Among 40 plant-derived biflavonoids with inhibitory potential against Eg5, morelloflavone from Garcinia dulcis leaves was selected for further testing based on in silico analysis of binding modes, molecular interactions, binding energies and functional groups that interact with Eg5. Computational models predicted that morelloflavone binds the putative allosteric pocket of Eg5, within the cavity surrounded by amino acid residues of Ile-136, Glu-116, Glu-118, Trp-127, Gly-117, Ala-133, Glu-215, Leu-214 and Tyr-211. Binding energy was -8.4 kcal/mol, with a single hydrogen bond formed between morelloflavone and Tyr-211. The binding configuration was comparable to that of a reference inhibitor, S-trityl-L-cysteine. Subsequent biochemical analysis in vitro confirmed that morelloflavone inhibited both the basal and microtubule-activated ATPase activity of Eg5 in a manner that does not compete with ATP binding. Morelloflavone also suppressed Eg5 gliding along microtubules. These results suggest that morelloflavone binds the allosteric binding site in Eg5 and thereby inhibits ATPase activity and motor function of Eg5.
Keywords: in silico; biflavonoids; kinesin Eg5; microtubule gliding; morelloflavone.
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