我们之前报道过bakuchiol,一种酚类类异戊二烯抗癌化合物,及其类似物发挥抗流感活性。然而,bakuchiol靶向的蛋白质仍不清楚。这里,我们调查了bakuchiol抗流感活性的化学结构,发现bakuchiol的所有官能团和C6手性都是其抗流感活性所必需的.基于这些结果,我们合成了一个分子探针,该探针包含与bakuchiol的C1位置结合的生物素标签。有了这个探测器,我们对Madin-Darby犬肾(MDCK)细胞裂解物进行了下拉测定,并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳纯化了特定的bakuchiol结合蛋白.采用纳米液相色谱-串联质谱分析,我们鉴定了阻抑素(PHB)2,电压依赖性阴离子通道(VDAC)1和VDAC2作为bakuchiol的结合蛋白。我们使用蛋白质印迹分析证实了bakuchiol与PHB1,PHB2和VDAC2的体外结合。免疫荧光分析表明,bakuchiol与细胞中的PHB和VDAC2结合,并共同定位在线粒体中。通过用特异性siRNA转染降低PHB或VDAC2,随着bakuchiol共同治疗,导致病毒感染的MDCK细胞的条件培养基中流感核蛋白(NP)表达水平和病毒滴度显着降低,与转染或单独治疗观察到的水平相比。这些发现表明,减少PHB或VDAC2蛋白,结合bakuchiol治疗,增加抑制流感病毒的生长。我们的发现表明,bakuchiol通过涉及这些线粒体蛋白的新机制发挥抗流感活性,为开发抗流感药物提供了新的见解。
We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.