Master of Science
Microbiology & Immunology
DISCOVERY AND CHARACTERIZATION OF CYTOMEGALOVIRUS INHIBITORS
USING REPORTER-BASED ANTIVIRAL ASSAYS
By Amine Ourahmane, MS
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University.
Virginia Commonwealth University, August 2017
Major Director: Michael McVoy, Ph.D.
Professor, Department of Pediatrics and Microbiology and Immunology
Human cytomegalovirus (HCMV), a member of the herpesvirus family, causes significant disease in immunocompromised patients and is the major infectious cause of birth defects when acquired congenitally. Current HCMV antivirals are suboptimal due to modest potency, significant toxicities, and emergence of resistance. Because HCMV does not infect non-human species, related animal cytomegaloviruses are used as animal models. Of the small animal cytomegaloviruses only guinea pig cytomegalovirus (GPCMV) has been found to cross the placenta to cause fetal infection and disease. Thus, the GPCMV/guinea pig model of congenital infection can be used to study the effectiveness of vaccines or small molecule inhibitors in preventing or treating congenital infections. However, not all antivirals that inhibit HCMV are active against GPCMV.
In Aim 1 of the current studies a luciferase-based assay was developed and used to determine the sensitivity of GPCMV to three novel inhibitory compounds, BDCRB, BAY 38-4766, and letermovir, which block DNA maturation of HCMV by targeting the viral terminase complex. BDCRB and BAY 38-4766 were active against GPCMV.Unfortunately, letermovir, which recently completed phase 3 clinical testing, was not active against GPCMV at concentrations up to 100 mM.
In Aim 2 the mechanism of action of BDCRB against GPCMV was explored by characterizing an L406P mutation in the GP89 terminase subunit that had been previously identified in a BDCRB-resistant GPCMV. In silico homology modeling was used to identify the location of the L406P mutation in a predicted 3-D structure of GP89. That it was not located near a putative BDCRB-binding pocket (which was predicted based on confirmed resistance mutations in the homologous HCMV UL89 subunit) suggested that L406P may not confer BDCRB resistance in GPCMV. That L406P does not confer BDCRB resistance was confirmed by genetic transfer of the L406 mutation into an otherwise wild type GPCMV background and demonstration, using the luciferase-based assay, that the IC50 of BDCRB was not significantly altered (i.e., the virus containing the L406 mutation was not resistant to BDCRB).
In Aim 3 a green fluorescent protein-based assay was used to evaluate four candidate compounds for antiviral activity against HCMV. These highly positively charged compounds, TriplatinNC, DiplatinNC, [Pt(dien)(Xan)]2+ and Werner’s Complex, were hypothesized to interfere with viral binding to cell surface glycosaminoglycans and thereby interfere with viral attachment and subsequent entry.
© Amine Ourahmane
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Date of Submission