GLP-1 agonists that induce prolonged GLP-1r activation

Abstract

Disclosed are GLP-1 agonists, and their use in the treatment of diseases such as diabetes, obesity, Alzheimer's disease, liver disease and cardiovascular diseases.

Description

[0001] GLP-1 AGONISTS THAT INDUCE PROLONGED GLP-1R ACTIVATION

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application Number 63 / 740,012, filed December 30, 2024; and U.S. Provisional Application Number 63 / 658,632, filed June 11, 2024. The contents of each of the aforementioned applications is incorporated by reference.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under GM130257, GM142448, DK131842, and AG061909 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0006] BACKGROUND

[0007] The Glucagon-like peptide- 1 (GLP-1) is a gut-derived peptide that mediates the release of insulin when glucose is ingested orally. The insulinotropic effects of GLP-1 are glucosedependent, thereby eliminating the risk of hypoglycemia making it a candidate for treating metabolic diseases. These benefits arise from the peptide ligand-induced stimulation of its cognate receptor, GLP-1R, resulting in the activation of downstream signaling pathways. However, the physiological effects of GLP-1 are rapidly tuned down by proteolytic inactivation, and via renal excretion. The ubiquitous serine protease, dipeptidyl-peptidase IV (DPP-IV) cleaves the N-terminal dipeptide 'His-Ala' from GLP-1, resulting in a shorter fragment that has a significantly diminished activity profile. Both intact and truncated peptides are also rapidly renally cleared. There are a couple of GLP-1 R agonists in the clinic that attempt to address these shortcomings.

[0008] SUMMARY

[0009] In some aspects, the present invention provides a peptide having the structure: wherein:

[0010] R1, R2and R3are each independently selected from H, fluoroalkyl, P1, and E1; wherein each P1independently comprises a lipid moiety; and each E1independently comprises an electrophilic moiety; provided that at least one of R1, R2and R3is E1;

[0011] X is a peptide moiety comprising the amino acid sequence EGTFTSDVSSYLEGQAA, or a sequence with at least 80% sequence identity thereto;

[0012] Y is a peptide moiety comprising the amino acid sequence EFIAWLV, or a sequence with at least 80% sequence identity thereto; and

[0013] Z is a peptide moiety comprising the amino acid sequence GR.

[0014] In some aspects, the present invention provides a method of treating diabetes, comprising administering to a subject in need thereof an effective amount of a peptide of the invention.

[0015] In some aspects, the present invention provides a method of treating obesity, comprising administering to a subject in need thereof an effective amount of a peptide of the invention.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 shows a timeline and representative concentration-response curves for washout at 4 h with the addition of Ex(9-39) for peptide variants. The luciferase assay was conducted with stably transfected HEK293 cells (GLP-1R); upon production of cAMP via receptor activation, luciferase is produced that is tightly correlated to the extent of receptor stimulation. After peptide addition, 3x washing steps were deployed at 4 h followed by the addition of Ex(9- 39) and a further incubation of 4 h. Control cells were not subjected to washing steps or to the addition of Ex(9-39). The total assay time was 8 h which encompassed the period between initial peptide addition through to cell lysis. The graphs represent the mean of one experiment performed in triplicate ± SEM. Fold-changes between control and washes are from Table 1. K34SulF, exendin-4, liraglutide, and semaglutide exhibited wash resistance whereas native GLP-1 and the derivatives with other warheads were unable to endure multiple washes.

[0018] FIGs. 2A-2C show representations of the volumes reactive partners occupy.

[0019] FIG. 2A shows that two atoms forming the covalent capture are assumed to fit in a sphere.

[0020] FIG. 2B shows that the motion of the warhead is restricted by the peptide binding to the receptor. For this reason, the assumption made was that it can occupy a half-sphere volume where the diameter is identified by the distance between the nucleophile and the electrophile.

[0021] FIG. 2C shows that the volume can get higher when a flexible linker is introduced.

[0022] FIGs. 3A & 3B show a timeline and representative concentration-response curve for luciferase assay conducted with stably transfected HEK293 cells (expressing GEP-1R + 6xCRElucif erase). Upon production of cAMP via receptor activation, luciferase is produced which is tightly correlated to the extent of receptor stimulation.

[0023] FIG. 3A shows the washout at 4 h experiment for GEP-1 and K34SulF. Cells were incubated with peptides for 4 h, followed by triple washes with serum-free expression media (100 pE / well). After the wash, all time points were incubated for an additional 4 h. Control peptides were not washed. The total assay time was 8 h which was the time from the addition of the peptide until cell lysis. The cell graphs represent the mean of one experiment performed in triplicate ± SEM. Fold-changes between control and washes are taken from Table 3. Warheaded peptide, K34SulF, was found to be more wash-resistant however the difference compared to the native GLP-1 was relatively modest.

[0024] FIG. 3B shows the graph represents the mean of one experiment performed in triplicate ± SEM. Ex(9-39) was found to be a partial agonist for GLP-1R.

[0025] FIG. 4 shows the experimental steps for the established washout assay. Peptides were initially incubated for 4 h. In order to detect whether there is covalent capture or not, cells were washed three times at 4 h, followed by orthosteric competitive binder Ex(9-39) addition to further displace the non-covalent peptides. Then, cells were incubated for an additional 4 h before readout. Control cells were incubated for 8 h without any treatment. GLP-1 was not fully removed by triple washes however it was further displaced by excess Ex(9-39) in the medium.

[0026] FIGs. 5A-5C show a timeline and representative concentration-response curves for combined washout and Ex(9-39) incubation. The luciferase assay was conducted with stably transfected HEK293 cells (expressing GLP-1R + 6xCREluciferase); upon production of cAMP via receptor activation, luciferase is produced that is tightly correlated to the extent of receptor stimulation. After peptide addition, 3x washing steps were deployed at different time points (at 4 h for part A, 12 h for part B, and 15 min for part C, followed by the addition of Ex(9-39) and a further incubation of 4 h. Control cells were not subjected to washing steps or to the addition of Ex(9-39). The total assay time was 8 h, 16 h, and 4 h 15 min for parts A, B, and C respectively, which encompassed the period between initial peptide addition through to cell lysis. The graphs represent the mean of one experiment performed in triplicate ± SEM.

[0027] FIG. 5A shows that adding a linker did not alter the results for PE and FPE warheads, suggesting the problem could be both sterics and low intrinsic reactivities towards solvent- exposed lysine.

[0028] FIG. 5B shows that incubating peptides for 12 h before the washout did not alter the results for K34Acr and K34PE; however, K34SulF retained its wash resistance.

[0029] FIG. 5C shows that all SulF analogues offered superior wash resistance compared to GLP-1, suggesting the covalent capture starts within 15 min. Inclusion of linkers between GLP- 1 and the SulF warhead had either no effect on or slightly decreased wash resistance.

[0030] FIG. 6 shows a timeline and representative concentration-response curves for the washout at 4 h experiment with N-AcGLPl and benzyl amine treated sample. The reaction mixture was used in luciferase assay which was conducted with stably transfected HEK293 cells (GLP-1R); upon production of cAMP via receptor activation, luciferase is produced that is tightly correlated to the extent of receptor stimulation. After peptide addition, 3x washing steps were deployed at 4 h, and a further incubation of 4 h. Control cells were not subjected to washing steps. The total assay time was 8 h which encompassed the period between initial peptide addition through to cell lysis. The graphs represent the mean of one experiment performed in triplicate ± SEM. Benzylamine incubation had no appreciable effect on N- AcGLPl potency after washing, reflecting no impact on (essentially absent) wash resistance as expected.

[0031] FIG. 7 shows structures of Exendin analogues with SulF warhead (top box) and lipidated GLP-1 analogues with N-terminal CH2CF3 modifications (bottom box).

[0032] FIG. 8A-8G show LC ESI-MS spectra of the covalent adducts from the reaction of .SWF-containing peptides and ECD of GLP-1 R. Molecular weights are average masses calculated using Peptide Mass Calculator v3.2 (Katholieke Universiteit, Leuven, Belgium).

[0033] FIG. 8A shows the calculated and observed molecular weights for ECD of the GLP- 1R (Mcaic 15546.33, Mobs15546.35). FIG. 8B shows the calculated and observed molecular weights for the adduct of the ECD of the GLP-1R with K34SulF (Mcaic19010.00, Mobs19009.68).

[0034] FIG. 8C shows the calculated and observed molecular weights for adduct of the ECD of the GLP-1R with K34_BetaAlaSulF (Mcaic19081.00, Mobs19081.38).

[0035] FIG. 8D shows the calculated and observed molecular weights for adduct of the ECD of the GLP-1R with K34_OEGSulF (Mcaic19155.00, Mobs19154.95).

[0036] FIG. 8E shows the calculated and observed molecular weights for adduct of the ECD of the GLP-1R with K34_OEGOEGSulF (Mcai19300.50, Mobs19300.10).

[0037] FIG. 8F shows the calculated and observed molecular weights for adduct of the GLP- 1R with Ex4_K27SulF (Mcaic19898.96, Mobs19899.34).

[0038] FIG. 8G shows the calculated and observed molecular weights for adduct of the ECD of the GLP-1R with Ex4_S33KSulF (Mcaic19940.05, Mobs19940.20).

[0039] FIG. 9 shows a timeline and representative concentration-response curves for combined washout at 4 h, Ex(9-39) and 1% HSA incubation for lipidated and SulF analogues. Luciferase assay was conducted with stably transfected HEK293 cells where luciferase was produced in a cAMP-dependent pathway that is correlated to receptor activation. Peptides were incubated for 4 h, followed by triple washes and subsequent incubation with (i) Ex(9-39), (ii) 1% HSA, (iii) Ex(9-39) in 1% HSA for an additional 4 h. Control peptides were not washed. The total assay time was 8 h which was the time passed from the initial peptide addition to cell lysis. The cell graphs represent the mean of one experiment performed in triplicate ± SEM. Warhead peptide was more wash-resistant under each condition.

[0040] FIG. 10 shows the number of cells left after the washout experiment. The graph represents the mean of the experiments performed in triplicate ± SEM. Each experiment has 48 wells as control and 48 wells treated with washes. After the washout experiment was done (triple washes with serum-free expression media), cells were treated with 100 pL / well 0.05% Trypsin / EDTA media for 5 min twice. Each time, wells were rinsed with growth solution (100 pL / well). After collecting, the standard procedure was followed to count cells with Countess (automated cell counter). The washout experiment does not lead to significant cell loss.

[0041] FIG. 11 shows a timeline and concentration response curves for GLP-1, K34SulF, and semaglutide in the presence (and absence) of human serum albumin (HSA, 0.01% and 2%). Fold-shifts for GLP-1 and K34SulF were negligible when incubated with 0.01% HSA and were less than 5-fold when incubated with 2% HSA. On the other hand, semaglutide suffers significant potency loss when incubated with 2% HSA (~600-fold). Fold shifts calculated from the average of two independent experiments. FIG. 12 is a scheme showing the synthesis of warheaded peptides where electrophiles were directly added to Lys34 on the peptide. For generating linker versions, OEG and BetaAla groups were added after Alloc deprotection and before warhead coupling.

[0042] FIG. 13 is a scheme showing the synthesis of C2K26DAC18_K34SulF that has trifluoroethyl on the N-terminus, a diacid C18 side chain on Lys26, and an arylsulfonyl fluoride warhead on Lys34.

[0043] DETAILED DESCRIPTION

[0044] While variants of GLP-1 have been clinically approved for both type 2 diabetes (T2D) and obesity, and have captured the imagination of the public and the popular press, there is plenty of room for improvement when it comes to drug-like properties. We hypothesized that inserting electrophilic warheads on GLP-1 to cross-link the peptide to the receptor can extend the residence time to engender long-lived activation of GLP-1 R. A covalently captured peptide would be protected from proteases when bound to the receptor and would not be renally cleared. Prolonged activation would yield receptors that are permanently in the 'on' state, the equivalent of a "constitutively active" receptor, in this case, the outcome triggered by a ligand. Using structure-guided design, GLP-1 was adorned with various electrophilic warheads to achieve crosslinking in a proximity-enabled manner that is propelled by high effective molarity. We highlight factors important for receptor activation engendered by covalent capture, as they vary when combined with N-terminal modifications and the addition of lipids to GLP-1.

[0045] Four electrophiles were chosen to function as 'warheads' to target Lysl 13 (on receptor) with reactivities in decreasing order: aryl sulfonyl fluoride (SulF) > p-fluorophenyl ester (FPE) > phenyl ester (PE) > acryloyl (Acr).19-24 The selected electrophiles have minimal or extremely sluggish reactivity with solvent water and neutral amines at physiological pH.19, 21, 23-27 The warheads were coupled to Lys34 on GLP-1, as well as in combination with (i) N- terminal modifications like acyl or trifluoroethyl incorporation, and (ii) lipid addition on Lys26. The activity profiles of GLP-1 analogues were measured by a luciferase assay. Briefly, peptides were incubated with HEK293 cells that were stably transfected with GLP-1R and CRE- luciferase. Upon receptor stimulation, downstream signaling results in the production of the enzyme luciferase, which can be quantified by the addition of substrate and measurement of chemiluminescence. In order to interrogate the ability of the electrophile decorated GLP-1 variants to be covalently captured by the receptor, we deployed washing steps at various times after incubation of the peptide with cells to remove any unreacted (free) peptide that remained. These experiments therefore provide an index of crosslinking (covalent capture by the receptor) where the warhead-bearing peptide is expected to be wash-resistant, and whereas the native ligand would not survive stringent washing steps. In order to make sure that a majority of the noncovalently bound peptides were removed, we introduced Ex(9-39) (an orthosteric competitive binder of GLP-1R) to the assay after the washout step.11, 28-30

[0046] The washout combined with the addition of Ex(9-39) resulted in a dramatic shift for native GLP-1 which was over five-thousand-fold increased EC50 value (concentration where half-maximal receptor stimulation is achieved). This suggests that less than 0.02% of the ligand remained bound to the receptor that enables agonist function. Neither the phenyl esters (K34PE, K34FPE) nor the acryloyl (K34Acr) group containing variants of GLP-1 exhibited any substantial increase in wash resistance when compared to the native peptide. Then, various linkers that differ in length, flexibility, and chemical functionality were added to K34PE and K34FPE to test the factors of sterics / geometry. Changes in linker properties however still did not result in a significant change in wash resistance suggesting that either the efforts to enable a beneficial geometry were unsuccessful or the slow reaction kinetics dictated the outcome. When the K34SulF derivative was tested under similar conditions with the washing step employed at 4 h with Ex(9-39), it exhibited only a 59-fold potency loss. This greatly reduced shift constitutes a dramatic difference when compared to the findings with native GLP-1, that suffered a much larger (5400-fold) diminution in potency when subjected to identical conditions (Table 1, FIG. 1). In addition, K34SulF still displays a regular sigmoidal profile in concentration-response curve, which is in stark contrast to unmodified GLP-1, and other tested derivatives. We also added linkers to SulF analogues to optimize the length and geometry. K34SulF is the most effective at resisting washouts (59-fold) and superior to K34_OEGSulF and K34_OEGOEGSulF that exhibited 384- and 443-fold lower EC50 values upon washing. Another linker containing variant K34_BetaAlaSulF had a minimal negative impact on peptide potency and also reduced washout resistance (166-fold potency loss). While some improvement in warheaded peptide potency by linker insertion could be achieved, all linkers that we tested led to reduced wash resistance possibly by suboptimal positioning of the SulF moiety.

[0047] We tested liraglutide and semaglutide for wash resistance which are FDA-approved GLP-1 analogues that are lipidated via side chain acylation. Semaglutide lost 63-fold in potency after washing followed by Ex(9-39) treatment, whereas liraglutide suffered a 210-fold loss. Given the observation that semaglutide displays superior wash resistance, we wanted to combine the positive attributes of its C18 diacid lipid chain with the promising SulF warhead. Despite the potency decrease with SulF addition, the wash resistance improved for K26DAC18_K34SulF compared to its non-warheaded control K26DAC18 (191- vs 1800-fold decrease, Table 1). Although the lipid addition and warhead addition separately provided more robust wash resistance compared to GLP-1 (K26DAC18 1800-fold and K34SulF with 59-fold potency losses, compared to 5400-fold for native GLP-1), the combination of these modifications did not yield the most promising compound (K26DAC18_K34SulF with a 191- fold drop after washout, Table 1). The lipid chain could force and potentially lock the peptide into certain futile conformations, thereby hampering the ability of its warhead to access nucleophilic residues on the receptor ECD. In summary, whereas incorporating lipidation as well as a warhead could both be successful strategies to increase agonist residence time on the receptor, the combination of these two modifications may not necessarily further improve wash resistance.

[0048] Next, we wanted to explore how N-terminal modifications influence wash resistance. Although incorporating a trifluoroethyl group (C2GLP-1) did not affect the potency of the native ligand dramatically during an 8 h incubation (EC50 values are 57 pM vs 88 pM for GLP- 1 and C2GLP-1, respectively), it was tougher to wash away (1200-fold decrease compared to 5400-fold for GLP-1, Table 1). The addition of the small trifluoroethyl moiety afforded similar outcomes for other peptides where C2K26DAC18, C2K26DAC18_K34SulF, and C2K26MAC16_K34R were found to be more wash -resistant compared to the N-terminally unmodified compound controls. Intriguingly, the semaglutide series did not follow the same trend. The addition of the N-trifluoroethyl group diminished the potency of semaglutide derivative C2K26DAC18_K34R (Aib8Ala) when assessed after an 8 h incubation and also did not result in improved wash resistance. It is likely that semaglutide is already at the cusp of most effective binding and does not benefit from additional cross -linking. In contrast, addition of the warhead improved the wash resistance for K26DAC18 significantly (1800- to 191-fold change, Table 1). The same trend was observed for C2K26DAC18 where the inclusion of the SulF group markedly improved the fold-change after wash from 102- to 47-fold (Table 1). The designed variant C2K26DAC18_K34SulF that flaunts three different decorations, an N- terminal trifluoroethyl that is protective of the frontline protease DDP4; a Cl 8 acid lipid acylation that helps bind albumin and delays renal elimination, can be even further improved by the addition of a chemical warhead that is capable of cross-linking with the receptor. This analogue is the most wash resistant compound among all the derivatives tested and even trumps the clinically approved compounds, liraglutide and semaglutide. This is a remarkable result and one that may help in further design and optimization of longer-lived GLP-1 R agonists. Table 1. Fold-change in activity after a washout experiment at 4 h with the addition of Ex(9-39).

[0049] Control6

[0050] Peptide" pECso ± SEM ECso (pM) n Fold-Shift

[0051] GLP-1 10.2 57 18 N / A

[0052] K34SulF 9.7 + 0.01 206 4 3.6 ’

[0053] K34_Acr 10.0 + 0.02 91 3 1.6 ’

[0054] K34_PE 9.9 + 0.003 120 3 2.1 ’

[0055] K34_FPE 10.0 + 0.08 94 2 1.6 ’

[0056] K34_BetaAl 9.5 + 0.008 288 4 5.0 ’ aSulF

[0057] K34_OEGS 10.0 + 0.01 109 4 1.9 ’ ulF

[0058] K34_OEGO 10.2 + 0.009 59 4 1.0 ’

[0059] EGSulF

[0060] Exendin-4 11.3 + 0.01 4.6 3 12 -

[0061] Liraglutide 10.8 + 0.01 17 3 3.4 -

[0062] Semaglutide 10.8 + 0.02 17 3 3.4 -

[0063] K26DAC18 10.6 + 0.002 27 8 2.1 -

[0064] K26DAC18. 9.8 + 0.003 156 7 2.7 ’

[0065] K34SulF

[0066] C2K26DAC 10.1 + 0.004 73 3 1.3 ’

[0067] 18

[0068] C2K26DAC

[0069] 9.2 + 0.02 633 3 11 ’

[0070] 18_K34SulF

[0071] C2GLP-1 10.0 + 0.01 101 3 1.8 ’

[0072] C2K26MAC 9.7 + 0.03 183 3 3.2 ’

[0073] 16.K34R

[0074] C2K26DAC

[0075] 10.2 + 0.01 61 3 1.1 ’

[0076] 18.K34R

[0077] Washed @4 h, Addition of Ex(9-39)cneFold-Shift(’ )s GLP-1 6.5 + 0.002 > 10518 5400

[0078] K34SulF 7.9 + 0.01 1.2 x lO43 59

[0079] K34_Acr 6.3 + 0.12 4.5 x 1053 5000

[0080] K34_PE 6.1 + 0.01 7.7 x lO53 6400

[0081] K34_FPE 6.0 + 0.02 1.0 x lO62 > 104

[0082] K34_BetaAlaS

[0083] 5.6 + 0.01 4.8 x lO44 166 ulF

[0084] K34_OEGSul

[0085] 7.2 + 0.01 4.2 x lO44 386

[0086] F

[0087] K34_OEGOE

[0088] 7.6 + 0.02 2.6 x lO44 443

[0089] GSulF

[0090] Exendin-4 10.5 + 0.01 31 3 6.8

[0091] Liraglutide 8.5 + 0.02 3200 3 193

[0092] Semaglutide 9.0 + 0.03 1000 3 63

[0093] K26DAC18 7.3 + 0.01 4.8 x 1047 1800

[0094] K26DAC18.K

[0095] 7.5 + 0.01 3.0 x lO46 191

[0096] 34SulF

[0097] C2K26DAC18 8.1 + 0.03 7500 3 102

[0098] C2K26DAC18

[0099] 7.5 + 0.01 2.9 x lO43 47

[0100] _K34SulF

[0101] C2GLP-1 7.0 + 0.03 9.2 x 1043 916

[0102] C2K26MAC1

[0103] 8.1 + 0.04 7900 3 43

[0104] 6.K34R

[0105] C2K26DAC18

[0106] 8.3 + 0.01 4700 3 78

[0107] _K34RaStructures of the peptides are displayed after text.bControl cells were incubated with peptide for 8 h without any further treatment.cCells were incubated with peptide for 4 h, then washed three times. After the washing step, 3 pM of the competitive binder Ex(9-39) was added, and cells were incubated for an additional 4 h before they were lysed.dECso(pM) is the concentration of peptide required for half-maximal activity of the targeted receptor. pECso = - log(ECso) ± standard error of the mean (SEM) of independent experiments.eNumber of independent experiments conducted. Fold- shift from native peptide, GLP-1 during the 8 h assay. Calculated as (EC50 ligand) / (ECso GLP-1).8Fold-shift from no-wash control. Calculated as (EC50 wash + Ex(9-39)) / (ECso no-wash control).

[0108] We report here a structure-guided design of GLP-1 analogues with the ability to induce prolonged receptor (GLP-1R) activation. We established a modified luciferase assay, one that includes washing steps combined with the addition of a competitive binder, Ex(9-39). This experiment allows linking the endurance to washouts as an index of covalent capture that prolongs ligand residence at the receptor. Among the various warheads with differential chemical reactivities and potential for cross-linking, the arylsulfonyl fluoride (SulF) group emerged as a suitable warhead that is efficiently captured, along with position Lys34 of the ligand as the site of attachment. Aside from improved pharmacokinetics, extended activation of the GLP-1R is useful for dissecting multi-pronged receptor-mediated downstream signaling cascades. Although GPCRs have been extensively studied mechanistically, many questions remain about what factors dictate downstream signaling and bias the system towards preferred versus undesirable outcomes. The chemical-biological approach described here could also be beneficial for fine-tuning receptor activation by modulating the equilibrium between distinct intracellular messenger pathways. Modifications as described in the current study could be used to further probe ligand- induced receptor activation, internalization, and biased signaling, as well as to generate next-generation GLP-1 therapeutics with an extended half-life, enhanced efficacy, and an improved safety profile.

[0109] In some aspects, the present invention provides a peptide having the structure: wherein:

[0110] R1, R2and R3are each independently selected from H, fluoroalkyl, P1, and E1; wherein each P1independently comprises a lipid moiety; and each E1independently comprises an electrophilic moiety; provided that at least one of R1, R2and R3is E1;

[0111] X is a peptide moiety comprising the amino acid sequence EGTFTSDVSSYLEGQAA, or a sequence with at least 80% sequence identity thereto; Y is a peptide moiety comprising the amino acid sequence EFIAWLV, or a sequence with at least 80% sequence identity thereto; and

[0112] Z is a peptide moiety comprising the amino acid sequence GR.

[0113] In certain embodiments, R1is H. In other embodiments, R1is fluoroalkyl. In yet other embodiments, R1is P1. In yet further embodiments, R1is E1. In certain embodiments, R3is:

[0114] In certain embodiments, R2is H. In other embodiments, R2is fluoroalkyl. In yet other embodiments, R2is P1. In yet other further embodiments, R2is E1.

[0115] In certain embodiments, R3is H. In other embodiments, R3is fluoroalkyl. In yet other embodiments, R3is P1. In yet further embodiments, R3is E1.

[0116] In certain embodiments, E1is independently selected from the group consisting of sulfonyl fluoride, acryloyl, and aryl carbamate. In certain embodiments, E1is independently selected from the group consisting of: wherein L’ is a bond or a linker moiety.

[0117] In certain embodiments, each other embodiments, each In yet other embodiments, each E1is . In yet other further embodiments, each

[0118] In certain embodiments, L’ is a linker moiety having the structure: wherein * indicates the point of attachment to the peptide moiety; and n is 1 to 4. is H . In yet other embodiments, L’ is a bond.

[0119] In certain embodiments, P1is: wherein m is 3 to 10; and n is 1 to 4.

[0120] In certain embodiments, m is 8.

[0121] In certain embodiments, n is 2.

[0122] In certain embodiments, R1is fluoroalkyl. In further embodiments, R1is 1,1,1- trifluoroethyl.

[0123] In certain embodiments, X is a peptide moiety having at least a 90% sequence identity to EGTFTSDVSSYLEGQAA. In certain embodiments, X is a peptide moiety having at least a 95% sequence identity to EGTFTSDVSSYLEGQAA. In certain embodiments, X is a peptide moiety having at least a 98% sequence identity to EGTFTSDVSSYLEGQAA. In certain embodiments, X is a peptide moiety comprising the amino acid sequence EGTFTSDVSSYLEGQAA.

[0124] In certain embodiments, Y is a peptide moiety having at least a 90% sequence identity to EFIAWL. In certain embodiments, Y is a peptide moiety having at least a 95% sequence identity to EFIAWL. In certain embodiments, Y is a peptide moiety having at least a 98% sequence identity to EFIAWL. In certain embodiments, Y is a peptide moiety comprising the amino acid sequence EFIAWL.

[0125] In certain embodiments, Z is the amino acid sequence GR.

[0126] In certain embodiments, R1is 1,1,1 -trifluoroethyl;

[0127] In certain embodiments, the peptide is selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

[0128] In further aspects, the present disclosure provides a pharmaceutical composition comprising a peptide disclosed herein; and a pharmaceutically acceptable excipient.

[0129] In yet further aspects, the present disclosure provides a method of treating diabetes in a subject in need thereof, comprising administering an effective amount of a peptide disclosed herein to the subject.

[0130] In yet further aspects, the present disclosure provides a method of treating obesity in a subject in need thereof, comprising administering an effective amount of a peptide disclosed herein to the subject.

[0131] Definitions

[0132] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

[0133] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0134] Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0135] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0136] It is understood that substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0137] As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O- alkyl, -OP(O)(O-alkyl)2 or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted.

[0138] Articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0139] As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to C1-C10 straight-chain alkyl groups or C1-C10 branched-chain alkyl groups. Preferably, the “alkyl” group refers to Ci-Ce straight-chain alkyl groups or Ci-Ce branched- chain alkyl groups. Most preferably, the “alkyl” group refers to C1-C4 straight-chain alkyl groups or C1-C4 branched-chain alkyl groups. Examples of “alkyl” include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1 -pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1 -hexyl, 2-hexyl, 3-hexyl, 1 -heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1 -octyl, 2-octyl, 3-octyl or 4-octyl and the like. The “alkyl” group may be optionally substituted.

[0140] The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0141] The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0142] The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0143] The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

[0144] The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl- O- alkyl.

[0145] The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Ci- 30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.

[0146] Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc.

[0147] The term “Cx.y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. Coalkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A Ci-6alkyl group, for example, contains from one to six carbon atoms in the chain.

[0148] The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group.

[0149] The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0150] The term “amide”, as used herein, refers to a group R9

[0151] R10•> wherein R9and R10each independently represent a hydrogen or hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0152] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by wherein R9, R10, and R10’ each independently represent a hydrogen or a hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0153] The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

[0154] The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group.

[0155] The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0156] The term “carbamate” is art-recognized and refers to a group wherein R9and R10independently represent hydrogen or a hydrocarbyl group.

[0157] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0158] The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct- 3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-lH- indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom.

[0159] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0160] The term “carbonate” is art-recognized and refers to a group -OCO2-.

[0161] The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H.

[0162] The term “ester”, as used herein, refers to a group -C(O)OR9wherein R9represents a hydrocarbyl group.

[0163] The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl- O-alkyl. The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

[0164] The terms “hetaralkyl” and “hetero aralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.

[0165] The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[0166] The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0167] The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.

[0168] The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0169] The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbonhydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof. The term “hydroxyalkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.

[0170] The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0171] The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

[0172] The term “sulfate” is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0173] The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae wherein R9and R10independently represents hydrogen or hydrocarbyl.

[0174] The term “sulfoxide” is art-recognized and refers to the group-S(O)-.

[0175] The term “sulfonate” is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0176] The term “sulfone” is art-recognized and refers to the group -S(O)2-.

[0177] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[0178] The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.

[0179] The term “thioester”, as used herein, refers to a group -C(O)SR9or -SC(O)R9. wherein R9represents a hydrocarbyl.

[0180] The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

[0181] The term “urea” is art-recognized and may be represented by the general formula wherein R9and R10independently represent hydrogen or a hydrocarbyl.

[0182] The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

[0183] The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0184] “Salt” is used herein to refer to an acid addition salt or a basic addition salt. Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01 / 062726.

[0185] Furthermore, certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers.

[0186] Some of the compounds may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

[0187] “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.

[0188] “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2- hydroxyethanesulfonic acid, benzenesulfonic acid, chlorobenzenesulfonic acid, 2- naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] -oct-2-ene-l -carboxylic acid, glucoheptonic acid , 3 -phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid , gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion , an alkaline earth ion , or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like. Salts further include, by way of example only, sodium potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functionality, salts of nontoxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0189] The term “pharmaceutically acceptable cation” refers to an acceptable cationic counterion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like (see, e. g., Berge, et al., J. Pharm. Sci. 66 (1): 1-79 (January 77).

[0190] “Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.

[0191] “Pharmaceutically acceptable metabolically cleavable group” refers to a group which is cleaved in vivo to yield the parent molecule of the structural formula indicated herein. Examples of metabolically cleavable groups include -COR, -COOR, -CONRR and -CH2OR radicals, where R is selected independently at each occurrence from alkyl, trialkylsilyl, carbocyclic aryl or carbocyclic aryl substituted with one or more of alkyl, halogen, hydroxy or alkoxy. Specific examples of representative metabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl, methoxymethyl and trimethylsilyl groups.

[0192] “Prodrugs” refers to compounds, including derivatives of the compounds of the invention, which have cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention which are pharmaceutically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N- alkylmorpholine esters and the like. Other derivatives of the compounds of this invention have activity in both their acid and acid derivative forms, but in the acid sensitive form often offers advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendant on the compounds of this invention are particular prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkylesters or (alkoxycarbonyl)oxy)alkylesters. Particularly the Ci-Cs alkyl, C2-C8 alkenyl, C2-C8 alkynyl, aryl, C7-C12 substituted aryl, and C7-C12 arylalkyl esters of the compounds of the invention. “Solvate” refers to forms of the compound that are associated with a solvent or water (also referred to as “hydrate”), usually by a solvolysis reaction. This physical association includes hydrogen bonding. Conventional solvents include water, ethanol, acetic acid and the like. The compounds of the invention may be prepared e.g., in crystalline form and may be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates, such as hydrates, and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates.

[0193] A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g, infant, child, adolescent) or adult subject (e.g., young adult, middle aged adult or senior adult) and / or a nonhuman animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. The terms “human,” “patient,” and “subject” are used interchangeably herein.

[0194] An “effective amount” means the amount of a compound that, when administered to a subject for treating or preventing a disease, is sufficient to effect such treatment or prevention. The “effective amount” can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. A “therapeutically effective amount” refers to the effective amount for therapeutic treatment. A “prophylatically effective amount” refers to the effective amount for prophylactic treatment.

[0195] “Preventing” or “prevention” or “prophylactic treatment” refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject not yet exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.

[0196] The term “prophylaxis” is related to “prevention,” and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization, and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high. “Treating” or “treatment” or “therapeutic treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting the disease or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, “treating” or “treatment” relates to slowing the progression of the disease.

[0197] As used herein, the term “isotopic variant” refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound. For example, an “isotopic variant” of a compound can contain one or more non-radio active isotopes, such as for example, deuterium (2H or D), carbon-13 (13C), nitrogen-15 (15N), or the like. It will be understood that, in a compound where such isotopic substitution is made, the following atoms, where present, may vary, so that for example, any hydrogen may be “2H / D, any carbon may be13C, or any nitrogen may be15N, and that the presence and placement of such atoms may be determined within the skill of the art. Likewise, the invention may include the preparation of isotopic variants with radioisotopes, in the instance for example, where the resulting compounds may be used for drug and / or substrate tissue distribution studies. The radio-active isotopes tritium, i.e.,3H, and carbon- 14, i.e.,14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Further, compounds may be prepared that are substituted with positron emitting isotopes, such asnC,18F,15O and13N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. All isotopic variants of the compounds provided herein, radioactive or not, are intended to be encompassed within the scope of the invention.

[0198] It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers.” Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.”

[0199] Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non- superimposable mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R - and S - sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+)- or (-)- isomers, respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”.

[0200] “Tautomers” refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of it electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci- and nitro-forms of phenylnitromethane, that are likewise formed by treatment with acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.

[0201] As used herein a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, in enantiomeric excess of the “R” form. The term “enantiomerically pure” or “pure enantiomer” denotes that the compound comprises more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight or more than 99.9% by weight, of the enantiomer. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound.

[0202] As used herein and unless otherwise indicated, the term “enantiomerically pure R- compound” refers to at least about 95% by weight R-compound and at most about 5% by weight S-compound, at least about 99% by weight R-compound and at most about 1% by weight S-compound, or at least about 99.9 % by weight R-compound and at most about 0.1% by weight S-compound. In certain embodiments, the weights are based upon total weight of compound.

[0203] As used herein and unless otherwise indicated, the term “enantiomerically pure S- compound” or “S-compound” refers to at least about 95% by weight S-compound and at most about 5% by weight R-compound, at least about 99% by weight S-compound and at most about 1% by weight R-compound or at least about 99.9% by weight S-compound and at most about 0.1% by weight R-compound. In certain embodiments, the weights are based upon total weight of compound. In the compositions provided herein, an enantiomerically pure compound or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising enantiomerically pure R-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R-compound. In certain embodiments, the enantiomerically pure R-compound in such compositions can, for example, comprise, at least about 95% by weight R-compound and at most about 5% by weight S-compound, by total weight of the compound. For example, a pharmaceutical composition comprising enantiomerically pure S-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S- compound. In certain embodiments, the enantiomerically pure S-compound in such compositions can, for example, comprise, at least about 95% by weight S-compound and at most about 5% by weight R-compound, by total weight of the compound. In certain embodiments, the active ingredient can be formulated with little or no excipient or carrier.

[0204] The compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)- stereoisomers or as mixtures thereof.

[0205] Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art.

[0206] One having ordinary skill in the art of organic synthesis will recognize that the maximum number of heteroatoms in a stable, chemically feasible heterocyclic ring, whether it is aromatic or non-aromatic, is determined by the size of the ring, the degree of unsaturation and the valence of the heteroatoms. In general, a heterocyclic ring may have one to four heteroatoms so long as the heteroaromatic ring is chemically feasible and stable.

[0207] EXAMPLES

[0208] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the compounds, compositions, materials, device, and methods provided herein and are not to be construed in any way as limiting their scope. Example 1 : Evaluation of Exemplary GLP-1 Agonists

[0209] General

[0210] Solid Phase Peptide Synthesis. All GLP-1 (7-36-NH2) analogues were assembled on solid support (Rink Amide MBHA Resin, Novabiochem 100-200 mesh, 0.66 mmol / g) using fast flow under high temperature (60 °C), high pressure conditions. The fast flow system was equilibrated with methanol for 20 minutes with a constant flow rate of 25 mL / min. Then, the resin was swollen for 20 minutes in A, A-di methyl formamide (DMF). Each A"- Fmoc amino acid (10 eq) was activated for 2 minutes with O-(7-azabenzotriazol-l-yl)-l, 1,3,3- tetramethyluroniumhexafluorophosphate (HATU, 0.36 M) and AA-diisopropylethylamine (DIEA, 500 pL) in dry DMF (2.5 mL). After 2 minutes, the activated amino acid (yellow) was loaded with a syringe to the fast flow chamber (<2 minutes). The system was washed with DMF for 3-4 minutes, and A“-Fmoc was deprotected with 50% piperdine / DMF (v / v) for 30 seconds. The resin was rinsed with DMF for 4 minutes before the next amino acid coupling. This cycle was repeated for Gly37-Ala8.

[0211] For warheaded analogues that end with a free A-terminus, the last amino acid coupled was N“-Boc-Histidine. For analogues with N-terminal acetylation, the last step was N“-Fmoc- Histidine(Trt) coupling, followed by Fmoc deprotection. For analogues with -CH2CF3 modification of the N-terminus, coupling was stopped after Fmoc deprotection of N“-Fmoc- Ala on position 8. After the last step in fast flow, the resin was washed with DMF for 20 minutes, followed by methanol wash for 20 minutes. Then, the resin was transferred to a reaction vessel from the fast flow chamber to proceed with selective side chain modifications or placed in a high vacuum chamber overnight before full cleavage.

[0212] As a general synthetic strategy (see below for details'), (1) N-terminal modifications (if any were present) were done as the first step after building the peptide scaffold with fast-flow synthesis. These were N-acetylation (N-AcGLPl, N-AcGLPl_K34SulF) or N-CH2CF3 modification (C2GLP-1). Then, (2) orthogonal groups were deprotected to perform (3) selective side chain modifications. For K26DAC18K34SulF, the following changes were made: NE-4-methyl-trityl (Mtt) deprotection, couplings of two OEGs, y-Glu, and Cl 8 diacid in order, followed by orthogonal AE-allyloxycarbonyl (Alloc) deprotection, and the addition of an arylsulfonyl fluoride warhead. For warheaded analogues, orthogonal Alloc deprotection was followed by (1) linker insertion if any were present (OEG, OEGx2 P-Ala) or (2) direct warhead addition (SulF, Acr, PE, FPE). Acetylation on the N-terminus

[0213] Acetic anhydride (0.75 mL) was mixed with dry DMF (5 mL) and DIEA (75 pL) for acetylating the free amine (0.1 mmol) on the N-terminus. The mixture was added to the reaction vessel and bubbled with N2 (g) for 30 minutes. After coupling, the resin was rinsed with DMF (3 x 5 mL). A Kaiser test was performed at the end to observe the completion of the reaction.

[0214] CH CFa-His modification on the N-terminus. N“-CH2CF3-His(Trt)-OH was synthesized as reported before. The modified amino acid (4 eq) was activated with HATU (3.6 eq) and DIEA (16 eq) in dry DMF for 2 minutes and was coupled manually for 90 minutes.

[0215] Removing Orthogonal Protecting Groups

[0216] The Mtt group was deprotected by bubbling the resin with 1 mL of 1.8% TFA / DCM solution for 3 minutes in N2 (g) and draining the mixture afterward. This step was repeated 9 times, where the solution color gradually changed from clear (first wash) to orange (last wash). Then, the beads were washed with DMF, DCM, and 0.1% DIEA / DCM (v / v), respectively. A Kaiser test was performed to confirm the deprotection. The Alloc protecting group was removed with catalyst Pd(PPh3)4 (60 mg) in a solution of phenyl silane (296 pL) and 5 mL dry dichloromethane (DCM), which were degassed beforehand for 5 minutes. The reaction vessel was shaken for 4 h to overnight in the dark. Then, the resin was decolorized by multiple washes with a mixture of sodium diethyldithiocarbamate trihydrate (100 mg) and 100 pL DIEA in 20 mL DMF (2 x 10 mL). Free amine formation was confirmed by purple beads in a Kaiser test.

[0217] Linker Additions

[0218] OEG, P-Ala, y-Glu were coupled manually in the same way as regular amino acids. Fmoc-8-amino-3,6-dioxaoctanoic acid for OEG (4 eq), Fmoc-Beta-Ala-OH for P-Ala (4 eq), or Fmoc-L-Glu-OtBu for y-Glu (4 eq) were activated with HATU (3.6 eq, 0.36 M) and DIEA (8 eq) in dry DMF for 2 minutes. Activated amino acids were coupled for Ih under N2(g). After a DMF wash (3 x 5 mL), the ri-Fmoc group was selectively removed with 20% piperdine / DMF (v / v) (1 x 5 mL for 5 min, 1 x 5 mL for 15 min). The resin was rinsed with DMF thereafter (3 x 5 mL). For OEGOEG compounds, OEG coupling was repeated twice with Fmoc deprotection in between. For diacid coupling, the resin was washed with 5 mL DCM beforehand. Then, Boc-C16-COOH (C18 diacid) was dissolved in dry DCM (4 eq, 0.8M), and HATU (3.6 eq, 0.72 M) was dissolved in dry DMF. HATU / DMF solution was added to the DCM / diacid solution and the mixture was transferred to the reaction vessel. The mixture was shaken for Ih, followed by DCM (3 x 5 mL) and DMF (3 x 5 mL) washes. SulF Coupling

[0219] Peptide (0.025 mmol) was synthesized from a previously assembled GLP-1 template where the orthogonal Alloc protecting group on the Lys34 side chain was selectively deprotected. HATU (3.6 eq) in dry DMF (150 pL) was mixed with 4-(fluorosulfonyl)benzoic acid (4 eq). The solution was combined with dry DMSO (150 pL) and vortexed vigorously. Warhead solution was activated with DIEA (4 eq) for 5 minutes (solution becomes orange) and transferred into the reaction vessel. The mixture was shaken for 40 minutes at ambient temperature (22 °C). After coupling, the resin was washed with dimethylsulfoxide (DMSO, 3 x 5 mL) and with DMF (3 x 5 mL). A Kaiser test was performed at the end to observe the completion of the reaction, and a mini cleavage was done to detect the mass of the final peptide before proceeding with the full cleavage.

[0220] PE and FPE Coupling

[0221] After orthogonal Alloc deprotection on Lys34, the resin (0.023 mmol) was treated with a mixture of dry DCM (400 pL) and DIEA (4 eq) and kept in the freezer (-20 °C) for 20 minutes. The mixture of phenyl chloroformate (2 eq) in dry DCM (50 pL) was added to the reaction vessel which was shaken at ambient temperature (22 °C) for 1 hour. The resin was rinsed with DCM (3 x 5 mL). The same conditions were followed for 4-fluorophenyl chloroformate coupling. Kaiser tests and mini cleavages were done to confirm coupling.

[0222] Acr Coupling

[0223] After orthogonal Alloc deprotection on Lys34, the resin (0.022 mmol) was treated with a mixture of dry DCM (163 pL) and DIEA (4 eq). The mixture of acryloyl chloride (2 eq) in dry DCM (240 pL) was added to the reaction vessel which was shaken at ambient temperature (22 °C) for 40 minutes. The resin was rinsed with DCM (3 x 5 mL). A Kaiser test and mini cleavage were done to confirm coupling.

[0224] Peptide Purification

[0225] After the side chain modifications were completed, the resin was washed with DMF (3 x 5 mL), and MeOH (3 x 5 mL) and dried in vacuo overnight. Peptides were cleaved from the resin using a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and H2O (95:2.5:2.5). After 90 minutes, TFA was removed using rotary evaporation and peptides were precipitated in cold Et2O. Crude peptides were lyophilized overnight and purified by reversephase high-performance liquid chromatography (RP-HPLC) using the following solvent system: A: 99% H2O, 1% CH3CN, 0.1% TFA, (v / v / v) and B: 10% H2O, 90% CH3CN, 0.07% TFA, (v / v / v). A linear gradient of 30-60% solvent B over 30 minutes was used to purify peptides using a flow rate of 2 mL / min on a Cl 8 column (Higgins Analytical, Cl 8 Proto 200, 250 x 10 mm, 5 pm) with detection of eluant at 230 nm. Purities were assessed by one of two methods: Method 1: Peptide purities (all >95%) were assessed by analytical C18 RP-HPLC (Higgins Analytical, Cl 8 Proto 200, 250 x 4.6 mm, 5 pm) using a linear gradient from 30% to 70% solvent B over 20 min. The identity of peptides was confirmed by electrospray ionization mass spectroscopy (ESI-MS, ThermoFisher Finnigan LTQ). Method 2 Alternatively, purities were assessed by C18 RP-HPLC (Agilent, C18 Zorbax, 2.1 x 50 mm, 1.8 pm) using a linear gradient from 30% to 60% of solvent B over 20 min. The solvent system employed for the latter LC-MS experiments was A: 100% H2O and 0.1% formic acid (v / v); B: CH3CN and 0.1% formic acid (v / v). Masses were confirmed by ESLMS on an Agilent 1260 Infinity II LC system in tandem with an Agilent 6230 LC-TOF (Conditions: Nozzle voltage: 1000 V ; drying gas: N2; sheath gas temperature: 355 °C; nebulizer: 35 psi). Pure peptide concentrations were calculated via absorbance of Tyr and Trp residues for use in the luciferase reporter assay.

[0226] Cell Culture

[0227] Human embryonic kidney (HEK) 293 cells were stably transfected with cDNAs encoding for (i) human GLP-1R, (ii) 6xCRE-promoted firefly luciferase reporter gene including luciferase destabilizer sequences to accelerate turnover of accumulated luciferase. Cells were grown in Dulbecco's Modified Eagle Medium (DMEM, high glucose, no glutamine; Gibco) supplemented with 10% tetracycline tested fetal bovine serum (FBS, Atlanta Biologicals), 2 mM L-glutamine (Gibco), and 100 U / mL penicillin- streptomycin (Gibco). The cells were incubated at 37 °C in a humid environment containing 5% CO2. In order to maintain the expression of the receptor and cAMP-responsive luciferase reporter gene, cells were selected for the corresponding encoded antibiotic-resistant markers. For selection, cells were grown in DMEM supplemented with 10% tetracycline-tested FBS (Atlanta Biologicals), 800 pg / mL Zeocin (Invivogen), and 1.25 pg / mL Puromycin (Invivogen) to 40-60% confluency. Before the receptor activity assay, cells were grown in the non-selection DMEM (with FBS, L-glutamine, and penicillin- streptomycin as described above). The cycle of selection and nonselection protocols was maintained throughout propagating cells for functional studies.

[0228] Counting Number of Cells After Washout

[0229] Washout experiments were carried out as reported in the experimental section (plated at 5,000 cells / well on a 96-well plate). On day 3, triple washes were done for the treated wells (48 wells) and 100 pL / well serum-free expression medium (with L-Gln and pen / strep) was added to be incubated for an additional 4 h. Control wells (48 wells) were incubated for 8 h without any treatment. At 8 h, the medium was aspirated for all wells. Cells were treated with 0.05% Trypsin in EDTA (Gibco, 100 pL / well) for 5 minutes at 37 °C. Then, cells were transferred into corresponding separate reservoirs for the control and washed cells. Trypsinized wells were then washed with growth DMEM (high glucose, no glutamine; Gibco) supplemented with 10% tetracycline tested fetal bovine serum (FBS, Atlanta Biologicals), 2 mM L-Glutamine (Gibco), and 100 U / mL penicillin-streptomycin (Gibco). Trypsin treatment and washes were repeated one more time. Finally, collected cells in the reservoirs were (i) transferred to separate falcon tubes, (ii) spun down, (iii) resuspended in growth DMEM, and (iv) counted with trypan blue on Countess (automated cell counter).

[0230] Washout Experiment with Human Serum Albumin Treatment

[0231] All procedures were followed as analogous to the standard washout experiment. Following 4 hours of cell incubation with ligand and subsequent triple washes, 100 pL / well of the defined medium was added including (i) 3 pL Ex(9-39), (ii) 1% (w / v) human serum albumin (HSA, Sigma Aldrich, A1887), or (iii) 3 pL Ex(9-39) in 1% HSA. All solutions were prepared in serum-free expression media (with L-Gln and pen / strep). Cells were then incubated for an additional 4 h at 37 °C. For control, cells did not have any washout treatment or Ex(9- 39) / HSA addition and were incubated with peptides for 8 h until cell lysis. The assay was stopped by adding steady lite® solution for both washout and control cells, followed by a readout of receptor-mediated, ligand-induced luciferase activity and data fitting. cAMP Assay in the Presence of Human Serum Albumin (HSA)

[0232] All procedures followed were the same as reported in the main manuscript ("Luciferase Reporter Assay” and “Data Analysis" in the Experimental Section) with an additional change: On day 3, existing medium was aspirated and 50 pL / well serum-free expression medium (with L-Gln and pen / strep) containing (i) 0.02% HSA (w / v), (ii) 4% HSA (w / v), or (iii) no HSA was added. Peptides at various concentrations in 50 pL / well serum-free expression medium ([HSA]finai = 0.01% or 2%) were incubated with cells for 4 h.

[0233] Detection of the Covalent Capture by LC-MS

[0234] A stock solution of the recombinant extracellular domain (ECD) of human GLP-1R expressed in E. coli (Biovender, RD 172243100) was prepared in 0.1 M pH 4.0 sodium acetate buffer (ECD: 0.5 mg / mL). The receptor (5 pg, 0.32 nmol) was combined with the target peptide (10 equivalents, 3.2 nmol for K34BetaAlaSulF and 25 equivalents, 8.1 nmol for the rest of the peptides tested) in a low protein binding eppendorf tube, and the mixture diluted with 400 mM potassium phosphate buffer (pH 8.4) to give a receptor concentration of 0.05 mg / mL. The final reaction mixture pH was 7.8. After overnight incubation at 25 °C, samples were analyzed by liquid chromatography-mass spectrometry (LC ESI-MS). Liquid chromatography was carried out at 0.5 mL / min on a C18 reverse-phase column (Agilent, Poroshell 120, EC-C18 2.7 p, 3.0 x 50 mm) with solvent A (100% H2O and 0.1% formic acid (v / v)) and solvent B (100% CH3CN and 0.1% formic acid (v / v)) (Agilent 1260 Infinity II LC). Each run (total of 21 mins) included a leader of isocratic solvent A (100%) for 3 minutes, followed by a linear gradient of 20-38% solvent B over 14 minutes. Then, the gradient was increased to 100% solvent B over 2 minutes and kept constant for another 2 minutes.

[0235] Masses were determined by use of an Agilent 6230B time-of-flight TOF LC / MS instrument (Conditions: Nozzle voltage: 1000 V; drying gas: N2; sheath gas temperature: 355 °C; nebulizer: 35 psi) and analyzed with Agilent MassHunter software.

[0236] Table SI. Kinetic data in the literature for warheads with shown nucleophiles and in reported conditions. a) Pseudo-first-order rate coefficients (kobs) for the aminolysis of p-fluorophenyl acetate with n-Butylaminc were converted to apparent second-order rate coefficients at pH 9.5 and 25°C (k2 = 30.2 x 10'2M'1s'1with n-Butylamine). b) Second-order rate constants were calculated for the modification of N-acetyltyrosine with amide-substituted aryl sulfonyl fluorides at pH 7.5 and 37 °C (k2= 328 M'1h'1). The value for the amide substituted group was estimated from the ratio of the same group with no substituted analogue for tyrosine modification (see part d, l<2 = 122 M'1h'1with N-acetyl lysine, estimated'), c) The apparent second-order rate constant for ammonolysis was obtained from pseudo-first-order rate constants for reactions with ammonia pH 7.5 and 30°C for -chlorophcnyl acetate, d) second- order rate constants were calculated for the modification of N-acetyltyrosine with aryl sulfonyl fluorides (k2 = 164 M'1h'1) and of N-acetylysine by aryl sulfonyl fluorides (k2 = 61 M^h'1) at pH 7.5 and 37 °C. (Mukherjee, 2017 #8) d) the apparent second-order rate constant for ammonolysis was obtained from pseudo-first-order rate constants for reactions with ammonia at pH 7.6 and 30°C for phenylacetate, e) Pseudo-first order was calculated from the reaction half-life with N-acetylysine at pH 10.2 and 37°C. The reported half-life for dimethyl acrylamide is 1 Ih.

[0237] Table S2. Approximate effective molarity calculation for the chosen warheads (SulF, PE, and Acr) on GLP-1.

[0238] Warhead on Distance C«jgr(M) Neat Warhead

[0239] Lys34 (dm) Concentration (M)

[0240] K34_SulF 2.5 x 10'951 5

[0241] K34_PE 3.0 x 10’929 8

[0242] K34_Acr 2.5 x 10'951 12

[0243] Warheads were attached at Lys34 on MOE via builder (PDB:5VAI) and the distances were calculated from the electrophilic center of the warhead and the amine of Lysl 13* on the receptor. The assumptions were made such that the warhead occupies a hemisphere where the halved distance between reactive partners is assigned as the radius. For the half-sphere volume, Ceff calculated where NA is Avogadro’s number and r is the radius of the hemisphere in dm. The distance from the electrophilic center of fluorophenyl carbamate was the same as that of phenyl carbamate so the Ceff is the same for both warheads. From approximate calculations, SulF and Acr displayed similar effective concentrations whereas the corresponding value for PE was approximately half of this. Neat warhead concentrations are calculated for neat warhead solutions. In order to get a rough number, the densities of starting warhead molecules were used. This was 4-(Fluorosulfonyl) benzoic acid for SulF, phenyl chloroformate for PE, acrylchloride for Acr. Densities of PE and Acr are d=1.25 and d=1.12 g / mE, respectively. 4- (Fluoro sulfonyl) benzoic acid is in solid form so the density of SulF is assumed to be d=l g / mE. Comparing Ceff and neat concentrations, SulF had the most dramatic increase of Ceff followed by Acr.

[0244] Table 3. Potency changes for GLP-1 and K34SulF upon (i) 8 h incubation, (ii) washing at 4 h, (iii) pre-incubation with Ex(9-39) for 30 min, (iv) combination of Ex(9-39) pre-incubation with washing at 4 h, (v) combination of wash at 4 h and Ex(9-39) addition after, (vi) combination of Ex(9-39) pre-incubation, washing at 4 h, and addition of Ex(9-39) after wash. a Structures of the peptides are displayed in Figure 2.bECso(pM) is the concentration of peptide required for half-maximal activity of the targeted receptor. pECso = -log(ECso) ± standard error of the mean (SEM) of independent experiments.cNumber of independent experiments conducted.dControl cells were incubated with peptide for 8 h without any further treatment. Fold- shift for K34SulF from native peptide was calculated as (EC50 K34SulF) / (ECso GLP-1).eCells were incubated with peptide for 4 h, then washed three times. After the washing step, cells were incubated for an additional 4 h before they were lysed. Fold-shifts were calculated as (EC50 wash) / (ECso control) to document the wash influence. Cells were preincubated with 6 pM Ex(9-39) for 30 min and the [Ex(9-39)] was reduced to 3 pM upon peptide addition. Cells were then incubated for 8 h before they were lysed. Fold-shifts were calculated as (EC50 pre-Ex(9-39)) / (ECso control) to observe effects of Ex(9-39) pre-incubation.8Cells were pre-incubated with 6 pM Ex(9-39) for 30 min and the [Ex(9-39)] was reduced to 3 pM upon peptide addition. After 4 h peptide incubation, cells were washed three times and incubated for additional 4 h before cell lysis. Fold-shifts were calculated as (EC50 pre-Ex(9-39) + wash) / (EC50 control) to observe the combined effects of Ex(9-39) pre-incubation and washout at 4 h.hCells were incubated with peptide for 4 h, then washed three times. After the washing step, 3 pM of Ex(9-39) was added, and cells were incubated for an additional 4 h before they were lysed. Fold-shifts were calculated as (EC50 wash + Ex(9-39)) / (ECso control).1Cells were pre-incubated with 6 pM Ex(9-39) for 30 min and the [Ex(9-39)] was reduced to 3 pM upon peptide addition. After 4 h peptide incubation, cells were washed three times, and 3 pM Ex(9-39) was added. Fold-shifts were calculated as (EC50 pre-Ex(9-39) + wash + Ex(9- 39)) / (ECso control).

[0245] Table S4. Fold-change in activity after a washout experiment at 4 h, 12 h, and 15 min with the addition of Ex(9-39). a Structures of the peptides are displayed in Figure 2.bControl cells were incubated with peptide without any further treatment. Total assay time is the duration from peptide addition until cell lysis.cCells were incubated with peptide for either 15 min, 4 h, or 12 h, then washed three times. After the washing step, 3 pM of the competitive binder Ex(9-39) was added, and cells were incubated for an additional 4 h before they were lysed. Control cells shown as (-) were incubated for the duration of the corresponding assay without any treatment.dECso(pM) is the concentration of peptide required for half-maximal stimulation of the targeted receptor. pECso = -log(ECso) ± standard error of the mean (SEM) of independent experiments.eNumber of independent experiments conducted. Fold- shift from native peptide, GLP-1 for the corresponding time point. Calculated as (EC50 ligand at t) / (ECso GLP-1 at t).8Fold-shift from no-wash control for the corresponding time point. Calculated as (EC50 wash + Ex(9-39) at t) / (ECso no-wash control at t).hFold-shift from Exendin-4 for an 8 h assay which is displayed in Table 1. Calculated as (EC50 ligand) / (ECso Ex-4).

[0246] Table 5. Potency changes for washout at 4 h experiment for N-AcGLPl after being incubated with benzyl amine.

[0247] ControlbWashed @4 hc

[0248] ECso pECso ± ECso

[0249] Peptide" pECso ± SEA I nene

[0250] (pM)dSEM4(pM)4

[0251] N-AcGLPl 10.2 + 0.003 60 5 7.0 + 0.03 9.3 x 1045

[0252] N-AcGLPl + 10.2 + 0.02 68 2 6.8 + 0.03 >1052

[0253] B11NH2 a Structures of the peptides are displayed in Figure 6.bControl cells were incubated for 8 h without any treatment.cCells were incubated for 4 h, then washed three times. After the wash, 100 pL / well serum-free expression media was added, and cells were incubated for an additional 4 h before cell lysis.dECso(pM) is the concentration of peptide required for half- maximal activity of the targeted receptor. pECso = -log(ECso) ± standard error of the mean (SEM) of independent experiments.eNumber of independent experiments conducted.

[0254] Table 6. Fold-change in activity after a washout experiment at 4 h with the addition of Ex(9-39) and / or 1% human serum albumin.

[0255] Washed Treatment pECso ± Avg ECso Fold-

[0256] Peptidean '

[0257] AtbAfter WashcSEAL Change

[0258] 10.6 +

[0259] - - 27 8 2.1

[0260] K26DAC18 0.002

[0261] 7.32 + 4 h 3 uM Ex(9-39) 4.8 x 1047 1800 ~s

[0262] K26DAC18 0.01

[0263] 7.73 + 4 h 1% HSA 1.9 x lO43 685 ’8

[0264] K26DAC18 0.02

[0265] 3 uM Ex(9-39) 7.73 +

[0266] 4 h 1.8 x lO44 677 -8

[0267] K26DAC18 in 1% HSA 0.02 9.81 +

[0268] - - 156 7 2.7 -f

[0269] K26DAC18_K34SulF 0.003

[0270] 7 53 ± 4 h 3 pM Ex(9-39) 3.0 x lO46 191 ’s

[0271] K26DAC18_K34SulF 0.01

[0272] 7.46 + 4 h 1% HSA 3.4 x lO42 220 ’s

[0273] K26DAC18_K34SulF 0.04

[0274] 3 pM Ex(9-39) 7.40 +

[0275] 4 h 3 9 x 1043 253 "g

[0276] K26DAC18_K34SulF in 1% HSA 0.02 a Structures of the peptides are displayed in Figure 7.bPeptides were incubated for 4 h, then washed three times with serum-free expression media (100 pE / well). Control peptides shown as (-) were not washed.cAfter the washout at 4 h, cells were treated with (i) 3 pM Ex(9- 39), (ii) 1% HSA, or (iii) 3 pM Ex(9-39) in 1% HSA. After treatment, cells were incubated for an additional 4 h. Control peptides shown as (-) were not treated with anything and incubated for 8 h straight.dECso(pM) is the concentration of peptide required for half-maximal activity of the targeted receptor. pECso = -log(ECso) ± standard error of the mean (SEM) of independent experiments. For graphs without a proper sigmoidal curve, the maximum readout was constrained to the plateau of the corresponding control peptide and EC50 was retrieved by Prism accordingly.eNumber of independent experiments conducted / Fold- shift from native peptide, GLP-1 during the 8 h assay. Calculated as (EC50 ligand) / (ECso GLP-1) Fold-shift from notreatment control. Calculated by (EC50 for wash + treatment) / (EC50 for no-wash control).

[0277] Table 7. Characterization of the peptides.

[0278] PeptideaMW (Calculated)bMW (Observed)c

[0279] N-AcGLPl 3368.69 3368.51

[0280] N-AcGLPl_K34SulF 3553.69 3554.41

[0281] K34SulF 3483.68 3484.13

[0282] K34Acr 3351.68 3350.95

[0283] K34PE 3417.68 3417.04

[0284] K34FPE 3435.68 3436.04

[0285] K34BetaAlaSulF 3554.68 3554.72

[0286] K34OEGSulF 3628.68 3628.75

[0287] K34OEGOEGSulF 3773.68 3775.23

[0288] K34BetaAlaPE 3488.68 3489.42 K34OEGFPE 3580.68 3581.01

[0289] K26DAC18 4013.58 4014.28

[0290] K26DAC18 K34SulF 4199.58 4200.32

[0291] C2K26DAC18 4095.58 4095.81

[0292] C2K26DAC18 K34SulF 4281.58 4281.54

[0293] Ex4 K27 SulF 4372.63 4373.19

[0294] Ex4 K27 OEG SulF 4517.63 4517.50

[0295] Ex4 S33K SulF 4413.73 4413.81

[0296] Ex4 S33K OEG SulF 4558.73 4558.97 a The purity (>95%) was analyzed by analytical C18 column (Higgins Analytical, C18

[0297] Proto 200, 250 x 4.6 mm, 5 pm) at a linear gradient from 30% to 70% solvent B over 20 min on HPLC (flow rate at 1 mL / min, absorbance at 230 nm). Alternatively, purify was assessed by C18 column (Agilent, C18 Zorbax, 2.1 x 50 mm, 1.8 pm) on LCMS (Agilent 1260 Infinity

[0298] II LC) at a linear gradient from 30% to 60% solvent B over 20 min (flow rate at 0.5 mL / min, absorbance at 230 nm) Molecular weights are average masses calculated using Peptide Mass

[0299] Calculator v3.2 (Katholieke Universiteit, Leuven, Belgium) / Molecular weight of each peptide was measured by either direct infusion to electrospray ionization mass spectroscopy - positive mode (ESLMS, ThermoFisher) in 50% solvent A:B (v / v) or injection to Agilent 1260 Infinity

[0300] II LC system in tandem with an Agilent 6230 LC-TOF - positive mode where masses were extracted from total ion chromatogram (TIC) and analyzed with Agilent MassHunter

[0301] Qualitative Analysis. K34PE:

[0302] K34BetaAlaPE:

[0303]

[0304] INCORPORATION BY REFERENCE

[0305] All U.S. and PCT patent publications and U.S. patents mentioned herein are hereby incorporated by reference in their entirety as if each individual patent publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0306] OTHER EMBODIMENTS

[0307] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

We claim:

1. A peptide having the structure:wherein:R1, R2and R3are each independently selected from H, fluoroalkyl, P1, and E1; wherein each P1independently comprises a lipid moiety; and each E1independently comprises an electrophilic moiety; provided that at least one of R1, R2and R3is E1;X is a peptide moiety comprising the amino acid sequence EGTFTSDVSSYLEGQAA, or a sequence with at least 80% sequence identity thereto;Y is a peptide moiety comprising the amino acid sequence EFIAWLV, or a sequence with at least 80% sequence identity thereto; andZ is a peptide moiety comprising the amino acid sequence GR.

2. The peptide of claim 1, wherein R1is H.

3. The peptide of claim 1, wherein R1is fluoroalkyl.

4. The peptide of claim 1, wherein R1is P1.

5. The peptide of claim 1, wherein R1is E1.

6. The peptide of any one of claims 1-5, wherein R2is H.

7. The peptide of any one of claims 1-5, wherein R2is fluoroalkyl.

8. The peptide of any one of claims 1-5, wherein R2is P1.

9. The peptide of any one of claims 1-5, wherein R2is E1.

10. The peptide of any one of claims 1-9, wherein R3is H.

11. The peptide of any one of claims 1-9, wherein R3is fluoroalkyl.

12. The peptide of any one of claims 1-9, wherein R3is P1.

13. The peptide of any one of claims 1-9, wherein R3is E1.

14. The peptide of any one of claims 1, 5, 9, and 13, wherein each E1is independently selected from the group consisting of sulfonyl fluoride, acryloyl, and aryl carbamate.

15. The peptide of any one of claims 1, 5, 9, and 13, wherein each E1is independently selected from the group consisting of:wherein L’ is a bond or a linker moiety.

16. The peptide of claim 15, wherein each17. The peptide of claim 15, wherein each E1is18. The peptide of claim 15, wherein each E1is20. The peptide of any one of claims 15-19, wherein L’ is a linker moiety having the structure:wherein * indicates the point of attachment to the peptide moiety; and n is 1 to 4.

21. The peptide of claim 20, wherein L’ isO22. The peptide of claim 20, wherein L’ is H23. The peptide of any one of claims 15-19, wherein L’ is a bond.

24. The peptide of any one of claims 1-23, wherein P1is:wherein m is 3 to 10; and n is 1 to 4.

25. The peptide of claim 24, wherein m is 8.

26. The peptide of claim 24 or 25, wherein n is 2.

27. The peptide of any one of claims 1-26, wherein R1is fluoroalkyl.

28. The peptide of any one of claims 1-26, wherein R1is 1,1,1-trifluoroethyl.

29. The peptide of any one of claims 1-9 and 14-28, wherein R3is:

30. The peptide of any one of claims 1-29, wherein X is a peptide moiety having at least a 90% sequence identity to EGTFTSDVSSYLEGQAA.

31. The peptide of any one of claims 1-29, wherein X is a peptide moiety having at least a 95% sequence identity to EGTFTSDVSSYLEGQAA.

32. The peptide of any one of claims 1-29, wherein X is a peptide moiety having at least a 98% sequence identity to EGTFTSDVSSYLEGQAA.

33. The peptide of any one of claims 1-29, wherein X is a peptide moiety comprising the amino acid sequence EGTFTSDVSSYLEGQAA.

34. The peptide of any one of claims 1-33, wherein Y is a peptide moiety having at least a 90% sequence identity to EFIAWL.

35. The peptide of any one of claims 1-33, wherein Y is a peptide moiety having at least a 95% sequence identity to EFIAWL.

36. The peptide of any one of claims 1-33, wherein Y is a peptide moiety having at least a 98% sequence identity to EFIAWL.

37. The peptide of any one of claims 1-33, wherein Y is a peptide moiety comprising the amino acid sequence EFIAWL.

38. The peptide of any one of claims 1-37, wherein Z is the amino acid sequence GR.

39. The peptide of any one of claims 1 and 30-38, wherein:R1is 1,1,1 -trifluoroethyl;40. The peptide of claim 1, wherein the peptide is selected from the group consisting of:; or a pharmaceutically acceptable salt thereof.

41. A pharmaceutical composition, comprising the peptide of claim any one of claims 1- 40; and a pharmaceutically acceptable excipient.

42. A method of treating diabetes, comprising administering to a subject in need thereof an effective amount of a peptide of any one of claims 1-40.

43. A method of treating obesity, comprising administering to a subject in need thereof an effective amount of a peptide of any one of claims 1-40.

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