Design of non-standard insulin analogs for the treatment of diabetes mellitus

Abstract

Structure-based protein design has enabled the engineering of insulin analogs with improved pharmacokinetic and pharmacodynamic properties. Exploiting classical structures of zinc insulin hexamers, the first insulin analog products focused on destabilization of subunit interfaces to obtain rapid-acting (prandial) formulations. Complementary efforts sought to stabilize the insulin hexamer or promote higher-order self-assembly within the subcutaneous depot toward the goal of enhanced basal glycemic control with reduced risk of hypoglycemia. Current products either operate through isoelectric precipitation (insulin glargine, the active component of Lantus; Sanofi-Aventis, Paris, France) or employ an albumin-binding acyl tether (insulin detemir, the active component of Levemir; Novo-Nordisk, Basværd, Denmark). In the past year second-generation basal insulin analogs have entered clinical trials in an effort to obtain ideal flat 24-hour pharmacodynamic profiles. The strategies employ non-standard protein modifications. One candidate (insulin degludec; Novo-Nordisk a/s) undergoes extensive subcutaneous supramolecular assembly coupled to a large-scale allosteric reorganization of the insulin hexamer (the TR transition). Another candidate (LY2605541; Eli Lilly and Co., Indianapolis, IN, USA) utilizes coupling to polyethylene glycol to delay absorption and clearance. On the other end of the spectrum, advances in delivery technologies (such as microneedles and micropatches) and excipients (such as the citrate/zinc-ion chelator combination employed by Biodel, Inc., Danbury, CT, USA) suggest strategies to accelerate PK/PD toward ultra-rapid-acting insulin formulations. Next-generation insulin analogs may also address the feasibility of hepatoselective signaling. Although not in clinical trials, early-stage technologies provide a long-range vision of "smart insulins" and glucose-responsive polymers for regulated hormone release.

Figure 1

(A) Ribbon model of wild-type insulin (B) Structure of insulin hexamer. Two axial zinc ions (red; overlaid at center) are coordinated by six histidine side chains (residue B10; black). The structure shown is the R₆ hexamer form characteristic of a pharmaceutical formulation [83]; coordinates were obtained from Protein Databank entry 1ZNJ. (C) Structure of insulin degludec showing the dicarboxylic acid attached to the ε-amino group of Lys^B29. (D) Structure of LY2605541 showing PEG attached to the ε-amino group of Lys^B28. (E) Exploiting the TR transition in supramolecular protein engineering: schematic representation of the mechanism of insulin degludec. Left, Insulin degludec is formulated at neutral pH as dimers of phenol- (or meta-cresol) stabilized R₆ zinc insulin hexamers (blue). The acyl modification of Lys^B29 is shown in schematic form as a black bar (in principle 6 per hexamer); for simplicity only two are shown. Center, On subcutaneous injection, diffusion of the phenolic ligand into cellular membranes triggers the R → T transition, leading in turn to linear polymerization of T₆ zinc hexamers (red). Classical hexamer reorganization is thus coupled to a change in mode of hexamer-hexamer assembly mediated in part by the B29-linked acyl group. Right, Upon entering the bloodstream as monomers (pink) insulin degludec binds to circulating albumin forming another depot that further protracts its action.