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Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation.

Jiang G, Mendes EA, Kaiser P, Wong DP, Tang Y, Cai I, Fenton A, Melcher GP, Hildreth JE, Thompson GR, Wong JK, Dandekar S - PLoS Pathog. (2015)

Bottom Line: Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed.Furthermore, PEP005 and the P-TEFb agonist, JQ1, exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone.This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of California, Davis, Davis, California, United States of America.

ABSTRACT
Although anti-retroviral therapy (ART) is highly effective in suppressing HIV replication, it fails to eradicate the virus from HIV-infected individuals. Stable latent HIV reservoirs are rapidly established early after HIV infection. Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed. We report that ingenol-3-angelate (PEP005), the only active component in a previously FDA approved drug (PICATO) for the topical treatment of precancerous actinic keratosis, can effectively reactivate latent HIV in vitro and ex vivo with relatively low cellular toxicity. Biochemical analysis showed that PEP005 reactivated latent HIV through the induction of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB signaling pathway. Importantly, PEP005 alone was sufficient to induce expression of fully elongated and processed HIV RNAs in primary CD4+ T cells from HIV infected individuals receiving suppressive ART. Furthermore, PEP005 and the P-TEFb agonist, JQ1, exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone. Conversely, PEP005 suppressed HIV infection of primary CD4+ T cells through down-modulation of cell surface expression of HIV co-receptors. This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.

No MeSH data available.


Related in: MedlinePlus

PEP005 induces reactivation of HIV latency in vitro.J-Lat A1 cells were exposed to different concentrations of PEP005 and virus reactivation was measured by GFP expression using flow cytometry. Cell viability was determined by Live/Dead dye staining. (A) Chemical Structure of PEP005. (B, C) PEP005 reactivates HIV in a dose-dependent manner and displays minimal cytotoxicity. (D) PEP005 potently reactivates latent HIV in cell lines compared with other LRAs. J-Lat A1 cells were treated with 10 nM PEP005, 500 nM SAHA, 2 μM JQ1, 2 μM GSK343, 10 ng/ml TNF, 2 μM Prostratin, or 5 ng/ml PMA for 24 hours and the percentage of GFP expression was evaluated using flow cytometry. *, p<0.05; **, p<0.01.
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ppat.1005066.g001: PEP005 induces reactivation of HIV latency in vitro.J-Lat A1 cells were exposed to different concentrations of PEP005 and virus reactivation was measured by GFP expression using flow cytometry. Cell viability was determined by Live/Dead dye staining. (A) Chemical Structure of PEP005. (B, C) PEP005 reactivates HIV in a dose-dependent manner and displays minimal cytotoxicity. (D) PEP005 potently reactivates latent HIV in cell lines compared with other LRAs. J-Lat A1 cells were treated with 10 nM PEP005, 500 nM SAHA, 2 μM JQ1, 2 μM GSK343, 10 ng/ml TNF, 2 μM Prostratin, or 5 ng/ml PMA for 24 hours and the percentage of GFP expression was evaluated using flow cytometry. *, p<0.05; **, p<0.01.

Mentions: In order to determine the potential of PEP005 to induce HIV expression, J-Lat A1 cells, an established HIV latency lymphocyte cell culture model in vitro [24,32,39], were treated with increasing concentrations (2–40 nM) of PEP005 (Fig 1A). PEP005 induced HIV expression in a dose-dependent manner and in the absence of any apparent cellular toxicity. A 7-fold increase in the reactivation of HIV latency was detected in J-Lat A1 cells at 20 nM of PEP005 as compared to untreated controls (Fig 1B and 1C). The effect of PEP005 on HIV expression was evident even at the 2 nM level. Compared to other compounds known to reactivate HIV from latency, PEP005 appeared to be more potent than SAHA (a histone deacetylase inhibitor), JQ1 (a BET bromodomain inhibitor) and GSK343 (an inhibitor of EZH2) (Fig 1D). At the 10 nM level, PEP005-induced HIV reactivation was similar to that induced by PMA and more potent than 2 μM Prostratin (p = 0.012). Interestingly, although EZH2 was shown to be critical for establishment of HIV latency through tri-methylation of H3K27 and inhibition of EZH2 by 3-deazaneplanocin A (DZNep) resulted in reactivation of latent HIV in vitro [40], the specific EZH2 inhibitor, GSK343 (recently developed by GSK) [41,42], failed to induce HIV expression in J-Lat A1 cells (Fig 1D). Taken together, these data show that PEP005 is highly potent in reactivating latent HIV in vitro.


Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation.

Jiang G, Mendes EA, Kaiser P, Wong DP, Tang Y, Cai I, Fenton A, Melcher GP, Hildreth JE, Thompson GR, Wong JK, Dandekar S - PLoS Pathog. (2015)

PEP005 induces reactivation of HIV latency in vitro.J-Lat A1 cells were exposed to different concentrations of PEP005 and virus reactivation was measured by GFP expression using flow cytometry. Cell viability was determined by Live/Dead dye staining. (A) Chemical Structure of PEP005. (B, C) PEP005 reactivates HIV in a dose-dependent manner and displays minimal cytotoxicity. (D) PEP005 potently reactivates latent HIV in cell lines compared with other LRAs. J-Lat A1 cells were treated with 10 nM PEP005, 500 nM SAHA, 2 μM JQ1, 2 μM GSK343, 10 ng/ml TNF, 2 μM Prostratin, or 5 ng/ml PMA for 24 hours and the percentage of GFP expression was evaluated using flow cytometry. *, p<0.05; **, p<0.01.
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Related In: Results  -  Collection

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ppat.1005066.g001: PEP005 induces reactivation of HIV latency in vitro.J-Lat A1 cells were exposed to different concentrations of PEP005 and virus reactivation was measured by GFP expression using flow cytometry. Cell viability was determined by Live/Dead dye staining. (A) Chemical Structure of PEP005. (B, C) PEP005 reactivates HIV in a dose-dependent manner and displays minimal cytotoxicity. (D) PEP005 potently reactivates latent HIV in cell lines compared with other LRAs. J-Lat A1 cells were treated with 10 nM PEP005, 500 nM SAHA, 2 μM JQ1, 2 μM GSK343, 10 ng/ml TNF, 2 μM Prostratin, or 5 ng/ml PMA for 24 hours and the percentage of GFP expression was evaluated using flow cytometry. *, p<0.05; **, p<0.01.
Mentions: In order to determine the potential of PEP005 to induce HIV expression, J-Lat A1 cells, an established HIV latency lymphocyte cell culture model in vitro [24,32,39], were treated with increasing concentrations (2–40 nM) of PEP005 (Fig 1A). PEP005 induced HIV expression in a dose-dependent manner and in the absence of any apparent cellular toxicity. A 7-fold increase in the reactivation of HIV latency was detected in J-Lat A1 cells at 20 nM of PEP005 as compared to untreated controls (Fig 1B and 1C). The effect of PEP005 on HIV expression was evident even at the 2 nM level. Compared to other compounds known to reactivate HIV from latency, PEP005 appeared to be more potent than SAHA (a histone deacetylase inhibitor), JQ1 (a BET bromodomain inhibitor) and GSK343 (an inhibitor of EZH2) (Fig 1D). At the 10 nM level, PEP005-induced HIV reactivation was similar to that induced by PMA and more potent than 2 μM Prostratin (p = 0.012). Interestingly, although EZH2 was shown to be critical for establishment of HIV latency through tri-methylation of H3K27 and inhibition of EZH2 by 3-deazaneplanocin A (DZNep) resulted in reactivation of latent HIV in vitro [40], the specific EZH2 inhibitor, GSK343 (recently developed by GSK) [41,42], failed to induce HIV expression in J-Lat A1 cells (Fig 1D). Taken together, these data show that PEP005 is highly potent in reactivating latent HIV in vitro.

Bottom Line: Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed.Furthermore, PEP005 and the P-TEFb agonist, JQ1, exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone.This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of California, Davis, Davis, California, United States of America.

ABSTRACT
Although anti-retroviral therapy (ART) is highly effective in suppressing HIV replication, it fails to eradicate the virus from HIV-infected individuals. Stable latent HIV reservoirs are rapidly established early after HIV infection. Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed. We report that ingenol-3-angelate (PEP005), the only active component in a previously FDA approved drug (PICATO) for the topical treatment of precancerous actinic keratosis, can effectively reactivate latent HIV in vitro and ex vivo with relatively low cellular toxicity. Biochemical analysis showed that PEP005 reactivated latent HIV through the induction of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB signaling pathway. Importantly, PEP005 alone was sufficient to induce expression of fully elongated and processed HIV RNAs in primary CD4+ T cells from HIV infected individuals receiving suppressive ART. Furthermore, PEP005 and the P-TEFb agonist, JQ1, exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone. Conversely, PEP005 suppressed HIV infection of primary CD4+ T cells through down-modulation of cell surface expression of HIV co-receptors. This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.

No MeSH data available.


Related in: MedlinePlus