Abstract

Insulin glargine, the active component of basal clinical pharmaceutical formulations Lantus(R) and Toujeo(R) (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 (ArgB31-ArgB32). In the first article in this series, we described pairwise substitution of CysA6 and CysA11 by seleno-cysteine (Sec; the 21st encoded amino acid) by solid-phase peptide synthesis. 1H-2H amide proton exchange, as monitored by 1H-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 ArgB31-ArgB32 or ThrB30-ArgB31-ArgB32) 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.