Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 7; pp. 2951 - 2956
• Main Authors: Dowal, Louisa, Sim, Derek S, Dilks, James R, Blair, Price, Beaudry, Sarah, Denker, Bradley M, Koukos, Georgios, Kuliopulos, Athan, Flaumenhaft, Robert
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 15.02.2011; National Acad Sciences
• Subjects: Aggregation; Agonists; Allosteric Regulation - physiology; Amino acids; Animals; Antithrombins - metabolism; Binding sites; Biological Sciences; Blood platelets; Calcium - metabolism; Cell Line; Dogs; Epinephrine; Flow Cytometry; Luciferases; Modulated signal processing; Molecules; P-Selectin - metabolism; Peptide Fragments - metabolism; Platelet activation; Platelet Aggregation; Platelets; Protein Structure, Secondary - physiology; Proteins; Receptor, PAR-1 - agonists; Receptor, PAR-1 - metabolism; Receptors; Receptors, G-Protein-Coupled - metabolism; Secretion; Signal transduction; Signal Transduction - physiology; Thrombosis; Thrombosis - metabolism
• DOI: 10.1073/pnas.1014863108
• ISSN: 0027-8424

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α₂A-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by Gαq but not Gα₁₂. The compound inhibited thrombus formation in vivo following vascular injury with an IC₅₀ of ~1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation.