Targeting Tat/TAR interactions with the superelongation complex to develop novel treatments for HIV/AIDS

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PROJECT SUMMARY (ABSTRACT) Despite the remarkable success of antiretroviral therapy against human immunodeficiency virus 1 (HIV-1), novel therapeutics are needed to address issues of resistance, tolerability, drug-drug interactions, and variable adherence to daily drug regiments. The objective of this application is to develop novel therapeutic strategies to expand the tools for HIV-treatment. The central hypothesis is that the interactions of TAR with the superelongation complex (SEC) can be inhibited by small-molecule drugs, and binding pockets on the Tat/Cyclin T surface close to TAR can be targeted by structure-based virtual screening for developing PROTAC molecules. The central hypothesis will be tested in a two-pronged approach. Aim 1: To identify and validate small-molecule inhibitors of TAR binding by high-throughput screening. After optimizing the fluorescence polarization binding assay for TAR binding to the SEC, we will extensively screen libraries from the Small Molecule Discovery Center at UCSF and characterize identified inhibitors functionally and structurally with transcription assays, crystallography to further optimize the ligands, and latency reversal assays. Preliminary results show the feasibility of a high-throughput FP assay with labelled TAR and purified SEC. We expect to identify several inhibitors of TAR binding by the end of the grant period. Aim 2: To identify Tat-specific ligands to Tat-AFF4-SEC with the goal of developing chimeric molecules that target Tat complexes for degradation (PROTACs). We identified several pockets that are mostly or partially defined by Tat residues. We will perform structure-based virtual screening (SBVS) to identify small molecule candidates for Tat-dependent binding to the Tat SEC. SBVS will be executed with the help of the Shoichet laboratory (UCSF). Top candidates will be tested in TAR binding assays with the expectation that ligand binding to surface pockets adjacent to Tat and TAR will inhibit TAR binding. We will determine the structures of ligand binding complexes by X-ray crystallography to guide further drug design studies. Such ligands will be a first step towards creating chimeric E3-ligase recruiting PROTACs. PROTACs offer a novel approach with the advantage of theoretically requiring lower affinity transient binding events that are of catalytic nature, avoiding high-level drug dosages. By targeting new HIV complexes with novel drug development methods, we intend to expand the repertoire of effective HIV drugs. The proposed research is expected to contribute to the development of novel therapeutics, targeting so-far neglected intracellular viral proteins and expanding druggability to additional intracellular HIV proteins and their complexes with host proteins. We expect our contributions will significantly advance the field as they are aimed at establishing robust screening assays for a novel HIV-1 target that regulates viral replication.