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 in vivo, complicating mealtime therapy and of unclear safety. 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 a companion article. The stability of the analog (ΔGu 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 hours at 75 °C. 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. These findings indicate that marked resistance to thermal inactivation in vitro is compatible with native duration of activity in vivo. Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.

DOI: 10.1074/jbc.M117.808626

Cite this paper

@article{Glidden2017AnUS, title={An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein.}, author={Michael D Glidden and Khadijah Aldabbagh and Nelson F. B. Phillips and Kelley Carr and Yen-Shan Chen and Jonathan Whittaker and Manijeh H Phillips and Nalinda P. Wickramasinghe and Nischay K Rege and Mamuni Swain and Yi Peng and Yanwu Yang and Michael C Lawrence and Vivien C. Yee and Faramarz Ismail-Beigi and Michael Wei\ss}, journal={The Journal of biological chemistry}, year={2017} }