Chen, Yen-ShanGleaton, JeremyYang, YanwuDhayalan, BalamuruganPhillips, Nelson B.Liu, YuleBroadwater, LaurieJarosinski, MarkChatterjee, DeepakLawrence, Michael C.Hattier, ThomasMichael, Dodson M.Weiss, Michael Aaron2022-10-252022-10-252021-05-03Chen YS, Gleaton J, Yang Y, et al. Insertion of a Synthetic Switch Into Insulin Provides Metabolite-Dependent Regulation of Hormone-Receptor Activation. J Endocr Soc. 2021;5(Suppl 1):A440. Published 2021 May 3. doi:10.1210/jendso/bvab048.899https://hdl.handle.net/1805/30410Insulin signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to large-scale structural reorganization. To test the coupling between insulin’s “opening” and receptor activation, we inserted an artificial ligand-dependent switch into insulin. Ligand binding disrupts an internal tether designed to stabilize the hormone’s native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the receptor. Similarly, metabolite-regulated signaling was not observed in control studies of (i) an unmodified insulin analog or (ii) an analog containing a diol sensor in the absence of internal tethering. Although as expected CD-detected secondary structure was unaffected by ligand binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and holoreceptor signaling. In addition to this basic finding, our results provide proof of principle for a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a “smart” insulin analog designed to mitigate risk of hypoglycemia in the treatment of diabetes mellitus.en-USAttribution-NonCommercial-NoDerivatives 4.0 InternationalInsulin signalingLigand bindingMonosaccharidesAnalogous glucose sensorArticle