Minimizing Mitogenic Potency of Insulin Analogues Through Modification of a Disulfide Bond.

The mechanisms by which insulin activates the insulin receptor to promote metabolic processes and cellular growth are still not clear. Significant advances have been gained from recent structural studies in understanding how insulin binds to its receptor. However, the way in which specific interactions lead to either metabolic or mitogenic signalling remains unknown. Currently there are only a few examples of insulin receptor agonists that have biased signalling properties. Here we use novel insulin analogues that differ only in the chemical composition at the A6-A11 bond, as it has been changed to a rigid, non-reducible C=C linkage (dicarba bond), to reveal mechanisms underlying signaling bias. We show that introduction of an A6-A11 cis-dicarba bond into either native insulin or the basal/long acting insulin glargine results in biased signalling analogues with low mitogenic potency. This can be attributed to reduced insulin receptor activation that prevents effective receptor internalization and mitogenic signalling. Insight gained into the receptor interactions affected by insertion of an A6-A11 cis-dicarba bond will ultimately assist in the development of new insulin analogues for the treatment of diabetes that confer low mitogenic activity and therefore pose minimal risk of promoting cancer with long term use.

Probing the correlation between insulin activity and structural stability through introduction of the rigid A6-A11 bond.

The development of fast-acting and highly stable insulin analogues is challenging. Insulin undergoes structural transitions essential for binding and activation of the insulin receptor (IR), but these conformational changes can also affect insulin stability. Previously, we substituted the insulin A6-A11 cystine with a rigid, non-reducible C=C linkage ("dicarba" linkage). A cis-alkene permitted the conformational flexibility of the A-chain N-terminal helix necessary for high-affinity IR binding, resulting in surprisingly rapid activity in vivo Here, we show that, unlike the rapidly acting LysB28ProB29 insulin analogue (KP insulin), cis-dicarba insulin is not inherently monomeric. We also show that cis-dicarba KP insulin lowers blood glucose levels even more rapidly than KP insulin, suggesting that an inability to oligomerize is not responsible for the observed rapid activity onset of cis-dicarba analogues. Although rapid-acting, neither dicarba species is stable, as assessed by fibrillation and thermodynamics assays. MALDI analyses and molecular dynamics simulations of cis-dicarba insulin revealed a previously unidentified role of the A6-A11 linkage in insulin conformational dynamics. By controlling the conformational flexibility of the insulin B-chain helix, this linkage affects overall insulin structural stability. This effect is independent of its regulation of the A-chain N-terminal helix flexibility necessary for IR engagement. We conclude that high-affinity IR binding, rapid in vivo activity, and insulin stability can be regulated by the specific conformational arrangement of the A6-A11 linkage. This detailed understanding of insulin's structural dynamics may aid in the future design of rapid-acting insulin analogues with improved stability.

Insulin in motion: The A6-A11 disulfide bond allosterically modulates structural transitions required for insulin activity.

The structural transitions required for insulin to activate its receptor and initiate regulation of glucose homeostasis are only partly understood. Here, using ring-closing metathesis, we substitute the A6-A11 disulfide bond of insulin with a rigid, non-reducible dicarba linkage, yielding two distinct stereo-isomers (cis and trans). Remarkably, only the cis isomer displays full insulin potency, rapidly lowering blood glucose in mice (even under insulin-resistant conditions). It also posseses reduced mitogenic activity in vitro. Further biophysical, crystallographic and molecular-dynamics analyses reveal that the A6-A11 bond configuration directly affects the conformational flexibility of insulin A-chain N-terminal helix, dictating insulin's ability to engage its receptor. We reveal that in native insulin, contraction of the Cα-Cα distance of the flexible A6-A11 cystine allows the A-chain N-terminal helix to unwind to a conformation that allows receptor engagement. This motion is also permitted in the cis isomer, with its shorter Cα-Cα distance, but prevented in the extended trans analogue. These findings thus illuminate for the first time the allosteric role of the A6-A11 bond in mediating the transition of the hormone to an active conformation, significantly advancing our understanding of insulin action and opening up new avenues for the design of improved therapeutic analogues.

Understanding the mechanism of insulin and insulin-like growth factor (IGF) receptor activation by IGF-II.

BACKGROUND: Insulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2. METHODOLOGY/PRINCIPAL FINDINGS: In this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation. CONCLUSIONS/SIGNIFICANCE: This study provides novel insight into the mechanism of receptor binding and activation by IGF-II, which may be important for the future development of inhibitors of its action for the treatment of cancer.

A novel approach to identify two distinct receptor binding surfaces of insulin-like growth factor II.

Very little is known about the residues important for the interaction of insulin-like growth factor II (IGF-II) with the type 1 IGF receptor (IGF-1R) and the insulin receptor (IR). Insulin, to which IGF-II is homologous, is proposed to cross-link opposite halves of the IR dimer through two receptor binding surfaces, site 1 and site 2. In the present study we have analyzed the contribution of IGF-II residues equivalent to insulin's two binding surfaces toward the interaction of IGF-II with the IGF-1R and IR. Four "site 1" and six "site 2" analogues were produced and analyzed in terms of IGF-1R and IR binding and activation. The results show that Val(43), Phe(28), and Val(14) (equivalent to site 1) are critical to IGF-1R and IR binding, whereas mutation to alanine of Gln(18) affects only IGF-1R and not IR binding. Alanine substitutions at Glu(12), Asp(15), Phe(19), Leu(53), and Glu(57) analogues resulted in significant (>2-fold) decreases in affinity for both the IGF-1R and IR. Furthermore, taking a novel approach using a monomeric, single-chain minimized IGF-1R we have defined a distinct second binding surface formed by Glu(12), Phe(19), Leu(53), and Glu(57) that potentially engages the IGF-1R at one or more of the FnIII domains.