Substrate-Guided Design of a Potent and Selective Kallikrein-Related Peptidase Inhibitor for Kallikrein 4 (original) (raw)

Human kallikrein-related peptidase 4 (hk4/prostase), a trypsin-like serine protease, is a potential target for prostate cancer treatment as a consequence of its proteolytic ability to activate many tumorigenic and metastatic pathways such as the protease activated receptors (PARs). Currently there are no KLK4specific small-molecule inhibitors available for therapeutic development. Here we re-engineer the naturally-occurring sunflower trypsin inhibitor (SFTI) to specifically block the proteolytic activity of KLK4 and prevent stimulation of PAR activity in cell based systems. The re-engineered inhibitor was designed using a combination of molecular modelling and sparse matrix substrate screening. The resulting inhibitor showed highly specific inhibition of KLK4 against the peptide substrate FVQR-para-nitroanilide (K i of 3.25 ± 1.60 nM) and the macromolecular substrate fibrinogen, blocked KLK4 initiated calcium flux from PAR2 and displayed remarkable stability in cell based assays with a half life of four days.