4S-fluorination of ProB29 in insulin lispro slows fibril formation

Abstract

Recombinant insulin is a life-saving therapeutic for millions of patients affected by diabetes mellitus. Standard mutagenesis has led to insulin variants with improved control of blood glucose; for instance, the fast-acting insulin lispro contains two point mutations that suppress dimer formation and expedite absorption. However, insulins undergo irreversible denaturation, a process accelerated for the insulin monomer. Here we replace ProB29 of insulin lispro with 4R-fluoroproline, 4S-fluoroproline, and 4,4-difluoroproline. All three fluorinated lispro variants reduce blood glucose in diabetic mice, exhibit similar secondary structure as measured by CD, and rapidly dissociate from the zinc- and resorcinol-bound hexamer upon dilution. Notably, however, we find that 4S-fluorination of ProB29 delays the formation of undesired insulin fibrils that can accumulate at the injection site in vivo and can complicate insulin production and storage. These results demonstrate how subtle molecular changes achieved through non-canonical amino acid mutagenesis can improve the stability of protein therapeutics.

Keywords: fibrillation; fluoroproline; insulin; insulin lispro; non-canonical amino acid; proline.

Figure 1

Proline mutagenesis at position B29 of insulin lispro.A, simplified representation of insulin and insulin lispro oligomerization. Disrupted oligomerization speeds the release of lispro into the bloodstream. B, the amino acid sequences of human insulin and insulin lispro. C, the structures of proline and the proline analogs used in this study. D–G, characterization of proline analog incorporation. Proinsulin was expressed in media supplemented with proline (D), 4R-F (E), 4S-F (F), 44-diF (G), digested with Glu-C, and analyzed by MALDI-TOF MS. The peptide-containing position B29 of mature lispro is ⁵⁰RGFFYTKPTRRE (expected m/z = 1557.8). H–K, MALDI-TOF characterization of mature insulin variants: insulin lispro (H), KP-4R-F (I), KP-4S-F (J), and KP-44-diF (K). The peaks at m/z ∼6050 correspond to adducts of the sinapic acid matrix. 44-diF, 4,4-difluoroproline; 4R-F, 4R-fluoroproline; 4S-F, 4S-fluoroproline.

Figure 2

Biological activity and CD spectroscopy of lispro variants.A, lispros were injected subcutaneously into diabetic mice, and blood glucose was measured over time after injection. The data presented here represent the mean ± SD of 3 to 6 biological replicates. B–E, far-UV circular dichroism spectra (60 μM lispro in 10 mM phosphate buffer, pH 8.0). The spectrum of lispro is overlaid with that of human insulin (B), KP-4R-F (C), KP-4S-F (D), and KP-44diF (E). 4R-F, 4R-fluoroproline; 4S-F, 4S-fluoroproline.

Figure 3

Hexamer dissociation kinetics of lispro variants.A, equilibrium CD spectra of lispro before and after dilution. To measure dissociation kinetics, the decrease in negative ellipticity at 222 nm was monitored over time after dilution. B–F, representative dissociation kinetics measurements for human insulin (B), lispro (C), KP-4R-F (D), KP-4S-F (E), KP-44diF (F). Note the extended x-axis for insulin in panel B. G, summary of dissociation half-life values; replicate measurements for each sample originate from at least two separate HPLC fractions, measured on different days. 4R-F, 4R-fluoroproline; 4S-F, 4S-fluoroproline.

Figure 4

Fibrillation of lispro variants.A, lispro variants (60 μM in 100 mM phosphate buffer, pH 8.0) were incubated at 37 °C with vigorous shaking; fibril formation was monitored by ThT fluorescence. Fibrillation runs were performed on two separate HPLC fractions, each in triplicate or quadruplicate, on two separate days. B–E, TEM images of lispro (B), KP-4R-F (C), KP-4S-F (D), and KP-44-diF (E) aggregates. F, ANS emission spectra of lispro variants (1 μM lispro variant labeled with 5 μM ANS in 100 mM phosphate buffer, pH 8.0). The data presented here indicate the mean ± SD for each variant from measurements of three separate HPLC fractions. 44-diF, 4,4-difluoroproline; 4R-F, 4R-fluoroproline; 4S-F, 4S-fluoroproline; ThT, thioflavin T.