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.Plasminogen activator inhibitor 1 (PAI-1) is a rapidly acting inhibitor of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) (Dellas and Loskutoff, 2005). PAI-1 is a member of the serpin class of serine protease inhibitors that characteristically produce SDS-stable complexes with their cognate protease targets (Silverman et al., 2001). Formation of the acyl-enzyme adduct between PAI-1 and the protease involves initial formation of a Michaelis-type noncovalent complex without significant conformational change, followed by reversible acylation and irreversible reactive loop conformational changes that trap the protease in a covalent complex (Olson et al., 2001). Two other conformation states of PAI-1 are known. First, the acyl-enzyme adduct between PAI-1 and tPA (or uPA) can be hydrolyzed to form cleaved (inactive) PAI-1 and regenerate active plasminogen activator (PA) (Declerck et al., 1992). Second, active PAI-1 can undergo a spontaneous large conformation change that gives rise to an inactive (latent) state of the inhibitor (Levin and Santell, 1987;Mottonen et al., 1992).PAI-1 plays a pivotal role in a myriad of physiological processes that involve activation of plasminogen (Dellas and Loskutoff, 2005). High ...

Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation

“…The background A 405 was subtracted from the final A 405 taken after 25 min. Quality control plates were interleaved with the screening plates containing multiple wells of a maximum effect control, diaplasinin, a known PAI-1 inhibitor (46), and a minimum effect control, DMSO. The extent of PAI-1 inhibition (Unknown) was determined by relating the activity in the compound well to the medians of the diaplasinin and vehicle control wells using the following equation, % Effect ϭ 100 ϫ (Unknown Ϫ min)/(max Ϫ min).…”

Can inactivators of plasminogen activator inhibitor alleviate the burden of obesity and diabetes? (Review)

“…A similar binding site was proposed for the XR5118 PAI-1 inactivator (98). A different binding site was postulated by Gardell et al (99). The PAI-749 small molecule inhibitor could block formation of the initial, reversible Michaelis complex between PAI-1 and its target protease; this indicates that binding in the proximity of Arg369 in the reactive center loop (RCL) of PAI-1 (100) or alerting the RCL conformation to disable the insertion of Arg into the specificity pocket of uPA or tPA.…”

Structure-Activity Relationships of New 2-Acylamino-3-thiophenecarboxylic Acid Dimers as Plasminogen Activator Inhibitor-1 Inhibitors

“…Furthermore, disruption of the PAI-1 gene does not result in any serious pathological defects and induces little sign of bleeding, a most common and critical adverse effect associated with various types of current anti-thrombotic drugs. [3][4][5] Although small molecule PAI-1 inhibitors such as PAI-039 (tiplaxtinin) and PAI-749 (diaplasinin) 4,6,7) have been reported to be effective in animal experiments, their clinical efficacy still remains unknown.…”