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Human cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes latent asymptomatic infections in healthy individuals but can cause serious infections in immunocompromised people, resulting in increased risk of morbidity and mortality. The current FDA-approved CMV drugs target late stages of the CMV life-cycle. While these drugs are effective in most cases, they have serious drawbacks, including poor oral bioavailability, dose-limiting toxicity, and a low barrier to resistance. Given the clinical relevance of CMV-associated diseases, novel therapies are needed. Thus, a novel class of compounds that inhibits the early stages of the CMV life-cycle was identified and found to block infection of different strains in physiologically relevant cell types. This class of compounds, N-arylpyrimidinamine (NAPA), demonstrated potent anti-CMV activity against ganciclovir-sensitive and -resistant strains in in vitro replication assays, a selectivity index30, and favorable in vitro ADME properties. Mechanism of action studies demonstrated that NAPA compounds inhibit an early step of virus infection. NAPA compounds are specific inhibitors of cytomegaloviruses and exhibited limited anti-viral activity against other herpesviruses. Collectively, we have identified a novel class of CMV inhibitor that effectively limits viral infection and proliferation.

Human cytomegalovirus (CMV) is a latent and persistent virus whose proliferation increases morbidity and mortality of immune-compromised individuals. The current anti-CMV therapeutics targeting the viral DNA polymerase or the major immediate-early (MIE) gene locus are somewhat effective at limiting CMV-associated disease. However, due to low bioavailability, severe toxicity, and the development of drug resistant CMV strains following prolonged treatment, current anti-CMV therapeutics are insufficient. To help address this shortfall, we established a high-content assay to identify inhibitors targeting CMV entry and the early steps of infection. The infection of primary human fibroblasts with a variant of the CMV laboratory strain AD169 expressing a chimeric IE2-yellow fluorescence protein (YFP) (AD169 IE2-YFP ) provided the basis for the high-content assay. The localization of IE2-YFP to the nucleus shortly following an AD169 IE2-YFP infection induced a robust fluorescent signal that was quantified using confocal microscopy. The assay was optimized to achieve outstanding assay fitness and high Z ’ scores. We then screened a bioactive chemical library consisting of 2080 compounds and identified hit compounds based on the decrease of fluorescence signal from IE2-YFP nuclear expression. The hit compounds likely target various cellular processes involved in the early steps of infection including capsid transport, chromatin remodeling, and viral gene expression. Extensive secondary assays confirmed the ability of a hit compound, convallatoxin, to inhibit infection of both laboratory and clinical CMV strains and limit virus proliferation. Collectively, the data demonstrate that we have established a robust high-content screen to identify compounds that limit the early steps of the CMV life cycle, and that novel inhibitors of early infection events may serve as viable CMV therapeutics.