Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: new insights into developing anti-viral agents.

We previously reported that bakuchiol, a phenolic isoprenoid anti-cancer 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 (MDCK) cell lysates and purified the specific bakuchiol-binding proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using nano-liquid chromatography with tandem mass spectrometry 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 co-localized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein (NP) expression levels and viral titers in the conditioned medium of virus-infected MDCK 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.

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Conflict of interest The authors declare that they have no conflicts of interest regarding the content of this article.