Insulin Analogues with Altered Insulin Receptor Isoform Binding Specificities and Enhanced Aggregation Stabilities

Abstract

Insulin is a lifesaver for millions of diabetic patients. There is a need for new insulin analogues with more physiological profiles and analogues that will be thermally more stable than human insulin. Here, we describe the chemical engineering of 48 insulin analogues that were designed to have changed binding specificities toward isoforms A and B of the insulin receptor (IR-A and IR-B). We systematically modified insulin at the C-terminus of the B-chain, at the N-terminus of the A-chain, and at A14 and A18 positions. We discovered an insulin analogue that has Cαcarboxyamidated Glu at B31 and Ala at B29 and that has a more than 3-fold-enhanced binding specificity in favor of the "metabolic" IR-B isoform. The analogue is more resistant to the formation of insulin fibrils at 37 °C and is also more efficient in mice than human insulin. Therefore, [Ala B29 ,Glu B31 ,amide B31 ]-insulin may be interesting for further clinical evaluation. Article pubs.acs.org/jmc

Modulation of the antagonistic properties of an insulin mimetic peptide by disulfide bridge modifications

“…Mouse embryonic fibroblasts (IR-A) derived from IGF-1R knockout mice and stably transfected with human IR-A, kindly provided by A. Belfiore (Catanzaro, Italy) and R. Baserga (Philadelphia, Pennsylvania, USA), were grown as described previously. 25 Ligand-dose response IR-A autophosphorylation levels for the analogs were determined using an in-cell Western assay adapted for chemiluminiscence as described in Machackova et al 28 Briefly, the IR-A cells were plated at 20,000 cells/well in white 96-well Brand plates cell grade (Brand GMBH, Germany) and incubated for 24 h. The cells were starved for Anti-actin (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33) antibody (Sigma-Aldrich, cat. A5060) was used as a loading control.…”

Insulin-like growth factor-1 signaling in the tumor microenvironment: Carcinogenesis, cancer drug resistance, and therapeutic potential

“…Interestingly, various reports, including a study aimed at developing new insulin analogues thermally more stable and with more physiological profiles than human insulin provided evidence for the possibility to develop IR-A/IGF-1R targeting agents with higher specificity ( 66 ). In this study, chemical engineering of insulin analogues allowed the development of an insulin analogue with more than 3-fold-enhanced binding specificity for the metabolic IR-B isoform ( 67 ), allowing to prevent the pro-tumorigenic effects of insulin mediated by IR-A and hybrids with IGF-1R or other receptor tyrosine kinases ( 68 , 69 ).…”

Omniligase-1-Mediated Phage-Peptide Library Modification and Insulin Engineering

“…25 Glucose responsiveness, 26−30 aggregation resistance, 31−37 and insulin receptor (IR) isoform selectivity have also been achieved through modifications to this region. 38,39 However, fully exploring the functional scope of the B25−30 region in insulin remains challenging due to a lack of a high-throughput synthetic approach. 23,40−43 Herein, we present a novel approach utilizing omniligase-1 to modify the phage peptide library and engineer novel insulin analogues from the library (Figure 1).…”

“…This is due to the necessity for each analogue to be individually synthesized or expressed for testing, which is a time-consuming and expensive process. − Insulin B chain C-terminal is critical to insulin bioactivity, and modifications in this region have been reported to enhance pharmacokinetic properties in FDA-approved insulin analogues . Glucose responsiveness, − aggregation resistance, − and insulin receptor (IR) isoform selectivity have also been achieved through modifications to this region. , However, fully exploring the functional scope of the B25–30 region in insulin remains challenging due to a lack of a high-throughput synthetic approach. ,− …”