Rational steering of insulin binding specificity by intra-chain chemical crosslinking

Abstract

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone's B-chain C-terminus for its IR-B specificity.

Figure 1. An overlay of human insulin structures that were key templates in this work.

All A chains (not relevant here) are in a translucent white; the wild-type B-chain (pdb 1mso) is in coral; the B-chain insulin structure attained in the complex with IR (pdb 4oga) is in blue; and the B-chain of one of the representative B26-turn-containing analogues (AsnB26-insulin, pdb 4 ung) is in gold.

Figure 2. Schematic representation of intra-chain crosslinked octapeptides in the insulin analogues 1–14 produced in this work.

Black dots (●) indicate the Cα carbons in the backbone and blue dots (●) indicate CH₂ atoms in the linkers. Dashed lines indicate alternative chirality. G in red indicates the positions of Gly mutations. mA in red indicates the position of N-methylated alanine. The binding affinities (in %) relative to native insulin are shown to the right. Triazole rings are shown as pentagrams with blue nitrogen atoms. nd stands for not determined. DOI is des(B23–B30)octapeptide-insulin.

Figure 3. Comparison of range of conformations of the B-chain observed in the structures of the wild-type human insulin (white), insulin in the IR complex (coral), and in the analogues discussed in this work (yellow):

(a) 2, (b) 10, (c) 11, (d) 12, and (e) 8. The carbon atoms of the crosslinks are in green, and the nitrogens in blue; crosslinked sites are labeled in red; in (c), the C-terminal carboxyl group (part of one crosslink precursor) is also given. The wide spectrum of crosslink-generated conformations of insulins is shown in (f ) by superposition of the analogues given in (a–e). The red letters at the analogues correspond to the structures in panels (a–e); wild-type (WT in white, pdb 1 mso) and IR-complexed insulin (coral, pdb 4 oga) are also given with all B7–B19-helices shown in white.

Figure 4. Two views of a putative fold of the B24–B30 crosslink-containing chain of the IR-B-specific analogue 8 modeled on the insulin:IR complex structure, assuming an invariant conformation of PheB24.

(a) View from the top of the IR L1 domain and (b) from the side (edge) of the IR L1 domain. Insulin A and B chains are in gold and coral, respectively, and the αCT IR-A segment in violet. The B24–B30 segment of 8 is in yellow with triazole nitrogens in blue. Red asterisk indicates the insertion site (716) of the exon 11-coded additional 12 amino acids of the αCT segment in the IR-B isoform.