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. The authors previously identified a family of viral insulin-like peptides (VILPs) with high homology to human insulin/IGF−1. Here, they report that one of these VILPs exhibits antagonist properties associated with a unique conformation of the IGF1R

Localization and functions of immunoactive sections of aphthous fever virus yp_1 protein

The investigation is concerned with the YP_1 protein of the aphthous fever virus of the O_1K and A_2_2 strains. The object of investigation is identification of the immunoactive sections in the sequence of the aphthous fever YP_1 protein. The researchers have identified the B- and T-epitopes of the immunodominant region of the YP_1 protein of aphthous fever of the O_1K and A_2_2 strains and revealed the new immunogenic fragments of the YP_1 protein and the viral specificity of the T-helper immune response to the YP_1 A_2_2 peptides 135-159 and 170-189. The investigators have substantiated the feasibility of including the 135-159, 197-213 sulfhydrylmodified peptide 170-189 in the synthetic anti-aphthous-fever (strain A_2_2 vaccine). The obtained results may find application in the spheres of molecular and cellular biologyAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Synthetic Route to Human Relaxin‑2 via Iodine-Free Sequential Disulfide Bond Formation

A new synthetic route to human relaxin-2 has been established through a sequential disulfide bond formation process in the absence of iodine. It is enabled by a combination of cysteine protection with penicillin G acylase-labile Phacm and a newly identified thiol activator bis­(5-(2-methoxyethoxy)-2-pyrimidinyl disulfide. The long-standing challenges in relaxin B-chain assembly and its poor solubility have been solved by the insertion of two isoacyl dipeptide segments. The overall yield was 25% from the B chain and 5.8% from the B-chain starting resin

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

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. Keywords: Glucose-dependent insulinotropic polypeptide (GIP), Agonism, Obesity, Diet-induced obese (DIO) mice, Pharmacolog

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. <i>In vitro</i> 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

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 <i>tert</i>-butylthiol (S<i>t</i>Bu), thiol-trimethoxyphenyl (STmp), trityl (Trt), 4-methoxytrityl (Mmt), S-acetamidomethyl (Acm), and <i>tert</i>-butyl (<i>t</i>Bu). This report describes chemistry that is broadly applicable to cysteine-rich peptides and the influence of a fourth disulfide bond on insulin bioactivity

Biomimetic Synthesis of Insulin Enabled by Oxime Ligation and Traceless “C-Peptide” Chemical Excision

For decades, insulin has represented a preeminent synthetic target. Recently introduced “biomimetic” strategies based on convertible single-chain precursors require incorporation of a chemical linker or a unique proteolytic site, which limits their practicality. In this approach the A- and B-chains are linked by two sequential oxime ligations followed by disulfide bond formation under redox conditions and linker excision by diketopiperazine (DKP) formation and ester hydrolysis, yielding native two-chain insulin. The method is expected to be applicable to any member of the insulin superfamily

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