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-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 alpha-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 alpha-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.
Andrade-Gordon
P
P
Drug Discovery, The R.W. Johnson Pharmaceutical Research Institute, Spring House, PA 19477, USA.
Maryanoff
B E
BE
Derian
C K
CK
Zhang
H C
HC
Addo
M F
MF
Darrow
A L
AL
Eckardt
A J
AJ
Hoekstra
W J
WJ
McComsey
D F
DF
Oksenberg
D
D
Reynolds
E E
EE
Santulli
R J
RJ
Scarborough
R M
RM
Smith
C E
CE
White
K B
KB
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D018716:Molecular Mimicry; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism
D017931:DNA Primers; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin