Fragment-Based Discovery of KLK6 and KLK7 Inhibitors

Abstract

Human tissue kallikreins are serine proteases implicated in the pathogenesis of neurodegenerative diseases, skin disorders, and various cancers. Despite their therapeutic relevance, the development of selective small-molecule inhibitors for these enzymes has been limited compared to other serine proteases. To address this, we implemented a fragment-based virtual screening (FBVS) strategy to identify chemically tractable starting points for the inhibition of KLK6 and KLK7. The computational workflow, combining structure-based docking tailored to serine protease active sites, was experimentally validated using in vitro enzymatic assays. Notably, the approach yielded hit rates (IC50 ≤ 100 μM) ranging from 4% (KLK7) to 21.6% (KLK6), underscoring both the robustness of the screening cascade and the druggability of these targets. Preliminary structure–activity relationship analysis identified fragment 69 as a potent and ligand-efficient KLK6 inhibitor (IC50 = 2.1 μM, LE = 0.43). For KLK7, a distinct neutral 2-hydroxyquinoline fragment (38) was validated as a ligand-efficient hit (IC50 = 43.3 μM; LE = 0.46). Overall, this study demonstrates that kallikrein-related peptidases are amenable to fragment-based discovery and provides chemically simple, highly ligand-efficient fragments that serve as promising starting points for lead optimization targeting kallikrein-driven pathologies.