ML354

Endothelial nitric oxide synthase (eNOS) is a critical regulatory enzyme that controls vascular tone via the production of nitric oxide. Although thrombin also modulates vascular tone predominantly via the activation of protease-activated receptors (PARs), the time course and mechanisms involved in how thrombin controls eNOS enzymatic activity are unknown. eNOS enzymatic activity is enhanced by the phosphorylation of eNOS-Ser1177 and reduced by the phosphorylation of eNOS-Thr495. In this study, we hypothesized that thrombin regulates vascular tone through the differential phosphorylation of eNOS. Using rat descending aorta, we show that thrombin modulates vascular tone in an eNOS-dependent manner via activated PAR-1. We also show that thrombin causes a temporal biphasic response. Protein kinase C (PKC) is associated with second phase of thrombin-induced response. Western blot analysis demonstrated thrombin phosphorylated eNOS-Ser1177 and eNOS-Thr495 in human umbilical vein endothelial cells. A PKC inhibitor suppressed the thrombin-induced phosphorylation of eNOS-Thr495, but not that of eNOS-Ser1177. Our results suggest that thrombin induces a temporal biphasic vascular response through the differential phosphorylation of eNOS via activated PAR-1. Thrombin causes transient vasorelaxation by the phosphorylation of eNOS-Ser1177, and subsequent attenuation of vasorelaxation by the phosphorylation of eNOS-Thr495 via PKC, leading to the modulation of vascular tone.

The detailed pharmacology and therapeutic potential of the central PAR4 receptors are poorly understood due to a lack of potent, selective, and brain-penetrant tool compounds. Despite this, robust data with biochemical and genetic tools show the therapeutic potential of PAR4 antagonists in traumatic brain injury, Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders with a neuroinflammatory component. Thus, we performed a functional HTS campaign, identified a fundamentally new PAR4 competitive inhibitor chemotype, optimized this new series (increased potency >45-fold), discovered enantiospecific activity (though opposing preference for human versus mouse PAR4), and engendered high central nervous system penetration (rat K_p's of 0.52 to 4.2 and K_p,uu's of 0.52 to 1.2).