Inhibitors of kinesin activity from structure-based computer screening

Kinesin motor proteins use ATP hydrolysis for transport along microtubules in the cell. We sought to identify small organic ligands to inhibit kinesin 's activity. Candidate molecules were identified by computational docking o f commercially available compounds using the computer program DOCK. Compoun ds were docked at either of two sites, and a selection was tested for inhib ition of microtubule-stimulated ATPase activity. Twenty-two submillimolar i nhibitors were identified. Several inhibitors appeared to be competitive fo r microtubule binding and not for ATP binding, and three compounds showed 5 0% inhibition down to single-digit micromolar levels. Most inhibitors group ed into four distinct classes (fluoresceins, phenolphthaleins, anthraquinon es, and naphthylene sulfonates). We measured the binding of one inhibitor, rose bengal lactone (RBL), to kinesin (dissociation constant 2.5 mu M) by i ts increase in steady-state fluorescence anisotropy. The RBL binding site o n kinesin was localized by fluorescent resonance energy transfer (FRET) usi ng a donor fluorophore (coumarin) covalently attached at unique, surface-ex posed cysteine residues engineered at positions 28, 149, 103, 220, or 330. RBL was found to bind in its original docked site: the pocket cradled by lo op 8 and beta-strand 5 in kinesin's three-dimensional structure. These resu lts confirm this region's role in microtubule binding and identify this poc ket as a novel binding site for kinesin inhibition.