APOPTOSIS-INDUCING ACTIVITY OF POTENTIAL INHIBITORS OF EPSHTEIN-BARR VIRUS PROTEIN – BHRF1
Zaremba P1,2 Zaremba A1,2 Naumenko K2 Platonov M3 Zagorodnya S.2
1ESC “Institute of Biology and Medicine” Taras Shevchenko National University
2D.K. Zabolotny Institute of Microbiology and Virology of NASU,
Department of virus reproduction
Epstein-Barr virus (EBV), which is a member of the Herpesvirus family, in addition to its wide distribution among the world’s population, has the ability to transform cells and induce cancer. This is facilitated by latency programs and other defense mechanisms to avoid the body’s immune response. One of these mechanisms is the inhibition of apoptosis in an infected cell by the viral protein BHRF1, which is a complete homologue of cellular Bcl-2. And since EBV is able to prevent apoptosis, the search for substances that can induce this process by suppressing viral effects is one step towards developing effective antiviral therapy.
The aim of this study was to determine the cytotoxicity and apoptosis-inducing activity of pre-selected in silico 11 chemically different compounds (Z-1 – Z-11), which are potential inhibitors of viral protein BHRF1. The studies were performed on virus-containing cell culture B95-8 (lymphocytes of cotton-top tamarin) and two reference cultures of different origin that do not contain EBV: Wish (human amnion cells) and MDCK (Madin-Darby canine kidney cells). Cytotoxicity was determined using the MTT assay, which is based on the determination of metabolic activity of cells. Apoptosis-inducing activity was determined by incubating B95-8 cells for 4 and 24 hours with selected compounds. Hoechst 33342 dye was used to visualize the results.
The CC50 values for some compounds differed significantly depending on cell culture. So the CC50 for Z-1 were: B95-8 – 48.88 μg/ml, Wish – 288.66 μg/mL, MDCK – 289.62 μg/mL. Substance Z-2 also showed a similar pattern: B95-8 – 47.49 μg/mL, Wish – 227.49 μg/mL, MDCK – 224.61 μg/mL. The CC50 of compound Z-6 were low for all cells: B95-8 – 48.81 μg/mL, Wish – 68.16 μg/mL, MDCK – 97.03 μg/mL, which may be associated with total toxicity of the compound.
Counting B95-8 in a fluorescent microscope (350 nm) showed that after incubation with compounds Z-1, Z-2, Z-6 the number of apoptotic cells increased. For substance Z-1 at a concentration of 80 μg/mL this increase occurred 2 times after 4 h of incubation and 1.8 times after 24 h compared to control cells. After incubation of B95-8 with Z-2 at the same concentration, the number of apoptotic cells increased in 2 (4 h) and 3 times (24 h). Compound Z-6 at a concentration of 60 μg/mL gave a significant increase in cells at the stage of apoptosis: 13.75 times after 4 h and 5.83 times after the day of incubation.
Thus, it can be concluded that of the 11 potential inhibitors of BHRF1 protein, three Z-1, Z-2, Z-6 – do show specific activity on EBV-containing culture. The substances are all of different origin: Z-1 is the derivative of tetrahydrobenzothiophen, Z-2 is the derivative of naphthalene‐2‐sulfonamide and Z-6 is the derivative of 4‐(benzyloxy)phenol. Among them, the compound Z-6 in addition to the specific effect on Epstein-Barr virus has a non-specific effect on virus-negative cultures. We plan to investigate the nature of this impact in more detail.