TOP3B as host factor and drug target

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Aim 1: Screening for TOP3B poisons based on our cellular assays: We have tested chemical lead candidate compounds (derived from inhibitors of the bacterial ortholog (TOP1A) in our TOP3B cellular and biochemical assays. Some of 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. We have used our engineered cell lines 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 also 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 Weidong Wang and Mariano Garcia-Blanco's groups. Identified drug leads, which we have patented and submitted for publication in the Proceedings of the National Academy of Sciences will be optimized with medicinal chemist colleagues. 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: Elucidating the structure of human TOP3B with and without its DNA and RNA substrates, protein cofactors and inhibitors: We have successfully obtained and published the structure of TOP3B with its single-stranded DNA and RNA substrates, discovered new catalytic residues involved in metal binding to the enzyme catalytic site, and developed new biochemical assays to study the catalytic steps of the catalytic reaction. We are currently developing additional substrate and have obtained the first structures of the C-terminal domain of the enzyme with its cofactor TDRD3. Aim 3: Elucidating the roles of TOP3B in DNA and RNA metabolism: Using our TOP3B knockout cell lines and our self-poisoning (R338W) TOP3B construct as well as wild-type and catalytic-dead constructs (Y336F), we have shown the importance of TOP3B in resolving R-loops. We observed elevated R-loops in TOP3B knockout cells (TOP3BKO), which are suppressed by TOP3B transfection. R-loop-inducing agents, the topoisomerase I inhibitor camptothecin, and the splicing inhibitor pladienolide-B also induce higher R-Loops in TOP3BKO cells. Camptothecin- and pladienolide-B-induced R-loops are concurrent with the induction of TOP3B cleavage complexes (TOP3Bccs). RNA/ DNA Hybrid IP-Western blotting show that TOP3B is physically associated with R-loops. Biochemical assays using recombinant TOP3B and oligonucleotides mimicking R-loops show that TOP3B cleaves the single-stranded DNA displaced by the R-loop RNA-DNA duplex. IP-Mass Spectrometry and IP-Western experiments reveal that TOP3B interacts with the R-loop helicase DDX5 independently of TDRD3. Finally, we demonstrate that DDX5 and TOP3B are epistatic in resolving R-loops in a pathway parallel with Senataxin. We proposed a decatenation model for R-loop resolution by TOP3B-DDX5 protecting cells from R-loop-induced damage. Aim 4: 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.