Chen Yixin, Duan Meitao, Xu Jianling, Duan Ao, Yang Haocheng, Tao Hongquan, Tian Shuo, Zhou Zishan, Li Wenzhang, Tao Huaming, Zhu Yongyan, Zhu Quanhong
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China; Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics, Guangzhou 510515, China.
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China; School of Pharmacy, Xiamen Medical College, Xiamen 361023, China.
Bioorg Chem. 2025 Jan;154:108054. doi: 10.1016/j.bioorg.2024.108054. Epub 2024 Dec 11.
The inhibition of HBV DNA and elimination of HBsAg has already been established as an indicator for HBV clinic cure, and a novel dual-targeting inhibitors of HBV polymerase/entry are designed and synthesized in this study. Pentacyclic triterpenes (PTs) scaffold of exhibiting a high affinity to NTCP, including glycyrrhitinic acid (GA), oleanolic acid (OA), ursolic acid (UA), and betulinic acid (BA) were linked neatly with the nucleoside drug zidovudine (AZT) through a molecular hybrid strategy to synthesize twenty of PTs-AZT conjugates for targeting HBV polymerase as well as sodium taurocholate cotransporting polypeptide (NTCP). The conjugates showed significant inhibitory effects on the secretion of HBsAg and HBeAg in HepG2.2.15 cells, and the activity on HBsAg were better. Moreover, HBV DNA replication was also notably suppressed after incubated with the conjugates. The IC value of BA-AZT1 on HBsAg inhibition was 0.65 ± 0.07 μM, and it was 284.2 times and 442.2 times higher comparing to corresponding parent compound BA and AZT. Additionally, the therapeutic index (TI) was also improved by 87.8 times than AZT. And the IC value of BA-AZT1 on inhibition of HBV DNA replication was 0.70 ± 0.02 μM, 10.4 times higher than that of AZT besides conspicuous TI. Molecular docking suggested that AZT skeleton of conjugate BA-AZT1 interacted with B region of HBV Polymerase reverse transcription region, and BA structure simultaneously targeted to C region of polymerase via hydrophobic chain, establishing strong binding interactions with the HBV Pol protein. In addition, docked with NTCP, BA-AZT1 with flat pentacyclic structure inserted into the interface and also formed hydrogen bonds, hydrophobic and van der Waals forces with the amino residue 157-165 of NTCP. Further SPR analysis demonstrated the binding affinity of BA-AZT1 to C region of polymerase was 19.55 μM, stronger than 53.21 μM of BA and 31.82 μM of AZT. BA-AZT1 selectively bound to the 157-165 epitopes of NTCP receptors in host cell but not PreS1 of virus. As a result, we deduced that the designed conjugates targeted NTCP and HBV polymerase, not only prevented HBV from entering host cells via selective binding NTCP, but also inhibited HBV DNA replication through obstructing the function of HBV polymerase, and it could potentially serve as a promising dual-functional and dual-target inhibitor with both replication and entry inhibition to exert anti-HBV activity.
抑制乙肝病毒（HBV）DNA及清除乙肝表面抗原（HBsAg）已被确立为HBV临床治愈的指标，本研究设计并合成了一种新型的HBV聚合酶/进入双靶点抑制剂。五环三萜（PTs）骨架对牛磺胆酸钠共转运多肽（NTCP）具有高亲和力，包括甘草次酸（GA）、齐墩果酸（OA）、熊果酸（UA）和桦木酸（BA），通过分子杂交策略将其与核苷类药物齐多夫定（AZT）巧妙连接，合成了20种PTs-AZT缀合物，以靶向HBV聚合酶及NTCP。这些缀合物对HepG2.2.15细胞中HBsAg和乙肝e抗原（HBeAg）的分泌具有显著抑制作用，对HBsAg的活性更佳。此外，与缀合物孵育后，HBV DNA复制也受到明显抑制。BA-AZT1对HBsAg抑制的半数抑制浓度（IC）值为0.65±0.07μM，与相应母体化合物BA和AZT相比，分别高284.2倍和442.2倍。此外，治疗指数（TI）也比AZT提高了87.8倍。BA-AZT1对HBV DNA复制抑制的IC值为0.70±0.02μM，除了显著的TI外，比AZT高10.4倍。分子对接表明，缀合物BA-AZT1的AZT骨架与HBV聚合酶逆转录区的B区相互作用，BA结构同时通过疏水链靶向聚合酶的C区，与HBV聚合酶蛋白建立了强结合相互作用。此外，与NTCP对接时，具有扁平五环结构的BA-AZT1插入界面，并与NTCP的157-165位氨基酸残基形成氢键、疏水作用和范德华力。进一步的表面等离子体共振（SPR）分析表明，BA-AZT1与聚合酶C区的结合亲和力为19.55μM，强于BA的53.21μM和AZT的31.82μM。BA-AZT1选择性地结合宿主细胞中NTCP受体的157-165表位，而非病毒的前S1区。因此，我们推断所设计的缀合物靶向NTCP和HBV聚合酶，不仅通过选择性结合NTCP阻止HBV进入宿主细胞，还通过阻碍HBV聚合酶的功能抑制HBV DNA复制，它有可能作为一种有前景的双功能双靶点抑制剂，兼具复制和进入抑制作用以发挥抗HBV活性。
