Mitotic kinesin Eg5 remains a validated target in antimitotic therapy because of its essential role in the formation and maintenance of bipolar mitotic spindles. Although numerous Eg5 inhibitors of synthetic origin are known, only a few inhibitors derived from natural products have been reported. In our study, we focused on identifying novel Eg5 inhibitors from medicinal plants, particularly Garcinia species. Herein, we report the inhibitory effect of kolaflavanone (KLF), a Garcinia biflavonoid, on the ATPase and microtubule-gliding activities of mitotic kinesin Eg5. Additionally, we showed the interaction mechanism between Eg5 and KLF via in vitro and in silico analyses. The results revealed that KLF inhibited both the basal and microtubule-activated ATPase activities of Eg5. The inhibitory mechanism is allosteric, without a direct competition with adenosine-5′-diphosphate for the nucleotide-binding site. KLF also suppressed the microtubule gliding of Eg5 in vitro. The Eg5–KLF model obtained from molecular docking showed that the biflavonoid exists within the α2/α3/L5 (α2: Lys111–Glu116 and Ile135–Asp149, α3: Asn206–Thr226; L5: Gly117–Gly134) pocket, with a binding pose comparable to known Eg5 inhibitors. Overall, our data suggest that KLF is a novel allosteric inhibitor of mitotic kinesin Eg5.

中文翻译：

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Kolaflavanone 是一种从药用植物藤黄中提取的双黄酮类化合物，是有丝分裂驱动蛋白 Eg5 的抑制剂

有丝分裂驱动蛋白 Eg5 仍然是抗有丝分裂治疗中经过验证的靶标，因为它在双极有丝分裂纺锤体的形成和维持中发挥着重要作用。尽管已知许多合成来源的 Eg5 抑制剂，但仅报道了少数源自天然产物的抑制剂。在我们的研究中，我们专注于从药用植物中鉴定新型 Eg5 抑制剂，尤其是藤黄属植物。在此，我们报告了可拉黄酮 (KLF)（一种藤黄素双黄酮类化合物）对有丝分裂驱动蛋白 Eg5 的 ATP 酶和微管滑动活性的抑制作用。此外，我们通过体外和计算机分析显示了 Eg5 和 KLF 之间的相互作用机制。结果表明，KLF 抑制 Eg5 的基础和微管激活的 ATP 酶活性。抑制机制是变构的，没有与腺苷-5'-二磷酸直接竞争核苷酸结合位点。KLF 还在体外抑制 Eg5 的微管滑动。从分子对接获得的 Eg5-KLF 模型表明，双黄酮存在于 α2/α3/L5（α2：Lys111-Glu116 和 Ile135-Asp149，α3：Asn206-Thr226；L5：Gly117-Gly134）口袋内，具有结合位姿与已知的 Eg5 抑制剂相当。总体而言，我们的数据表明 KLF 是有丝分裂驱动蛋白 Eg5 的新型变构抑制剂。