A Therapeutic Strategy to Combat HIV-1 Latently Infected Cells With a Combination of Latency-Reversing Agents Containing DAG-Lactone PKC Activators

Abstract

Advances in antiviral therapy have dramatically improved the therapeutic effects on HIV type 1 (HIV-1) infection. However, even with potent combined antiretroviral therapy, HIV-1 latently infected cells cannot be fully eradicated. Latency-reversing agents (LRAs) are considered a potential tool for eliminating such cells; however, recent in vitro and in vivo studies have raised serious concerns regarding the efficacy and safety of the "shock and kill" strategy using LRAs. In the present study, we examined the activity and safety of a panel of protein kinase C (PKC) activators with a diacylglycerol (DAG)-lactone structure that mimics DAG, an endogenous ligand for PKC isozymes. YSE028, a DAG-lactone derivative, reversed HIV-1 latency in vitro when tested using HIV-1 latently infected cells (e.g., ACH2 and J-Lat cells) and primary cells from HIV-1-infected individuals. The activity of YSE028 in reversing HIV-1 latency was synergistically enhanced when combined with JQ1, a bromodomain and extra-terminal inhibitor LRA. DAG-lactone PKC activators also induced caspase-mediated apoptosis, specifically in HIV-1 latently infected cells. In addition, these DAG-lactone PKC activators showed minimal toxicity in vitro and in vivo. These data suggest that DAG-lactone PKC activators may serve as potential candidates for combination therapy against HIV-1 latently infected cells, especially when combined with other LRAs with a different mechanism, to minimize side effects and achieve maximum efficacy in various reservoir cells of the whole body.

Keywords: HIV-1; HIV-1 latently infected cells; HIV-1 reservoirs; diacylglycerol-lactone; protein kinase C activator.

Figure 1

Structures of diacylglycerol (DAG)-lactone derivatives.

Figure 2

Reversal of HIV-1 latency with DAG-lactone derivatives in vitro. ACH-2 and U1 cells were exposed to a DAG-lactone derivative and prostratin. The expression of intracellular HIV-1 p24 protein (A) and production of p24 in the supernatant (B) were measured after 24 and 48 h of incubation, respectively. (C) J-Lat 10.6 cells were exposed to different concentrations of YSE028 or JQ1 or a combination of both, and the change in the number of green fluorescent protein (GFP)-positive cells was analyzed after 24 h by flow cytometry. (D) Synergism in drug combinations was examined using CompuSyn software. Combination index (CI) values <1 indicate synergistic effects. Data are shown as means ± standard deviations of three independent experiments.

Figure 3

YSE028 reactivates HIV-1 in CD4⁺ T cells from HIV-1-infected individuals. (A) Human CD4⁺ T cells purified from seven HIV-1-infected individuals undergoing cART (Table 1) were treated with 10 μM YSE028, 1 μM JQ1, a combination of YSE028 and JQ1, or 100 nM PMA plus 2 μM ionomycin for 24 h. Intracellular HIV-1 mRNA levels were detected by quantitative real-time PCR (qRT-PCR) and compared to those in untreated controls. (B) Statistical significance was determined using a Mann-Whitney U test, where a value of p < 0.05 was considered to be significant.

Figure 4

Diacylglycerol-lactone derivatives specifically induce caspase-3 activation in HIV-1 latently infected cells. The active form of caspase-3 was measured by flow cytometry. (A) ACH-2 and A3.01 cells were exposed to different concentrations of DAG-lactone derivatives and prostratin for 24 h. (B) The histogram shows representative data for caspase-3 activation with exposure to 10 μM reagent in ACH-2 and A3.01 cells. (C) U1 and U937 cells were exposed to different concentrations of DAG-lactone derivatives and prostratin for 24 h. (D) The histogram shows representative data for caspase-3 activation with exposure to 10 μM reagent in U1 and U937 cells. Data are shown as means ± SDs of three independent experiments.

Figure 5

The effect of YSE028 on T cell activation. PBMCs from three healthy donors were exposed to different concentrations of a reagent for 24 h. Changes in CD69 expression on CD4⁺ or CD8⁺ primary T cells (A), CD8⁺PD1⁺ primary T cells, CD8⁺CD38⁺ primary T cells, and CD8⁺PD1⁺CD38⁺ primary T cells (B) were analyzed by flow cytometry. Data are shown as means ± SDs. Statistical significance was determined using a paired T-test, where a value of p < 0.05 was considered to be significant.

Figure 6

In vivo toxicity and pharmacokinetic analyses of YSE028. (A) The experimental scheme is illustrated. BALB/c mice were challenged with increasing concentrations of PEP005 or YSE028 by intraperitoneal injection, with five animals in each group. (B) The survival rate of PEP005- and YSE028-injected mice. (C) Plasma concentration of YSE028 in BALB/c mice. The concentrations were measured by LC-MS/MS at 0.5, 1, 3, 6, 12, and 24 h after administration of YSE028 at a dose of 10 mg/kg. Data are shown as medians with interquartile ranges, n = 5. (D) The experimental scheme for short-term exposure to YSE028. (E) J-Lat 10.6 cells were exposed to YSE028 for 0.5 or 1 h, and then reagent was washed-out. The number of GFP-positive cells was analyzed after 24 h by flow cytometry. Data are shown as means ± SDs of three independent experiments.