A viral insulin-like peptide inhibits IGF-1 receptor phosphorylation and regulates IGF1R gene expression.

OBJECTIVE: The insulin/IGF superfamily is conserved across vertebrates and invertebrates. Our team has identified five viruses containing genes encoding viral insulin/IGF-1 like peptides (VILPs) closely resembling human insulin and IGF-1. This study aims to characterize the impact of Mandarin fish ranavirus (MFRV) and Lymphocystis disease virus-Sa (LCDV-Sa) VILPs on the insulin/IGF system for the first time. METHODS: We chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin like) forms of MFRV and LCDV-Sa VILPs. Using cell lines overexpressing either human insulin receptor isoform A (IR-A), isoform B (IR-B) or IGF-1 receptor (IGF1R), and AML12 murine hepatocytes, we characterized receptor binding, insulin/IGF signaling. We further characterized the VILPs' effects of proliferation and IGF1R and IR gene expression, and compared them to native ligands. Additionally, we performed insulin tolerance test in CB57BL/6 J mice to examine in vivo effects of VILPs on blood glucose levels. Finally, we employed cryo-electron microscopy (cryoEM) to analyze the structure of scMFRV-VILP in complex with the IGF1R ectodomain. RESULTS: VILPs can bind to human IR and IGF1R, stimulate receptor autophosphorylation and downstream signaling pathways. Notably, scMFRV-VILP exhibited a particularly strong affinity for IGF1R, with a mere 10-fold decrease compared to human IGF-1. At high concentrations, scMFRV-VILP selectively reduced IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation (Ras/MAPK pathway), while leaving Akt phosphorylation (PI3K/Akt pathway) unaffected, indicating a potential biased inhibitory function. Prolonged exposure to MFRV-VILP led to a significant decrease in IGF1R gene expression in IGF1R overexpressing cells and AML12 hepatocytes. Furthermore, insulin tolerance test revealed scMFRV-VILP's sustained glucose-lowering effect compared to insulin and IGF-1. Finally, cryo-EM analysis revealed that scMFRV-VILP engages with IGF1R in a manner closely resembling IGF-1 binding, resulting in a highly analogous structure. CONCLUSIONS: This study introduces MFRV and LCDV-Sa VILPs as novel members of the insulin/IGF superfamily. Particularly, scMFRV-VILP exhibits a biased inhibitory effect on IGF1R signaling at high concentrations, selectively inhibiting IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation, without affecting Akt phosphorylation. In addition, MFRV-VILP specifically regulates IGF-1R gene expression and IGF1R protein levels without affecting IR. CryoEM analysis confirms that scMFRV-VILP' binding to IGF1R is mirroring the interaction pattern observed with IGF-1. These findings offer valuable insights into IGF1R action and inhibition, suggesting potential applications in development of IGF1R specific inhibitors and advancing long-lasting insulins.

Interaction of a viral insulin-like peptide with the IGF-1 receptor produces a natural antagonist.

Lymphocystis disease virus-1 (LCDV-1) and several other Iridoviridae encode viral insulin/IGF-1 like peptides (VILPs) with high homology to human insulin and IGFs. Here we show that while single-chain (sc) and double-chain (dc) LCDV1-VILPs have very low affinity for the insulin receptor, scLCDV1-VILP has high affinity for IGF1R where it can antagonize human IGF-1 signaling, without altering insulin signaling. Consequently, scLCDV1-VILP inhibits IGF-1 induced cell proliferation and growth hormone/IGF-1 induced growth of mice in vivo. Cryo-electron microscopy reveals that scLCDV1-VILP engages IGF1R in a unique manner, inducing changes in IGF1R conformation that led to separation, rather than juxtaposition, of the transmembrane segments and hence inactivation of the receptor. Thus, scLCDV1-VILP is a natural peptide with specific antagonist properties on IGF1R signaling and may provide a new tool to guide development of hormonal analogues to treat cancers or metabolic disorders sensitive to IGF-1 without affecting glucose metabolism.

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.

Efficacy of glucagon-like peptide-1 and estrogen dual agonist in pancreatic islets protection and pre-clinical models of insulin-deficient diabetes.

We study the efficacy of a glucagon-like peptide-1 (GLP-1) and estrogen dual agonist (GLP1-E2) in pancreatic islet protection. GLP1-E2 provides superior protection from insulin-deficient diabetes induced by multiple low-dose streptozotocin (MLD-STZ-diabetes) and by the Akita mutation in mice than a GLP-1 monoagonist. GLP1-E2 does not protect from MLD-STZ-diabetes in estrogen receptor-α (ERα)-deficient mice and fails to prevent diabetes in Akita mice following GLP-1 receptor (GLP-1R) antagonism, demonstrating the requirement of GLP-1R and ERα for GLP1-E2 antidiabetic actions. In the MIN6 ß cell model, GLP1-E2 activates estrogen action following clathrin-dependent, GLP-1R-mediated internalization and lysosomal acidification. In cultured human islet, proteomic bioinformatic analysis reveals that GLP1-E2 amplifies the antiapoptotic pathways activated by monoagonists. However, in cultured mouse islets, GLP1-E2 provides antiapoptotic protection similar to monoagonists. Thus, GLP1-E2 promotes GLP-1 and E2 antiapoptotic signals in cultured islets, but in vivo, additional GLP1-E2 actions in non-islet cells expressing GLP-1R are instrumental to prevent diabetes.

A viral insulin-like peptide is a natural competitive antagonist of the human IGF-1 receptor.

OBJECTIVE: Natural sources of molecular diversity remain of utmost importance as a reservoir of proteins and peptides with unique biological functions. We recently identified such a family of viral insulin-like peptides (VILPs). We sought to advance the chemical methods in synthesis to explore the structure-function relationship within these VILPs, and the molecular basis for differential biological activities relative to human IGF-1 and insulin. METHODS: Optimized chemical methods in synthesis were established for a set of VILPs and related analogs. These modified forms included the substitution of select VILP chains with those derived from human insulin and IGF-1. Each peptide was assessed in vitro for agonism and antagonism at the human insulin and the human insulin-like growth factor 1 receptor (IGF-1R). RESULTS: We report here that one of these VILPs, lymphocystis disease virus-1 (LCDV1)-VILP, has the unique property to be a potent and full antagonist of the IGF-1R. We demonstrate the coordinated importance of the B- and C-chains of the VILP in regulating this activity. Moreover, mutation of the glycine following the first cysteine in the B-chain of IGF-1 to serine, in concert with substitution to the connecting peptide of LCDV1-VILP, converted native IGF-1 to a high potency antagonist. CONCLUSIONS: The results reveal novel aspects in ligand-receptor interactions at the IGF-1 receptor and identify a set of antagonists of potential medicinal importance.

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.

Addition of Sialic Acid to Insulin Confers Superior Physical Properties and Bioequivalence.

Native insulin is susceptible to biophysical aggregation and fibril formation, promoted by manual agitation and elevated temperatures. The safety of the drug and its application to alternative forms of administration could be enhanced through the identification of chemical modifications that strengthen its physical stability without compromising its biological properties. Complex polysialic acids (PSAs) exist naturally and provide a means to enhance the physical properties of peptide therapeutics. A set of insulin analogues site-specifically derivatized with sialic acid were prepared in an overall yield of 50-60%. Addition of a single or multiple sialic acids conferred remarkable enhancement to the biophysical stability of human insulin while maintaining its potency. The time to the onset of fibrillation was extended by more than 10-fold relative to that of the native hormone. These results demonstrate that simplified sialic acid conjugates represent a viable alternative to complex natural PSAs in increasing the stability of therapeutic peptides.

A Disulfide Scan of Insulin by [3 + 1] Methodology Exhibits Site-Specific Influence on Bioactivity.

Insulin is the principal hormone involved in the regulation of metabolism and has served a seminal role in the treatment of diabetes. Building upon advances in insulin synthetic methodology, we have developed a straightforward route to novel insulins containing a fourth disulfide bond in a [3 + 1] fashion establishing the first disulfide scan of the hormone. All the targeted analogs accommodated the constraint to demonstrate an unexpected conformational flexibility of native insulin. The bioactivity was established for the constrained (4-DS) and unconstrained (3-DS) analogs by in vitro methods, and extended to in vivo study for select peptides. We also identified residue B10 as a preferred anchor to introduce a tether that would regulate insulin bioactivity. We believe that the described [3 + 1] methodology might constitute the preferred approach for performing similar disulfide scanning in peptides that contain multiple disulfides.

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.

The islet-expressed Lhx1 transcription factor interacts with Islet-1 and contributes to glucose homeostasis.

The LIM-homeodomain (LIM-HD) transcription factor Islet-1 (Isl1) interacts with the LIM domain-binding protein 1 (Ldb1) coregulator to control expression of key pancreatic ß-cell genes. However, Ldb1 also has Isl1-independent effects, supporting that another LIM-HD factor interacts with Ldb1 to impact ß-cell development and/or function. LIM homeobox 1 (Lhx1) is an Isl1-related LIM-HD transcription factor that appears to be expressed in the developing mouse pancreas and in adult islets. However, roles for this factor in the pancreas are unknown. This study aimed to determine Lhx1 interactions and elucidate gene regulatory and physiological roles in the pancreas. Co-immunoprecipitation using ß-cell extracts demonstrated an interaction between Lhx1 and Isl1, and thus we hypothesized that Lhx1 and Isl1 regulate similar target genes. To test this, we employed siRNA-mediated Lhx1 knockdown in ß-cell lines and discovered reduced Glp1R mRNA. Chromatin immunoprecipitation revealed Lhx1 occupancy at a domain also known to be occupied by Isl1 and Ldb1. Through development of a pancreas-wide knockout mouse model ( Lhx1∆Panc), we demonstrate that aged Lhx1∆Panc mice have elevated fasting blood glucose levels, altered intraperitoneal and oral glucose tolerance, and significantly upregulated glucagon, somatostatin, pancreatic polypeptide, MafB, and Arx islet mRNAs. Additionally, Lhx1∆Panc mice exhibit significantly reduced Glp1R, an mRNA encoding the insulinotropic receptor for glucagon-like peptide 1 along with a concomitant dampened Glp1 response and mild glucose intolerance in mice challenged with oral glucose. These data are the first to reveal that the Lhx1 transcription factor contributes to normal glucose homeostasis and Glp1 responses.

Optimized GIP analogs promote body weight lowering in mice through GIPR agonism not antagonism.

OBJECTIVE: Structurally-improved GIP analogs were developed to determine precisely whether GIP receptor (GIPR) agonism or antagonism lowers body weight in obese mice. METHODS: A series of peptide-based GIP analogs, including structurally diverse agonists and a long-acting antagonist, were generated and characterized in vitro using functional assays in cell systems overexpressing human and mouse derived receptors. These analogs were characterized in vivo in DIO mice following acute dosing for effects on glycemic control, and following chronic dosing for effects on body weight and food intake. Pair-feeding studies and indirect calorimetry were used to survey the mechanism for body weight lowering. Congenital Gipr-/- and Glp1r-/- DIO mice were used to investigate the selectivity of the agonists and to ascribe the pharmacology to effects mediated by the GIPR. RESULTS: Non-acylated, Aib2 substituted analogs derived from human GIP sequence showed full in vitro potency at human GIPR and subtly reduced in vitro potency at mouse GIPR without cross-reactivity at GLP-1R. These GIPR agonists lowered acute blood glucose in wild-type and Glp1r-/- mice, and this effect was absent in Gipr-/- mice, which confirmed selectivity towards GIPR. Chronic treatment of DIO mice resulted in modest yet consistent, dose-dependent decreased body weight across many studies with diverse analogs. The mechanism for body weight lowering is due to reductions in food intake, not energy expenditure, as suggested by pair-feeding studies and indirect calorimetry assessment. The weight lowering effect was preserved in DIO Glp-1r-/- mice and absent in DIO Gipr-/- mice. The body weight lowering efficacy of GIPR agonists was enhanced with analogs that exhibit higher mouse GIPR potency, with increased frequency of administration, and with fatty-acylated peptides of extended duration of action. Additionally, a fatty-acylated, N-terminally truncated GIP analog was shown to have high in vitro antagonism potency for human and mouse GIPR without cross-reactive activity at mouse GLP-1R or mouse glucagon receptor (GcgR). This acylated antagonist sufficiently inhibited the acute effects of GIP to improve glucose tolerance in DIO mice. Chronic treatment of DIO mice with high doses of this acylated GIPR antagonist did not result in body weight change. Further, co-treatment of this acylated GIPR antagonist with liraglutide, an acylated GLP-1R agonist, to DIO mice did not result in increased body weight lowering relative to liraglutide-treated mice. Enhanced body weight lowering in DIO mice was evident however following co-treatment of long-acting selective individual agonists for GLP-1R and GIPR, consistent with previous data. CONCLUSIONS: We conclude that peptide-based GIPR agonists, not peptide-based GIPR antagonists, that are suitably optimized for receptor selectivity, cross-species activity, and duration of action consistently lower body weight in DIO mice, although with moderate efficacy relative to GLP-1R agonists. These preclinical rodent pharmacology results, in accordance with recent clinical results, provide definitive proof that systemic GIPR agonism, not antagonism, is beneficial for body weight loss.

Structurally Constrained Insulin Analogs by Directed Stepwise Crosslinking.

BACKGROUND: Research has been directed at the optimization of insulin for medicinal purposes. An insulin analog that could be reversibly activated might provide more precise pharmacokinetic control and broaden the inherent therapeutic index of the hormone. The prospect of using intramolecular structural constraint to reversibly inactive insulin might constitute the first step to achieving such an optimized analog. Chemically crosslinked insulin analogs have been reported where two amines are covalently linked by reaction with symmetrical bifunctional active esters. There is little selectivity in this synthetic approach to molecular constraint with multiple derivatives being formed. OBJECTIVE: To systematically evaluate the synthesis of covalently crosslinked insulin analogs by asymmetric methods and the biological consequences. METHOD: We report synthesis of amine crosslinked insulin analogs via a two-step procedure. The stepwise approach was initiated by amide bond formation and followed by second site alkylation to produce site-specific, cross-linked insulin analogs. RESULTS: A set of unique insulin analogs crosslinked at the two of the three native amines were synthesized. They were chemical characterized and assessed by in vitro bioanalysis to result in a significant and reasonably consistent reduction in biological potency. CONCLUSION: We achieved an unambiguous two-step synthesis of several crosslinked insulin analogs differing in location of the chemical tether. Bioanalysis demonstrated the ability of the molecular constraint to reduce bioactivity. The results set the stage for in vivo assessment of whether such a reduction in potency can be used pharmacologically to establish a constrained hormone upon which reversible tethering might be subsequently introduced.

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.

Viral insulin-like peptides activate human insulin and IGF-1 receptor signaling: A paradigm shift for host-microbe interactions.

Viruses are the most abundant biological entities and carry a wide variety of genetic material, including the ability to encode host-like proteins. Here we show that viruses carry sequences with significant homology to several human peptide hormones including insulin, insulin-like growth factors (IGF)-1 and -2, FGF-19 and -21, endothelin-1, inhibin, adiponectin, and resistin. Among the strongest homologies were those for four viral insulin/IGF-1-like peptides (VILPs), each encoded by a different member of the family Iridoviridae VILPs show up to 50% homology to human insulin/IGF-1, contain all critical cysteine residues, and are predicted to form similar 3D structures. Chemically synthesized VILPs can bind to human and murine IGF-1/insulin receptors and stimulate receptor autophosphorylation and downstream signaling. VILPs can also increase glucose uptake in adipocytes and stimulate the proliferation of fibroblasts, and injection of VILPs into mice significantly lowers blood glucose. Transfection of mouse hepatocytes with DNA encoding a VILP also stimulates insulin/IGF-1 signaling and DNA synthesis. Human microbiome studies reveal the presence of these Iridoviridae in blood and fecal samples. Thus, VILPs are members of the insulin/IGF superfamily with the ability to be active on human and rodent cells, raising the possibility for a potential role of VILPs in human disease. Furthermore, since only 2% of viruses have been sequenced, this study raises the potential for discovery of other viral hormones which, along with known virally encoded growth factors, may modify human health and disease.

Synthesis and Characterization of the R27S Genetic Variant of Insulin-like Peptide 5.

We report the synthesis and in vitro bioactivity assessment for an insulin-like peptide 5 (INSL5) analogue that was recently discovered as a genetic mutation in an Amish population. The mutation was associated with improved metabolic status, and receptor-based antagonism was proposed as a potential mechanism for the altered phenotype. We determined the specific peptide analogue to be fully potent and of maximal efficacy at the human relaxin family peptide receptor 4 (RXFP4), suggesting an alternative basis for the observed effect. In preparation of this synthetically challenging hormone, we have introduced several improvements such as implementation of isoacyl chemistry for high-efficiency preparation of INSL5 B-chain and selective intramolecular A6-11 disulfide formation as a first step in sequential disulfide assembly.

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.

Synthetic Advances in Insulin-like Peptides Enable Novel Bioactivity.

Insulin is a miraculous hormone that has served a seminal role in the treatment of insulin-dependent diabetes for nearly a century. Insulin resides within in a superfamily of structurally related peptides that are distinguished by three invariant disulfide bonds that anchor the three-dimensional conformation of the hormone. The additional family members include the insulin-like growth factors (IGF) and the relaxin-related set of peptides that includes the so-called insulin-like peptides. Advances in peptide chemistry and rDNA-based synthesis have enabled the preparation of multiple insulin analogues. The translation of these methods from insulin to related peptides has presented unique challenges that pertain to differing biophysical properties and unique amino acid compositions. This Account presents a historical context for the advances in the chemical synthesis of insulin and the related peptides, with division into two general categories where disulfide bond formation is facilitated by native conformational folding or alternatively orthogonal chemical reactivity. The inherent differences in biophysical properties of insulin-like peptides, and in particular within synthetic intermediates, have constituted a central limitation to achieving high yield synthesis of properly folded peptides. Various synthetic approaches have been advanced in the past decade to successfully address this challenge. The use of chemical ligation and metastable amide bond surrogates are two of the more important synthetic advances in the preparation of high quality synthetic precursors to high potency peptides. The discovery and application of biomimetic connecting peptides simplifies proper disulfide formation and the subsequent traceless removal by chemical methods dramatically simplifies the total synthesis of virtually any two-chain insulin-like peptide. We report the application of these higher synthetic yield methodologies to the preparation of insulin-like peptides in support of exploratory in vivo studies requiring a large quantity of peptide. Tangentially, we demonstrate the use of these methods to study the relative importance of the IGF-1 connecting peptide to its biological activity. We report the translation of these finding in search of an insulin analog that might be comparably enhanced by a suitable connecting peptide for interaction with the insulin receptor, as occurs with IGF-1 and its receptor. The results identify a unique receptor site in the IGF-1 receptor from which this enhancement derives. The selective substitution of this specific IGF-1 receptor sequence into the homologous site in the insulin receptor generated a chimeric receptor that was equally capable of signaling with insulin or IGF-1. This novel receptor proved to enhance the potency of lower affinity insulin ligands when they were supplemented with the IGF-1 connecting peptide that similarly enhanced IGF-1 activity at its receptor. The chimeric insulin receptor demonstrated no further enhancement of potency for native insulin when it was similarly prepared as a single-chain analogue with a native IGF-1 connecting peptide. These results suggest a more highly evolved insulin receptor structure where the requirement for an additional structural element to achieve high potency interaction as demonstrated for IGF-1 is no longer required.

Synthesis of relaxin-2 and insulin-like peptide 5 enabled by novel tethering and traceless chemical excision.

This report presents an entirely chemical, general strategy for the synthesis of relaxin-2 and insulin-like peptide 5. Historically, these two peptides have represented two of the more synthetically challenging members of the insulin superfamily. The key synthetic steps involve two sequential oxime ligations to covalently link the individual A-chain and B-chain, followed by disulfide bond formation under aqueous, redox conditions. This is followed by two chemical reactions that employ diketopiperazine cyclization-mediated cleavage and ester hydrolysis to liberate the connecting peptide and the heterodimeric product. This approach avoids the conventional iodine-mediated disulfide bond formation and enzyme-assisted proteolysis to generate biologically active two-chain peptides. This novel synthetic strategy is ideally suited for peptides such as relaxin and insulin-like peptide 5 as they possess methionine and tryptophan that are labile under strong oxidative conditions. Additionally, these peptides possess multiple arginine and lysine residues that preclude the use of trypsin-like enzymes to obtain biologically active hormones. This synthetic methodology is conceivably applicable to other two-chain peptides that contain multiple disulfide bonds. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.