Abstract

<

p>Protease-activated receptor 2 (PAR2) is an attractive target for the treatment of inflammation and cancer. But screening and characterization of PAR2-targeting compounds is challenging, since most (if not all) cells also express PAR1. HT-29 cells are commonly used for studying PAR2 since they mainly express PAR2 and only little PAR1 – but they still respond to PAR1 agonists. Moreover, PAR1/PAR2 heterodimerization might alter the pharmacology of a given PAR2-targeting compound. Here we used CRISPR to completely knock out PAR1 in HT-29 cells, thereby generating a perfect new cell model for studying PAR2 without any possible interference from PAR1. Using functional Ca²⁺ assays we confirm that HT-29 WT cells do not discriminate between PAR1 vs. PAR2 agonists, while our new HT-29 PAR1 knock out (KO) cells respond only to PAR2, but not to PAR1 agonists any more. For further characterization, due to poor commercial availability of PAR2 antagonists we then first synthesized the described PAR2 antagonist AZ8838, and then tested different PAR1 vs. PAR2 antagonists against different PAR1 vs. PAR2 agonist on our HT-29 WT vs. PAR1 KO cells. Most remarkably, against a mixed PAR1/2 agonist, PAR2 antagonists displayed only partial inhibition in the HT-29 WT, but full inhibition in our PAR1 KO cells, confirming again the successfull knock-out.