WO2023159052A2 - Stapled peptides, methods of making same, and uses thereof - Google Patents

Abstract

Stapled peptides, methods of making same, and uses thereof. In various examples, a stapled peptide comprises two staples (which may be referred to as crosslinks). In various examples, a composition comprises one or more stapled peptide(s) and optionally, one or more permeation enhancer(s). In various examples, a method of making a stapled peptide comprises reacting a peptide comprising two cysteines at selected positions and two protected cysteines at selected positions with a crosslinking agent, deprotecting singly- crosslinked peptide and reacting the deprotected singly-crosslinked peptide with a crosslinking agent to form a stapled peptide comprising two staples. In various examples, a stapled peptide or stapled peptides are used in methods of lowering the glucose in an individual and/or weight loss methods.

Description

STAPLED PEPTIDES, METHODS OF MAKING SAME, AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/310,430, filed February 15, 2022, the contents of the above-identified application are hereby fully incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] This application contains a sequence listing filed in electronic form as an xml file entitled TTL-0100WP_ST26.xml, created on February 14, 2023, and having size of 134,535 bytes. The content of the sequence listing is incorporated herein in its entirety.

BACKGROUND OF THE DISCLOSURE

[0003] Glucagon-like peptide-1 receptor (GLP-1R) agonists have been successfully developed for the treatment of type 2 diabetes mellitus (T2DM) due to its ability to lower blood glucose level and reduce body weight. Several GLP-1R agonist drugs including exenatide, dulaglutide, liraglutide, and semaglutide have been used in the clinic for the treatment of T2DM. Recently, several preclinical studies and clinical trial results showed that peptides that effectively activate both GLP-1R and glucose-dependent insulinotropic peptide receptor (GIPR) provide superior efficacy in lowering blood glucose level and reducing body weight than peptides that activate GLP-1R alone. However, these dual agonist peptides are not suitable for oral administration mainly due to their poor resistance to digestive enzymes and poor absorption in the gastrointestinal tract. Although oral delivery of semaglutide was achieved by formulation with salcaprozate sodium (SNAC), studies have shown that this formulation approach may not offer the same delivery efficiency for other peptide analogs. There remains an unmet need of designing GLP-1R/GIPR agonist peptides that are potent and intrinsically stable to the digestive enzymes.

SUMMARY OF THE DISCLOSURE

[0004] The present disclosure provides, inter alia, a method for enhancing proteolytic stability of peptide dual agonists based on double biaryl stapling, and use of this method to generate potent and proteolytically stable GLP-1R/GIPR agonists suitable for oral delivery. [0005] In an aspect, the present disclosure provides compounds. The compounds are stapled peptides. Compounds are also referred to herein as peptides or stapled peptides. In various examples, a compound comprises two staples. In various examples, a compound is made by a method of the present disclosure.

[0006] In various examples, a compound (e.g., a stapled peptide) comprising the following structure: Y-X1-E-G-T-X2-T-S-D-Y-S-I-X3-L-D-K-X4-A-X5-X6-X7-X8-V-X9- W-L-X10-A-G (SEQ ID NO: 1), where XI is Aib; X2 is Phe or a-MePhe; X3 is Tyr or Aib; X4 is Gin or He; X5 is Ala or Gin; X6 is Aib or Lys; X7 is Glu or Ala; X8 is Phe or a- MePhe; X9 is Asn or Gin; XI 0 is Leu or He; and two amino acids at position i and i+7 are replaced with cysteines and crosslinked with a crosslinking group, and another two amino acids at position j and j+7 are also replaced with cysteines and crosslinked with a crosslinking group. In various examples, the staples of any of these peptides are independently chosen at each occurrence from bismethylene aryl (BMA) crosslinking groups. The crosslinking group rings can independently be substituted or unsubstituted (e.g., the aryl and/or heteroaryl ring(s) of the crosslinking group are independently substituted or unsubstituted). A stapled peptide may be PEGylated. In various examples, one or more stapled peptide(s) activate GLP-1R and/or GIPR (e.g., in an individual).

[0007] In an aspect, the present disclosure provides compositions comprising one or more compound(s) (stapled peptide(s)) of the present disclosure. A composition may also comprise one or more additional component(s), one or more or all of which may be pharmaceutically acceptable components or the like. In various examples, a composition is suitable for oral administration.

[0008] In an aspect, the present disclosure provides methods of making compounds (stapled peptides) of the present disclosure. In various examples, a method of the present disclosure provides a compound (peptide) comprising two staples (which may be referred to crosslinks). In various examples, a method of the present disclosure provides a compound (peptide) of the present disclosure. It is considered that any peptide, with appropriately placed cysteines (naturally occurring, introduced, etc.) can be stapled. Various crosslinking agents can be used. Combinations of crosslinking agents may be used.

[0009] In an aspect, the present disclosure provides uses of compounds (stapled peptides) of the present disclosure. In various examples, the present disclosure provides methods of treating diabetes and/or lowering the blood glucose level of an individual and/or inducing weight loss in an individual. In various examples, a method of treating diabetes, such as, for example, type 2 diabetes mellitus or the like, comprises administration to an individual an effective amount of one or more stapled peptide(s). In various examples, a method of lowering the blood glucose levels of an individual comprises administration to the individual an effective amount of one of or more stapled peptide(s). In various examples, a method of inducing weight loss in an individual comprises administration to the individual an effective amount of one of or more stapled peptide(s). In various examples, a method of treating diabetes, such as, for example, type 2 diabetes mellitus or the like, and/or lowering the blood glucose levels of an individual and/or inducing weight loss in an individual comprises administration to the individual an effective amount of one of or more compound(s) (stapled peptide(s)).

BRIEF DESCRIPTION OF THE FIGURES

[0010] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0011] Figure 1 shows dose-response curves of selected doubly stapled analogs for GLP- 1 receptor and GIP receptor activation.

[0012] Figure 2 shows stability of selected double-stapled peptide analogs in 1% simulated gastric fluid (SGF; 100-fold dilution).

[0013] Figure 3 shows stability of selected double-stapled peptide analogs in 100% SGF.

[0014] Figure 4 shows stability of selected doubly stapled peptides and their half-lives

(Z1/2) in the presence of 1% simulated intestine fluid (SIF; 100-fold dilution).

[0015] Figure 5 shows stability of (A) TT201 and (B) TT205 and half-lives (Z1/2) thereof in the presence of diluted simulated intestine fluid (SIF).

[0016] Figure 6 shows stability of desmopressin and selected double-stapled peptide analogs in 100% SIF.

[0017] Figure 7 shows stability of semaglutide and selected double-stapled peptide analogs in mouse serum.

[0018] Figure 8 shows oral glucose tolerance test and exposure PK of selected doublestapled analogs. C57BL/6 male (age 10 weeks; n = 4) were injected with the peptide at a dosage of 0.4 mpk subcutaneously 1 hour before the glucose challenge. (A) Subcutaneous Oral Glucose Tolerance Test (S.C. OGTT). Mouse blood glucose concentrations measured by glucometer at 0, 15, 30, 60, and 90 minutes. (B) Bar graph showing total glucose amounts in the treated mice by measuring the area under the curve (AUC) over the monitoring period.

(C) Exposure Pharmacokinetics (Exposure PK). Peptide plasma concentration at 3 hours post-injection measured by the cell-based luciferase reporter assay. [0019] Figure 9 shows representative peptide plasma concentration-time profiles in rats following IV or ID administration. (A) Single intravenous (IV) administration of TT226 dosed at 0.02 mg/kg (dissolved in PBS containing 0.2% DMSO, 0.5 mg/mL Sodium Chenodeoxycholate, and 0.25 mg/mL Propyl Gallate). (B) Single intraduodenal administration (ID) of TT226 dosed at 2.0 mg/kg (dissolved in PBS containing 2% DMSO, 5 mg/mL Sodium Chenodeoxycholate, and 2.5 mg/mL Propyl Gallate). (C) Single intraduodenal administration (ID) of semaglutide dosed at 2.0 mg/kg (dissolved in PBS containing 2% DMSO, 5 mg/mL Sodium Chenodeoxycholate, and 2.5 mg/mL Propyl Gallate). The peptide concentrations in rat plasma were determined using the GLP-1R activation assay.

[0020] Figure 10 (Scheme 1) show structures of selected double-stapled peptides.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0021] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.

[0022] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also, unless otherwise stated, include individual values (e.g., about 1%, about 2%, about 3%, about 4%, etc.) and the sub-ranges (e.g., about 0.5% to about 1.1%, about 0.5% to about 2.4%, about 0.5% to about 3.2%, about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0023] As used herein, unless otherwise indicated, “about” or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including those within experimental error (which can be determined by e.g. given data set, art accepted standard, and/or with e.g. a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as variations of +/- 10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations and variations in the alternatives are appropriate to perform in the instant disclosure . As used herein, the terms “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0024] As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like). Illustrative examples of groups include:

[0025] As used herein, unless otherwise indicated, the term “alkyl group” refers to branched or unbranched saturated hydrocarbon groups. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, and the like. In various examples, the alkyl group is Cl to C20, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Cl, C2, C3, C4, C5, C6, C7, C8, C9, CIO, Cll, C12, C13, C14, C15, C16, C17, C18, C19, and C20). The alkyl group may be unsubstituted or substituted with one or more substituent(s). Examples of substituents include, but are not limited to, halide groups (-F, -Cl, -Br, and -I), aryl groups, halogenated aryl groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, silyl ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and any combination thereof. [0026] As used herein, unless otherwise indicated, the term “aryl group” refers to C5 to C30 aromatic or partially aromatic carbocyclic groups, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., C5, C6, C7, C8, C9, C10, Cll, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, and C30). In various examples, an aryl group is an aromatic group. Aryl groups include groups such as, for example, fused ring, biaryl groups, or a combination thereof. In various examples, an aryl group is multicyclic (e.g., bicyclic, or tricyclic). An aryl group may be unsubstituted or substituted with one or more substituent(s). Examples of substituents include, but are not limited to, halide groups (-F, -Cl, -Br, and -I), alkyl groups, halogenated alkyl groups (e.g., trifluoromethyl group and the like), alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, silyl ether groups, alcohol groups, and the like, and any combination thereof. Aryl groups may contain hetero atoms, such as, for example, nitrogen (e.g., pyridinyl groups and the like). Such group may be referred to as heteroaryl groups. Examples of aryl groups include, but are not limited to, phenyl groups, biaryl groups (e.g., biphenyl groups and the like), fused ring groups (e.g., naphthyl groups and the like), hydroxybenzyl groups, tolyl groups, xylyl groups, furanyl groups, benzofuranyl groups, indolyl groups, imidazolyl groups, benzimidazolyl groups, pyridinyl groups, and the like.

[0027] Unless otherwise indicated, the term “alpha(a)-amino acid” or simply “amino acid” refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated as the a-carbon. Suitable amino acids include, but are not limited to, both the D- and L-isomers of the amino acids and amino acids prepared by organic synthesis or other metabolic routes. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include amino acid analogs. Non-limiting examples of suitable amino acids include, “naturally occurring amino acids” or canonical amino acids, which refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0028] Unless otherwise indicated, 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 crosslinked peptide. Amino acid analogs include, without limitation, compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additional methylene group(s) between the amino and carboxyl group (e.g., P-amino 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 the carboxy group with an ester), or a, a-di substituted amino acids (e.g., a-alkyl substituted amino acids, such as, for example, a-methylcysteine and the like). Unless the context specifically indicates otherwise, the term amino acid, is intended to include amino acid analogs.

[0029] Unless otherwise indicated, the term “amino acid residue” refers to an amino acid that is part of a peptide. The residues are amino acids connected to other amino acid resides through a peptidic bond or bonds to form a peptide. Unless the context specifically indicates otherwise, the term amino acid, is intended to include amino acid resides.

[0030] Unless otherwise indicated, the term “stapled” or “staple” as used herein refers to the intramolecular connection of two cysteine residues, two a-alkylcysteine residues (e.g., a- methylcysteine residues), or a cysteine residue and an a-alkylcysteine residues (e.g., a- methylcysteine residue). As an illustrative example, the residues are at the i and z+7 positions of a peptide, i is an integer and denotes the location of a cysteine residue in the peptide sequence and i+7 denotes the location of a cysteine residue 7 amino acid residues away from the cysteine at the i position (there are six (6) amino acids between the two cysteine residues). [0031] Unless otherwise indicated, the term “peptide” as used herein refers to an amino acid chain, where the chain has from 8 to 50 amino acid residues, including all integer number of amino acids and ranges therebetween.

[0032] Unless otherwise indicated, the term “protein” as used herein refers to an amino acid chain, where the chain has greater than 50 amino acid residues, which can be obtained, for example, from either chemical synthesis or DNA-based recombinant methods.

[0033] Unless otherwise indicated, the term “enzymatic stability” as used herein refers to the ability of the peptides to stay intact in the presence of an enzyme having proteolytic activity such as, for example, trypsin and chymotrypsin in biological buffers or a mixture of proteolytic enzymes present in simulated or native gastric fluid or simulated intestine fluid in human serum. In various examples, the proteolytic stability of the peptides is measured by liquid chromatography-mass spectrometry (LC-MS), or the like.

[0034] The present disclosure provides compounds (stapled peptides) and uses thereof. The present disclosure also provides methods of making compounds (stapled peptides). [0035] The present disclosure describes, inter alia, compounds (stapled peptides) (e.g., stapled GLP-1R/GIPR dual agonist peptide analogs) that exhibit potent activity in activating both GLP1-R and/or GIPR, and desirable stability against the digestion enzymes in the intestine fluid or the like.

[0036] In an aspect, the present disclosure provides compounds. The compounds are stapled peptides. In various examples, a peptide comprises two staples. In various examples, a compound (stapled peptide) is made by a method of the present disclosure. Non-limiting examples of compounds (stapled peptides) are described herein.

[0037] In various examples, a compound (e.g., a peptide) comprises the following structure: Y-X1-E-G-T-X2-T-S-D-Y-S-I-X3-L-D-K-X4-A-X5-X6-X7-X8-V-X9-W-L-X10- A-G (SEQ ID NO: 1), where XI is Aib; X2 is Phe or a-MePhe; X3 is Tyr or Aib; X4 is Gin or He; X5 is Ala or Gin; X6 is Aib or Lys; X7 is Glu or Ala; X8 is Phe or a-MePhe; X9 is Asn or Gin; XI 0 is Leu or He; and two amino acids at position i and i+7 are replaced with cysteines and crosslinked with a crosslinking group, and another two amino acids at position j and j+7 are also replaced with cysteines and crosslinked with a crosslinking group. In various examples, i is an integer (such as, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) and/or j is an integer (such as, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) and/or the portions of the compound (e.g., peptide) defined by the i and i+7 amino acids and j and i+7 amino acids are mutually exclusive (i.e., the portions do not share an amino acid).

[0038] In various examples, a compound (stapled peptide) comprises (or has) the following sequence: Y-X1-E-G-T-X2-T-S-D-Y-S-I-X3-L-D-K-X4-A-X5-X6-X7-X8-V-X9-W-L-X10-A-G (SEQ ID NO: 1), at least 80%, at least 85%, at least 90% homology, or at least 95% homology with SEQ ID NO: 1, where:

(1) XI is Aib, X2 is Phe, X3 is Tyr, X4 is Gin, X5 is Ala, X6 is Aib, X7 is Glu, X8 is Phe, X9 is Asn, XI 0 is Leu, position 10 and 17 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups, and position 18 and 25 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups;

(2) XI is Aib, X2 is Phe, X3 is Tyr, X4 is Gin, X5 is Ala, X6 is Aib, X7 is Glu, X8 is Phe, X9 is Asn, XI 0 is Leu, position 10 and 17 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups, and position 21 and 28 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups;

(3) XI is Aib, X2 is Phe, X3 is Aib, X4 is He, X5 is Gin, X6 is Lys, X7 is Ala, X8 is Phe, X9 is Gin, XI 0 is He, position 10 and 17 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups, and position 18 and 25 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups;

(4) XI is Aib, X2 is Phe, X3 is Aib, X4 is lie, X5 is Gin, X6 is Lys, X7 is Ala, X8 is Phe, X9 is Gin, XI 0 is He, position 10 and 17 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups, position 21 and 28 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups;

(5) XI is Aib, X2 is a-MePhe, X3 is Aib, X4 is He, X5 is Gin, X6 is Lys, X7 is Ala, X8 is Phe, X9 is Gin, X10 is He, position 10 and 17 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups, position 21 and 28 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups;

(6) XI is Aib, X2 is a-MePhe, X3 is Aib, X4 is He, X5 is Gin, X6 is Lys, X7 is Ala, X8 is Phe, X9 is Gin, X10 is He, optionally, Lys (e.g., at position 20) is conjugated with ([2-(2- amino-ethoxy)-ethoxy]-acetyl)2-(YGlu)-CO-(CH2)i6-COOH on the epsilon-amino group of its side chain, position 10 and 17 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups, and position 21 and 28 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups; or

(7) XI is Aib, X2 is a-MePhe, X3 is Aib, X4 is He, X5 is Gin, X6 is Lys, X7 is Ala, X8 is Phe, X9 is Gin, X10 is He, optionally, Lys (e.g., at position 20) is conjugated with ([2-(2- amino-ethoxy)-ethoxy]-acetyl)2-(YGlu)-CO-(CH2)i6-COOH on the epsilon-amino group of its side chain is appended at position 30, position 10 and 17 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups, position 21 and 28 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups; or

(8) XI is Aib; X2 is Phe or a-MePhe; X3 is Tyr or Aib; X5 is Ala or Gin; X6 is Aib or Lys; X8 is Phe or a-MePhe; X9 is Asn or Gin; X10 is Leu or He, optionally, Lys (e.g., at position 20) is conjugated with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(YGlu)-CO-(CH2)i6-COOH on the epsilon-amino group of its side chain is appended at position 30, position 10 and 17 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups, position 21 and 28 or 18 and 25 are replaced with cysteines where the cysteine side chains are stapled by crosslinking groups. In various examples, the staples of any of these peptides are independently chosen at each occurrence from the following crosslinking groups (which may be referred to in the alternative as bismethylene aryl (BMA) crosslinking groups):

like. The crosslinking group rings can independently be substituted or unsubstituted (e.g., the aryl and/or heteroaryl ring(s) of the crosslinking group are independently substituted or unsubstituted). When the aryl and/or heteroaryl ring(s) are unsubstituted, the aryl rings and/or heteroaryl ring(s) do not has/have any substituents (e.g., R^x substituent(s)). When the aryl and/or heteroaryl ring(s) are substituted, depending on the aryl and/or heteroaryl ring, the individual aryl and/or heteroaryl ring has/have one or more substituent(s) (e.g., one or more R^x substituent(s) or the like). In various examples, in the crosslinking groups shown above, each aryl and/or heteroaryl ring of the structure has/have 1, 2, 3, or 4 (where Z is a carbon) R^x substituents (e.g., x is independently at each occurrence 1, 2, 3, or 4). In various examples, the R^x groups are independently chosen at each occurrence from halide groups (-F, -Cl, -Br, or -I), alkyl groups, aryl groups, nitro group, amino group, alkylamino groups (e.g., where the alkyl group(s) of the alkylamino group, independently, has/have from 1 to 15 carbons, including all integer values of carbons and ranges therebetween), PEG groups, lipid groups (e.g., lipid-like groups and the like), PEG groups conjugated to one or more fatty diacid(s), and the like, and Z is independently at each occurrence chosen from nitrogen, carbon, and the like. A crosslinking group may be symmetrically substituted or asymmetrically substituted. In various examples, a PEG group has a molecular weight of about 20 to about 40 kD, including all 0.1 kD values and ranges therebetween. In an example, a crosslinking group comprises a PEG group having a molecular weight of about 40 kD. In another example, a crosslinking group comprises two PEG groups and each of the PEG groups, independently, has a molecular weight of about 20 kD. Examples of fatty diacids that can be separated from the crosslinking group by a spacer (e.g., a flexible spacer such as, for example, a PEG group spacer) include, but are not limited to,

m is 14, 16, 18, 20, or the like.

[0039] One having skill in the art will understand that the terminal amino acids of the peptide have an amino group or carboxylic acid (or a salt thereof). In various examples, in the structures herein when x is 0 that terminus of the peptide is an amino group, and when y is 0 that terminus of the peptide is a carboxylic acid.

[0040] A compound (a stapled peptide) may be PEGylated. Accordingly, in an example, a compound (a stapled peptide) comprises one or more PEG group(s) conjugated (e.g., covalently bound) to the peptide. Suitable PEG polymers are typically commercially available or may be made by techniques well-known to those skilled in the art. The polyethylene glycol (PEG) groups may have an average mass of about 20 kD to about 40 kD, including all integer and 0.1 g/mol values and ranges therebetween. The PEG groups can be linear or branched. In various examples, PEG group(s) is/are conjugated to the C-terminus, the N-terminus, amino acid sidechains, or the like, or any combination thereof.

[0041] Strategies for conjugating PEG to peptides are known in the art (see, e.g., Veronese, Biomaterials 22:405-417, 2001). Those skilled in the art, will therefore be able to utilize well-known techniques for linking PEG to a compound (a stapled peptide) described herein. [0042] A lipid group can be a substituent comprising 8 to 40 carbons, including all integer number of carbons therebetween. The lipid group may further comprise an amino group, at least one free carboxylic acid group, a negatively charged functional group, or the like, or any combination thereof. The lipid group may be a straight chain fatty acid that has an amino group. The lipid may contain a spacer. In an example, succinic acid, glutamic acid, and aspartic acid may be used as spacers. When succinic acid is used as a spacer, one of its carboxyl groups can form an amide bond with an amino group in the N-terminal amino acid of the parent peptide while the other carboxyl group can form an amide bond with an amino group contained in the bulk lipophilic group. When glutamic acid or aspartic acid is used as a spacer, one of the carboxyl groups can form an amide bond with an amino group in the N- terminal amino acid of the parent peptide while the bulk lipophilic substituent may be the alkyl group of a straight chain fatty acid. In another example, an additional free carboxy group on the fatty acid may form an amide bond with lysine or an ester bond with serine. [0043] Lipid-like groups are non-lipid organic structures that bind to proteins (such as, for example, serum albumin proteins (e.g., human serum albumin and the like) and the like. In various examples, a lipid-like group is functionally equivalent to a lipid or lipid group. In various examples, a lipid-like group extends the half-life of a peptide. In various examples, a compound (stapled peptide) comprising lipid-like group(s) exhibit an extended half-life.

[0044] A peptide may be present in a fusion protein. In various examples, a fusion protein comprises a peptide, a peptide linker, and a protein. The protein can be any suitable protein (e.g., transferrin, human IgG Fc variant, human serum albumin, or the like).

[0045] In various examples, one or more stapled peptide(s) activate GLP-1R and/or GIPR, or the like (e.g., in an individual). In various examples, the effective concentration of one or more stapled peptide(s) (in the aggregate) effecting a 50% or greater rise of maximal activity, EC50, ranges from 0.001 nM to 10 nM, including all 0.0005 nM values and ranges therebetween, in one or more luciferase-based reporter assay(s).

[0046] In various examples, the present disclosure provides a means of activating GLP- 1R and/or GIPR (e.g., in an individual) or the like. In various examples, a means effects a 50% or greater rise of maximal activity, EC50, at a concentration of 0.001 nM to 10 nM, including all 0.0005 nM values and ranges therebetween, in one or more luciferase-based reporter assay (s).

[0047] In an aspect, the present disclosure provides compositions comprising one or more compound(s) (stapled peptide(s)) of the present disclosure. Non-limiting examples of compositions are described herein. [0048] A composition may also comprise one or more additional component(s), one or more or all of which may be pharmaceutically acceptable components or the like. In various examples, a composition is a pharmaceutical composition comprising one or more pharmaceutically acceptable component(s) or the like.

[0049] As used herein, unless otherwise indicated, the term “pharmaceutically acceptable” refers to those components and dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans or animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Non-limiting examples of materials that can be used as additional component(s) in a composition include sugars, such as, for example, lactose, glucose, sucrose, and the like; starches, such as, for example, corn starch, potato starch, and the like; cellulose, and its derivatives, such as, for example, sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, and the like; powdered tragacanth; malt; gelatin; talc; excipients, such as, for example, cocoa butter, suppository waxes, and the like; oils, such as, for example, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, soybean oil, and the like; glycols, such as, for example, propylene glycol and the like; polyols, such as, for example, glycerin, sorbitol, mannitol, polyethylene glycol, and the like; esters, such as, for example, ethyl oleate, ethyl laurate, and the like; agar; buffering agents, such as, for example, magnesium hydroxide, aluminum hydroxide, and the like; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. (See, e.g., REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).

[0050] In various examples, a composition is suitable for oral administration. Such a composition may exhibit desirable oral absorption. In various example, a composition, which may be for oral administration, comprises one or more permeation enhancer(s) or the like. Non-limiting examples of permeation enhancer(s) include salcaprozate sodium (SNAC), sodium decanoate (Na caprate), octyl gallate, sodium octanoate (Na caprylate), lauryl gallate, dodecylmaltoside (DDM), sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC), tetradecylmaltoside (TDM), palmitoyl carnitine, sodium chenodeoxycholate (NaCDC), polyoxyl 15 hydroxystearate (Kolliphor HS 15), sodium deoxycholate, polyoxyethylene (10) oleyl ether (Brij 10), sodium tetraglycocholate, polyethylene glycol hexadecyl ether, sodium ursodeoxy cholate, polyoxyethylene (10) cetyl ether, sodium glycocholate, Purebright, sodium taurocholate, diethylene glycol monoethyl ether (Transcutol ES), sodium cholate, polyoxyl- 35 castor oil (Kolliphor EL), chenodeoxycholic acid, lauroglycol FCC, ethyl gallate, trimethyl chitosan, propyl gallate (PG), chitosan, and the like, and any combinations thereof. In various examples, an oral administration composition comprises sodium chenodeoxycholate (NaCDC) and propyl gallate (PG).

[0051] In an aspect, the present disclosure provides methods of making compounds (stapled peptides) of the present disclosure. In various examples, a method of the present disclosure provides a compound (a stapled peptide) comprising two staples. In various examples, a method of the present disclosure provides a compound (a stapled peptide) of the present disclosure. Non-limiting examples of making compounds (stapled peptides) are described herein.

[0052] It is considered that any peptide, with appropriately placed cysteines (naturally occurring, introduced, etc.) can be stapled. It may be desirable that a peptide exhibits biological activity. Examples of suitable peptides include, but are not limited to, bioactive peptides, such as, for example, peptide hormones and the like, fragments thereof, and the like. [0053] A peptide may be present in a fusion protein. In various examples, a fusion protein comprises a peptide, a peptide linker, and a protein. The protein can be any suitable protein (e.g., transferrin, human IgG Fc variant, human serum albumin, or the like) or the like.

[0054] In various examples, a peptide has one or more asymmetric center(s), and thus, occur as racemates and racemic mixtures, single enantiomers, individual diastereomers or diastereomeric mixtures. All such isomeric forms of these peptides (which may be present in a fusion protein) are included in the present disclosure unless expressly provided otherwise. The peptides may be represented in multiple tautomeric forms, in such instances, the disclosure includes all tautomeric forms of the compounds described herein. All such isomeric forms of such compounds are included in the present disclosure unless expressly provided otherwise. All crystal forms of the compounds described herein are included in the present disclosure unless expressly provided otherwise.

[0055] In various examples, the cysteine residues used to form an individual staple comprise a-alkyl groups having from 1 to 15 carbons, including all integer numbers of carbons and ranges therebetween, at the a-carbon of the cysteine residues. Examples of a suitable alkyl group include, but are not limited to, methyl group, ethyl group, propyl groups, isopropyl group, butyl groups, and the like.

[0001] Various crosslinking groups may staple sets of cysteine residues. While not intending to be bound by any particular theory, it is considered that stapling stabilizes a peptide and/or increases its efficacy, or the like. Non-limiting examples of crosslinking groups are described herein. [0056] A compound (a stapled peptide) comprises two staples, or crosslinks, each of which may be formed by, for example, a rigid, distance matching crosslinking group or the like. The individual staples form a macrocyclic ring, which is not part of the core (i.e., it is exogeneous) or inherent structure of the peptide. The macrocyclic ring is comprised of a crosslinking group and includes 8 amino acids of the peptide, two of which are cysteine residues connected to the crosslinking group through thioether bonds.

[0057] It is considered that the crosslinking groups are of a size and/or rigidity that provide desirable stabilization of a helical motif of a peptide. Without intending to be bound by any particular theory, it is considered the distance matching and/or rigidity of crosslinking group is such that the stapled peptide has increased helicity and/or increased cell permeability relative to peptides that are not stapled according to the present disclosure.

[0058] A crosslinking group (which may be referred to in the alternative as BMA crosslinking group) comprises an aryl group and is connected to the peptide through two thioether bonds. In various examples, a crosslinking group comprises two phenyl rings connected by a single bond. In various examples, a crosslinking group has the following structure:

the like, where Z is nitrogen or carbon. The crosslinking group rings can independently be substituted or unsubstituted (e.g., the aryl and/or heteroaryl ring(s) of the crosslinking group are independently substituted or unsubstituted). When the aryl and/or heteroaryl ring(s) are unsubstituted, the aryl rings and/or heteroaryl ring(s) do not have any substituents (e.g., R^x substituent(s) or the like). When the aryl and/or heteroaryl ring(s) are substituted, depending on the aryl and/or heteroaryl ring, the individual aryl and/or heteroaryl ring has/have one or more substituent(s) (e.g., one or more R^x substituent(s) or the like). In various examples, in the crosslinking groups shown above, each aryl and/or heteroaryl ring of the structure has/have 1, 2, 3 or 4 (where Z is a carbon) R^x substituents (e.g., x independently at each occurrence 1, 2, 3, or 4). In various examples, the R^x groups are independently chosen at each occurrence from halide groups (-F, -Cl, -Br, or -I), alkyl groups, aryl groups, nitro group, amino group, alkylamino groups (e.g., where the alkyl group(s) of the alkylamino group, independently, has/have from 1 to 15 carbons, including all integer values of carbons and ranges therebetween), PEG groups, lipid groups (e.g., lipid-like groups and the like), PEG groups conjugated to one or more fatty diacid(s), and the like, and Z is independently at each occurrence chosen from nitrogen, carbon, and the like. A crosslinking group may be symmetrically substituted or asymmetrically substituted. Non-limiting examples of PEG groups are described herein.

[0059] In various examples, two R^x groups (one from each aryl and/or heteroaryl ring) of a crosslinking group above are joined to form a fused ring structure. In various examples, the ring formed by the joining of two R^x groups has 5, 6, or 7 carbons. Non-limiting examples of such structures are represented in the following:

, , or the like.

In various examples, the aryl and/or heteroaryl rings of these structures are substituted or unsubstituted as described herein.

[0060] It may be desirable that the aryl structure is symmetrical so that only one isomer (e.g., regioisomer or the like) of the stapled peptide is formed due to symmetry-related degeneration. In various examples, in the structures where Z is nitrogen, it is desirable the aryl portion (e.g., aryl and/or heteroaryl ring(s)) of the group be symmetrical (e.g., biphenyl, bipyridine, and phenanthroline, etc.) and not unsymmetrical (e.g., benzoisoquinoline or the like) as to not form isomers.

[0061] The individual stapling reactions (e.g., first stapling reaction and/or second stapling reaction) can be performed using suitable reaction conditions that is within the purview of one skilled in the art. As an illustrative example, crosslinking (i.e., stapling) reactions (e.g., first stapling reaction and/or second stapling reaction) are performed by incubating a suitable peptide (or a singly crosslinked peptide) with a slight excess (e.g., 1.01- 1.5 equivalent) of a crosslinking agent (e.g., a brominated analog of a crosslinking group of the present disclosure) where the crosslinking agent is a in a buffered media (e.g., a mixture of 1 :4 to 2:3 acetonitrile/water containing 30 mM NH4HCO3), with stirring for several hours. The product can be recovered through methods known in the art such as, for example, sublimation (e.g., lyophilization or the like). Further purification can be performed using methods known in the art such as, for example, chromatography (e.g., HPLC) or the like. As an example, a stapled peptide is purified by washing the lyophilized residue with an organic solvent (e.g., diethyl ether), followed by purification of the rinsed material by preparative HPLC.

[0062] Various crosslinking agents can be used. Combinations of crosslinking agents may be used. In various examples, a crosslinking agent (which may be referred to in the alternative as a bismethylene aryl (BMA) crosslinking agent) comprises an aryl group and two suitable leaving groups. In various examples, a crosslinking agent comprises two phenyl rings connected by a single bond. In various examples, a crosslinking agent has the following structure:

suitable leaving group (LG) substituent and Z is independently at each occurrence nitrogen or carbon. In various examples, the leaving group substituent is independently at each occurrence a chloride group, a bromide group, an iodide group, a tosylate group, or the like. The crosslinking agent rings can independently be substituted or unsubstituted (e.g., the aryl and/or heteroaryl ring(s) of the crosslinking agent are independently substituted or unsubstituted). When the aryl and/or heteroaryl ring(s) are unsubstituted, the aryl rings and/or heteroaryl ring(s) do not have any substituents (e.g., R^x substituent(s) or the like). When the aryl and/or heteroaryl ring(s) are substituted, depending on the aryl and/or heteroaryl ring, the individual aryl and/or heteroaryl ring has/have one or more substituent(s) (e.g., one or more R^x substituent(s) or the like). In various examples, in the crosslinking groups shown above, each aryl and/or heteroaryl ring of the structure has/have 1, 2, 3 or 4 (where Z is a carbon) R^x substituents (e.g., x is independently at each occurrence 1, 2, 3, or 4). In various examples, the R^x groups are independently chosen at each occurrence from halide groups (-F, -Cl, -Br, or -I), alkyl groups, aryl groups, nitro group, amino group, alkylamino groups (e.g., where the alkyl group(s) of the alkylamino group, independently, has/have from 1 to 15 carbons, including all integer values of carbons and ranges therebetween), PEG groups, lipid groups (e.g., lipid-like groups and the like), PEG groups conjugated to one or more fatty diacid(s), and the like, and Z is independently at each occurrence chosen from nitrogen, carbon, and the like. A crosslinking agent may be symmetrically substituted or asymmetrically substituted. It may be desirable that the aryl structure is symmetrical so that only one isomer (e.g., regioisomer or the like) of the stapled peptide is formed due to symmetry-related degeneration. Non-limiting examples of PEG groups are described herein.

[0063] In various examples, two R^x groups (one from each aryl and/or heteroaryl ring) of a crosslinking agent above are joined to form a fused ring structure. In various examples, the ring formed by the joining of two R^x groups has 5, 6, or 7 carbons. Non-limiting examples of such structures are represented in the following:

, or the like, where X and Z are defined above. The aryl and/or heteroaryl ring(s) of these structures may be substituted or unsubstituted as described herein. It may be desirable that a structure be symmetrical so that isomers (e.g., diastereomers) of the crosslinked peptide are not formed. [0064] In each stapling reaction, the peptide (or single stapled peptide or precursor compound or peptide, or the like) and crosslinking agent(s) are contacted under conditions (e.g., reaction time, reaction temperature and reaction atmosphere) that result in formation of at least two carbon-sulfur bonds providing a crosslink or staple as described herein which comprises at least two thioether linkages. Determination of suitable conditions is within the purview of one having skill in the art.

[0065] While the crosslink or staple can be formed by the reaction as described in the method above, it is considered that the crosslink or staple can be formed by reaction of crosslinking agent(s) with functional groups other than methylene halides described herein, such as, for example, bismethylene aryl tosylates and the like.

[0066] A compound (e.g., a stapled peptide, a precursor compound or peptide, which is stapled or is not stapled, or the like) can be made by making two or more fragments of the compound (the stapled peptide), which independently may be stapled (e.g., comprise a crosslinking group), and joining the fragments (such as, for example, by a condensation reaction or the like) to make a precursor compound (or peptide), which may comprise one crosslinking group or no crosslinking groups or the compound (a stapled peptide). In various examples, a method of making a compound (a stapled peptide) comprises making two or more precursor compounds (or peptides) individually corresponding to a fragment of the desired compound (stapled peptide), which individually comprise or do not comprise a crosslinking group; and joining the fragments (such as, for example, by a condensation reaction or the like) to form a precursor compound (or peptide), which may comprise one crosslinking group or no crosslinking groups or the compound (a stapled peptide); and, in the case of where at least one precursor compound (or peptide) does not comprise a crosslinking group or no crosslinking groups is formed; subjecting the precursor compound to at least one (e.g., one or two) stapling reactions, as necessary, to form the compound (a stapled peptide). Identification of suitable fragments is within the purview of one having skill in the art. It may be desirable to use this method in large scale production of a compound (a stapled peptide). [0067] A compound (a stapled peptide), which comprises two staples, can have improved properties as compared to the parent non-crosslinked peptide and/or parent peptides with a single staple. In various examples, a compound (a stapled peptide) exhibits increased enzymatic stability (e.g., proteolytic stability against proteases, such as, for example, trypsin, chymotrypsin, or the like, or any combination thereof, stability in gastric and/or intestinal fluids, or the like), and/or improved biological activity (such as, for example, inhibitory activities against protein-protein interactions, agonist activities toward cell surface receptors, or the like).

[0068] In an aspect, the present disclosure provides uses of compounds (stapled peptides) of the present disclosure. In various examples, the present disclosure provides methods of treating diabetes and/or lowering the blood glucose level of an individual and/or inducing weight loss in an individual. Non-limiting examples of uses of the compounds (stapled peptides) are described herein.

[0069] In various examples, a method of treating diabetes, such as, for example, type 2 diabetes mellitus or the like, comprises administration to an individual an effective amount of one or more compound(s) (stapled peptide(s)). In various examples, a method of lowering the blood glucose levels of an individual comprises administration to the individual an effective amount of one of or more compound(s) (stapled peptide(s)). In various examples, a method of inducing weight loss in an individual comprises administration to the individual an effective amount of one of or more compound(s) (stapled peptide(s)). In various examples, a method of treating diabetes, such as, for example, type 2 diabetes mellitus or the like, and/or lowering the blood glucose levels of an individual and/or inducing weight loss in an individual comprises administration to the individual an effective amount of one of or more compound(s) (stapled peptide(s)). In various examples, the stapled peptides are GLP- 1R/GIPR dual agonists.

[0070] Clinicians will be able to assess individuals who are in need of being treated for these conditions or individuals themselves may be able to assess a need for intake of these compositions. The present stapled peptides and compositions may be used in combination with other therapeutic approaches for the conditions. The additional therapeutic approaches can be carried out sequentially or simultaneously with the treatment involving the present compositions.

[0071] As used herein, “treatment” of diabetes is not limited to treatment, but encompasses alleviation of one or more or all of the symptom(s) of diabetes, management of blood glucose levels, and the like.

[0072] An individual may be a human or a non-human animal. An individual may be a mammal. Non-limiting examples of non-human animals or mammals include cows, pigs, goats, mice, rats, rabbits, other agricultural mammals, cats, dogs, pets, service animals, and the like.

[0073] Administration of stapled peptides or compositions comprising stapled peptides as described herein can be carried out using any suitable route of administration known in the art. In various examples, the stapled peptides or the compositions are administered via intravenous, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, oral, topical, inhalation routes, or the like. The compositions may be administered parenterally or enterically. The compositions may be introduced as a single administration or as multiple administrations or may be introduced in a continuous manner over a period of time. In various examples, the administration(s) can be a pre-specified number of administrations or daily, weekly or monthly administrations, which may be continuous or intermittent, as may be clinically needed and/or therapeutically indicated.

[0074] The following Statements describe various examples of compounds (e.g., peptide) and methods of the present disclosure and are not intended to be in any way limiting: Statement 1. A compound (e.g., a peptide) comprising the following structure: Y-Xl-E-G-T- X2-T-S-D-Y-S-I-X3-L-D-K-X4-A-X5-X6-X7-X8-V-X9-W-L-X10-A-G (SEQ ID NO: 1), where XI is Aib; X2 is Phe or a-MePhe; X3 is Tyr or Aib; X4 is Gin or He; X5 is Ala or Gin; X6 is Aib or Lys; X7 is Glu or Ala; X8 is Phe or a-MePhe; X9 is Asn or Gin; X10 is Leu or He; and two amino acids at position i and i+7 are replaced with cysteines and crosslinked with a crosslinking group, and another two amino acids at position j and j+7 are also replaced with cysteines and crosslinked with a crosslinking group.

Statement 2. A compound according to Statement 1, where the crosslinking groups are independently at each occurrence chosen from:

and the like, where Z is nitrogen or carbon, x is independently at each occurrence 1, 2, 3, 4 (where Z is carbon), and the R^x groups are each optionally present and, when present, is/are independently chosen at each occurrence from halide groups (-F, -Cl, -Br, or -I), alkyl groups, aryl groups, nitro group, amino group, alkylamino groups (e.g., where the alkyl group(s) of the alkylamino group, independently, has/have from 1 to 15 carbons, including all integer values of carbons and ranges therebetween), polyethylene (PEG) groups, lipid groups (e.g., lipid-like groups and the like), PEG groups conjugated to one or more fatty diacid(s), and the like.

Statement 3. A compound according to Statement 1 or 2, where i is at position 10 and j is at position 18 or 21; and the crosslinking group is Bph or Bpy.

Statement 4. A compound according to Statement 3, where j is at position 18.

Statement 5. A compound according to Statement 3, where j is at position 21.

Statement 6. A compound according to Statement 3, where: XI is Aib; X2 is Phe or a- MePhe; X3 is Tyr or Aib; X5 is Ala or Gin; X6 is Aib or Lys; X7 is Glu or Ala; X8 is Phe or a-MePhe; X9 is Asn or Gin; XI 0 is Leu or He; and the crosslinking group is Bph or Bpy.

Statement 7. A compound according to Statement 4, where XI is Aib; X2 is Phe or a-MePhe; X3 is Tyr or Aib; X4 is Gin or He; X5 is Ala or Gin; X6 is Aib or Lys; X8 is Phe or a- MePhe; X9 is Asn or Gin; X10 is Leu or He; and the crosslinking group is Bph or Bpy. Statement 8. A compound according to Statement 7, where Lys at position 20 is conjugated with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(YGlu)-CO-(CH2)i6-COOH on the epsilon-amino group of its side chain. Statement 9. A compound according to Statement 7, where a Lys conjugated with ([2-(2- amino-ethoxy)-ethoxy]-acetyl)2-(YGlu)-CO-(CH2)i6-COOH on the epsilon-amino group of its side chain is appended at position 30.

Statement 10. A composition comprising one or more compound(s) of the present disclosure (e.g., compound(s) of any of the preceding Statements).

Statement 11. A composition according to Statement 10, where the composition comprises one or more additional component(s).

Statement 12. A composition according to Statement 10 or 11, where the additional component(s) is/are permeation enhancer(s).

Statement 13. A composition according to Statement 12, where the permeation enhancer(s) is/are chosen from salcaprozate sodium (SNAC), sodium decanoate (Na caprate), octyl gallate, sodium octanoate (Na caprylate), lauryl gallate, dodecylmaltoside (DDM), sodium N- (8-[2-hydroxybenzoyl]amino)caprylate (SNAC), tetradecylmaltoside (TDM), palmitoyl carnitine, sodium chenodeoxy cholate (NaCDC), polyoxyl 15 hydroxystearate (Kolliphor HS 15), sodium deoxy cholate, polyoxyethylene (10) oleyl ether (Brij 10), sodium tetraglycocholate, polyethylene glycol hexadecyl ether, sodium ursodeoxycholate, polyoxyethylene (10) cetyl ether, sodium glycocholate, Purebright, sodium taurocholate, di ethylene glycol monoethyl ether (Transcutol ES), sodium cholate, polyoxyl-35 castor oil (Kolliphor EL), chenodeoxy cholic acid, lauroglycol FCC, ethyl gallate, trimethyl chitosan, propyl gallate (PG), chitosan, and the like, and any combination thereof.

Statement 14. A method of lowering blood glucose level in an individual (which may be an individual in need of treatment for a high glucose level) comprising administering one or more compound(s) and/or composition(s) (e.g., one or more compound(s) and/or composition(s) of any of the preceding Statements) to the individual, where the blood glucose level of the individual is lowered (e.g., the administration results in reduction of the blood glucose level of the individual).

Statement 15. A method of inducing weight loss of an individual (which may be an individual in need of weight loss) comprising administering one or more compound(s) and/or composition(s) (e.g., one or more compound(s) and/or composition(s) of any of the preceding Statements) (which may be an effective amount of the compound(s)) to the individual, where the individual loses weight (e.g., the administration results in the individual losing weight).

Statement 16. A method of activating GLP-1R and/or GIPR in individual (which may be an individual in need of treatment for a high glucose level and/or in need of weight loss) comprising administering one or more compound(s) and/or composition(s) (e.g., one or more compound(s) and/or composition(s) of any of the preceding Statements) to the individual (which may be an effective amount of the compound(s)), where the GLP-1R and/or GIPR are activated in the individual (e.g., the administration results in activation of the GLP-1R and/or GIPR in the individual).

Statement 17. A method for making a stapled peptide (which may be a stapled peptide of any of the preceding Statements) comprising two staples, comprising: providing a peptide having the following structure:

where [Xaa] is any amino acid or amino acids sequence; Ri, R2, R3, and R4 are each independently a hydrogen or an alkyl group; b = 6 and d = 6, or b + c = 5 and c + d = 5; when b = 6 and d = 6, R5 and Rs are hydrogens, and R7 and Rs are protecting groups (e.g., TFA-resistant thiol protecting groups, such as, for example, A'-acetamidom ethyl group (Acm) or the like) or vice versa; when b + c = 5 and c + d = 5, R5 and R7 are hydrogens, and Rs and Rs are protecting groups (e.g., TFA-resistant thiol protecting groups, such as, for example, S- acetamidomethyl group (Acm) or the like), or vice versa; contacting the peptide with a first crosslinking agent/agents, where a first stapled peptide/peptides is/are formed (e.g., stapling cysteines with the free thiols with a crosslinking group, such as, for example, a crosslinking group having the following structure:

, or the like, where Z is nitrogen or carbon, x is independently at each occurrence 1, 2, 3, 4 (where Z is carbon), and the R^x groups are each optionally present and, when present, is/are independently chosen at each occurrence from halide groups (-F, -Cl, -Br, or -I), alkyl groups, aryl groups, nitro group, amino group, alkylamino groups (e.g., where the alkyl group(s) of the alkylamino group, independently, has/have from 1 to 15 carbons, including all integer values of carbons and ranges therebetween), PEG groups, lipid groups (e.g., lipid-like groups and the like), PEG groups conjugated to one or more fatty diacid(s), and the like; deprotecting the first stapled peptide(s) (e.g., deprotecting the TFA-resistant thiol protecting groups on cysteines of the first stapled peptide(s) using an appropriate reagent, such as, for example, mercury (II) acetate or the like, for removing Acm); and contacting the first peptide(s) with a second crosslinking agent/agents, where a second stapled peptide/peptides having two crosslinks, or staples, is/are formed (e.g., stapling cysteines with the free thiols with a second crosslinking group/groups, such as, for example, a crosslinking group/groups described above with respect to the first crosslinking group).

Statement 18. A method according to Statement 17, where the crosslinking agent is, independently at each occurrence, chosen from

like, where X is a suitable leaving group (LG) chosen independently at each occurrence from a chloride group, a bromide group, an iodide group, a tosylate group, and the like, Z is independently at each occurrence nitrogen or carbon, x is independently at each occurrence 1, 2, 3, 4 (where Z is carbon) and the R^x groups are each optionally present and, when present, is/are independently chosen at each occurrence from halide groups (-F, -Cl, - Br, or -I), alkyl groups, aryl groups, nitro group, amino group, alkylamino groups (e.g., where the alkyl group(s) of the alkylamino group, independently, has/have from 1 to 15 carbons, including all integer values of carbons and ranges therebetween), PEG groups, lipid groups (e.g., lipid-like groups and the like), PEG groups conjugated to one or more fatty diacid(s), and the like.

Statement 19. A method according to Statement 17 or 18, where the stapling results in formation of a crosslink, or staple, comprising, independently at each occurrence, the following structure:

, , or the like, where Z is nitrogen or carbon, x is independently at each occurrence 1, 2, 3, 4 (where Z is carbon), and the R^x groups are each optionally present and, when present, is/are independently chosen, when present, at each occurrence from halide groups (-F, -Cl, -Br, or - I), alkyl groups, aryl groups, nitro group, amino group, alkylamino groups (e.g., where the alkyl group(s) of the alkylamino group, independently, has/have from 1 to 15 carbons, including all integer values of carbons and ranges therebetween), PEG groups, lipid groups (e.g., lipid-like groups and the like), PEG groups conjugated to one or more fatty diacid(s), and the like. [0075] The steps of the methods described in the various embodiments and examples disclosed herein are sufficient to produce a stapled peptide or carry out a use of the stapled peptide(s) of the present disclosure. Thus, in various examples, a method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, a method consists of such steps.

[0076] The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any manner.

EXAMPLE 1

[0077] The following describes non-limiting examples of stapled peptides of the present disclosure and characterization thereof.

[0078] Synthesis and characterization of stapled GLP-1R/GIPR dual agonists (Tablet, Table 2, Figure 10).

[0079] Synthesis of the linear peptides. The linear peptides containing both free and acetaminomethyl (Acm)-protected cysteines are either purchased from commercial sources or synthesized using a Tribute peptide synthesizer (Protein Technology). Rink amide resin (0.49 mmol/g, 100-200 mesh; 80 mg, 40 pmol) was swelled in DCM for 15 min (m = minute(s)) and then drained. The resin was washed with DMF (3 x 1.5 mL). The Fmoc group was removed by treating the resin with 1.5 mL 20% piperidine in DMF (two times: first 2 min, second 12 min). The resin was washed with DMF (3 x 1.5 mL). To couple each amino acid, Fmoc-protected amino acids (3 eq relative to the resin loading) were dissolved in DMF (2 mL) and mixed with O-(7-benzotriazol-l-yl)-7V,7V,7V’,7V’-tetramethyluronium hexafluorophosphate (HBTU; 3 eq) and diisopropylethylamine (DIPEA; 3 eq). The solution was mixed for 3 min prior to being added to the resin and agitated for 40 min at room temperature. For synthesis of TT213/214, O-(7-azabenzotriazol-l-yl)-7V,7V,7V’,7V’- tetramethyluronium hexafluorophosphate (HATU) was used instead of HBUT for coupling of the 6^th and 12^th residues, and double coupling was performed (first 3 h, second 12 h). Then, the resin was drained and washed with DMF (3 x 1.5 mL). The above process was repeated until the desired peptide sequence was obtained, and the last Fmoc was removed. Upon completion of peptide synthesis, the resin was washed with DCM before it was transferred into a 15-mL centrifuge tube. The peptide was cleaved from the resin, along with the trifluoroacetic acid (TFA) labile protecting groups, by mixing the resin with a 3-mL cleavage cocktail containing 95% TFA, 2.5% triisopropylsilane (TIPS), and 2.5% deionized water for 3 h (h = hour(s)) with occasional agitation. The resulting cleavage solution was separated from the resin through filtration, and the filtrate was transferred into a 50-mL centrifuge tube. A cooled solution of diethyl ether/hexanes (1 : 1; 45 mL) was added to the filtrate, and the peptide was allowed to precipitate in a -20 °C freezer for 10 min. The mixture was centrifuged at 3000 RPM for 5 min and the supernatant was removed to yield the crude peptide. The materials were dissolved in 10 mL acetonitrile (I)-water (1 :1) and lyophilized. Peptides were purified using a Gilson semipreparative reverse-phase HPLC system equipped with a Phenom enex Cl 8 column at a flow rate of 5 mL/min and a gradient of 30-60% acetonitrile/JLO while monitoring at 220 and 254 nm.

[0080] Synthesis of the stapled peptides. The two free cysteines were stapled using the following procedure. The linear peptides (2 mg) were dissolved in 600 pL acetonitrile/NH4HCO3 buffer (30 mM, pH = 8.5; 1 : 1). A solution of 4, 4’-bis-bromomethyl- biphenyl (Bph) or 4, 4’-bis-bromomethyl-bipyridyl (Bpy) in acetonitrile (1.2 eq) were added to the above peptide solution and the mixture was stirred at room temperature for 2-3 h. When over 95% peptides were reacted as indicated by LC-MS, the mixture was lyophilized overnight. Next, the Acm protecting groups were removed using the following procedure. The lyophilized peptide was dissolved in 600 pL acetonitrile-water (1 : 1) solution. Then, 30 pL acetic acid was added to the peptide solution, followed by the addition of mercury (II) acetate (dissolved in acetic acid, 1.5 eq per Acm group) for Acm deprotection. When >95% peptide underwent deprotection reaction as indicated by QTOF-LC/MS, 100 pL 0- mercaptoethanol was added to the reaction mixture to dissociate mercury from the peptide. When over 90% peptide was free of mercury as indicated by LC/MS, the peptide intermediate that contains two free cysteines and two stapled cysteines was purified by preparative HPLC. The stapling of the remaining free cysteines was performed using the same procedure as described above, followed by another round of HPLC purification.

[0081] Table 1. MS characterization data.

Calculated exact mass Observed exact mass

Name

[M + 3H ], m/z [M + 3H⁺], m/z

TT201 1164.1896 1164.1914

TT202 1165.5166 1165.5135

TT203 1164.8531 1164.8501

TT204 1164.8531 1164.8478

TT205 1156.8774 1156.8755

TT206 1158.2044 1158.2009

TT207 1157.5409 1157.5447 TT208 1157.5409 1157.5392

TT209 1183.2019 1183.2063

TT210 1183.8653 1183.8612

TT211 1195.2248 1195.2259

TT212 1195.8883 1195.8889

TT213 1199.8966 1199.8975

TT214 1200.5601 1200.5640

TT215 1438.3649 1438.3718

TT216 1439.0296 1439.0353

TT217 1481.0701 1481.0661

TT218 1481.7336 1481.7284

TT219 1204.5685 1204.5697

TT220 1205.2320 1205.2326

TT221 1485.7420 1485.7415

TT222 1486.4055 1486.4041

TT223 1201.2236 1201.2251

TT224 1439.6988 1439.6989

TT225 1482.3971 1482.3978

TT226 1205.8955 1205.8964

TT227 1487.0690 1487.0683

TT228 1443.0437 1443.0469

TT229 1443.7072 1443.7117

TT230 1444.3707 1444.3752

EXAMPLE 2

[0082] The following describes non-limiting examples of characterization and use of stapled peptides of the present disclosure. [0083] Receptor activity of stapled GLP-1R/GIPR dual agonist analogs. Experiments were carried out as follows to determine the activity of the stapled GLP-1R/GIPR dual agonist analogs in activating GIPR and GLP-1R. In vitro receptor activation reporter assays: HEK293-GLP-1R-CRE and HEK293-GIPR-CRE cells were seeded in a white 384-well plate at a density of 5,000 cells per well and cultured for 24 hours in DMEM with 10% FBS at 37°C with 5% CO2. Cells were treated with different peptides at varying concentrations.

After 24 hours, 10 pL of Bright-Glo reagent (Promega, Madison, WI) was added to each well and luminescence was determined using an Envision multilabel plate reader (PerkinElmer, Waltham, MA). The EC50 of each peptide (Figure 1, Table 2) was calculated using GraphPad Prism 9.2 software (San Diego, CA). Oral glucose tolerance test (OGTT) and in vivo pharmacokinetics (Figure 8): C57BL/6 male mice (age 10 weeks) were fasted overnight and then administered with peptides through s.c. injection at a dosage of 0.4 mpk. After 1 hour, mice were orally administered with 2 g of glucose solution per kg body weight and their tail blood glucose levels were measured before (0 min) and after glucose challenge for 90 min. Food was provided to mice 2 hours post glucose administration. Blood was extracted into heparinized tubes and centrifuged at 3,000 g for 15 min. The resulting supernatant plasma was stored at -80 °C. Peptide concentration in the plasma was determined by in vitro GLP-1R activation reporter assay.

[0084] Oral bioavailability of dual agonists with permeation enhancers in vivo (Figure 9). Male Sprague Dawley rats (Charles River, Shrewsbury, MA, USA) weighing 250-275 g were housed with ad libitum access to standard chow and water. Test peptides were administered via either an intravenous or a duodenal catheter (3 rats per group) after overnight fasting, and food was returned following 4 h blood collection. Blood was collected via a jugular vein catheter at 4 h predose and 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h after intravenous dosing, or for intraduodenal groups at 4 h predose and 0.25, 0.5, 0.75, 1, 2, 4, 6, 8 and 24 h postdose. Blood was collected in K2EDTA tubes and kept on wet ice prior to 10 min centrifugation (3000 * g) at 5 °C ± 3 °C. Plasma samples were stored at - 70 °C until analysis.

[0085] Table 2. Sequence of the double-stapled peptides and their agonist activity in GLP-1R and GIPR-mediated luciferase reporter assays ^a

Agonist activity (nM)

Name Sequence GLP- GIP

_ 1R R

Semaglutide HXEGTFTSDVSSYLEGQAAK’EFIAWLVRGRG^b (SEQ 0.04 NA

ID NO: 2) hGIP YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDW NA 1.77

KHN (SEQ ID NO: 3)

Tirzepatide YXEGTFTSDYSIXLDKIAQK”AFVQWLIAGGPSSGAPP 0.10 0.03

PS^C (SEQ ID NO: 4)

TT201 YXEGTFTSDC’SIYLDKC’C’AXEFVNC’LLAG^d (SEQ ID 0.33 0.08

NO: 5)

TT202 YXEGTFTSDC”SIYLDKC”C”AXEFVNC”LLAG^e (SEQ 0.31 0.09

ID NO: 6)

TT203 YXEGTFTSDC”SIYLDKC”C’ AXEFVNC’LLAG (SEQ ID 0.09 0.07

NO: 7)

TT204 YXEGTFTSDC’SIYLDKC’C” AXEFVNC’LLAG (SEQ ID 0.19 0.03

NO: 8)

TT205 YXEGTFTSDC’SIXLDKC’C’QKAFVQC’LIAG (SEQ ID 0.03 0.10

NO: 9) TT206 YXEGTFTSDC”SIXLDKC”C”QKAFVQC”LIAG (SEQ ID 0.06 0.19 NO: 10)

TT207 YXEGTFTSDC”SIXLDKC”C’QKAFVQC’LIAG (SEQ ID 0.02 0.09

NO: 11)

TT208 YXEGTFTSDC’SIXLDKC’C”QKAFVQC”LIAG (SEQ ID 0.05 0.19

NO: 12)

TT209 YXEGTFTSDC’SIYLDKC’AAXC’FVNWLLC’G (SEQ ID 19.35 1.64

NO: 13)

TT210 YXEGTFTSDC”SIYLDKC”AAXC’FVNWLLC’G (SEQ 3.26 0.42

ID NO: 14)

TT211 YXEGTFTSDC’SIXLDKC’AQKC’FVQWLIC’G (SEQ ID 0.48 0.34

NO: 15)

TT212 YXEGTFTSDC”SIXLDKC”AQKC’FVQWLIC’G (SEQ ID 0.15 0.14

NO: 16)

TT213 YXEGTF’TSDC’SIXLDKC’AQKC’FVQWLIC’G^f(SEQ ID 0.08 0.06

NO: 17)

TT214 YXEGTF’TSDC”SIXLDKC”AQKC’FVQWLIC’G (SEQ 0.03 0.05

ID NO: 18)

TT215 YXEGTF’TSDC’SIXLDKC’AQK’C’FVQWLIC’G (SEQ 0.74 0.06

ID NO: 19)

TT216 YXEGTF’TSDC”SIXLDKC”AQK’C’FVQWLIC’G (SEQ 0.16 0.03

ID NO: 20)

TT217 YXEGTF’TSDC’SIXLDKC’AQKC’FVQWLIC’GK’ (SEQ 0.81 0.71

ID NO: 21)

TT218 YXEGTF’TSDC”SIXLDKC”AQKC’FVQWLIC’GK’ (SEQ 0.34 0.32

ID NO: 22)

TT219 YXEGTF’TSDC’SIXLDKC’AQKC’F’VQWLIC’G (SEQ 2.90 0.53

ID NO: 23)

TT220 YXEGTF’TSDC”SIXLDKC”AQKC’F’VQWLIC’G (SEQ 0.74 0.17

ID NO: 24)

TT221 YXEGTF’TSDC’SIXLDKC’AQKC’F’VQWLIC’GK’ (SEQ 5.20 0.87

ID NO: 25)

TT222 YXEGTF’TSDC”SIXLDKC”AQKC’F’VQWLIC’GK’ 1.60 0.54

(SEQ ID NO: 26)

TT223 YXEGTF’TSDC”SIXLDKC”AQKC”FVQWLIC”G (SEQ 0.07 0.07

ID NO: 27)

TT224 YXEGTF’TSDC”SIXLDKC”AQK’C”FVQWLIC”G (SEQ 0.58 0.08

ID NO: 28)

TT225 YXEGTF’TSDC”SIXLDKC”AQKC”FVQWLIC”GK’ 0.24 0.12

(SEQ ID NO: 29)

TT226 YXEGTF’TSDC”SIXLDKC”AQKC”F’VQWLIC”G (SEQ 0.04 0.04

ID NO: 30)

TT227 YXEGTF’TSDC”SIXLDKC”AQKC”F’VQWLIC”GK’ 0.37 0.11

(SEQ ID NO: 31)

TT228 YXEGTF’TSDC’SIXLDKC’AQK’C’F’VQWLIC’G (SEQ 9.20 0.45

ID NO: 32)

TT229 YXEGTF’TSDC”SIXLDKC”AQK’C’F’VQWLIC’G (SEQ 1.50 0.11

ID NO: 33)

TT230 YXEGTF’TSDC”SIXLDKC”AQK’C”F’VQWLIC”G (SEQ 0.56 0.06

ID NO: 34) ^a All peptides contain free N-terminus and aminated C-terminus. ^b K’ denotes lysine modified by PEG3-GI11-C18 fatty diacid; X = 2-aminoisobutyric acid (Aib). ^c K” denotes lysine modified by PEG3-GIU-C20 fatty diacid. ^d C’ denotes cysteine residue stapled with Bph. ^e C” denotes cysteine residue stapled with Bpy. ^f F’ = a-methyl-L-phenylalanine.

EXAMPLE 3

[0086] The following describes non-limiting examples of characterization and use of crosslinked peptides of the present disclosure.

[0087] Stability of the stapled GLP-1R/GIPR dual agonists in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Studies were carried out to examine the stability of stapled GLP-1R/GIPR agonist analogs in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) (Figures 2-6, Table 3).

[0088] SGF was prepared by dissolving 0.32 g pepsin (Promega, WI) with 100 mL 70 mM HC1 solution containing 0.2 g NaCl. Notably, 0.025% Tween-20 was included in SGF to improve the solubility of the peptides. For preparation of SIF, monobasic potassium phosphate (0.68 g) was dissolved in 25 mL water. A solution of 0.2 N NaOH (7.7 mL) was added to adjust the pH to 6.8. Then, 1 g pancreatin (Sigma-Aldrich, P3292) was added to the above solution. The mixture was shaken gently for 15 min before the volume was adjusted to 100 mL with water. After thorough mixing, undissolved material from the pancreatin was spun down with centrifugation and only the supernatant was used for the stability assay. Like SGF, 0.025% tween 20 was also included in the SIF to improve the solubility of the peptides. [0089] To test the stability of the double stapled peptide in SGF and SIF, 1 pL peptide stock solution in DMSO was mixed with 1 pL metoprolol water solution (as reference) and 98 pL SGF or SIF solution. The final peptide concentration is 30 pM. The mixture was incubated at 37 ° C with shaking. Samples (3 pL) were withdrawn at different intervals and mixed with 200 pL I-water (1 : 1) solvent. Samples were analyzed by QTOF-LC/MS.

[0090] Stability of the stapled GLP-1R/GIPR dual agonists in mouse serum. Studies were carried out to examine the stability of stapled GLP-1R/GIPR agonist analogs in mouse serum (Figure 7). One pL of peptide stock solution (3 mM in DMSO) was added to 99 pL fresh mouse serum (Invitrogen), and the mixture was incubated at 37 ° C with shaking. Samples (3 pL) were withdrawn at different intervals and mixed with 200 pL cold I-water (1 : 1) solvent, and the mixture was placed on ice for 30 min. Then, the sample mixture was centrifuged at 13,200 rpm for 5 min, and the supernatant was analyzed by QTOF-LC/MS.

[0091] Table 3. Stability of the doubly stapled peptides in the simulated gastric fluid and intestine fluid analyzed by LC-MS ^a.

1% SGF SGF 1% SIF SIF

Name , . . , . . , . . , . .

0/2 (mm) 0/2 (mm) 0/2 (mm) 0/2 (mm)

Desmopressin 201

TT201 29 <5 29

TT202 <10 3.8

TT203 <10 5.1

TT204 <10 3.3

TT205 11 78 12

TT206 <10 <2

TT207 <10 5-10

TT208 <10 6.3

TT209 <10 33 10

TT210 <10

TT211 <2 10

TT212 <10

TT213 <5 100

TT214 12

TT215 103

TT216 21

TT217 126

TT218 37

TT219 41 360

TT220 180

TT223 <3 4.4

TT224 4.2

TT225 8.8

TT226 <3 32

[0092] Although the present disclosure has been described with respect to one or more particular embodiment s) and/or examples, it will be understood that other embodiments and/or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS:

1. A compound comprising the following structure:

Y-X1-E-G-T-X2-T-S-D-Y-S-I-X3-L-D-K-X4-A-X5-X6-X7-X8-V-X9-W-L-X10-A-G (SEQ

ID NO: 1), wherein

XI is Aib;

X2 is Phe or a-MePhe;

X3 is Tyr or Aib;

X4 is Gin or He;

X5 is Ala or Gin;

X6 is Aib or Lys;

X7 is Glu or Ala;

X8 is Phe or a-MePhe;

X9 is Asn or Gin;

Xl 0 is Leu or He; and two amino acids at position i and i+7 are replaced with cysteines and crosslinked with a crosslinking group, and another two amino acids at position j and j+7 are also replaced with cysteines and crosslinked with a crosslinking group.

2. The compound of claim 1, wherein the crosslinking groups are independently at each

wherein Z is nitrogen or carbon, x is independently at each occurrence 1, 2, 3, 4 (wherein Z is carbon), and the R^x groups are each optionally present and, when present, is/are independently chosen at each occurrence from halide groups, alkyl groups, aryl groups, nitro group, amino group, alkylamino groups, polyethylene glycol (PEG) groups, lipid groups, and PEG groups conjugated to one or more fatty diacid(s).

3. The compound of claim 1, wherein i is at position 10 and j is at position 18 or 21; and the crosslinking group is Bph or Bpy.

4. The compound of claim 3, wherein j is at position 18.

5. The compound of claim 3, wherein j is at position 21.

6. The compound of claim 3, wherein:

XI is Aib;

X2 is Phe or a-MePhe;

X3 is Tyr or Aib;

X5 is Ala or Gin;

X6 is Aib or Lys;

X7 is Glu or Ala;

X8 is Phe or a-MePhe;

X9 is Asn or Gin; XI 0 is Leu or He; and the crosslinking group is Bph or Bpy.

7. The compound of claim 4, wherein

XI is Aib;

X2 is Phe or a-MePhe;

X3 is Tyr or Aib;

X4 is Gin or He;

X5 is Ala or Gin;

X6 is Aib or Lys;

X8 is Phe or a-MePhe;

X9 is Asn or Gin;

X10 is Leu or He; and the crosslinking group is Bph or Bpy.

8. The compound of claim 7, wherein Lys at position 20 is conjugated with ([2-(2-amino- ethoxy)-ethoxy]-acetyl)2-(YGlu)-CO-(CH2)i6-COOH on the epsilon-amino group of its side chain.

9. The compound of claim 7, wherein a Lys conjugated with ([2-(2-amino-ethoxy)-ethoxy]- acetyl)2-(YGlu)-CO-(CH2)i6-COOH on the epsilon-amino group of its side chain is appended at position 30.

10. The compound of claim 5, wherein

XI is Aib;

X2 is Phe or a-MePhe;

X3 is Tyr or Aib;

X5 is Ala or Gin;

X6 is Aib or Lys;

X8 is Phe or a-MePhe;

X9 is Asn or Gin;

X10 is Leu or He; and the crosslinking group is Bph or Bpy.

11. The compound of claim 10, wherein Lys at position 20 is conjugated with ([2-(2-amino- ethoxy)-ethoxy]-acetyl)2-(yGlu)-CO-(CH2)i6-COOH on the epsilon-amino group of its side chain.

12. The compound of claim 10, wherein a Lys conjugated with ([2-(2-amino-ethoxy)- ethoxy]-acetyl)2-(yGlu)-CO-(CH2)i6-COOH on the epsilon-amino group of its side chain is appended at position 30.

13. A composition comprising one or more compound(s) of claim 1.

14. The composition of claim 13, wherein the composition comprises one or more additional component(s).

15. The composition of claim 13, wherein the additional component(s) is/are permeation enhancer(s).

16. The composition of claim 15, wherein the permeation enhancer(s) is/are chosen from salcaprozate sodium (SNAC), sodium decanoate (Na caprate), octyl gallate, sodium octanoate (Na caprylate), lauryl gallate, dodecylmaltoside (DDM), sodium N-(8-[2- hydroxybenzoyl]amino)caprylate (SNAC), tetradecylmaltoside (TDM), palmitoyl carnitine, sodium chenodeoxy cholate (NaCDC), polyoxyl 15 hydroxystearate (Kolliphor HS 15), sodium deoxy cholate, polyoxyethylene (10) oleyl ether (Brij 10), sodium tetraglycocholate, polyethylene glycol hexadecyl ether, sodium ursodeoxy cholate, polyoxyethylene (10) cetyl ether, sodium glycocholate, Purebright, sodium taurocholate, diethylene glycol monoethyl ether (Transcutol ES), sodium cholate, polyoxyl-35 castor oil (Kolliphor EL), chenodeoxycholic acid, lauroglycol FCC, ethyl gallate, trimethyl chitosan, propyl gallate (PG), chitosan, and any combination thereof.

17. A method of lowering blood glucose level in an individual comprising administering one or more compound(s) of claim 1 to the individual, wherein the blood glucose level of the individual is lowered.

18. A method of inducing weight loss of an individual comprising administering one or more compound(s) of claim 1 to the individual, wherein the individual loses weight.

19. A method of activating GLP-1R and/or GIPR in individual comprising administering one or more compound(s) of claim 1 to the individual, wherein the GLP-1R and/or GIPR are activated in the individual.

20. A method for making a stapled peptide comprising two crosslinks, comprising: providing a peptide having the following structure:

wherein

[Xaa] is any amino acid or amino acids sequence;

Ri, R2, R3, and R4 are each independently a hydrogen or an alkyl group; b = 6 and d = 6, or b + c = 5 and c + d = 5; when b = 6 and d = 6, R5 and Rs are hydrogens, and R7 and Rs are protecting groups or vice versa; when b + c = 5 and c + d = 5, R5 and R7 are hydrogens, and Re and Rs are protecting groups, or vice versa; contacting the peptide with a first crosslinking agent/agents, wherein a first stapled peptide/peptides is/are formed; deprotecting the first stapled peptide(s); and contacting the first peptide(s) with a second crosslinking agent/agents, wherein a second stapled peptide/peptides having two crosslinks, or staples, is/are formed.

21. The method of claim 20, wherein the crosslinking agent is, independently at each occurrence, chosen from

wherein X is a suitable leaving group (LG) chosen independently at each occurrence from a chloride group, a bromide group, an iodide group, and a tosylate group, Z is independently at each occurrence nitrogen or carbon, x is independently at each occurrence 1, 2, 3, 4 (wherein Z is carbon) and the R^x groups are each optionally present and, when present, is/are independently chosen at each occurrence from halide groups, alkyl groups, aryl groups, nitro group, amino group, alkylamino groups, polyethylene glycol (PEG) groups, lipid groups, and PEG groups conjugated to one or more fatty diacid(s).

22. The method of claim 20, wherein the stapling results in formation of a crosslink, or staple, comprising, independently at each occurrence, the following structure:

wherein Z is nitrogen or carbon, x is independently at each occurrence 1, 2, 3, 4 (wherein Z is carbon), and the R^x groups are each optionally present and, when present, is/are independently chosen, when present, at each occurrence from halide groups, alkyl groups, aryl groups, nitro group, amino group, alkylamino group(s), PEG groups, lipid groups, and PEG groups conjugated to one or more fatty diacid(s).