Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind ‘decoy’ PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This ‘substrate-capturing’ KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 ‘proteinase substrate capture’ approach to regulate proteinase-mediated PAR1 signaling.
Bibliographical noteFunding Information:
The authors thank Dr. Uzi Hadad for his technical assistance and Prof. Carmit Levi and Dr. Isam Khalaila for providing cells. Flow-cytometry experiments were performed at the Ilse Katz Institute for Nanoscale Science & Technology. N.P. acknowledges support from the European Research Council “Ideas program” ERC-2013-StG (contract grant number: 336041) and the US-Israel Binational Science Foundation (BSF). Studies were also supported in part by a Grant from the Canadian Institutes of Health Research (to M.H.).
© 2021, The Author(s).