"Shock and kill" effects of class I-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoximine in cell line models for HIV-1 quiescence

Abstract

Latently infected, resting memory CD4+ T cells and macrophages represent a major obstacle to the eradication of HIV-1. For this purpose, "shock and kill" strategies have been proposed (activation of HIV-1 followed by stimuli leading to cell death). Histone deacetylase inhibitors (HDACIs) induce HIV-1 activation from quiescence, yet class/isoform-selective HDACIs are needed to specifically target HIV-1 latency. We tested 32 small molecule HDACIs for their ability to induce HIV-1 activation in the ACH-2 and U1 cell line models. In general, potent activators of HIV-1 replication were found among non-class selective and class I-selective HDACIs. However, class I selectivity did not reduce the toxicity of most of the molecules for uninfected cells, which is a major concern for possible HDACI-based therapies. To overcome this problem, complementary strategies using lower HDACI concentrations have been explored. We added to class I HDACIs the glutathione-synthesis inhibitor buthionine sulfoximine (BSO), in an attempt to create an intracellular environment that would facilitate HIV-1 activation. The basis for this strategy was that HIV-1 replication decreases the intracellular levels of reduced glutathione, creating a pro-oxidant environment which in turn stimulates HIV-1 transcription. We found that BSO increased the ability of class I HDACIs to activate HIV-1. This interaction allowed the use of both types of drugs at concentrations that were non-toxic for uninfected cells, whereas the infected cell cultures succumbed more readily to the drug combination. These effects were associated with BSO-induced recruitment of HDACI-insensitive cells into the responding cell population, as shown in Jurkat cell models for HIV-1 quiescence. The results of the present study may contribute to the future design of class I HDACIs for treating HIV-1. Moreover, the combined effects of class I-selective HDACIs and the glutathione synthesis inhibitor BSO suggest the existence of an Achilles' heel that could be manipulated in order to facilitate the "kill" phase of experimental HIV-1 eradication strategies.

Figure 1

Potencies of different HDACIs in terms of activation of HIV-1 replication in U1 and ACH-2 cells, and toxicity in uninfected Jurkat T-cells. Panel A: Cells were incubated with the test compounds (1 μM), and p24 production was measured by ELISA in cell culture supernatants at 72 hours post-infection (means ± SEM; 3 experiments). Asterisks show the significant differences in comparison to untreated control cultures according to repeated-measures ANOVA using Dunnet's multiple comparison post-test (a Log transformation of p24 values was applied to restore normality). Panel B: Uninfected Jurkat T cells were incubated for 72 h under similar conditions, and toxicity was measured by the methyl tetrazolium (MTT) method. Results are presented as a percentage of the O.D. (λ = 550) of untreated controls subtracted of background (means ± SEM; 3 experiments). Asterisks show the significant differences in comparison to untreated control cultures according to repeated-measures ANOVA using Dunnet's multiple comparison post-test.

Figure 2

Dose-dependent activation of HIV-1 replication by class I-selective HDACIs and corresponding toxicity in U1 and ACH-2 cells. Panels A, B: Concentration-dependent stimulation of HIV-1 p24 production in the latently infected cell lines U1 (A) and ACH-2 (B) at 72 hours of incubation with MS-275, MC2211, MC2113 (class I-selective HDACIs) and SAHA (a non-class-selective HDACI used as a positive control). Mean values are from three independent experiments (error bars are not shown for better clarity). Dotted lines represent the average p24 levels found in untreated controls in the same experiments. Panel C. Effective concentrations for increasing viral replication to 500% of the basal levels of untreated controls (EC₅₀₀). Panel D: Cell viability of ACH-2 cells, as measured by the methyl tetrazolium (MTT) method. Results are presented as a percentage of the O.D. (λ = 550) of untreated controls subtracted for background (means ± SEM; 3 experiments). Panel E: Cell viability of uninfected Jurkat T cells incubated for 72 hours with the same drugs is shown as comparison. Panel F. 50% cytotoxic concentrations (CC₅₀). For the symbols in panels D, E, the reader should refer to those of panels A, B.

Figure 3

Structural characteristics of HIV-1 activating HDACIs. Panel A: Docking of the HDACI MC2211 at the catalytic cavity of HDAC2, a class I enzyme. The different portions of the inhibitor [i.e. the CAP portion (CAP), the connection unit (CU), the hydrophobic spacer (HS), and the zinc-binding group (ZBG)] are mapped to the molecule represented in the picture. The enzyme is shown as semi-transparent Connolly surface. The Zn⁺⁺ ion embedded in the catalytic cavity is shown as a dotted sphere. The inhibitor is shown according to CPK colouring. Panels B, C: General formulas for HDACIs capable of inducing HIV-1 activation from quiescence. Panel D: Structural superimposition of the best docking poses for the HDACIs MC2113 and MC2211 within the catalytic cavity of HDAC2. Inhibitors are shown in CPK (MC2113: carbon backbone in white; MC2211: carbon backbone in cyan). The enzyme backbone is shown as cartoons. The Zn⁺⁺ ion is shown as a gray sphere. Amino acids D100, H141 and G150 (important for hydrogen bonding with the inhibitors) are shown as orange sticks.

Figure 4

Effects on HIV-1 replication and cell viability of class I-selective HDACIs, MS-275 and buthionine sulfoximine (BSO), alone or in combination. Panel A: HIV-1 p24 concentrations in ACH-2 cell culture supernatants at 24 hours of incubation with the drugs. Panels B-D: Cell viability values at 72 hours of incubation, as determined by the methyl tetrazolium (MTT) method: ACH-2 cells (B), Jurkat 6.3 cells (C), uninfected Jurkat cells (D). Results are presented as percentages of the absorbance (λ = 550) in untreated controls subtracted for background (means ± SEM; 3 experiments). Asterisks show the significant differences found between BSO treatments and matched treatments in the absence of BSO (* P < 0.05; ** P < 0.01; *** P < 0.001). Statistical significance was calculated using repeated-measures, two-way ANOVA and Bonferroni's post-test, following an appropriate transformation to restore normality, where necessary. The higher drug concentrations adopted in Panels C, D serve as comparisons with the experiment in Figure 5.

Figure 5

Stimulation of HIV-1 LTR-controlled expression of green fluorescent protein (GFP) by MS-275 and buthionine sulfoximine (BSO), alone or in combination in a Jurkat cell clone (A1). The A1 cell clone, derived from T-lymphoid Jurkat cells, is a model for latent HIV-1 infection. This clone has an integrated GFP/Tat construct under the control of the HIV-1 LTR and displays a basal proportion of cells expressing GFP, which increase following stimuli activating the HIV-1 promoter. A1 cells were incubated for 72 hours with the different treatments, and GFP expression was monitored by standard flow-cytometric techniques and assessed as the percentage of fluorescent cells (indicated for each histogram) beyond the threshold value established using control non-transfected Jurkat cells. One experiment out of three with similar results is shown. The histograms derived from double-drug treatments were found to be significantly different (P < 0.01) from those derived from treatments with a single drug at matched concentrations (Kolmogorov-Smirnoff statistics). Differences between the drug concentrations adopted in this experiment and that in Figure 4A are derived from adjustments due to the different nature of the cell lines adopted.