title: Biochemical characterization of a novel high-affinity and specific plasma kallikrein inhibitor.
abstract: OBJECTIVE
  Kallikrein acts on high molecular weight kininogen (HK) to generate HKa (cleaved HK) and bradykinin (BK). BK exerts its effects by binding to B(2) receptors. The activation of B(2) receptors leads to the formation of tissue plasminogen activator, nitric oxide (NO) and prostacyclin (PGI(2) ). An elevated kallikrein-dependent pathway has been linked to cardiovascular disease risk. The aim of this study was to investigate whether our novel plasma kallikrein inhibitor abolishes kallikrein-mediated generation of BK from HK and subsequent BK-induced NO and PGI(2) formation, thereby influencing endothelial pathophysiology during chronic inflammatory diseases.
  
  
  METHODS
  Kinetic analysis was initially used to determine the potency of PF-04886847. Biochemical ligand binding assays, immunological methods and calcium flux studies were used to determine the selectivity of the kallikrein inhibitor. In addition, the effect of PF-04886847 on BK-induced relaxation of the rat aortic ring was determined in a model of lipopolysaccharide-induced tissue inflammation.
  
  
  RESULTS
  Evidence was obtained in vitro and in situ, indicating that PF-04886847 is a potent and specific inhibitor of plasma kallikrein. PF-04886847 efficiently blocked calcium influx as well as NO and PGI(2) formation mediated through the BK-stimulated B(2) receptor signalling pathway. PF-04886847 blocked kallikrein-induced endothelial-dependent relaxation of isolated rat aortic rings pre-contracted with phenylephrine.
  
  
  CONCLUSIONS
  PF-04886847 was shown to be the most potent small molecule inhibitor of plasma kallikrein yet described; it inhibited kallikrein in isolated aortic rings and cultured endothelial cells. Overall, our results indicate that PF-04886847 would be useful for the treatment of kallikrein-mediated inflammatory disorders.
authors.affiliation: Department of Pharmacology, School of Pharmacy, University of Mississippi, University, MS, USA CVMED Exploratory, Pfizer Global Research and Development, Groton, CT, USA.
authors.forename: D
authors.initials: D
authors.lastname: Kolte
authors.forename: Jw
authors.initials: J
authors.lastname: Bryant
authors.forename: D
authors.initials: D
authors.lastname: Holsworth
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: P
authors.initials: P
authors.lastname: Akbari
authors.forename: Gw
authors.initials: G
authors.lastname: Gibson
authors.forename: Z
authors.initials: Z
authors.lastname: Shariat-Madar
mesh_terms: D062365:Aminobenzoates / Q000494:pharmacology*; D000631:Aminopyridines / Q000494:pharmacology*; D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D011464:Epoprostenol / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000188:drug therapy / Q000201:enzymology; D007700:Kinetics; D019679:Kininogen, High-Molecular-Weight / Q000037:antagonists & inhibitors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism; D010656:Phenylephrine / Q000378:metabolism / Q000494:pharmacology; D020842:Plasma Kallikrein / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity
chemical_list: D062365:Aminobenzoates; D000631:Aminopyridines; D004791:Enzyme Inhibitors; D019679:Kininogen, High-Molecular-Weight; D008070:Lipopolysaccharides; C559289:PF 04886847; D010656:Phenylephrine; D009569:Nitric Oxide; D011464:Epoprostenol; D020842:Plasma Kallikrein; D001920:Bradykinin