A Novel Bromodomain Inhibitor Reverses HIV-1 Latency through Specific Binding with BRD4 to Promote Tat and P-TEFb Association

Abstract

While combinatory antiretroviral therapy (cART) can effectively reduce HIV-1 viremia, it cannot eliminate HIV-1 infection. In the presence of cART, viral reservoirs remain latent, impeding the cure of HIV-1/AIDS. Recently, latency-reversing agents (LRAs) have been developed with the intent of purging latent HIV-1, providing an intriguing strategy for the eradication of the residual viral reservoirs. Our earlier studies show that the first-generation, methyl-triazolo bromodomain, and extra-terminal domain inhibitor (BETi), JQ1, facilitates the reversal of HIV-1 latency. BETis have emerged as a new class of compounds that are promising for this HIV-1 “shock and kill” eradication approach. However, when used as a single drug, JQ1 only modestly reverses HIV-1 latency, which complicates studying the underlining mechanisms. Meanwhile, it has been widely discussed that the induction of latent proviruses is stochastic (Ho et al., 2013). Thus, new BETis are currently under active development with focus on improving potency, ease of synthesis and structural diversity. Using fluorous-tagged multicomponent reactions, we developed a novel second-generation, 3,5-dimethylisoxazole BETi based on an imidazo[1,2-a] pyrazine scaffold, UMB-32. Furthermore, we screened 37 UMB-32 derivatives and identified that one, UMB-136, reactivates HIV-1 in multiple cell models of HIV-1 latency with better efficiency than either JQ1 or UMB-32. UMB-136 enhances HIV-1 transcription and increases viral production through the release of P-TEFb. Importantly, UMB-136 enhances the latency-reversing effects of PKC agonists (prostratin, bryostatin-1) in CD8-depleted PBMCs containing latent viral reservoirs. Our results illustrate that structurally improved BETis, such as UMB-136, may be useful as promising LRAs for HIV-1 eradication.

Curaxin CBL0100 Blocks HIV-1 Replication and Reactivation through Inhibition of Viral Transcriptional Elongation

“…The Planelles model of HIV-1 latency was used to generate latently infected CD4 + T cells with slight modifications (Bosque and Planelles, 2009 ; Hayashi et al, 2017 ; Huang et al, 2017 ). Briefly, naïve CD4 + T cells were isolated from two healthy PBMC donors and were stimulated with anti-CD3/CD28 antibodies that were precoated on a Nunc-Immuno Maxi Sorp plate (Thermo Scientific).…”

Screening of an FDA-approved compound library identifies levosimendan as a novel anti-HIV-1 agent that inhibits viral transcription

“…PBMCs were isolated from fresh whole blood of cART-treated, HIV-infected aviremic patients as described previously (Huang et al, 2017). The CD4 + T cells isolated from PBMCs (2 × 10 6 cells per well) were then stimulated with anti-CD3/CD28 antibodies (1 μg/ml of each) in the presence of LSM (10 μM) or SPR (10 μM).…”

“…On day 3, media and drugs were replaced. On day 6, the supernatant was harvested and subjected to RNA extraction followed by ultra-sensitive qPCR assay to quantify reactivated HIV viruses as described previously (Huang et al, 2017; Mousseau et al, 2015a). A serial dilution of HIV-1 IIIB RNAs with known copy numbers were used to create standard curve for calculation of copy number of HIV RNAs in supernatant.…”

Diversity of small molecule HIV‐1 latency reversing agents identified in low‐ and high‐throughput small molecule screens

“…Latently infected cells harboring transcriptionally silent provirus theoretically would not express TAT protein . However, the fact that BET inhibitors, and Cdk9 agonists (Figure , BETi, Cdk9), induce HIV‐1 provirus expression supports the contention that latent HIV‐1 must produce sporadic transcripts to maintain low levels of virus gene products in these cells . In this context, perhaps the most effective combination of LRAs, to produce an effective “shock,” would include agents that elevate basal HIV‐1 provirus transcription, plus simultaneously stimulating activity of HIV‐1 TAT protein.…”

“…HIV‐1 does encodes its own transactivator TAT (Figure , Tat), but which functions by binding the nascent TAR RNA to activate elongation of paused RNA Polymerase II complexes . Several LRAs function to indirectly enhance TAT activity by upregulating function of Cdk9/cyclin T of the pTEFb elongation complex (Figure , pTEFb); however, these factors also regulate numerous cellular genes, and consequently compounds affecting this mechanism do not specifically target HIV‐1 transcription. Consequently, development of agents capable of enhancing TAT function, or inhibiting factors that negatively regulate TAT, would provide a highly specific means of enhancing provirus expression.…”