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. 2021 Oct 9;13(10):2037.
doi: 10.3390/v13102037.

The Novel PKC Activator 10-Methyl-Aplog-1 Combined with JQ1 Induced Strong and Synergistic HIV Reactivation with Tolerable Global T Cell Activation

Ayaka Washizaki  1 , Megumi Murata  1 , Yohei Seki  1 , Masayuki Kikumori  2 , Yinpui Tang  1 , Weikeat Tan  1 , Nadita P Wardani  1   3 , Kazuhiro Irie  2 , Hirofumi Akari  1   4
Affiliations

The Novel PKC Activator 10-Methyl-Aplog-1 Combined with JQ1 Induced Strong and Synergistic HIV Reactivation with Tolerable Global T Cell Activation

Ayaka Washizaki et al. Viruses. .

Abstract

The presence of latent human immunodeficiency virus (HIV) reservoirs is a major obstacle to a cure. The "shock and kill" therapy is based on the concept that latent reservoirs in HIV carriers with antiretroviral therapy are reactivated by latency-reversing agents (LRAs), followed by elimination due to HIV-associated cell death or killing by virus-specific cytotoxic T lymphocytes. Protein kinase C (PKC) activators are considered robust LRAs as they efficiently reactivate latently infected HIV. However, various adverse events hamper the intervention trial of PKC activators as LRAs. We found in this study that a novel PKC activator, 10-Methyl-aplog-1 (10MA-1), combined with an inhibitor of bromodomain and extra-terminal domain motifs, JQ1, strongly and synergistically reactivated latently infected HIV. Notably, higher concentrations of 10MA-1 alone induced the predominant side effect, i.e., global T cell activation as defined by CD25 expression and pro-inflammatory cytokine production in primary CD4+ T lymphocytes; however, JQ1 efficiently suppressed the 10MA-1-induced side effect in a dose-dependent manner. Considering the reasonable accessibility and availability of 10MA-1 since the chemical synthesis of 10MA-1 requires fewer processes than that of bryostatin 1 or prostratin, our results suggest that the combination of 10MA-1 with JQ1 may be a promising pair of LRAs for the clinical application of the "shock and kill" therapy.

Keywords: 10-methyl-aplog-1; HIV; PKC activator; latency-reversing agents; shock and kill.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structure of 10MA-1 and related PKC activators. Chemical structures of PMA, prostratin, DPP, bryostatin 1, and 10MA-1 are shown.
Figure 2
Figure 2
Reactivation of HIV by LRAs in Jurkat 1G5 and J-Lat 9.2. (A) Jurkat 1G5 was treated with LRAs, including prostratin (Pro), DPP, bryostatin 1 (Bryo), 10MA-1, JQ1, and SAHA. Fold changes of RLU between 1G5 treated with and without the LRAs for 24 h are shown. (B) J-Lat 9.2 was treated with the same LRAs as shown in (A) for 48 h, then (B) p24 amount in the culture supernatants and (C) GFP expression was measured. We performed experiments in triplicate, and averages and standard deviations of the results were indicated. The LRAs were used in these assays at the concentration of 10 nM, 32 nM, 100 nM, 320 nM, 1 µM, or 3.2 µM. PMA was used as a positive control at the concentration of 0.1 nM, 0.32 nM, 1 nM, 3.2 nM, 10 nM, or 32 nM.
Figure 3
Figure 3
Combined treatment of 10MA-1 and JQ1 synergistically reactivated latent HIV. (A) Jurkat 1G5 was treated for 24 h with prostratin (Pro) or 10MA-1 with or without JQ1, as indicated in the figure. Fold changes of RLU in 1G5 and synergy of the combination treatment are shown. (B) J-Lat 9.2 was treated with the same LRAs as shown in (A) for 48 h, then p24 amount in the culture supernatants and synergy of the combined treatment was measured. We performed experiments in triplicate; averages and standard deviations of the results are shown. The LRAs were used in (A,B) at a concentration of 320 nM, 1 µM, or 3.2 µM for each LRA with or without 3.2 µM of JQ1. PMA was used as a positive control at a concentration of 3.2 nM, 10 nM, or 32 nM. (C,D) J-Lat 9.2 was treated with 10MA-1 (C) or prostratin (Pro) (D) with or without JQ1 for 48 h, then p24 amount in the culture supernatants and synergy of the combination treatment were measured. Experiments were performed in triplicate; averages and standard deviations of the results are shown. The LRAs were used in (C,D) at a concentration of 10 nM, 32 nM, 100 nM, 320 nM, 1 µM or 3.2 µM for each LRA or along with 320 nM, 1 µM, or 3.2 µM of JQ1, respectively. PMA was used as a positive control at a concentration of 0.1 nM, 0.32 nM, 1 nM, 3.2 nM, 10 nM, or 32 nM. The Bliss independence model statistically calculated synergies of the combined treatment. If Δfaxy > 0, the combination displays synergy. Alternatively, if ∆faxy < 0, the combination displays antagonism.
Figure 4
Figure 4
Cytotoxicity of LRAs in T cell lines. (A) Jurkat and (B) M8166 were treated for 24 h with each LRA or along with 3.2 µM of JQ1, then relative numbers of living cells were measured by Cell Counting Kit-8. We performed experiments in triplicate; averages and standard deviations of the results are shown. Moreover, the relative cell viability was calculated by dividing the average absorbance at 450 nm in the culture media of the treated cells by that of untreated cells. The LRAs were used in these assays at a concentration of 320 nM, 1 µM, or 3.2 µM, with or without 3.2 µM of JQ1. PMA was used as a positive control at a concentration of 3.2 nM, 10 nM, or 32 nM.
Figure 5
Figure 5
T cell activation induced by the combined treatment of LRAs. (A) PBMCs were untreated or treated with increasing concentrations of PMA (0.1 nM, 1 nM, or 10 nM), JQ1 (10 nM, 100 nM, or 1 µM), 10MA-1 (1 nM, 10 nM, 100 nM, or 1 µM), or JQ1 (10 nM, 100 nM, or 1 µM) and 10MA-1 (1 nM, 10 nM, 100 nM, or 1 µM) for 24 h. The CD4+ T lymphocytes were measured for the positivity of CD69 (left) and CD25 (right) by flow cytometry. We performed experiments in triplicate; the averages and standard deviations of the results obtained from the PBMCs of a macaque (MF-1) are shown. (B) Representative dot plot data by flow cytometry for (A) are shown. (C) PBMCs were untreated or treated with an increasing concentration of PMA (0.1 nM, 1 nM, or 10 nM), JQ1 (320 nM, 1 µM, 3.2 µM, or 10 µM), 10MA-1 (320 nM, 1 µM, 3.2 µM, or 10 µM), or JQ1 (320 nM, 1 µM, 3.2 µM, or 10 µM) and 10MA-1 (320 nM, 1 µM, 3.2 µM, or 10 µM) for 24 h. CD4+ T lymphocytes were examined for the positivity of CD69 (left) and CD25 (right) by flow cytometry. We performed experiments in triplicate; the averages and standard deviations of the results obtained from the PBMCs of a macaque (MF-1) are shown. (D) Averages of the results obtained from PBMCs of three monkeys regarding CD69 and CD25 expression in the CD4+ T lymphocytes treated along with LRAs. The averages of the data from PBMCs of three monkeys in each experimental setting employed in A and C are indicated as D and E, respectively. The data were calculated as relative percentages of CD69 or CD25-positive cells; the 1 µM or 10 µM 10MA-1 treatment data were set to 100%. (E) Averages of the results obtained from PBMCs of three monkeys regarding CD69 and CD25 expression in the CD4+ T lymphocytes treated along with LRAs. The averages of the data from PBMCs of three monkeys in each experimental setting employed in C are indicated. The data were calculated as relative percentages of CD69 or CD25-positive cells; the 1 µM or 10 µM 10MA-1 treatment data were set to 100%.
Figure 5
Figure 5
T cell activation induced by the combined treatment of LRAs. (A) PBMCs were untreated or treated with increasing concentrations of PMA (0.1 nM, 1 nM, or 10 nM), JQ1 (10 nM, 100 nM, or 1 µM), 10MA-1 (1 nM, 10 nM, 100 nM, or 1 µM), or JQ1 (10 nM, 100 nM, or 1 µM) and 10MA-1 (1 nM, 10 nM, 100 nM, or 1 µM) for 24 h. The CD4+ T lymphocytes were measured for the positivity of CD69 (left) and CD25 (right) by flow cytometry. We performed experiments in triplicate; the averages and standard deviations of the results obtained from the PBMCs of a macaque (MF-1) are shown. (B) Representative dot plot data by flow cytometry for (A) are shown. (C) PBMCs were untreated or treated with an increasing concentration of PMA (0.1 nM, 1 nM, or 10 nM), JQ1 (320 nM, 1 µM, 3.2 µM, or 10 µM), 10MA-1 (320 nM, 1 µM, 3.2 µM, or 10 µM), or JQ1 (320 nM, 1 µM, 3.2 µM, or 10 µM) and 10MA-1 (320 nM, 1 µM, 3.2 µM, or 10 µM) for 24 h. CD4+ T lymphocytes were examined for the positivity of CD69 (left) and CD25 (right) by flow cytometry. We performed experiments in triplicate; the averages and standard deviations of the results obtained from the PBMCs of a macaque (MF-1) are shown. (D) Averages of the results obtained from PBMCs of three monkeys regarding CD69 and CD25 expression in the CD4+ T lymphocytes treated along with LRAs. The averages of the data from PBMCs of three monkeys in each experimental setting employed in A and C are indicated as D and E, respectively. The data were calculated as relative percentages of CD69 or CD25-positive cells; the 1 µM or 10 µM 10MA-1 treatment data were set to 100%. (E) Averages of the results obtained from PBMCs of three monkeys regarding CD69 and CD25 expression in the CD4+ T lymphocytes treated along with LRAs. The averages of the data from PBMCs of three monkeys in each experimental setting employed in C are indicated. The data were calculated as relative percentages of CD69 or CD25-positive cells; the 1 µM or 10 µM 10MA-1 treatment data were set to 100%.
Figure 6
Figure 6
Pro-inflammatory cytokine production induced by the combined treatment of LRAs. PBMCs were treated with increasing concentrations of PMA (0.1 nM, 1 nM, or 10 nM), JQ1 (10 nM, 100 nM, or 1 µM), 10MA-1 (1 nM, 10 nM, 100 nM, or 1 µM), or JQ1 (10 nM, 100 nM, or 1 µM) and 10MA-1 (1 nM, 10 nM, 100 nM, or 1 µM) for 24 h. IL-8 (A) and TNF-α (B) in the culture supernatants were measured by ELISA. Experiments were performed in triplicate; the averages and standard deviations of the results obtained from the treated PBMCs are shown.
Figure 7
Figure 7
A kinetic model for the safety margin in the combined treatment of 10MA-1 with JQ1. The combined treatment of 10MA-1 with JQ1 leads to both synergistic HIV reactivation and reduced pro-inflammatory cytokine expression. As demonstrated by the relative difference in their activities of combined treatment, the safety margin is ameliorated dose-dependently of JQ1, whereas 10MA-1 alone merely exhibits the safety margin, irrespective of the dose of 10MA-1.
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