Achieving protease substrate-specific inhibition by mAb dual functional selections.

Authors

Ki Baek Lee, Sangcheon Lee, Bharati Reddi, Sushma Krishnan, Zening Wang, Xin Ge

Published in

Protein science : a publication of the Protein Society. Volume 35. Issue 7. Pages e70688.

Abstract

Proteases are promiscuous enzymes acting on multiple substrates. Inhibiting pathogenic proteolysis while sparing physiological ones is essential for the development of effective and safe inhibitors. By engineering β-lactamase and levansucrase as selection and counterselection markers in E. coli periplasm, this study established functional positive and negative selections for the facile discovery of protease inhibitory mAbs rendering substrate specificity. Isolated anti-matrix metalloproteinase (MMP)-14 inhibitory mAbs exhibited nanomolar binding affinities and inhibitory potencies, exclusive selectivity, high proteolysis stability, and substrate-specific (SS) inhibition, that is, blocking MMP-14 mediated proteolysis of pro-tumorigenic syndecan-1 (Sdc-1) while allowing cleavage of anti-inflammatory macrophage chemoattractant protein 3 (MCP-3). Mechanistic studies suggested that the isolated Fabs were active-site competitive inhibitors and their substrate specificity was achieved by recognizing the protease's prime subsites shared with Sdc-1 but distinct to that of MCP-3. Given the pharmaceutical significance of SS inhibition, we envision the dual functional selection can be widely applicable to important protease targets.

PMID:
42321978
Bibliographic data and abstract were imported from PubMed on 20 Jun 2026.

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