A novel type of antiviral agent for human cytomegalovirus (HCMV) is required, because the appearance of ganciclovir (GCV) resistant viruses has been reported. Tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone) has been shown to suppress significantly HCMV replication in human embryonic lung (HEL) fibroblast cells. Recently, we revealed that the action of tricin is different from that of GCV and cyclin-dependent kinase 9 (CDK9) is one of the target proteins of tricin. These results suggested that tricin is considered as a novel type of anti-HCMV agent. However, its anti-HCMV potency is not greater than that of GCV. This study tried to develop novel compounds with much greater anti-HCMV activity than GCV. We first made modifications to tricin by introducing fluorine atom, and then performed molecular docking simulations using the designed compounds and CDK9. The calculated binding energies showed that 6F-tricin (6-fluoro-4′-hydroxy-3′,5′-dimetoxyflavone) binds to CDK9 much stronger than tricin. Based on these results, 6F-tricin was synthesized, and then its anti-HCMV effect was analyzed in HEL cell cultures. As a result, 6F-tricin strongly suppressed HCMV replication in a dose-dependent manner. The anti-HCMV activity with a 50% effective concentration (EC₅₀) was 0.126 nM, corresponding to about 1/200 and 1/400 of EC₅₀ of GCV (27.5 nM) and tricin (54.3 nM), respectively. Moreover, 6F-tricin had no cytotoxicity against HEL cells at concentrations up to 10 μM. We further performed detailed analysis on the amino acid contributions to the binding energies and found that the strong binding affinity for 6F-tricin to CDK9 is attributed to the specific binding orientation of 6F-tricin in the ATP-binding site. These results suggest that 6F-tricin is a promising candidate for anti-HCMV drug development.

由于已报道了更昔洛韦（GCV）耐药病毒的出现，因此需要一种新型的人类巨细胞病毒（HCMV）抗病毒剂。Tricin（4'，5,7-三羟基-3'，5'-二甲氧基黄酮）已显示可显着抑制人胚肺（HEL）成纤维细胞中的HCMV复制。最近，我们发现，tricin的作用不同于GCV，并且细胞周期蛋白依赖性激酶9（CDK9）是tricin的目标蛋白之一。这些结果表明曲霉素被认为是新型的抗HCMV剂。但是，其抗HCMV效力不大于GCV。这项研究试图开发出具有比GCV更强的抗HCMV活性的新型化合物。我们首先通过引入氟原子对Tricin进行了修饰，然后使用设计的化合物和CDK9进行分子对接模拟。计算出的结合能表明，6F-肌动蛋白（6-氟-4'-羟基-3'，5'-二甲氧基黄酮）与CDK9的结合力比三辛酸强得多。基于这些结果，合成了6F-肌动蛋白，然后在HEL细胞培养物中分析了其抗HCMV作用。结果，6F-肌动蛋白以剂量依赖性方式强烈抑制HCMV复制。有效浓度为50％的抗HCMV活性（EC₅₀）为0.126 nM，分别对应于GCV（27.5 nM）和曲霉素（54.3 nM）的EC ₅₀的约1/200和1/400 。此外，浓度高达10μM的6F-tricin对HEL细胞无细胞毒性。我们进一步分析了氨基酸对结合能的贡献，发现对6F-tricin与CDK9的强结合亲和力归因于6F-tricin在ATP结合位点的特异性结合方向。这些结果表明6F-肌动蛋白是抗HCMV药物开发的有前途的候选者。