Biophysical Optimization of a Therapeutic Protein by Non-Standard Mutagenesis. STUDIES OF AN IODO-INSULIN DERIVATIVE [Molecular Biophysics]

July 3rd, 2014 by Pandyarajan, V., Phillips, N. B., Cox, G. P., Yang, Y., Whittaker, J., Ismail-Beigi, F., Weiss, M. A.

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 non-standard mutagenesis. Our studies employed LysB28, ProB29-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 (TyrB26) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5(±0.2) kcal/mole, (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C prolonged by fourfold), (iii) enhances affinity for the insulin receptor (1.5(±0.1)-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. 1H-NMR studies suggest that the bulky iodo-substituent packs within a non-polar inter-chain crevice. Remarkably, the 3-iodo-TyrB26 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-TyrB26-lispro thus illustrates how a non-standard amino-acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages.