The Natural Stilbenoid (–)-Hopeaphenol Inhibits HIV Transcription by Targeting Both PKC and NF-κB Signaling and Cyclin-Dependent Kinase 9

Despite effective combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to harbor replication-competent and transcriptionally active virus in infected cells, which in turn can lead to ongoing viral antigen production, chronic inflammation, and increased risk of age-related comorbidities. To identify new agents that may inhibit postintegration HIV beyond cART, we screened a library of 512 pure compounds derived from natural products and identified (–)-hopeaphenol as an inhibitor of HIV postintegration transcription at low to submicromolar concentrations without cytotoxicity. Using a combination of global RNA sequencing, plasmid-based reporter assays, and enzyme activity studies, we document that hopeaphenol inhibits protein kinase C (PKC)- and downstream NF-κB-dependent HIV transcription as well as a subset of PKC-dependent T-cell activation markers, including interleukin-2 (IL-2) cytokine and CD25 and HLA-DRB1 RNA production. ABSTRACT Despite effective combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to harbor replication-competent and transcriptionally active virus in infected cells, which in turn can lead to ongoing viral antigen production, chronic inflammation, and increased risk of age-related comorbidities. To identify new agents that may inhibit postintegration HIV beyond cART, we screened a library of 512 pure compounds derived from natural products and identified (–)-hopeaphenol as an inhibitor of HIV postintegration transcription at low to submicromolar concentrations without cytotoxicity. Using a combination of global RNA sequencing, plasmid-based reporter assays, and enzyme activity studies, we document that hopeaphenol inhibits protein kinase C (PKC)- and downstream NF-κB-dependent HIV transcription as well as a subset of PKC-dependent T-cell activation markers, including interleukin-2 (IL-2) cytokine and CD25 and HLA-DRB1 RNA production. In contrast, it does not substantially inhibit the early PKC-mediated T-cell activation marker CD69 production of IL-6 or NF-κB signaling induced by tumor necrosis factor alpha (TNF-α). We further show that hopeaphenol can inhibit cyclin-dependent kinase 9 (CDK9) enzymatic activity required for HIV transcription. Finally, it inhibits HIV replication in peripheral blood mononuclear cells (PBMCs) infected in vitro and dampens viral reactivation in CD4+ cells from PLWH. Our study identifies hopeaphenol as a novel inhibitor that targets a subset of PKC-mediated T-cell activation pathways in addition to CDK9 to block HIV expression. Hopeaphenol-based therapies could complement current antiretroviral therapy otherwise not targeting cell-associated HIV RNA and residual antigen production in PLWH.

[1]  D. V. van Bockel,et al.  Navigating the complexity of chronic HIV-1 associated immune dysregulation. , 2022, Current opinion in immunology.

[2]  Yoshinori Uekusa,et al.  Elucidation of the inhibitory effect of (+)-hopeaphenol on polyinosinic-polycytidylic acid-induced innate immunity activation in human cerebral microvascular endothelial cells. , 2022, Journal of pharmacological sciences.

[3]  G. Jiang,et al.  Human Immunodeficiency Virus-1 Latency Reversal via the Induction of Early Growth Response Protein 1 to Bypass Protein Kinase C Agonist-Associated Immune Activation , 2022, Frontiers in Microbiology.

[4]  S. Lewin,et al.  Pembrolizumab induces HIV latency reversal in people living with HIV and cancer on antiretroviral therapy , 2022, Science Translational Medicine.

[5]  Shuwen Liu,et al.  A synthetic resveratrol analog termed Q205 reactivates latent HIV-1 through activation of P-TEFb. , 2021, Biochemical pharmacology.

[6]  J. Rodríguez-Fernández,et al.  The Actin Cytoskeleton at the Immunological Synapse of Dendritic Cells , 2021, Frontiers in Cell and Developmental Biology.

[7]  L. Montaner,et al.  The Natural Stilbenoid (–)-Hopeaphenol Inhibits Cellular Entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7, and B.1.351 Variants , 2021, bioRxiv.

[8]  S. Valente,et al.  The Block-and-Lock Strategy for Human Immunodeficiency Virus Cure: Lessons Learned from Didehydro-Cortistatin A. , 2021, The Journal of infectious diseases.

[9]  L. Montaner,et al.  Flavonoid-based inhibition of cyclin-dependent kinase 9 without concomitant inhibition of histone deacetylases durably reinforces HIV latency. , 2021, Biochemical pharmacology.

[10]  Li Ding,et al.  Design strategies for long-acting anti-HIV pharmaceuticals. , 2020, Current opinion in pharmacology.

[11]  L. Montaner,et al.  The African natural product knipholone anthrone and its analogue anthralin (dithranol) enhance HIV-1 latency reversal , 2020, The Journal of Biological Chemistry.

[12]  C. Nelson,et al.  Synthesis of a Unique Psammaplysin F Library and Functional Evaluation in Prostate Cancer Cells by Multiparametric Quantitative Single Cell Imaging. , 2020, Journal of natural products.

[13]  T. Richard,et al.  Screening of Natural Stilbene Oligomers from Vitis vinifera for Anticancer Activity on Human Hepatocellular Carcinoma Cells , 2020, Antioxidants.

[14]  J. Safrit,et al.  Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells , 2020, Nature.

[15]  B. Bell,et al.  Cat and Mouse: HIV Transcription in Latency, Immune Evasion and Cure/Remission Strategies , 2019, Viruses.

[16]  P. Palma,et al.  Immune Activation, Inflammation, and Non-AIDS Co-Morbidities in HIV-Infected Patients under Long-Term ART , 2019, Viruses.

[17]  Tetsuro Ito,et al.  Resveratrol oligomer C-glucosides and anti-viral resveratrol tetramers isolated from the stem bark of Shorea uliginosa , 2018, Phytochemistry Letters.

[18]  David E. Williams,et al.  Identification of Novel HIV-1 Latency-Reversing Agents from a Library of Marine Natural Products , 2018, Viruses.

[19]  M. Murray,et al.  A Review of Long-Term Toxicity of Antiretroviral Treatment Regimens and Implications for an Aging Population , 2018, Infectious Diseases and Therapy.

[20]  M. Wainberg,et al.  Inhibition of NF‐&kgr;B‐dependent HIV‐1 replication by the marine natural product bengamide A , 2018, Antiviral research.

[21]  T. Okamoto,et al.  HIV Tat/P-TEFb Interaction: A Potential Target for Novel Anti-HIV Therapies , 2018, Molecules.

[22]  V. Avery,et al.  Screening a Natural Product-Based Library against Kinetoplastid Parasites , 2017, Molecules.

[23]  Chi-I Chang,et al.  Anti-Inflammatory Effects of Vitisinol A and Four Other Oligostilbenes from Ampelopsis brevipedunculata var. Hancei , 2017, Molecules.

[24]  Shuwen Liu,et al.  Resveratrol Reactivates Latent HIV through Increasing Histone Acetylation and Activating Heat Shock Factor 1. , 2017, Journal of agricultural and food chemistry.

[25]  Amit Kumar,et al.  Targeting TNF and TNF Receptor Pathway in HIV-1 Infection: from Immune Activation to Viral Reservoirs , 2017, Viruses.

[26]  T. Morikawa,et al.  Quantitative Determination of Stilbenoids and Dihydroisocoumarins in Shorea roxburghii and Evaluation of Their Hepatoprotective Activity , 2017, International journal of molecular sciences.

[27]  Nanxi Wang,et al.  Controlling Multicycle Replication of Live-Attenuated HIV-1 Using an Unnatural Genetic Switch. , 2017, ACS synthetic biology.

[28]  T. Hayakawa,et al.  Inhibitory Effects of Oligostilbenoids from the Bark of Shorea roxburghii on Malignant Melanoma Cell Growth: Implications for Novel Topical Anticancer Candidates. , 2016, Biological & pharmaceutical bulletin.

[29]  C. Schwartz,et al.  Improving combination antiretroviral therapy by targeting HIV-1 gene transcription , 2016, Expert opinion on therapeutic targets.

[30]  F. Benvenuti.  The Dendritic Cell Synapse: A Life Dedicated to T Cell Activation , 2016, Front. Immunol..

[31]  Jonathan B Baell,et al.  Feeling Nature's PAINS: Natural Products, Natural Product Drugs, and Pan Assay Interference Compounds (PAINS). , 2016, Journal of natural products.

[32]  V. Avery,et al.  Solving the supply of resveratrol tetramers from Papua New Guinean rainforest anisoptera species that inhibit bacterial type III secretion systems. , 2014, Journal of natural products.

[33]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[34]  R. Quinn,et al.  The Resveratrol Tetramer (-)-Hopeaphenol Inhibits Type III Secretion in the Gram-Negative Pathogens Yersinia pseudotuberculosis and Pseudomonas aeruginosa , 2013, PloS one.

[35]  D. Montefiori,et al.  Mitigation of variation observed in a peripheral blood mononuclear cell (PBMC) based HIV-1 neutralization assay by donor cell pooling. , 2013, Virology.

[36]  D. Havlir,et al.  The distribution of HIV DNA and RNA in cell subsets differs in gut and blood of HIV-positive patients on ART: implications for viral persistence. , 2013, The Journal of infectious diseases.

[37]  M. Doyle,et al.  CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes , 2012, Retrovirology.

[38]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[39]  Hiroki Sato,et al.  Antidiabetogenic oligostilbenoids and 3-ethyl-4-phenyl-3,4-dihydroisocoumarins from the bark of Shorea roxburghii. , 2012, Bioorganic & medicinal chemistry.

[40]  Hiroki Sato,et al.  Anti-hyperlipidemic constituents from the bark of Shorea roxburghii , 2012, Journal of Natural Medicines.

[41]  Colin N. Dewey,et al.  RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome , 2011, BMC Bioinformatics.

[42]  S. Deeks,et al.  HIV infection, inflammation, immunosenescence, and aging. , 2011, Annual review of medicine.

[43]  R. Furler,et al.  Signaling through the P38 and ERK pathways: a common link between HIV replication and the immune response , 2010, Immunologic research.

[44]  S. Moro,et al.  Quinalizarin as a potent, selective and cell-permeable inhibitor of protein kinase CK2. , 2009, The Biochemical journal.

[45]  G. Spear,et al.  Donor variability in HIV binding to peripheral blood mononuclear cells , 2008, Virology Journal.

[46]  D. Nickle,et al.  Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy. , 2008, The Journal of infectious diseases.

[47]  O. Bensaude,et al.  Inhibition of HIV-1 replication by P-TEFb inhibitors DRB, seliciclib and flavopiridol correlates with release of free P-TEFb from the large, inactive form of the complex , 2007, Retrovirology.

[48]  Todd M. Allen,et al.  Use of a novel GFP reporter cell line to examine replication capacity of CXCR4- and CCR5-tropic HIV-1 by flow cytometry. , 2006, Journal of virological methods.

[49]  F. Kasolo,et al.  Predominance of three NF-kappaB binding sites in the long terminal repeat region of HIV Type 1 subtype C isolates from Zambia. , 2005, AIDS research and human retroviruses.

[50]  E. Verdin,et al.  HIV reproducibly establishes a latent infection after acute infection of T cells in vitro , 2003, The EMBO journal.

[51]  Y. Benjamini,et al.  Controlling the false discovery rate in behavior genetics research , 2001, Behavioural Brain Research.

[52]  T. Rana,et al.  DSIF and NELF Interact with RNA Polymerase II Elongation Complex and HIV-1 Tat Stimulates P-TEFb-mediated Phosphorylation of RNA Polymerase II and DSIF during Transcription Elongation* , 2001, The Journal of Biological Chemistry.

[53]  B. Berkhout,et al.  Subtype-specific sequence variation of the HIV type 1 long terminal repeat and primer-binding site. , 2000, AIDS research and human retroviruses.

[54]  G. Hunt,et al.  Occurrence of additional NF-kappaB-binding motifs in the long terminal repeat region of South African HIV type 1 subtype C isolates. , 2000, AIDS research and human retroviruses.

[55]  M. Iinuma,et al.  Antitumor agents 200. Cytotoxicity of naturally occurring resveratrol oligomers and their acetate derivatives. , 1999, Bioorganic & medicinal chemistry letters.

[56]  D. Hazuda,et al.  P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. , 1997, Genes & development.

[57]  M. Malim,et al.  Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes , 1988, Nature.

[58]  H. Gendelman,et al.  Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone , 1986, Journal of virology.