Rhodamine B-based fluorescent probes for molecular mechanism study of the anti-influenza activity of pentacyclic triterpenes.

The antiviral activity of pentacyclic triterpenes has attracted increasing attention. However, the detailed antiviral mechanism remains fully unclear. In the present study, four C28 or C30 modified pentacyclic triterpene probes via conjugating with rhodamine B were designed and synthesized, and their anti-influenza virus activity was evaluated. The results indicated that two compounds 14 and 23 showed significant antiviral activity to influenza A/WSN/33 (H1N1) virus in Madin-Darby canine kidney (MDCK) cells with IC50 values of 8.36 and 8.24 μM, respectively. The mechanism of action studies of representative probe 23 indicated that it could inhibit the membrane fusion by binding with influenza virus hemagglutinin (HA), and the apparent dissociation constant (KD) value for probe 23-HA interaction was successfully evaluated (1.78 × 10-5 M) using surface plasmon resonance spectroscopy. In addition, the subcellular localization of probe 23 in MDCK cells was determined by confocal microscopy and flow cytometry, and the results suggested that fluorescent probe 23 was rapidly taken up in MDCK cells and accumulated in cytoplasm, but no antiviral activity was observed after its entry into cells. The present study further confirmed our previous finding that pentacyclic triterpenes could tightly bind to the viral envelope HA protein, thus blocking the virus entry into host cells.