Elimination of HIV-1 Latently Infected Cells by Gnidimacrin and a Selective HDAC Inhibitor

Abstract

We have previously reported gnidimacrin (GM), a protein kinase C (PKC) agonist, significantly reduces the frequency of HIV-1 latently infected cells in peripheral blood mononuclear cells (PBMCs) from patients undergoing successful antiretroviral therapy at low picomolar concentrations ex vivo, which is distinct from other latency reversing agents. In this study, we demonstrate that strong viral reactivation by GM is a mechanism for elimination of latently infected cells, and a histone deacetylase inhibitor (HDACI), a thiophenyl benzamide (TPB), further potentiated the efficacy of GM against latent HIV-1. The effect of GM on latent HIV-1 activation was potentiated by TPB in cell models by 2-3-fold. The GM/TPB combination further decreased the frequency of HIV-infected cells in latently infected patient PBMCs over 3-fold when compared with GM alone, which caused a 5-fold reduction compared with the solvent control. Thus, GM/TPB is a unique combination that may reduce latent HIV-1 reservoirs at nontoxic concentrations.

Figure 1

FACS analysis of the percentage of GFP⁺ J-Lat cells. J-Lat (A2) cells were incubated with GM (80 pM), ingenol-3A (ING) (0.5 nM), TPB (0.3 μM), TPyB (1.0 μM), GM (80 pM)/TPB (0.3 μM), GM (80 pM)/TPyB (1.0 μM), ING (0.5 nM)/TPB (0.3 μM), and ING (0.5 nM)/TPyB (1.0 μM) for 72 h. (A) Frequency of GFP-expressing cells. (B) Percent of cell viability. The data were derived from two independent experiments. *p ≤ 0.05 and **p = 0.005 (one-tailed t test).

Figure 2

Elimination of U1 cells by LRA(s) from U1/U937 cell mixture. GM (26 pM), TPyB (0.57 μM), GM (26 pM)/TPyB (0.57 μM), and no drug control (DMSO) were incubated with latent HIV-1-infected U1 and uninfected U937 (1:4 ratio) in the presence of T20 (1 μg/mL) for 18 days. For post-treatment reactivation, cells at day 18 were reactivated with GM (129 pM) for additional 72 h. (A) P24 level in cell culture supernatants. (B) Reduction of proviral DNA (HIV-1 pol) of U1/U937 coculture in the presence of LRAs. M, Marker; G, GM; T, TPyB; G+T, GM/TPyB combination. *The numbers in panel B are ratios of DNA quantity of compound-treated sample/DMSO-treated control, analyzed using a Kodak molecular imaging system and software. Beta-2-microglobulin level was measured as control.

Figure 3

Effect of TPB on GM-induced IFN-γ production. Normal PBMCs were treated with GM (2000 pM, the bars in blue), GM (20 pM, the bars in red), or control (no GM, the bars in light green) with/without 3 μM TPB (TPB-3), 1 μM TPB (TPB-1), or 0.3 μM vorinostat (SAHA) for 2 days. Anti-CD3/CD28 antibodies (1 μg/mL) were used as positive control. The culture supernatants were quantified for IFN-γ using a Qiagen ELISA kit. The data were derived from three independent experiments.