FIELD OF THE INVENTION

The present invention provides relaxin-3 B-chain antagonist peptides, compositions comprising same, and their use for the treatment of disorders, such as over-eating leading to obesity, which would benefit from a level of RXFP3 inhibition. Also provided are methods of production of relaxin-3 B-chain antagonist peptides of the invention.

BACKGROUND OF THE INVENTION

Relaxin-3 is a neuropeptide and is a member of relaxin/insulin superfamily. Relaxin-3 binds and activates its cognate receptor RXFP3 and thus shows a promising pharmacological target for the treatment of eating and associated neurological disorders.

Both the ligand and receptor are highly conserved from fish to human [Chen J., et al., The Journal of pharmacology and experimental therapeutics 312(1): 83-95 (2005)] and are predominantly expressed in brain regions involved in feeding, stress, and sensory perception [Liu C., et al., The Journal of biological chemistry 278(50): 50754-64 (2003); Sutton S.W., et al. Neuroendocrinology 82(3-4): 139-50 (2005); Ma S., et al., Neuroscience 144(1): 165-90 (2007)]. In-vivo studies of RXFP3 have been impeded by the lack of selective tools for the RXFP3 receptor. The development of potent and selective RXFP3 antagonist that is synthetically tractable has been an important challenge for relaxin researchers in the last decade. The development of the single chain antagonist R3 B1-22R was the first example of a variant that retained native like binding affinity in a single chain peptide [Haugaard-Kedstrom L.M., et al., J Am Chem Soc 133(13): 4965-74 (201 1)]. Data showing modulation of the relaxin- 3/RXFP3 system control important behaviors highlight R3 B1-22R as a potential therapeutic lead. However, further improvements of this peptide are required as unstructured peptide can be easily degraded by proteases and current studies have relied on intracerebroventricular administration. For the peptide to be a viable candidate for further preclinical studies it must be modified to be able to pass the blood-brain barrier to engage its neuronal target after systemic administration.

Stapled peptides were first described [Schafmeister C.E., et al., Journal of the American Chemical Society 122(24): 5891-5892 (2000)] as a novel chemistry approach to create macrocyclic a-helical peptides via all hydrocarbon cross-links using ruthenium- catalyzed olefin metathesis between two a-methyl-substituted, non-proteogenic amino acids bearing olefinic side chains. Numerous studies have shown that stapled peptides can modulate such intracellular protein-protein interactions, including BH3, Notch, Hif-1 a, b- catenin, and Ras [Walensky L.D., et al. , Science 305(5689): 1466-70 (2004); Danial N.N., et al., Nature Medicine 14: 144 (2008); Patgiri A., et al., Nat Chem Biol 7(9): 585-7 (201 1); Moellering R.E., et al. , Nature 462: 182 (2009)], to exemplify some key targets for cancer therapy. With respect to relaxin-3, Jayakody et al and Hojo et al applied this strategy to generate stabilized a-helical peptide derived from relaxin-3 B-chain that exhibits a high affinity towards RXFP3 (Jayakody T. et al., Peptides 84: 44-57 (2016); Hojo K., et al., Journal of medicinal chemistry 59(16): 7445-56 (2016)].

The development of novel relaxin-3 antagonist peptide drugs for the clinical translation has been hindered by lack of readily available high-affinity antagonists for RXFP3 and their inability to cross the blood-brain barrier.

There is a need for relaxin-3 antagonist peptide analogues that are an improvement over known antagonists for RXFP3.

SUM MARY OF THE INVENTION

Eating disorders and obesity are major healthcare concerns. Stapled peptide antagonists of RXFP3 receptors target central nervous system pathways associated with feeding behaviours and stress-induced increases in feeding. Thus, stapled relaxin-3 B chain antagonists are a potential novel therapeutic approach to treatment of obesity and eating disorders. Our optimized delivery of relaxin-3 peptides via the intranasal route will enable future clinical trials.

According to a first aspect, the present invention provides a single chain relaxin-3 polypeptide or analog, derivative, fragment or mimetic thereof having RXFP3 receptor binding and antagonism, comprising a relaxin B-chain polypeptide stapled at amino acid positions 14 and 18 or at positions 14 and 21 of the relaxin-3 B-chain; wherein the amino acids at positions 14 and 18 or at positions 14 and 21 are covalently cross-linked.

In preferred embodiments, the staple is an all-hydrocarbon staple.

In preferred embodiments, the staple is an 8- or 1 1 -carbon cross-link.

In preferred embodiments, the B-chain polypeptide sequence has the Formula 1 ; R A A P Y G V R L S G R E X1 I R A V I F X2 S R (SEQ ID NO: 16)

wherein X1 and X2 are cross-linked amino acids.

In other embodiments, the B-chain polypeptide sequence has the Formula 2;

R A A P Y G V R L S G R E X1 I R A X2 I F T S R (SEQ ID NO: 17)

wherein X1 and X2 are cross-linked amino acids.

In some embodiments, the Serine residues at positions 10 and 22 are replaced by Cysteine residues.

In other embodiments, the stapled amino acids at positions 14 and 18 or at positions 14 and 21 are a, a-di-substituted amino acids. An example of a suitable amino acid is Alanine. In preferred embodiments, the B-chain polypeptide sequence has the Formula 2 (SEQ ID NO: 17).

In preferred embodiments, the cross-linking amino acids which generate i+7 or /+ 4 stapled peptide are a-methyl, a-alkenyl amino acids, wherein the first amino acid contains a C5 alkenyl, preferably, a 4’-pentenyl. The C5 alkenyl, preferably a 4’-pentenyl, is advantageous in R configuration.

In other preferred embodiments, the cross-linking amino acids which generate i+7 or /+ 4 stapled peptide are a-methyl, a-alkenyl amino acids, wherein the first amino acid contains a C8 alkenyl, preferably a 7’-octenyl, is advantageously in S configuration.

Such a type of stapled peptide, known as all-hydrocarbon stapled alpha-helical peptides, are disclosed in [Schafmeister C.E., et al. , Journal of the American Chemical Society 122(24): 5891-5892 (2000); Verdine G.L. and Hilinski G.J., Drug Discovery Today: Technologies 9(1): e41-e47 (2012)]. Other types of stapled peptides which are within the skill of one in the art can be used in the invention. In particular, a double hydrocarbon staple as well as multiple contiguous staples (recently called stitched peptides) [Hilinski G.J. et al., J Am Chem Soc 136: 12314-12322 (2014)] could be used.

In other embodiments, other cross-linking stabilizing secondary structure or a-helical conformation could be also inserted in the sequence, selected from the group comprising or consisting of lactam, disulphide, thioether, azobenzene, hydrazine, triazole, biphenyl, bis- triazoylyl, oxime, perfluoroaryl and carbamate. In preferred embodiments the stapled polypeptide has a-helical conformation.

In another aspect of the present invention, the cross-linker may be proteogenic or non- proteogenic. The linker may be as simple as a covalent bond (e.g. carbon-carbon double bond, disulphide bond, carbon-heteroatom bond, etc.), or it may be more complicated such as a polymeric linker (e.g. polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid (e.g. glycine, ethanoic acid, alanine, beta-alanine, 3-aminoproapanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx).

In other embodiments, the linker comprises amino acids and may include functionalized moieties to facilitate attachment of nucleophile (e.g. thiol, amino) from the peptide to linker.

In more preferred embodiments, the B-chain polypeptide sequence is from human relaxin-3 (SEQ ID NO: 1 ; RAAPYGVRLCGREFI RAVI FTCGGSRW) with removal of the Tryptophan residue from the C-terminus and up to five additional amino acid residues from the C-terminus with or without addition of a terminal Arginine residue. Examples of such peptides comprise or consist of an amino acid sequence set forth in the group comprising: SEQ ID NO: 8 (RLCGRE-S5IRA-S5I FTCR),

SEQ ID NO: 9 (RLSGRE-S5IRA-S5IFTSR),

SEQ ID NO: 10 (RAAPYGVRLCGRE-S5IRA-S5I FTCR),

SEQ ID NO: 1 1 (RAAPYGVRLSGRE-S5I RA-S5I FTSR),

SEQ ID NO: 12 (RAAPYGVRLCGRE-R5IRAVIF-S8CR), and

SEQ ID NO: 13 (RAAPYGVRLSGRE-R5IRAVIF-S8SR).

In other embodiments, the B-chain polypeptide sequence is from human relaxin-3 with removal of the Tryptophan residue from the C-terminus and replacement of up to five additional amino acid residues from the C-terminus with a chain of up to five Glycine or Serine residues, or a combination of Glycine or Serine residues, with or without a terminal Arginine residue.

In other embodiments, the B-chain polypeptide sequence is from human relaxin-3 with the N-terminus truncated by up to nine amino acids, and the Tryptophan residue and up to five additional amino acid residues removed from the C-terminus with or without addition of a terminal Arginine residue. Examples of such peptides comprise or consist of an amino acid sequence set forth in the group comprising SEQ ID NO: 8 and SEQ ID NO: 9.

In more preferred embodiments, the B-chain sequence is from human relaxin-3 and the cysteine residues at positions 10 and 22 of the native human relaxin-3 sequence are replaced by serine residues. Examples of such peptides comprise or consist of an amino acid sequence set forth in the group comprising:

SEQ ID NO: 6 (RAAPYGVRLSGRE-S5IRA-S5I FTSGGSRW),

SEQ ID NO: 7 (RLSGRE-S5I RA-S5I FTSGGSRW),

SEQ ID NO: 9, SEQ ID NO: 1 1 and SEQ ID NO: 13.

In more preferred embodiments, the alpha carbon of the residue at position 14 has (R) stereochemistry and the alpha carbon of the residue at position 21 has (S) stereochemistry. In more preferred embodiments, the alpha carbon of the residue at position 14 has (S) stereochemistry and the alpha carbon of the residue at position 18 has (S) stereochemistry. In more preferred embodiments, the B-chain polypeptide sequence is from human relaxin-3 and the amino acids at positions 14 and 21 are replaced by (R)-2-(4’-pentenyl) alanine and (S)-2-(7’-octenyl) alanine, respectively, as set forth in SEQ ID NO: 12 or SEQ ID NO: 13.

In more preferred embodiments, the B-chain polypeptide sequence is from human relaxin-3 and the amino acids at positions 14 and 18 are replaced by (S)-2-(4-pentenyl) alanine). Examples of such peptides comprise or consist of an amino acid sequence set forth in the group comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 14 (RAAPYGVRLCGRE-S5IRA-S5I FTSGGSRW) and

SEQ ID NO: 15 (RLCGRE-S5I RA-S5I FTCGGSRW).

In more preferred embodiments, the polypeptide has alpha-helical and/or any helical secondary structure.

In more preferred embodiments, a stapled polypeptide of the invention has an amino acid sequence and structure set forth in SEQ ID NO: 10, SEQ ID NO: 11 , or SEQ ID NO: 12.

According to another aspect, the present invention provides a pharmaceutically acceptable salt form of the polypeptide of the invention.

In more preferred embodiments, the polypeptide decreases stress-related and/or anxiety-related and/or depressive-like behavior and/or increases feeding activity.

According to another aspect, the present invention provides a pharmaceutical composition comprising a stapled polypeptide according to any aspect of the invention or a pharmaceutically acceptable salt thereof, optionally together with one or more pharmaceutically acceptable carriers, excipients or diluents for the treatment of eating disorders, addiction, stress-related eating disorders, binge eating, obesity, eating disorders and weight gain associated with psychiatric disorders, or eating, weight gain or obesity associated with taking drugs used to treat psychiatric disorders such as anxiolytic drugs, antipsychotic drugs, and antidepressant drugs. In certain embodiments, the pharmaceutical compositions described herein include a therapeutically effective amount of a polypeptide comprising an amino acid sequence set forth in Formula 1 , or Formula 2, or a pharmaceutically acceptable salt thereof.

In some embodiments, the Serine residues at positions 10 and 22 are replaced by Cysteine residues.

In some embodiments, the pharmaceutical composition comprises a stapled polypeptide represented by an amino sequence selected from the group comprising SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15. In a more preferred embodiment the pharmaceutical composition comprises a stapled polypeptide represented by an amino sequence and structure set forth in SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12.

Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

In preferred embodiments of the invention, the pharmaceutical composition is formulated for nasal delivery of a stapled polypeptide according to any aspect of the invention. In preferred embodiments of the invention, the pharmaceutical composition comprises: (a) the said stapled polypeptide; (b) a bioavailability enhancing agent selected from the group consisting of a fatty acid, a sugar ester of a fatty acid and a mixture thereof and (c) a poly(alkylene oxide) chain, wherein the chain is a PEG (polyethylene glycol) or a poly(ethylene oxide) PEO.

Preferred poly(alkylene oxides) are selected from the group consisting of alpha- substituted poly(alkylene oxide) derivatives, PEG homopolymers and derivatives, polypropylene glycol) (PPG) homopolymers and derivatives, poly(ethylene oxides) (PEO) polymers and derivatives, bis-poly(ethylene oxides) and derivatives, copolymers of poly(alkylene oxides), and block copolymers of poly(alklyene oxides), poly(lactide-co- glycolide) and derivatives, or activated derivatives. Preferably, the polymer is water-soluble and has a molecular weight of about 200 to about 40,000 Da. The preferred water-soluble polymers are poly(alkylene oxides), most preferably PEG or poly(ethylene oxide) (PEO).

In another preferred embodiment, the composition comprises: (a) the said peptide; (b) sucrose laurate; (c) a citrate-based bioavailability enhancing agent.

In another aspect of the invention, the intranasal composition in accordance with the present invention is an aqueous or dry powder composition.

According to another aspect, the present invention provides a method for the prophylaxis or treatment of a disorder or adverse effects of drug treatment for a disorder, the method comprising administering to a subject in need thereof an effective amount of a polypeptide according to any aspect of the invention, or a pharmaceutical composition according to any aspect of the invention.

Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

In some embodiments the said polypeptide comprises a stapled polypeptide represented by an amino sequence and structure selected from the group comprising the set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15. In a more preferred embodiment the said polypeptide comprises a stapled polypeptide represented by an amino sequence and structure set forth in SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12.

Exemplary disorders are eating disorders, stress-related eating disorders, eating disorders and weight gain associated with psychiatric disorders, binge eating, obesity, addiction, generalized anxiety disorders, major depressive disorders, eating disorders or any stress-related neuropsychiatric disorders. It is considered that stress-related eating disorders and eating disorders secondary to other psychiatric illness are important specific types of eating disorders that targeting RXFP3 is likely to be especially helpful for.

Exemplary adverse effects of drug treatments for disorders are eating, weight gain or obesity associated with taking drugs such as anxiolytic drugs, antipsychotic drugs, and antidepressant drugs used to treat psychiatric disorders.

In other embodiments, the administration is by the intranasal or the intravenous route.

In more preferred embodiments the administration is by the intranasal route.

In a further aspect, the composition according to any aspect of the invention may be administered using a nasal metered dose spray, metered dose inhaler or measured dose inhaler.

According to another aspect, the present invention provides the use of a polypeptide according to any aspect of the invention, or a pharmaceutical composition according to any aspect of the invention for the manufacture of a medicament for the treatment of eating disorders, stress-related eating disorders, binge eating, obesity, eating disorders and weight gain associated with psychiatric disorders, or eating, weight gain or obesity associated with taking drugs such as anxiolytic drugs, antipsychotic drugs, and antidepressant drugs used to treat psychiatric disorders.

In some embodiments the said polypeptide comprises an amino acid sequence set forth in Formula 1 , or Formula 2, or a pharmaceutically acceptable salt thereof. In some embodiments, the Serine residues at positions 10 and 22 are replaced by Cysteine residues.

In some embodiments the said polypeptide comprises a stapled polypeptide represented by an amino sequence and structure selected from the group comprising the set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15. In a more preferred embodiment the said polypeptide comprises a stapled polypeptide represented by an amino sequence and structure set forth in SEQ ID NO: 10, SEQ ID NO: 1 1 , or SEQ ID NO: 12.

The present invention provides various advantages, which are discussed below. Stapling on relaxin-3 B-chain in which peptides are chemically stabilized by crosslinking with small molecules, has been proposed a breakthrough solution to address important yet currently undruggable targets. We have designed a series of stapled relaxin-3 B-chain peptides. This generates smaller RXFP3 antagonists. We achieved i+4 and i+7 stapling strategies for hydrocarbon crosslinking, generating peptides with alpha-helical content with this stapling strategy.

The ability of stapled peptides to bind and block RXFP3 was assessed in HEK - RXFP3 cell lines and in a rat model. It was evident stapled peptides tested on ligand binding and cell signaling assays have an increased ligand binding affinity and potency on RXFP3 receptor.

One of the biggest hurdles with the existing relaxin-3 antagonist peptides has been that they do not cross the blood brain barrier and so have to be administered directly into the brain. We established intranasal delivery as a non-invasive and convenient method which rapidly targets the therapeutic to the CNS, bypassing the BBB and minimizing the systemic exposure. This approach is shown herein in an animal model to deliver the peptides systemically into the brain and lead to reduced weight gain.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1 B show the structural analysis of stapled peptides. Circular dichroism spectra of stapled antagonist peptides demonstrating the observed range of a-helical stabilization by hydrocarbon stapling as shown in the corresponding tables. (A) Comparison of a-helix stabilization between /, i+4 and /, i+7 stapled antagonist peptides. (B) Effect of N- and C-terminus truncation on the helix stabilization in 14s18 stapled peptide. Bar diagram showing the percent helicity of all peptides studied.

Figure 2 shows the determination of binding affinity of stapled antagonist analogues. Competition binding curves for stapled antagonist analogues and R3 B1-22R peptide in HEK- RXFP3 cell lines using Eu-DTPA-R3B1-22R as the labelled ligand. Data points are shown as mean ± SEM of triplicate determinations from three independent experiments.

Figure 3 shows agonist or antagonist activities of relaxin-3 analogues as measured in inhibition of forskolin induced cAMP assay. Various concentrations of relaxin-3 (R3), R3 13- chain, R3 B1-22R and stapled antagonist analogues (0.1 pM to 10 mM) were tested in HEK- RXFP3 cells. Data points are shown as mean ± SEM of triplicate determinations from three to four independent experiments.

Figure 4 shows the ability of R3 B1-22R to antagonize relaxin-3 inhibition of cAMP activity in HEK-RXFP3 cells. The antagonistic effect of R3 B1-22R is shown with a rightward shift of the R3 relaxin dose-response curve in the presence of 10 nM and 100 nM R3 B1-22R. Data points are shown as mean ± SEM of triplicate determinations from three to four independent experiments.

Figure 5 shows pharmacological characterization of stapled analogues of R3 B1-22R peptide as an antagonist for RXFP3. The antagonistic effect of stapled analogues is shown with a rightward shift of relaxin-3 (R3) dose-response curve in the presence of 10 nM 14s18R, 14s21 R peptide and 10 nM of R3 B1-22R peptide. Data points are shown as mean ± SEM of triplicate determinations from three to four independent experiments.

Figure 6 shows ribbon diagram structures of both 14s18R and 14s21 R stapled antagonist analogues of R3 B1-22R peptide. Image shows ribbon diagrams with cyan-colored a-helices. Linker stapled is shown in green color with hydrogen atoms in white. /,/+ 4 linker staple is shown in green color at (A) 14s18 and (B) 14s21 positions. Residues important in RXFP3 binding and activation are highlighted in the peptide ribbon structure.

Figure 7 shows the effect of Cys to Ser residue mutation and C-terminus truncation on the helix stabilization in 14s21 stapled peptide as shown in Circular dichroism spectra and in corresponding table. Bar diagram showing the percent helicity of all the peptides studies.

Figure 8 shows food intake in male SD rats after daily intranasal application of relaxin-

3 for 2 weeks. Day 0 represents the baseline food intake between the groups before giving the drug treatment. Solid circles, Vehicle group; solid squares, 0.1 pmol relaxin-3. Data are mean ± SEM. Two-way repeated measures ANOVA followed by Bonferroni’s t test (post hoc). Significance of changes: *P < 0.05; **P < 0.01 , ***P < 0.001 with respect to vehicle. N = 4 rats/gp.

Figure 9 shows food intake after daily intranasal administration of 50 pmol and 100 pmol concentration of R3 B1-22R antagonist peptide in SD rats. Solid circles, Vehicle group; solid squares, 50 pmol R3 B1-22R peptide; and Solid triangles, 100 pmol R3 B1-22R antagonist peptide. Data are mean ± SEM. Two-way repeated measures ANOVA followed by Bonferroni’s t test (post hoc). Significance of changes: **P < 0.01 , with respect to vehicle. N =

4 rats/gp. Figure 10 shows the effect of R3 B1-22R peptide on relaxin-3 induced feeding in adult Sprague-Dawley rats. Relaxin-3 (0.1 pmol) significantly increased food intake until 7 ^th day of drug administration. This increase was blocked by daily pre-treatment with R3 B1-22R antagonist (100 pmol). Data are mean ± SEM. Two-way repeated measures ANOVA followed by Bonferroni’s t test (post hoc). Significance of changes: *P < 0.05; **P < 0.01 , ***P < 0.001 with respect to vehicle. N = 4 rats/gp.

Figure 11 shows body weight in male SD rats after daily intranasal administration of relaxin-3 and R3 B1-22R antagonist peptide for 2 weeks. Solid circles, Vehicle group; solid squares, 0.1 pmol relaxin-3. Solid triangles, showing the R3 B1-22R treatment post relaxin-3 (R3) administration. Data are mean ± SEM. Two-way repeated measures ANOVA followed by Bonferroni’s t test (post hoc). Significant effect of interaction (Day X Time): F (30, 144) = 0.098, P > 0.99. N = 4 rats/gp.

Figure 12 shows a graph of cumulative body weight change (mean ± sem) against days of intranasal treatment with the staple peptide antagonist (red squares) and saline vehicle control (black circles). The reduction of body weight gain by the stapled peptide antagonist was significant (two-way ANOVA: Fi _{, 144} =22.60, p < 0.0001) without causing any significant loss of body weight below the normal starting body weight.

DETAILED DESCRIPTION OF THE INVENTION

Bibliographic references mentioned in the present specification are for convenience listed at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference for the material contained in them that is discussed in the sentence in which the reference is relied upon.

Definitions

Certain terms employed in the specification, examples and appended claims are collected here for convenience.

As used in the specification and the appended claims, the singular forms“a,”“an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

The terms "amino acid" or "amino acid sequence," as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule. Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

As used herein, the terms“polypeptide”,“peptide” or“protein” refer to one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or peptide can comprise a plurality of chains non- covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. A“polypeptide”, “peptide” or“protein” can comprise one (termed “a monomer”) or a plurality (termed “a multimer”) of amino acid chains.

As used herein, terms in the phrase“single chain relaxin-3 polypeptide or analog, derivative, fragment or mimetic thereof” are defined as follows. The term "amino acid analog" refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptide or peptidomimetic macrocycle. Amino acid analogs include compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additional methylene groups between the amino and carboxyl group (e.g., a-amino _/ 3-carboxy acids), or for the substitution of the amino or carboxy group by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution or the carboxy group with an ester).

The term“derivative” can be used to describe the amino acid derivative and peptide derivative. Peptide derivatives contain one or more substituents separately linked by an amide, amino or sulfonamide bond to an amino group on either the N-terminal end or side chain of a biologically active peptide moiety. The peptide derivatives have relatively enhanced biological activity when compared to the corresponding peptide alone. In the modified form, the derivatives may have more potent and prolonged biological activity than the corresponding unmodified peptide.

The term “mimetic” or“peptide mimetic” can be used to describe functionally and structurally similar organic compounds to the reference peptides. Peptide mimetics may have several potential advantages over native peptides, such as increased stability, increased lipophilicity, increased rigidity, decreased size, and affordability of production.

Various methods exist for developing peptide mimetics. These include computational as well as experimental screening methods. One method is to identify small peptides that are essential for the interactions of the protein. Subsequently, mimetics for these peptides are designed that can be used as drugs. On the basis of a known protein structure, scaffolding templates for binders can also be constructed and then optimized using different methods.

A person skilled in the art will appreciate that the present invention may be practiced without undue experimentation according to the methods given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology textbooks. Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001).

EXAMPLE 1

Cell lines and culture

HEK293T cells stably expressing RXFP3 (HEK-RXFP3) were provided by Prof. Roger Summers (Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, Monash University). HEK-RXFP3 cells were maintained in DMEM (Life Technologies, USA), 10% (v/v) fetal bovine serum (FBS) (Life Technologies, South America) and 1X penicillin/streptomycin (100,00 U/mL) (Life Technologies, USA) at 37°C in a 5% CO2 humidified environment. Cell lines were passaged using TripLE (Life Technologies, USA) at 80% confluency. Poly-L-Lysine (PLL), 3-isobutyl-1-methylxanthine (IBMX) were from Sigma (Saint Louis, USA). Dimethyl sulfoxide (DMSO) was from MP Biomedicals (Solon-Ohio, USA). Forskolin was from Tocris Biosciences (Bristol, UK). The cyclic AMP (cAMP) enzyme immunoassay (EIA) kit was from Cayman Chemicals (Ann Arbour, Ml, USA) and hydrochloric acid was from BDH Chemicals Ltd. (UK).

Stable RXFP3-expressing HEK293T cells (HEK-RXFP3) were maintained in 75 cm ² flasks Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) FBS, 1X penicillin/streptomycin (10,000 U/ml). The cells were grown in an incubator at 37° C in 5% CO2. Cells were harvested at 90% cell confluency for plating onto PLL pre-coated well plates for receptor-ligand binding and receptor activation assays.

EXAMPLE 2

Design of stapled relaxin-3 peptide antagonists and images

We designed the hydrocarbon stapled antagonist analogues of relaxin-3 B-chain based on the in-silico modeling prediction and a comprehensive staple scanning approach to derive optimized stapled analogues for the testing [Jayakody T. et al., Peptides 84: 44-57 (2016; Harder E., et al., Journal of chemical theory and computation 12(1): 281-96 (2016); Shivakumar D., et al., Journal of chemical theory and computation 8(8): 2553-8 (2012)]. Based on previously published structure-activity studies, amino acid residues Arg8, Arg12, Ile15, Arg16, Ile19 and Phe20 were reported to be important in the helix region of relaxin-3 B-chain for RXFP3 binding [Kuei C., et al., The Journal of biological chemistry 282(35): 25425-35 (2007); Bathgate R.A., et al., Frontiers in endocrinology 4: 13 (2013)]. Removal of the C- terminal Arg26-Trp27 activation domain was reported to be an antagonist peptide of relaxin-3 B-chain [Haugaard-Kedstrom L.M., et al., J Am Chem Soc 133(13): 4965-74 (2011); Kuei C., et al., The Journal of biological chemistry 282(35): 25425-35 (2007)]. Based on this knowledge and in-silico modeling prediction studies, we chose two different stapling sites in the relaxin-3 B-chain antagonist peptide. These two stapled peptides were stapled using two different stapling approaches and were stapled at (/, /+ 4) 14 ^th and 18 ^th positions (14s18) and at (/, i+7) 14 ^th and 21 ^st positions (14s21) (Table 1). Residues chosen for stapling were not reported to be involved in receptor binding and activation. The peptide ribbon renderings were made using Maestro (wwwdotschrodingerdotcom) software. Stapled H3 B-chain analogues are shown in Table 1.

S5: (S)-2-(4-pentenyl) alanine), R5. (R)-2-(4’-pentenyl) alanine, S8. (S)-2-(7’-octenyl) alanine

EXAMPLE 3

Peptide synthesis and characterization

Hydrocarbon stapled peptides and R3 B1-22R antagonist peptide were purchased from Biosynthesis Inc, USA. Staples were incorporated by placing the 8-carbon metathesized cross-link in (S)-configuration at /( 14) , /+4(18) positions with (S)2-(4'-pentenyl)alanine (Ss) in R3 B1-22R peptide sequence. Hydrocarbon stapling for i, i+7 peptides was carried out with an 11 -carbon cross-link by using the combination of R-2-(4’-pentenyl) alanine (Rs) and S-2- (7’-octenyl) alanine (Ss) at /( 14), /+ 7(21) positions. Peptides were purified by the manufacturer using high-performance liquid chromatography to >95% purity. We further verified the molecular weight and purity of the peptides by mass spectrometry and RP- HPLC analysis (Table 3). The purity of each peptide was confirmed to be > 95% by using analytical RP-HPLC peak area integration. Peptide molecular weights were confirmed by Matrix-assisted laser desorption/ionization time-of-f light (MALDI-TOF) mass spectrometry for all peptides were re analyzed on reverse-phase high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS).

Table 3. Analytical Data and Mass profiles of the synthesized peptides. Theoretical and observed masses of each analog.

EXAMPLE 4

Circular dichroism spectroscopy

Circular dichroism (CD) spectra was acquired using a Jasco 815 spectrometer (Tokyo, Japan). All peptide samples were measured in water with the peptide concentration ranging from 50 to 65 mM. The final peptide concentration was kept constant (0.20 mg/ml) for all the peptides studied. CD spectra were recorded from 190 to 260 nm using a 0.1 cm path length quartz cuvette. We used a continuous scan with a 20 nm/min scanning speed with a response time of 0.5 s, 0.5 nm step resolution and 1 nm spectral band width. The data were converted to per residue molar ellipticity. The percentage of helicity was calculated as described previously [Jayakody T. et ai, Peptides 84: 44-57 (2016); incorporated herein by reference] EXAMPLE 5

Ligand-receptor binding assay

Determination of receptor binding affinity was carried out in HEK-RXFP3 cells as previously described (Jayakody T. et al. , Peptides 84: 44-57 (2016)). Briefly, HEK-RXFP3 cells were seeded into 96 well plates. Increasing concentrations of non-labelled R3 B1-22R, R3 relaxin, and stapled analogues (1 pM to 100 pM) and a fixed concentration of Eu-DTPA-R3B1- 22R (8 nM) was utilized using identical procedure and conditions. The fluorescence measurements were taken on a Wallac VICTOR ³ instrument using the standard Eu(lll) TRL measurements (300 nm excitation, 400 ps delay, and emission collection for 400 ps at 615 nm). The europium binding curves were fitted using non-linear regression and a one-site binding model and the pK, calculated using the K _d value of 34.64 nM. Values were expressed as the mean ± SEM of at least three independent experiments with triplicate determinations within each assay. The data from three experiments were analyzed using one-way ANOVA followed by Tukey’s post hoc test in GraphPad Prism.

EXAMPLE 6

Inhibition of c AMP accumulation assay

The peptides were tested for their ability to inhibit cAMP activity in HEK-RXFP3 cells as were assayed as per manufacturer’s instructions (Cyclic AMP EIA Kit, Cayman Chemicals, Ann Arbor, Ml) and previously described (Jayakody T. et al., Peptides 84: 44-57 (2016); Harris G.L., et al. , PloS one 7(4): e35129 (2012)]. Briefly HEK-RXFP3 cells were plated in PLL coated well plates and were stimulated with 5 mM forskolin as previously described. R3 B1-22R was tested for its ability to produce a rightward-shift in the relaxin-3 dose-response curve in the presence of 10 nM and 100 nM R3 B1-22R to demonstrate functional antagonism. To test the antagonism of R3 B1-22R and stapled antagonist peptides, different concentrations of relaxin- 3 were added to cells in the presence of 10 nM, 100 nM R3 B1-22R peptide or 10 nM of stapled antagonist peptides. The results were analyzed and plotted based on a sigmoidal dose-response curve by using Graph Pad Prism and presented as mean ± SEM.

EXAMPLE 7

Data analysis

For the ligand binding assays, all statistical analysis was evaluated with GraphPad Prism using appropriate non-linear regression analysis. Competitive binding experiments were fitted to a one-site binding model and pK, calculated using the K _d value of 34.64 nM determined from the saturation binding. Binding data are presented as mean ± SEM. In the inhibition of forskolin-induced cAMP assay, the cAMP concentrations were calculated using a cAMP standard curve (03-750 nM). The data from three experiments were analyzed using on-way ANOVA followed by Tukey’s post hoc analysis using GraphPad Prism.

EXAMPLE 8

Hydrocarbon stapling in R3 B1-22R peptide sequence reinforces a-helicity

We previously reported the development of /, /+ 4 stapled 14s18 peptide and conformational analysis revealed that the insertion of 14s18 hydrocarbon linker enhanced a- helicity in the native sequence of R3 B-chain peptide (Jayakody T. et al., Peptides 84: 44-57 (2016)). Subsequently, we expanded the utility of hydrocarbon stapling technology and showed that (/, i+7) cross-linked hydrocarbon staple cross-linked over the critical hydrophobic binding surfaces served as more powerful nucleator of a-helical structure throughout a much longer sequence (unpublished). In this report, we installed the /, /+ 4 and /, i+7 hydrocarbon stapling approaches in R3 B1-22R antagonist peptide. The hydrocarbon-stapled R3 B1-22R antagonist peptides were synthesized by substituting the amino acids at i(14) and i+4(18) positions with (S)2-(4’-pentenyl)alanine or i(14) and i+7(21) positions by using the combination of R-2-(4’-pentenyl) alanine (R ₅ ) and S-2-(7’-octenyl) alanine (Ss) (Table 1 , Figure 6). Conformational analysis of stapled 14s18R (/, /+ 4) antagonist peptide revealed the preservation of a-helicity and overall percent a-helical content was increased up to 10.31 % compared to the 0.81 % of unmodified R3 B1-22R antagonist peptide (Figure 1A). Circular dichroism analysis of stapled 14s21 R (/, i+7) antagonist peptide also displayed the preservation of a-helicity and overall a-helical content was increased up to 9.50% (Figure 1 A). We previously showed that /, i+7 stapling has significantly increased the a-helical content into linear R3 B-chain peptide and improvement in helicity was superior to /, /+ 4 stapling strategy (Unpublished). In contrast to agonist, both the stapling strategies were able to introduce the a-helicity in R3 B1-22R antagonist peptide with approx similar ability (Figure 1A).

EXAMPLE 9

C-terminal of relaxin-3 B-chain is essential to maintain optimal a-helicity in full length 14s18 staple peptide

We further compared the full length stapled 14s18 stapled peptide, N-terminus truncated 14s18 peptide (A14s18) and C-terminus truncated 14s18R antagonist peptide (Figure 1 B). CD analysis showed that 14s18 stapled peptide has increased the a-helical content up to 18.5% compared to the 1.81 % of linear B-chain peptide. Further, N-terminus 7- residue truncation in 14s18 stapled peptide resulted into the partial loss of a-helicity and it was reduced to 13.2%. Interestingly, 5-residues truncation at C-terminus with Gly23 replaced to Arg23, of 14s18 stapled antagonist peptide reported the major loss in the overall a-helical content in 14s18 peptide resulting into only 10.81 % a-helical, thus highlighting the role of C- terminus in contributing the optimal a-helical structure in 14s18 peptide (Figure 1 B). Similarly, compared to the previously shown 23.5% of a-helicity of 14s21 agonist peptide, C-terminus truncation resulted into 9.50% a-helical content in 14s21 R antagonist peptide, emphasizing again the importance of C-terminus in maintaining the optimal a-helicity across the full-length B-chain peptide (Figure 7).

EXAMPLE 10

Binding affinity of stapled antagonist peptide towards RXFP3 receptor

R3B1-22R has been reported to be selective RXFP3 ligand [Haugaard-Kedstrom L.M., et al., JAm Chem Soc 133(13): 4965-74 (201 1)]. The pharmacological profile of Eu-DTPA-R3 B1-22R was characterized by us in both saturation and competition binding assays (Jayakody T. et al., Peptides 84: 44-57 (2016)). To rule out any significant difference in agonist and antagonistic binding affinities, we first studied the competition binding using various concentrations (1 pM to 10 mM) of unlabelled R3B1-22R peptide and intact human relaxin-3 (Jayakody T. et al., Peptides 84: 44-57 (2016)). The property of relaxin-3 B-chain antagonist peptides was also evaluated in competition binding assays in RXFP3 expressing cell lines. R3B1-22R demonstrated a pK, of 7.16 ± 0.15 (n=3), which was not significantly different from intact human relaxin-3 with a pK, of 7.38 ± 0.1 1 (n=3) (p > 0.05 by one-way ANOVA and Tukey’s post hoc comparisons; Table 4). In the similar manner, we carried out the competition binding assays using various concentration (1 pM to 10 mM) of the stapled antagonist peptides (14s18R and 14s21 R) (Figure 2, Table 4). 14s18R stapled peptide displayed improvement on the binding affinity over the unmodified R3 B1-22R peptide (pK, = 7.37 ± 0.15) (Figure 2) (Table 4). Similarly, 14s21 R peptide also showed improvement in the binding affinity towards the RXFP3 receptor (pK, = 7.40 ± 0.18) (Figure 2, Table 4). Thus, both the stapled antagonist peptides demonstrated almost equal improvement on the binding affinity towards RXFP3 receptor.

Table 4: Competitive binding activity (p ) and functional in-vitro cAMP activity (pECso) values for R3 B-chain analogues.

**p < 0.01 , vs R3 B-chain; ^# p < 0.05, ^## p < 0.01 , ^### p < 0.001 vs R3 B-chain

EXAMPLE 11

Agonist or antagonist activity of stapled peptides at receptor RXFP3

Activation of adenylate cyclase by forskolin and inhibition of adenylate cyclase by G _ai has been reported earlier [Taussig R., et al., Science 261 :(51 18) 218-21 (1993)]. Peptides were further tested for their ability to activate receptors in a functional cAMP assay by use of HEK-RXFP3 cell lines. Since RXFP3 are coupled to G _ai , the activity of the analogues was measured as inhibition of forskolin-induced cAMP activity. Relaxin-3 dose dependently inhibit the forskolin-induced cAMP levels and thus displayed the agonistic activity (pECso = 9.34 ± 0.09) (Figure 3, Table 2). None of the truncated analogues including R3 B1-22R, stapled 14s18R and 14s21 R peptides showed any ability to activate RXFP3 receptor (Figure 3). EXAMPLE 12

Ability of R3 B1-22R peptide to antagonize relaxin-3 inhibition of cAMP activity

We measured the ability of R3 B1-22R to antagonize relaxin-3 induced inhibition of cAMP activity. The competitive antagonistic behavior of R3 B1-22R peptide was confirmed by monitoring the activation of RXFP3 by relaxin-3 in the presence of different concentrations of 10 nM and 100 nM of R3 B1-22R peptide, which resulted in a rightward shift of the relaxin-3 activation curves (Figure 4).

EXAMPLE 13

Ability of hydrocarbon stapled 14s18R and 14s21R peptide to antagonize relaxin-3 inhibition of cAMP activity

We demonstrated the ability of 10 nM and 100 nM of R3 B1-22R peptide to antagonize the relaxin-3 inhibition of cAMP activity (Figure 4). Given the improved binding profile of 14s18R and 14s21 R antagonistic peptides over R3 B1-22R peptide, we chose to compare the 10 nM of stapled peptides to understand whether they can antagonize the relaxin-3 induced inhibition of cAMP activity. Our results showed that both 14s18R and 14s21 R dose- dependently shifted the relaxin-3 agonism curves to the right (Figure 5). In contrast to 14s21 R peptide, 14s18 peptide demonstrated major shift of the relaxin-3 agonism curves for RXFP3 and thus displayed stronger antagonist potency. Next, in vivo efficacy was tested.

EXAMPLE 14

Animals for in vivo study

Experimentally naive male Sprague-Dawley rats, weighing 150 - 180 g, (for intranasal drug administration) were utilized in this study. The animals were acclimated for 14 days, two per cage, and then randomly assigned to the experimental groups (8-12 animals each) and housed one per cage. The body weight was monitored during the experimental procedures. All efforts were made to minimize the number of animals used and their discomfort. All animals had free access to food and water throughout the experiment. The animal colony was maintained at 22 ± 2 ^° C during a 12-h light/12-h dark cycle with light on from 7:00 a.m. to 7:00 p.m. All behavioral testing occurred during the light phase between 8:00 a.m. and 1 :00 p.m.

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC). The procedures conducted followed the National Advisory Committee for Laboratory Animal Research (NACLAR), Singapore, and were in accordance with the Guide for the Care and Use of Laboratory Animals, National Research Council of the National Academies, USA. Animal studies are reported in compliance with the ARRIVE international guideline and the BJP guidelines [McGrath J.C., Lilley E., Br J Pharmacol 172(13): 3189-93 (2015)]. Intranasal drug administration

Intranasal delivery in Sprague- Dawley rats was carried out manually without anesthesia as previously described [Thorne R.G., et al., Neuroscience 127(2): 481-96 (2004).; Lukas M., Neumann I.D., Neuropharmacology 62(1): 398-405 (2012), incorporated herein by reference) with slight modifications. To minimize the non-specific stress response during the application procedure, rats were habituated to the handler 10 mins daily prior to the day of experiment. In detail, the conscious rat head was restrained in a supine position and two pads were placed under the dorsal neck to extend the head back towards the supporting surface. All rats were treated with relaxin-3 or R3 B1-22R antagonist peptide. Peptide or saline/vehicle was delivered over both nares alternatively using a 100 pi pipette. To minimize respiratory distress and swallowing of the dose, the total volume of 50 mI dose solution as 8-10 m I drops, alternating between each nares every 1-2 min, over a total of 6.5 to 7 mins was delivered until the drugs were naturally sniffed in by the rat [Migliore M.M., et al., Neuroscience 274: (2014) 1 1-23] The rat was held for an additional 30-60 seconds to ensure the fluid was inhaled. Rats were returned to their home cage thereafter until behavioral testing started 25-35 mins later.

Food and water intake, body weight and locomotory activity assessment after intranasal drug administration

All behavioral testing occurred during the light phase between 8:00 a.m. and 1 :00 p.m. Before the start of experiment trial, rats were singly housed and were acclimatized to the behavior room for 1 h before the experiments. Rats were given repeated intranasal administrations of relaxin-3 (H3), antagonist (50 pmol or 100 pmol) or saline (vehicle) for 15 days. Body weight, food intake, water intake was measured after 2 hours of intranasal drug administration. Food intake was calculated according to the weight change of the chow box. Water intake was measured according to the weight change in drinking water bottles. Body weight was measured at 24 h intervals.

EXAMPLE 15

Effect of repeated intranasal administration of relaxin-3 peptide in adult SD rats

We previously reported that acute administration of intranasal relaxin-3 can increase food intake within first 80 min post peptide administration. Relaxin-3 at 0.1 pmol dose was shown to significantly increase the food intake within the first 80 min after peptide administration. Here we were interested to understand whether chronic treatment of intranasal relaxin-3 peptide can further enhance the food consumption and body weight. We measured the food intake after 2 hours of intranasal relaxin-3 administration up to 15 days.

There were significant differences in post intranasal food intake between the two groups (FI , 96 = 85.01 , P < 0.001); Significant effect of interaction (Day x Treatment): (Fi5,96 = 5.55, P < 0.001) (Figure 1). On day 2, animals exhibited compensatory hyperphagia, eating significantly more food than in the baseline monitoring period (post-test, p < 0.001). The significant increase in the food intake was observed up to the 9 ^th day of intranasal relaxin-3 administration. Soon after the 10 ^th day, the food consumption rate started declining and came to basal level at the last day of drug administration (Figure 8).

All data are expressed as a mean ± SEM for each group. Significant differences between groups were assessed by repeated measures two-way ANOVA followed by Bonferroni’s post hoc for comparisons on the same day. Statistical analysis was defined p < 0.05. All analyses were performed using GraphPad Prism version 7 (GraphPad Prism Software, La Jolla, CA, USA).

EXAMPLE 16

Effect of chronic intranasal administration of different doses of antagonist in rats

To understand whether R3 B1-22R can inhibit food intake behavior, we chose the higher dose of R3B1-22R antagonist peptide. We previously attempted to use 0.1 pmol, 1 pmol and 10 pmol R3 B1-22R peptide but none of these doses were shown to be effective in modulating the food intake behavior in our pilot experimental studies. So, we attempted the higher doses of 50 pmol and 100 pmol to further investigate the food consumption effects of R3 B1-22R peptide. Food intake was measured after intranasal administration of R3 B1-22R peptide up to 15 days. There was no significant difference in food intake measurement observed up to 7 days post intranasal peptide administration by either dose. Afterwards, there was a trend in reduced food intake until the last day of peptide treatment at both the doses. However, the trend was more strongly reflected in the 100 pmol dose compared to the 50 pmol of R3 B1-22R antagonist peptide treatment. There was significant effect of treatment (/¾ _{, 144} = 4.52, P = 0.012), significant effect of days (Fis _{, i44} = 4.471 , P < 0.001) and significant effect of interaction (Day x Treatment): (/½ _{, 144} ) = 1.833, P = 0.010) observed. Post hoc comparison showed the effect of 100 pmol R3 B1-22R peptide on food intake on day 13 (post-test, p < 0.01), day 14 (post-test, p < 0.05) and day 15 (post-test, p < 0.001) on SD rats (Figure 9). EXAMPLE 17

Intranasal administration of R3 B1-22R antagonist did not affect locomotor activity

Intranasal administration of R3 B1-22R peptide (100 pmol) did not alter the locomotor activity analyzed using the LABORAS platform during the period of 4 hours post administration (324.8 ± 23.5 m, n = 4) relative to vehicle controls (337 ± 26.4 m), suggesting an absence of any“sedative” effects of antagonist peptide. Given the chronic action of the antagonist peptide, this finding is unlikely to be physiologically relevant.

EXAMPLE 18

Effect of R3 B1-22R peptide on relaxin-3 induced feeding in adult Sprague-Dawley rats After the significant reduction observed in food intake by 100 pmol R3 B1-22R peptide from day 13 of intranasal treatment, we chose this dose to further observe the effect on the inhibition on relaxin-3 induced feeding behavior. Pre-treatment with 100 pmol R3 B1-22R peptide completely inhibited the food intake induced by 0.1 pmol of relaxin-3. There was a significant effect of treatment (/¾ _, = 60.9, P < 0.001), significant effect of days ^5 _{, U} 4 = 3.448, P < 0.001) and significant effect of interaction (Day x Treatment): (/½ _, 144) = 4.513, P < 0.010) observed (Figure 10). Interestingly, a significant difference between relaxin-3 treatment (R3) and R3 B1-22R peptide infused before relaxin-3 peptide (R3 + R3 B1-22R) was observed on the first day of experiment (R3 vs. R3 + R3 B1-22R, day 1 , post-hoc, p < 0.01 vs relaxin- 3). The significant inhibition of food intake by R3 + R3 B1-22R peptide treatment remained elevated until day 6 of intranasal administration (day 2, p < 0.001 ; day 3, p < 0.05; day 4, p < 0.001 ; day 5, p < 0. 001 ; day 6, p < 0.001). But soon after the 6 ^th day, the activity of both the peptides started declining, reaching to basal level at day 15 of intranasal treatment. Although there was a significant difference in food intake stimulated by relaxin-3 alone or with R3 B1- 22R antagonist pre-treatment observed within 2 hours of intranasal administration, the basal levels of daily 24-hour food intake remain unaffected.

EXAMPLE 19

Changes in body-weight in response to agonist and antagonist treatment in SD rats

To examine the effect of chronic intranasal administration with relaxin-3 on long-term food intake, we studied food intake and body weight gain during a 14-day intranasal infusion with vehicle, 0.1 pmol relaxin-3 (R3), relaxin-3 with prior infusion of 100 pmol antagonist peptide (R3 + R3 B1-22R). There was no significant difference in body weight between vehicle or relaxin-3 treated groups at intranasal administration time points. Cumulative body weight by the vehicle or peptide treatment is shown in Figure 11. In the vehicle group, intranasal administration and handling of the animals did not change the normally observed level of food consumption. In animals receiving relaxin-3, the expected initial fall in feeding was not seen, and their body-weight gain was slightly elevated compared to the control groups but did not result in a significant increase over the vehicle control values during the 15-day treatment as expected. Animals receiving the R3 + R3 B1-22R treatment showed a slight drop in body- weight at day 3 of intranasal treatment but their basal body-weight gain remained unaffected and was not significantly different from vehicle or relaxin-3 treatment.

EXAMPLE 20

In vivo testing of stapled peptide RXFP3 antagonist 14s18R (Ser)

Male Sprague- Dawley rats (n=14) weighing 300 to 350g at the time of arrival were acquired from InVivos Pte Ltd, Singapore. The animal colony was maintained at 22 ± 2°C under a 12-hr light/12-hr dark cycle with light on from 7:00 am to 7:00 pm with ad libitum access to standard laboratory chow. The rats were acclimatized for one week to the housing conditions and subsequently acclimatized to daily intranasal administration of saline for a further week. The rats were then randomly assigned to receive a total volume of 50 mI solution as 8-10 mI drops, alternating between each nares every 1-2 min, of either 572 mM [i.e. 28.6 nmol] stapled peptide 14s18R (Ser): RAAPYGVRLSGRE-S5I RA-S5I FTSR (SEQ ID NO: 1 1) or vehicle (saline). The animals were weighed daily and the difference in weight from baseline was determined. Statistical analysis by two-way ANOVA was conducted using Graphpad Prism 6 software (Graphpad Software, Inc).

The stapled peptide RXFP3 antagonist significantly reduced the weight gain of the rats (Fi _{, 144} =22.60, p < 0.0001) without causing any significant loss of weight below the normal starting body weight (Figure 12). These in vivo data confirm that the stapled peptide 14s18R (Ser) is bioavailable on intranasal administration in rodents and reduces body weight gain in adult male rats.

Summary

Pharmacological studies

We applied hydrocarbon stapling technology to the R3 B1-22R peptide to develop a structurally stable and potent antagonist of RXFP3. Stapling enhanced helicity of the peptides. The stapled peptides showed an improved binding affinity compared to the unmodified R3 B1- 22R peptide.

Given the clear differences in binding contributions from individual amino acids between relaxin-3 and R3 B1-22R peptide, we further investigated the antagonistic activity of these peptides in a popular inhibition of forskolin induced cAMP assay. We first examined the antagonistic activity of R3 B1-22R peptide and confirmed the right shift on relaxin-3 dose- response curve. We further tested the ability of the hydrocarbon stapled peptides to antagonize the relaxin-3 dose-response curve.

Both 14s18R and 14s21 R stapled peptides were truncated at the C-terminus and despite good binding to RXFP3 none of them was able to activate RXFP3 demonstrating that they are effective antagonists. Although both the stapled peptides showed approximately similar binding affinity towards RXFP3 receptor, the observed antagonistic shift caused by 14s18R peptide was slightly more potent than the 14s21 R peptide.

The final affinity and the potency of the best stapled peptide is further improved by means of i, i+4 stapling at 14 and 18 positions and, as such, 14s18R provides a good exemplar of a stapled peptide antagonist at RXFP3. Previously reported structure-activity relationship studies by installing several means of helix introducing strategies reflect the difficulty in designing high affinity RXFP3 antagonist peptides and highlight the importance of our peptide [Wong L.L.L., et al., Journal of Biological Chemistry 293(41): 15777-15789 (2018)]. It is important to note that (1) inserting a staple at any given position does not guarantee structural reinforcement [Bernal F., et al., Journal of the American Chemical Society 129(9): 2456-7 (2007)] and (2) maximizing a-helicity does not guarantee optimal pharmacological or biological activity. Therefore, our discovery of exemplars of functional stapled peptide antagonist designs provides a foundation for developing new RFXP3 antagonists.

In vivo studies

The major finding was that a stapled peptide antagonist, 14s18R (Ser), was able to work via intranasal administration to reduce body weight gain in male SD rats. This provides support for the possibility of using intranasal relaxin-3 antagonist peptide as a method of non- invasive peptide administration that can modulate feeding behaviour and weight gain in human and non-human animals.

As the targets for RXFP3 antagonist regulation of weight gain are likely in the brain, and in particular in the hypothalamus, the in vivo effect of the stapled peptide RXFP3 antagonist indicates actions in the brain. For example, effects on feeding can be explained by the fact that relaxin-3 activates orexin neurons in the lateral hypothalamus and neuropeptide Y neurons in the dorsomedial hypothalamus [Ganella D.E., et al., Frontiers in endocrinology 4: 128 (2013)], which already contains the orexigenic peptides. On the other hand, chronic infusion of ICV administered relaxin-3 has shown significant increase in body weight gain, and with higher plasma concentrations of leptin and insulin [Hida T., et al., Journal of receptor and signal transduction research 26(3): 147-58 (2006)]. Thus, the fact that intranasal stapled peptide RXFP3 antagonist reduced body weight gain suggests that it crossed the blood-brain barrier and had an effect in the brain. These results suggest that intranasal administration is an effective route whereby stapled peptide RXFP3 antagonists can exert effects as anti obesity drugs.

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