i,8)-NH2 were prepared in sterile physiological 0.9% saline solution.
  • Test compounds or vehicle (saline) were s.c. administered in a volume of 10 pL/g body weight.
  • DRG Dorsal Root Ganglion
  • DRG neurons were cultured in DMEM supplemented with 100 mg/mL streptomycin/penicillin, 1% L-glutamine and 100 ng/mL mouse nerve growth factor (Alomone Labs, Israel). Neurons were cultured at 37°C and 5% CO2 for 15 to 30 h.
  • the coverslips were incubated with Fura-2 AM ester (Biotium, USA) for 30 min at 37°C and 5% CO2, before placement in glass-bottom 35 mm dishes in extracellular solution (in mM: 145 NaCI, 5 KCI, 10 glucose, 10 HEPES, 1.25 CaCl2, 1 MgCb, pH 7.4, 300 mOsm). After a recovery period of 10 min, dishes were mounted onto an Olympus IX73-inverted microscope (Olympus, Tokyo, Japan) and imaged using a lOx objective. Cells were permanently superfused with extracellular solution using a software-controlled 8-channel, gravity-driven, common-outlet system (ALA Scientific Instruments Inc, USA).
  • Fluorescence emission was long-pass filtered at 495 nm, and pairs of images were acquired at a rate of 1 Hz with a 4.2-megapixel 16-bit CCD camera (6.5 pm pixel edge length, 18.8 mm sensor diameter, Prime BSI; Teledyne Photometries, USA).
  • the hardware was controlled by the pManager 1.4 plugin in ImageJ. (Schneider, Nat Methods 9, 2012, 671-675)
  • the background intensity was subtracted before calculating the ratio between the fluorescence emitted when the dye was excited at 340 nm and at 385 nm (F340/F385 nm). The time course of this ratio was analyzed for regions of interest adapted to individual cells.
  • the radiant heat tail-flick test was performed using an UB 37360 Ugo Basile analgesiometer (Ugo Basile s.r.l., Varese, Italy) as described previously (Dumitascuta, Sci Rep 10, 2020, 5653).
  • the reaction time required by the mouse to remove its tail after application of the radiant heat was measured and defined as the tail-flick latency (in seconds).
  • Tail-flick latencies were measured before and after s.c. administration of saline (control) or U50,488 (5 mg/kg) (i.e. 30 and 60 min) (test latency, TL).
  • CSD-CH2(i,8)-NH2 (20 mg/kg) or nor-BNI (10 mg/kg) were s.c.
  • mice were habituated to the equipment in two training sessions (30 min apart) one day before testing. On the experimental day, mice were placed on the rotarod, and treadmill was accelerated from 4 to 40 rpm over a period of 5 min. The time spent on the drum was recorded for each mouse before (baseline) and at 30 min afer s.c. administration of saline (control) or U50,488 (5 mg/kg).
  • CSD-CH2(i,8)-NH2 (20 mg/kg) or nor-BNI (10 mg/kg) were s.c. administered 15 min or 24 h, respectively, before U50,488 (5 mg/kg).
  • Decreased latencies to fall in the rotarod test indicate impaired motor performance.
  • a 300 sec cut-of time was used.
  • Each experimental group included 5-6 mice.
  • the rotarod data are expressed as percentage (%) changes from the rotarod latencies obtained before (baseline, BL) and after drug administration (test, TL) were calculated as 100 x (TL/BL).
  • Cysteine staples have emerged as a late-stage functionalization strategy that can be used to modulate GPCR pharmacology (White, J Med Chem 65, loc. cit.), but hitherto have not been investigated in the design of KOR ligands.
  • Applied herein was a cysteine stapling strategy to constrain a dynAi-13 peptide template bearing a native C-terminal carboxylic acid (cf. Goldstein, Proc Natl Acad Sci U 5 A 76, 1979, 6666-70).
  • a cysteine residue was introduced by substituting either isoleucine at position 8, or proline at position 10, and a second thiol was manually coupled onto the N-terminus using 3-(tritylthio)propionic acid.
  • a C-terminal amidation was introduced to the most promising peptides, i.e. CSD-CH2(i,8)-OH, CSD-CH2(i,io)-OH and CSD-ace(i,s)-OH in order to further improve their desired pharmacological properties at the KOR.
  • Peptides containing an amidated C-terminus were synthesized analogous to peptides with the carboxyl group at the C-terminus ( Figure 1). Following synthesis, peptides were subject to pharmacological characterization. The affinity of C-terminally amidated peptides was first assessed in radioligand binding studies.
  • CSD-CH 2 (i,8)-NH 2 was inactive at the KOR on its own compared to CSD-CH2(i,io)-NH2, which acted as a partial agonist following C-terminal amidation with an EC50 and Emax of 540 nM and 49%, respectively (Figure 2B, Table 1).
  • peptides were screened in the kinetic p-arrestin-2 recruitment assay. Like previously characterized analogs, all three peptides did not or only partially recruit p-arrestin-2 at concentrations of 10 pM ( Figure 2C, Figure 6A-C).
  • Example 6 In vivo KOR Antagonism and Site of Action of CSD-CH2(i,s)-NH2 after s.c. Administration in Mice
  • mice were pretreated with CSD- CH 2 (i,8)-NH 2 (20 mg/kg) prior to U50,488.
  • Administration of CSD-CH2(i,8)-NH2 to mice significantly antagonized the effect of U50,488 in the rotarod test, comparable to the effect produced by nor-BNI ( Figure 5B).
  • BRET bioluminescence resonance energy transfer
  • cAMP cyclic adenosine monophosphate
  • CSD cysteine stappled dynorphin
  • DOR 6-opioid receptor
  • DRG dorsal root ganglion
  • dynA dynorphin A
  • EGFP enhanced green fluorescence protein
  • ESI-MS electrospray ionization mass spectrometry
  • GPCR G protein-coupled receptor
  • KOR K-opioid receptor
  • MALDI-MS matrix- assisted laser desorption ionization mass spectrometry
  • MOR p-opioid receptor
  • nor-BNI nor- binaltorphimine
  • RP-HPLC reversed-phase high performance liquid chromatography
  • s.c. subcutaneous.

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Abstract

La présente invention concerne un nouveau peptide cyclique/dynorphine cyclisé un analogue (analogue de dynA) qui agit sur un antagoniste spécifique du récepteur opioïde kappa (KOR). La présente invention concerne en outre une composition pharmaceutique comprenant le nouveau peptide cyclique/analogue de dynA cyclisé selon l'invention. De plus, la présente invention concerne en outre une composition pharmaceutique comprenant le nouveau peptide cyclique/analogue de dynA cyclisé selon l'invention destiné à être utilisé dans le traitement, l'inhibition ou la prévention de la dépression (trouble dépressif majeur (MDD)), une maladie neuropsychiatrique (liée à la dépression), des troubles liés au stress (par exemple, un trouble de stress post-traumatique et/ou des phobies), l'anxiété, et/ou une toxicomanie (comme l'opiacé, la méthamphétamine, l'alcool, la nicotine, l'ecstasy et/ou la dépendance à la cocaïne/un dérivé de cocaïne). La présente invention concerne également un procédé de préparation du nouveau peptide cyclique/analogue de dynA cyclisé, par exemple par fonctionnalisation (étape tardive) avec des agrafes cystéine.
PCT/EP2024/073297 2023-08-21 2024-08-20 Antagonistes sélectifs du récepteur kappa-opioïde de peptide cyclisé Pending WO2025040657A1 (fr)

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