GLP2 RECEPTOR AGONISTS AND METHODS OF USE

BACKGROUND OF THE INVENTION

The development of therapeutic agents is often hampered by short half-lives. The biological half-life of an agent is the time it takes for the agent to lose half of its pharmacologic, physiologic, or radiologic activity. As a result, patients are often administered higher dosages of a therapeutic agent more frequently, which can lead to reduced compliance, higher costs and greater risk of side effects. Accordingly, there is a need for generation of therapeutic agents with extended half-lives.

SUMMARY OF THE INVENTION

Disclosed herein is a peptide conjugate comprising:

- a) a peptide that modulates the GLP-2 receptor; and
- b) a staple attached to the peptide at a first amino acid and a second amino acid.

In some embodiments, the staple is of Formula (I):

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wherein

- A is an optionally substituted alkylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted —NR³-alkylene-NR³—, or —N—;
- X¹and X²are independently a bond, —C(═O)—, -alkylene-C(═O)—, —C(═O)-alkylene, or -alkylene-C(═O)NR³—, -alkylene-C(═O)NR³-alkylene-;
- wherein X¹is attached to a first amino acid of the peptide, and X²is attached to a second amino acid of the peptide;
- R is hydrogen or -(L)_s-Y;
- each L is independently —(CR¹R²)_v—, -alkylene-O—, —O-alkylene-, —C(═O)-alkylene-, -alkylene-C(═O)—, —NR³-alkylene-, -alkylene-NR³—, —S-alkylene-, -alkylene-S—, —S(═O)-alkylene-, -alkylene-S(═O)—, —S(═O)₂-alkylene, -alkylene-S(═O)₂—, —C(═O)—, —C(═O)NR³—, —NR³C(═O)—, —NR³C(═O)NR³—, —NR³C(═O)NR³-alkylene-, —NR³C(═O)-alkylene-NR³—, -alkylene-C(═O)NR³—, —C(═O)NR³-alkylene-, -alkylene-NR³C(═O)—, or —NR³C(═O)-alkylene-;
- v is 2-20;
- each R¹or R²is independently hydrogen, halogen, —CN, —OR^a, —SR^a, —S(═O)R^b, —NO₂, —NR^cR^d, —S(═O)₂R^d, —NR^aS(═O)₂R^d, —S(═O)₂NR^cR^d, —C(═O)R^b, —OC(═O)R^b, —CO₂R^a, —OCO₂R^a, —C(═O)NR^cR^d, —OC(═O)NR^cR^d, —NR^aC(═O)NR^cR^d, —NR^aC(═O)R^b, —NR^aC(═O)OR^a, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₅heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OR^a, or —NR^cR^d; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OR^a, —NR^cR^d,
- or R¹and R²are taken together to form a C₁-C₆cycloalkyl or C₁-C₆heterocycloalkyl;
- each R³is independently hydrogen, —S(═O)R^b, —S(═O)₂R^a, —S(═O)₂NR^cR^d, —C(═O)R^b, —CO₂R^a, —C(═O)NR^cR^d, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OR^a, or —NR^cR^d; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OR^a, or —NR^cR^d;
- Y is hydrogen, C₁-C₆alkyl, —CO₂H, —CO₂(C₁-C₆alkyl), —CO₂NH₂, —CO₂N(alkyl)₂, or —CO₂NH(alkyl); and
- s is 0-20;
- R^ais hydrogen, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- R^bis C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- each R^cand R^dis independently hydrogen, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- or R^cand R^d, together with the nitrogen atom to which they are attached, form a heterocycloalkyl or heteroaryl; wherein the heterocycloalkyl and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂.

Also disclosed herein is a pharmaceutical composition comprising a peptide conjugate described herein and a pharmaceutically acceptable excipient.

Also disclosed herein is a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a peptide conjugate described herein.

Also described herein is a staple of Formula:

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wherein

- A is an optionally substituted alkylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted —NR³-alkylene-NR³—, or —N—;
- X¹and X²are independently a bond, —C(═O)—, -alkylene-C(═O)—, —C(═O)-alkylene, -alkylene-C(═O)NR³—, or -alkylene-C(═O)NR³-alkylene-;
- Y¹and Y²are independently halogen, —COOH,

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or are independently the —S— of a sulfhydryl-containing amino acid or —CONH— wherein the —NH— is part of an amine-containing amino acid in a peptide that modulates the GLP-2 receptor;

- R is hydrogen or -(L)_s-Y;
- each L is independently —(CR¹R²)_v—, -alkylene-O—, —O-alkylene-, —C(═O)-alkylene-, -alkylene-C(═O)—, —NR³-alkylene-, -alkylene-NR³—, —S-alkylene-, -alkylene-S—, —S(═O)-alkylene-, -alkylene-S(═O)—, —S(═O)₂-alkylene, -alkylene-S(═O)₂—, —C(═O)—, —C(═O)NR³—, —NR³C(═O)—, —NR³C(═O)NR³—, —NR³C(═O)NR³-alkylene-, —NR³C(═O)-alkylene-NR³—, -alkylene-C(═O)NR³—, —C(═O)NR³-alkylene-, -alkylene-NR³C(═O)—, or —NR³C(═O)-alkylene-;
- v is 2-20;
- each R¹or R²is independently hydrogen, halogen, —CN, —OR^a, —SR^a, —S(═O)R^b, —NO₂, —NR^cR^d, —S(═O)₂R^a, —NR^aS(═O)₂R^d, —S(═O)₂NR^cR^d, —C(═O)R^b, —OC(═O)R^b, —CO₂R^a, —OCO₂R^a, —C(═O)NR^cR^d, —OC(═O)NR^cR^d, —NR^aC(═O)NR^cR^d, —NR^aC(═O)R^b, —NR^aC(═O)OR^a, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₅cycloalkyl, C₂-C₅heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OR^a, or —NR^cR^d; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OW, —NR^cR^d,
- or R¹and R²are taken together to form a C₁-C₆cycloalkyl or C₁-C₆heterocycloalkyl;
- each R³is independently hydrogen, —S(═O)R^b, —S(═O)₂R^a, —S(═O)₂NR^cR^d, —C(═O)R^b, —CO₂R^a, —C(═O)NR^cR^d, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OR^a, or —NR^cR^d; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OR^a, or —NR^cR^d;
- Y is hydrogen, C₁-C₆alkyl, —CO₂H, —CO₂(C₁-C₆alkyl), —CO₂NH₂, —CO₂N(alkyl)₂, or —CO₂NH(alkyl); and
- s is 0-20;
- R^ais hydrogen, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- R^bis C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- each R^cand R^dis independently hydrogen, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- or R^cand R^d, together with the nitrogen atom to which they are attached, form a heterocycloalkyl or heteroaryl; wherein the heterocycloalkyl and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂.

In some embodiments, A is optionally substituted alkylene. In some embodiments, A is —(CH₂)_t—, wherein t is 1-12. In some embodiments, A is optionally substituted arylene. In some embodiments, A is —NR³-alkylene-NR³—. In some embodiments, A is —N—.

In some embodiments, X¹and X²are —C(═O)—. In some embodiments, X¹and X²are -alkylene-C(═O)—. In some embodiments, X¹and X²are —CH₂—C(═O)—. In some embodiments, X¹and X²are independently -alkylene-C(═O)NR³—. In some embodiments, X¹and X²are independently —CH₂—C(═O)NR³—. In some embodiments, X¹and X²are independently -alkylene-C(═O)NR³-alkylene-. In some embodiments, X¹and X²are independently —CH₂—C(═O)NR³—CH₂CH₂—.

In some embodiments, >A-R has the following structure:

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- wherein r1 and r2 are each independently 0-4.

In some embodiments, >A-R has the following structure:

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In some embodiments, >A-R has the following structure:

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- wherein p1 is 1-5.

In some embodiments, >A-R has the following structure:

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In some embodiments, >A-R has the following structure:

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In some embodiments, s is 1-15. In some embodiments, s is 1-10. In some embodiments, s is 5-15. In some embodiments, s is 5-10.

In some embodiments, Y is hydrogen or —CO₂H.

In some embodiments, each L is independently —(CR¹R²)_v—, -alkylene-O—, —C(═O)—, —C(═O)NR³—, —NR³C(═O)—, -alkylene-C(═O)NR³—, or -alkylene-NR³C(═O)—; and v is 2-20.

In some embodiments, Y¹and Y²are halogen. In some embodiments, Y¹and Y²are —COOH. In some embodiments, Y¹and Y²are the —S— of two sulfhydryl-containing amino acids in a peptide that modulates the GLP-2 receptor.

In some embodiments, Y¹and Y²are the —S— of two sulfhydryl-containing amino acids in a peptide

In some embodiments, Y¹and Y²are —CONH— wherein the —NH— is part of two amine-containing amino acids in a peptide that modulates the GLP-2 receptor.

In some embodiments, Y¹and Y²—CONH— wherein the —NH— is part of two amine-containing amino acids in a peptide that modulates the GLP-2 receptor which are 7 amino acids apart.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A depicts a concentration-response curve of teduglutide and the long-acting GLP2R agonists to the human GLP2R with no serum added.

FIG. 1B depicts a concentration-response curve of teduglutide and the long-acting GLP2R agonists to the human GLP2R with serum added.

FIG. 2 depicts a concentration-response curve of teduglutide, the long-acting GLP2R agonists, and apraglutide to the mouse GLP2R.

FIG. 3 depicts a concentration-response curve of teduglutide and the long-acting GLP2R agonists to the cyno monkey GLP2R.

FIG. 4A depicts a concentration-response curve of teduglutide and the long-acting GLP2R agonists to GLP1R.

FIG. 4B depicts a concentration-response curve of teduglutide and the long-acting GLP2R agonists to GCGR.

FIG. 4C depicts a concentration-response curve of teduglutide and the long-acting GLP2R agonists to GIPR.

FIG. 5A depicts the thermal stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) over 4 days at 4° C.

FIG. 5B depicts the thermal stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) over 4 days at 25° C.

FIG. 5C depicts the thermal stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) over 4 days at 37° C.

FIG. 5D depicts the thermal stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) over 4 days at 70° C.

FIG. 6A depicts the stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) over 4 days at a pH of 3.3 and a temperature of 4° C.

FIG. 6B depicts the stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) over 4 days at a pH of 3.3 at room temperature.

FIG. 6C depicts the stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) over 4 days at a pH of 37.5 and a temperature of 4° C.

FIG. 6D depicts the stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) over 4 days at a pH of 37.5 at room temperature.

FIG. 6E depicts the stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) over 4 days at a pH of 8.9 and a temperature of 4° C.

FIG. 6F depicts the stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) over 4 days at a pH of 8.9 at room temperature.

FIG. 7A depicts the hepatic stability of long-acting GLP2-2G-1-EX4-L5A over 120 minutes.

FIG. 7B depicts the hepatic stability of long-acting GLP2-2G-10Nle-1-EX4-L5A over 120 minutes.

FIG. 7C depicts the hepatic stability of long-acting GLP2-2G-10Nle-1K-EX4-K5 over 120 minutes.

FIG. 8 depicts the mean plasma concentration of GLP2-2G-1-EX4-L5A over 96 hours in mice.

FIG. 9 depicts the mean plasma concentration of GLP2-2G-1-EX4-L5A over 504 hours in cyno monkeys.

FIG. 10 depicts the mean plasma concentration of GLP2-2G-10Nle-1-EX4-L5A over 96 hours in mice.

FIG. 11 depicts the mean plasma concentration of GLP2-2G-10Nle-1-EX4-L5A over 504 hours in cyno monkeys.

FIG. 12 depicts the mean plasma concentration of GLP2-2G-10Nle-1K-EX4-K5 over 96 hours in mice.

FIG. 13 depicts the mean plasma concentration of GLP2-2G-10Nle-1K-EX4-K5 over 504 hours in cyno monkeys.

FIG. 14A depicts the normalized length of the small intestine in wildtype mice that received no treatment, GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5A) and GLP2-2G-5-L5A (GLP2-2G-10Nle-1-EX4-L5A).

FIG. 14B depicts the normalized length of the small intestine in wildtype mice that received no treatment, GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5A) and GLP2-2G-5-L5A (GLP2-2G-10Nle-1-EX4-L5A).

FIG. 14C depicts the bodyweight over 11 days of wildtype mice that received no treatment, GLP2-2G-1-L5A (GLP2-2G-1-EX4) and GLP2-2G-5-L5A (GLP2-2G-10Nle-1-EX4).

FIG. 15A depicts the length of the small intestine in wildtype mice treated with GLP2-2G-10Nle-1K-EX4-K5 and untreated wildtype mice.

FIG. 15B depicts the weight of the small intestine in wildtype mice treated with GLP2-2G-10Nle-1K-EX4-K5 and untreated wildtype mice.

FIG. 15C depicts the length of the colon in wildtype mice treated with GLP2-2G-10Nle-1K-EX4-K5 and untreated wildtype mice.

FIG. 15D depicts the weight of the colon in wildtype mice treated with GLP2-2G-10Nle-1K-EX4-K5 and untreated wildtype mice.

FIG. 16A depicts the body weight over 12 days of mice with induced acute colitis that received no treatment, that were treated with GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5), and with cyclosporin A.

FIG. 16B depicts the normalized colon weight of mice with induced acute colitis that received no treatment, that were treated with GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5), and with cyclosporin A.

FIG. 16C depicts the normalized small intestine weight of mice with induced acute colitis that received no treatment, that were treated with GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5), and with cyclosporin A.

FIG. 16D depicts the crypt depth in the colon of wildtype mice and mice with induced acute colitis that received no treatment and that were treated with GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5).

FIG. 16E depicts the jejunum villi length of wildtype mice and mice with induced acute colitis that received no treatment and that were treated with GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5).

FIG. 17A depicts the body weight over 10 days of mice with induced acute colitis that received no treatment, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A.

FIG. 17B depicts the colon length of mice with induced acute colitis that received no treatment, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A.

FIG. 17C depicts the small intestine length of mice with induced acute colitis that received no treatment, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A.

FIG. 17D depicts the small intestine weight of mice with induced acute colitis that received no treatment, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A.

FIG. 17E depicts the height of the jejunum villi of mice with induced acute colitis that received no treatment, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A.

FIG. 17F depicts the proliferation index in the jejunum of mice with induced acute colitis that received no treatment, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A.

FIG. 17G depicts the pharmacokinetics of GLP2-2G-10Nle-1K-EX4-K5 and teduglutide in the mouse.

FIG. 18A depicts the change in percent of bodyweight over 8 days of mice with induced acute colitis that received no treatment, GLP2-2G-10Nle-L5A, and cyclosporin A.

FIG. 18B depicts the change in percent of bodyweight over 8 days of mice with induced acute colitis that received no treatment, GLP2-2G-1-EX4-L5A, and cyclosporin A.

FIG. 18C depicts the change in percent of bodyweight over 8 days of mice with induced acute colitis that received no treatment, GLP2-2G-10Nle-1K-EX4-K5, cyclosporin A.

FIG. 18D depicts the colon length of mice with induced acute colitis that received no treatment, that were treated with different long-acting GLP2R agonists, and cyclosporin A.

FIG. 18E depicts the colon weight of mice with induced acute colitis that received no treatment, long-acting GLP2R agonists, and cyclosporin A.

FIG. 18F depicts the small intestine length of mice with induced acute colitis that received no treatment, different long-acting GLP2R agonists, cyclosporin A.

FIG. 18G depicts the small intestine weight of mice with induced acute colitis that received no treatment, different long-acting GLP2R agonists, and cyclosporin A.

FIG. 18H depicts the gall bladder enlargement of mice with induced acute colitis that received no treatment, different long-acting GLP2R agonists, cyclosporin A.

FIG. 18I depicts the amount of occult blood in the stool of mice with induced acute colitis that received no treatment, different long-acting GLP2R agonists, cyclosporin A.

FIG. 18J depicts the pharmacokinetics of the long-acting GLP2R agonists at the 0.03 mg/kg dose.

FIG. 18K depicts the pharmacokinetics of the long-acting GLP2R agonists at the 0.1 mg/kg dose.

FIG. 18L depicts the pharmacokinetics of GLP2-2G-10Nle-L5A at both doses in an acute colitis mouse model.

FIG. 18M depicts the pharmacokinetics of GLP2-2G-1-EX4-L5A at both doses in an acute colitis mouse model.

FIG. 18N depicts the pharmacokinetics of GLP2-2G-10Nle-1K-EX4-K5 at both doses in an acute colitis mouse model.

FIG. 19A depicts the absolute change in bodyweight of mice with induced chronic colitis that received no treatment, that were treated with GLP2-2G-1-EX4-L5A, that were treated with cyclosporin, and that were treated with teduglutide.

FIG. 19B depicts the colon length of mice with induced chronic colitis that received no treatment, GLP2-2G-1-EX4-L5A, cyclosporin, and teduglutide.

FIG. 19C depicts the colon weight of mice with induced chronic colitis that received no treatment, GLP2-2G-1-EX4-L5A, cyclosporin, and teduglutide.

FIG. 19D depicts the small intestine weight of mice with induced chronic colitis that received no treatment, GLP2-2G-1-EX4-L5A, cyclosporin, and teduglutide.

FIG. 20A depicts the colon length of mice with induced chronic colitis that received no treatment, GLP2-2G-10Nle-1-EX4-L5A, cyclosporin, and teduglutide.

FIG. 20B depicts the colon weight of mice with induced chronic colitis that received no treatment, GLP2-2G-10Nle-1-EX4-L5A, cyclosporin, and teduglutide.

FIG. 20C depicts the small intestine length of mice with induced chronic colitis that received no treatment, GLP2-2G-10Nle-1-EX4-L5A, cyclosporin, and teduglutide.

FIG. 21A depicts serum levels of ALT in choline deficient mice that were untreated and that received GLP2-2G-5-EX4-L5A treatment, and in mice fed a normal diet.

FIG. 21B depicts serum levels of AST in choline deficient mice that were untreated and that received GLP2-2G-5-EX4-L5A treatment, and in mice fed a normal diet.

FIG. 21C depicts fibrosis scores in choline deficient mice that were untreated and that received GLP2-2G-5-EX4-L5A treatment, and in mice fed a normal diet.

FIG. 21D depicts steatosis in choline deficient mice that were untreated and that received GLP2-2G-5-EX4-L5A treatment, and in mice fed a normal diet.

FIG. 21E depicts lobular inflammation in choline deficient mice that were untreated and that received GLP2-2G-5-EX4-L5A treatment, and in mice fed a normal diet.

FIG. 22A depicts the body weight of male mice weaned to a conventional diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5.

FIG. 22B depicts the body weight of female mice weaned to a conventional diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5.

FIG. 22C depicts the body weight of male mice weaned to a deficient diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5.

FIG. 22D depicts the body weight of female mice weaned to a deficient diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5.

FIG. 22E depicts the normalized small intestine weight of male mice weaned to a conventional diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5.

FIG. 22F depicts the normalized small intestine weight of female mice weaned to a conventional diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5.

DETAILED DESCRIPTION OF THE INVENTION

Glucagon-like peptide 2 (GLP-2) is a hormone secreted from gut endocrine cells. GLP-2 stimulates intestinal growth, increases nutrient absorption and blood flow, decreases gut permeability and motility, and reduces epithelial cell apoptosis and inflammation. Due to the intestinotrophic effects of GLP-2, GLP-2 and related analogs may be useful for the treatment of GI disorders. In humans, the short plasma half-life of native GLP-2 requires higher doses and frequent injections or infusions to achieve a clinical efficacy, which can negatively affect patient compliance. Approaches have been utilized to extend the half-life of GLP-2, including PEGylation and fusion to polypeptides to increase molecular weight and hydrodynamic radius, and decrease the clearance rate through renal filtration. However, resulting analogs have suffered from reduced in vitro potency and, as a result, require higher doses to be effective in vivo.

Peptide Conjugates

In one aspect, disclosed herein are peptide conjugates comprising a peptide that modulates the GLP-2 receptor. In exemplary cases, the peptide that modulates the GLP-2 receptor comprises two amino acids connected by a staple. Non-limiting examples of amino acids for use in conjugation include cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, 2-amino-6-mercaptohexanoic acid, lysine, ornithine, diaminobutyric acid, diaminopropionic acid, homolysine, other sulfhydryl containing amino acids, or other amine containing amino acids. For a peptide that modulates the GLP-2 receptor comprising two amino acids connected by a staple, the two amino acids are about or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more amino acids apart. For example, the first amino acid has position i, and the second amino acid has position i+7, i+11, i+13, i+15, or i+16. For example, the first amino acid has a position i in the peptide and the second amino acid has a position i+n in the peptide, wherein n is 4-16. For example, the first amino acid has a position i in the peptide and the second amino acid has a position i+7 in the peptide. For example, the first amino acid has a position i in the peptide and the second amino acid has a position i+11 in the peptide. For example, the first amino acid has a position i in the peptide and the second amino acid has a position i+15 in the peptide. For example, the first amino acid has a position i in the peptide and the second amino acid has a position i+16 in the peptide.

Peptide that Modulates the GLP-2 Receptor

In one aspect, provided herein are peptide conjugates comprising a peptide that modulates the GLP-2 receptor. In some embodiments, a peptide that modulates the GLP-2 receptor is a GLP-2 receptor agonist.

The binding affinity of the peptide conjugate as described herein may be within about 5% of the binding affinity of an unmodified form of the GLP-2 peptide (e.g., the unconjugated GLP-2 peptide). The binding affinity of the peptide conjugate as described herein may be within about 10% of the binding affinity of an unmodified form of the GLP-2 peptide. The binding affinity of the peptide conjugate as described herein may be within about 15% of the binding affinity of an unmodified form of the GLP-2 peptide. The binding affinity of the peptide conjugate as described herein may be within about 20% of the binding affinity of an unmodified form of the GLP-2 peptide.

The peptide that modulates the GLP-2 receptor may comprise at least a portion of a wild-type GLP-2 peptide and may comprise one or more amino acid mutations. The one or more amino acid mutations may comprise a deletion, substitution, addition or a combination thereof. The one or more amino acid mutations may comprise adding one or more amino acid residues to a wild-type GLP-2 peptide. The one or more amino acid mutations may comprise deletion of one or more amino acid residues of the wild-type GLP-2 peptide. The one or more amino acid mutations may comprise substitution of one or more amino acid residues of the wild-type GLP-2 peptide. The one or more amino acid mutations may comprise substituting one or more amino acid residues of the wild-type GLP-2 peptide with one or more cysteine, lysine or other sulfhydryl or amine containing residues. The one or more amino acid mutations may comprise substituting one or more amino acid residues of the wild-type GLP-2 peptide with one or more D-amino acid residues. The one or more amino acid residues of the GLP-2 wild-type peptide may comprise one or more alanines, methionines, arginines, serines, threonines, and tyrosines.

The peptide that modulates the GLP-2 receptor may be modified with, for example, acetylation, phosphorylation, and methylation. The peptide modification may comprise a chemical modification. Peptide modifications may occur on the N-terminus of the peptide. Peptide modifications may comprise acetyling the amino group at the N-terminus of the peptide. Alternatively, or additionally, peptide modifications may occur on the C-terminus of the peptide. Peptide modifications may occur at one or more internal amino acids of the peptide. Peptide modifications may comprise replacing the carboxyl group at the C-terminus of the peptide. Peptide modifications may comprise modifying the carboxyl group at the C-terminus of the peptide. The carboxyl group at the C-terminus of the peptide may be modified to produce an amide group. The carboxyl group at the C-terminus of the peptide may be modified to produce an amine group.

Non-limiting examples of a peptide that modulates the GLP-2 receptor are shown in Table 1.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of any one of SEQ ID NOs: 1-40. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any one of SEQ ID NOs: 1-40. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to any one of SEQ ID NOS: 1-40.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of any one of SEQ ID NOs: 1-9. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any one of SEQ ID NOs: 1-9. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to any one of SEQ ID NOS: 1-9.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 1. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 1. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 1.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 2. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 2. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 2.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 3. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 3. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 3.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 4. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 4. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 4.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 5. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 5. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 5.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 6. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 6. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 6.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 7. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 7. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 7.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 8. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 8. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 8.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 9. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 9. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 9.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of any one of SEQ ID NOs: 10-20. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any one of SEQ ID NOs: 10-20. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to any one of SEQ ID NOS: 10-20.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 10. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 10. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 10.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 11. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 11. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 11.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 12. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 12. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 12.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 13. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 13. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 13.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 14. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 14. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 14.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 15. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 15. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 15.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 16. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 16. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 16.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 17. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 17. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 17.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 18. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 18. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 18.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 19. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 19. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 19.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 20. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 20. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 20.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of any one of SEQ ID NOs: 21-29. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any one of SEQ ID NOs: 21-29. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to any one of SEQ ID NOS: 21-29.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 21. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 21. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 21.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 2. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 22. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 22.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 23. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 23. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 23.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 24. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 24. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 24.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 25. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 25. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 25.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 26. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 26. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 26.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 27. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 27. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 27.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 28. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 28. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 28.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 29. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 29. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 29.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of any one of SEQ ID NOs: 30-40. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any one of SEQ ID NOs: 30-40. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to any one of SEQ ID NOS: 30-40.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 30. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 30. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 30.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 31. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 31. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 31.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 32. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 32. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 32.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 33. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 33. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 33.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 34. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 34. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 34.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 35. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 35. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 35.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 36. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 36. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 36.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 37. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 37. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 37.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 38. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 38. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 38.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 39. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 39. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 39.

In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence of SEQ ID NO: 40. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 40. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 40.

TABLE 1

SEQ ID Table

SEQ ID

NO.
Name
Sequence

1
GLP2-2G-1-EX4
HGDGSFSDEMNTILDNCAARDFICWLIQTKITDPSS

GAPPPS

2
GLP2-2G-10Nle-1-EX4
HGDGSFSDE(Nle)NTILDNCAARDFICWLIQTKITDP

SSGAPPPS

3
GLP2-2G-10L-1-EX4
HGDGSFSDELNTILDNCAARDFICWLIQTKITDPSS

GAPPPS

4
GLP2-2G-10I-1-EX4
HGDGSFSDEINTILDNCAARDFICWLIQTKITDPSSG

APPPS

5
GLP2-2G-10Aib-1-EX4
HGDGSFSDE(Aib)NTILDNCAARDFICWLIQTKITDP

SSGAPPPS

6
GLP2-2G-10Nva-1-EX4
HGDGSFSDE(Nva)NTILDNCAARDFICWLIQTKITDP

SSGAPPPS

7
GLP2-2G-10V-1-EX4
HGDGSFSDEVNTILDNCAARDFICWLIQTKITDPSS

GAPPPS

8
GLP2-2G-5-EX4
HGDGSFSDCMNTILDCLAARDFINWLIQTKITDPSS

GAPPPS

9
GLP2-2G-6-EX4
HGDGSFSDEMCTILDNLCARDFINWLIQTKITDPSS

GAPPPS

10
GLP2-2G-10Nle-1K-EX4
HGDGSFSDE(Nle)NTILDNKAARDFIKWLIQTKITDP

SSGAPPPS

11
GLP2-2G-10Nle-11f-1K-
HGDGSFSDE(Nle)(D-

EX4
Phe)TILDNKAARDFIKWLIQTKITDPSSGAPPPS

12
GLP2-2G-10Nle-12S-13L-
HGDGSFSDE(Nle)NSLLDNKAARDFIKWLIQTKITD

1K-EX4
PSSGAPPPS

13
GLP2-2G-10Nle-13A-
HGDGSFSDE(Nle)NTA(Nle)DNKAARDFIKWLIQTKI

14Nle-1K-EX4
TDPSSGAPPPS

14
GLP2-2G-10Nle-13Nle-
HGDGSFSDE(Nle)NT(Nle)(Nle)DNKAARDFIKWLIQ

14Nle-1K-EX4
TKITDPSSGAPPPS

15
GLP2-2G-10Nle-11A-13A-
HGDGSFSDE(Nle)ATA(Nle)DAKAARDFIKWLIQTKI

14Nle-16A-1K-EX4
TDPSSGAPPPS

16
GLP2-2G-1K-EX4
HGDGSFSDEMNTILDNKAARDFIKWLIQTKITDPSS

GAPPPS

17
GLP2-2G-11f-1K-EX4
HGDGSFSDEM(D-

Phe)TILDNKAARDFIKWLIQTKITDPSSGAPPPS

18
GLP2-2G-5K-EX4
HGDGSFSDKMNTILDKLAARDFINWLIQTKITDPSS

GAPPPS

19
GLP2-2G-10Nle-5K-EX4
HGDGSFSDK(Nle)NTILDKLAARDFINWLIQTKITDP

SSGAPPPS

20
GLP2-2G-10m-EX4
HGDGSFSDEMNTILDN(Orn)AARDFI(Orn)WLIQTKI

TDPSSGAPPPS

21

HGDGSFSDEMNTILDNCAARDFICWLIQTKITD

22

HGDGSFSDE(Nle)NTILDNCAARDFICWLIQTKITD

23

HGDGSFSDELNTILDNCAARDFICWLIQTKITD

24

HGDGSFSDEINTILDNCAARDFICWLIQTKITD

25

HGDGSFSDE(Aib)NTILDNCAARDFICWLIQTKITD

26

HGDGSFSDE(Nva)NTILDNCAARDFICWLIQTKITD

27

HGDGSFSDEVNTILDNCAARDFICWLIQTKITD

28

HGDGSFSDCMNTILDCLAARDFINWLIQTKITD

29

HGDGSFSDEMCTILDNLCARDFINWLIQTKITD

30

HGDGSFSDE(Nle)NTILDNKAARDFIKWLIQTKITD

31

HGDGSFSDE(Nle)(D-

Phe)TILDNKAARDFIKWLIQTKITD

32

HGDGSFSDE(Nle)NSLLDNKAARDFIKWLIQTKITD

33

HGDGSFSDE(Nle)NTA(Nle)DNKAARDFIKWLIQTKI

TD

34

HGDGSFSDE(Nle)NT(Nle)(Nle)DNKAARDFIKWLIQ

TKITD

35

HGDGSFSDE(Nle)ATA(Nle)DAKAARDFIKWLIQTKI

TD

36

HGDGSFSDEMNTILDNKAARDFIKWLIQTKITD

37

HGDGSFSDEM(D-

Phe)TILDNKAARDFIKWLIQTKITD

38

HGDGSFSDKMNTILDKLAARDFINWLIQTKITD

39

HGDGSFSDK(Nle)NTILDKLAARDFINWLIQTKITD

40

HGDGSFSDEMNTILDN(Orn)AARDFI(Orn)WLIQTKI

TD

embedded image

Staples

Disclosed herein are peptide conjugates comprising a staple.

In some embodiments, the staple attached to the peptide is of Formula (I):

embedded image

wherein

- A is an optionally substituted alkylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted —NR³-alkylene-NR³—, or —N—;
- X¹and X²are independently a bond, —C(═O)—, -alkylene-C(═O)—, —C(═O)-alkylene-, -alkylene-C(═O)NR³—, -alkylene-NR³C(═O)—, —C(═O)NR³-alkylene-, —NR³C(═O)-alkylene-, -alkylene-C(═O)NR³-alkylene-, or -alkylene-NR³C(═O)-alkylene-;
- wherein X¹is attached to a first amino acid of the peptide, and X²is attached to a second amino acid of the peptide;
- R is hydrogen or —X³-(L)_s-Y;
- X³is bond, —C(═O)—, -alkylene-C(═O)—, —C(═O)-alkylene, -alkylene-C(═O)NR³—, or -alkylene-C(═O)NR³-alkylene-;
- each L is independently —(CR¹R²)_v—, -alkylene-O—, —O-alkylene-, —C(═O)-alkylene-, -alkylene-C(═O)—, —NR³-alkylene-, -alkylene-NR³—, —S-alkylene-, -alkylene-S—, —S(═O)-alkylene-, -alkylene-S(═O)—, —S(═O)₂-alkylene, -alkylene-S(═O)₂—, —C(═O)—, —C(═O)NR³—, —NR³C(═O)—, —NR³C(═O)NR³—, —NR³C(═O)NR³-alkylene-, —NR³C(═O)-alkylene-NR³—, -alkylene-C(═O)NR³—, —C(═O)NR³-alkylene-, -alkylene-NR³C(═O)—, or —NR³C(═O)-alkylene-;
- v is 2-20;
- each R¹or R²is independently hydrogen, halogen, —CN, —OR^a, —SR^a, —S(═O)R^b, —NO₂, —NR^cR^d, —S(═O)₂R^a, —NR^aS(═O)₂R^d, —S(═O)₂NR^cR^d, —C(═O)R^b, —OC(═O)R^b, —CO₂R^a, —OCO₂R^a, —C(═O)NR^cR^d, —OC(═O)NR^cR^d, —NR^aC(═O)NR^cR^d, —NR^aC(═O)R^b, —NR^aC(═O)OR^a, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OR^a, or —NR^cR^d; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OR^a, —NR^cR^d;
- or R¹and R²are taken together to form a C₁-C₆cycloalkyl or C₁-C₆heterocycloalkyl;
- each R³is independently hydrogen, —S(═O)R^b, —S(═O)₂R^a, —S(═O)₂NR^cR^d, —C(═O)R^b, —CO₂R^a, —C(═O)NR^cR^d, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OR^a, or —NR^cR^d; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OR^a, or —NR^cR^d;
- Y is hydrogen, C₁-C₆alkyl, —CO₂H, —CO₂(C₁-C₆alkyl), —CO₂NH₂, —CO₂N(alkyl)₂, or —CO₂NH(alkyl); and
- s is 0-20;
- R^ais hydrogen, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- R^bis C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- each R^cand R^dis independently hydrogen, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- or R^cand R^d, together with the nitrogen atom to which they are attached, form a heterocycloalkyl or heteroaryl; wherein the heterocycloalkyl and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂.

In some embodiments, the staple attached to the peptide is of Formula (I):

embedded image

wherein

- A is an optionally substituted alkylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted —NR³-alkylene-NR³—, or —N—;
- X¹and X²are independently a bond, —C(═O)—, -alkylene-C(═O)—, —C(═O)-alkylene, -alkylene-C(═O)NR³—, or -alkylene-C(═O)NR³-alkylene-;
- wherein X¹is attached to a first amino acid of the peptide, and X²is attached to a second amino acid of the peptide;
- R is hydrogen or —X³-(L)_s-Y;
- X³is bond, —C(═O)—, -alkylene-C(═O)—, —C(═O)-alkylene, -alkylene-C(═O)NR³—, or -alkylene-C(═O)NR³-alkylene-;
- each L is independently —(CR¹R²)_v—, -alkylene-O—, —O-alkylene-, —C(═O)-alkylene-, -alkylene-C(═O)—, —NR³-alkylene-, -alkylene-NR³—, —S-alkylene-, -alkylene-S—, —S(═O)-alkylene-, -alkylene-S(═O)—, —S(═O)₂-alkylene, -alkylene-S(═O)₂—, —C(═O)—, —C(═O)NR³—, —NR³C(═O)—, —NR³C(═O)NR³—, —NR³C(═O)NR³-alkylene-, —NR³C(═O)-alkylene-NR³—, -alkylene-C(═O)NR³—, —C(═O)NR³-alkylene-, -alkylene-NR³C(═O)—, or —NR³C(═O)-alkylene-;
- v is 2-20;
- each R¹or R²is independently hydrogen, halogen, —CN, —OR^a, —SR^a, —S(═O)R^b, —NO₂, —NR^cR^d, —S(═O)₂R^a, —NR^aS(═O)₂R^d, —S(═O)₂NR^cR^d, —C(═O)R^b, —OC(═O)R^b, —CO₂R^a, —OCO₂R^a, —C(═O)NR^cR^d, —OC(═O)NR^cR^d, —NR^aC(═O)NR^cR^d, —NR^aC(═O)R^b, —NR^aC(═O)OR^a, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₅cycloalkyl, C₂-C₅heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OW, or —NR^cR^d; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OR^a, —NR^cR^d;
- or R¹and R²are taken together to form a C₁-C₆cycloalkyl or C₁-C₆heterocycloalkyl;
- each R³is independently hydrogen, —S(═O)R^b, —S(═O)₂R^a, —S(═O)₂NR^cR^d, —C(═O)R^b, —CO₂R^a, —C(═O)NR^cR^d, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OR^a, or —NR^cR^d; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OR^a, or —NR^cR^d;
- Y is hydrogen, C₁-C₆alkyl, —CO₂H, —CO₂(C₁-C₆alkyl), —CO₂NH₂, —CO₂N(alkyl)₂, or —CO₂NH(alkyl); and
- s is 0-20;
- R^ais hydrogen, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- R^bis C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- each R^cand R^dis independently hydrogen, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl, or heteroaryl; wherein the alkyl, alkenyl, alkynyl, and heteroalkyl is optionally substituted with one, two, or three of halogen, —OH, —OMe, or —NH₂; and the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂;
- or R^cand R^d, together with the nitrogen atom to which they are attached, form a heterocycloalkyl or heteroaryl; wherein the heterocycloalkyl and heteroaryl is optionally substituted with one, two, or three of halogen, C₁-C₆alkyl, C₁-C₆haloalkyl, —OH, —OMe, or —NH₂.

In some embodiments, A is optionally substituted alkylene. In some embodiments, A is —(CH₂)_t—, wherein t is 1-12. In some embodiments, A is —(CH₂)_t—, wherein t is 1-10. In some embodiments, A is —(CH₂)_t—, wherein t is 1-8. In some embodiments, A is —(CH₂)_t—, wherein t is 1-6. In some embodiments, A is —(CH₂)_t—, wherein t is 1-4.

In some embodiments, A is optionally substituted arylene. In some embodiments, A is arylene optionally substituted with halogen, alkyl, or haloalkyl. In some embodiments, A is arylene.

In some embodiments, A is —NR³-alkylene-NR³—.

In some embodiments, A is —N—.

In some embodiments, X¹and X²are identical. In some embodiments, X¹and X²are different.

In some embodiments, X¹and X²are —C(═O)—. In some embodiments, X¹and X²are independently -alkylene-C(═O)— or —C(═O)alkylene-. In some embodiments, X¹and X²are independently —CH₂—C(═O)— or —C(═O)—CH₂—. In some embodiments, X¹and X²are independently -alkylene-C(═O)NR³— or —C(═O)NR³-alkylene-. In some embodiments, X¹and X²are independently —CH₂—C(═O)NR³— or —C(═O)NR³—CH₂—. In some embodiments, X¹and X²are independently -alkylene-C(═O)NR³-alkylene- or -alkylene-NR³C(═O)-alkylene-. In some embodiments, X¹and X²are independently —CH₂—C(═O)NR³—CH₂CH₂— or —CH₂—NR³C(═O)—CH₂CH₂—. In some embodiments, X¹and X²are independently —CH₂—C(═O)NH—CH₂CH₂— or —CH₂—NHC(═O)—CH₂CH₂—.

In some embodiments, each R³is independently hydrogen or C₁-C₆alkyl. In some embodiments, each R³is hydrogen.

In some embodiments, >A-R has the following structure:

embedded image

wherein r1 and r2 are each independently 0-4.

In some embodiments, r1 and r2 are each independently 0-2. In some embodiments, r1 and r2 are each 0. In some embodiments, r1 and r2 are each 1. In some embodiments, r1 and r2 are each 3.

In some embodiments, >A-R has the following structure:

embedded image

In some embodiments, >A-R has the following structure:

embedded image

wherein p1 is 1-5.

In some embodiments, p1 is 1-3. In some embodiments, p1 is 1-2. In some embodiments, p1 is 1. In some embodiments, p1 is 2. In some embodiments, p1 is 3. In some embodiments, p1 is 4. In some embodiments, p1 is 5.

In some embodiments, >A-R has the following structure:

embedded image

In some embodiments, >A-R has the following structure:

embedded image

In some embodiments, s is 1-15. In some embodiments, s is 1-10. In some embodiments, s is 5-15. In some embodiments, s is 5-10. In some embodiments, s is 5-20.

In some embodiments, Y is hydrogen or —CO₂H. In some embodiments, Y is hydrogen. In some embodiments, Y is —CO₂H.

In some embodiments, each L is independently —(CR¹R²)_v—, -alkylene-O—, —C(═O)—, —C(═O)NR³—, —NR³C(═O)—, -alkylene-C(═O)NR³—, or -alkylene-NR³C(═O)—; and v is 2-16.

In some embodiments, v is 2-16. In some embodiments, v is 2-5. In some embodiments, v is 5-16. In some embodiments, v is 5 or 16. In some embodiments, v is 2 or 16.

In some embodiments, each R¹or R²is independently hydrogen, halogen, —CN, —OW, —NR^cR^d, —C(═O)R^b, —CO₂R^a, —C(═O)NR^cR^d, or C₁-C₆alkyl.

In some embodiments, each R¹or R²is independently hydrogen, halogen, —CO₂R^a, —C(═O)NR^cR^d, or C₁-C₆alkyl. In some embodiments, each R¹or R²is independently hydrogen, —CO₂R^a, or —C(═O)NR^cR^d. In some embodiments, each R¹or R²is independently hydrogen or —CO₂R^a.

In some embodiments, the staple is

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