J Biol Chem. 2014 Aug 22;289(34):23367-81.

Biophysical optimization of a therapeutic protein by nonstandard mutagenesis: studies of an iodo-insulin derivative.

Pandyarajan V1, Phillips NB1, Cox GP2, Yang Y1, Whittaker J1, Ismail-Beigi F3, Weiss MA4.
  • 1From the Departments of Biochemistry.
  • 2Physiology and Biophysics.
  • 3Physiology and Biophysics, Medicine, and.
  • 4From the Departments of Biochemistry, Medicine, and Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106 michael.weiss@case.edu.



Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed Lys(B28), Pro(B29)-insulin (“lispro”) as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (Tyr(B26)) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5 ± 0.2 kcal/mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. (1)H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-Tyr(B26) modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R6 zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T6 hexamer). By exploiting this allosteric switch, 3-iodo-Tyr(B26)-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages.

KEYWORDS: Diabetes; Hormone; Insulin; Nonstandard Mutagenesis; Nuclear Magnetic Resonance (NMR); Protein Allostery; Protein Design; Protein Stability

PMID: 24993826



Insulin is a protein hormone that is released by the pancreas and plays a critical role in maintaining metabolic homeostasis in vertebrate animals. Lack of, or an inability to effectively utilize, this vital hormone results in the development of diabetes mellitus (DM): a condition classically characterized by hyperglycemia and other metabolic perturbations. Patients who cannot maintain adequate blood glucose control are prone to the long-term development of a number of life-threatening micro- and macrovascular complications, including renal disease, atherosclerosis and blindness. Although insulin injections have been the mainstay of therapy for Type 1 DM (an autoimmune disease formerly called “juvenile-onset”) and essential for many patients with Type 2 DM (formerly called “adult-onset”), it was appreciated in the 1980s that natural insulins (such as recombinant human insulin or animal insulins) are not optimal as drugs. For more than two decades protein-engineering efforts have therefore focused on how to improve the insulin molecule to optimize its duration of action, safety and efficacy. In a new study published in the Journal of Biological Chemistry, V. Pandyarajan and colleagues in the laboratory of Dr. Michael A. Weiss in the Case Western Reserve University School of Medicine demonstrated how the unique chemistry of iodine may be employed to stabilize a rapid-acting insulin analog. Their strategy exploited an iodo-aromatic modification of a conserved tyrosine residue at the hormone-receptor interface.

Insulin is stored in pancreatic β-cells as a zinc-coordinated hexamer. Such self-assembly plays a key role in enhancing the resistance of insulin to chemical degradation (i.e., alteration of chemical bonds) and physical (misfolding of the hormone leading to a loss of function) degradation. These advantages unfortunately come at the cost of delayed absorption from a patient’s subcutaneous depot following injection. As insulin monomers and dimers are more readily absorbed than are hexamers, it was quickly realized that the rate-limiting step for insulin onset of action was determined by the rate of insulin hexamer disassembly. Insulin analogs were thus developed in the 1990s to circumvent this barrier by including amino acid substitutions at key interfaces that would accelerate the disassembly of insulin hexamers. Although such structure-based mutagenesis was a triumph in the field of rational protein design, these alterations also came with a cost. Such formulations show accelerated physical and chemical degradation (especially upon dilution and above room temperature) relative to wild-type insulin. This trade-off between insulin’s protective self-assembly and accelerated hexamer disassembly within the subcutaneous depot constitutes a “Catch-22,” a critical barrier that has motivated exploration of other technologies that could overcome such therapeutic limitations. Pandyarajan and colleagues sought to investigate whether the trade-off between stability and pharmacokinetics could be overcome by utilizing a nonstandard amino acid (3-iodotyrosine). Although not ordinarily found in proteins, a precedent for use of iodo-tyrosine is provided by the unrelated biosynthetic pathway of thyroid hormone, which in part contains tyrosine-derived iodo-aromatic groups.

                Based on the structure/function relationships elucidated through decades of structural studies of insulin, Pandyarajan and colleagues hypothesized that specific placement of this nonstandard amino acid within the structural context of insulin’s dimer interface might bestow improvements in both stability and pharmacokinetic properties (1). Conventional mutagenesis (using the canonical 20 amino acids) may have reached the limits of its utility, reasoned the investigators, but non-standard amino acids might offer a route to go beyond what nature has allowed in protein design. The team therefore synthesized a derivative of a rapid-acting insulin analog in current clinical use (insulin lispro, the active component of Humalog®; Eli Lilly and Co.). The modified form of insulin lispro contains an iodine atom at a key position within insulin’s dimer interface and within its receptor-binding surface. Biochemical, biophysical and function characterization of the modified protein demonstrated that the iodo-aromatic modification circumvents the stability-disassembly trade-off without loss of biological activity.

An ideal insulin analog would be extremely stable within a pharmaceutical formulation while simultaneously retaining a favorable pharmacokinetic profile on subcutaneous injected subcutaneous injection. Clinical formulations of insulin are protected from forming fibrils (comprised of β-amyloid) through native self-assembly. To test the ability of insulin formulations to form fibrils, vials containing insulin in pharmaceutical formulation are typically subjected to elevated temperatures and gentle agitation. The CWRU team demonstrated that the accelerated fibrillation of insulin lispro analog was mitigated by the iodo-modification. Further, the chemical- and physical stabilities of the modified insulin monomer were also enhanced. To test in vivo function, the modified hormone was tested in diabetic rats. In these animal studies the rapid action of insulin lispro was unaltered.

To elucidate molecular mechanisms, Pandyarajan and colleagues undertook studies of self-assembly using size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS). This technique probed the assembly of insulin under (i) conditions that mimic the subcutaneous space or (ii) conditions relevant to pharmaceutical formulation. These studies demonstrated that in the presence of additives (“excipients”) that promote allosteric assembly of insulin in pharmaceutical formulations (typically small cyclic alcohols), the modified insulin exhibited a greater tendency to form hexamers. Remarkably, this effect weakened when the additives were removed (as occurs quickly in a subcutaneous depot). In essence, the mechanism of hexamer stabilization was observed to be dependent on the presence of the excipients. Together, these results rationalized why protective assembly may be enhanced only where and when needed.

The rapid degradation of diluted insulin formulations (as often used in children) highlights the clinical need for better and more stable insulin formulations. Such formulations are not recommended for use in insulin pumps due to the risk of catheter occlusion: the protein’s constant exposure to changes in temperature and motion accelerate amyloid formation. The results of Pandyarajan and colleagues thus provide a rational route to solve this practical problem.  A single iodo-aromatic modification, exploiting general chemical principles underlying the function of thyroid hormone, may markedly improve the biophysical properties of a globular protein.



(1) Baker, Edward N., Thomas L. Blundell, John F. Cutfield, Susan M. Cutfield, Eleanor J. Dodson, Guy G. Dodson, Dorothy M. Crowfoot Hodgkin et al. “The structure of 2Zn pig insulin crystals at 1.5 resolution.” Philos Trans Royal Soc Lond B Biol Sci (1988): 369-456.


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