A Regulatory Hydrophobic Area in the Flexible Joint Region of Plasminogen Activator Inhibitor-1, Defined with Fluorescent Activity-neutralizing Ligands

Abstract

We have characterized the neutralization of the inhibitory activity of the serpin plasminogen activator inhibitor-1 (PAI-1) by a number of structurally distinct organochemicals, including compounds with environmentsensitive spectroscopic properties. In contrast to latent and reactive center-cleaved PAI-1 and PAI-1 in complex with urokinase-type plasminogen activator (uPA), active PAI-1 strongly increased the fluorescence of the PAI-1-neutralizing compounds 1-anilinonaphthalene-8-sulfonic acid and 4,4-dianilino-1,1-bisnaphthyl-5,5-disulfonic acid. The fluorescence increase could be competed by all tested nonfluorescent neutralizers, indicating that all neutralizers bind to a common hydrophobic area preferentially accessible in active PAI-1. Activity neutralization proceeded through two consecutive steps as follows: first step is conversion to forms displaying substrate behavior toward uPA, and second step is to forms inert to uPA. With some neutralizers, the second step was associated with PAI-1 polymerization. Vitronectin reduced the susceptibility to the neutralizers. Changes in sensitivity to activity neutralization by point mutations were compatible with the various neutralizers having overlapping, but not identical, binding sites in the region around ␣-helices D and E and ␤-strand 1A, known to act as a flexible joint when ␤-sheet A opens and the reactive center loop inserts as ␤-strand 4A during reaction with target proteinases. The defined binding area may be a target for development of compounds for neutralizing PAI-1 in cancer and cardiovascular diseases.Plasminogen activator inhibitor-1 (PAI-1) 1 is a fast and specific inhibitor of the serine proteinases urokinase-type (uPA) and tissuetype plasminogen activator (tPA) and, as such, an important regulator of extracellular proteolysis in turn over of extracellular matrix and in fibrinolysis (for reviews see Refs. 1 and 2). PAI-1 binds with high affinity to vitronectin (for reviews see Refs. 3 and 4) and may regulate cell migration and adhesion by inhibition of vitronectin binding of integrins and the uPA receptor (5-10). The PAI-1 level in malignant tumors is one of the most informative biochemical markers of a poor prognosis (for reviews see Refs. 11 and 12), and PAI-1 seems to be causally involved in tumor invasion and angiogenesis (13). A high PAI-1 level in blood plasma is a risk factor for ischemic cardiovascular disease and venous thromboembolism (for review see Ref. 14). PAI-1 is therefore a potential target for both anti-cancer and anti-thrombotic therapy.PAI-1 belongs to the serpin superfamily. Serpins are composed of 3 ␤-sheets and 9 ␣-helices. Serpins and their target proteinases form stable complexes by interaction of the active site of the proteinases with the reactive center peptide bond (P 1 -P 1 Ј) in the solvent-exposed, ϳ20-amino acid long peptide loop, the reactive center loop (RCL) (for reviews see Refs. 2 and 15-17). There is both structural and biochemical evidence that complex formation is associated with the P 1 -P 1 Ј bond being cle...

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

“…The second site was consistent with a previously described organochemical compound binding site, bordered by β-strand 1A, strand 2A, and α-helix D (sA/hD pocket; Fig. S2) (9). However, as discussed below, biochemical and mutational analyses of these two regions revealed that the sB/sC pocket site most likely represented the high-affinity functional binding site for CDE-096 and suggested that the sA/hD pocket interaction was likely a result of the high concentrations of CDE-096 used for crystal soaking studies.…”

“…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.…”

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

“…Several other small molecules have been determined to bind in the same, or similar, region of PAI-1, e.g. ANS, bis-ANS, and XR5118 (51). Among the small molecule inhibitors, AZ3976 is unique because it (a) binds preferentially to latent PAI-1 over active PAI-1 and (b) inhibits PAI-1 by accelerating latency transition.…”

“…This part of PAI-1 includes what is referred to as the flexible joint region (i.e. ␣-hD, ␣-hF, and ␤-s1A) because it participates in structural rearrangements going from active PAI-1 to the irreversible PAI1⅐tPA complex (51). The presence of five hydrogen bonds indicates specific interactions between PAI-1 and AZ3976.…”

Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor-1

“…The flexible structure of PAI-1, the lack of a rigid active site, and its multiple functions all contribute to the difficulties in identifying and designing small-molecule PAI-1 inactivators. Despite these obstacles, several small-molecule PAI-1 inhibitors have been described (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36); however, each has significant limitations that have reduced their potential for further drug development.…”