Mechanism of Inactivation of Plasminogen Activator Inhibitor-1 by a Small Molecule Inhibitor

Abstract

The inactivation of plasminogen activator inhibitor-1 (PAI-1) by the small molecule PAI-1 inhibitor PAI-039 (tiplaxtinin) has been investigated using enzymatic analysis, direct binding studies, site-directed mutagenesis, and molecular modeling studies. Previously PAI-039 has been shown to exhibit in vivo activity in various animal models, but the mechanism of inhibition is unknown. PAI-039 bound specifically to the active conformation of PAI-1 and exhibited reversible inactivation of PAI-1 in vitro. SDS-PAGE indicated that PAI-039 inactivated PAI-1 predominantly through induction of PAI-1 substrate behavior. Preincubation of PAI-1 with vitronectin, but not bovine serum albumin, blocked PAI-039 activity while analysis of the reciprocal experiment demonstrated that preincubation of PAI-1 with PAI-039 blocked the binding of PAI-1 to vitronectin. Together, these data suggest that the site of interaction of the drug on PAI-1 is inaccessible when PAI-1 is bound to vitronectin and may overlap with the PAI-1 vitronectin binding domain. This was confirmed by site-directed mutagenesis and molecular modeling studies, which suggest that the binding epitope for PAI-039 is localized adjacent to the previously identified interaction site for vitronectin. Thus, these studies provide a detailed characterization of the mechanism of inhibition of PAI-1 by PAI-039 against free, but not vitronectin-bound PAI-1, suggesting for the first time a novel pool of PAI-1 exists that is vulnerable to inhibition by inactivators that bind at the vitronectin binding site.

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

“…Arg-76 and Tyr-79 in ␣-hD and Thr-93, Thr-94, and Asp-95 in ␤-s2A have been suggested to be involved in binding (see Fig. 9A) (53). Notably, all of these residues are within 5 Å from the AZ3976 ligand in the present structure (Fig.…”

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

“…The most extensively studied PAI-1 inactivating compound to date, tiplaxtinin, binds to active but not latent PAI-1 (11). This selectivity is a consequence of structural differences in the binding site between the two serpin conformations.…”

“…These associations have made PAI-1 an attractive pharmaceutical target. However, despite extensive studies, only a few small molecule inhibitors have been identified thus far (7)(8)(9)(10)(11)(12)(13)(14)(15)(16), and the majority of these are poor pharmaceutical candidates as they have relatively low affinity for PAI-1 and are unable to inactivate PAI-1 bound to its plasma cofactor vitronectin.…”

Chronic relapsing experimental allergic encephalomyelitis (CREAE) in plasminogen activator inhibitor‐1 knockout mice: the effect of fibrinolysis during neuroinflammation

“…In summary, inhibition of PAI-1 could be therapeutically beneficial in treating MS, using small molecular weight PAI-1 inhibitors now available [53,54].This would provide a dual palliative approach whereby leucocyte migration and inflammation are reduced, allowing efficient removal of fibrin, which could potentially decrease axonal/neuronal damage and reduce frequency of disease relapses in MS patients.…”