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.

This work demonstrates for the first time the concept that small synthetic sulfated agents could effectively inhibit HCMV entry into host cells. Although previous work has demonstrated the concept that certain sulfated, natural, or unnatural polysaccharides (e.g., dextran sulfate, pentosan polysulfate, heparin, copolymers of acrylic acid) can inhibit HCMV infectivity in CHO-K1 and MRC-5 cells [71] , the foundation for this activity was based on mimicking the polymeric scaffold of heparan sulfate, which has now been shown to be critical for HCMV entry [72] [73] [74] [75] . In fact, the plausible molecular basis for this competitive inhibition was the interaction of sulfated polymers to viral glycoprotein gB of HCMV [25] [26] [27] . More specifically, the competitive inhibition was predicted to arise from mimicking the structure of certain heparan sulfates, e.g., 3-O sulfated and 6-O sulfated species [74] . In stark contrast, SPGG is a much smaller sulfated entity that performs the mimicking function with excellent potency. This is important because a smaller molecular scaffold is easier to transform into clinical drug candidates. Whereas polymers are difficult to synthesize, characterize, purify, analyze, monitor, and administer, smaller molecules are the exact opposite. Thus, SPGG provides the first small molecule lead toward discovery of anti-HCMV drugs. mechanism behind SPGG's inhibition of HCMV entry. Spots containing 5 × 10 5 PFU of HCMV AD169 strain and dilutions of SPGG were applied to nitrocellulose membrane and an immunoassay was carried out. The signal was then quantified for size and pixel density using ImageJ gel analysis function and the profile plot was generated with each curve representing a respective spot on the original blot (panel A). The area under each curve in (A) was then calculated and plotted in (panel B). Panel (C): Cell-ELISA was used to assess the affinity of SPGG for HCMV gB over-expressed in CHO-K1 cells. Each experimental condition was tested in at least triplicate with an N = 4. The errors in (B) and (C) correspond to + SD. ANOVA determined significant differences among means with a P-value of 0.0273 (*) in (B). Further analysis of (B) using Dunnett's multiple comparison test found a significant difference in the 100 µM treatment when compared to the mean of the positive control with a P-value of 0.0117 (*). T-test analysis was used to determine significance in (C). (****) signifies a P-value of < 0.0001.