Poster Presentation Australasian RNA Biology and Biotechnology Association 2024 Conference

Antivirals Targeting the Conserved HIV-1 TIM-TAM Riboswitch Specifically Reactivate HIV-1 from Latency through Modulating Viral RNA-biology. (#160)

Leigh Harty 1 , Jonathan jacobson 1 , Marvin Holtz 1 , Michelle Lee 1 , William Nguyen 2 , Brad Sleebs 2 , Sharon Lewin 3 , Georges Khoury 1 , Vincent Corbin 1 , Damian FJ Purcell 1
  1. Microbiology and Immunology, Doherty Institute, University of Melbourne, Melbourne, VIC, Australia
  2. Walter and Eliza Hall Institute, Parkville, VIC, Australia
  3. Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia

RNA genome viruses evolve in response to selective pressures to favour viral replication and transmission. Viral RNAs fold into “breathing” RNA structures that impact on viral replication and host defences. We pursued HIV latency reversing agents (LRAs) that target a highly conserved RNA structure overlapping the HIV-1 Tat coding sequence that we called the Tat IRES modulator of tat mRNA (TIM-TAM). TIM-TAM functions as a riboswitch regulating mRNA splicing in the nucleus and translation of Tat mRNA in the cytoplasm, impacting HIV-1 RNA transcription, protein translation, miRNA biogenesis, and the switch between productive and latent infection states. In RNA-pulldown experiments the TIM-TAM riboswitch binds to SRP14 and HMGB3 that respectively increase or diminish expression of Tat and consequent productive HIV replication. We made TIM-TAM riboswitch reporter cells, FlipInFM, that were used to screen 114,000 compounds culminating in a novel amidothiazole that specifically reactivated HIV in primary patient cells. Progressive rounds of med-chem increased the potency from EC50=23.6uM to 30.9nM. The amidothiazoles synergise with BRD4 inhibitor JQ1 (+) in primary latent-infected T-cells (Bliss Independence (BI)=0.196). In silico docking predicted the amidothiazoles bind directly to a conserved structured pocket within the TIM-TAM RNA with thermodynamic reactivities that matched the potency and structure activity relationship of the series. The RNA binding site for these compounds overlapped the AUG start codon for the Tat protein, Tat mRNA-splice control elements and a DRACH (AAACU) sequence loop that contains a site for m6A modification. Using STM2457, a potent catalytic inhibitor of the METTL3 writer protein for m6A modification of DRACH was confirmed to modulate the function of the TIM-TAM motif. The DRACH motif in TIM-TAM also overlaps the binding site for HMGB3 and SRP14, indicating control through this riboswitch involves both RNA methylation and structure modification, impacting cellular protein binding and viral Tat protein expression.