Se-Glargine II. Native Function of a Basal Insulin Analog Stabilized by an Internal Diselenide Bridge

Insulin glargine, the active component of basal clinical pharmaceutical formulations Lantus® and Toujeo® (Sanofi), provides a model for principles of therapeutic protein design. Formulated in solution at pH 4, insulin glargine exhibits a shifted isoelectric point (from pH 5.3 to neutral pH) due to a basic dipeptide B-chain extension (Arg B31 -Arg B32 ). In the first article in this series, we described pairwise substitution of Cys A6 and Cys A11 by seleno-cysteine (Sec; the 21 st encoded amino acid) by solid-phase peptide synthesis. 1 H- 2 H amide proton exchange, as monitored by 1 H-NMR spectroscopy, provides evidence that substitution of internal cystine A6-A11 by a diselenide bridge stabilizes the protein and damps segmental conformational fluctuations. Further, this analog and its major metabolites M1 and M2 (respectively denoting proteolytic derivatives lacking Arg B31 -Arg B32 or Thr B30 -Arg B31 -Arg B32 ) exhibit native hormonal activity in mammalian cell-based assays measuring dose-dependent autophosphorylation of the insulin receptor (pIR/IR ratio) and metabolic gene regulation in human liver-derived HepG2 cells. The internal diselenide bridge also did not alter respective baseline mitogenicities of insulin glargine or its proteolytic products as evaluated by a qPCR-based assay of the balance between proliferative and antiproliferative cyclin gene expression; the assays employed L6 myoblasts over-expressing mitogenic IR isoform A. Given such native function, shelf life—and hence global access to insulin in the developing world—may be enhanced by stabilizing diselenide chemistry.