Insulin formulation chemistry represents a cross roads between protein science and applied biotechnology. In accord with the dual perspective of the NIH Roadmap, we propose to exploit general principles of protein engineering to improve the stability of insulin formulations. This application thus seeks clinical translation of basic insights into the folding and unfolding of proteins. Present insulin formulations must be stored in refrigerator and discarded after 28 days. In addition, patients are urged to monitor vials for discoloration, frosting of the glass, or precipitation: signs of chemical or physical degradation of the protein. Of principal concern is insulin fibrillation, a misfolding process analogous to amyloid formation. The Food & Drug Administration has set stringent standards to protect patients from inadvertent injection of insulin fibrils. We seek to develop novel insulin analogs and formulations that are highly active but resistant to fibrillation even after prolonged storage at elevated temperatures. Two novel design ideas are proposed based on preliminary studies of an insulin amyloidogenic intermediate: chiral stabilization of a B-chain ?-turn by D-amino acids and introduction of an ancillary zinc-binding site in the A chain. Thermodynamic, kinetic and structural studies of these insulin analogues will be pursued to validate the design goals. ? ? The proposed studies, if successful, would represent the exciting application of structural principles to a problem of enormous medical and economic significance. Insulin formulations of prolonged stability and extended shelf life would be of particular advantage in the developing world where refrigeration is not consistently available. Our analog design strategies, although focused on structural features of insulin, employ concepts and principles of broad application in protein science. Accordingly, our strategy may generalize to enhance the utility of diverse protein pharmaceuticals in the post-genomic era. ? ?
