Kallikrein-related peptidase 6 (KLK6) is highly up-regulated in several types of cancer, where it is involved in a tissue-specific proteolysis of numerous endogenous substrates and considered a regulatory protease with key signaling properties, promoting cancer invasion and metastasis. In this study, we present a human amyloid precursor protein Kunitz protease inhibitor domain (APPI) variant, the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity, up to 560-fold

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... reater specificity, and 13-fold improved proteolytic stability compared with wild-type APPI (APPI WT ). To elucidate the mechanism of binding and inhibition of APPI-4M to KLK6, and to compare it with that of APPI WT , we crystallized each inhibitor in complex with KLK6 and solved the crystal structures. Our crystal structures reveal altered interactions of APPI-4M at the enzyme interface, primarily involving the optimization of primed side interactions with the S2′, S3′, and S4′ subsites of KLK6. Notably, these subsites are largely shaped by KLK6 residues 39-41, representing a stretch of sequence that is highly unique to KLK6 and is poorly conserved between KLKs and other trypsin-like proteases. The improved specificity of APPI-4M toward KLK6 suggests that an optimization of the S2′-S4′ subsite contacts may represent a general strategy for developing selective KLK6 inhibitors.