Protease-activated receptor-1 (PAR1) is a widely expressed G protein-coupled receptor (GPCR) that functions in thrombus formation, inflammation, and mitogenesis. Like other GPCRs, PAR1 can assume multiple conformations and possess multiple binding sites. We hypothesized that the binding of small molecules to sites outside of the tethered ligand binding pocket can stabilize these alternative conformations, resulting in novel signaling properties. To identify allosteric modulators of PAR1, we screened >300,000 compounds for the ability to inhibit PAR1-mediated dense granule release. Following the identification of potent compounds with a 1,3-diaminophenyl scaffold, 81 analogs were synthesized or procured and tested for inhibition of PAR1-mediated granule secretion. Structural features that were required for optimal inhibition included a 4-carbon chain at the “western end” and a 2'-substituted benzamide at the “eastern end” of the molecule. The most potent compound, ML161, inhibited PAR1-mediated platelet activation with an IC50 of 300 nM, >10-fold more potently than JF5, a previously identified allosteric modulator of PAR1. ML161 inhibited platelet activation induced by the PAR1 agonists SFLLRN and thrombin, but not other platelet agonists including the PAR4 agonist AYPGKF, PMA, ionophore, collagen, or ADP. ML161 inhibited SFLLRN-induced Ca2+ flux in platelets and HEK293 cells overexpressing PAR1, confirming activity at PAR1. Modeling of multiple Ca2+ flux curves at different ML161 concentrations over a range of SFLLRN doses indicated an allosteric mode of inhibition. A similar analysis of the effect of ML161 on PAR1-mediated P-selectin expression confirmed an allosteric mechanism. In platelet aggregation studies, ML161 inhibited SFLLRN-induced aggregation, but not shape change, raising the possibility that it inhibits Gαq-mediated, but not Gα12/13-mediated pathways. Shape change in the presence of ML161 was sensitive to the Rho kinase inhibitor Y27632, indicating involvement of Rho kinase in ML161-resistant signaling. ML161 failed to inhibit PAR1-mediated decreases in transepithelial resistance (TER) in MDCK cells overexpressing Gα12. In contrast, orthosteric inhibitors of PAR1, 3,5-difluoro aminoisoxazole and SCH79797, blocked PAR1-mediated shape change in platelets and PAR1-mediated decrease in TER in MDCK cells. These results confirmed that PAR1 modified by ML161 couples to Gα12/13, but loses coupling to Gαq. Although ML161 did not inhibit activation through human PAR4, whose 8th helix lacks a palmitoylation site and contains amino acids that disrupt helices, it inhibited activation through mouse PAR4, which like human PAR1 possesses a constrained 8th helix with a C-terminal palmitoylation site. Consistent with its ability to inhibit mouse PAR4-mediated activation of mouse platelets, 5 mg/kg ML161 inhibited by >90% platelet accumulation during thrombus formation following laser-induced injury of the cremaster arteriole in mice. These results demonstrate that ML161 acts as an allosteric modulator of PAR1 that blocks coupling to Gαq, but not Gα12/13. ML161 demonstrates saturable inhibition as well as selective blockade of Gα subunits coupling and could provide improved control of PAR1 function in the setting of thrombotic disease.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.