“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.
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.
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.
Nonreceptor tyrosine phosphatases (NTPs) play an important role regulating protein phosphorylation and have been proposed as attractive therapeutic targets for different diseases. We have previously identified that 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhanced STAT activation upon cytokine treatment. Here, we demonstrated that HODHBt interacts and inhibits the catalytic domain of the NTPs PTPN1 and PTPN2. We confirmed that PTPN1 and PTPN2 specifically control the phosphorylation of STATs. PTPN1 and PTPN2 have been identified as intracellular checkpoint inhibitors that limit immune effector function. We validated that HODHBt enhanced CD8 and γδ-T cells immune effector functions. Finally, we identified a new family of compounds that act as dual PTPN1/PTPN2 inhibitors. Thus, our studies provide a new series of scaffold molecules for the development of specific PTPN1/PTPN2 inhibitors.
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 SimoaTM planar array technology that can detect as low as a single HIV-1 particle and a single HIV-1 infected cell. 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.
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