Design of an Insulin Analog with Enhanced Receptor Binding Selectivity

Abstract

Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor (IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical interest in relation to the association between hyperinsulinemia and colorectal cancer. Here, we employ nonstandard mutagenesis to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR. Unnatural amino acids were introduced by chemical synthesis at the N-and C-capping positions of a recognition ␣-helix (residues A1 and A8). These sites adjoin the hormone-receptor interface as indicated by photocross-linking studies. Specificity is enhanced more than 3-fold on the following: Epidemiological studies have demonstrated an association between the metabolic syndrome and type 2 diabetes mellitus (DM) 4 with enhanced risk of colorectal cancer (CRC) (1-3). Animal models suggest that this risk is due to hyperinsulinemia (4, 5). An increased risk of CRC is conferred by treated-derived hyperinsulinemia, either as a direct consequence of insulin replacement therapy or as an indirect response to sulfonylurea secretagogues (6 -9). A reduced CRC risk is by contrast associated with metformin treatment, which results in lower circulating insulin concentrations (7). Because of the global epidemic of type 2 DM (10, 11), it would be of interest to develop a form of insulin therapy that does not elevate CRC risk in this clinical setting.A variety of evidence suggests that the association between hyperinsulinemia and CRC is due to aberrant activation of the type 1 IGF receptor (IGFR) (for review see Refs. 5,12). Although underlying molecular mechanisms are incompletely understood, two models have been proposed (5, 12). The first posits cross-binding of insulin to IGFR in colonic epithelia and neoplastic clones, driving proliferation and tumorigenesis; the second proposes indirect IGFR activation through perturbation of the IGF-I axis (including expression of the IGF-binding proteins) (13,14). Whereas consistent changes in bioactive IGF-I have been difficult to document (4), evidence favoring the direct model is provided by studies of epithelial proliferation in rats (15). Exogenous administration of insulin with a euglycemic clamp causes an acute dose-dependent increase in the proliferation rate of colonic epithelia despite decreased IGF-I expression. Such increased proliferation was not observed as a consequence of hyperglycemia or following infusion of a triglyceride emulsion (15).Insulin binds with high affinity (K d ϳ0.05 nM) to the insulin receptor (IR) and with low but significant affinity (K d Ј ϳ9.0 nM) to IGFR (16). Such cross-binding reflects the structural homology between insulin and IGF-I (17) and between IR and IGFR (18). The structural basis for receptor discrimination by insulin and IGF-I has been described recently (19). As a first step toward dissecting the molecular mechanism of insulin-dependent proliferation of colonic epithelia c...

Protein Phylogenetic Analysis of Ca2+/cation Antiporters and Insights into their Evolution in Plants

“…It is likely that both halves of the protein formed following an ancient duplication event. While split halves of NCX or CAX proteins are not able to function alone (Ottolia et al, 2001; Zhao et al, 2009), it is not inconceivable that an ancestral form was useful as part of a related protein domain if this event occurred very early in the evolution of the gene family.…”

Selectivity of Cell Signaling in the Neuronal Response Based on NGF Mutations and Peptidomimetics in the Treatment of Alzheimers Disease

“…This attempt is a true challenge with a multifunctional, pleiotropic cytokine such as TNF; nevertheless some progress has been made. Similarly, insulin has been engineered to improve its stability as a single chain (Hua, et al, 2008), its stability as a zinc 'stapled' hexamer with even longer lasting attributes (Phillips, et al, 2010), and its selectivity for the insulin receptor over the non-cognate IGF-1 receptor (Zhao, et al, 2009). A reported 3-fold improvement in receptor selectivity has the potential of reducing colorectal cancer risk for diabetic patients using insulin replacement therapy (Zhao, et al, 2009).…”

Supramolecular Protein Engineering

“…Wild-type insulin, insulin glargine, and IGF-I provided controls. Data were analyzed by non-linear regression using a two-site sequential model (21). The percentage of tracer bound in the absence of competing ligand was Ͻ15% to avoid ligand depletion artifacts.…”

“…Receptor Binding Assays-IR (isoform B) and IGF-1R binding assays were performed by a microtiter plate antibody capture assay (supplemental Methods) (21). Wild-type insulin, insulin glargine, and IGF-I provided controls.…”

“…Positions A4 and A8 were chosen as sites of modification to avoid structural perturbation, preserve activity (18), and maintain classical zinc assembly (4,19). Choice of this A-chain ␣-helix was further motivated by potentially favorable effects of its modification on the ratio of IR:IGF-1R binding (20,21). Zincstapled insulin hexamers exemplify a structure-based approach to the design of a subcutaneous protein depot.…”