Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Protease-activated receptors (PARs) represent a unique family of seven-tran smembrane G protein-coupled receptors, which are enzymatically cleaved to e xpose a truncated extracellular N terminus that acts as a tethered activati ng ligand. PAR-1 is cleaved and activated by the serine protease alpha-thro mbin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, prolifer ation, and tissue injury. We have discovered a series of potent peptide-mim etic antagonists of PAR-I, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-I agonist peptide epitope SF LLRN were employed to design and synthesize candidate ligands with appropri ate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical lib raries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid o f PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interf eres with PAR-1 calcium mobilization and cellular function (platelet aggreg ation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 act ivation and internalization, without affecting N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation respo nses in human vascular cells, albeit not in human platelets; whereas, at hi gh concentrations of SFLLRN-NH2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-I, i.e., through PAR-4, which we confirmed by PCR analysis. Selective P AR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and resteno sis.