Insulin Analogues with Modifications at Position B26. Divergence of Binding Affinity and Biological Activity

Abstract

In this study, we prepared several shortened and full-length insulin analogues with substitutions at position B26. We compared the binding affinities of the analogues for rat adipose membranes with their ability to lower the plasma glucose level in nondiabetic Wistar rats in vivo after subcutaneous administration, and also with their ability to stimulate lipogenesis in vitro. We found that [NMeHisB26]-DTI-NH 2 and [NMeAlaB26]-DTI-NH 2 were very potent insulin analogues with respect to their binding affinities (214 and 465%, respectively, compared to that of human insulin), but they were significantly less potent than human insulin in vivo. Their full-length counterparts, [NMeHisB26]-insulin and [NMeAlaB26]-insulin, were less effective than human insulin with respect to binding affinity (10 and 21%, respectively) and in vivo activity, while [HisB26]-insulin exhibited properties similar to those of human insulin in all of the tests we carried out. The ability of selected analogues to stimulate lipogenesis in adipocytes was correlated with their biological potency in vivo. Taken together, our data suggest that the B26 residue and residues B26-B30 have ambiguous roles in binding affinity and in vivo activity. We hypothesize that our shortened analogues, [NMeHisB26]-DTI-NH 2 and [NMeAlaB26]-DTI-NH 2, have different modes of interaction with the insulin receptor compared with natural insulin and that these different modes of interaction result in a less effective metabolic response of the insulin receptor, despite the high binding potency of these analogues.

Implications for the active form of human insulin based on the structural convergence of highly active hormone analogues

“…Chemical changes were systematically introduced in the insulin molecule to achieve high-activity. Semisynthesis (21) of several unique and some previously reported (22,23) (Tables S1-S4) in order to identify the underpinning structural features and to get further insight into the structural origins of the active conformation of insulin.…”

“…The most active (465%) insulin presented in this study: N B26 -methylated and B 26 -truncated analogue ½NMeAla B26 -DTI-NH 2 (22), was crystallized as a monomer with a unique conformation at B 24 -B 26 . Its most striking feature is a type II β-turn at Phe B24 -NMeAla B26 referred to here as the B26 turn ( Fig.…”

Insulin analogues for insulin receptor studies and medical applications

“…Substitutions of each of these residues has created analogues of both high and low affinities 75,100,150 .…”

“…Subsequently novel mutations can lead to improved insulin binding affinity 100 . To achieve these series of unique semisynthetic insulin analogues were created to expose the binding epitope; previously described semisynthetic methods have been employed in making of these analogues 73,92,150 .…”

“…Replacing the Tyr with another amino acid can lead to analogues with a range of affinities between less than 1%, up to 4-5 times higher than that of WT insulin 150 .…”