Signaling Mechanisms of Ultra-Stable and Glucose-Regulated Insulin Analogs

About This Grant

Project Summary Insulin, central to the hormonal regulation of metabolism, is foundational to the management of Type 1 diabetes (T1D). This MPI application focuses on (a) ultra-stable single-chain insulin (SCI) analogs as probes of structure- activity relationships and their implications for design of (b) a novel class of glucose-responsive insulin analogs (GRIs). The latter would be transformational to mitigate the hypoglycemic risk of insulin therapy in T1D. Our Approach builds on respective cryo-EM-based structures of (i) an insulin fibril and (ii) signaling complexes between the hormone and the insulin receptor (IR). Each class of structures highlights the importance of conformational change. On the one hand, the major barrier to the stability of insulin above room temperature is posed by fibrillation, conformational conversion to a cross-β assembly (amyloid). On the other hand, partial unfolding of the B chain in the hormone-IR complex triggers receptor reorganization to achieve an active signaling conformation. The connecting (C) domain of SCIs provides a “molecular ruler” to probe these respective mechanisms of conformational change, enabled by analogs of successive C-domain length. Aim 1 seeks to resolve fundamental discrepancies between two recent 3D models of an insulin fibril. Decisive tests will be provided by residue-specific photo-cross-linking and 13C-directed solid-state NMR. Aim 2 investigates why foreshortened SCIs are intrinsically refractory to fibrillation: we hypothesize that a “sweet spot” exists in C-domain length: too short to permit cross-β assembly, sufficiently long to enable receptor binding and activation. Selective photo-reactive residues and 13C labels will be inserted by chemical protein synthesis. Cryo- EM structures will be sought of representative active SCI-IR ectodomain complexes and threshold SCI fibrils. Aim 3 re-conceptualizes the chain topology of an SCI as a platform for GRI design. Motivated by the profound clinical significance of hypoglycemia as a complication of insulin therapy, we envision a novel class of GRIs based on a glucose-dependent switch between an insulin antagonist (under hypoglycemic conditions) and an insulin agonist (under hyperglycemic conditions). This mechanism was anticipated in our studies of a fructose- responsive insulin prototype (FRI; Chen, Y.-S., et al. Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone-receptor activation. PNAS 118(3):e210351818 (2021)); see also a concurrent Commentary (Blundell, T.L. PNAS 118(33):e2111313118 (2021)). Here, we extend this strategy from FRI to GRI via a chemical glucose sensor. Remarkably, the proposed antagonist-agonist switch has been visualized in cryo-EM structures depicting glucose-dependent activation of a GRI-IR ectodomain complex and functionally validated in a rat model of T1D. To our knowledge, this scheme is unprecedented in the GRI field. Our Aims thus integrate basic and translational perspectives: ultra-stable, fibrillation-resistant SCIs would enhance insulin access in the face of an emerging diabetes pandemic (Weiss, M.A. Lancet Diabetes Endocrin. 11:307-9 (2023)) whereas GRIs promise to fundamentally transform the safety and efficacy of insulin therapy.