Flow cytometric evaluation of the cytotoxicity of novel antiviral compounds

Two acyclic analogs of bromotubericidin were tested for cytotoxic effects on uninfected cells by monitoring cell growth and measuring cell cycle perturbations using flow cytometry. As reported elsewhere, 5-bromotubercidin analogs in which ribose was replaced by 2-hydroxyethoxymethyl (compound 102) or by 1,3-dihydroxypropoxymethyl (compound 183) were potent inhibitors of human cytomegalovirus (HCMV) replication in vitro (Pudlo et al.: Journal of Medicinal Chemistry 31:2086–2092, 1988). Because these compounds also inhibited the growth of uninfected cells, we performed kinetic studies with an established neoplastic line of human cells (KB) using flow cytometry. Growth of KB cells treated with either compound 102 or 183 were inhibited in a dose-dependent manner. Growth inhibition by compound 183, however, was not fully expressed for at least 24 h. DNA analysis by flow cytometry showed that a 4-h incubation with 10 μM compound 102 caused a decrease of cells in G2/M phase. Cells began to accumulate in early S phase by 12 h of incubation, leading to mid S phase accumulation at 21 h. Compound 183 at 10 μM slightly decreased the number of cells in G2/M phase after a 4-h incubation, and led to accumulation of DNA in S phase after a 12-h incubation. By 24 and 30 h, DNA histograms appeared similar to those of control cells but with a slight accumulation of the population in early S phase. In separate experiments, drugs were removed following a 24-h incubation. After removal of compound 102, KB cell growth resumed with a normal population doubling time. In contrast, the effects of compound 183 were not reversible, suggesting the two compounds acted by different biochemical mechanisms.