Insulin in motion: The A6-A11 disulfide bond allosterically modulates structural transitions required for insulin activity

The structural transitions required for insulin to activate its receptor and initiate regulation of glucose homeostasis are only partly understood. Here, using ring-closing metathesis, we substitute the A6-A11 disulfide bond of insulin with a rigid, non-reducible dicarba linkage, yielding two distinct stereo-isomers (cis and trans). Remarkably, only the cis isomer displays full insulin potency, rapidly lowering blood glucose in mice (even under insulin-resistant conditions). It also posseses reduced mitogenic activity in vitro. Further biophysical, crystallographic and molecular-dynamics analyses reveal that the A6-A11 bond configuration directly affects the conformational flexibility of insulin A-chain N-terminal helix, dictating insulin's ability to engage its receptor. We reveal that in native insulin, contraction of the Cα-Cα distance of the flexible A6-A11 cystine allows the A-chain N-terminal helix to unwind to a conformation that allows receptor engagement. This motion is also permitted in the cis isomer, with its shorter Cα-Cα distance, but prevented in the extended trans analogue. These findings thus illuminate for the first time the allosteric role of the A6-A11 bond in mediating the transition of the hormone to an active conformation, significantly advancing our understanding of insulin action and opening up new avenues for the design of improved therapeutic analogues.A.J.R., B.E.F. and M.C.L. acknowledge funding from National Health and Medical Research Council (Project Grant APP1069328 and Project Grant APP1058233) and Australian Research Council. Te Walter and Eliza Hall Institute of Medical Research acknowledges Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS

Insulin in motion: The A6-A11 disulfide bond allosterically modulates structural transitions required for insulin activity

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.The structural transitions required for insulin to activate its receptor and initiate regulation of glucose homeostasis are only partly understood. Here, using ring-closing metathesis, we substitute the A6-A11 disulfide bond of insulin with a rigid, non-reducible dicarba linkage, yielding two distinct stereo-isomers (cis and trans). Remarkably, only the cis isomer displays full insulin potency, rapidly lowering blood glucose in mice (even under insulin-resistant conditions). It also posseses reduced mitogenic activity in vitro. Further biophysical, crystallographic and molecular-dynamics analyses reveal that the A6-A11 bond configuration directly affects the conformational flexibility of insulin A-chain N-terminal helix, dictating insulin’s ability to engage its receptor. We reveal that in native insulin, contraction of the Cα-Cα distance of the flexible A6-A11 cystine allows the A-chain N-terminal helix to unwind to a conformation that allows receptor engagement. This motion is also permitted in the cis isomer, with its shorter Cα-Cα distance, but prevented in the extended trans analogue. These findings thus illuminate for the first time the allosteric role of the A6-A11 bond in mediating the transition of the hormone to an active conformation, significantly advancing our understanding of insulin action and opening up new avenues for the design of improved therapeutic analogues

Chemical synthesis and structure-function relationship study of insulin-like peptide 5 (INSL5)

© 2012 Dr. Alessia BelgiInsulin-like peptide 5 (INSL5) is a member of the insulin/relaxin superfamily of peptides. It is composed of two chains, A and B, linked together by two interchain disulfide bonds. A third intrachain disulfide bond exists within the A-chain. It has been identified as the cognate ligand for the G-protein-coupled receptor RXFP4. Although the complete physiological role of this naturally occurring peptide is still under investigation, there is evidence that it acts to both stimulate appetite and activate colon motility. Therefore both peptide agonists and antagonists may have potential important therapeutic applications. To further investigate the biochemical role of this peptide and because of the ready availability of the mouse as an experimental animal, the chemical synthesis of mouse INSL5 was undertaken. Due to its complex structure and the intractable nature of the two constituent chains, different solid phase synthesis strategies were investigated including the use of a temporary B-chain solubilising tag. Unfortunately, none provided significantly improved yield of purified mouse INSL5 which reflects the complexity of this peptide. A mouse INSL5 analogue with its two chains as C-terminal amides was also prepared. It was found that mouse INSL5 amide was substantially less potent than the native acid form highlighting the necessity for free C-terminal carboxylates for function. The structure-function relationship study continued with the design of two additional mouse INSL5 analogues. One had mutations targeting residues potentially involved in receptor activation (mouse INSL5 A-B(R24A, W25A)) and the other with the substitution of the residues potentially involved in the binding (mouse INSL5 A-B(K6A, R14A, Y18A)). The bioassays results showed that the Arg-Trp pair at the C-terminus of the B-chain is indeed important for activation and that LysB6, ArgB14 and TyrB18 are some of the key residues for receptor binding. The synthesis of the point-mutated analogues was again very challenging despite the application of various strategies. To potentially bypass this ongoing difficulty, biologically active single chain analogues were sought. A series of single B- and A-chain analogues were synthesized but none showed biological activity within the concentration range tested suggesting that INSL5 likely requires two chains for bioactivity. Next, minimization of native two-chain mouse INSL5 was examined. Mouse INSL5 A(C7A, C12A)-B and mouse INSL5 A(8-21, C12A)-B were prepared. The intramolecular disulfide bond was removed from both analogues and one had an additional N-terminus truncation on the A-chain. The synthesis of both analogues was simpler compared to other INSL5 peptides and they were also able to rescue both affinity and activity compared to the single chain analogues. They were also active in vivo, representing attractive templates for further modification. Additionally, a parallel study on INSL5 and the closely related relaxin-3 was undertaken. INSL5 was made more relaxin-3-like and, conversely, relaxin-3 more INSL5-like. These modifications generated unexpected results suggesting that the two peptides may not interact with their receptors using the same mechanism as initially assumed. Overall this project achieved important preliminary findings of the structure-function relationship of mouse INSL5 peptide opening the way to further biochemical investigation

Characterisation of Elevenin-Vc1 from the Venom of <i>Conus victoriae</i>: A Structural Analogue of α-Conotoxins

Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11–18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1β1εδ, α3β2, α3β4, α4β2, α7, and α9α10) at 1 µM concentrations

Dicarba analogues of α-conotoxin RgIA. Structure, stability, and activity at potential pain targets

α-Conotoxin RgIA is both an antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype and an inhibitor of high-voltage-activated N-type calcium channel currents. RgIA has therapeutic potential for the treatment of pain, but reduction of the disulfide bond framework under physiological conditions represents a potential liability for clinical applications. We synthesized four RgIA analogues that replaced native disulfide pairs with nonreducible dicarba bridges. Solution structures were determined by NMR, activity assessed against biological targets, and stability evaluated in human serum. [3,12]-Dicarba analogues retained inhibition of ACh-evoked currents at α9α10 nAChRs but not N-type calcium channel currents, whereas [2,8]-dicarba analogues displayed the opposite pattern of selectivity. The [2,8]-dicarba RgIA analogues were effective in HEK293 cells stably expressing human Cav2.2 channels and transfected with human GABAB receptors. The analogues also exhibited improved serum stability over the native peptide. These selectively acting dicarba analogues may represent mechanistic probes to explore analgesia-related biological receptors

Dicarba α-conotoxin Vc1.1 analogues with differential selectivity for nicotinic acetylcholine and GABAB receptors

Conotoxins have emerged as useful leads for the development of novel therapeutic analgesics. These peptides, isolated from marine molluscs of the genus Conus, have evolved exquisite selectivity for receptors and ion channels of excitable tissue. One such peptide, α-conotoxin Vc1.1, is a 16-mer possessing an interlocked disulfide framework. Despite its emergence as a potent analgesic lead, the molecular target and mechanism of action of Vc1.1 have not been elucidated to date. In this paper we describe the regioselective synthesis of dicarba analogues of Vc1.1 using olefin metathesis. The ability of these peptides to inhibit acetylcholine-evoked current at rat α9α10 and α3β4 nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes has been assessed in addition to their ability to inhibit high voltage-activated (HVA) calcium channel current in isolated rat DRG neurons. Their solution structures were determined by NMR spectroscopy. Significantly, we have found that regioselective replacement of the native cystine framework with a dicarba bridge can be used to selectively tune the cyclic peptide\u27s innate biological activity for one receptor over another. The 2,8-dicarba Vc1.1 isomer retains activity at γ-aminobutyric acid (GABAB) G protein-coupled receptors, whereas the isomeric 3,16-dicarba Vc1.1 peptide retains activity at the α9α10 nAChR subtype. These singularly acting analogues will enable the elucidation of the biological target responsible for the peptide\u27s potent analgesic activity

Minimum Active Structure of Insulin-like Peptide 5

Insulin-like peptide 5 (INSL5) is a complex two-chain peptide hormone constrained by three disulfide bonds in a pattern identical to insulin. High expression of INSL5 in the colon suggests roles in activation of colon motility and appetite control. A more recent study indicates it may have significant roles in the regulation of insulin secretion and β-cell homeostasis. This peptide thus has considerable potential for the treatment of eating disorders, obesity, and/or diabetes. However, the synthesis of INSL5 is extremely challenging either by chemical or recombinant means. The A-chain is very poorly soluble and the B-chain is highly aggregating in nature which, together, makes their postsynthesis handling and purification very difficult. Given these difficulties, we have developed a highly active INSL5 analogue that has a much simpler structure with two disulfide bonds and is thus easier to assemble compared to native INSL5. This minimized peptide represents an attractive new mimetic for investigating the functional role of INSL5