title: Characterization of a small molecule inhibitor of plasminogen activator inhibitor type 1 that accelerates the transition into the latent conformation.
abstract: A novel class of small molecule inhibitors for plasminogen activator inhibitor type 1 (PAI-1), represented by AZ3976, was identified in a high throughput screening campaign. AZ3976 displayed an IC(50) value of 26 μm in an enzymatic chromogenic assay. In a plasma clot lysis assay, the compound was active with an IC(50) of 16 μm. Surprisingly, AZ3976 did not bind to active PAI-1 but bound to latent PAI-1 with a K(D) of 0.29 μm at 35 °C and a binding stoichiometry of 0.94, as measured by isothermal calorimetry. Reversible binding was confirmed by surface plasmon resonance direct binding experiments. The x-ray structure of AZ3976 in complex with latent PAI-1 was determined at 2.4 Å resolution. The inhibitor was bound in the flexible joint region with the entrance to the cavity located between α-helix D and β-strand 2A. A set of surface plasmon resonance experiments revealed that AZ3976 inhibited PAI-1 by enhancing the latency transition of active PAI-1. Because AZ3976 only had measurable affinity for latent PAI-1, we propose that its mechanism of inhibition is based on binding to a small fraction in equilibrium with active PAI-1, a latent-like prelatent form, from which latent PAI-1 is then generated more rapidly. This mode of action, with induced accelerated latency transition of active PAI-1 may, together with supporting x-ray data, provide improved opportunities for small molecule drug design in the hunt for therapeutically useful PAI-1 inhibitors.
authors.affiliation: Department of Medicinal Chemistry, AstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden. ola.fjellstrom@astrazeneca.com
authors.forename: Ola
authors.initials: O
authors.lastname: Fjellström
authors.forename: Johanna
authors.initials: J
authors.lastname: Deinum
authors.forename: Tove
authors.initials: T
authors.lastname: Sjögren
authors.forename: Carina
authors.initials: C
authors.lastname: Johansson
authors.forename: Stefan
authors.initials: S
authors.lastname: Geschwindner
authors.forename: Viveca
authors.initials: V
authors.lastname: Nerme
authors.forename: Anne
authors.initials: A
authors.lastname: Legnehed
authors.forename: Jane
authors.initials: J
authors.lastname: McPheat
authors.forename: Karolina
authors.initials: K
authors.lastname: Olsson
authors.forename: Cristian
authors.initials: C
authors.lastname: Bodin
authors.forename: Amalia
authors.initials: A
authors.lastname: Paunovic
authors.forename: David
authors.initials: D
authors.lastname: Gustafsson
mesh_terms: D000818:Animals; D001384:Azetidines / Q000737:chemistry / Q000494:pharmacology*; D016466:CHO Cells; D002151:Calorimetry; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008958:Models, Molecular; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry*; D011487:Protein Conformation; D011744:Pyrimidinones / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: C579128:AZ3976; D001384:Azetidines; D017395:Plasminogen Activator Inhibitor 1; D011744:Pyrimidinones