Characterization of the antiviral activity of four novel inhibitors of Human Cytomegalovirus Immediate-Early 2 protein

The Human Cytomegalovirus (HCMV) is a widespread pathogen responsible for generally asymptomatic infections in healthy people. However, it can cause life-threatening diseases in immunocompromised individuals, such as transplant recipients and AIDS patients. HCMV is also the major viral cause of congenital infections, developmental abnormalities and fetal death. To date, only a limited number of inhibitors of the viral DNA polymerase have been approved for treatment of HCMV infections in immunocompromised patients. However, despite their clinical benefits, these drugs suffer from several drawbacks including long-term toxicity, unfavorable pharmacokinetic properties and emergence of viral resistance. Moreover, no antiviral drugs have been approved for treatment of HCMV congenital infections and, although attempts to develop a vaccine have been pursued, no one is currently available. For all these reasons, there is a urgent need to develop new, safe antiviral compounds, possibly endowed with a new mechanism of action to avoid cross-resistance and to decrease the risk of selection for resistant viral strains. Among HCMV proteins, the Immediate-Early 2 (IE2) protein can be proposed as a new ideal virus-specific target for development of anti-HCMV agents, since the activities of this viral transcription factor are essential for the progression of the virus replicative cycle and play a major role in HCMV pathogenesis. To identify small molecule inhibitors of IE2 activities, we have recently screened a chemical library by means of a specific IE2-activatable EGFP cell-based assay. The screening was performed in the context of a Drug Repurposing approach with a library of FDA-approved bioactive molecules as well as drugs under development. Drug Repurposing represents in fact a cost-effective way to search for side activities among molecules for which bioavailability and toxicity studies have already been performed, and which have been proven usefulness for the treatment of different diseases in humans. Thus, by exploiting existing knowledge, a Drug Repurposing strategy can offer a faster and cheaper approach to develop antiviral molecules than traditional drug discovery strategies. In this thesis work, the antiviral activity of four of the hits selected from the library was further characterized. The four molecules were: Nitazoxanide, Deguelin, Alexidine dihydrochloride, and Thioguanosine. All these four hits confirmed their antiviral activity in dose-dependent experiments against a laboratory strain of HCMV. Analysis of their effect on the progression of the viral replicative cycle demonstrated that hits were unable to affect Immediate-Early (IE) protein expression, whereas they reduced significantly the content of Early (E) proteins of the virus, and prevented both viral DNA synthesis and Late (L) protein expression. These findings suggested that the IE2-dependent expression of E genes might be the target of the four compounds. This hypothesis was then proved through transfection/transduction experiments that demonstrated the ability of the selected hits to inhibit the transcriptional activation of two different HCMV E gene promoters and in two different experimental cell-based reporter systems. Altogether, these results indicate that the ability of the four selected hits to inhibit the IE2-mediated transactivation of E gene promoters contributes to the overall anti-HCMV activity.