High-throughput screening of a GlaxoSmithKline protein kinase inhibitor set identifies an inhibitor of human cytomegalovirus replication that prevents CREB and histone H3 post-translational modification

Abstract

To identify new compounds with anti-human cytomegalovirus (HCMV) activity and new anti-HCMV targets, we developed a high-throughput strategy to screen a GlaxoSmithKline Published Kinase Inhibitor Set. This collection contains a range of extensively characterized compounds grouped into chemical families (chemotypes). From our screen, we identified compounds within chemotypes that impede HCMV protein production and identified kinase proteins associated with inhibition of HCMV protein production that are potential novel anti-HCMV targets. We focused our study on a top 'hit' in our screen, SB-734117, which we found inhibits productive replication of several HCMV strains. Kinase selectivity data indicated that SB-734117 exhibited polypharmacology and was an inhibitor of several proteins from the AGC and CMCG kinase groups. Using Western blotting, we found that SB-734711 inhibited accumulation of HCMV immediate-early proteins, phosphorylation of cellular proteins involved in immediate-early protein production (cAMP response element-binding protein and histone H3) and histone H3 lysine 36 trimethylation (H3K36me3). Therefore, we identified SB-734117 as a novel anti-HCMV compound and found that inhibition of AGC and CMCG kinase proteins during productive HCMV replication was associated with inhibition of viral protein production and prevented post-translational modification of cellular factors associated with viral protein production.

Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

“…Histone proteins are key components of chromatin, acting as spools around which DNA winds, and play a significant role in gene regulation. Additionally, their function in viral infections is being increasingly acknowledged [ 51 , 52 , 53 , 54 ]. Currently, there are five major families of histones: H1/H5, H2A, H2B, H3, and H4.…”

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

“…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][355][356][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][356][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.…”

“…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.…”

“…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.…”

Mutli-Features Prediction of Protein Translational Modification Sites

“…ost translation modifications (PTMs) are of pivotal importance for understanding protein functionalities in the field of bioinformatics and machine learning [1][2][3]. PTMs lie in the crucial functional regions of protein; they maintain the stability of protein-protein interactions and other protein functions [4].…”