Identification of isoform-selective hydroxamic acid derivatives that potently reactivate HIV from latency

Abstract

Objectives: Current antiretroviral therapy can suppress HIV replication, increase CD4 count and result in increased lifespan. However, it cannot eradicate the virus due to the presence of latent provirus in cellular reservoirs, such as resting CD4+ T cells. Using combination latency-reversing agents to shock the virus out of latency for elimination through immune clearance or viral cytopathic effect is one of the most promising strategies for HIV eradication. Specifically, recent evidence shows that isoform-selective histone deacetylase inhibitors may be more effective than their non-selective counterparts. Therefore, identification and characterisation of new isoform-selective compounds are of prime importance. Here, we sought to determine the ability of two new isoform-targeted hydroxamic acid derivatives to reactivate HIV from latency.

Methods: We used cell lines and infected primary resting CD4+ T cells. These were treated with these compounds with HIV reactivation measured using fluorescence-activated cell sorting, Western blots and luciferase luminescence. Isoform selectivity and acetylation of the HIV promoter were measured by Western blotting and chromatic immunoprecipitation.

Results and conclusions: The two new hydroxamic acid derivatives, MC2625 and MC1742, potently reactivate HIV from latency. These compounds are isoform-selective histone deacetylate inhibitors that increase the levels of histone acetylation at the HIV promoter. In addition, they synergise effectively with the protein kinase C modulators bryostatin-1 and INDY, an inhibitor of the dual-specificity tyrosine phosphorylation regulated kinase 1A. We conclude that the combinations of new hydroxamic acid derivatives and bryostatin-1 or INDY could be a new tool for HIV reactivation in the cure efforts.

Keywords: HIV; histone deacetylase inhibitors; isoform-targeted; latency.

Figure 1.

Structure of (a) SAHA (vorinostat) compared to the new hydroxamic acid derivatives (b) MC2625 and (c) MC1742 presented in this paper. SAHA: suberoylanilide hydroxamic acid

Figure 2.

MC2625 and MC1742 reactivate HIV from latency. (a) J-Lat 10.6 cells were incubated with compounds at indicated concentrations for 24 hours. Percent green fluorescent protein positive (GFP+) GFP+ cells were measured by FACS and were used as a measure of HIV reactivation from latency. SAHA and DMSO were used as positive and negative controls, respectively. (b) J-Lat 10.6 cells were incubated with the indicated compounds for 24 hours, and HIV-1 mRNA was quantified by quantitative reverse-transcriptase polymerase chain reaction with primers for gag, using GAPDH as internal control. Fold change is relative to DMSO. (c) J-Lat 10.6 cells were incubated with the compounds for 24 hours, and Western blots were performed for HIV-1 gag and actin. (d) J-Lat 10.6 cells were incubated with the indicated compounds for 24 hours, and MTT toxicity assay was performed as described in Materials and methods. (e,f) Measurement of EC50 for MC2625 and MC1742. J-Lat 10.6 cells were incubated with increasing concentrations of compounds and FACS for GFP+ cells performed previously. Data show means, and error bars indicate ±SEM, n = 3. **P < 0.001, *P < 0.05. Student's t-test was used for pairwise comparisons with DMSO. DMSO: dimethylsulphoxide; FACS: fluorescence-activated cell sorting; MTT: 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; SAHA: suberoylanilide hydroxamic acid; TNF-α:, tumour necrosis factor-α

Figure 3.

MC1742 and MC2625 induce HIV reactivation in a primary cell latency model (Greene model). (a) Resting CD4+ T cells were infected with replication-competent HIV-Luc and were treated with the protease inhibitor darunavir for 2 days. Cells were incubated with the compounds shown for 24 hours in the presence of raltegravir (see Materials and methods for details). HIV reactivation was measured using luciferase luminescence in infected cell lysates normalised to total protein concentration. (b) Toxicity profile of MC1742 and MC2625 in resting T cells. Primary resting T cells were incubated with the indicated compounds for 48 hours, and the MTT toxicity assay was performed as described in Materials and methods. (c) Primary resting CD4+ T cells prepared as in (a) were reactivated for 24 hours, and Western blots were performed for gag and actin. Data indicate means, and error bars indicate ±SEM, n = 3. **P < 0.001, *P < 0.05. Student's t-test was used for pairwise comparisons with DMSO. DMSO: dimethylsulphoxide; MTT: 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; SAHA: suberoylanilide hydroxamic acid

Figure 4.

Isoform selectivity for hydroxamic acid compounds and accumulation of acetylated histone H3 at the HIV promoter. (a) Compounds were incubated with resting primary CD4+ T cells for 8 hours. Acetylated histone H3 (a measure of class I HDAC) and acetylated tubulin (a measure of class II HDAC) were resolved on WBs. Quantification of acetylated tubulin over actin was normalised to SAHA. (b) MC2625 and MC1742 do not inhibit NF-κB activation. Primary CD4+ T cells were incubated with the indicated compounds with or without 5 nM of byrostatin-1 for 24 hours, and WB was performed for NF-κB p65 and actin. Quantification of p65 over actin was normalised to byrostatin-1. (c,d) Chromatin immunoprecipitation using Ac-H3 antibody for HIV LTR (c) or glyceraldehyde-3 phosphate dehydrogenase promoter (d). J-Lat 10.6 cells were incubated with DMSO or the indicated compounds for 24 hours, and ChIP was performed using specific antibody for acetylated histone H3 or IgG. Data indicate means, and error bars indicate ±SEM, n = 3. **P < 0.001. Student's t-test was used for pairwise comparisons with IgG. DMSO: dimethylsulphoxide; HDAC: histone deacetylase; NF-κB: nuclear factor-kappa B; SAHA: suberoylanilide hydroxamic acid; WB: Western blot

Figure 5.

New hydroxamic acid-derived compounds synergise with bryostatin-1 and dual-specificity tyrosine phosphorylation-regulated kinase 1A inhibitor INDY. (a,b) Dose response for byrostatin-1 and INDY in J-Lat 10.6 cells. (c,d) J-Lat 10.6 cells were incubated with increasing HDACi concentration and constant concentration of either 1 nm of bryostatin-1 or 50 μM of INDY. The reactivation of HIV was measured by fluorescence-activated cell sorting of green fluorescent protein (GFP+) cells. (e) J-Lat 10.6 cells were incubated with the indicated compounds, and HIV reactivation was measured by qRT-PCR of gag mRNA. HDACi concentrations were 0.33 μM for SAHA and 0.60 μM for MC2625 and MC1742. Red lines indicate predicted effect by the Bliss independence model. (f–i) Isobole curves for synergy HDAC inhibitors and INDY or bryostatin-1. Red lines are predictions from the model if the compounds were additive. Data indicate means, and error bars indicate ±SEM, n = 3. **P < 0.001, analysis of variance. DMSO, dimethylsulphoxide; HDACi, histone deacetylase inhibitor; SAHA, suberoylanilide hydroxamic acid

Figure 6.

Combinations of hydroxamic acid derivatives with INDY or bryostatin-1 do not activate primary resting T cells. (a) Primary resting CD4+CD25− cells were isolated from HIV-negative blood donors and incubated with the indicated compounds for 24 hours. Cells were incubated with CD25 or CD69 antibody, and fluorescence-activated cell sorting was performed. (b) Toxicity profile of the latency-reversing agent combinations in primary T cells. Resting T cells were incubated with the indicated compounds, and MTT toxicity assay was performed as in Materials and methods. DMSO: dimethylsulphoxide; MTT: 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; SAHA: suberoylanilide hydroxamic acid