Targeted small molecule recruitment to suppress the HIV transcriptionally active reservoir - PROJECT SUMMARY/ABSTRACT Antiretroviral therapy (ART), introduced nearly 40 years ago, can durably suppress HIV replication but fails to target the latent reservoir composed of resting CD4+ T cells and myeloid cells, such as microglia in the brain. The intertwined nature of HIV and substance use disorder (SUD) exacerbates this issue, as addictive substances increase the risk of HIV acquisition, persistence, and reactivation, especially in the brain, and a cure is still missing for 1.4 million people worldwide who live with HIV and SUD. The long-term goal is to develop therapeutically effective latency-promoting agents (LPAs) that “block” HIV reactivation in people with SUD and “lock” HIV in a silenced state even after treatment interruptions. The overall objectives of this application are to turn chemical inhibitors of the host CDK9, essential for transcription of the HIV genome, into LPAs and increase their HIV specificity while reducing cytotoxic off-target effects by (i) using them in combination with protein phosphatase 2A (PP2A) activators and (ii) developing targeted chemical recruitment strategies to specifically deliver them to the HIV genome. These objectives will be accomplished through three specific aims: (1) Determine the mechanism(s) by which CDK9 inhibition and PP2A activation block and lock HIV reactivation; (2) Develop a targeted small molecule recruitment strategy for HIV silencing using a bifunctional system relying on the specific interaction between FKBP12F36V and its ligand ortho-AP1867, and (3) Establish Tat/TAR binders for the targeted small molecule recruitment strategy. In Aim 1, the efficacy and potency of existing CDK9 inhibitors (AZD4573, SNS-032) and PP2A activators (SMAP-2), currently in clinical trials for cancer treatment, will be evaluated for blocking and locking HIV reactivation in Jurkat cell lines, primary CD4+ T cells, and iPSC-derived microglia, in the context of addictive substances. Underlying epigenetic and molecular mechanisms will be studied in chromatin immunoprecipitation and RNA-seq experiments. In Aim 2, a targeted small molecule recruitment strategy developed by the applicants, wherein the HIV transactivator Tat is genetically fused to FKBP12F36V and the CDK9 inhibitor SNS-032 is chemically linked to an FKBP12F36V-directed ligand, ortho- AP1867, will be developed and evaluated as an LPA to determine whether it can increase the local concentration of the drug at the HIV promoter with minimal off-target effects in the presence of addictive substances. In Aim 3, known chemical molecules naturally binding Tat or its RNA recruitment module TAR will be fused to CDK9 inhibitors and tested in different latency models to recruit CDK9 inhibitors to the HIV promoter without the need for the FKBP12F36V tag, blocking transcription of untagged HIV. The central hypothesis is that when used in combination and targeted specifically to HIV, CDK9 inhibitors act as LPAs that block HIV reactivation in the presence of addictive substances without cytotoxicity, allowing durable “locking” even after treatment pauses. The proposed research combines deep expertise in HIV biology and targeted kinase inhibition using modern chemical genetics, paving the way for a functional cure for people living with HIV and SUD.
