Depsipeptides Featuring a Neutral P1 Are Potent Inhibitors of Kallikrein-Related Peptidase 6 with On-Target Cellular Activity

Kallikrein-related peptidase 6 (KLK6)
is a secreted serine protease
that belongs to the family of tissue kallikreins (KLKs). Many KLKs
are investigated as potential biomarkers for cancer as well as therapeutic
drug targets for a number of pathologies. KLK6, in particular, has
been implicated in neurodegenerative diseases and cancer, but target
validation has been hampered by a lack of selective inhibitors. This
work introduces a class of depsipeptidic KLK6 inhibitors, discovered
via high-throughput screening, which were found to function as substrate
mimics that transiently acylate the catalytic serine of KLK6. Detailed
structure–activity relationship studies, aided by in silico
modeling, uncovered strict structural requirements for potency, stability,
and acyl-enzyme complex half-life. An optimized scaffold, DKFZ-251,
demonstrated good selectivity for KLK6 compared to other KLKs, and
on-target activity in a cellular assay. Moreover, DKFZ-633, an inhibitor-derived
activity-based probe, could be used to pull down active endogenous
KLK6.