TOP3B as host factor and drug target

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Aim 1: Screening for TOP3B poisons based on our cellular assays: We are testing chemical lead candidate compounds (derived from inhibitors of the bacterial ortholog (TOP1A) in our TOP3B cellular and biochemical assays. These compounds were discovered by our collaborator Dr. Yuk-Ching Tse-Dinh (FIU). We are is in the process of having them tested in parallel by Dr. Mariano Garcia-Blanco as antivirals. We have engineered cloned cell lines with reporters (green-GFP and red-mCherry) allowing concurrent cultures of TOP3B wild-type (TOP3B+/+) and knockout (TOP3B-KO = TOP3B-/-) cells. Our engineered cell lines are used for high throughput screening. The 2,500 compound NCATS Library has been screened in our lab. A transfer of our TOP3B-engineered cells to the CCR Molecular Target Laboratory has been set up with Barry O'Keefe. We are moving forward and testing the NCI libraries including natural products. Identified drugs are further tested in our biochemical assays with recombinant TOP3B as we have the assays running to study the biochemical and structural determinants of nucleic acid cleavage by TOP3B. Anti-SARS-CoV-2 activity will be tested by our collaborators in Mariano Garcia-Blanco's group. Identified drug leads will be optimized with medicinal chemist colleagues unless they are already FDA-approved. Co-crystal studies can be performed as TOP3B can be crystallized. Our collaborator Yuk-Ching Tse-Dinh has the expertise, and we have a batch of enzyme for our collaboration with David Waugh (NCI-Frederick). Aim 2: Mapping the TOP3B sites in SARS-CoV-2: We have already generated the constructs to map TOP3B cleavage complexes (TOP3Bcc) in the genomic DNA and the cytoplasmic RNA of human cells. Our tagged constructs for wild-type TOP3B and the self-poisoning TOP3B (R338W) have been shipped to our collaborators growing the virus (Dr. Garcia-Bianco, UTMB). We are working with them to optimize the assay to map TOP3B sites in the RNA of SARS-CoV-2. The TOP3-DPC (DNA-protein crosslinks) can be directly pulled down and the viral RNA sequence covalently bound to TOP3B will be sequenced at the NIH and mapped. We will perform biochemical experiments with our recombinant TOP3B to map the TOP3B cleavage sites in purified SARS-CoV-2 RNA fragments. TOP3B tagged-constructs, which have also generated, can potentially be used to visualize whether TOP3B colocalizes with SARS-CoV-2 viral particles. Our constructs have been shipped and the experiments ongoing in collaboration with the laboratories of our collaborators growing the virus. Aim 3: In vivo studies in TOP3B knockout mice: The Baric group at UNC recently published a murine model to study SARS-CoV-2 infection. We have established our own colony of TOP3B knockout mice to be shipped to the Baric group at UNC. We work together with Tim Sheahan and Ralph Baric to determine whether and to what extent TOP3B contributes to the replication cycle of SARS-CoV-2. If these experiments confirm the resistance seen in cell lines, additional viruses can be tested in murine models. Also, the impact of TOP3B on antiviral drugs could be determined, as well as the potential role of TOP3B as a regulator of the innate immune responses.