This R43 proposal answers the call of the RFA PA-22-176 for assay development and chemical probe screening by addressing the unmet need for development of antiviral treatments for Ebola patients through an innovative high-content small molecule screen for antagonists of the Ebola VP40 matrix protein. There is a need for a fast-acting therapy that is independent of the immune system and targets essential viral proteins. Such a novel therapeutic is anticipated to enhance the survival probability for infected people in hot zones of an Ebola outbreak. We chose to target EBOV VP40 because it is absolutely required for EBOV particle assembly at the cell membrane, is capable of budding virus- like particles (VLPs) when expressed in isolation and VP40 protein-protein interaction domains have been structurally determined. Further guiding this application is our published pilot screen which established proof-of-concept by demonstrating the accessibility of Ebola VP40 protein-protein interactions to sangivamycin, a dual acting small molecule antagonist of both EBOV VP40 assembly of virions and the viral replication machinery. Given this success and due to anticipated complications with efficacy and MOA studies inherent to compounds with dual targets, our Specific Aims propose to screen a library of ~123,000 small molecule compounds to identify antagonists of VP40 accumulation at the cell membrane for VLP formation and release from cells through a fully automated, quantitative, and high-content assay. The assay has been vetted to quantify the effect of small molecules on the cellular distribution of a fluorescent VP40 expressed in 293T cells. Hits validated as dose-dependent by qHTS and displaying low cytotoxicity will be further prioritized based on their absolute requirement for VP40 in antiviral mechanism of action through counter screening with the VP40- independent minigenome assay. Lead compounds also will be prioritized by their favorable ADMET profiles. Our proposed critical path anticipates identifying 2-4 dose-dependent, VP40- selective antagonists with potent antiviral activity that display low cytotoxicity for future medicinal chemistry and preclinical development.
Public Health Relevance Statement: PROJECT NARRATIVE This proposal addresses the RFA (PA-22-176) call for assay development and screening of chemical probes or therapeutic agents. The urgent need for novel therapeutic intervention strategies is underscored by what has become regular outbreaks of Ebola virus (EBOV), each threatening a potential pandemic of global proportions. Given the rapid progression of the virus and the vulnerabilities in the immune response, a fast- acting, immune system-independent medical countermeasure is proposed that targets EBOV matrix protein (i.e. VP40) as an essential viral protein to produce infectious viruses. This novel approach for a potent, fast-acting therapeutic that is a selective antagonist of Ebola VP40 and viral particle assembly will enhance survival for infected people in hot zones of an Ebola outbreak. We have published our validated drug discovery assay for compounds that will inhibit the function of EBOV VP40 in viral particle assembly. With this proof-of-concept we seek to identify VP40-selective inhibitors with greater antiviral potency that act through a unique mechanism of action on a single target for preclinical development that will address the current sparse pipeline of Ebola therapeutics.
Project Terms: Promega; Pro-Mega; sangivamycin; Area; Filoviridae; Filovirus; Link; Chemicals; Evaluation; Individual; Viral Activity; Viral Function; Viral Physiology; viral assembly; Virus Assembly; Critical Paths; Critical Pathways; Immunological response; host response; immune system response; immunoresponse; Immune response; Cell-Mediated Lympholysis; Cellular Cytotoxicity; Lymphocyte Cytotoxicity; Lymphocytotoxicity; cell mediated cytotoxicity; Cell-Mediated Cytolysis; Therapeutic; Therapeutic Agents; Reporter; Life; Complex; cell type; Location; Country; Viral; Services; cytotoxicity; Lytotoxicity; Membrane; membrane structure; mutant; particle; success; membrane assembly; Structure; novel; social; Property; protein protein interaction; assay development; Intervention; Intervention Strategies; interventional strategy; drug discovery; Zaire Ebola virus; ZEBOV; Zaire ebolavirus; Homo; virus-like nanoparticles; viruslike particle; Virus-like particle; small molecule; Address; Dose; Symptoms; cytotoxic; Diversity Library; Dryness; Immune Targeting; Protein Binding Domain; Protein Binding Motif; Protein-Protein Interaction Domain; Transcript; Characteristics; Gene variant; allele variant; allelic variant; genomic variant; genetic variant; BSL-4 facility; BSL4 facility; biosafety level 4 facility; new approaches; novel approaches; novel strategy; novel strategies; Population; migration; innovate; innovative; innovation; new drug treatments; new drugs; new pharmacological therapeutic; new therapeutics; new therapy; next generation therapeutics; novel drug treatments; novel drugs; novel pharmaco-therapeutic; novel pharmacological therapeutic; novel therapy; novel therapeutics; protein crosslink; new therapeutic approach; new therapeutic intervention; new therapeutic strategies; new therapy approaches; new treatment approach; new treatment strategy; novel therapeutic approach; novel therapeutic strategies; novel therapy approach; novel therapeutic intervention; effective treatment; effective therapy; drug candidate; screenings; screening; targeted drug therapy; targeted drug treatments; targeted therapeutic; targeted therapeutic agents; targeted therapy; targeted treatment; imaging system; small molecule inhibitor; extracellular vesicles; pre-clinical development; preclinical development; Immunize; medical countermeasure; Ebola; high-throughput drug screening; antagonist; antagonism; antiviral drug development; anti-viral development; anti-viral drug development; anti-viral therapeutic development; anti-viral therapy development; antiviral development; antiviral therapeutic development; antiviral therapy development; developing anti-viral agent; developing anti-viral drug; developing anti-viral therapeutic; developing anti-viral therapy; developing antiviral agent; developing antiviral drug; developing antiviral therapeutic; developing antiviral therapy; pandemic potential; pandemic concern; pandemic risk; pandemic threat; new outbreak; emergent outbreak; emerging outbreak; novel outbreak; previous outbreak; past outbreak; prior outbreak; inhibitor; Antiviral Agents; Antiviral Drugs; Antivirals; anti-viral agents; anti-viral compound; anti-viral drugs; anti-viral medication; anti-viral therapeutic; anti-virals; antiviral compound; antiviral medication; antiviral therapeutic; Back; Dorsum; Biogenesis; Origin of Life; Biological Assay; Assay; Bioassay; Biologic Assays; Cause of Death; Cell Cycle; Cell Division Cycle; Cell membrane; Cytoplasmic Membrane; Plasma Membrane; plasmalemma; Cells; Cell Body; Pharmaceutical Chemistry; Medicinal Chemistry; Pharmaceutic Chemistry; Communities; Cytoplasm; Disease Outbreaks; Outbreaks; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Ebola virus; EBOV; Ebola-like Viruses; ebolavirus; Future; Recording of previous events; History; histories; Immune system; In Vitro; indexing; Infection; Lead; Pb element; heavy metal Pb; heavy metal lead; Libraries; Persons; Pathology; Patients; Plasma Cells; Blood Plasma Cell; Plasmacytes; plasmocyte; Powder dose form; Powders; Probability; Production; Proteins; Publishing; Research; Risk; Testing; Texas; Genetic Transcription; Gene Transcription; RNA Expression; Transcription; Transfection; Travel; Triage; Vaccination; Viral Genome; virus genome; Viral Proteins; Viral Gene Products; Viral Gene Proteins; virus protein; Virion; Virus Particle; Virus Replication; viral multiplication; viral replication; virus multiplication; Virus; World Health Organization; Democratic Republic of the Congo; Belgian Congo; Zaire
