Insulin analogs for the treatment of diabetes mellitus: therapeutic applications of protein engineering

Abstract

The engineering of insulin analogs represents a triumph of structure-based protein design. A framework has been provided by structures of insulin hexamers. Containing a zinc-coordinated trimer of dimers, such structures represent a storage form of the active insulin monomer. Initial studies focused on destabilization of subunit interfaces. Because disassembly facilitates capillary absorption, such targeted destabilization enabled development of rapid-acting insulin analogs. Converse efforts were undertaken to stabilize the insulin hexamer and 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) or employ an albumin-binding acyl tether (insulin detemir, the active component of Levemir(®); Novo-Nordisk). To further improve pharmacokinetic properties, modified approaches are presently under investigation. Novel strategies have recently been proposed based on subcutaneous supramolecular assembly coupled to (a) large-scale allosteric reorganization of the insulin hexamer (the TR transition), (b) pH-dependent binding of zinc ions to engineered His-X(3)-His sites at hexamer surfaces, or (c) the long-range vision of glucose-responsive polymers for regulated hormone release. Such designs share with wild-type insulin and current insulin products a susceptibility to degradation above room temperature, and so their delivery, storage, and use require the infrastructure of an affluent society. Given the global dimensions of the therapeutic supply chain, we envisage that concurrent engineering of ultra-stable protein analog formulations would benefit underprivileged patients in the developing world.

Figure 1

Wild-type insulin hexamer. The A chain (residues A1–A21) is shown in black and the B chain in blue (residues B1–B6) or green (residues B7–B30). Two axial zinc ions (purple; overlaid at center) are coordinated by six histidine side chains (residue B10; white). The structure shown is the R₆ hexamer form characteristic of a pharmaceutical formulation; coordinates were obtained from Protein Databank entry 1EV3.

Figure 2

Exploiting the TR transition in supramolecular protein engineering. (A) Schematic representation of the three types of zinc insulin hexamers, designated T₆, T₃R^f₃, and R₆. Residues B1–B8 exhibit a change in secondary structure as shown in black. T-state protomers are otherwise shown in red, and R-state protomers in blue. (B) Corresponding ribbon representation of wild-type crystal structures. Axial zinc ions are shown in blue-gray. Coordinates were obtained from Protein Databank entries 4INS, 1TRZ, and 1ZNJ, respectively. This figure is reprinted by Wan and colleagues with permission of the authors.

Figure 3

Structure of prandial insulin analogs. (A) Ribbon model of insulin lispro (the active component of Humalog^®) as a phenol-stabilized T₃R^f₃ zinc hexamer (PDB entry 1LPH). The A- and B-chains of T-state protomers are shown in light and dark red, respectively; the A- and B-chains of R^f-state protomers are show in light and dark blue, respectively. Axial zinc ions are shown as purple spheres. (B) Corresponding model of insulin aspart (the active component of Novolog^®) as a phenol-stabilized R₆ zinc hexamer (Asp^B28-insulin; PDB entry 1ZEG). The A- and B-chains of R-state protomers are shown in light and dark blue, respectively. (C and D) Expanded region of dimer contacts in prandial insulin analogs versus wild-type insulin: dimer-related β-sheet (residues B24–B28) and inter-strand hydrogen bonds (dotted lines). (C) Insulin lispro versus wild-type T₃R^f₃ zinc hexamer (PDB entry 1TRZ). The A- and B-chains of wild-type insulin are shown in light and dark gray, respectively; the color code is otherwise as in panel A. T-state of lispro are shown in light and dark red, and R state in light and dark blue. (D) Insulin aspart versus wild-type T₃R^f₃ zinc hexamer (PDB entry 1ZNJ). The wild-type shading is as in panel C, and color scheme as in panel B. In panels C and D the variant and wild-type structures were aligned according to the main-chain atoms of residues B3–B28 and A3–A20. Hydrogen-bond lengths and angles are given in Table 2.

Figure 4

Molecular details of B28-related dimer contact. (A) Stereo models of wild-type insulin and insulin lispro (in corresponding T₃R^f₃ hexamers): enlargement of the interface between the C-terminal residues of one B-chain (B28–B30) and the B20–B23 β-turn of the dimer-related B-chain. The wild-type B-chain is shown in dark gray; residues belonging to the T- or R^f-state protomers of insulin lispro are shown in red or blue, respectively. (B) Corresponding alignment of wild-type insulin and insulin aspart (in corresponding R₆ hexamers). The coloring scheme is as in panel A. (C) Corresponding interface in classical wild-type T₆ hexamer (PDB entry 4INS). The site of closest contact between the dimer-related B-chains at this site are shown in Corey-Pauling-Koltun (CPK) representation; respective residues belong to the A- and B-chains are otherwise shown as light and dark gray sticks. The side chain of Phe^B24 (which anchors the β-turn to the central B-chain α-helix) is also shown. The proteins were in each case aligned as in Figure 2.

Figure 5

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 LysB29 is shown in schematic form as a black bar (in principle 6 per hexamer); for simplicity only two are shown. Right, 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. Panels A and B are reprinted by Wan and colleagues with permission of the authors.

Figure 6

Supramolecular assembly. (A) Schematic representation of supramolecular assembly of monomers, each containing complementary surfaces. Such assembly is often exploited in biology to form diverse architectures, including multi-layered ropes (as in the collagen of ligaments and tendons), hollow tubes (as in microtubules), and cages (as in viral assembly). (B) Ligand-linked mechanism of supramolecular assembly. One possible realization of this strategy is provided by metal ions. Each monomer (blue) presents half of a metal-ion binding site; successive coordination by metal ions (purple) mediates polymerization. (C) Assembly of a microtubule from tubulin monomers (this panel was kindly provided by Dr. Danton H. O’Day, Professor of Cell and Systems Biology, University of Toronto Mississauga, Ontario, Canada.) (D) Southern bean mosaic virus. Presence of divalent cations directs the swelling from left to right (this panel was kindly provided by Dr. Donald L. Caspar, Professor Emeritus of Biological Sciences, Florida State University, Tallahassee FL).

Figure 7

Classical zinc-finger motif. The peptide backbone is shown in black (N-terminal β-hairpin and central loop) or blue (C-terminal α-helix); the encaged zinc ion is shown in magenta. The interior Zn²⁺ coordination site of the C₂H₂ motif comprises the thiolate groups of two cysteine side chains (green) and imidazole rings of two histidine side chains (red). The structure shown is domain 2 of Zif268; coordinates were obtained from Protein Databank entry 1ZAA.

Figure 8

Zinc-stapled insulin analogs. (A) Schematic model showing successive stacking of insulin hexamers (gray cylinders) bridged by layers of zinc ions (purple spheres). Upper and lower surfaces of insulin analog hexamer present three α-helix-related HX₃H half-coordination sites; each site recapitulates the C-terminal half of the classical zinc finger (see Fig. 3). (B) Ribbon model of variant insulin monomer showing designed HX₃H sites (red) at residue positions A4 (left) and A8 (right) in the N-terminal A-chain α-helix. The A chain is otherwise shown in black, and the B chain in green. (C) Crystal structure of variant R₆ insulin hexamer. The three novel interfacial zinc ions are shown in magenta whereas the two classical axial zinc ions (coordinated by His^B10; black) are shown in purple (overlaid in center). The color code is otherwise as in Figure 1 with the A chain in black and B chain in blue (residues B1–B6) or green (residues B7–B30). In the crystal lattice successive insulin hexamers are stacked as in panel A with intervening layers of zinc ions. Coordinates were obtained from Protein Databank entry 3KQ6.