Protease‐Activated Receptor 1‐Selective Antagonist SCH79797 Inhibits Cell Proliferation and Induces Apoptosis by a Protease‐Activated Receptor 1‐Independent Mechanism

Abstract

Thrombin, a key mediator of blood coagulation, exerts a large number of cellular actions via activation of a specific G-protein-coupled receptor, named protease-activated receptor 1 (PAR1). Several studies in experimental animals have demonstrated a therapeutic potential of small molecules with PAR1 antagonistic properties for treatment of diseases such as vascular thrombosis and arterial restenosis. We have studied the biological actions of one highly potent, selective PAR1 antagonist, SCH79797 (in vitro , and found that this compound was able to interfere with the growth of several human and mouse cell lines, in a concentration-dependent manner. The ED 50 for growth inhibition was 75 nM, 81 nM and 116 nM for NIH 3T3, HEK 293 and A375 cells, respectively. Moreover, in NIH 3T3 cells, SCH79797 inhibited serum-stimulated activation of p44/p42 mitogen-activated protein kinases (MAPK) at low concentrations and induced apoptosis at higher concentrations. However, the antiproliferative and pro-apoptotic effects of SCH79797 are likely not mediated by PAR1 antagonism, as they were also observed in embryonic fibroblasts derived from PAR1 null mice. These data suggest that, in view of the development of PAR1-selective antagonists as therapeutic agents, effects potentially unrelated to PAR1 inhibition should be carefully scrutinized.Thrombin, a trypsin-like serine protease, is the most potent agonist for platelet aggregation and plays a central role in haemostatic processes [1]. Thrombin catalyses the conversion of fibrinogen to fibrin by cleaving the peptide bond between an arginine and a glycine residue in the fibrinogen sequence [2]; it is also responsible for proteolytic activation of factors V, VIII, XI, XIII and protein C [1]. However, in addition to its role in blood coagulation, thrombin also stimulates mitogenic events in several cell types including fibroblasts, smooth muscle cells and astrocytes [3], therefore playing a central role in tissue repair, fibrosis, inflammation, neurodegeneration, atherosclerosis and restenosis [4][5][6][7].All cellular actions of α -thrombin are mediated by specific G-protein-coupled receptors, named protease-activated receptors (PAR). Activation of PARs by thrombin and other trypsin-like serine proteases is based on a novel mechanism: the protease cleaves part of the N-terminal domain of the receptor, releasing a 'tethered ligand' that subsequently binds to an extracellular loop of the receptor and activates the G-protein-coupled signal transduction [8]. Four PARs have now been cloned [9]; in humans, PAR1 is considered the primary α -thrombin receptor, although thrombin can also activate PAR3 and PAR4 [10]. Thrombin cleaves PAR1 between Arg 41 and Ser 42 , unmasking the N-terminal recognition motif 'SFLLRN' [11].Several small molecules capable of blocking the α -thrombin active site have been characterized over the years as antithrombotic agents, starting with hirudin, a natural leech-derived peptide [12]. However, the identification of the many biological actions of α -t...

PAR1 contributes to influenza A virus pathogenicity in mice

“…Accordingly, the natural PAR1 activator thrombin was generated in IAV-infected lungs (45), and elevated levels of PAR1 were observed in the airways of IAV-infected mice (17). It is worth noting, however, that SCH79797 is known to have off-target effects on cell proliferation and survival (46,47); thus, we cannot exclude PAR1-independent effect of SCH79797. However, SCH79797 was capable of inhibiting PAR1 signaling ( Figure 4A and ref.…”

Protease-activated receptor 1 knockout reduces experimentally induced liver fibrosis

“…Indeed, since rodent platelets do not express PAR-1 but instead use PAR-3 together with PAR-4, PAR-1 antagonists do not alter blood coagulation (9) and their protective effects are thus the consequence of the blockage of PAR-1 function in other cells such as fibro/ myofibroblasts. However, the use of pharmacological antagonists may lead to improper results because of incomplete effects on one hand or nonspecific effects on the other (13). Thus in this study we used mice in which PAR-1 expression was abolished via homologous recombination to clarify the implication of PAR-1 in liver fibrogenesis.…”

Challenges and Opportunities in Protease-Activated Receptor Drug Development

“…The most commonly used are SCH79797 and SCH203009, which exhibit potent inhibition of PAR1-mediated events in multiple cell types (36,37) and are effective in vivo (37)(38)(39). However, these agents have inferior efficacy and selectivity to atopaxar and vorapaxar, and there has been some concern regarding their PAR1-independent off-target effects (40,41). Consequently, the two clinically tested agents, atopaxar and vorapaxar, have become the gold-standard small-molecule PAR1 antagonists for experimental studies.…”

“…The mechanism of PAR activation is best understood for PAR1. Thrombin (α-Th) binds to and cleaves the extracellular N terminus of PAR1 at a specific arginine (R) residue located at position 41. This results in the generation of a new N terminus that binds intramolecularly to the receptor to trigger transmembrane signaling mediated by heterotrimeric G proteins comprising α and βγ subunits.…”