Abstract:
The HIV-1 transactivator protein Tat interacts with the transactivation response element (TAR), a 59 nucleotide, non-coding, stem loop structure present in 5’ LTR of HIV-1 genome at 3 nucleotide UCU bulge to facilitate the recruitment of positive transcription elongation factor-b (P-TEFb) and induce the transcription of the integrated proviral genome. Therefore, unique to the virus, the Tat-TAR interaction is a promising target for developing antiviral therapeutics. There are no FDA-approved drugs against HIV-1 transcription to date, suggesting the need to develop novel inhibitors that specifically target HIV transcription. Here, we have screened a panel of small molecules that have been previously found to noncanonically bind HIV-1 Trans Activation Response (TAR) element, a 5’ RNA element that is required for activated viral transcription. We have identified five potential candidates that effectively inhibit viral transcription in myeloid and T-cells without toxicity. Additionally, we observed that the infected cells of lymphoid and myeloid origin responded differently to the same TAR binding molecules. Among five candidates, two molecules showed inhibition of viral protein expression. Furthermore, a biotinylated RNA pulldown assay was used to test the effect of these molecules on Tat-TAR RNA interaction. Additionally, a molecular docking and molecular dynamics (MD) simulation approach was used to determine the mechanism of action by these small molecules on TAR RNA in the presence of the P-TEFb complex. The complex dynamics elucidated how TAR binding molecule 110FA targets Tat/TAR interface and prevent Tat binding to TAR RNA. The top candidate showed significant viral transcription inhibition in peripheral blood mononuclear cells infected with HIV-1(89.6). Collectively, our study identified a potential transcription inhibitor that could potentially complement existing cART drugs to address the current therapeutic gap in current regimens. In my work, I used an interdisciplinary approach to answer a biological question where computational simulation studies validated the wet-lab experiments. The protein-RNA binding dynamics were studied using accelerated sampling technologies, which improved sample efficiency and magnified interaction changes. The method can be used to investigate multi-body interactions in a complex biomolecular system. Furthermore, an RNA binding inhibitor 110FA that targets the Tat/TAR interface can prevent TAR from pulling down Tat from P-TEFb protein, thereby showing effective viral transcription inhibition.
