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 (IC50 values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent Kd (254 nM) was similar to the IC50 (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.
Footnotes
-
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
-
doi:10.1124/mol.107.037010.
-
ABBREVIATIONS: PAI, plasminogen activator inhibitor; PAI-749, 1-benzyl-3-pentyl-2-[6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-1H-indole; PA, plasminogen activator; tPA, single-chain tissue-type plasminogen activator; 2c-tPA, two-chain tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; NBD, N,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); PAI-NBD119, PAI-1 tagged with NBD at amino acid position 119; Fl, fluorescence; PAB, p-aminobenzamidine; Apo-10, dimethyldecylphosphine oxide; HNEP, HEPES/NaCl/EDTA/PEG-8000; HNEPA, HEPES/NaCl/EDTA/PEG-8000/apo-10; DMSO, dimethylsulfoxide; PAGE, polyacrylamide gel electrophoresis; BN-PAGE, blue native polyacrylamide gel electrophoresis; VN, vitronectin; pNA, p-nitroanilide; BSA, bovine serum albumin; LDS, lithium dodecyl sulfate.
- Received April 13, 2007.
- Accepted July 10, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|