Novel histone deacetylase inhibitors CG05 and CG06 effectively reactivate latently infected HIV-1
Choi, Byeong-Suna; Lee, Hak Sunga; Oh, You-Takea; Hyun, Young-Lanb; Ro, Sunggub; Kim, Sung Soona; Hong, Kee-Jonga
aDivision of AIDS, Center for Immunology and Pathology, Korea National Institute of Health, Korea
bCrystal Genomics, Seoul, Korea.
Received 20 April, 2009
Revised 30 September, 2009
Accepted 2 October, 2009
Correspondence to Kee-jong Hong, PhD, Division of AIDS, Center for Immunology and Pathology, Korea National Institute of Health, 194 Tongillo, Eunpyung-gu, Seoul, Republic of Korea. Tel: +82 2 380 2154; fax: +82 2 359 1397; e-mail: email@example.com
Histone deacetylase plays an important role in HIV latency. Novel histone deacetylase inhibitors, CG05 and CG06, were evaluated for their roles in HIV latency using ACH2 cells. Both inhibitors were highly efficient in reactivation of provirus and exerted lesser toxicity compared with other known histone deacetylase inhibitors. Histone acetylation increased when proviruses were reactivated by the compounds. These new inhibitors may contribute to the reduction of the HIV reservoir when used in conjunction with highly active antiretroviral therapy.
HIV-1 latency is the major obstacle to HIV-1 eradication and a critical source for rebound of viral load after the interruption of highly active antiretroviral therapy (HAART) . Latency can be established either by defects in the virus genome or by host cellular factors modulating viral gene silencing [2,3]. Latently infected cells have a limited repertoire of viral factors that can act as therapeutic targets as the viral factors are poorly expressed in vivo in these cells [4,5]. Therefore, there exists an urgent need to develop novel therapeutic drugs that modulate the interaction between host factors and viral replication thereby activating ‘silent’ viruses.
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are the enzymes that regulate host cellular factors maintaining the control of viral gene expression, and chromatin remodeling through histone acetylation/deacetylation. This has been proposed as a potential mechanism for the development of chronic viral reservoir, including HIV . Histone acetylation in the integrated HIV-1 genome is essential for viral gene expression [7–9], and purified HAT stimulates histone acetylation, which enhances HIV-1 transcription from preassembled nucleosomal templates [10,11]. Recently, HDACs have been reported to play an important role in HIV reservoir development by inhibiting the transcriptional initiation of the HIV-1 promoter . Additionally, HDAC inhibitors (HDACis) are also considered as potential therapeutic agents for the reduction of the latent HIV-1 reservoirs [13,14]. HDACis such as valproic acid (VPA), suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA) have been shown to break the chronic stage of HIV infection in vitro. However, VPA was not very effective in clinical studies and TSA showed high cytotoxicity [12,15–18]. Thus, there is an urgent need for the development of HDACis with improved efficacy and safety to selectively reactivate the latent proviral HIV while avoiding high cytotoxicity.
In the current study, we first synthesized novel HDACis, CG05 and CG06 (Fig. 1a; Crystal Genomics, Seoul, Korea), and evaluated their ability to reactivate provirus from the HIV reservoir in the latently infected ACH2 cells. Next, we investigated histone H3 acetylation in ACH2 cells to evaluate HDACi activity of these CG compounds. As shown in Fig. 1(b), both CG05 and CG06 showed dose-dependent cytotoxicity and reactivation efficacy in ACH2 cells.
Viability of ACH2 cells was measured using MTT assay to determine the cytotoxicity of the novel HDACi. SAHA  was used as a control compound with mild toxicity and PXD101 , another hydroxamate type HDACi, as another control with strong HDACi activity. CG05 demonstrated slightly lower cytotoxicity than SAHA in ACH2 cells, whereas CG06 showed similar cytotocixity to SAHA after 48 h of treatment (Fig. 1c). The 50% cytotoxic dose (CD50) for CG05 was 0.96 μmol/l, 0.14 μmol/l for CG06, and 0.18 μmol/l for SAHA when measured using MTT assays. Results from a Trypan blue assay also showed dose-dependent cell viability with higher CD50 levels than the MTT assay (CD50 was >1.0 μmol/l for both CG05 and CG06, data not shown).
As a marker of reactivation efficacy, HIV p24 antigen production levels in supernatant were measured after 48 h of HDACi treatment (Fig. 1d). Enzyme linked immunosorbent assay (ELISA) results showed that CG06 reactivated the HIV-1 provirus more efficiently than PXD101, and CG05 also showed considerable reactivation activity; the 50% efficacy dose (ED50) was 0.11 μmol/l for CG05, 0.04 μmol/l for CG06, and 0.06 μmol/l for PXD101. When the cytotoxicity and reactivation efficacy were compared at 0.14 μmol/l, which is CD50 for CG06, both CG compounds showed higher HIV-1 reactivation efficacy than SAHA and less toxicity compared with PXD101. In J1.1 cells, which are another latently HIV-infected T cells derived from Jurkat T lymphocytes, cytotoxicity and reactivation efficacy showed similar trends (dose-dependent decrease of cell viability, 0.1–0.2 μmol/l range ED50; data not shown). There is a small difference in the chemical structure between CG05 and CG06, which leads to stronger HDACi activity of CG06 than CG05 (data not shown). Cell viability and reactivation efficacy also can be influenced by this structural difference. Considering cell viability and reactivation efficacy, CG05 and CG06 are less toxic and stronger reactivators for breaking the HIV latency. Based on the safety and efficacy data, cells were treated with 0.14 μmol/l HDACi as an optimal concentration for further experiments.
To compare the inhibitory activity of each HDACi during HIV reactivation, histone H3 acetylation was measured by ELISA following HDACi treatment (24 h). Acetylation was increased considerably, especially with CG05 and CG06. When the acetylation of lysine residues on histone H3  was investigated by western blot analysis, Lys9 and Lys27 were highly acetylated by HDACi (data not shown). Based on the results from ELISA and western blot analysis, histone acetylation seemed to be associated with the reactivation of HIV provirus by the HDACi activity of CG05 and CG06. Future investigation of histone modification including H2A, H2B, and H4 will help to elucidate the detailed working mechanism of CG05 and CG06 action.
Then, the proportion of live ACH2 cells expressing intracellular p24 antigen was measured using flow cytometry to confirm that HIV antigen was indeed produced through true HIV-1 proviral reactivation instead of direct release from dead cells due to chemical toxicity (Fig. 1e, upper row). The number of cells showing high intensity of HIV-1 p24 antigen (shift to white peak) was remarkably increased by CG05 and CG06 (approximately > 50%) compared with untreated cells (gray area), whereas SAHA or PXD101 induced low-level HIV-1 reactivation. The reactivation by CG compounds was maintained for 48 h postinfection (data now shown). These intracellular p24 levels implied that CG05-mediated and CG06-mediated p24 production was indeed due to true HIV reactivation efficacy, whereas p24 increase in supernatant of PXD101-treated cells could result from the toxic effects of chemicals.
Taken together, our findings suggest that CG05 and CG06 are very efficient and less toxic reactivation agents, and the use of these HDACis in latently HIV-infected cells enhances HIV transcriptional activation. These results lead to the speculation that novel CG inhibitors may promote a reduction in the HIV reservoir when used in conjunction with HAART protocols.
K-J.H. designed the study and conducted the whole procedure of experiments, including manuscript writing. B-S.C. mainly participated in the data analysis and manuscript writing. H.S.L. produced most parts of data and Y-T.O. took part in the acetylation assay experiments and western blot analysis. S.S.K. advised the experimental procedure and participated in the data analysis. Y-L.H. and S.R. performed the synthesis of new HDAC inhibitors.
Byeong-Sun Choi and Hak Sung Lee contributed equally to this study.
The authors would like to thank Dr Shilpa Buch of University of Nebraska Medical School and Dr Hyewon Youn of Seoul National University Medical School for carefully reading this manuscript.
The present study was supported by a grant from the Korean National Institute of Health (Grant No. 2008-N00387-00).
The authors have no conflicts of interest to declare.
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