*7.1. IE2 Inhibitors*

Compounds that inhibit IE2 function have been identified and offer promise as a potential new class of HCMV inhibitors (Figure 2). IE2 has been targeted, as it is an essential multifunctional protein that regulates critical events in HCMV replication including transactivation of early and late genes and auto-regulation of the MIEP. IE2 has also been linked to broad dysregulation of host gene expression affecting cell cycle progression, immunomodulation and pathogenesis (see Section 3.1). The first compound demonstrated to directly inhibit IE2 function was WC5, a 6-aminoquinolone derivative [374]. WC5 was tested based on evidence that compounds within this chemical group exhibit antiviral activity against HIV-1 by inhibiting Tat transactivation [375,376]. WC5 specifically inhibits HCMV but not a selection of other herpesviruses [376,377]. In addition to IE, early and late gene expression profiles, which suggest inhibition of IE2 function, WC5 has been shown to directly inhibit IE2- s transactivating activity via a cell-based assay in which an EGFP reporter gene was placed under the control of IE2-dependent early gene promoters [374]. In these assays, WC5 significantly inhibited IE2-mediated transcriptional activation of early gene promoters UL54 and UL112/113. A minimal region of the UL54 promoter composed of a 150-bp segment upstream of the transcriptional start site has been demonstrated to be sufficient to mediate the inhibitory activity of WC5 [378]. Within this 150-bp segment is the IR-1 signal (8-bp inverted repeat element 1), a *cis*-acting sequence with an established role in IE2-dependent transactivation, yet the IR-1 signal has been shown not to be required for WC5- s inhibitory activity [378]. In addition, two key protein interactions, IE2 dimerization and its interaction with TBP, known to be involved in IE2-dependent transactivation of viral promoters have also been discounted as WC5- s target [378]. Intriguingly, WC5- s activity appears to be specifically confined to the regulation of HCMV promoters, as the compound exhibits no effect on a variety of cellular promoters regulated either by IE2 protein interactions or a direct IE2 interaction with promoter DNA [378]. In addition to inhibiting IE2 transactivation of viral promoters, a second mechanism of action by which WC5 inhibits a different IE2 function has been identified [378]. WC5 specifically disrupts IE2- s direct interaction with the crs within the MIEP (Figure 1). Disruption of the IE2-crs interaction abolishes IE2- s auto-repression of its own promoter, a function essential for viral replication. Although WC5 has been demonstrated to inhibit two IE2 functions, transactivation of viral early and late genes and MIEP auto-regulation, the exact molecular mechanisms require further elucidation.

WC5- s unique activity offers the possibility to develop a new mechanistic class of anti-HCMV compounds. Towards this goal, WC5 potently and selectively inhibits HCMV replication in the sub-micromolar range irrespective of testing against laboratory or clinical isolates, and its activity is comparable to GCV [376]. Unsurprisingly, given WC5- s novel mechanisms of action, the compound similarly inhibits isolates resistant to clinically approved anti-herpesvirus DNA polymerase inhibitors [376]. Further, when WC5 is combined with GCV, synergistic activity against HCMV replication was observed without significant increases in cellular cytotoxicity [374]. WC5 also inhibits

MCMV replication, albeit with ~10-fold lower activity compared to HCMV [376]. Importantly, WC5- s mechanism of action against HCMV and MCMV appears to be conserved, as it has been shown to block MCMV early gene transactivation mediated by the MCMV IE2 homolog ie3 [378]. Thus, it has been suggested that the murine model may be used to test WC5 activity in vivo as a prerequisite to clinical development [378]. Structure–activity relationship studies have been conducted with the aim of improving WC5- s potency [374,379]. These studies gained insight into chemical groups required for WC5 activity, and identified an analogue with an improved selectivity index compared to WC5 without compromising antiviral activity. However, analogues with significantly improved potency were not identified [379].

WC5- s discovery together with its novel mechanism of action provided proof-of-principle that IE2 is a valid target for drug discovery and encouraged a screening campaign to identify new compounds targeting IE2 [364,380]. A screen has been performed employing essentially the same cell-based assay used to determine WC5- s mechanism of action as an inhibitor of IE2-mediated transactivation of early gene expression [374]. Assay optimization identified conditions using the stable cell-line that expresses EGFP under the control of the IE2-dependent UL54 early promoter as suitable for screening purposes [364]. A 2320 bioactive compound library including all FDA-approved drugs was screened and six hit compounds have so far been selected for further study [364,381,382]. These hit compounds are deguelin (DGN), nitazoxanide (NTZ), thioguanosine (TGN), alexidine dihydrochloride (AXN), manidipine dihydrochloride (MND) and berberine (BBR). All hits inhibited HCMV replication with EC50 values in the low micromolar range and lacked significant toxicity. This antiviral activity was observed for laboratory, clinical and drug-resistant HCMV isolates. Further, MND was shown to be inactive against a selection of other DNA and RNA viruses and is thus likely to be a specific anti-HCMV compound [381]. The antiviral mechanism of these compounds was confirmed to be inhibition of IE2-mediated viral early gene transactivation and, like with WC5, a minimal 150-bp segment of the UL54 promoter is sufficient for inhibitory activity. However, the precise mechanism of action has not been elucidated, although prior knowledge of these bioactive compounds has led to the proposal that they are likely to interfere with pathways in HCMV-infected cells that are required for the switch from the IE to early phase of viral replication [364]. Despite the lack of a precise mechanism of action, repurposing of bioactive compounds for anti-HCMV activities may allow compound development to be fast-tracked, especially in the case of MND, as it is already an FDA-approved drug used in the treatment of hypertension [381].
