title: Characterization of a small molecule PAI-1 inhibitor, ZK4044.
abstract: Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. In animal studies, inhibition of PAI-1 activity prevents arterial and venous thrombosis, indicating that PAI-1 inhibitors may be used as a new class of antithrombotics. In this study, we characterize a small molecule PAI-1 inhibitor, ZK4044, which was identified by high throughput screening and chemically optimized. In a chromogenic substrate-based urokinse (uPA)/PAI-1 assay and a tissue-type plasminogen activator (tPA)-mediated clot lysis assay, ZK4044 inhibited human PAI-1 activity with IC50 values of 644+/-255 and 100+/-90 nM, respectively. ZK4044 had no detectable inhibitory activity toward other serpins such as antithrombin III, alpha1-antitrypsin and alpha2-antiplasmin, indicating that ZK4044 is a specific PAI-1 inhibitor. ZK4044 was shown to bind directly to PAI-1 and prevent the binding of PAI-1 to tPA in a dose-dependent manner in surface plasmon resonance Biacore-based experiments. ZK4044 also prevented PAI-1/tPA complex formation, as analyzed by SDS/PAGE. ZK4044 had little effect on elastase-mediated cleavage of active PAI-1, indicating that the primary mode of action of ZK4044 is most likely to directly block the PAI-1/tPA interaction rather than to convert active PAI-1 to latent PAI-1. In the chromogenic substrate-based uPA/PAI-1 assay, ZK4044 was approximately 2-fold less potent against a mutant PAI-1 (14B-1), which contains four mutations at N150H, K154T, Q319L and M354I, compared with wild-type PAI-1, suggesting that the ZK4044 binding site on the surface of PAI-1 is close to these mutant residues. Together, our data show that ZK4044 represents a new class of small molecule PAI-1 inhibitors with anti-thrombotic potential.
authors.affiliation: Departments of Cardiovascular Research, Molecular Pharmacology and Medicinal Chemistry, Berlex Biosciences, Richmond, CA 94804, United States. amy_liang@berlex.com
authors.forename: Amy
authors.initials: A
authors.lastname: Liang
authors.forename: Faye
authors.initials: F
authors.lastname: Wu
authors.forename: Kathy
authors.initials: K
authors.lastname: Tran
authors.forename: Steven W
authors.initials: SW
authors.lastname: Jones
authors.forename: Gary
authors.initials: G
authors.lastname: Deng
authors.forename: Bin
authors.initials: B
authors.lastname: Ye
authors.forename: Zhuchun
authors.initials: Z
authors.lastname: Zhao
authors.forename: R Michael
authors.initials: RM
authors.lastname: Snider
authors.forename: William P
authors.initials: WP
authors.lastname: Dole
authors.forename: John
authors.initials: J
authors.lastname: Morser
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Wu
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001565:Benzoates / Q000737:chemistry / Q000494:pharmacology*; D001779:Blood Coagulation Factors / Q000037:antagonists & inhibitors*; D004353:Drug Evaluation, Preclinical; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000187:drug effects / Q000378:metabolism; D020349:Surface Plasmon Resonance; D010959:Tissue Plasminogen Activator / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D001565:Benzoates; D001779:Blood Coagulation Factors; D005343:Fibrinolytic Agents; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D015843:Serpins; C502734:ZK 4044; D010959:Tissue Plasminogen Activator