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 IC50 values 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.
Highlights
From the ‡Department of Microbiology & Immunology, §Department of Pharmacology, ¶Lineberger Comprehensive Cancer Center, ʈDepartment of Biostatistics, **UNC Michael Hooker Proteomics Core Facility University of North Carolina, Chapel Hill, 27599 North Carolina
Our multiplexed kinase inhibitor bead-mass spectrometry (MIB-mass spectrometry (MS)) columns were composed of a four inhibitor (PP58, Purvalanol B, VI16832, and UNC21474) bead mix that was optimized for kinome capture as previously reported [27] (East, unpublished observations)
Our ultimate goal was to use this methodology to identify kinase inhibitors that could potentially be repurposed as novel Human cytomegalovirus (HCMV) antivirals
Summary
From the ‡Department of Microbiology & Immunology, §Department of Pharmacology, ¶Lineberger Comprehensive Cancer Center, ʈDepartment of Biostatistics, **UNC Michael Hooker Proteomics Core Facility University of North Carolina, Chapel Hill, 27599 North Carolina. The most potent inhibitor of HCMV replication was OTSSP167 (IC50 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. Repurposing of existing drugs for use as antivirals provides an alternative to the traditional drug development process and leverages the fact that viruses manipulate many of the same cellular pathways dysregulated in other diseases states. There are currently thousands of FDA-approved drugs whose effect on virus replication has not been examined As these drugs have already been tested for safety and bioavailability in humans, they could be rapidly repurposed for clinical use [2]. New antiviral drugs are greatly needed to curtail HCMV disease
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