*6.6. Cardiac Glycosides*

Discovery of novel small molecules that inhibit major IE gene expression can be accomplished using cell-based assays designed to target the early steps of HCMV replication including IE expression but also virus attachment, entry and capsid transport steps [361,362]. One such assay utilizes an engineered variant of the HCMV laboratory strain AD169 that expresses IE2 with a C-terminal yellow fluorescent protein tag (AD169IE2-YFP) [362]. IE2-YFP levels in the nucleus of infected cells are quantified using high-content confocal microscopy and hit inhibitory compounds identified via a decrease in nuclear fluorescent signal and therefore a decrease in IE2-YFP protein levels. This IE2-YFP cell-based reporter assay was used to screen a 2080 bioactive compound library and identified one lead compound, the cardiac glycoside convallatoxin. This compound exhibited potent anti-HCMV activity (EC50 values in the low nanomolar range) without significant cellular cytotoxicity [362,363]. However, it should be noted that convallatoxin has been discounted as a hit from a different screen due to toxicity [364]. Interestingly, other cardiac glycosides (ouabain, β-antiarin, digoxin, digitoxin) have also been reported to exhibit anti-HCMV activity [363,365–367]. Inhibition of HCMV by cardiac glycosides is effective against clinical and GCV-resistant strains and exhibits additive activity when administered to cells in combination with GCV [362,363,365,366]. Members of this compound family have been used clinically for the treatment of heart conditions such as congestive heart failure, although toxicity and dosage issues mean that they are increasingly replaced with synthetic drugs such as ACE inhibitors and beta-blockers [368]. Clinical development as antiviral drugs has not yet been undertaken, but medicinal chemistry approaches have demonstrated the ability to improve antiviral activity and selectivity [369].

A common feature of cardiac glycoside treatment is reduction in IE1/2 protein levels [362,363,365]. Mechanism of action studies demonstrated that convallatoxin does not inhibit IE2 mRNA levels but instead inhibits global translation of viral and host cell proteins [363]. Cellular translation machinery is not directly inhibited by convallatoxin; instead the compound reduces methionine transport into the cell, limiting the intracellular pool of this essential amino acid for translation. Convallatoxin has been proposed to mediate this indirect mechanism of translation inhibition by its ability to bind to and inhibit the cellular sodium-potassium ATP pump (NA+,K+-ATPase) [363]. In this model, inhibition of the pump causes a reduction in the sodium gradient across the cell membrane, leading to a decrease in sodium-dependent methionine transport [363]. Despite inhibition of global translation, minimal cellular cytotoxicity was observed at the nanomolar concentrations of convallatoxin required for antiviral effect. This observation suggests that, whilst the cell can tolerate a reduction in protein synthesis, HCMV is unable to compensate for reductions in viral protein levels, particularly in IE proteins which are required for early and late protein production and are thus essential for virus replication [363]. Convallatoxin-induced inhibition of viral protein translation by methionine transport reduction is not the only mechanism attributed to the antiviral activity of cardiac glycosides. Alternate mechanisms of action are based on the ability of these compounds to modulate cell signalling pathways [370]. For example, cardiac glycoside digitoxin has been reported to inhibit HCMV through induction of cellular autophagy following activation of the regulatory kinase AMPK via a novel NA+,K+-ATPase subunit α1-AMPK-ULK1 pathway [173]. In addition to inhibiting HCMV, cardiac glycosides act as antivirals against a range of clinically important DNA and RNA viruses. This broad-spectrum activity has been attributed to a range of host-directed mechanisms [371].

#### *6.7. Novel Miscellaneous Compounds*

A cell-based screen designed to monitor IE2 nuclear translocation was used to identify the cardiac glycoside convallatoxin discussed in Section 6.6. A second screening approach targeting IE2 gene expression utilized a reporter cell-line in which the IE2-activated HCMV TRL4 promoter drives luciferase expression [361]. The reporter cell-line assay was used to screen a 9600 compound library for inhibitors of early phase HCMV infection [361]. Two hit compounds arising from the screen, 1-(3,5-dichloro-4-pyridyl)piperidine-4-carboxamide and 2,4-diamino-6-(4-methoxyphenyl)pyrimidine termed DPCC and 35C10, respectively, have been demonstrated to potently inhibit HCMV replication as effectively as GCV [361,372]. DPCC was also independently identified as having potent anti-HCMV activity in an unrelated high throughput screen designed to target IE1 IFN antagonist function (assay concept described in Section 7.2) [373]. In this screen, DPPC was alternatively termed StA-IE1-3, and a further novel hit compound with similar anti-HCMV activity termed StA-IE1-2 (1-(3-nitrophenyl)-2-(pyrido[3,2-d][1,3]thiazol-2-ylthio)ethan-1-one) was also identified [373]. All three structurally diverse compounds act after viral entry but before IE expression, with significantly decreased IE1 and IE2 expression at both the mRNA and protein levels [361,372,373]. Like the majority of compounds that have been shown to inhibit HCMV IE gene expression, the precise mechanism of action of these three compounds has not been elucidated. However, it has been postulated that they may target a cellular transcription factor or upstream signalling protein required for activation of the HCMV MIEP [373].

#### *6.8. Summary*

Overall, a variety of approaches utilizing existing knowledge to repurpose known compounds or screening campaigns to discover novel compounds has successfully identified a wide variety of IE gene expression inhibitors. These inhibitors exploit host cell factors and signalling pathways utilized by HCMV to activate the MIEP and thus offer the hypothetical advantage of a reduced risk of drug resistance. A major challenge associated with the development of these compounds is the complexity in elucidating their relevant host cell targets and mechanism of action. Characterization of these compounds has been predominantly conducted in vitro. However, a few compounds have been progressed to in vivo testing using the MCMV model, and clinical testing of artesunate produced mixed clinical outcomes that warrant further investigation. Overall, compounds that inhibit HCMV IE gene expression merit future investigation and development as potential antivirals.

#### **7. Inhibition of Major IE Protein Functions**
