Developing PAR1 Inhibitors based on KLK4 Protease for Clinical Translation as Therapeutic Anticancer Agents

Proteinase activated receptor 1 (PAR1) is a member of the G-coupled protein receptor family. Thrombin, the cognate activator, cleaves PAR1 at the extracellular N-terminal region, forcing the new terminus to act as a tethered ligand and activate PAR1. Activation of PAR1 promote blood coagulation as part of the homeostasis regulation function in normal conditions, however in cancer it was found to promote survival and metastasis in breast and melanoma cancer cells, angiogenesis and progression in ovarian cancer. In addition, higher expression levels of PAR1 correlates to poor prognosis for prostate cancer patients. Therefore, interfering with PAR1 activation might provide an opportunity for the development of new, mechanism- based strategies for cancer diagnosis, prevention and treatment.

In this study, we are developing a novel PAR1 high affinity antagonist based on the structure of human kallikrein 4 (KLK4), in order to block PAR1 cleavage site. Using computational prediction, we were able identified two promising positions that by introducing new random mutations should increase the affinity of catalytically inactive KLK4 mutant toward PAR1 while not destabilizing its structure. Mutant variants are expressed separately using Pichia Pastoris yeast strain as soluble proteins and using competition assays we are screening for inactive KLK4 variant to block PAR1 activation.

We are introducing an advanced usage of our method combining computational analysis of protein-protein interaction and the usage of experimental combinatorial mutant libraries, and implement these complementary approaches for the purpose of engineering novel PAR1 antagonists for cancer therapy.