title: Modulation of PAR(1)  signalling by benzimidazole compounds.
abstract: OBJECTIVE
  Recently, a small molecule (Q94) was reported to selectively block PAR(1) /Gα(q)  interaction and signalling. Here, we describe the pharmacological properties of Q94 and two analogues that share its benzimidazole scaffold (Q109, Q89). Q109 presents a modest variation from Q94 in the substituent group at the 2-position, while Q89 has quite different groups at the 1- and 2-positions.
  
  
  METHODS
  Using human microvascular endothelial cells, we examined intracellular Ca(2+)  mobilization and inositol 1,4,5-trisphosphate accumulation as well as isoprenaline- or forskolin-stimulated cAMP production in response to thrombin.
  
  
  RESULTS
  Q89 (10 µM) produced a leftward shift in the thrombin-mediated intracellular Ca(2+)  mobilization concentration-response curve while having no effect on the E(max) . Both Q94 (10 µM) and Q109 (10 µM) reduced intracellular Ca(2+)  mobilization, leading to a decrease in E(max)  and an increase in EC(50)  values. Experiments utilizing receptor-specific activating peptides confirmed that Q94 and Q109 were selective for PAR(1)  as they did not alter the Ca(2+)  response mediated by a PAR(2)  activating peptide. Consistent with our Ca(2+)  results, micromolar concentrations of either Q94 or Q109 significantly reduced thrombin-induced inositol 1,4,5-trisphosphate production. Neither Q94 nor Q109 diminished the inhibitory effects of thrombin on cAMP production, indicating they inhibit signalling selectively through the G(q)  pathway. Our results also suggest the 1,2-disubstituted benzimidazole derivatives act as 'allosteric agonists' of PAR(1) .
  
  
  CONCLUSIONS
  The Q94 and Q109 benzimidazole derivatives represent a novel scaffold for the development of new PAR(1)  inhibitors and provide a starting point to develop dual signalling pathway-selective positive/negative modulators of PAR(1) .
authors.affiliation: Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Asteriti
authors.forename: S
authors.initials: S
authors.lastname: Daniele
authors.forename: F
authors.initials: F
authors.lastname: Porchia
authors.forename: M T
authors.initials: MT
authors.lastname: Dell'Anno
authors.forename: A
authors.initials: A
authors.lastname: Fazzini
authors.forename: I
authors.initials: I
authors.lastname: Pugliesi
authors.forename: M L
authors.initials: ML
authors.lastname: Trincavelli
authors.forename: S
authors.initials: S
authors.lastname: Taliani
authors.forename: C
authors.initials: C
authors.lastname: Martini
authors.forename: M R
authors.initials: MR
authors.lastname: Mazzoni
authors.forename: A
authors.initials: A
authors.lastname: Gilchrist
mesh_terms: D001562:Benzimidazoles / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D001562:Benzimidazoles; D044463:Receptor, PAR-1; D005576:Colforsin; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D007545:Isoproterenol; D002118:Calcium