Structure-Activity Relationship Analysis of Benzotriazine Analogues as HIV-1 Latency-Reversing Agents.

"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.

The HIV latency reversing agent HODHBt inhibits the phosphatases PTPN1 and PTPN2.

Nonreceptor tyrosine phosphatases (NTPs) play an important role regulating protein phosphorylation and have been proposed as attractive therapeutic targets for cancer and metabolic diseases. We have previously identified that 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhanced STAT activation upon cytokine stimulation leading to increased reactivation of latent HIV and effector functions of NK and CD8 T cells. Here, we demonstrated that HODHBt interacts with and inhibits the NTPs PTPN1 and PTPN2 through a mixed inhibition mechanism. We also confirmed that PTPN1 and PTPN2 specifically control the phosphorylation of different STATs. The small molecule ABBV-CLS-484 (AC-484) is an active site inhibitor of PTPN1 and PTPN2 currently in clinical trials for advanced solid tumors. We compared AC-484 and HODHBt and found similar effects on STAT5 and immune activation albeit with different mechanisms of action leading to varying effects on latency reversal. Our studies provide the first specific evidence that enhancing STAT phosphorylation via inhibition of PTPN1 and PTPN2 is an effective tool against HIV.

IL-15 and N-803 for HIV Cure Approaches.

In spite of the advances in antiretroviral therapy to treat HIV infection, the presence of a latent reservoir of HIV-infected cells represents the largest barrier towards finding a cure. Among the different strategies being pursued to eliminate or reduce this latent reservoir, the γc-cytokine IL-15 or its superagonist N-803 are currently under clinical investigation, either alone or with other interventions. They have been shown to reactivate latent HIV and enhance immune effector function, both of which are potentially required for effective reduction of latent reservoirs. In here, we present a comprehensive literature review of the different in vitro, ex vivo, and in vivo studies conducted to date that are aimed at targeting HIV reservoirs using IL-15 and N-803.

AP-1/c-Fos supports SIV and HIV-1 latency in CD4 T cells infected in vivo.

Persistent HIV-1 reservoirs of infected CD4 T cells are a major barrier to HIV-1 cure, although the mechanisms by which they are established and maintained in vivo remain poorly characterized. To elucidate host cell gene expression patterns that govern virus gene expression, we analyzed viral RNA+ (vRNA) CD4 T cells of untreated simian immunodeficiency virus (SIV)-infected macaques by single-cell RNA sequencing. A subset of vRNA+ cells distinguished by spliced and high total vRNA (7-10% of reads) expressed diminished FOS, a component of the Activator protein 1 (AP-1) transcription factor, relative to vRNA-low and -negative cells. Conversely, FOS and JUN, another AP-1 component, were upregulated in HIV DNA+ infected cells compared to uninfected cells from people with HIV-1 on suppressive therapy. Inhibiting c-Fos in latently infected primary cells augmented reactivatable HIV-1 infection. These findings implicate AP-1 in latency establishment and maintenance and as a potential therapeutic target to limit HIV-1 reservoirs.

The latency-reversing agent HODHBt synergizes with IL-15 to enhance cytotoxic function of HIV-specific T cells.

IL-15 is under clinical investigation toward the goal of curing HIV infection because of its abilities to reverse HIV latency and enhance immune effector function. However, increased potency through combination with other agents may be needed. 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhances IL-15-mediated latency reversal and NK cell function by increasing STAT5 activation. We hypothesized that HODHBt would also synergize with IL-15, via STAT5, to directly enhance HIV-specific cytotoxic T cell responses. We showed that ex vivo IL-15 + HODHBt treatment markedly enhanced HIV-specific granzyme B-releasing T cell responses in PBMCs from antiretroviral therapy-suppressed (ART-suppressed) donors. We also observed upregulation of antigen processing and presentation in CD4+ T cells and increased surface MHC-I. In ex vivo PBMCs, IL-15 + HODHBt was sufficient to reduce intact proviruses in 1 of 3 ART-suppressed donors. Our findings reveal the potential for second-generation IL-15 studies incorporating HODHBt-like therapeutics. Iterative studies layering on additional latency reversal or other agents are needed to achieve consistent ex vivo reservoir reductions.

An ultrasensitive planar array p24 Gag ELISA to detect HIV-1 in diverse biological matrixes.

Human immunodeficiency virus-1 (HIV-1) persistence in the presence of antiretroviral therapy (ART) has halted the development of curative strategies. Measuring HIV persistence is complex due to the low frequency of cells containing virus in vivo. Most of the commercially available assays to date measure nucleic acid. These assays have the advantage of being highly sensitive and allow for the analysis of sequence diversity, intactness of the HIV genome or evaluation of diverse RNA species. However, these assays are limited in evaluating translational competent viral reservoirs. In here, we developed an ultrasensitive p24 ELISA that uses the Simoa planar array technology that can detect HIV-1 virions and HIV-1 infected cell with limit of detection similar to nucleic acid assays. Furthermore, the assay is optimized to measure very low levels of p24 in different biological fluids without a major loss of sensitivity or reproducibility. Our results demonstrate that the 'homebrew' planar p24 ELISA immunoassay is a broadly applicable new tool to evaluate HIV persistence in diverse biological fluids and cells.