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

Abstract

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease ␣-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; 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 activation and internalization, without affecting N-terminal cleavage. At high concentrations of ␣-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH 2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

Thrombin Responses in Human Endothelial Cells

“…BMS200261 is one such antagonist. Others have been described recently (47,52). On platelets, the efficacy of this type of inhibitor is limited by the presence of PAR4.…”

Protease-activated receptors in cancer: A systematic review

“…Efforts to develop receptor inhibitors as compared to targeting thrombin are currently regarded as a priority. As outlined in Table V ( [134][135][136][137][138][139][140][141][142][143][144][145][146][147][148][149][150][151][152], substantial success has been achieved in the development of PAR1 and PAR4 antagonists.…”

Protease activated receptors: theme and variations

“…Cathepsin G (Sambrano et al, 2000). g PAR1 antagonists: (Bernatowicz et al, 1996;Hoekstra et al, 1998;McComsey et al, 1999;Andrade-Gordon et al, 1999;Kawabata et al, 1999) sion, a meticulous, tissue by tissue approach may be required to better understand the role of PARs in development and homeostasis. Determining which receptor mediates protease responses in a particular cell or tissue is not necessarily straight-forward.…”