Inhibition of Human Cytomegalovirus Entry into Host Cells through A Pleiotropic Small Molecule

Elste, James; Kaltenbach, Dominik; Patel, Vraj R.; Nguyen, Max T.; Sharthiya, Harsh; Tandon, Ritesh; Mehta, Satish K.; Volin, Michael V.; Fornaro, Michele; Tiwari, Vaibhav; Desai, Umesh R.

To first assess whether SPGG is toxic to cells typically targeted by HCMV, we studied dose-dependence of cell viability. We utilized lactate dehydrogenase (LDH) assay as a surrogate for cell viability, as described in the literature [62] . Briefly, rupture of plasma membranes due to apoptosis, necrosis, and other forms of cellular damage results in release of LDH into the cell culture supernatant, which can be quantified by an NADH coupled chromogenic reaction. We selected two different cell lines, Human Foreskin Fibroblasts (HFF-1) and neuroepithelioma cells (SK-N-MC), as it is well-established that HCMV infects and replicates in fibroblasts and neuronal cell types [18, 19, 63] . Figure 2 shows viability of both cell lines as a function of SPGG concentrations reaching up to 100 µM. Even after a 24-h incubation period, both cell lines exhibited essentially no morphological changes from the mock-treatment control (not shown). Likewise, neither cell line displayed any measurable increase in LDH activity even at the highest dose of SPGG. These results suggested that SPGG displayed no measurable cytotoxicity up to 100 µM in the tested cell lines. In fact, the LC 50 (lethal concentration for 50% cell death) is likely to be several fold higher because of complete absence of increase in LDH activity at 100 µM. necrosis, and other forms of cellular damage results in release of LDH into the cell culture supernatant, which can be quantified by an NADH coupled chromogenic reaction. We selected two different cell lines, Human Foreskin Fibroblasts (HFF-1) and neuroepithelioma cells (SK-N-MC), as it is well-established that HCMV infects and replicates in fibroblasts and neuronal cell types [18, 19, 63] . Figure 2 shows viability of both cell lines as a function of SPGG concentrations reaching up to 100 µM. Even after a 24-h incubation period, both cell lines exhibited essentially no morphological changes from the mock-treatment control (not shown). Likewise, neither cell line displayed any measurable increase in LDH activity even at the highest dose of SPGG. These results suggested that SPGG displayed no measurable cytotoxicity up to 100 µM in the tested cell lines. In fact, the LC50 (lethal concentration for 50% cell death) is likely to be several fold higher because of complete absence of increase in LDH activity at 100 µM.