An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein

Abstract

Thermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling , of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (Δ 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for >140 days at 45 °C and >48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation is compatible with native duration of activity Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1-7 mm Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.

A thing of beauty: Structure and function of insulin's “aromatic triplet”

“…The receptor‐bound open conformation of the hormone may be particularly susceptible to such degradation, including formation of amyloid (insulin fibrillation). A promising structure‐based route to the engineering of ultra‐stable analogs is provided by foreshortened C domains . In such single‐chain insulins (SCIs) the aromatic triplet resides in a closed conformation—tethered by a foreshortened connecting peptide—and yet sufficient “play” is provided to enable its detachment on receptor binding.…”

“…A promising structure-based route to the engineering of ultra-stable analogs is provided by foreshortened C domains. 104,105 In such singlechain insulins (SCIs) the aromatic triplet resides in a closed conformation-tethered by a foreshortened connecting peptide-and yet sufficient "play" is provided to enable its detachment on receptor binding. It would be of future interest to investigate crystal-or cryo-EM structures of SCI-receptor complexes as probes of the closedopen transition.…”

Solution structure of an ultra-stable single-chain insulin analog connects protein dynamics to a novel mechanism of receptor binding

“…In the accompanying article (9), we described an ultrastable single-chain insulin analog (labeled SCI-a; Fig. 1C) with appropriate biological activity (including duration of signaling) in a rat model (9). The crystal structure of SCI-a, determined as a non-canonical zinc-free hexamer, revealed native-like component dimers with apparent disorder in the engineered C domains and adjoining B domain residues.…”

“…Intravenous (i.v.) bolus injection of SCI-b led, for unknown reasons, to protracted insulin action (9), similar to that of ultra-stable two-chain insulin analog cross-linked by an additional disulfide bridge (3). The monophasic pharmacodynamics (PD) profile of SCI-a by contrast resembled that of wild-type (WT) insulin (9).…”

Novel four-disulfide insulin analog with high aggregation stability and potency

“…Further, we report A22-B22-4SS-Ins as a novel insulin analog with retained potency, increased aggregation stability, and a crystal structure that is closely similar to native insulin. Our ndings thereby complement recent efforts in generating four-disulde insulin 9,10 and single-chain insulin, 23,24 and advance efforts to develop a much more stable therapeutic insulin. Further investigations regarding insulin signaling pathway activation in vitro and in vivo are underway.…”