High-throughput Screening Uncovers a Compound That Activates Latent HIV-1 and Acts Cooperatively with a Histone Deacetylase (HDAC) Inhibitor

Abstract

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cellbased system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4 ؉ T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.The ability of human immunodeficiency virus type 1 (HIV-1) to establish a latent infection results in life-long virus persistence even after long-term antiretroviral therapy (ART). 4 The role that latency plays in preventing sustained clearance of the virus infection has become evident in recent years. Patients that have been successfully treated with ART, having undetectable levels of viral RNA (below 50 copies/ml) in the plasma for years, experienced rapid virus rebound upon withdrawal of therapy (1, 2). Moreover, it was found that after T cell activation, virus could be isolated from CD4 ϩ T cells taken from these patients, underscoring the need to eliminate the latently infected cells to eradicate the virus (3-5).Activation of latent proviruses from infected cells in combination with ART is part of a therapeutic strategy that may lead to the complete elimination of HIV infection. Prior attempts to "flush out" the virus by activation of latently infected resting CD4 ϩ T cells with the administration of IL-2 and/or anti-CD3 monoclonal antibodies were ultimately unsuccessful, probably because of its inability to reach all of the latent viral reservoirs and the toxicity of the regimen (6 -10). A more promising approach to complete viral clearance is the use of small molecules with pharmacological properties that allow them to access the viral reservoirs and to specifically reactivate the latent proviruses. The concept of small molecule activation of latent HIV-1 has been tested in a clinical study using the histone deacetylase (HDAC) inhibitor valproic acid (VA) (11). However, it is questionable whether VA alone can be used as a supplement to ART for succe...

Combination of Biological Screening in a Cellular Model of Viral Latency and Virtual Screening Identifies Novel Compounds That Reactivate HIV-1

“…A recently reported high-throughput screening using SupT1 cells latently infected with a GFP and secretable alkaline phosphatase encoding HIV identified a new activator (AV6) of HIV-1 which required NFAT and synergized with the HDAC inhibitor valproic acid (55). Our data have shown that MMQO activity on HIV is independent of NFAT activation, as it was not affected by cyclosporine or FK506 (data not shown).…”

“…S2 in the supplemental material). Its structure was a new variation, different from the class 1 quinolin-8-ol and the MMQO quinolin-4-ol, and it partially resembles the AV6 compound recently reported elsewhere (55). Due to the structural coincidence between MMQA and the quinolinamine moiety of AV6, we cannot rule out the possibility that MMQA acts on a different pathway than that of MMQO.…”

Cyclin T1 and CDK9 T-Loop Phosphorylation Are Downregulated during Establishment of HIV-1 Latency in Primary Resting Memory CD4+T Cells

“…The observations presented here also have implications for screening efforts geared toward the discovery of antilatency drugs (52)(53)(54). In particular, screening methods that use cells in division may favor the discovery of substances that elicit dissociation of P-TEFb from the 7SK RNP complex, for example, HMBA.…”

RNA stability regulates human T cell leukemia virus type 1 gene expression in chronically-infected CD4 T cells

“…RNA stability was reported to be important for activation of latent HIV (Micheva-Viteva et al, 2011), however cis-acting RNA stability-regulating sequences were identified in gag-pol , but not in tat mRNA (Schwartz et al, 1992). More recently, viral RNA stability as a key mediator of latency and reactivation was also described for the Kaposi’s associated herpesvirus (KSHV).…”