Therapeutic Disruption of HIV Transcription by HIV-1 Tat Protein-Degrading Molecules - Abstract Despite effective antireroviral therapy (ART) suppression of HIV-1 replication to below the detection limit, latent proviruses can reinitiate viral production upon cell stimulation or treatment interruption. The HIV-1 Tat protein plays a pivotal role in regulating the transition between viral latency and active replication, enhancing transcript elongation from the HIV-1 promoter through a positive feedback mechanism. Tat inhibitors hold exceptional promise due to several reasons: 1) Tat is expressed early in the virus replication cycle; 2) No homologs of Tat exist in host cells; 3) Inhibition of Tat disrupts the essential positive transactivation feedback loop required for viral activation; 4) Disruption of this loop leads to epigenetic modifications at the HIV promoter, thus stably repressing viral reactivation. Tat also causes neurotoxicity and disrupts the blood-brain barrier causing neuroinflammation. Consequently, there is considerable interest in developing Tat inhibitors to complement ART. Furthermore, the block-and-lock HIV functional cure strategy relies on specific HIV transcriptional inhibitors to promote epigenetic silencing of proviral expression, locking the virus in a profound state of latency, from which reactivation is highly improbable post-ART interruption. This principle was demonstrated with the Tat inhibitor didehydro-Cortistatin A (dCA); however, clinical studies are pending due to dCA’s complex structure and costly synthesis. Therefore, additional structurally distinct candidates with equivalent bioactivity are essential in the pre-clinical pipeline. To discover novel Tat inhibitors with more cost-effective and simpler synthesis paths, we conducted a cell based high-throughput screening of several compound libraries. Using counter- screens and multiple orthogonal techniques to exclude non-specific and toxic molecules, we identified three small molecules with a therapeutic index (TI) greater than 10 and favorable chemical properties. Interestingly, all three compounds uniquely promote Tat protein degradation by activating the ubiquitin-proteasome pathway, effectively functioning as molecular glues. Here, we propose to carry out hit-to-lead validation and characterization, following a logical pathway for compound progression. The synthesis of novel analogs in each series, utilizing both traditional medicinal chemistry techniques and computational molecular dynamics, will be carried out by Dr. Bannister's medicinal chemistry group at UF Scripps. The structure-activity relationship (SAR) studies will be conducted collaboratively by the medicinal chemists and Dr. Valente’s virology group. Dr. Valente will evaluate the anti-HIV activity in human primary cells and conduct in depth characterization of the mechanism of action (MOA) of all the top-performing compounds. Additionally, ADME studies will be carried out by Dr. Cameron’s group at UF Scripps, and the antiviral efficacy of the compounds will be assessed using humanized mouse models of HIV infection in Dr. Garcia’s laboratory at the University of Alabama. At the conclusion of this study, we expect to have specific Tat inhibitors/degraders with good metabolic and pharmacokinetic properties that prove highly effective in reducing HIV transcription in studies involving humanized mice.
