Targeting the insulin receptor with hormone and peptide dimers

Insulin is a key hormone involved in the regulation of overall energetic homeostasis of the organism. The dimeric character of the receptor for insulin evokes ideas about its activation or inhibition with peptide dimers that could either trigger or block the structural transition of the insulin receptor, leading to its activation. Herewith, we present the chemical engineering and biological characterization of several series of insulin dimers or dimers of specific peptides that should be able to bind receptors for insulin or insulin growth factor 1. The hormones or peptides in the dimers were interconnected with different linkers, consisting of triazole moieties and 3, 6, 8, 11, or 23 polyethylene glycol units. The prepared dimers were weaker in binding to insulin receptors than human insulin. However, some of the insulin dimers showed preferential binding specificity toward the isoform A of the insulin receptor, and the insulin dimers also stimulated the insulin receptor more strongly than would be consistent with their binding affinities. Our results suggest that designing insulin dimers may be a promising strategy for modulating the ability of the hormone to activate the receptor or to alter its specificity toward insulin receptor isoforms.     

Structural Investigations of Full-Length Insulin Receptor Dynamics and Signalling

Insulin regulates glucose homeostasis via binding and activation of the insulin receptor dimer at two distinct pairs of binding sites 1 and 2. Here, we present cryo-EM studies of full-length human insulin receptor (hIR) in an active state obtained at non-saturating, physiologically relevant insulin conditions. Insulin binds asymmetrically to the receptor under these conditions, occupying up to three of the four possible binding sites. Deletion analysis of the receptor together with site specific peptides and insulin analogs used in binding studies show that both sites 1 and 2 are required for high insulin affinity. We identify a homotypic interaction of the fibronectin type III domain (FnIII-3) of IR resulting in tight interaction of membrane proximal domains of the active, asymmetric receptor dimer. Our results show how insulin binding at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer. We propose an insulin binding and activation mechanism, which is sequential, exhibits negative cooperativity, and is based on asymmetry at physiological insulin concentrations with one to three insulin molecules activating IR.     

胰岛素折叠、结合与稳定性的核磁共振研究(英)

Insulin is one of the most important hormonal regulators of metabolism. Since the diabetes patients increase dramatically, the chemical properties, biological and physiological effects of insulin had been extensively studied. In last decade the development of NMR technique allowed us to determine the solution structures of insulin and its variety mutants in various conditions, so that the knowledge of folding, binding and stability of insulin in solution have been largely increased. The solution structure of insulin monomers is essentially identical to those of insulin monomers within the dimer and bexamer as determined by X-ray diffraction. The studies of insulin mutants at the putative residues for receptor binding explored the possible conformational change and fitting between insulin and its receptor. The systematical studies of disulfide paring coupled insulin folding intermediates revealed that in spite of the conformational variety of the intermediates, one structural feature is always remained: a “native-like B chain super-secondary structure“, which consists of B9-B19 helix with adjoining B23-B26 segment folded back against the central segment of B chain, an internal cystine A20-B19 disulfide bridge and a short a-helix at C-terminal of A chain linked. The “super-secondary structure“ might be the “folding nucleus“ in insulin folding mechanism. Cystine A20-B19 is the most important one among three disulfides to stabilize the nascent polypeptide in early stage of the folding. The NMR structure of C. elegans insulin-like peptide resembles that of human insulin and the peptide interacts with human insulin receptor. Other members of insulin superfamily adopt the “insulin fold“ mostly. The structural study of insulin-insulin receptor complex, that of C elegans and other invertebrate insulin-like peptide, insulin fibril study and protein disulfide isomerase (PDI) assistant proinsulin folding study will be new topics in future to get insight into folding, binding, stability, evolution and fibrillation of insulin in detail.     

Preparation and characterization of two LysB29 specifically labelled fluorescent derivatives of human insulin.

The preparation and characterization of two novel LysB29 selectively labelled fluorescent derivatives of human insulin are described. Two probes were chosen: 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD) and 7-methoxycoumarin-4-acetic acid (MCA), which have a relatively small, compact structure and are able to react with amino groups to form highly fluorescent derivatives. The combination of solid phase peptide synthesis and enzymatic semisynthesis was chosen for preparation of these fluorescent derivatives. Using two different protocols of solid-phase peptide synthesis, two fluorescent octapeptides were prepared corresponding to the position B23-B30 of human insulin, each with a different fluorescent label, NBD or MCA, on the epsilon-amino group of lysine. Then, the fluorescent octapeptides were coupled to desoctapeptide-(B23-B30)-insulin by a trypsin catalysed reaction. The receptor binding affinities of two novel fluorescent derivatives of human insulin with NBD and MCA (HI-NBD and HI-MCA) were determined on rat adipose tissue plasma membranes. Both fluorescent insulins, HI-NBD and HI-MCA, had only slightly reduced binding affinity and will be used for studying the interaction of insulin with its receptor.     

Functional reconstitution of insulin receptor binding site from non-binding receptor fragments

We have previously shown that a minimized insulin receptor (IR) consisting of the first 468 amino acids of the insulin receptor fused to 16 amino acids from the C terminus of the alpha-subunit (CT domain) bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Sch?ffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780-17786). In the present study, we show that a smaller construct that has the first 308 residues fused to the CT domain also binds insulin. Insulin receptor fragments consisting of the first 468 or 308 residues did not bind insulin. However, when these fragments were mixed with a synthetic peptide corresponding to the CT domain, insulin binding was detectable. At concentrations of 10 microm CT peptide, insulin binding was fully reconstituted yielding apparent affinities of 9-11 nm. To further investigate the minimum requirement for the length of the N terminus of IR, we tested smaller receptor fragments for insulin binding in the presence of the CT peptide and found that a fragment consisting of the first 255 amino acids of IR was able to fully reconstitute the insulin binding site, yielding an apparent affinity of 11 +/- 4 nm for insulin.     

Structural models of viral insulin-like peptides and their analogs

The insulin receptor (IR), the insulin-like growth factor-1 receptor (IGF1R), and the insulin/IGF1 hybrid receptors (hybR) are homologous transmembrane receptors. The peptide ligands, insulin and IGF1, exhibit significant structural homology and can bind to each receptor via site-1 and site-2 residues with distinct affinities. The variants of the Iridoviridae virus family show capability in expressing single-chain insulin/IGF1 like proteins, termed viral insulin-like peptides (VILPs), which can stimulate receptors from the insulin family. The sequences of VILPs lacking the central C-domain (dcVILPs) are known, but their structures in unbound and receptor-bound states have not been resolved to date. We report all-atom structural models of three dcVILPs (dcGIV, dcSGIV, and dcLCDV1) and their complexes with the receptors (μIR, μIGF1R, and μhybR), and probed the peptide/receptor interactions in each system using all-atom molecular dynamics (MD) simulations. Based on the nonbonded interaction energies computed between each residue of peptides (insulin and dcVILPs) and the receptors, we provide details on residues establishing significant interactions. The observed site-1 insulin/μIR interactions are consistent with previous experimental studies, and a residue-level comparison of interactions of peptides (insulin and dcVILPs) with the receptors revealed that, due to sequence differences, dcVILPs also establish some interactions distinct from those between insulin and IR. We also designed insulin analogs and report enhanced interactions between some analogs and the receptors.     

Design, synthesis and pharmacological evaluation of cyclic mimetics of the insulin-like peptide 3 (INSL3) B-chain.

Insulin-like peptide 3 (INSL3) is a peptide hormone belonging to the relaxin-insulin superfamily of peptides that plays important roles in testes descent, oocyte maturation and the control of male germ cell apoptosis. These actions are mediated via a specific G-protein coupled receptor, LGR8. Previous structure-activity studies have shown that the key binding site of INSL3 is situated within its B-chain. Recent studies in our laboratory have led to the identification of a cyclic peptide mimetic 2 of the INSL3 B-chain, which we have shown to compete with the binding of [33P]-relaxin to LGR8 expressed in HEK293T cells, and to inhibit cAMP-mediated signaling in these cells, i.e. it is an antagonist of INSL3. In order to further define the structure-activity relationships of cyclic analogues of the INSL3 B-chain, we used a structure-based approach to design a series of cyclic, disulfide-constrained INSL3 B-chain mimetics. To do this, we first created a model of the 3D structure of INSL3 using the crystal structure of human relaxin as a template. This model of INSL3 was then used as a template to design a series of disulfide-constrained mimetics of the INSL3 B-chain. The peptides were synthesized by solid-phase peptide synthesis using pseudoproline dipeptides to improve the synthesis outcome. Of the seven prepared INSL3 B-chain mimetics, three compounds were found to have partial displacement activity, while four were able to completely displace [33P]-relaxin from LGR8, including compounds that were markedly shorter than compound 2. The best of these, mimetic 6, showed significantly greater affinity for LGR8 than compound 2, but still displayed around 1000-fold less affinity for LGR8 than native INSL3. Analysis of selected mimetics for their alpha-helical content using circular dichroism (CD) spectroscopy revealed that, generally, the mimetics showed less than expected helicity. The inability of the compounds to display true native INSL3 structure is likely contributing to their reduced receptor binding affinity. We are currently examining alternative INSL3 B-chain mimetics that might better present key receptor binding residues in the native INSL3-like conformation.     

The road to the first,fully active and more stable human insulin variant with an additional disulfide bond

Insulin, a small peptide hormone, is crucial in maintaining blood glucose homeostasis. The stability and activity of the protein is directed by an intricate system involving disulfide bonds to stabilize the active monomeric species and by their non‐covalent oligomerization. All known insulin variants in vertebrates consist of two peptide chains and have six cysteine residues, which form three disulfide bonds, two of them link the two chains and a third is an intra‐chain bond in the A‐chain. This classical insulin fold appears to have been conserved over half a billion years of evolution. We addressed the question whether a human insulin variant with four disulfide bonds could exist and be fully functional. In this review, we give an overview of the road to engineering four‐disulfide bonded insulin analogs. During our journey, we discovered several active four disulfide bonded insulin analogs with markedly improved stability and gained insights into the instability of analogs with seven cysteine residues, importance of dimerization for stability, insulin fibril formation process, and the conformation of insulin binding to its receptor. Our results also open the way for new strategies in the development of insulin biopharmaceuticals. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses

The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.     

Identification of a disulfide bridge connecting the alpha-subunits of the extracellular domain of the insulin receptor.

The alpha 2 beta 2 structure of the insulin receptor has previously been shown to involve one disulfide bridge between the alpha-subunits in the region containing Cys435, Cys468 and Cys524. We have digested the soluble extracellular domain of the insulin receptor with succinylated trypsin, partially separated the resulting peptides, and sequenced a number of fractions. The peptides containing Cys435 and Cys468 appeared in the same fraction, indicating that these two form a disulfide bond, and in another fraction we found the sequence of the peptide containing Cys524. Since it has been shown that the extracellular domain of the insulin receptor has no free thiols and since no other sequences containing cysteine were found in these fractions, we conclude that Cys524 forms a disulfide bond to the Cys524 in the other alpha-subunit.     

Insulin/IGF-I receptor hybrids: a mechanism for increasing receptor diversity.

Insulin and IGF-I receptors are homologous disulfide linked alpha 2 beta 2 tetramers. These tetramers are formed biosynthetically when proreceptors containing alpha and beta subunits in a single uninterrupted linear peptide form disulfide linked homodimers and are subsequently proteolytically cleaved at the alpha-beta junctions. Cells expressing both receptors also express hybrid receptors that contain one insulin receptor alpha and beta subunit, and one IGF-I receptor alpha and beta subunit. These presumably form by the association of mixed proreceptors. Hybrid receptors greatly expand the possible repertoire of cellular responses to hormonal stimulation. Although not yet examined in detail, both the hormone binding and the signaling properties of the hybrid receptor appear to be different from that of either insulin or IGF-I receptor. Regulatory mechanisms that involve either insulin or IGF-I receptor, at the level of expression or subsequently, could alter the expression or function of the hybrid receptor or the other receptor. Similarly, pathology in one receptor could affect both the hybrid and other receptor, or perhaps be partially compensated for by a hybrid receptor. The magnitude of these effects could vary greatly in different tissues depending upon the relative level of expression of the different receptor forms. These postulated responses might explain some of the complex heterogeneity and linkage of these receptors that have been observed previously.     

Structure-activity relationships of a peptide inhibitor of the human FcRn:human IgG interaction

A family of five peptides was previously discovered by phage display techniques that binds to the human neonatal Fc receptor (FcRn) and inhibits the human IgG:human FcRn protein-protein interaction [Proc. Nat. Acad. Sci. U.S.A.2008, 105, 2337-2342]. The consensus peptide motif consists of the sequence GHFGGXY where X is preferably a hydrophobic amino acid, and also includes a disulfide bridge enclosing 11-amino acids in varying positions about the consensus sequence. We describe herein the structure-activity relationships of one of the five peptides in binding to FcRn using surface plasmon resonance and IgG:FcRn competition ELISA assays. Modifications of the peptide length, cyclization, and the incorporation of amino acid substitutions and dipeptide mimetics were studied. The most potent analogs exhibited a 50- to 100-fold improvement of in vitro activity over that of the phage-identified peptide sequence.     

The relationship between the connecting peptide of recombined single chain insulin and its biological function

To investigate the relationship between the biological activity of recombined single chain insulin and the length of the connecting peptide, we designed and prepared three single chain insulin molecules, namely, PIP, [A]₅PIP and [A]₁₀PIP, by site-directed mutagenesis, in which B30 and A1 were linked through dipeptide A-K, heptapeptide A-A-A-A-A-A-K, and dodecapeptide A-A-A-A-A-A-A-A-A-A-A-K, respectively. Their receptor binding capacities were 0.14%, 14.3% and 11.1% of that of insulin respectively and theirin vivo biological activities were in consistence with their receptor binding capacity; whereas their growth promoting activities were 17%, 116.3% and 38% of that of insulin. These results suggested the following conclusions. (i) The recombined single chain insulin could also possess the same metabolic and mitogenic function as insulin. (ii) The receptor binding capacity of recombined single chain insulin to insulin receptor was closely related to the length and amino acid composition of the connecting peptide and could change from 0 to 100% of insulin depending on the different connecting peptides. This result further illustrated the necessity of B chain C-terminus swaying away from A chain N-terminus when insulin binds to its receptor. (iii) The mitogenic activity of recombined single chain insulin also depended on the length and the amino acid composition of the connecting peptide and was higher than its metabolic activity.     

Diabetes mellitus caused by mutations in human insulin: analysis of impaired receptor binding of insulins Wakayama,Los Angeles and Chicago using pharmacoinformatics

Several naturally occuring mutations in the human insulin gene are associated with diabetes mellitus. The three known mutant molecules, Wakayama, Los Angeles and Chicago were evaluated using molecular docking and molecular dynamics (MD) to analyse mechanisms of deprived binding affinity for insulin receptor (IR). Insulin Wakayama, is a variant in which valine at position A3 is substituted by leucine, while in insulin Los Angeles and Chicago, phenylalanine at positions B24 and B25 is replaced by serine and leucine, respectively. These mutations show radical changes in binding affinity for IR. The ZDOCK server was used for molecular docking, while AMBER 14 was used for the MD study. The published crystal structure of IR bound to natural insulin was also used for MD. The binding interactions and MD trajectories clearly explained the critical factors for deprived binding to the IR. The surface area around position A3 was increased when valine was substituted by leucine, while at positions B24 and B25 aromatic amino acid phenylalanine replaced by non-aromatic serine and leucine might be responsible for fewer binding interactions at the binding site of IR that leads to instability of the complex. In the MD simulation, the normal mode analysis, rmsd trajectories and prediction of fluctuation indicated instability of complexes with mutant insulin in order of insulin native insulin < insulin Chicago < insulin Los Angeles < insulin Wakayama molecules which corresponds to the biological evidence of the differing affinities of the mutant insulins for the IR.     

Studies on the biological activity of an insulin-stimulating peptide from a tryptic digest of bovine serum albumin

Summary A two-chain polypeptide, which corresponds to amino acid residues 115–143 and 144–184(185) of bovine serum albumin, connected to each other by a disulfide bridge, potentiated the effects of insulin on glucose transport and glucose metabolism in isolated rat adipocytes. Although the peptide alone had little activity, it shifted the concentration-response curves of insulin-stimulated D-[I-¹⁴C]glucose oxidation, 2-deoxyglucose transport, and lipid synthesis from D-[U-¹⁴C]glucose to lower insulin concentrations. It also increased the maximal responses of these parameters to insulin. However, it did not affect insulin binding to adipocytes. The peptide protected insulin considerably from degradation, but this effect alone cannot account for its effect in increasing the maximal responses to the hormone, and even when degradation of a submaximal concentration of insulin was suppressed by bacitracin, the peptide still had an enhancing effect. These results suggest not only that the peptide influences a step distal to receptor-mediated insulin binding but also that inhibition of insulin degradation alone cannot explain its total effect.The peptide lost its insulin-stimulating activity completely when it was further digested with V8 or lysinespecific endopeptidase, or when it was reduced and then carboxamidomethylated or oxidized with performic acid. Similar active tryptic fragments were obtained from human and rat albumins.Insulin-stimulating peptides should be useful in studies on the mechanisms of insulin action including both the sensitivities and responsiveness of target cells to the hormone.Abbreviations ISP insulin-stimulating peptide - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - HPLC high-performance liquid chromatography - SDS sodium dodecyl sulfate     

Histidine 186 of the nicotinic acetylcholine receptor alpha subunit requires the presence of the 192-193 disulfide bridge to interact with alpha-bungarotoxin

We have previously shown that two histidine residues of the nicotinic acetylcholine receptor are relevant for alpha-bungarotoxin binding. This paper studies: (1) the interaction between alpha-bungarotoxin and the peptide alpha173-202--synthesized according to the sequence of the Torpedo californica receptor alpha subunit--and between the toxin and the same peptide containing His186 modified with ethoxyformic anhydride or substituted by Ala; (2) the influence of the presence of Cys192-Cys193 disulfide bridge on such interactions. Solid-phase and in-solution competition assays were performed: ethoxyformylation of His186 or its substitution by Ala led to a significant drop in the toxin binding capacity only for peptides containing the bridge. Circular dichroism and fourth derivate spectra of all peptides were also analyzed. Results strongly indicate the involvement of His186 in the toxin binding to those peptides with the bridge--also present in the native receptor molecules--but not to their reduced forms; on the other hand, they give further support to the already established premise that, though the bridge does not participate directly in receptor-toxin binding, its presence is relevant to define the appropriate conformation of the interaction area.     

Identification of a molecular activator for insulin receptor with potent anti-diabetic effects

Insulin exerts its actions through the insulin receptor (IR) and plays an essential role in diabetes. The inconvenient daily injection and undesirable side-effects associated with insulin injection demand novel drugs for the diseases. To search for bioactive insulin mimetics, we developed an in vitro screening assay using phospho-IR ELISA. After screening the small molecule chemical libraries, we have obtained a compound (5,8-diacetyloxy-2,3-dichloro-1,4-naphthoquinone) that provokes IR activation by directly binding to the receptor kinase domain to trigger its kinase activity at micromolar concentrations. This compound selectively activates IR but not other receptors and sensitizes insulin's action. Moreover, it elevates glucose uptake in adipocytes and has oral hypoglycemic effect in wild-type C57BL/6J mice and db/db and ob/ob mice without demonstrable toxicity. Hence, this promising compound mimics the biological functions of insulin and is useful for further drug development for diabetes treatment.     

Insulin analog with additional disulfide bond has increased stability and preserved activity

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα‐Cα distances, solvent exposure, and side‐chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C‐terminus of the B‐chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild‐type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R‐state conformation and thus showing that the R‐state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.     

Synthetic peptides corresponding to sequences of snake venom neurotoxins and rabies virus glycoprotein bind to the nicotinic acetylcholine receptor

Peptides corresponding to portions of loop 2 of snake venom curare-mimetic neurotoxins and to a structurally similar region of rabies virus glycoprotein were synthesized. Interaction of these peptides with purified Torpedo electric organ acetylcholine receptor was tested by measuring their ability to block the binding of 125I-labeled alpha-bungarotoxin to the receptor. In addition, inhibition of alpha-bungarotoxin binding to a 32-residue synthetic peptide corresponding to positions 173-204 of the alpha-subunit was determined. Neurotoxin and glycoprotein peptides corresponding to toxin loop 2 inhibited labeled toxin binding to the receptor with IC50 values comparable to those of nicotine and the competitive antagonist d-tubocurarine and to the alpha-subunit peptides with apparent affinities between those of d-tubocurarine and alpha-cobratoxin. Substitution of neurotoxin residue Arg37, the proposed counterpart of the quaternary ammonium of acetylcholine, with a negatively charged Glu residue reduced the apparent affinity about 10-fold. Peptides containing the neurotoxin invariant residue Trp29 and 10- to 100-fold higher affinities than peptides lacking this residue. These results demonstrate that relatively short synthetic peptides retain some of the binding ability of the native protein from which they are derived, indicating that such peptides are useful in the study of protein-protein interactions. The ability of the peptides to compete alpha-bungarotoxin binding to the receptor with apparent affinities comparable to those of other cholinergic ligands indicates that loop 2 of the neurotoxins and the structurally similar segment of the rabies virus glycoprotein act as recognition sites for the acetylcholine receptor. Invariant toxin residues Arg37 and Trp29 and their viral homologs play important, although not essential, roles in binding, possibly by interaction with complementary anionic and hydrophobic subsites on the acetylcholine receptor. The alpha-subunit peptide most likely contains all of the determinants for binding of the toxin and glycoprotein peptides present on the alpha-subunit, because these peptides bind to the 32-residue alpha-subunit peptide with the same or greater affinity as to the intact subunit.