Engineering of insulin receptor isoform-selective insulin analogues

Abstract

Background: The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues.

Methodology/principal findings: Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity.

Conclusions/significance: We have discovered a new class of IR isoform-selective insulin analogues with 2-4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.

Figure 1. Representative IR competition binding curves.

Displacement of ¹²⁵I-labelled insulin by human insulin and insulin analogues from the (A) A-isoform or the (B) B-isoform of the IR. The amount of bound ¹²⁵I-labelled insulin as a percentage of bound ¹²⁵I-labelled insulin in the absence of unlabelled analogue is plotted against the concentration of unlabelled analogue. Data points are means ± SEM of four measurements within one assay (n = 1). The ¹²⁵I-labelled insulin was displaced with purified human insulin standard (*) or insulin analogues [A8H, B25H, B27E] (▴), [A8H, B25N, B27E] (▾), and [A8H, B25N] (•).

Figure 2. Structure of the [B25H, B27E] insulin analogue.

(A) Overview of the [B25H, B27E] insulin analogue with the A chain and B chain depicted in light- and dark blue, respectively. The segment shown in panel B and C is indicated by a gray dotted line in A. In (B), the 2Fo-Fc electron density is shown for residues B25H to B27E contoured at 1 σ. A hydrogen bond interaction is mediated by a water molecule (red sphere), but no direct interaction is shown between B25H and B27E. In (C), a modelled B27E residue is shown (yellow) in another rotamer orientation to indicate that a direct interaction between B25H and B27E could be possible. Distances are shown in Ångstrom.