Contents
Protease-Activated Receptors (PARs)
Guest Editor: Bruce E. Maryanoff
Peptide-Derived Protease-Activated Receptor-1
(PAR-1) Antagonists Pp.1-11
Steven M. Seiler and Michael S. Bernatowicz
[Abstract]
Discovery of Potent Peptide-Mimetic
Antagonists for the Human Thrombin Receptor, Protease-Activated Receptor-1
(PAR-1) Pp.13-36
Bruce E. Maryanoff, Han-Cheng Zhang,
Patricia Andrade-Gordon and Claudia K. Derian
[Abstract]
Potent Non-Peptide Thrombin Receptor
Antagonists Pp.37-45
Samuel Chackalamannil, Ho-Sam Ahn, Yan Xia,
Darío Doller and Carolyn Foster
[Abstract] [Full text article]
Non-Peptidic Small-Molecule Antagonists of
the Human Platelet Thrombin Receptor PAR-1 Pp.47-59
H.G. Selnick, J.C. Barrow, P.G. Nantermet
and T.M. Connolly
[Abstract] [Full text article]
Proteinase-Activated Receptor-2:
Physiological and Pathophysiological Roles Pp.61-72
Anne-Marie Coelho, Valeria Ossovskaya and Nigel W. Bunnett
[Abstract] [Full text article]
Protease-Activated Receptor-2 Antagonists and
Agonists Pp.73-82
Robert M. Scarborough
[Abstract] [Full text article]
Abstracts
[Back to top] Peptide-Derived Protease-Activated Receptor-1
(PAR-1) Antagonists
Steven M. Seiler and Michael S. Bernatowicz
Protease activated receptor–1 (PAR-1) is a G-coupled receptor cleaved by thrombin and other proteases to expose a new N-terminus, a “tethered ligand”, that activates the receptor. Independently of proteolytic cleavage, peptides similar to the new N-terminus also activate the receptor, and structure activity relationships for the activating peptides have been extensively studied. Modification of activating peptides led to rationally designed peptide antagonists. The more potent peptide antagonists were N-terminal and 3-position modifications of the agonist peptides. The resulting PAR- 1 antagonists have proved useful in pharmacological studies resolving the contribution of PAR-1 signaling mechanisms relative to other PARs in platelets, vascular endothelial and other cell types. High affinity peptide agonists and antagonists have been radiolabled and proven useful in binding assays. Screening of combinatorial libraries and compound collections using the radioligands have identified non-peptide antagonists of several different chemotypes. When the “thrombin receptor” (PAR-1) was first cloned and its mechanism of activation elucidated, there was great enthusiasm for the receptor as a drug target. The use of peptide agonists and antagonists has made possible much progress in our understanding of the role of this receptor.
[Back to top] Discovery of Potent Peptide-Mimetic
Antagonists for the Human Thrombin Receptor, Protease-Activated Receptor-1
(PAR-1)
Bruce E. Maryanoff, Han-Cheng Zhang, Patricia
Andrade-Gordon and Claudia K. Derian
Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease a-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. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-mimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of a-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.
[Back to top] Potent Non-Peptide Thrombin Receptor
Antagonists
Samuel Chackalamannil, Ho-Sam Ahn, Yan Xia,
Darío Doller and Carolyn Foster
Protease activated receptor-1 (PAR-1), also known as thrombin receptor, is present in a variety of cell types such as platelets and endothelial cells. PAR-1 is proteolytically activated by thrombin by cleavage at its extracellular domain, unmasking a new amino terminus, which internally binds to the proximal receptor, eliciting cellular activation. Inhibition of the cellular activation by thrombin is a potentially promising therapeutic approach for the treatment of thrombotic and vascular proliferative disorders such as atherosclerosis and restenosis. Reported herein is the pharmacology of potent, low molecular weight thrombin receptor antagonists from pyrroloquinazoline, benzimidazole, and himbacine series. In the radioligand binding assay, these compounds inhibited PAR-1 in a competitive manner. They also inhibited thrombin and agonist peptide induced human platelet aggregation in a dose-dependent manner. Additionally, these compounds showed dose-dependent inhibition of agonist-induced cytosolic Ca+2 transients and thymidine incorporation in human coronary artery smooth muscle cells (hCASMC). The most potent compound among these antagonists showed a Ki of 12 nM in the radioligand binding assay and an IC50 of 70 nM in the platelet aggregation inhibition assay.
[Back to top] Non-Peptidic Small-Molecule Antagonists of
the Human Platelet Thrombin Receptor PAR-1
H.G. Selnick, J.C. Barrow, P.G. Nantermet and
T.M. Connolly
The thrombin receptor on human platelets is the first member identified of a new family of G-protein coupled receptors referred to as protease activated receptors (PARs). These receptors are activated by a unique mechanism involving proteolytic cleavage of a portion of the extracellular domain to generate a new N-terminus which then acts as a tethered or intramolecular ligand (agonist) for the receptor. The hexapeptide SFLLRN-NH2 comprising the new Nterminus is referred to as the Thrombin Receptor Activating Peptide, or “TRAP”. Thrombin is the most potent agonist for platelet aggregation and selective blockade of the intramolecular activation step without effecting the proteolytic activity of thrombin should result in moderation of platelet activation and aggregation without interfering with the other coagulation cascade effects of thrombin. Screening of combinatorial libraries identified a novel, non-peptide PAR-1 thrombin receptor antagonist. Examination of structure-activity relationships revealed that portions of the molecule could be replaced resulting in simpler molecules of lower molecular weight that were at the same time more potent. Molecules in this series were effective antagonists of TRAP-stimulated platelet activation, but had limited activity when thrombin was the agonist. Additional directed screening and subsequent lead refinement resulted in a second series of isoxazole based compounds. Some of the resultant molecules were potent PAR-1 antagonists that were effective against both TRAP- and thrombin-stimulated receptor activation. These compounds do not inhibit the proteolytic effects of thrombin but rather interfere with the intramolecular binding of the tethered ligand (SFLLRN) to the transmembrane portion of the thrombin receptor. They represent promising leads for future explorations of antithrombotic activity of thrombin receptor antagonists.
[Back to top]
Proteinase-Activated Receptor-2: Physiological and Pathophysiological
Roles
Anne-Marie Coelho, Valeria Ossovskaya and Nigel
W. Bunnett
Protease-activated receptor 2 (PAR2) is the second member of a new subfamily of G-protein coupled receptors: the protease-activated receptors (PARs). At present, four different PARs have been cloned and all of them share the same basic mechanism of activation. A serine protease cleaves the extended, extracellular N-terminus of the receptor at a specific site within the protein chain to expose an N-terminal tethered ligand domain, which binds to and activates the cleaved receptor. In this manner, trypsin and mast cell b-tryptase activate PAR2. PARs are single use receptors because proteolytic activation is irreversible and the cleaved receptors are degraded in lysosomes. Thus, PARs play important roles in emergency situations, such as trauma and inflammation. Emerging evidence indicates that PAR2 is involved in the cardiovascular, pulmonary and gastrointestinal systems, where it controls inflammation and nociception. Work with selective agonists and knockout animals suggests a contribution of PAR2 to certain inflammatory diseases. Therefore, selective antagonists or agonists of these receptors may be useful therapeutic agents for the treatment of human diseases.
[Back to top] Protease-Activated Receptor-2 Antagonists and
Agonists
Robert M. Scarborough
[Full
text article]
Interest in the development of specific antagonists of the
protease-activated receptors are significant, however, achieving such goals remain
extremely challenging. Considerable efforts have been directed at developing
specific antagonists of the first elucidated member of this receptor family,
namely the thrombin receptor, PAR-1. However, significantly less effort has
been directed at the second member of the family, PAR-2 due in part to lack of
clarity concerning its activating protease(s), and uncertainty concerning its
physiological and pathophysiological roles in disease pathways. This review
will briefly summarize what is known about the activating protease(s), the
potential (patho)physiological roles for PAR-2 and structure-activity
relationships that have been developed for PAR-2 agonists and antagonists in
relationship to agonists and antagonists developed for the other protease-activated
receptors.