David J. Edmonds

Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists.

Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal β-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small molecule agonists of the GLP-1 receptor to treat type 2 diabetes.

Improving on Nature: Making a Cyclic Heptapeptide Orally Bioavailable†

The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR-derived structures, amide H-D exchange rates, and temperature-dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N-methylation.

Cyclic Penta- and Hexaleucine Peptides without N-Methylation Are Orally Absorbed

Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with N-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no N-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4-17%) approaching that of the heavily N-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for N-methylation or modified amino acids.

Selective Activation of AMPK β1-Containing Isoforms Improves Kidney Function in a Rat Model of Diabetic Nephropathy.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the β1 subunit. After confirming that human and rodent kidney predominately express AMPK β1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.

Demonstration of the Innate Electrophilicity of 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP), a Small-Molecule Positive Allosteric Modulator of the Glucagon-Like Peptide-1 Receptor

4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP) represents a novel small-molecule activator of the glucagon-like peptide-1 receptor (GLP-1R), and exhibits glucose-dependent insulin secretion in rats following i.v. (but not oral) administration. To explore the quantitative pharmacology associated with GLP-1R agonism in preclinical species, the in vivo pharmacokinetics of BETP were examined in rats after i.v. and oral dosing. Failure to detect BETP in circulation after oral administration of a 10-mg/kg dose in rats was consistent with the lack of an insulinotropic effect of orally administered BETP in this species. Likewise, systemic concentrations of BETP in the rat upon i.v. administration (1 mg/kg) were minimal (and sporadic). In vitro incubations in bovine serum albumin, plasma, and liver microsomes from rodents and humans indicated a facile degradation of BETP. Failure to detect metabolites in plasma and liver microsomal incubations in the absence of NADP was suggestive of a covalent interaction between BETP and a protein amino acid residue(s) in these matrices. Incubations of BETP with glutathione (GSH) in buffer revealed a rapid nucleophilic displacement of the ethylsulfoxide functionality by GSH to yield adduct M1, which indicated that BETP was intrinsically electrophilic. The structure of M1 was unambiguously identified by comparison of its chromatographic and mass spectral properties with an authentic standard. The GSH conjugate of BETP was also characterized in NADPH- and GSH-supplemented liver microsomes and in plasma samples from the pharmacokinetic studies. Unlike BETP, M1 was inactive as an allosteric modulator of the GLP-1R.

Cyclic alpha-conotoxin peptidomimetic chimeras as potent GLP-1R agonists

Type 2 diabetes mellitus (T2DM) results from compromised pancreatic β-cell function, reduced insulin production, and lowered insulin sensitivity in target organs resulting in hyperglycemia. The GLP-1 hormone has two biologically active forms, GLP-1-(7-37) and GLP-1-(7-36)amide, which are equipotent at the glucagon-like peptide-1 receptor (GLP-1R). These peptides are central both to normal glucose metabolism and dysregulation in T2DM. Several structurally modified GLP-1 analogues are now approved drugs, and a number of other analogues are in clinical trials. None of these compounds is orally bioavailable and all require parenteral delivery. Recently, a number of smaller peptidomimetics containing 11-12 natural and unnatural amino acids have been identified that have similar insulin regulating profiles as GLP-1. The α-conotoxins are a class of disulfide rich peptide venoms isolated from cone snails, and are known for their highly constrained structures and resistance to enzymatic degradation. In this study, we examined whether 11-residue peptidomimetics incorporated into α-conotoxin scaffolds, forming monocyclic or bicyclic compounds constrained by disulfide bonds and/or backbone cyclization, could activate the GLP-1 receptor (GLP-1R). Several compounds showed potent (nanomolar) agonist activity at GLP-1R, as evaluated via cAMP signaling. In addition, HPLC retention times and in silico calculations suggested that mono- and bicyclic compounds had more favorable n-octanol/water partition coefficients according to the virtual partition coefficient model (vLogP), while maintaining a smaller radius of gyration compared to corresponding uncyclized peptidomimetics. Our findings suggest that cyclic peptidomimetics provide a potential avenue for future design of potent, compact ligands targeting GLP-1R and possessing improved physicochemical properties.

Chapter Nine - Oral GLP-1 Modulators for the Treatment of Diabetes

Abstract This review covers recent (2009–Jan 2013) developments in the field of orally administered glucagon-like peptide 1 receptor (GLP-1R) agonists for the treatment of diabetes. Several approaches are discussed, namely formulation approaches for oral administration of peptides, the use of transporters for active uptake of peptide drugs, and small molecule GLP-1R agonists.

Truncated Glucagon-like Peptide-1 and Exendin-4 α-Conotoxin pl14a Peptide Chimeras Maintain Potency and α-Helicity and Reveal Interactions Vital for cAMP Signaling in Vitro.

Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22–27) directing the binding of Phe22 into a hydrophobic pocket on the GLP-1R.

Helixconstraints and amino acid substitution in GLP-1 increase cAMP and insulin secretion but not beta-arrestin 2 signaling

Glucagon-like peptide (GLP-1) is an endogenous hormone that induces insulin secretion from pancreatic islets and modified forms are used to treat diabetes mellitus type 2. Understanding how GLP-1 interacts with its receptor (GLP-1R) can potentially lead to more effective drugs. Modeling and NMR studies of the N-terminus of GLP-1 suggest a β-turn between residues Glu9-Phe12 and a kinked alpha helix between Val16-Gly37. N-terminal turn constraints attenuated binding affinity and activity (compounds 1-8). Lys-Asp (i, i+4) crosslinks in the middle and at the C-terminus increased alpha helicity and cAMP stimulation without much effect on binding affinity or beta-arrestin 2 recruitment (compounds 9-18). Strategic positioning of helix-inducing constraints and amino acid substitutions (Tyr16, Ala22) increased peptide helicity and produced ten-fold higher cAMP potency (compounds 19-28) over GLP-1(7-37)-NH2. The most potent cAMP activator (compound 23) was also the most potent inducer of insulin secretion.