“Shock and kill” therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues. First, we characterized the essential motifs within these molecules that are required for their biological activity. Second, we identified three benzotriazine analogues with similar activity. We demonstrated that these three compounds are able to increase STAT5 phosphorylation and transcriptional activity. All active analogues reactivate latent HIV in a primary cell model of latency and enhance the ability of interleukin-15 to reactivate latent HIV in cells isolated from aviremic participants. Third, this family of compounds also promote immune effector functions in vitro in the absence of toxicity or global immune activation. Finally, initial studies in mice suggest lack of acute toxicity in vivo. A better understanding of the biological activity of these compounds will help in the design of improved LRAs that work via inhibition of STAT5 SUMOylation.

Antiretroviral drugs that target various stages of the Human Immunodeficiency Virus (HIV) life cycle have been effective in curbing the AIDS epidemic. However, drug resistance, offtarget effects of antiretroviral therapy (ART), and varying efficacy in prevention underscore the need to develop novel and alternative therapeutics. In this study, we investigated whether targeting the signaling molecule Sphingosine-1-phosphate (S1P) would inhibit HIV-1 infection and generation of the latent reservoir in primary CD4 T cells. We show that FTY720 (Fingolimod), an FDA-approved functional antagonist of S1P receptors, blocks cellfree and cell-to-cell transmission of HIV and consequently reduces detectable latent virus. Mechanistically, FTY720 impacts the HIV-1 life cycle at two levels. Firstly, FTY720 reduces the surface density of CD4, thereby inhibiting viral binding and fusion. Secondly, FTY720 decreases the phosphorylation of the innate HIV restriction factor SAMHD1 which is associated with reduced levels of total and integrated HIV, while reducing the expression of Cyclin D3. In conclusion, targeting the S1P pathway with FTY720 could be a novel strategy to inhibit HIV replication and reduce the seeding of the latent reservoir.

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