Se-Glargine I. Chemical Synthesis of a Basal Insulin Analog Stabilized by an Internal Diselenide Bridge

Insulin, a small globular protein, has long provided a model for studies of biophysical principles with therapeutic application. The safety and efficacy of insulin replacement therapy for the treatment for diabetes mellitus have been enhanced by protein engineering. Here, we describe the chemical synthesis of a basal insulin analog stabilized by the substitution of an internal cystine (A6-A11) by a diselenide bridge. The studies focused on insulin glargine, the active component of clinical products Lantus(R) and Toujeo(R) (Sanofi). Formulated in solution at pH 4 in the presence of zinc ions, insulin glargine exhibits a shifted isoelectric point (from pH 4.5 to neutral pH) due to a basic extension of the B chain (ArgB31-ArgB32). Subcutaneous injection of such an acidic formulation leads to pH-dependent precipitation of protein-zinc complexes to form a long-lived depot. Pairwise substitution of CysA6 and CysA11 by selenocysteine (Sec; the 21st encoded amino acid) was effected by solid-phase peptide synthesis. The modified A chain also contained substitution of AsnA21 by Gly, introduced in glargine to avoid acid-catalyzed deamidation of the A21 carboxamide group in the formulation. Although classical chain combination of the di-Arg-extended B chain and modified A chain exhibited lower yield than does wild-type chain combination, substantial product was obtained through repeated reactions and successive purification. This strategy exemplifies the rational optimization of protein stability and may be generalizable to diverse disulfide-stabilized proteins of therapeutic interest.