Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation |
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Authors: | Ola Fjellstr?m Johanna Deinum Tove Sj?gren Carina Johansson Stefan Geschwindner Viveca Nerme Anne Legnehed Jane McPheat Karolina Olsson Cristian Bodin Amalia Paunovic David Gustafsson |
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Affiliation: | From the Departments of ‡Medicinal Chemistry.;§Bioscience, and ;¶Discovery Sciences, AstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden |
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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 IC50 value of 26 μm in an enzymatic chromogenic assay. In a plasma clot lysis assay, the compound was active with an IC50 of 16 μm. Surprisingly, AZ3976 did not bind to active PAI-1 but bound to latent PAI-1 with a KD 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. |
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Keywords: | Drug Discovery High Throughput Screening (HTS) Isothermal Titration Calorimetry Plasminogen Regulation Surface Plasmon Resonance (SPR) X-ray Crystallography PAI-1 Inhibitor Plasma Clot Lysis Prelatent PAI-1 |
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