Abstract
Proteases are one of attractive therapeutic targets to play key roles in pharmacological action. There are many protease inhibitors in nature, and most of them structurally have cystine knot motifs. Their structures are favorable for recognition of active pockets of proteases, leading to the potent inhibition. However, they also have drawbacks, such as broad cross-reactivity, on the therapeutic application. To create therapeutic proteins derived from a disulfide-rich scaffold, we selected human serine protease inhibitor Kazal type 2 (SPINK2) through a scaffold screening, as a protein scaffold with requirements for therapeutic proteins. We then constructed a diverse library of the engineered SPINK2 by introducing random mutations into its flexible loop region with the designed method. By phage panning against four serine proteases, we isolated potent inhibitors against each target with picomolar KD and sub-nanomolar Ki values. Also, they exhibited the desired specificities against target proteases without inhibiting non-target proteases. The crystal structure of kallikrein related peptidase 4 (KLK4)-engineered SPINK2 complex revealed the interface with extensive conformational complementarity. Our study demonstrates that engineered SPINK2 can serve as a scaffold to generate therapeutic molecules against target proteins with groove structures.
Highlights
Many engineered proteins have emerged as potential therapeutic agents, and have been evaluated in pre-clinical and clinical studies[1,2,3]
serine protease inhibitor Kazal-type 2 (SPINK2) and Lympho-epithelial Kazal-type-related inhibitor (LEKTI) 15th domain displayed on phages mainly showed monomeric forms fused to bacteriophage M13 gIII protein, others contained multimeric forms showing inter-molecular disulfide bonds (Supplementary Fig. 1)
These results indicated both Kazal-type inhibitors tend to form a monomeric form with proper intra-molecular disulfide bonds in E.coli
Summary
Many engineered proteins have emerged as potential therapeutic agents, and have been evaluated in pre-clinical and clinical studies[1,2,3]. Along with the progress of protein engineering, it has been reported that antibodies and Nanobody could be specific protease inhibitors by entirely or partially binding to the pocket of proteases, as well as allosteric www.nature.com/scientificreports/. Given side-effects resulted from broad cross-reactivities of inhibitors on therapeutic usages, they must selectively recognize just a target protease notwithstanding high similarities among proteases, to reduce adverse effects. Engineered proteins derived from a scaffold require potent activities and as high specificities as possible for therapeutic usages. We aimed to generate therapeutic proteins targeting proteases with high specificity by engineering a protein scaffold. By screening of scaffolds which met requirements as therapeutic proteins, we selected serine protease inhibitor Kazal-type 2 (SPINK2) as a protein scaffold, followed by constructing an engineered SPINK2 library. Generation of a crystal structure provided insights into the mechanism underlying the high affinity specific binding of those inhibitors for their target molecules, suggesting that the engineered SPINK2 inhibitors preferentially bind to the groove of target proteases via the engineered flexible loop, supported by its constrained structure
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