Peptide mimetic for modulating the activity of incretin receptors, compositions and methods thereof

Abstract

The present disclosure provides agonist peptides against incretin hormone receptors for the management of metabolic disorders including Type 2 Diabetes, Obesity, and / or co-morbidities. Also provided are compositions and methods for treatment and management of Type 2 Diabetes, Obesity, and / or co-morbidities.

Description

PEPTIDE MIMETIC FOR MODULATING THE ACTIVITY OF INCRETIN RECEPTORS, COMPOSITIONS AND METHODS THEREOF FIELD OF INVENTION

[0001] The present disclosure broadly relates to the field of peptidomimetics. In particularly, the invention relates to peptides having agonist activity for therapeutic application. The present invention also relates to compositions and methods for treatment and management of Type 2 Diabetes, Obesity, and / or comorbidities.BACKGROUND OF INVENTION

[0002] Peptide-based therapies have emerged as promising avenues in recent times. Incretins plays an essential role in the regulation of blood glucose levels, energy expenditure and fat deposition. These hormones stimulate the ? -cells in pancreas to release insulin upon sensing the presence of excess glucose in the intestinal lumen.

[0003] T2DM, characterized by insulin resistance and relative insulin deficiency, frequently coexists with obesity, marked by excessive adiposity with about two thirds of the T2DM cases diagnosed already having obesity. Historically, managing T2DM and obesity has relied on lifestyle modifications, pharmacotherapy, and, in severe cases, bariatric surgery. However, existing interventions exhibit limitations in efficacy, tolerability, and long-term adherence. Weight loss alone through medication or lifestyle changes is not enough to reduce the progression of T2DM and Obesity. Consequently, there is a burgeoning interest in innovative therapeutic approaches targeting the intricate interplay between T2DM and obesity. Exogeneous insulin therapy was one promising approach to overcome hyperglycemia, but it led to weight gain.

[0004] Peptidomimetics, particularly incretin mimetics and analogues have been implemented to manage Type 2 Diabetes (T2DM) and Obesity. Incretin mimetics having mono agonist and dual agonist activity have previously been developed.However, there is a need for effective, stabilized, and long-acting peptides and therapeutic approaches for enhancing quality of life for affected individuals.SUMMARY OF THE INVENTION

[0005] In an initial aspect of the present disclosure, there is provided a peptide having an amino acid sequence of SEQ ID NO. 1, wherein Xi is selected from Histidine (H) or Tyrosine (Y), X2 is selected from Glycine (G), Serine (S), or a non-natural amino acid, X12 is selected from lysine (K) or modified lysine (Km), X13 is selected from Tyrosine (Y) or a-methylated amino acid, wherein a hydrocarbon staple is positioned to link non-natural amino acids at positions i, i+4, preferably non-natural amino acids at positions 30 and 34; 31 and 35; or 29 and 33, or a pharmaceutically acceptable salt thereof.

[0006] In another aspect of the present disclosure, there is provided a composition comprising the peptide as disclosed herein, and at least one pharmaceutically acceptable carrier.

[0007] In yet another aspect of the present disclosure, there is provided a method for modulating Incretin receptors, preferably glucagon-like peptide- 1 receptor (GLP1R), gastric inhibitory polypeptide receptor GIPR, and / or glucagon receptor GCGR, comprising: providing an effective amount of the peptide or the composition as disclosed herein.

[0008] In an aspect of the present disclosure, there is provided a method for treatment or management of obesity in a subject, comprising providing an effective amount of the peptide as disclosed herein, to the subject.

[0009] In an aspect of the present disclosure, there is provided a method for treatment or management of Type 2 Diabetes in a subject, comprising providing an effective amount of the peptide as disclosed herein, to the subject.

[0010] In an aspect of the present disclosure, there is provided a method for preparation of a peptide, said method comprising: (a) synthesizing the peptide by solid-phase peptide synthesis and optionally including at least one non-naturalamino acid; (b) optionally incorporating at least one modified amino acid residue; and (c) cleaving and recovering the peptide to obtain the peptides of the present disclosure.

[0011] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF FIGURES

[0012] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0013] Figure 1 depicts a schematic representation of the amino acid structures of the peptides, in accordance with the embodiments herein.

[0014] Figure 2 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R, GIPR and GCGR, where (A)-(D) depict the activation of GLP-1R in presence of peptides represented by the SEQ ID NO. 5 -SEQ ID NO. 7 along with the positive control peptide Retatrutide, and (E)- (F) depict the activation of GIPR and GCGR in presence of peptide represented by the SEQ ID NO. 7, in accordance with the embodiments herein.

[0015] Figure 3 shows the change in body weight in diet-induced obese (DIO) mice after twice weekly subcutaneous dosing of SEQ ID NO: 7 (ND-MC-ZB-48) at 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0016] Figure 4 shows the results of Oral Glucose Tolerance test (OGTT) at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with SEQ IDNO: 7 (ND-MC-ZB-48), 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0017] Figure 5 shows the results of Insulin Tolerance Test (ITT) at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with SEQ ID NO: 7 (ND-MC-ZB-48), 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0018] Figure 6 depicts the results of glucose levels, total cholesterol, triglyceride, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels in treated mice, at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with SEQ ID NO: 7 (ND-MC-ZB-48), 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0019] Figures 7 show the results of serum clinical metabolic parameters: leptin, at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with SEQ ID NO: 7 (ND-MC-ZB-48), 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0020] Figure 8 shows the results of retroperitoneal fat accumulation at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with SEQ ID NO: 7 (ND-MC-ZB-48), 0.5 mg / kg body weight., in accordance with the embodiments herein.

[0021] Figure 9 shows the results of hepatic steatosis at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with SEQ ID NO: 7 (ND-MC-ZB-48), 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0022] Figure 10 shows the results of a Surface Plasmon Resonance (SPR) sensorgram showing immobilisation of GIPR onto a CM5 chip (Fc=l) via amine coupling, with sequential baseline, EDC / NHS activation, receptor injection, and post-immobilisation stabilisation phases, in accordance with the embodiments herein.

[0023] Figure 11(a) shows the results of a single-concentration binding SPR sensorgram of LY (positive control) at 50 pM and 5 pM injected over immobilised GIPR. Figure 11(b) shows the results of multi-concentration kinetic SPR sensorgram of LY (0, 4.1, 12, 37, 111, 333, 1000 nM) binding to immobilised GIPR, with 1:1 Langmuir fitting used to determine ka, kd, and KD in accordance with the embodiments herein.

[0024] Figure 12(a) shows the results of a single-concentration binding sensorgram of Peptide 20 (positive control) at 50 pM and 5 pM injected over immobilised GIPR. Figure 12(b) shows the results of multi-concentration kinetic sensorgram of Peptide 20 (20, 61, 185, 555, 1666, 5000 nM) binding to immobilised GIPR, in accordance with the embodiments herein.

[0025] Figure 13(a) shows the results of single-concentration binding sensorgram of SEQ ID NO: 7 (ND-MC-ZB-48) at 50 pM and 5 pM injected over immobilised GIPR. Figure 13(b) shows the results of multi-concentration kinetic sensorgram of SEQ ID NO: 7 (ND-MC-ZB-48) (0, 20, 61, 185, 555, 1666, 5000 nM) binding to immobilised GIPR, in accordance with the embodiments herein.

[0026] Figure 14 shows the results of a SPR sensorgram showing immobilisation of GLP-1R onto a CM5 chip (Fc=3) via amine coupling, with sequential baseline, EDC / NHS activation, receptor injection, and post-immobilisation stabilisation phases, in accordance with the embodiments herein.

[0027] Figure 15 shows the results of multi-concentration kinetic sensorgram of LY (positive control; 4.1, 12, 37, 111, 333, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0028] Figure 16 shows the results of multi-concentration kinetic sensorgram of Peptide 20 (0, 20, 61, 185, 555, 1666, 5000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0029] Figure 17 shows the results of multi-concentration kinetic sensorgram of SEQ ID NO: 7 (ND-MC-ZB-48) (0, 20, 61, 185, 555, 1666, 5000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0030] Figure 18 shows the results of a SPR sensorgram showing immobilisation of GCGR (GCGR-lig 3) onto a CM5 chip (Fc=l) via amine coupling, with sequential baseline, EDC / NHS activation, receptor injection, and postimmobilisation stabilisation phases, in accordance with the embodiments herein.

[0031] Figure 19 shows the results of multi-concentration kinetic sensorgram of LY (positive control; 0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0032] Figure 20 shows the results of multi-concentration kinetic sensorgram of Peptide 20 (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR. The low maximal binding response (~8 RU) is indicative of weak binding affinity to this receptor, in accordance with the embodiments herein.

[0033] Figure 21 shows the results of multi-concentration kinetic sensorgram of SEQ ID NO: 7 (ND-MC-ZB-48) (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR. The low maximal binding response (~8 RU) reflects weak binding of ND48 to GCGR under these assay conditions, in accordance with the embodiments herein.DETAILED DESCRIPTION OF THE INVENTION

[0034] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.Definitions

[0035] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0036] The articles “a”, “an” and “the” are used to refer to one or more than one (i.e., to at least one) of the grammatical object of the article.

[0037] The terms “comprise” or “contain” and “comprising” or “containing” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0038] The term "at least one" is used to mean one or more and thus includes individual components as well as mixtures / combinations.

[0039] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0040] The term “including” is used to mean “including but not limited to”, “including” and “including but not limited to” are used interchangeably.

[0041] The term “peptide”, as used herein, broadly refers to a molecule having two or more amino acids residues joined together by peptide bonds. The term “peptide” and “polypeptide” are used interchangeably herein.

[0042] The term “agonist”, as used herein, refers to a substance which promotes the biological activity of another. The term agonist encompasses substances that bind to receptors and substances which promote receptor function without bindingthereto. For example, a Glucagon-like peptide- 1 (GLP-1) analog binds to GLP-1 receptor (GLP-1R) to agonize GLP-1R.

[0043] Amino acid residues are generally represented by single letter and three letter abbreviations, for example “Alanine” is represented by single letter code “A” and three letter code “ala” or “Ala”. The expression “X” as used in amino acid sequences refers to any amino acid residue, including natural, non-natural, and modified amino acid residues. In the expression “Xi”, the number ‘1’ refer to the position of X amino acid in the amino acid sequence of the peptide. For example, “Xi”, denotes X amino acid position 1, “X2” denotes X amino acid position 2, and so on. Such representations are generally used and well understood by a person skilled in the art. The present disclosure in describing the present invention employs such representations or phrases which is intended to mean the generally acceptable meaning in the art.

[0044] The term “peptide staple” refers to a brace, preferably a covalent or non-covalent bond, positioned in a peptide to link two amino acids including natural or non-natural amino acids in a peptide. The term “stapled peptide” or “cyclized peptide” refers to a peptide comprising a peptide staple. It refers to a peptide wherein side chains of two amino acids including natural or non-natural amino acids in the peptide are linked. The term “hydrocarbon staple” refers to a brace positioned in a peptide to link hydrocarbon side chains of two amino acids including natural or non-natural amino acids in the peptide. Hydrocarbon stapling is a technique capable of promoting secondary structure formation, and / or stabilizing secondary structure of peptide. Hydrocarbon stapling helps promote and stabilize an alpha-helical secondary structure in peptides. Hydrocarbon staples may be suitably positioned to link natural or non-natural amino acids in peptides between various positions such as i, i+4, i+7, etc. The terms “i,”, and “i+4”, as used herein, refers to the positions of the natural or non-natural amino acids within the peptide that become linked to one another upon stapling. The “i” position refers to the position of the amino acid that is nearest to the N-terminal ofthe peptide. The “i+4” position is 4 amino acids downstream (4 amino acids further towards the C-terminal of the peptide).

[0045] Embodiments herein disclose peptidomimetic molecules, particularly agonist peptides, for therapeutic application. The agonist peptides, according to embodiments herein, are incretin mimetics having agonist activity on receptors. The term “incretin”, as used herein, broadly refers to group of metabolic hormones including glucagon -like peptide- 1 (GLP-1) and glucose dependent insulinotropic peptide (GIP). Incretin hormones are naturally-occurring peptide hormones. Embodiments herein provide agonist peptides that are capable of mimicking the activity of incretin hormones. Embodiments herein provide incretin mimetics having agonist activity on receptors, particularly incretin hormone receptors, including glucagon-like peptide-1 receptor (GLP-1R), gastric inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR. In a preferred embodiment, the agonist peptides as disclosed herein are agonists which exhibit agonist activity on at least three receptors including glucagon-like peptide-1 receptor (GLP1R), gastric inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR. Further embodiments herein provide dimers or multimer, conjugates, and compositions comprising the peptides as disclosed herein. Also disclosed are methods for use and preparation of the peptide disclosed herein. The agonist peptide, according to embodiments herein, are capable of regulating blood glucose and body weight in subjects. Accordingly, embodiments herein include methods for modulating receptors including GLP-1R, GIPR and GCGR, methods for treating and / or management of metabolic disorders including diabetes, obesity and / or co-morbidities.

[0046] The present inventors have, by application of computational structural biology, designed unimolecular agonist peptides capable of agonist activity on at least three receptors. With the application of the machine learning (ML), molecular modelling and molecular dynamics simulations (MD), and classical physics based binding free energy calculations, the fine intricacies of peptide-receptor complex formation and protein-protein interactions were studied. Thiswas instrumental in enhancing the binding efficiency of the designed peptides with the three receptors, i.e. GLP-1R, GIPR and GCGR, and fine tuning the agonistic properties of the designed peptides. Accordingly, the present inventors have been able to achieve agonist peptides having improved properties.

[0047] The agonist peptides, according to embodiments herein, are capable of binding and having selectivity to incretin hormone receptors. The agonist peptides, as disclosed herein, are long acting with good serum half-life and stability. The agonist peptides, as disclosed herein, have stabilized alpha-helix, comprising non-natural amino acids linked by a peptide staple, preferably hydrocarbon staple. It is observed that the peptides, as disclosed herein, have a primary structure comprising non-natural amino acids which help in promoting and maintaining secondary structure, particularly alpha helical secondary structure. In general, serum half-life is mainly determined by the two critical factors viz. clearance rate by the kidney and enzyme mediated degradation. It is observed that the peptides of the present invention have increased stability and solubility, showing enhanced activation of GLP-1R, reduced clearance rate, and lower chances of enzyme mediated degradation.Peptides

[0048] Embodiments herein disclose agonist peptides having agonist properties. The agonist peptides, according to embodiments herein, are incretin mimetics having specificity for incretin hormone receptors. The agonist peptides, as disclosed herein, have amino acid sequences comprising natural, non-natural, and / or modified amino acid residues. Further, the non-natural amino acids may further include side chains that facilitate peptide stapling. The peptides, as disclosed herein, is such that it exhibits enhanced stability, specificity and binding affinity to incretin receptors, and prolonged serum half-life of the peptides.

[0049] In an embodiment, the agonist peptide comprises a peptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO.8. In an embodiment, the agonist peptide comprises a peptide having an amino acid sequence as set forth in SEQ ID NO. 1 :X1X2QGTFTSDYSX12X13LDERAAQDFVQWLLDX29X30X31SX33X34X35PPPS(SEQ ID NO. 1); orX1X2QGTFTSDYS X12X13LDERAAQDFVQWLLDGG X31SSGX35PPPS(SEQ ID NO. 8);wherein, Xi, X2, X12, X13, X29, X30, X31, X33, X34, and X35 are amino acids selected from natural, non-natural, or modified amino acid residues. Examples of natural, non-natural, and modified amino acid residues include, but are not limited to, histidine (H), tyrosine (Y), lysine (K), arginine (R), isoleucine (I), alanine (A), glutamic acid (E) or glutamine (Q), aspartic acid (D, serine (S), Alphaaminobutyric acid (Aib), Alpha-Methyl Leucine (crMeL), HomoSerine (hSer), Norvaline (nVal), NorLeucine (nLeu), Diaminopimelic acid (DAP), alpha-4-pentenyl alanine, Bis-Pentenyl glycine, S-octenyl alanine, R-octenyl alanine, (S)-N-Fmoc-a-(4-pentenyl)alanine, Methionine sulfoxide (MetO), Selenocysteine (Sec), Methylalanine, or acylated forms thereof. Acylated forms include, but are not limited to, modified amino acids having an acyl group conjugated to the amino acid.

[0050] In an embodiment, there is provided an agonist peptide, wherein the peptide has an amino acid sequence as set forth in SEQ ID NO. 1, wherein, Xi, X2, X12, X13, X29, X30, X31, X33, X34 and X35 are amino acids selected from a group consisting of histidine (H) or tyrosine (Y), lysine (K), arginine (R), isoleucine (I), alanine (A), glutamic acid (E), glutamine (Q), aspartic acid (D), and serine (S).

[0051] In an embodiment, there is provided an agonist peptide, wherein the peptide has an amino acid sequence as set forth in SEQ ID NO. 1, wherein, Xi is selected from Histidine (H) or Tyrosine (Y), X2 is selected from Glycine (G), Serine (S), or a non-natural amino acid, X12 is selected from lysine (K) or modified lysine (Km), X13 is selected from Tyrosine (Y) or a-methylated aminoacid, wherein a peptide staple, preferably hydrocarbon staple, is positioned to link non-natural amino acids at positions 30 and 34; 31 and 35; or 29 and 33, or a pharmaceutically acceptable salt thereof.Modifications

[0052] The peptides, according to embodiments herein, include modified amino acid residues. These modifications are made to amino acids in the peptides at certain positions to promote and maintain secondary structure of peptide, particularly alpha-helical secondary structure. Further, the peptide as disclosed herein include modification for improving the serum half-life, solubility and stability of the peptide. The term “acylated amino acid”, as used herein, refers to an amino acid that have at least one acyl group attached to the amino acid. The acylated amino acid may be attached to the acyl group by one or more linker and / or spacers. Accordingly, the term “acylated lysine” or “modified lysine”, used interchangeably herein, refers to lysine having at least one acyl group (for eg: lipid) attached to, preferably via one or more linker and / or spacers. The acyl group may be fatty acid or fatty diacid. The modification, as disclosed herein, facilitate in improving the serum half-life, solubility, and stability of the peptide. In an embodiment, the amino acid residue is modified or acylated by conjugating with a lipid (also referred to herein as “lipid tail”) via a linker and a spacer. The lipid tail may bind (reversibly) to serum albumin which increases the serum half-life of the peptide.

[0053] The term “lipid”, as used herein, broadly refers to organic compounds that are soluble in non-polar solvents. The term lipid includes fatty acids and fatty diacids, and their substituted or unsubstituted forms. The term “lipid tail” and “lipid” are used interchangeably herein, and include, substituted or unsubstituted, tail of a fatty acid moiety or tail of a diacid moiety. In an embodiment, the lipid is selected from Cs- C22 fatty acids or diacids. Examples of lipid include, but are not limited to, caprylic acid (Cs), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (Cie), stearic acid (Cis), arachidic acid (C20), dodecanedioicacid (C12), tridecanedioic acid (C13), tetradecanedioic acid (C14), pentadecanoic acid (C15), hexadecanedioic acid (Cie), heptadecanedioic acid (C17), octadecanedioic acid (Cis), eicosanedioic Acid (C20), and Docosanedioate (C22). In an embodiment, the lipid is selected from a compound of Formula (a) to (i) depicted in Table 1. In an embodiment, the lipid is selected from a compound of Formula (a), (b), (c), (d), (e), (f), (g), (h), and (i) depicted in Table 1. The lipid may be attached to the amino acid via a linker and / or spacer, in accordance with the embodiment herein.

[0054] The lipid, in various embodiments herein, may comprise a compound of Formula I. In an embodiment, the lipid is a compound of Formula I.CO2H-(CH2)a-CO- Formula Iwherein ‘a’ is in the range of 8 to 22, preferably 12 to 20, more preferably 16 to 18.

[0055] The lipid, in various embodiments herein, may comprise a compound of Formula I(i). In an embodiment, the lipid is a compound of Formula I(i).CH3-(CH2)a-CO- Formula I(i)wherein ‘a’ is in the range of 8 to 22, preferably 12 to 20, more preferably 16 to 18.

[0056] In an embodiment, the lipid is a compound of Formula I or Formula I(i).

[0057] The linker, in various embodiments herein, is attached to the lipid, preferably at the hydroxyl end of the lipid, at one end and a spacer at the other end. The linker, in some embodiments herein, at one end, preferably at the hydroxyl end of the lipid, is attached to the lipid, and at the other end, is attached to the amino acid. Examples of linkers include, but are not limited to, one or more gamma-glutamic acid (yGlu) moieties, and Gamma-aminobutyric acid (GABA). In an embodiment, the linker is selected from one or more yGlu moieties, Gamma-aminobutyric acid (GABA), or combination thereof. The linker may comprise ofone or more moieties of yGlu. In alternate embodiments, the linker may be one or more moieties selected from benzyl-PAla, yGlu, or alpha-aminobutyric acid-yGlu (Abu-yGlu). The yGlu may be D-yGlu or L-yGlu. Accordingly, in an embodiment, the linker is at least one yGlu linker. In an embodiment, the linker is a compound of Formula (1) depicted in Table 1. In an embodiment, the linker comprises a yGlu linker, wherein the lipid is preferably attached to the yGlu linker at its a -amino group. In another embodiment, the linker comprises two yGlu moieties, wherein the lipid is preferably attached to the a -amino group of one yGlu moiety which is further attached at the carboxyl end to another yGlu moiety. Similarly, the linker may comprise two or more yGlu moieties. The linker is further attached to a spacer.

[0058] The spacer, in various embodiments herein, is attached to the linker at one end and the amino acid residue at the other end. The spacer, in some embodiments herein, is attached to the lipid at one end and the amino acid residue at the other end. Examples of spacers include, but are not limited to, Aminoethylethanolamine (AEEA) moeities, y-Carboxylate moieties, GGGGS repeats, and one or more PEG moieties. In an embodiment, the spacer comprises a moiety selected from Aminoethylethanolamine (AEEA), y-Carboxylate moieties, GGGGS repeats, one or more polyethylene glycol (PEG) moieties, or combination thereof.

[0059] The term “PEG”, as used herein, refers to any water-soluble polyethylene oxide). The term “PEG” encompasses “Oligo(ethylene glycol)” or “OEG”, and is used interchangeably herein. Typically, PEG comprises the following structure — (OCH2CH2)b — , (also represented herein as (PEG)b), wherein ‘b’ ranges from 1 to 4000. In an embodiment herein, PEG comprises the following structure — (PEG)b — wherein ‘b’ is in the range of 1 to 8 or 2 to 8, preferably 2 to 6.

[0060] The spacer, in various embodiments herein, may comprise a compound of Formula II.

[0061] In an embodiment, the spacer is a compound of Formula II.-(amino-(PEG / AEEA)b-CH2CO2H)c- Formula II

[0062] In an embodiment, the spacer is a compound of Formula II and is selected from(i) -(amino-(PEG)b-CH2CO2H)c- ; or (ii) -(amino-(AEEA)b-CH2CO2H)c- , wherein ‘b’ is in the range of 1 to 10, preferably 2 to 10, more preferably 2 to 8, and ‘c’ is in the range of 1 to 10, preferably 1 to 8, more preferably 1 to 5.

[0063] In an embodiment, the spacer is selected from a compound of Formula (j) to (k) depicted in Table 1. Examples of AEEA moieties include, but is not limited to, Aminoethylethanolamine, 17-Amino-10-oxo-3,6,12,15-tetraoxa-9azaheptadecanoic acid, among others.

[0064] The spacer, according to embodiments herein, is covalently attached to the amino acid residue. In an embodiment, the amino acid residue is lysine (K). The spacer is attached to the epsilon amino group of the lysine side chain. Accordingly, in an embodiment, the modified lysine (Km) is lysine (K) attached to a lipid by a linker and spacer at the s-amino group of the lysine side chain. In a preferable embodiment, the modified lysine is lysine (K) attached to a lipid, preferably a diacid of Formula I, by at least one yGlu linker and a spacer of Formula II.

[0065] The term “linker and spacer”, as used herein, refers to the combination of linker and spacer according to embodiments herein. In an embodiment, the linker and spacer is selected from yGlu-PEG, D-yGlu-(PEG)2, (PEG)2- yGlu, yGlu-(PEG)s yGlu-(PEG)s, benzyl-PAla-(PEG)2, (yGlu)2-(PEG)2, (yGlu) 3-(PEG)2, Abu-yGlu-PEG, Abu-(yGlu)2-PEG, or Abu-(PEG)2.

[0066] In an embodiment, the linker and spacer is selected from a compound of Formula (m) to (x) depicted in Table 1. In an embodiment, the linker and spacer is selected from a compound of Formula (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w) and (x) depicted in Table 1.

[0067] In an embodiment, the modified amino acid is modified lysine of Formula III.Formula III

[0068] In another embodiment, the modified amino acid is modified lysine of Formula IV.Formula IV

[0069] In another embodiment, the modified amino acid is modified lysine of Formula V.Formula V

[0070] The modified amino acid, preferably modified lysine, may be present at one or more positions in the peptide. In an embodiment, the modified amino acid may be present at a position selected from a group consisting of 2, 10, 12, 17, 20,21, 28, 30, and 32. In an embodiment, the modified amino acid is modified lysine (Km) present at a position selected from a group consisting of 2, 10, 12, 17, 20, 21, 28, 30, and 32.

[0071] In an embodiment, the modified ammo acid is modified lysine (I ) present at a position selected from a group consisting of 10, 12, and 20. In an embodiment, the peptide comprises modified lysine (Km) at position 12 of the peptide, wherein the modified lysine is lysine (K) attached to a lipid, preferably a diacid, by a linker and a spacer, wherein the linker is selected from one or more yGlu moieties, Gamma-aminobutyric acid (GABA), or combination thereof, and wherein the spacer comprises a moiety selected from Aminoethylethanolamine (AEEA), y-Carboxylate moieties, GGGGS repeats, one or more PEG moieties, combination thereof.Non-natural amino acid

[0072] The peptides, according to embodiments herein, include one or more nonnatural amino acids. The non-natural amino acid helps decrease / avoid enzyme mediated degradation of the peptide, thereby contributing to improve serum half-life of the peptide. Examples of non-natural amino acids include, but are not limited to, Alpha-aminobutyric acid (Aib), a-methylated amino acid, Alpha-MethylLeucine (aMeL), HomoSerine (hSer), Norvaline (nVal), NorLeucine (nLeu), Diaminopimelic acid (DAP), Methionine sulfoxide (MetO), Selenocysteine (Sec), Methylalanine, or derivatives thereof. Accordingly, in an embodiment, the peptide comprises a non-natural amino acid selected from a group consisting of Alpha-aminobutyric acid (Aib), Alpha-MethylLeucine (aMeL), HomoSerine (hSer), Norvaline (nVal), NorLeucine (nLeu), Diaminopimelic acid (DAP), Methionine sulfoxide (MetO), Selenocysteine (Sec), and Methylalanine. In a preferred embodiment, the non-natural amino acid is Aib. In another preferred embodiment, the non-natural amino acid is a-methylated amino acid, preferably Alpha-Methyl Leucine (aMeL). In an embodiment, the non-natural amino acid is selected from Aib, aMeL, or combination thereof. In anembodiment, the peptide comprises a non-natural amino acid at a position selected from 2 or 13. In an embodiment, the peptide comprises a non-natural amino acid at a position selected from 2 or 13, wherein the non-natural amino acid is selected from Aib or Alpha-Methyl Leucine (aMeL). In an embodiment, the peptide comprises a non-natural amino acid at position 2, wherein the non-natural amino acid is Aib. In an embodiment, the peptide comprises a non-natural amino acid at position 13, wherein the non-natural amino acid is aMeL.

[0073] In an embodiment, there is provided an agonist peptide, wherein the peptide has an amino acid sequence selected from SEQ ID NO. 1, wherein X2 is Aib. In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 1, wherein X13 is aMeL. In another embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 2, and SEQ ID NO. 4. In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO.1, wherein X2 is Glycine (G). In another embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 1, wherein X2 is Serine (S). In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO.1, wherein X13 is Tyrosine (Y).Peptide stapling

[0074] The peptides, according to embodiments herein, include a peptide staple. The peptide staple in the peptide, according to embodiments herein, may be positioned to link the amino acids including natural or non-natural amino acids in the peptide. The peptide staple, in an embodiment, is a brace that links the amino acids residues to promote and maintain secondary structure of the peptide. The peptide staple may be positioned at a suitable position in the peptide. In an embodiment, the peptide comprises a stabilized alpha-helix with the non-natural amino acids linked by a peptide staple, preferably hydrocarbon staple. In an embodiment, the peptide staple links non-natural amino acids. In another embodiment, the peptide staple links or is positioned to link non-natural amino acids at positions i, i+4, preferably non-natural amino acids at positions 30 and34; 31 and 35; or 29 and 33. Accordingly, in an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 1, wherein a peptide staple, preferably hydrocarbon staple, is positioned to link non-natural amino acids at positions i, i+4 in the peptide.

[0075] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 1 or 8, wherein a peptide staple, preferably hydrocarbon staple, is positioned to link non-natural amino acids at positions 30 and 34; 31 and 35; or 29 and 33. Accordingly, in an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, wherein a peptide staple, preferably hydrocarbon staple, is positioned to link non-natural amino acids at positions i, i+4 in the peptide.

[0076] The non-natural amino acids for peptide stapling, according to embodiments herein, may be selected from alpha-4-pentenyl alanine or derivatives thereof, Bis-Pentenyl glycine, S-octenyl alanine, R-octenyl alanine, (S)-N-Fmoc-a-(4-pentenyl)alanine, Lactam bridge, Copper catalyzed azide-alkyne cycloaddition (CuAAC), or Triazole-based cyclization.

[0077] In an embodiment, the non-natural amino acids at positions i, i+4, preferably the non-natural amino acids at positions 30 and 34; 31 and 35; or 29 and 33, are selected from alpha-4-pentenyl alanine, Bis-Pentenyl glycine, (S)-N-Fmoc-a-(4-pentenyl)alanine, S-octenyl alanine, and R-octenyl alanine, preferably (S)-N-Fmoc-a-(4-pentenyl)alanine.

[0078] In an embodiment, the non-natural amino acids for peptide stapling in the peptide, according to embodiments herein, is alpha-4-pentenyl alanine or (S)-N-Fmoc-a-(4-pentenyl)alanine, preferably of Formula VI.Formula VI

[0079] Further, the peptide, as disclosed herein, is amidated at the C-terminal end. The peptide having amino acid sequence as set forth in SEQ ID NO. 1 comprises a serine residue at the C-terminal end which is amidated, to preferably eliminate the negative charge and stabilize the peptide. The term “amidation”, as used herein, refers to a modification of the C-terminus of a peptide to include an amine group. Amidation may be performed by methods generally known in the field including: Rink Amide Resin-Based Approach, Pre-Functionalized Resins based approach, or On-Resin Amidation via Carbodiimide Coupling. In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO. 1, wherein the serine residue at position 39 is amidated at the C-terminal end.

[0080] In an embodiment, the peptide has an amino acid sequence as set forth inX1X2QGTFTSDYSX12X13LDERAAQDFVQWLLDX29X30X31SX33X34X35PPPS(SEQ ID NO. 1); orX1X2QGTFTSDYS X12X13LDERAAQDFVQWLLDGG X31SSGX35PPPS(SEQ ID NO. 8);wherein, Xi is selected from Histidine (H) or Tyrosine (Y); X2 is selected from Glycine (G), Serine (S), or a non-natural amino acid; X12 is selected from lysine (K) or modified lysine (Km); X13 is selected from Tyrosine (Y) or a-methylated amino acid; and wherein a peptide staple, preferably hydrocarbon staple, is positioned to link non-natural amino acids at positions i, i+4, or apharmaceutically acceptable salt thereof. In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO. 1, wherein X29, X30, X31, X33, X34 and X35 are, independently, selected from Glycine (G), Serine (S), Proline (P), Alanine (A), or non-natural amino acids.

[0081] In an embodiment, the peptide has an amino acid sequence as set forth inX1X2QGTFTSDYSX12X13LDERAAQDFVQWLLDX29X30X31SX33X34X35PPPS(SEQ ID NO. 1); orX1X2QGTFTSDYS X12X13LDERAAQDFVQWLLDGG X31SSGX35PPPS(SEQ ID NO. 8);wherein, Xi is selected from Histidine (H) or Tyrosine (Y); X2 is Serine (S); X12 is modified lysine (Km); X13 is a-methylated amino acid, preferably a-methylated leucine; and wherein a peptide staple, preferably hydrocarbon staple, is positioned to link non-natural amino acids at positions i, i+4, or a pharmaceutically acceptable salt thereof.

[0082] In another embodiment, there is provided an agonist peptide, wherein the peptide has an amino acid sequence selected from:X1X2QGTFTSDYSX12X13LDERAAQDFVQWLLDX29X30X31SX33X34X35PPPS(SEQ ID NO. 1); orwherein, Xi is selected from Histidine (H) or Tyrosine (Y); X2 is Glycine (G); X12 is modified lysine (Km); X13 is Tyrosine (Y); and wherein a peptide staple, preferably hydrocarbon staple, is positioned to link non-natural amino acids at positions i, i+4, preferably non-natural amino acids at positions 30 and 34; 31 and 35; or 29 and 33; or a pharmaceutically acceptable salt thereof. In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO. 1, wherein X29, X30, X31, X33, X34 and X35 are, independently, selected from Glycine (G), Serine (S), Proline (P), Alanine (A), or non-natural amino acids.

[0083] In an embodiment, there is provided an agonist peptide, wherein the peptide has an amino acid sequence as set forth inX1X2QGTFTSDYS X12X13LDERAAQDFVQWLLDGG X31SSGX35PPPS(SEQ ID NO. 8);wherein, Xi is selected from Histidine (H) or Tyrosine (Y); X2 is Aib; X12 is modified lysine (Km); X13 is Tyrosine (Y); and wherein a peptide staple, preferably hydrocarbon staple, is positioned to link non-natural amino acids at positions 31 and 35, or a pharmaceutically acceptable salt thereof.

[0084] In an embodiment, there is provided an agonist peptide, wherein the peptide has an amino acid sequence selected from the group consisting of:HAibQGTFTSD YSKmYLDERAAQDF VQWLLDGG X31SSG X35PPPS (SEQ ID NO. 2);HGQGTFTSD YSKmYLDERAAQDF VQWLLDGG X31 S SGX35PPPS(SEQ ID NO. 3);HSQGTFTSDYSKmaMeLLDERAAQDFVQWLLDGGX3iSSG X35PPPS(SEQ ID NO. 4);HAibQGTFTSD YSKmYLDERAAQDF VQWLLDGGX31 S SGX35PPPS-NH2(SEQ ID NO. 5);HGQGTFTSD YSKmYLDERAAQDF VQWLLDGGX31 S SG X35PPPS-NH2 (SEQ ID NO. 6); andHSQGTFTSDYSKmaMeLLDERAAQDFVQWLLDGGX3iSSG X35PPPS-NH2(SEQ ID NO. 7),whereinX31 is selected from alpha-4-pentenyl alanine or derivatives thereof, Bis-Pentenyl glycine, S-octenyl alanine, R-octenyl alanine, or (S)-N-Fmoc-a-(4-pentenyl)alanine, preferably (S)-N-Fmoc-a-(4-pentenyl)alanine,X35 is selected from alpha-4-pentenyl alanine or derivatives thereof, Bis-Pentenyl glycine, S-octenyl alanine, R-octenyl alanine, or (S)-N-Fmoc-a-(4-pentenyl)alanine, preferably (S)-N-Fmoc-a-(4-pentenyl)alanine, andKmis modified lysine.

[0085] In an embodiment, there is provided an agonist peptide, wherein the peptide has an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8.

[0086] In an embodiment, there is provided an agonist peptide, wherein the peptide has an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.

[0087] In an embodiment, there is provided an agonist peptide, wherein the peptide has an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, wherein the serine residue at position 39 is amidated at the C-terminal end.

[0088] In an embodiment, there is provided an agonist peptide, wherein the peptide has an amino acid sequence selected from SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, wherein the serine residue at position 39 is amidated at the C- terminal end.

[0089] Table 1

[0090] Various embodiments of the peptide are disclosed herein. In an embodiment, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO. 2; SEQ ID NO. 3; SEQ ID NO.4; SEQ ID NO. 5, SEQ ID NO. 6; and SEQ ID NO. 7.

[0091] Table 2 depicts the amino acid sequences of peptides in accordance with the embodiments herein.Compositions

[0092] Embodiments herein provide compositions comprising the agonist peptides as disclosed herein. In an embodiment, the composition comprises at least one agonist peptide; and at least one pharmaceutically acceptable excipient. The excipient may be a carrier, diluent, buffering agent, a tonicity modifier, an antimicrobial agent, a surfactant, and / or salt. The excipient may vary and depends on the dosage form, mode of delivery, intended therapeutic application, etc. In some embodiments, the buffering agent is selected from disodium hydrogen phosphate dihydrate, di sodium hydrogen phosphate heptahydrate, sodium phosphate monobasic, potassium phosphate dibasic, sodium acetate, citric acid, sodium citrate, L-histidine, histidine hydrochloride, sodium succinate, sodium lactate, tris(hydroxymethyl)aminomethane, or combinations thereof. The tonicity modifier is selected from the group consisting of sodium chloride, glycerol, sucrose, mannitol, trehalose, propylene glycol, or combinations thereof. The antimicrobial preservative is selected from m-cresol, phenol, benzyl alcohol, or combinations thereof. The surfactant is selected from polysorbate 20, polysorbate 80, pol oxamer 188, pol oxamer 407, or combinations thereof.

[0093] In an embodiment, the composition may comprise 0.5 % w / w to 5% w / w of the agonist peptides as disclosed herein.

[0094] In an embodiment, the composition comprises 0.05% w / w to 5% w / w of tonicity modifier with respect to the total weight of the composition, wherein the tonicity modifier comprises 0.05% w / w to 0.9% or 0.3% w / w to 0.9% w / w of ionic tonicity modifier, and / or 0.05% w / w to 0.9% or 0.3% w / w to 2.5% w / w of nonionic tonicity modifier.

[0095] In an embodiment, the buffering agent is in a concentration range of 0.01 % w / w % w / w to 2% w / w or 0.01 % w / w to 1% w / w, in respect of the total composition.

[0096] In an embodiment, the antimicrobial preservative is in a concentration range of 0.15% w / w to 0.35% w / w, in respect of the total composition.

[0097] In an embodiment, the surfactant is in a concentration range of 0.0001% w / w to 0.2 % w / w, in respect of the total composition.

[0098] Further examples of excipients include, but are not limited to, phosphate buffer saline (PBS), sodium or potassium salts, sugars, cyclodextrins, nanoparticles, and / or polymers (eg: PEG). Various other excipients are known to a person skilled in the art and may be used in various embodiments herein.

[0099] The composition may further be formulated in various dosage forms such as parenteral including subcutaneous, intravenous, intraperitoneal, or intramuscular; oral dosage forms including tablets, capsules, powders, or suspensions; transdermal; sprays, etc.Methods

[0100] The peptides, according to embodiment herein may be used for therapeutic applications. Accordingly, embodiments herein provide a method for stimulating incretin hormone receptors including glucagon -like peptide- 1 receptor (GLP-1R), or modulating gastric inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR. In an embodiment, the method for stimulating incretin hormone receptors including glucagon-like peptide-1 receptor (GLP-1R), gastric1inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR comprises providing an effective amount of the peptide as disclosed herein.

[0101] Further embodiment herein provides a method for treating a metabolic disorder. Embodiments herein further include a method for treatment and management of blood glucose levels, and reduce adiposity. Embodiments herein further include a method for treatment and management of comorbidities of diabetes including cardiovascular disease, kidney disease, retinopathy, renal diseases, non-alcoholic fatty liver, neuropathy, and mental health issues.

[0102] In an embodiment, the method for treatment and / or management of obesity in a subject, comprising administering an effective amount of the peptide as disclosed herein. In an embodiment, the method for treatment and / or management of Type 2 Diabetes in a subject, comprising administering an effective amount of the peptide as disclosed herein. The peptide as disclosed herein may be administered in combination with a therapeutic agent. Accordingly, in an embodiment, the method comprises administering a therapeutic agent selected from anti-diabetic agents, sodium-Glucose Transport Protein (SGLT) inhibitors, amylin analogues, dipeptidyl peptidase IV (DPP-4) inhibitors, thiazolidinediones (TZD’s), anti-obesity drugs, bile acid sequestrants, leptin analogues, or combination thereof. Non-limiting examples of anti-diabetic agent included metformin; SGLT inhibitors includes empagliflozin, dapagliflozin, and canagliflozin; amylin analogues include cagrilintide, and pramlintide; DPP-4 inhibitors include sitagliptin, and saxagliptin; thiazolidinediones (TZD’s) include Pioglitazone. Further, Non-limiting examples of anti-obesity drugs include; phentermine-topiramate, and naltrexone-bupropion; bile acid sequestrants include Colesevelam; and Leptin analogues include metreleptin.

[0103] Embodiments herein may also be used to management of Type 2 Diabetes and / or obesity including management of symptoms and / or comorbidities, thereof.

[0104] The term “effective amount” or “therapeutically effective amount”, used interchangeably herein, is an amount that is capable of exhibiting the desired effect in a subject. The desired effect may be stimulatory or modulatory or agonist effect on at least one incretin hormone receptors including glucagon-like peptide-1 receptor (GLP-1R), gastric inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR; or therapeutic effect. The effective amount may vary and depend on various factors including age of subject, severity of the diabetes / obesity, mode of delivery, etc.

[0105] The term “subject”, as used herein, refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include non-human primates, dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, mice, rats, hamsters, guinea pigs, and etc. The terms “patient” or “subject” are used herein interchangeably. In an embodiment, the subject is human. In an embodiment, the subject is a patient having or suspected of a metabolic disorder selected from having diabetes including Type-2 Diabetes, comorbidities of diabetes (including cardiovascular disease, kidney disease, retinopathy, renal diseases, non-alcoholic fatty liver, neuropathy, and mental health issues); and obesity; or symptoms thereof.

[0106] The agonist peptide or peptide, according to embodiments herein, may be prepared by synthesizing the amino acid chain by methods generally known in the field. Exemplary methods of peptide synthesis include, but is not limited to, solid phase synthesis, vapor-phase synthesis, and liquid-phase synthesis. The amino acid chain is amidated to replace the C-terminal carboxyl (-COOH) group with an amide (-CONEE). Exemplary methods that may be used for amidation of the amino acid chain include Rink Amide Resin-Based Approach; Pre-Functionalized Resins; and On-Resin Amidation via Carbodiimide Coupling. The selection of amidation method may vary and depend on Peptide sequence complexity, Desired purity and yield, and Synthetic feasibility and reagent availability.

[0107] In an example, the peptide assembly may be performed using Fmoc-SPPS on Rink Amide Resin. Standard deprotection, coupling cycles, and capping may be performed until full sequence elongation is achieved. Further, cleavage may be carried out using trifluoroacetic acid (TFA) to remove the peptide from the resin while leaving the C-terminal amidation intact. The amidated peptide may then be purified using HPLC, followed by lyophilization

[0108] In another example, a pre-functionalized resin based on the desired peptide properties maybe selected. Fmoc-based SPPS was performed, ensuring standard deprotection, coupling cycles, and capping steps. Upon peptide completion, TFA cleavage may be used to release the peptide with a C-terminal amide (-CONH2). The amidated peptide may then be purified using HPLC, followed by lyophilization

[0109] In yet another embodiment, the peptide may be assembled using Wang Resin or another acid-labile resin. The C-terminal carboxyl (-COOH) may be activated, before cleavage, using carbodiimide coupling agents, such as: HBTU / HOBt(O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), DIC / DMAP (Diisopropylcarbodiimide / 4- Dimethylaminopyridine). The amide donor (e.g., NFb in MeOH or DMF) may then be introduced to convert -COOH to -CONH2. Upon peptide completion, TFA cleavage may be used to release the peptide with a C-terminal amide (-CONH2). The amidated peptide may then be purified using HPLC, followed by lyophilization.

[0110] The amidated amino acid chain may then be acylated by lipidation of the lysine residue. The s-amino group (-NH2) in lysine provides a site for selective acylation without modifying the peptide backbone. In an example, regents such as fatty acid chlorides or NHS esters (e.g., palmitoyl chloride, stearic acid-NHS), diisopropylcarbodiimide (DIC) or l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-Hydroxysuccinimide (NHS) activation, or Base catalyst DIPEA (N,N-Diisopropylethylamine) and TEA(Triethylamine); and spacers and / or linker as described herein may be used in acylation of the amino acid chain.

[0111] In an embodiment, there is provided a method for preparation of the peptide, said method comprising: (a) synthesizing the peptide by solid-phase peptide synthesis and optionally including at least one non-natural amino acid; (b) optionally incorporating at least one modified amino acid residue; and (c) cleaving and recovering the peptide to obtain the peptide of the present disclosure.

[0112] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.EXAMPLES

[0113] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.Example 1 : Peptide synthesis

[0114] Peptide construction: The peptide sequences of the present disclosure were generated by solid-phase peptide synthesis using a Fmoc / tBustrategy on a PurePep™ Chorus or Prelude X peptide synthesizer (Gyros Protein Technologies). The synthesis was started from Tentagel Rink Amide resin to accommodate amidation at the C-terminus. During each standard coupling cycle, 4.8 equivalents of amino acid were activated using 2-cyano-2 (hydroxyimino)acetate (OxymaPure®) and diisopropylcarbodiimide (DIC) and the reaction mixture was agitated at 80 °C for 5 min. In the case of unnatural amino acids, an extended coupling time of 15 min was used. Capping was performed using acetic anhydride (0.5 M in DMF) to avoid the formation of impurities due to amino acid deletions. Fmoc-deprotection was conducted using piperidine (20% v / v in DMF). To allow for site-specific incorporation of the fatty acid moiety at a later stage in the synthetic process, Fmoc-Lys(ivDde)-OH (CAS 204777-78-6) was incorporated at the required K residues of the backbone sequence.

[0115] Orthogonal ivDde-deprotection of the appropriate lysine residue was conducted by agitation of the resin-bound peptide in a solution of hydrazine in DMF (5% v / v). Stepwise assembly of the side chain was then performed at this position by solid phase peptide synthesis on the PurePep™ Chorus or Prelude X synthesizer using the standard OxymaPure® / DIC extended coupling chemistry described above.

[0116] Cleavage: The peptides were suspended in cleave cocktail (TFA-H2O-TIS-EDT; 90.5:5.0:2.5:2.0) and stirred at room temperature for 4 h. The peptides were precipitated into ice-cold Et2O and centrifuged. The pellet was then washed with Et2O to give the crude peptide as a white solid.

[0117] Purification: The crude peptides were purified to >98% purity by reverse-phase HPLC using a Luna Cl 8 column (Phenom enex) and a water / acetonitrile gradient (containing 0.1% v / v TFA). Fractions containing the product that met purity were pooled and lyophilised. Peptide purity was examined by analytical reverse-phase HPLC (Luna C18 column; 2-90% B over 34 min;Buffer A: Water + 0.1% TFA; Buffer B: MeCN + 0.1% TFA; 214 nm) and peptide identity was confirmed by ESI-MS.

[0118] Figure 1 depicts a schematic representation of the amino acid chains of the peptides ND-MC-46 (SEQ ID NO. 5), ND-MC-47 (SEQ ID NO. 6), and ND-MC-48 (SEQ ID NO. 7) along with their respective modifications including amidation, acylation, and non-natural amino acid substitutions, in accordance with the embodiments herein.Example 2 : In vitro studies

[0119] GLP-1R activation was assessed in-vitro for ND-MC-46 (SEQ ID NO. 5), ND-MC-47 (SEQ ID NO. 6), and ND-MC-48 (SEQ ID NO. 7) by performing cAMP assay carried out on GLP-1R positive CHO cell lines. Receptor activation efficacy of the test peptides against the glucagon -like peptide- 1 receptor (GLP-1R) was assessed in vitro using the cAMP-Glo™ Max Assay (Promega, Cat. No. VI 681) in Chinese Hamster Ovary (CHO) cells stably expressing human GLP-1R (generated by in-house stable transduction). Cells were seeded at a density of 5,000 cells per well in white, opaque 96-well plates and allowed to adhere overnight. Prior to the assay, test peptides and Retatrutide (positive control) were serially diluted in assay buffer to achieve a final concentration range of 2* 101M to 3.2* 105M. Cells were treated with the respective peptide concentrations and incubated for 1 hour at 37°C. Following incubation, cAMP accumulation was measured according to the manufacturer's protocol. Luminescence was detected using a PerkinElmer VICTOR Nivo multiplate reader. Concentration-response data were analysed and ECso values were determined by nonlinear regression using a three-parameter logistic curve fit in GraphPad Prism. Data represent three independent biological replicates, each performed in triplicate technical wells. Results are expressed as relative luminescence units (RLU) plotted against the log of peptide concentration (M).

[0120] Observations: Figure 2 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R. Plots shown depict theactivation of GLP-1R in presence of peptides represented by the SEQ ID NO. 5 - SEQ ID NO. 7 along with the positive control peptide Retatrutide. All three peptides demonstrated good receptor activation efficacy against GLP-1R even at lower molar concentrations as compared to the control. This cAMP assay was carried out on GLP-1R positive CHO cell lines.

[0121] In addition, SEQ ID NO. 7 (ND-MC-48) was tested in the cAMP assay for further validation against all three receptors GLP-1R, GIPR, and GCGR. As shown in Table 3, overall SEQ ID NO. 7 demonstrated similar or better binding affinity as well as potency against GLP1R compared to the positive control, with similar affinity for the other two receptors GIPR and GCGR.Table 3: Results of GLP-1R, GIPR, and GCGR activation by SEQ ID NO. 7 (ND-MC-48) as measured by cAMP AssayExample 3: Preliminary Pharmacology study on body weight and glucose levels in diet-induced obese (DIO) mice:

[0122] To evaluate the efficacy of the GLP-l / GCGR / GIPR agonist ND- MC-48 (SEQ ID NO. 7) on weight loss and metabolic dysfunction, diet-induced obese (DIO) mice were treated with 0.5 mg / kg subcutaneously twice weekly with ND-MC-48 (SEQ ID NO. 7) dissolved in 20 mM Tris HC1 (pH 8.0) or vehicle control, 20mM Tris HC1 (pH 8.0) for four weeks. The study compared treatment effects against the vehicle control through body weight monitoring, metabolic tolerance tests (OGTT and ITT).

[0123] Observations: Change in body weight: Figure 3 shows the change in body weight in DIO treated mice. At Day 0, both the control (Vehicle Control) and treated (ND-MC-ZB-48) groups started with comparable baseline body weights. The ND-MC-ZB-48-treated group showed a rapid and pronounced reduction in body weight early in the treatment period, with a substantial decrease evident by approximately Day 12. The weight reduction stabilized between Day 15 and Day 22, forming a plateau that suggests a steady-state effect during this period. Toward the end of the study (Day 26), a slight increase in body weight was observed in the treatment group, indicating minor late-stage recovery or physiological adaptation. In contrast, the vehicle control group remained relatively stable initially and gradually increased in body weight over time, while treated animals consistently maintained lower body weight throughout the study period.

[0124] Effect on Glucose Tolerance (OGTT): As shown in Figure 4, Oral Glucose Tolerance Test (OGTT) performed at day 28 showed that ND-MC-ZB-48 (SEQ ID NO. 7) treatment significantly attenuated glucose excursions following an oral glucose challenge. The treated group exhibited a markedly lower peak and a faster return to baseline. The overall glucose exposure (AUC) was reduced significantly indicating a profound improvement in glucose tolerance.

[0125] Effect on Glucose Response During Insulin Tolerance Test (ITT):Figure 5 demonstrates the effect of treatment with ND-MC-ZB-48 (SEQ ID NO: 7) on the Insulin Tolerance Test (ITT) taken at day 28 after treatment. As shown in Figure 5, treatment with the peptide significantly enhanced insulin sensitivity. Following insulin administration, the treated group showed a much more pronounced and sustained reduction in blood glucose compared to the vehicle control, which remained relatively resistant to the insulin challenge.Example 4: Preliminary pharmacological studies on metabolic markers in diet-induced obese (DIO) mice:

[0126] To evaluate the efficacy of the GLP-l / GCGR / GIPR agonist ND-MC-ZB-48 (SEQ ID NO. 7) (dissolved in 20 mM Tris HC1 (pH 8.0) on majorbiomarkers of metabolism, DIO mice were treated with 0.5 mg / kg of the peptide subcutaneously administered twice weekly for four weeks. The study compared treatment effects against the vehicle control, dissolved in 20 mM Tris HC1 (pH 8.0), through serum biochemistry, and histopathological analysis taken at day 28 after the treatment.

[0127] Observations: Effect on Clinical Metabolic Parameters: Figure 6 demonstrates the effect of treatment with ND-MC-ZB-48 (SEQ ID NO: 7) on major metabolic parameters including glucose levels, total cholesterol, triglycerides, HDL (high-density lipoprotein) and LDL (low-density lipoprotein) levels taken at day 28 after treatment. As shown in Figure 6, twice weekly treatment of DIO mice with ND-MC-ZB-48 (SEQ ID NO: 7)( 0.5 mg / kg body weight) was associated with lower blood glucose levels compared with vehicle controls, suggesting improved glucose homeostasis. Total cholesterol, triglycerides, and LDL concentrations were reduced in treated animals, indicating potential improvements in systemic lipid metabolism and decreased atherogenic lipid fractions. HDL levels appeared slightly lower in the treatment group, although substantial overlap in individual measurements between groups suggests no consistent treatment-related change in this parameter.

[0128] Effect on Liver Enzymes and Renal Markers: Serum biochemical analysis was also conducted, the results of which are shown in Table 4. The serum biochemical analysis of ND-MC-ZB-48 demonstrated a stable safety profile with renal and hepatic parameters remaining largely comparable to vehicle controls. Renal function was maintained, as evidenced by creatinine levels that remained virtually identical between groups, though a minor numerical increase in urea was noted in the treated animals. Hepatic synthetic function and nutritional status remained robust, with total protein and albumin levels showing only negligible variations. Regarding liver enzymes, AST values were comparable across both groups, while ALT levels showed a moderate increase in the treated group. Overall, the absence of consistent, high-magnitude shifts in these markers suggestthat ND-MC-ZB-48 is well-tolerated at the tested dose without inducing significant organ toxicity.Table 4: Summary of Liver and Renal function test markers at day 28 after twice weekly 0.5 mg / kg body weight ND-MC-ZB-48 treatmentExample 5: Preliminary pharmacological studies on Serum Leptin, Adiponectin and Glucagon Levels in diet-induced obese (DIO) mice:

[0129] To evaluate the efficacy of the GLP-l / GCGR / GIPR agonist ND-MC-48 (SEQ ID NO. 7) on serum leptin, adiponectin and glucagon levels, DIO mice were treated with 0.5 mg / kg body weight of the peptide dissolved in 20 mM Tris HC1 (pH 8.0) and administered subcutaneously twice weekly for four weeks. The study compared treatment effects against the vehicle control 20 mM Tris HC1 (pH 8.0) through serum biochemistry, and histopathological analysis taken at day 28 after the treatment.

[0130] Observations: As shown in Figure 7, treatment with ND-MC-ZB-48 (SEQ ID NO:7) (0.5 mg / kg) resulted in a marked reduction in leptin levels, decreasing from approximately -105-115 ng / mL in the vehicle control group to -20-30 ng / mL, with the decrease reported as statistically significant (p< 0.001).Example 6: Preliminary pharmacological studies on organ weight of diet-induced obese (DIO) mice:

[0131] To evaluate the efficacy of the GLP-l / GCGR / GIPR agonist ND-MC-48 (SEQ ID NO. 7) on organ weight (retroperitoneal fat) and hepatic steatosis, DIO mice were treated with 0.5 mg / kg body weight of the peptide dissolved in 20 mM Tris HC1 (pH 8.0) and administered subcutaneously twice weekly for four weeks. The study compared treatment effects against the vehicle control 20 mM Tris HC1 (pH 8.0) and the results were taken at day 28 after the treatment.

[0132] Observations: Treatment with ND-MC-ZB-48 (SEQ ID NO: 7)(0.5 mg / kg) resulted in a marked reduction in retroperitoneal adipose tissue mass (Figure 8) compared with vehicle controls. The retroperitoneal fat mass decreased from -1.25 g to -0.25 g, representing an approximately 80% reduction in adipose tissue mass.

[0133] As shown in Figure 9, the mean hepatic steatosis score decreased from -2.8 in controls to -1.5 in treated animals, representing an approximately 46% reduction in liver fat accumulation. Individual scores shifted from predominantly moderate-to-severe steatosis in the control group to mild steatosis in the treatment group, with a high level of statistical significance. Overall, the findings indicate that ND-MC-ZB-48 markedly improves hepatic lipid accumulation and visceral fat deposition, and may contribute to improved metabolic liver health under the study conditions.Example 7: Surface Plasmon binding studies

[0134] Binding kinetics of test peptides to human GLP-1R, GIPR, and GCGR were determined by Surface Plasmon Resonance (SPR) using a BiacoreT200 instrument (Cytiva). Each receptor was covalently immobilised onto a CM5 sensor chip via standard amine coupling chemistry (EDC / NHS activation) in HBS-EP+ running buffer. A reference flow cell treated with EDC / NHS but without receptor was used for background subtraction. Test peptides and the reference compound LY were serially diluted in HBS-EP+ and injected over the receptor-immobilised flow cells at multiple concentrations at a flow rate of 30 pl / min during an association phase (~60 s) followed by a dissociation phase (~60 s). Referring to Figures 10 to 21, all the sensorgrams are plotted as baseline-subtracted binding response (Resonance Units, RU) versus time (seconds). Multi -concentration sensorgrams were globally fitted to a 1:1 Langmuir binding model to derive the association rate constant (ka, 1 / Ms), dissociation rate constant (kd, 1 / s), and equilibrium dissociation constant (KD, M).

[0135] Observations: The binding kinetics studies depicted in Figure 10-21 characterize the similar or better binding affinity for the peptides under evaluation with respect to the positive controls against all three receptor classes included GLP1R, GIPR and GCGR. These observations could be inferenced based on the association and dissociation profiles of these peptides relative to the positive control.Advantages of the present disclosureThe present disclosure provides agonist peptides against incretin hormone receptors, including glucagon-like peptide-1 receptor (GLP-1R), gastric inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR. These peptides demonstrate good serum half-life, stability, and binding kinetics. The peptides have been designed with specific modifications that enable stabilized alpha-helix structures and contribute to the overall stability of the peptides. The peptides are also effective in vivo in stabilizing glucose levels and enhancing insulin sensitivity and ameliorating the effects of metabolic disorders such as lowering cholesterol levels and adipose tissue accumulation, among others. Also disclosed are methods for use and preparation of the peptides disclosed herein.

Claims

/ We Claim1. An agonist peptide comprising a peptide having an amino acid sequence of at least 90% identity to an amino acid sequence as set forth in SEQ ID NO. 1 : X1X2QGTFTSDYS X12X13LDERAAQDFVQWLLDX29X30X31SX33X34X35PPPS (SEQ ID NO. 1),whereinXi is selected from Histidine (H) or Tyrosine (Y);X2 is selected from Glycine (G), Serine (S), or a non-natural amino acid; X12 is selected from lysine (K) or modified lysine (Km);X13 is selected from Tyrosine (Y) or a-methylated amino acid; and wherein a peptide staple, preferably hydrocarbon staple, is positioned to link non-natural amino acids at positions i, i+4, preferably non-natural amino acids at positions 30 and 34; 31 and 35; or 29 and 33;or a pharmaceutically acceptable salt thereof.

2. The agonist peptide as claimed in claim 1, wherein X29, X30, X31, X33, X34 and X35 are, independently, selected from Glycine (G), Serine (S), Proline (P), Alanine (A), or non-natural amino acids.

3. The agonist peptide as claimed in claim 1 or claim 2, wherein the non-natural amino acids are at positions 29, 30, 31, 33, 34, and 35 and are selected from Alpha-aminobutyric acid (Aib), Alpha-Methyl Leucine (crMeL), HomoSerine (hSer), Norvaline (nVal), NorLeucine (nLeu), Diaminopimelic acid (DAP), alpha-4-pentenyl alanine, Bis-Pentenyl glycine, S-octenyl alanine, R-octenyl alanine, (S)-N-Fmoc-a-(4-pentenyl)alanine, Methionine sulfoxide (MetO), Selenocysteine (Sec), Methylalanine, or acylated forms thereof.

4. The agonist peptide as claimed in claim 3, wherein the non-natural amino acids are at positions 31 and 35 and are selected from alpha-4-pentenylalanine, Bis-Pentenyl glycine, (S)-N-Fmoc-a-(4-pentenyl)alanine, S-octenyl alanine, and R-octenyl alanine, preferably (S)-N-Fmoc-a-(4-pentenyl)alanine.

5. The agonist peptide as claimed in claim 1, wherein the peptide comprises modified lysine (Km) at a position 12, and wherein the modified lysine is lysine (K) attached to a lipid, preferably a diacid, by a linker and / or a spacer, wherein the linker is selected from one or more yGlu moieties, Gamma- aminobutyric acid (GABA), or combination thereof, andwherein the spacer comprises a moiety selected from Aminoethylethanolamine (AEEA), GGGGS repeats, y-Carboxylate moieties, one or more polyethylene glycol (PEG) moieties, or combination thereof.

6. The agonist peptide as claimed in claim 5, wherein the modified lysine is lysine (K) attached to a diacid of Formula I by at least one yGlu linker and a spacer of Formula IICO2H-(CH2)a-CO- Formula I-(amino-(PEG / AEEA)b-CH2CO2H)c- Formula II wherein ‘a’ is in the range of 8 to 22, preferably 12 to 20, ‘b’ is in the range of 1 to 10, and ‘c’ is in the range of 1 to 10.

7. The agonist peptide as claimed in claim 6, wherein the spacer is a compound of Formula II and is selected from(i) -(amino-(PEG)b-CH2CO2H)c- ; or (ii) -(amino-(AEEA)b-CH2CO2H)c- ,wherein ‘b’ is in the range of 1 to 10, and ‘c’ is in the range of 1 to 10.

8. The agonist peptide as claimed in claim 5, wherein the modified lysine is of Formula IIIFormula III.

9. The agonist peptide as claimed in claim 1, having an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3, and (SEQ ID NO. 4.

10. The agonist peptide as claimed in claim 1, wherein the serine residue at position 39 is amidated at the C-terminal end.

11. The agonist peptide as claimed in claim 1, wherein the peptide is having an amino acid sequence selected from the group consisting of: SEQ ID NO. 2; SEQ ID NO. 3; SEQ ID NO. 4; SEQ ID NO. 5; SEQ ID NO. 6; SEQ ID NO.7, and SEQ ID NO.8.

12. The agonist peptide as claimed in claim 1, wherein the peptide exhibits agonist activity on glucagon-like peptide- 1 receptor (GLP1R), gastric inhibitory polypeptide receptor GIPR, and / or glucagon receptor GCGR.

13. A composition comprising the at least one agonist peptide as claimed in claim 1, and at least one pharmaceutically acceptable excipient.

14. A method for modulating glucagon-like peptide-1 receptor (GLP1R), gastric inhibitory polypeptide receptor GIPR, and / or glucagon receptor GCGR, comprising providing an effective amount of the agonist peptide as claimed in claim 1, or the composition as claimed in claim 13.

15. A method for treatment or management of obesity in a subject, comprising providing an effective amount of the agonist peptide as claimed in claim 1 to said subject.

16. The method as claimed in claim 15, further comprising administering a therapeutic agent selected from anti-obesity drugs, bile acid sequestrants, leptin analogues, or combination thereof, to said subject.

17. A method for treatment or management of Type 2 Diabetes in a subject, comprising providing an effective amount of the agonist peptide as claimed in claim 1 to said subject.

18. The method as claimed in claim 17, further comprising administering a therapeutic agent selected from anti-diabetic agents, sodium-Glucose Transport Protein (SGLT) inhibitors, amylin analogues, dipeptidyl peptidase IV (DPP-4) inhibitors, thiazolidinediones (TZD’s), or combination thereof, to said subject.

19. A method for preparation of the agonist peptide as claimed in claim 1, said method comprising:(a) synthesizing the peptide by solid-phase peptide synthesis and optionally including at least one non-natural amino acid;(b) optionally incorporating at least one modified amino acid residue;and(c) cleaving and recovering the agonist peptide to obtain the peptide as claimed in claim 1.

Images