title: Neutralization of plasminogen activator inhibitor I (PAI-1) by the synthetic antagonist PAI-749 via a dual mechanism of action.
abstract: PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC(50) values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent K(d) (254 nM) was similar to the IC(50) (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min (sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog (producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation.
authors.affiliation: Wyeth Research, N2274, 500 Arcola Road, Collegeville, PA 19426, USA. gardels@wyeth.com
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Gardell
authors.forename: Julie A
authors.initials: JA
authors.lastname: Krueger
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Antrilli
authors.forename: Hassan
authors.initials: H
authors.lastname: Elokdah
authors.forename: Scott
authors.initials: S
authors.lastname: Mayer
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Orcutt
authors.forename: David L
authors.initials: DL
authors.lastname: Crandall
authors.forename: George P
authors.initials: GP
authors.lastname: Vlasuk
mesh_terms: D001704:Biopolymers / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006801:Humans; D006868:Hydrolysis; D007211:Indoles / Q000494:pharmacology*; D008823:Micelles; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D013777:Tetrazoles / Q000494:pharmacology*; D019096:Vitronectin / Q000378:metabolism / Q000494:pharmacology
chemical_list: D001704:Biopolymers; D007211:Indoles; D008823:Micelles; D017395:Plasminogen Activator Inhibitor 1; D013777:Tetrazoles; D019096:Vitronectin; C524408:1-benzyl-3-pentyl-2-(6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl)-1H-indole