A Novel Protein Scaffold, Engineered SPINK2, for Generation of Inhibitors with High Affinity and Specificity Against Target Proteases

摘要

With the development of protein engineering, many engineered proteins have emerged in clinical stages. Few studies, however, are available on generation of engineered proteins that can inhibit proteases with high potency and specificity. In this study, we report that we designed a novel protein scaffold, engineered SPINK2, to generate potent inhibitors with high specificity against various proteases. The SPINK2 scaffold is one of cystine knot proteins which contain disulfide bonds, and we constructed a random and diverse library for engineered SPINK2. Through screening against several proteases, we succeeded in obtaining some inhibitors with picomolar KD and sub-nanomolar Ki values against each target molecule. In addition, the result of a crystal structure of a target-engineered SPINK2 provided deep insight into the mechanisms underlying the high affinity and specificity of the inhibitors. We demonstrate that engineered SPINK2 can serve as a novel protein scaffold to generate therapeutic tools against proteins with groove structures.