Inhibitors of Kinesin Activity from Structure-Based Computer Screening

Abstract

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 of commercially available compounds using the computer program DOCK. Compounds were docked at either of two sites, and a selection was tested for inhibition of microtubulestimulated ATPase activity. Twenty-two submillimolar inhibitors were identified. Several inhibitors appeared to be competitive for microtubule binding and not for ATP binding, and three compounds showed 50% inhibition down to single-digit micromolar levels. Most inhibitors grouped into four distinct classes (fluoresceins, phenolphthaleins, anthraquinones, and naphthylene sulfonates). We measured the binding of one inhibitor, rose bengal lactone (RBL), to kinesin (dissociation constant 2.5 µM) by its increase in steady-state fluorescence anisotropy. The RBL binding site on kinesin was localized by fluorescent resonance energy transfer (FRET) using a donor fluorophore (coumarin) covalently attached at unique, surface-exposed 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 loop 8 and -strand 5 in kinesin's three-dimensional structure. These results confirm this region's role in microtubule binding and identify this pocket as a novel binding site for kinesin inhibition.

Myosin Va and microtubule-based motors are required for fast axonal retrograde transport of tetanus toxin in motor neurons

“…To selectively impair MT-based motors, we used the wellestablished dynein inhibitor EHNA (Penningroth et al, 1982;Reynolds et al, 1998) and the kinesin inhibitor aurintricarboxylic acid (AA) (Hopkins et al, 2000). AA has been recently identified via a structure-based computer screening for kinesin-binding molecules using human conventional kinesin as template (Hopkins et al, 2000).…”

“…To selectively impair MT-based motors, we used the wellestablished dynein inhibitor EHNA (Penningroth et al, 1982;Reynolds et al, 1998) and the kinesin inhibitor aurintricarboxylic acid (AA) (Hopkins et al, 2000). AA has been recently identified via a structure-based computer screening for kinesin-binding molecules using human conventional kinesin as template (Hopkins et al, 2000). It has been shown to have a broad specificity for members of the kinesin superfamily (conventional kinesin, ncd C-terminal kinesin and Unc104/KIF1) (Vale, 2003) and an IC50 in the low micromolar range (0.5-1.4 µM), which is comparable to that of the only other known kinesin inhibitor, adocia sulfate-2 (Sakowicz et al, 1998).…”

Bringing flexibility to virtual screening for enzymatic inhibitors on the grid

“…The molecular docking application DOCK [15] was chosen for being a nearly all-in-one virtual screening solution and because of its past successes in identifying effective inhibitory molecules [2,16]. Other appealing features of the DOCK software package are the inclusion of a number of different docking algorithms, which allows for consensus scoring, and the MPI functionality.…”

Structure-based design of inhibitors of CXCR4.

“…Virtual screening presents the capability to prescreen vast databases, such as the Zinc compound library, a 13 million compound virtual database [208], by targeting a three dimensional structure to determine those compound which best fit, or dock, into a chosen allosteric or ligand binding site. This method has proven successful in the identification of a wide variety of targets [209][210][211][212][213][214][215][216][217][218][219][220][221][222][223][224][225].…”

“…Photolabeling studies of radiolabeled T140 identified TM4 as the most important segment of CXCR4 involved in binding [207]. [212], retinoic acid receptor [213], farnesyl transferase [214], kinesin [215], hypoxanthine-guanine-xanthine phosphoribosyl transferase [216], DNA gyrase [217], H IV -1 RNA transactivation response element [218], aldose reductase [219], rac GTPase [220], checkpoint kinase-1 [221], alpha amylase [222], BCR-ABL tyrosine kinase [223], protein kinase CK2 [224], and alpha-reductase [225]. In one documented case virtual screening targeting DNA gyrase identified lead compounds where experimental high throughput screening of libraries was unsuccessful [181,217].…”