Development of kallikrein-related peptidase 7 (KLK7) inhibitors and use in a species-adapted model for ovarian cancer

The serine protease KLK7 contributes to several skin disorders and tumorigenesis, making it an attractive drug target. Owing to structural similarities in the S1 binding pocket between human KLK7 and certain trypsin-like serine proteases harboring an Ala at position 190, compounds containing a chlorine–substituted benzylamide as P1 group were screened for KLK7 inhibition. Further optimization yielded the substrate analog inhibitor Bzls- d -hTyr-Pro-2-aminomethyl-5-chloro-benzylamide ( K i value 29.3 nM), which, however, had insufficient selectivity against the tested clotting proteases. Guided by a published KLK7 crystal structure, a second, more selective non-peptide inhibitor series was synthesized. The best derivatives ( K i values <100 nM) contain a chlorine-substituted aromatic P1 group and additionally address the non-prime region of KLK7. Murine Klk7 is poorly inhibited by these compounds; however, mutating threonine 190 in the S1 pocket to alanine restored the inhibitory potency. The new inhibitors effectively block KLK7-mediated functions, including chemokine cleavage and moesin gene upregulation, without cytotoxicity towards murine ovarian cancer cells. Moreover, tumors derived from ovarian cancer cells overexpressing either wild-type or mutated Klk7 reduced overall survival in mice compared to vector control cells. Together, these findings establish a robust inhibitor–enzyme system to evaluate human KLK7 inhibitors in a preclinical mouse model.