This grant is in response to PAR-12-060, Solicitation of Validated Hits for the Discovery of in vivo Chemical Probes. Platelets play a critical role in thrombosis, the major cause of mortality and morbidity. Medical management of acute coronary syndrome and cerebrovascular injury is centered on anti-platelet therapies. Thrombin receptor antagonists (TRAs) are highly awaited in clinical medicine but Vorapaxar, a TRA that targets PAR1, was recently shown in Phase III clinical trials to increase clinically significant bleeding including intracranial hemorrhage. PAR4 is an attractive target for anti-platelet therapy in thrombosis and cerebrovascular injury. Because of PAR4's low affinity for thrombin, it is activated locally at the site of the 3D clot as more thrombin builds up. Because of this delay in activation of PAR4, we hypothesize that PAR4 antagonism might not affect hemostasis as potently and thus may be a better therapeutic target than PAR1. Our major hypothesis is that inhibition of PAR4 is a potential target for platelet inhibition in thrombosis and cerebrovascular injury. However, there are no PAR4 antagonists that are efficacious in vivo to study the role of PAR4 in thrombosis, ischemic stroke and neurodegeneration. Due to the lack of tool compounds, the field's understanding of the role of PAR4 in physiological environments is limited. Inhibition of PAR4 would not perturb signaling through the PAR1 receptor, which is essential for basic hemostasis during injury. The PAR4 antagonist YD-3 has poor physiochemical properties and virtually no solubility in acceptable vehicles;therefore, it is not suitable for in vivo studies. We propose to optimize solubility, potency, and selectivity of YD- 3 and related molecules in order to better understand the role of PAR4 in thrombosis and hemostasis. We propose to 1) optimize a small molecule antagonist of PAR4 using a library approach, 2) determine selectivity of optimized compounds for PAR4, 3) determine the metabolism and disposition of novel PAR4 antagonists using in vitro and in vivo DMPK techniques, and 4) test the optimized compound in models of thrombosis. An optimized compound will allow us to determine basic pharmacological information about PAR4 including receptor density, mechanism of action for PAR4 antagonists and agonists, and effects on in vivo models of thrombosis, data which currently cannot be collected. These studies should provide a valuable tool compound which will enable studies that may reveal PAR4 as an alternative target for preventive and therapeutic suppression of pathologic thrombus formation.
Thrombin receptor antagonists (TRA) are potential novel anti-platelet agents, but recently vorapaxar, a TRA that targets PAR1, was shown to increase clinically significant bleeding including intracranial hemorrhage. To determine if PAR4 would be a better target, we propose to optimize novel PAR4 antagonists for in vivo use to determine basic pharmacological information about PAR4 and perform proof of concept studies of in vivo thrombosis.
