title: Kinome Profiling Identifies Druggable Targets for Novel Human Cytomegalovirus (HCMV) Antivirals.
abstract: Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. There is thus an urgent need for new strategies to treat HCMV infection. Repurposing existing drugs as antivirals is an attractive approach to limit the time and cost of new antiviral drug development. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity, which led us to hypothesize that defining the complement of kinases (the kinome), whose abundance or expression is altered during infection would identify existing kinase inhibitors that could be repurposed as new antivirals. To this end, we applied a kinase capture technique, multiplexed kinase inhibitor bead-mass spectrometry (MIB-MS) kinome, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with a laboratory-adapted (AD169) or clinical (TB40E) HCMV strain. MIB-MS profiling identified time-dependent increases and decreases in MIB binding of multiple kinases including cell cycle kinases, receptor tyrosine kinases, and mitotic kinases. Based on the kinome data, we tested the antiviral effects of kinase inhibitors and other compounds, several of which are in clinical use or development. Using a novel flow cytometry-based assay and a fluorescent reporter virus we identified three compounds that inhibited HCMV replication with IC50values of <1 μm, and at doses that were not toxic to uninfected cells. The most potent inhibitor of HCMV replication was OTSSP167 (IC50<1.2 nm), a MELK inhibitor, blocked HCMV early gene expression and viral DNA accumulation, resulting in a >3 log decrease in virus replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs.
authors.affiliation: From the ‡Department of Microbiology & Immunology.|¶Lineberger Comprehensive Cancer Center.
authors.forename: Kyle C
authors.initials: KC
authors.lastname: Arend
authors.affiliation: From the ‡Department of Microbiology & Immunology.|¶Lineberger Comprehensive Cancer Center.
authors.forename: Erik M
authors.initials: EM
authors.lastname: Lenarcic
authors.affiliation: From the ‡Department of Microbiology & Immunology.|¶Lineberger Comprehensive Cancer Center.
authors.forename: Heather A
authors.initials: HA
authors.lastname: Vincent
authors.affiliation: ¶Lineberger Comprehensive Cancer Center.|‖Department of Biostatistics.
authors.forename: Naim
authors.initials: N
authors.lastname: Rashid
authors.affiliation: From the ‡Department of Microbiology & Immunology.|¶Lineberger Comprehensive Cancer Center.
authors.forename: Eric
authors.initials: E
authors.lastname: Lazear
authors.affiliation: §Department of Pharmacology.
authors.forename: Ian M
authors.initials: IM
authors.lastname: McDonald
authors.affiliation: §Department of Pharmacology.
authors.forename: Thomas S K
authors.initials: TS
authors.lastname: Gilbert
authors.affiliation: §Department of Pharmacology.
authors.forename: Michael P
authors.initials: MP
authors.lastname: East
authors.affiliation: §Department of Pharmacology.|**UNC Michael Hooker Proteomics Core Facility University of North Carolina, Chapel Hill, 27599 North Carolina.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Herring
authors.affiliation: §Department of Pharmacology.
authors.forename: Gary L
authors.initials: GL
authors.lastname: Johnson
authors.affiliation: §Department of Pharmacology.|**UNC Michael Hooker Proteomics Core Facility University of North Carolina, Chapel Hill, 27599 North Carolina.
authors.forename: Lee M
authors.initials: LM
authors.lastname: Graves
authors.affiliation: From the ‡Department of Microbiology & Immunology, nmoorman@med.unc.edu.|¶Lineberger Comprehensive Cancer Center.
authors.forename: Nathaniel J
authors.initials: NJ
authors.lastname: Moorman
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000187:drug effects* / Q000378:metabolism; D058492:Drug Repositioning; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D047428:Protein Kinase Inhibitors