Histone deacetylase inhibitors containing a benzamide functional group and a pyridyl cap are preferentially effective human immunodeficiency virus-1 latency-reversing agents in primary resting CD4+ T cells

Abstract

Antiretroviral therapy (ART) can control human immunodeficiency virus-1 (HIV-1) replication in infected individuals. Unfortunately, patients remain persistently infected owing to the establishment of latent infection requiring that ART be maintained indefinitely. One strategy being pursued involves the development of latency-reversing agents (LRAs) to eliminate the latent arm of the infection. One class of molecules that has been tested for LRA activity is the epigenetic modulating compounds histone deacetylases inhibitors (HDACis). Previously, initial screening of these molecules typically commenced using established cell models of viral latency, and although certain drugs such as the HDACi suberoylanilide hydroxamic acid demonstrated strong activity in these models, it did not translate to comparable activity with patient samples. Here we developed a primary cell model of viral latency using primary resting CD4+ T cells infected with Vpx-complemented HIV-1 and found that the activation profile using previously described LRAs mimicked that obtained with patient samples. This primary cell model was used to evaluate 94 epigenetic compounds. Not surprisingly, HDACis were found to be the strongest activators. However, within the HDACi class, the most active LRAs with the least pronounced toxicity contained a benzamide functional moiety with a pyridyl cap group, as exemplified by the HDACi chidamide. The results indicate that HDACis with a benzamide moiety and pyridyl cap group should be considered for further drug development in the pursuit of a successful viral clearance strategy.

Figures

Fig. 1.

Protocol for establishing latent virus infection in primary resting CD4+ T cells. (a) Construct gGn-p6* is a replication competent vector with the Vpx binding motif (DPAVDLL) inserted within p6 as indicated. gluc, Gaussialuciferase gene; T2A, T2A peptide promoting ‘ribosomal skipping’;EGFP, enhanced green fluorescent protein gene. (b) Primary resting CD4+ T cells were isolated from leukocyte-enriched healthy donor samples using a Ficoll gradient and negative selection. The isolated T cells were infected by spinoculation with gGn-p6* virus containing Vpx protein. Three days following infection, cultures were treated with test compounds followed 48 h later by flow cytometric analyses. Where indicated, resting cells were co-cultured with the H80 glioma cell line at day 1 after infection. (c) Percentage of activation-marker positive cells in isolated resting CD4+ T cells. Cells from three independent healthy donors or those cells activated with anti-CD3/CD28 beads (Life Technologies) were stained with FITC-conjugated antibodies for each marker and analysed via flow cytometry. Each column shows the mean of three experiments. Error bars show standard deviations.

Fig. 2.

Evaluation of the primary cell model. (a) Flow cytometric analysis of uninfected resting cells. (b) Flow cytometric analysis of cells infected with gGn-p6* virus. (c) Flow cytometric analysis of infected cells treated with ionomycin and PMA. (d) Low-level virus production from resting cells infected with gGn-p6*. HeLaT4 cells were inoculated with supernatants from gGn-p6* infected resting T cells or activated T cells. The number of infected GFP+ HeLaT4 cells was measured via flow cytometry and normalized to the number of GFP+ virus producer cells (resting cells or activated cells). Each mean was calculated from triplicate samples from three independent blood donors 1–3, as indicated on the horizontal axis. Error bars show standard deviations. AU, arbitrary unit.

Fig. 3.

Activation pattern of latent virus in the primary cell model using previously described LRAs. Resting T cells infected with gGn-p6* were treated with 400 nM ionomycin plus 20 ng PMA ml−1, 1 µM JQ1, 30 nM panobinostat, 40 nM romidepsin, 335 nM SAHA, 10 nM bryostatin or 300 nM prostratin. Relative mRNA copy number (a) and GFP mean channel fluorescence (b) from the infected cells were measured 24 or 48 h later, respectively. Relative mRNA copy number per infected cell was determined by dividing the value from quantitative PCR by the GFP-positive cell number ascertained via FACS analysis. Each value is obtained from four or five healthy donor samples indicated with symbols. NC, negative control. Error bars show standard deviations. AU, arbitrary unit.

Fig. 4.

Distribution of EGFP MCF from gGn-p6*-infected resting T cells treated with drugs in the Epigenetics Screening Library. EGFP MCF from infected resting cells treated with drugs is depicted in descending order. The data were obtained using cells isolated from three donors. Numbers on thex-axis indicate ranking position of the compounds. Red, blue and orange columns indicate HDACi, negative control and SAHA, respectively. The top three activators are noted in each graph. AU, arbitrary unit.

Fig. 5.

Scatter plot of MCF and gated cell ratio from infected resting T cells treated with drugs in the Epigenetics Screening Library. Graphs from cells treated with drugs at 20 µM (a) and 2 µM (b). The data were obtained using cells isolated from three donors. MCF data and the gated ratio of resting cells are indicated on the y-axis andx-axis, respectively. Modulator drugs that plotted distantly from each graph’s trend line are noted. Green, orange and light blue circles indicate HDACis with a pyridine cap group, SAHA and negative controls, respectively. AU, arbitrary unit.

Fig. 6.

Scatter plot of EGFP MCF from infected primary resting CD4+ T cells plotted against GFP+ cell percentage of the 24STNLEG cell line treated with drugs in the Epigenetics Screening Library. The data were obtained using cells isolated from three donors compared to a representative experiment from a single culture of the cell line. Graphs are from cells treated with 20 µM for each molecule (a) and 2 µM for each molecule (b). MCF data from resting cells and percentage data from the cell line 24STNLEG are indicated on the y-axis andx-axis, respectively. Green and orange circles represent HDACis with benzamide groups and SAHA, respectively. In (a), data from chaetocin, UNC1999 and Tenovin-6 are not included because of cell death. AU, arbitrary unit.