Discovery of a potent GIPR peptide antagonist that is effective in rodent and human systems.

OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) is one of the two major incretin factors that regulate metabolic homeostasis. Genetic ablation of its receptor (GIPR) in mice confers protection against diet-induced obesity (DIO), while GIPR neutralizing antibodies produce additive weight reduction when combined with GLP-1R agonists in preclinical models and clinical trials. Conversely, GIPR agonists have been shown to promote weight loss in rodents, while dual GLP-1R/GIPR agonists have proven superior to GLP-1R monoagonists for weight reduction in clinical trials. We sought to develop a long-acting, specific GIPR peptide antagonist as a tool compound suitable for investigating GIPR pharmacology in both rodent and human systems. METHODS: We report a structure-activity relationship of GIPR peptide antagonists based on the human and mouse GIP sequences with fatty acid-based protraction. We assessed these compounds in vitro, in vivo in DIO mice, and ex vivo in islets from human donors. RESULTS: We report the discovery of a GIP(5-31) palmitoylated analogue, [Nα-Ac, L14, R18, E21] hGIP(5-31)-K11 (γE-C16), which potently inhibits in vitro GIP-mediated cAMP generation at both the hGIPR and mGIPR. In vivo, this peptide effectively blocks GIP-mediated reductions in glycemia in response to exogenous and endogenous GIP and displays a circulating pharmacokinetic profile amenable for once-daily dosing in rodents. Co-administration with the GLP-1R agonist semaglutide and this GIPR peptide antagonist potentiates weight loss compared to semaglutide alone. Finally, this antagonist inhibits GIP- but not GLP-1-stimulated insulin secretion in intact human islets. CONCLUSIONS: Our work demonstrates the discovery of a potent, specific, and long-acting GIPR peptide antagonist that effectively blocks GIP action in vitro, ex vivo in human islets, and in vivo in mice while producing additive weight-loss when combined with a GLP-1R agonist in DIO mice.

Optimization of Truncated Glucagon Peptides to Achieve Selective, High Potency, Full Antagonists.

Antagonism of glucagon's biological action is a proven strategy for decreasing glucose in diabetic animals and patients. To achieve full, potent, and selective suppression, we chemically optimized N-terminally truncated glucagon fragments for the identification and establishment of the minimum sequence peptide, [Glu9]glucagon(6-29) amide (11) as a full antagonist in cellular signaling and receptor binding (IC50 = 36 nM). Substitution of Phe6 with l-3-phenyllactic acid (Pla) produced [Pla6, Glu9]glucagon(6-29) amide (21), resulting in a 3-fold improvement in receptor binding (IC50 = 12 nM) and enhanced antagonist potency. Further substitution of Glu9 and Asn28 with aspartic acid yielded [Pla6, Asp28]glucagon amide (26), which demonstrated a further increase in inhibitory potency (IC50 = 9 nM), and improved aqueous solubility. Peptide 26 and a palmitoylated analogue, [Pla6, Lys10(γGluγGlu-C16), Asp28]glucagon(6-29) amide (31), displayed sustained duration in vivo action that successfully reversed glucagon-induced glucose elevation in mice.

Insulin-like peptide 5 fails to improve metabolism or body weight in obese mice.

Insulin-like peptide 5 (INSL5) is a member of the insulin-like family of peptides. It has been reported to be orexigenic in rodent models of obesity with impaired glucose metabolism. We attempted to confirm this property as a first step in establishing the ability of INSL5 to successfully integrate with other agents more proven in their ability to reverse obesity and improve metabolism. INSL5 was chemically synthesized by two alternative methods to a native form and one that was site-specifically conjugated to a 20 KDa polyethylene glycol (PEG) polymer. The pharmacology of each peptide was assessed by high-dose chronic administration in normal and obese mice. INSL5 failed to produce pharmacologically relevant effects on food intake, body weight or glucose control indicative of a negligible role of the peptide in the control of feeding and glucose metabolism.

High-Yield Synthesis of Human Insulin-Like Peptide 5 Employing a Nonconventional Strategy.

A simplified route to synthesis of INSL5 is reported, where the elimination of intermediate purification steps and nonconventional disulfide pairing results in final yields that are an order of magnitude higher than in previously reported stepwise syntheses. The intramolecular disulfide of A-chain was produced by a thiol displacement of StBu-protected cysteine, and was followed by an A-B chain disulfide formation in dimethylsulfoxide (DMSO). The final disulfide was formed by deprotection of StBu-cysteines in hydrofluoric acid (HF) at room temperature, which is a historical approach infrequently employed today, followed by oxidation using 2,2-dithiobis(5-nitropyridine) (DTNP) in acidic aqueous buffer. Throughout the synthesis, an isoacyl surrogate to a midsequence native amide bond was utilized to enhance solubility of the intermediate compounds.

Controlled intramolecular antagonism as a regulator of insulin receptor maximal activity.

In the treatment of insulin-dependent diabetes the risk of a fatal insulin overdose is a persistent fear to most patients. In order to potentially reduce the risk of overdose, we report the design, synthesis, and biochemical characterization of a set of insulin analogs designed to be fractionally reduced in maximal agonism at the insulin receptor isoforms. These analogs consist of native insulin that is site-specifically conjugated to a peptide-based insulin receptor antagonist. The structural refinement of the antagonist once conjugated to insulin provided a set of partial agonists exhibiting between 25 and 70% of the maximal agonism of native insulin at the two insulin receptor isoforms, with only slight differences in inherent potency. These rationally-designed partial agonists provide an approach to interrogate whether control of maximal activity can provide glycemic control with reduced hypoglycemic risk.

Max Bergmann award lecture:Macromolecular medicinal chemistry as applied to metabolic diseases.

This review presents the scope of research presented in an October 2016 lecture pertaining to the award of the 2015 Max Bergmann Medal. The advancement in synthetic and biosynthetic chemistry as applied to the discovery of novel macromolecular drug candidates is reviewed. The evolution of the technology from the design, synthesis, and development of the first biosynthetic peptides through the emergence of peptide-based incretin agonists that function by multiple biological mechanisms is exemplified by the progression of such peptides from preclinical to clinical study. A closing section highlights recent progress made in total chemical synthesis of insulin and related peptides.

Synthesis of Four-Disulfide Insulin Analogs via Sequential Disulfide Bond Formation.

Naturally occurring, multiple cysteine-containing peptides are a structurally unique class of compounds with a wide range of therapeutic and diagnostic applications. The development of reliable, precise chemical methods for their preparation is of paramount importance to facilitate exploration of their utility. We report here a straightforward and effective approach based on stepwise, sequentially directed disulfide bond formation, exemplified by the synthesis of four-disulfide bond-containing insulin analogs. Cysteine protection consisted of tert-butylthiol (StBu), thiol-trimethoxyphenyl (STmp), trityl (Trt), 4-methoxytrityl (Mmt), S-acetamidomethyl (Acm), and tert-butyl (tBu). This report describes chemistry that is broadly applicable to cysteine-rich peptides and the influence of a fourth disulfide bond on insulin bioactivity.

Chemical synthesis of insulin analogs through a novel precursor.

Insulin remains a challenging synthetic target due in large part to its two-chain, disulfide-constrained structure. Biomimetic single chain precursors inspired by proinsulin that utilize short peptides to join the A and B chains can dramatically enhance folding efficiency. Systematic chemical analysis of insulin precursors using an optimized synthetic protocol identified a 49 amino acid peptide named DesDi, which folds with high efficiency by virtue of an optimized structure and could be proteolytically converted to bioactive two-chain insulin. In subsequent applications, we observed that the folding of the DesDi precursor was highly tolerant to amino acid substitution at various insulin residues. The versatility of DesDi as a synthetic insulin precursor was demonstrated through the preparation of several alanine mutants (A10, A16, A18, B12, B15), as well as ValA16, an analog that was unattainable in prior reports. In vitro bioanalysis highlighted the importance of the native, hydrophobic residues at A16 and B15 as part of the core structure of the hormone and revealed the significance of the A18 residue to receptor selectivity. We propose that the DesDi precursor is a versatile synthetic intermediate for the preparation of diverse insulin analogs. It should enable a more comprehensive analysis of function to insulin structure than might not be otherwise possible through conventional approaches.

Discovery of high potency, single-chain insulin analogs with a shortened B-chain and nonpeptide linker.

A series of novel, single chain insulin analogs containing polyethylene glycol based connecting segments were synthesized by native chemical ligation and tested for biological activity. While the full length single chain insulin analogs exhibited low potency, deletion of amino acids B26-B30 unexpectedly generated markedly higher activity. This observation is unprecedented in all previous studies of single chain insulin analogs and is consistent with the presumption that in the native hormone this sequence must translocate to achieve high potency insulin receptor interaction. Optimization of the sequence yielded an insulin analog with potency and selectivity comparable to that of native insulin. These results establish a basis for discovery of novel higher potency, single chain insulin analogs of shortened length.

Functional association of the N-terminal residues with the central region in glucagon-related peptides.

GLP-1 is an incretin peptide involved in the regulation of glucose metabolism and the glucose-dependent stimulation of insulin secretion. Ex-4 is a paralog of GLP-1 that has comparable GLP-1R potency but extended physiological action. GLP-1 and Ex-4 are helical peptides that share ∼50% sequence homology but differ at several residues, notably the second amino acid which controls susceptibility to DPP-IV cleavage. This single amino acid difference yields divergent receptor potency when studied in the context of the two hormone sequences. Ex-4 uniquely tolerates Gly2 through select amino acid differences in the middle region of the peptide that are absent in GLP-1. We report that substitution of Ex-4 amino acids Glu16, Leu21, and Glu24 to the GLP-1 sequence enabled Gly2 tolerance. The coordination of the N-terminus with these central residues shows an interaction of substantial importance not only to DPP-IV stability but also to receptor activation. Extension of this observation to glucagon-based co-agonist peptides showed different structural requirements for effective communication between the N-terminus and the mid-section of these peptides in achieving high potency agonism at the GLP-1 and GCGRs.

A novel human-based receptor antagonist of sustained action reveals body weight control by endogenous GLP-1.

Ex-4 (9-39)a is a well characterized GLP-1 receptor antagonist that suffers from two notable limitations, its nonhuman amino acid sequence and its relatively short in vivo duration of action. Comparable N-terminal shortening of human GLP-1 lessens agonism but does not provide a high potency antagonist. Through a series of GLP-1/Ex-4 hybrid peptides, the minimal structural changes required to generate a pure GLP-1-based antagonist were identified as Glu16, Val19, and Arg20, yielding an antagonist of approximately 3-fold greater in vitro potency compared with Ex-4 (9-39)a. The structural basis of antagonism appears to result from stabilization of the α helix combined with enhanced electrostatic and hydrophobic interactions with the extracellular domain of the receptor. Site-specific acylation of the human-based antagonist yielded a peptide of increased potency as a GLP-1 receptor antagonist and 10-fold greater selectivity relative to the GIP receptor. The acylated antagonist demonstrated sufficient duration of action to maintain inhibitory activity when administered as a daily subcutaneous injection. The sustained pharmacokinetics and enhanced human sequence combine to form an antagonist optimized for clinical study. Daily administration of this antagonist by subcutaneous injection to diet-induced obese mice for 1 week caused a significant increase in food intake, body weight, and glucose intolerance, demonstrating endogenous GLP-1 as a relevant hormone in mammalian energy balance in the obese state.

Optimization of the native glucagon sequence for medicinal purposes.

BACKGROUND: Glucagon is a life-saving medication used in the treatment of hypoglycemia. It possesses poor solubility in aqueous buffers at or near physiological pH values. At low and high pH, at which the peptide can be formulated to concentrations of a milligram or more per milliliter, the chemical integrity of the hormone is limited, as evidenced by the formation of multiple degradation-related peptides. Consequently, the commercial preparation is provided as a lyophilized solid with an acidic diluent and directions for rendering it soluble at the time of use. Any unused material is recommended for disposal immediately after initial use. METHODS: A set of glucagon analogs was prepared by solid-phase peptide synthesis to explore the identification of a glucagon analog with enhanced solubility and chemical stability at physiological pH. The physical properties of the peptide analogs were studied by solubility determination, high-performance chromatography, and mass spectral analysis. The biochemical properties were determined in engineered human embryonic kidney cell line 293 (HEK293) cells that overexpressed either the human glucagon or glucagon-like peptide-1 (GLP-1) receptors linked to a luciferase reporter gene. RESULTS: We observed the previously characterized formation of glucagon degradation products upon incubation of the peptide in dilute acid for extended periods or elevated temperature. Lowering the isoelectric point of the hormone through the substitution of asparagine-28 with aspartic acid significantly increased the solubility at physiological pH. Similarly, the C-terminal extension (Cex) of the hormone with an exendin-based, 10-residue, C-terminal sequence yielded a peptide of dramatically enhanced solubility. These two glucagon analogs, D28 and Cex, maintained high potency and selectivity for the glucagon receptor relative to GLP-1 receptor. CONCLUSIONS: Glucagon presents unique structural challenges to the identification of an analog of high biological activity and selectivity that also possesses sufficient aqueous solubility and stability such that it might be developed as a ready-to-use medicine. The glucagon analogs D28 and Cex demonstrated all of the chemical, physical, and biochemical properties supportive of further study as potential clinical candidates for treatment of hypoglycemia.