@article {Rabinovitch2021-jb,
	title = {A KLK4 proteinase substrate capture approach to antagonize PAR1},
	journal = {Sci. Rep.},
	volume = {11},
	number = {1},
	year = {2021},
	month = {aug},
	pages = {16170},
	publisher = {Springer Science and Business Media LLC},
	abstract = {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 {\textquoteright}decoy{\textquoteright} 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 {\textquoteright}substrate-capturing{\textquoteright} KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 {\textquoteright}proteinase substrate capture{\textquoteright} approach to regulate proteinase-mediated PAR1 signaling.},
	author = {Rabinovitch, Eitan and Mihara, Koishiro and Sananes, Amiram and Zaretsky, Marianna and Heyne, Michael and Shifman, Julia and Aharoni, Amir and Hollenberg, Morley D and Papo, Niv}
}
