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

Joseph P Dougherty,Yoshifumi Kobayashi,Jamie Breslin,Yacov Ron,Zhengxian Gu,Joseph M Colacino,Bradley D Phelan,Hui-Ling Rose Lee,Stuart W Peltz,Leia K Miller,Annmarie L Pacchia,Jason D Graci,Sofiya Micheva-Viteva,Leonard C Edelstein

doi:10.1074/jbc.m110.195537

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 cell-based 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.

Highlights

We present the optimization and utilization of our latency model in an high-throughput screen (HTS) of a library of small molecules that led to identification of compounds capable of reactivating latent HIV-1
We used the 24STNLSG cell line to establish optimal conditions for latent HIV-1 activation in a 384-well format and obtained a Z-factor of 0.7 when TNF␣ was used as an activator (Fig. 1B)
Our latency system was adapted for a 384-well plate format assay yielding excellent reliability, as reflected by the Z-factor values, and was utilized in HTS of a small molecule library of 200,000 compounds

Summary

Introduction

We present data demonstrating that one of the reactivating compounds requires NFAT activity for early gene expression while affecting RNA stability. To test whether AV6 activated latent virus as a result of host cell proliferation, we performed a set of experiments monitoring de novo DNA synthesis or expression of activation markers in PBMC isolated from healthy blood donors.

Results

Conclusion