Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region

Yanjie Li, Yuehua Wei, Mark Ultsch, Wei Li, Wanjian Tang, Benjamin Tombling, Xinxin Gao, Yoana Dimitrova, Christian Gampe, Jakob Fuhrmann, Yingnan Zhang, Rami N. Hannoush () and Daniel Kirchhofer ()
Additional contact information
Yanjie Li: Genentech Inc.
Yuehua Wei: Genentech Inc.
Mark Ultsch: Genentech Inc.
Wei Li: Genentech Inc.
Wanjian Tang: Genentech Inc.
Benjamin Tombling: Genentech Inc.
Xinxin Gao: Genentech Inc.
Yoana Dimitrova: Genentech Inc.
Christian Gampe: Genentech Inc.
Jakob Fuhrmann: Genentech Inc.
Yingnan Zhang: Genentech Inc.
Rami N. Hannoush: Genentech Inc.
Daniel Kirchhofer: Genentech Inc.

Nature Communications, 2024, vol. 15, issue 1, 1-17

Abstract: Abstract Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1’ site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.

Date: 2024
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DOI: 10.1038/s41467-024-48655-w

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