*6.4. Kinase Inhibitors*

A general theme in host cell signalling pathway inhibitors is to target kinases, which mediate regulatory post-translational phosphorylation modifications of pathway components. There is an abundance of kinase inhibitors, which have been identified and developed for a wide range of applications particularly cancer treatment [353], which can potentially be repurposed as anti-HCMV compounds. Indeed, examples of kinase inhibitors (AS602868, LY294002) with activity against HCMV have already been discussed in Section 6.3. A further example of a kinase inhibitor repurposed for anti-HCMV testing is the multi-targeted anti-cancer tyrosine kinase inhibitor sorafenib (Nexavar), which has been shown to inhibit MIEP activity, IE expression and also later stages of HCMV replication [172]. The mechanism by which sorafenib inhibits HCMV was not fully elucidated due to its multitude of known kinase targets. However, inhibition of RAF1 activation was implicated but via a mechanism independent of MAPK/ERK signalling [172]. Inhibitors of CDKs also have potential as antiviral drug candidates; for example, the CDK7 inhibitor LDC4297 blocked HCMV replication with EC50 values in the nanomolar range [175]. The compound's mode of action was concluded to be multifaceted but occurs at the level of IE gene expression and interferes with HCMV-mediated inactivation of the retinoblastoma (Rb) protein, which controls progression through the G1 phase of the cell cycle via its phosphorylation state and ability to bind transcription factor complexes [175]. Promisingly, LDC4297 has been shown to possess in vivo antiviral activity in the mouse model. MCMV replication was significantly reduced and restricted to the site of infection, preventing organ dissemination without adverse effects [329].

In addition to directly repurposing known kinase inhibitors, a number of cell-based screens have been performed against various targeted kinase inhibitor compound libraries [354–357]. Compound-treated HCMV-infected cells were monitored for antiviral effects via expression of a green fluorescent protein (GFP) reporter [354] or late viral protein pp28 from the HCMV genome [355–357]. These screens have identified a number of interesting kinase inhibitors with anti-HCMV activity against laboratory and clinical strains that target a variety of cellular kinases without causing significant cytotoxicity. The lack of kinase inhibitor target specificity has made full elucidation of mechanism of action challenging, although in all cases discussed here antiviral activity has been linked to interference with IE expression or protein production without affecting viral entry. Several c-Jun N-terminal kinase (JNK) inhibitors were identified following a 600 compound kinase inhibitor library screen and the SP600125 inhibitor was shown to inhibit JNK activation and suppress IE gene transcription [354]. XMD7 5-aminopyrazine compounds were identified upon screening of the Gray kinase inhibitor library. These compounds target a range of cellular protein kinases to inhibit HCMV replication via a reduction in genome-wide transcription and a defect in the production of certain HCMV proteins including IE2 (86kDa, 60kDa and 40kDa species) [355]. The proposed mechanism of action for XMD7 compounds is consistent with IE2- s role as a viral transcriptional activator, but it is not clear why only a subset of HCMV proteins is affected. CMGC kinase inhibitor RO0504985, an oxindole compound with anti-HCMV activity identified by screening a Roche kinase inhibitor library, also inhibited IE2 and pp28 protein levels [356]. Screening of the GlaxoSmithKline kinase inhibitor set identified SB-734117, a furazan benzimidazole compound, which inhibits several proteins from the AGC and CMCG kinase groups [357]. SB-734117 inhibited IE protein production and reduced phosphorylation of host cell transcription factor CREB and histone H3. However, disappointingly these effects did not lead to any defects in transcription from the MIEP and thus the compound's mechanism of action remains undetermined [357]. Overall, the wealth of preexisting kinase inhibitors and accompanying knowledge offers good potential for the identification and development of novel anti-HCMV compounds.

### *6.5. Histone Modifying Enzyme Inhibitors*

Major IE gene expression is also regulated by "epigenetic" modifications, including histone post-translational methylation, which can result in repressed gene expression [358]. Histone demethylases are required to remove repressive "epigenetic" marks to promote IE gene expression and hence HCMV productive infection or reactivation from latency [358]. Histone demethylase inhibitors (e.g., ML324, a JMJD2 demethylase family inhibitor) have been shown to potently inhibit HCMV IE gene expression [123,359,360]. These demethylase inhibitors also repress IE gene expression from the related Herpes simplex virus type 1 (HSV-1), and importantly they have been shown to potently inhibit HSV-1 infection and reactivation from latency [123,359,360]. These results suggest that compounds that target histone demethylases and possibly other histone modifying or chromatin remodeling enzymes may have potential as HCMV inhibitors [358].
