Morelloflavone as a novel inhibitor of mitotic kinesin Eg5

Abstract

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.

Insights into the Molecular Mechanisms of Eg5 Inhibition by (+)-Morelloflavone

“…However, it appears that the tubulin-binding region gains flexibility of residues after MF interaction (Figure 6E), and MF may interfere with Eg5-microtubule interaction as one of its possible Eg5-inhibitory mechanisms. This is in agreement with our earlier report that MF significantly affects microtubule binding to Eg5 and inhibited its microtubule-activated ATPase activities [29].…”

“…Both inhibitors moved closer to loop5 as confirmed from the interaction modes and the distance from loop5 (Figure 4F). This behavior marks the likely similar mode of action of these molecules, which is compatible with their similar binding configuration [29].…”

“…STLC and ispinesib reportedly both bind the allosteric pocket formed by the loop5/α2/α3, despite having diverse chemical structures [23,35,36]. Based on an Eg5-MF model generated by molecular docking, it was predicted that MF also binds at the allosteric loop5/α2/α3 pocket, ~12 Å from the nucleotide-binding site [29]. Here, we sought to unveil the possible effect of MF binding on the loop5/α2/α3 pocket in comparison with the effects of STLC and ispinesib.…”

“…Morelloflavone (MF), a biflavonoid isolated from Garcinia spp., has been reported for its antitumor effect in vivo [28]. Recently, our research team identified Eg5 as a possible target for the anti-proliferative effect of the biflavonoid [29]. MF inhibited Eg5 basal and microtubule-activated ATPase activities.…”

“…MF inhibited Eg5 basal and microtubule-activated ATPase activities. The inhibitor also suppressed the microtubule gliding of Eg5 in vitro [29]. The inhibitory mechanism appears to involve direct binding to an allosteric site on Eg5 to structurally alter the ATP-binding pocket, thus inhibiting its enzymatic functions.…”