Discovery and Mechanistic Study of HIV-1 Transcriptional Inhibitors from Natural and Synthetic Products

by Cole Schonhofer

B.Sc., Simon Fraser University, 2015

Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science

in the Master of Science Program Faculty of Health Sciences

© Cole Schonhofer 2019 SIMON FRASER UNIVERSITY Fall 2019

Copyright in this work rests with the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation. Approval

Name: Cole Schonhofer Degree: Master of Science Title: Discovery and Mechanistic Study of HIV-1 Transcriptional Inhibitors from Natural and Synthetic Products Examining Committee: Chair: Tim Takaro Professor, Associate Dean Ian Tietjen Senior Supervisor Assistant Professor Zabrina Brumme Supervisor Associate Professor Jonathan Choy Supervisor Associate Professor Timothy Beischlag Examiner Professor

Date Defended/Approved: December 17, 2019

ii Ethics Statement

iii Abstract

There is currently no safe, scalable cure for HIV-1 infection due to the persistence of a latent viral reservoir. The Block-and-Lock strategy to achieve drug-free HIV remission requires promotion of a durable, deep latent state from which dormant HIV does not reactivate. However, there are currently no licensed HIV inhibitors that target expression from the latent reservoir. Factors that are required for efficient viral transcription, such as the HIV protein Tat and the host factor CDK9, are potential targets for Block-and-Lock approaches. My thesis describes an effort to discover novel antivirals that prevent expression from latently infected cells and inhibit viral replication. I describe the identification of natural and synthetic inhibitors of HIV Tat-mediated transcription, the investigation of their molecular mechanisms, and their potential as Block-and-Lock agents. The work presented here informs the development of future HIV drug-free remission strategies and identifies CDK9 as a potential target for Block-and-Lock strategies.

Keywords: HIV; Block-and-Lock; CDK9; flavonoids; Tat

iv Dedication

To my family, thank you for everything, all the support to help me reach this stage. I couldn’t have done it without the support of my mother, father, wife, sister, grandparents, several cats, and a few dogs.

v Acknowledgements

While individual contributions are noted in the relevant data chapters, I’d like to use this space to thank my supervisor Dr. Ian Tietjen for all of his support, mentorship, training, and the opportunity to work on these projects. I’d also like to thank every member of the Tietjen, Brumme, and Brockman labs who assisted me throughout my time as a Master’s student. Additionally, thank you to the members of all the labs who collaborated with us on these projects. Finally, I’d also like to acknowledge the financial support provided to me by the Faculty of Health Sciences at SFU and the Canadian Institute for Health Reasearch, in the form of scholarships and travel awards.

This work was supported by an SFU Presidential Research Startup Grant, a Canadian Institutes of Health Project Grant (PJT-153057), the Sub-Saharan African Network for TB/HIV Research Excellence (SANTHE), a DELTAS African Initiative (grant # DEL-15- 006), the Canadian Foundation for AIDS Research (CANFAR), and the New Frontiers in Research Fund – Exploration (NFRFE-2018-01386).

vi Table of Contents

Approval ...... ii Ethics Statement ...... iii Abstract ...... iv Dedication ...... v Acknowledgements ...... vi Table of Contents ...... vii List of Tables ...... x List of Figures...... xi List of Acronyms ...... xii

Chapter 1. Introduction ...... 1 1.1. The Global HIV-1 Epidemic ...... 1 1.2. HIV Genome and Life Cycle ...... 1 1.3. HIV Pathogenesis ...... 5 1.4. Antiretroviral Therapies: Drug Classes, Mechanisms, and Limitations ...... 6 1.5. The HIV Latent Reservoir: Barrier to a Cure ...... 9 1.6. HIV Cure or Remission Strategies ...... 11 1.7. Conclusion...... 16 1.8. Thesis Objectives ...... 16 1.9. References ...... 17

Chapter 2. Identification of Flavonoids that Suppress HIV-1 Latency Reversal from African Natural Products ...... 31 2.1. Abstract ...... 31 2.2. Introduction ...... 31 2.3. Materials and Methods ...... 32 2.3.1. Cells and Reagents ...... 32 2.3.2. Plasmids ...... 33 2.3.3. Virus Production ...... 33 2.3.4. HIV-1 Replication Assays and Compound Screening ...... 34 2.3.5. Apoptosis Detection Assays ...... 35 2.3.6. Inhibition of Latency Reversal Assays ...... 35 2.3.7. GFP Quenching Assay ...... 36 2.3.8. HIV-1 Tat Inhibition Assays...... 36 2.3.9. Block and Lock Assays ...... 36 2.3.10. Data and Statistical Analysis ...... 37 2.4. Results ...... 37 2.4.1. Identification of Flavonoids that Inhibit HIV Replication...... 37 2.4.2. Flavonoids Inhibit HIV Replication in PBMC ...... 42 2.4.3. Flavonoids Inhibit Replication of ARV-Resistant Viruses ...... 44 2.4.4. Flavonoids Inhibit Latency Reversal in Multiple Cell Lines ...... 47 2.4.5. Aglycone Flavones, but not L7G, Inhibit HIV-1 Tat Function ...... 51

vii 2.4.6. Apigenin, Chrysin, and Luteolin Reversibly Reinforce Latency ...... 55 2.5. Discussion ...... 57 2.6. Contributions ...... 60 2.7. References ...... 60

Chapter 3. Mechanistic Investigation of Post Integration Activities of Flavonoids 65 3.1. Abstract ...... 65 3.2. Introduction ...... 65 3.3. Methods ...... 69 3.3.1. Reagents ...... 69 3.3.2. Plasmids ...... 69 3.3.3. Kinase Activity Assays ...... 70 3.3.4. Quantitation of Intracellular HIV mRNA Levels...... 70 3.3.5. Screens for Flavonoids that Synergistically Promote Latency Reversal ...... 71 3.3.6. HIV Rev Function Assay ...... 71 3.3.7. HDAC Activity Assay ...... 72 3.3.8. Block and Lock Assays ...... 72 3.4. Results ...... 73 3.4.1. Aglycone Flavones are More Potent Inhibitors of CDK9 than L7G ...... 73 3.4.2. CK2 Inhibition by Flavones is Not Responsible for Tat Inhibition Activity or Inhibition of Latency Reversal ...... 75 3.4.3. Flavonoids Alter HIV Splicing Patterns in J-Lat 9.2 Cells ...... 78 3.4.4. Flavonoids do not Inhibit HIV Rev Function ...... 79 3.4.5. Flavonoids Isolated from Natural Products Synergise with a PKC Agonist, but not an HDAC Inhibitor, to Reverse Latency ...... 81 3.4.6. Flavonoids Inhibit HDAC Activity in Jurkat Cells ...... 83 3.4.7. Specific CDK9 Inhibition by Flavopiridol Promotes Latency ...... 85 3.5. Discussion ...... 87 3.6. Contributions ...... 91 3.7. References ...... 91

Chapter 4. Discovery and Mechanistic Study of Novel Inhibitors of HIV Tat Function ...... 98 4.1. Abstract ...... 98 4.2. Introduction ...... 98 4.3. Materials and Methods ...... 99 4.3.1. Reagents ...... 99 4.3.2. High-Throughput Microscopy and Flow Cytometry Screen ...... 100 4.3.3. Tat inhibition Assays ...... 101 4.3.4. Latency Reversal Assays ...... 101 4.3.5. CDK9 inhibition Assay ...... 101 4.3.6. NFκB-reporter Assays ...... 101 4.3.7. HIV LTR-RL Reporter Assays ...... 101 4.3.8. Viability/Apoptosis Assays ...... 102

viii 4.3.9. PBMC Assays...... 102 4.4. Results ...... 103 4.4.1. Discovery of Novel Tat Inhibitors by HTS in JurTat Cells ...... 103 4.4.2. A7 and C11 Inhibit Tat Function in JurTat Cells and CEM-GXR Cells ...... 104 4.4.3. A7 and C11 Inhibit Latency Reversal in Multiple Cell Lines...... 106 4.4.4. C11, but not A7, Inhibits CDK9 Activity ...... 108 4.4.5. A7 and C11-Mediated Suppression of NFκB Activity is not Essential for Inhibition of Tat-driven Transcription ...... 109 4.4.6. A7 and C11 Inhibit HIV Replication in PBMCs ...... 112 4.4.7. A7 and C11 Prevent Viral Transfer from HIV-Infected Cells Reactivated from Latency 113 4.5. Discussion ...... 115 4.6. Contributions ...... 118 4.7. References ...... 118

Chapter 5. Conclusion ...... 122 5.1. Summary ...... 122 5.2. References ...... 124

ix List of Tables

Table 2.1. Summary of EC50s, CC50s, and Selectivity Indices of flavonoids in CEM GXR cells and PBMC ...... 44 Table 2.2. Summary of ARV and flavonoid activities against ARV-resistant HIV strains...... 47

Table 2.3. Summary of EC50s of latency reversal inhibition by flavonoids in J-Lat 9.2 and OM-10.1 cells ...... 50

Table 2.4. Summary of flavonoid EC50s against Tat function in JurTat and CEM-GXR cells ...... 54

Table 4.1. Summary of A7 and C11 EC50s against latency reversal in J-Lat 9.2 and OM-10.1 cells ...... 106

Table 4.2. Summary of EC50s of A7 and C11 against NFκB signaling in HEK 293T cells and Tat-driven luciferase production in Jurkat cells ...... 112

x List of Figures

Figure 1.1. Transcription from integrated proviruses requires Tat-mediated recruitment of cellular P-TEFb ...... 3 Figure 2.1. Identification of flavonoids that inhibit HIV replication ...... 38 Figure 2.2. Flavonoids inhibit HIV replication in vitro ...... 41 Figure 2.3. Flavonoids inhibit HIV replication in PBMCs ...... 43 Figure 2.4. Flavonoids inhibit ARV-resistant HIV replication in CEM-GXR cells ...... 46 Figure 2.5. Flavonoids antagonize HIV latency reversal in multiple cell lines ...... 50 Figure 2.6. Aglycone flavones, but not L7G, inhibit HIV Tat-induced gene expression ...... 52 Figure 2.7. Apigenin, chrysin, and luteolin reversibly promote latency ...... 56 Figure 3.1. Aglycone flavones are more potent inhibitors of CDK9 than L7G ...... 74 Figure 3.2. Activity of a CK2-specific inhibitor compared to flavonoids ...... 76 Figure 3.3. Flavonoids alter abundance of HIV mRNA spliceforms ...... 78 Figure 3.4. Flavonoids do not inhibit HIV Rev function ...... 80 Figure 3.5. Discovery of novel flavonoids that synergistically reactivate latency ...... 83 Figure 3.6. Flavonoids inhibit HDAC activity ...... 84 Figure 3.7. The CDK9 inhibitor Flavopiridol promotes latency in J-Lat 10.6 cells ...... 86 Figure 4.1. Identification of novel Tat inhibitors by high-throughput screening of JurTat cells ...... 103 Figure 4.2. A7 and C11 inhibit HIV Tat function...... 105 Figure 4.3. A7 and C11 inhibit latency reversal in multiple cell lines ...... 108 Figure 4.4. C11, but not A7, inhibits CDK9 activity ...... 109 Figure 4.5. A7 and C11-mediated suppression of NFκB signalling is not required for Tat inhibition ...... 111 Figure 4.6. A7 and C11 inhibit HIV replication in PBMCs ...... 113 Figure 4.7. A7 and C11 treatment reduces viral transfer from stimulated HIV+ PBMCs ...... 114

xi List of Acronyms

3M6PA 3-methoxy-6-prenyl Apigenin 3TC Lamivudine 6PA 6 prenyl apigenin AIDS Acquired Immunodeficiency Syndrome ARV Antiretroviral AZT Zidovudine Brd4 Bromodomain-containing protein 4 cART Combination Antiretroviral Therapy

CC50 50% Cytotoxic Concentration CCR5 C-C Chemokine Receptor Type 5 CD32 Cluster of Differentiation 32 CD4 Cluster of Differentiation 4 CD8 Cluster of Differentiation 8 CDK9 Cyclin Dependant Kinase 9 CK2 Casein Kinase II CRM1 Chromosomal Maintainance 1/Exportin 1 CXCR4 C-X-C Chemokine Receptor Type 4 dCA Didehydro Cortistatin A DMSO Dimethyl sulfoxide DNA Deoxyribonucleic Acid DRB 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole DSIF DRB Sensitivity Inducing Factor

EC50 50% Effective Concentration EFV Efavirenz Env Envelope Gp Glycoprotein HDAC Histone Deactylase HIV-1 Human Immunodeficiency Virus Type 1 HLA Human leukocyte antigen HMBA Hexamethylene Bisacetamide

IC50 50% Inhibitory Concentration IND Indinavir

xii INSTI Integrase Strand Transfer Inhibitor L7G Luteolin-7-Glucoside LMB Leptomycin B LRA Latency Reversing Agent LTR Long Terminal Repeat MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NELF Negative Elongation Factor NFκB Nuclear Factor kappa-light-chain-enhancer of activated B cells NNTRI Non-Nucleoside Reverse Transcriptase Inhibitor NRTI Nucleoside Reverse Transcriptase Inhibitor pANAPL Pan-African Natural Products Library PBMC Peripheral Blood Mononuclear Cell PI Protease Inhibitor PII Post Integration Inhibitor PKC Protein Kinase C PLWH People Living With HIV PMA Phorbol 12-myristate 13-acetate P-TEFb Positive Transcription Elongation Factor b QN Quinalizarin RAL Raltegravir RNA Ribonucleic Acid RNAP II RNA Polymerase II RRE Rev Response Element RT Reverse Transcriptase SI Selectivity Index SMAC Second Mitochondrial Activator of Caspases snRNA small nuclear Ribonucleic Acid TAR Trans-Activation Response Element TNFα Tumour necrosis Factor α

xiii Chapter 1. Introduction

1.1. The Global HIV-1 Epidemic

Human Immunodeficiency Virus (HIV) is the infectious agent responsible for Acquired Immunodeficiency Syndrome (AIDS), a condition that has been responsible for approximately 32 million deaths worldwide since 1981 [1]. As of 2018 there are an estimated 37.9 million people living with HIV (PLWH), with the largest infection burden in sub-Saharan Africa. In Canada, approximately 75,500 people are living with HIV as of 2014 [2]. HIV-associated deaths have declined by over 50% since 2004, and new HIV infections have declined almost 40% worldwide since 1997 [1], thanks in large part to effective combination Antiretroviral Therapies (cART). cART suppresses HIV replication to below detectable limits, preventing progression to AIDS and greatly reducing transmission risk [3][4]. However, there are no vaccines or cures available and cART must be maintained for life. While generally well tolerated, long term cART is associated with an increased risk of comorbities such as cardiovascular disease, metabolic diseases, and cancer [5]. Additionally, due in part to inefficiencies in cost and distribution of antiretrovirals (ARVs), only 62% of PLWH were accessing treatment. Furthermore, HIV status remains stigmatized, affecting quality of life for many individuals and reducing access to care [4]. Progress in HIV cure strategies and the development of an effective vaccine represent the best hope for ending the global HIV pandemic.

1.2. HIV Genome and Life Cycle

HIV belongs to a genus of retroviruses known as lentiviruses, and is classified into two main types, HIV-1 and -2. HIV-1 is further classified into groups M, N, O, and P. HIV-1 group M is responsible for the majority of global infections and features higher mortality and transmission rates than HIV-2 [6][7]. The HIV-1 genome is positive-sense, single-stranded RNA that encodes 9 proteins: The structural proteins Gag and Envelope (Env); the enzymatic proteins Protease, Reverse Transcriptase (RT), and Integrase; the regulatory proteins Tat and Rev; and the accessory proteins Vif, Vpr, Vpu, and Nef (Figure 1.1A).

1 HIV-1 particles contain two copies of the RNA genome enclosed within a capsid (viral protein p24) that is further surrounded by the viral envelope. The HIV envelope is comprised of a host-derived lipid bilayer studded with the viral glycoprotein Env. Mature Env features two subunit proteins, a surface, heavily glycosylated cap protein, gp120, and a hydrophobic stem subunit, gp41 [8].

Viral entry into target cells requires binding of the gp120 subunit to the main entry receptor, the host protein CD4. Immune cells that express high levels of CD4, primarily T-helper cells, are the major target for HIV infection, although the virus is also capable of infecting immune cells with lower levels of CD4 expression such as dendritic cells and macrophages [9][10]. Initial binding of gp120 to CD4 triggers a conformational change in Env that allows binding to an additional coreceptor, CCR5 or CXCR4. Coreceptor binding stabilizes virion attachment and allows insertion of the hydrophobic gp41 into the host cell membrane, the formation of a fusion pore, and the delivery of the viral capsid into the host cell [8].

Inside the capsid, the HIV genome is packaged with several viral proteins, including RT and Integrase. RT is required to convert the single-strand RNA genome into double-stranded DNA, which is then transported to the nucleus and integrated into the host genome by Integrase. Integration preferentially occurs in actively-transcribing genes near the edge of the nucleus [11][12]. In general, transcription, virion assembly, and progeny release begins shortly following integration; however, in rare cases proviruses enter a largely transcriptionally silent, latent state [13][14]. These rare instances result in the formation of a latent reservoir, which represents a major barrier to an HIV cure.

HIV integration is an inefficient process, especially in resting T cells, and levels of unintegrated HIV DNA accumulate in infected cells. Linear HIV cDNA is subject to degradation by the host cell, but can also undergo processes other than integration. For example, it can undergo autointegration reactions wherein Integrase catalyzes the 3’ end of the viral genome to attack sites within the cDNA itself, creating a non-replicative circular form. Additionally, 1-LTR and 2-LTR circular cDNA products can be produced by end-joining recombination, with host factor assistance. In particular, production of 2-LTR circles can serve as a marker of spreading infections, as levels increase transiently in newly infected cells [15]. Importantly, 2-LTR circles can be detected in patients on long

2 term cART, suggesting that even when viral loads are suppressed below detectable limits, low-level HIV replication and infection of new target cells can still occur. These de novo replication events may contribute to the robustness of the latent reservoir; however, the existence of ongoing replication in the presence of cART remains controversial [16][17][18]. Instead, clonal expansion of latently infected cells is a more accepted method of reservoir maintenance [19].

Figure 1.1. Transcription from integrated proviruses requires Tat-mediated recruitment of cellular P-TEFb (A) The HIV genome is approximately 9,700 base pairs and encodes 9 genes. Following integration, transcription initiates at the 5’ LTR, and viral proteins are produced by alternative splicing, frameshifting during translation, and proteolytic cleavage of polyproteins. Shown are the genes encoding for the structural proteins Gag and Env (orange), the enzymatic polyprotein Pol (yellow), the regulatory proteins Tat and Rev (green), and the accessory proteins Vif, Vpu, Vpr, and Nef (blue). Nucleotide positions correspond to the HIV-1 reference strain HXB2. (B) The viral protein Tat is required for efficient HIV transcription. Following integration, transcription factors such as NFκB, AP-1, and SP1 prime the HIV LTR for transcription initiation by RNAPII. However, transcription elongation is inefficient due to the presence of negative elongation factors NELF and

3 DSIF, as well as hypo-phosphorylation of Serine 2 in the C Terminal Domain (CTD). The HIV regulatory protein Tat is initially produced in small quantities from rare transcripts that are able complete transcription. Following translation, Tat re-enters the nucleus and recruits P-TEFb to the LTR by binding to TAR, a hairpin RNA structure formed by nascent LTR transcripts. P-TEFb phosphorylates NELF and DSIF (red arrows), removing their inhibitory effects. P-TEFb also hyper-phosphorylates the RNAPII CTD at Ser2, causing conformational changes that stimulate transcriptional elongation and greatly increase viral expression.

Following integration, cellular transcription factors such as NFκB bind to the HIV promoter within the 5’ Long Terminal Repeat (LTR) and initiate transcription [20]. However, early viral transcription is inefficient and terminates early due to the presence of 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) sensitivity inducing factor (DSIF) and the negative elongation factor (NELF) [21]. DSIF and NELF are negative transcription elongation factors that bind to RNA Polymerase II (RNAP II) and promote transcriptional pausing and termination. To overcome this transcriptional block, the HIV regulatory protein Tat binds to the Trans-Activating Response Element (TAR) in the 5’ LTR, a stem-loop secondary RNA structure formed by folding of nascent HIV transcripts [22][23][24]. Simultaneously, Tat recruits a Super-Elongation-Complex (SEC), which includes the Positive Transcription Elongation Factor b (P-TEFb) [25]. P-TEFb consists of Cyclin Dependant Kinase 9 (CDK9) and its cyclin partner Cyclin T1. Tat stimulates P- TEFb to phosphorylate RNAP II at Serine 2 of the protein’s C Terminal Domain, promoting a switch to productive elongation [26]. P-TEFb also phosphorylates DSIF and NELF, causing them to disassociate from RNAP II and further stimulating transcriptional elongation [21] (Figure 1.1B).

While Tat activity massively increases productive transcription of the HIV genome, full-length transcripts are rapidly spliced by the host spliceosome, resulting in the loss of intron regions such as gag-pol. The HIV genome is highly ordered and contains 16 splice sites capable of producing over 40 different mRNA transcripts [27]. As such, full expression of all viral genes requires alternative splicing in a tightly regulated pattern [28]. The HIV genome is transcribed in full, then multiply spliced to produce Tat, Rev, and Nef transcripts [29]. However, splicing of the HIV genome is often incomplete due to several suboptimal splice sites, resulting in nuclear accumulation of incompletely spliced and unspliced mRNAs [30][31]. Because these mRNAs contain introns, they are not exported by typical nuclear export factors. Instead, nuclear export and translation of these single-spliced and unspliced mRNAs requires the intervention of the HIV regulatory protein Rev. Rev recognizes and binds to the Rev-Response Element (RRE)

4 sequence within incompletely spliced mRNAs and, via the CRM1 pathway, facilitates nuclear export before these transcripts are completely spliced. Rev action allows translation of proteins from single-spliced transcripts (Env, Vpu, Vif, and Vpr) and unspliced, full length transcripts (Gag and Gag-Pol polyprotein).

While Gag features its own initiation and termination codons, Pol translation requires a -1 translation frameshift to produce a Gag-Pol polyprotein. After translation, HIV virion assembly occurs at the plasma membrane and is mediated by Gag and Gag- Pol. Meanwhile, Env is processed by host proteases from a precursor form (gp160) to a mature form (gp120 and gp41), which is then delivered to and inserted into the host cell plasma membrane. Emerging, immature virions bud out from the cell membrane, capturing a lipid bilayer envelope that includes Env. Packaged within the virions is Gag, Gag-Pol, 2 copies of the HIV RNA genome, and several other factors [32].

During the budding process, immature virions mature by proteolytic cleavage. Protease present in Gag-Pol polyproteins auto-processes to release itself, then further catalyzes release of RT, Integrase, and more Protease. Protease also processes Gag into the capsid proteins p17 (Matrix), p24 (capsid), p7 (Nucleocapsid), and p6. The processed proteins then rearrange in a structured manner to create a mature virion. Infection of new cells requires maturation of the virion, and can occur via several mechanisms. Cell-free viral particles can directly infect new cells, but HIV can also be transmitted through cell-to-cell mechanisms during close contact between infected and target cells [33].

1.3. HIV Pathogenesis

HIV replication in actively-infected cells is cytopathic through a variety of pathways including direct cytotoxicity of viral proteins, induced apoptosis, and immune- mediated clearing [34][35]. Because HIV predominately infects and kills CD4+ T helper cells, which are important in directing and regulating immune responses, untreated HIV infection compromises the immune system and enhances progression to AIDS [36]. At this stage, the immune system is severely weakened and the risk of mortality from opportunistic infections and AIDS-associated cancers is greatly increased. Fortunately, progression to AIDS is halted by treatment with cART, which can restore CD4+ T cell counts to normal levels [37]. However, cART does not represent a cure due to the

5 presence of the HIV latent reservoir. Latently infected cells can reactivate to produce infectious virus at any time. As such, within weeks of treatment interruption, viral rebound from the reservoir allows viremia to reach pre-treatment levels. Therefore, current treatment guidelines recommend that cART be initiated immediately after HIV diagnosis and continued for the entire lifetime of the patient [38][39].

1.4. Antiretroviral Therapies: Drug Classes, Mechanisms, and Limitations

There are currently seven classes of approved HIV drugs: Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), Protease Inhibitors (PIs), Fusion inhibitors, CCR5 antagonists, and Integrase strand transfer inhibitors (INSTIs). Each class targets a different process necessary for the HIV replication cycle. The first licensed ARV, the NRTI azidothymidine (AZT), was approved in 1987. While AZT and other early NRTIs were associated with limited effectiveness and poor tolerability, the development of NNRTIs and PIs in the next decade represented a step forward in drug development, allowing combination triple therapies that for the first time could durably reduce viral replication to levels undetectable by conventional RT-PCR-based assays [40]. The development of cART transformed HIV infection from a fatal infection to a manageable chronic disease. More recently, new classes of ARVs including INSTIs have improved the diversity of treatments available for HIV. Currently, first-line cART is recommended as a triple therapy consisting of 2 NRTIs plus an INSTI (Dolutegravir) or 2 NRTIs plus an NNRTI (Efavirenz, EFV) [41].

RT inhibitors make up two classes of ARVs, NRTIs and NNRTIs. NRTIs mimic nucleosides during the reverse transcription process, but lack a 3’ hydroxyl on the sugar group. Normally during reverse transcription, incoming nucleosides form 5’ – 3’ linkages with the previously-incorporated nucleoside, resulting in a growing viral DNA strand. However, when RT instead incorporates an NRTI into the growing HIV DNA strand, incoming nucleosides are unable to form 5’ – 3’ linkages due to the lack of a 3’ hydroxyl. This results in early strand termination and greatly limits HIV reverse transcription and replication. Conversely, NNRTIs such as Efavirenz (EFV) inhibit reverse transcription by binding directly to RT near the active site and inducing a conformational change in the substrate binding site, greatly reducing enzymatic activity [42].

6 PIs such as Indinavir (IND) are peptide-like small molecules designed to mimic HIV Protease substrates and thus occupy the active site, preventing enzymatic activity [43]. Protease function is necessary for virion maturation and is thus required for the production of infectious virions. During treatment with PIs, HIV virions continue to bud off from infected cells, but are unable to mature and are thus unable to infect new target cells [43].

INSTIs such as Raltegravir (RAL) are a relatively new class of ARVs that target the active site of HIV Integrase to prevent enzymatic activity [44]. Normally, Integrase catalyzes nucleophilic attack of the host chromosome with the 3’ hydroxyl of HIV DNA strands, leading to insertion of the viral sequence into the host genome. INSTIs block that process and prevent integration of HIV into the host genome. In particular, the INSTI Cabotegravir is highly potent and pharmokinetically stable, and, in combination with the NNRTI Rilpivirine, is currently in clinical trials as a once-monthly injection [45]. Long- Acting ART would represent a progression away from daily pill-based therapies, a direction that is welcomed by PLWH [46][47].

Several steps of the HIV entry process are targeted by licensed therapies. HIV Fusion inhibitors, such as the peptide Enfuvirtide [48], bind directly to gp41 and block conformational changes needed for viral membrane fusion with the host cell. These inhibitors are designed to mimic gp41 and thus inhibit fusion via dominant-negative binding [49]. Alternatively, CCR5 antagonists prevent gp120 binding to the coreceptor CCR5, preventing HIV attachment and membrane fusion. Unlike other ARVs which target viral proteins, the CCR5 antagonist Maraviroc (MVC) binds directly to a host protein (CCR5) to induce a conformational change that prevents gp120 recognition [50].

Current ARVs are generally well tolerated, effective medications, and have transformed HIV infection into a chronically manageable health condition. Unfortunately, side-effects, growing viral resistance, and stigma continue to be an issue for patients. For example, long-term use of PIs is associated with metabolic disorders such as dyslipidemia [51][52], while HIV-stigma is associated with lower treatment adherence, depression, and anxiety in PLWH [53][54]. Additionally, as the population of PLWH ages, comorbidities such as renal disorders, cancers, and cardiovascular disease, among others, occur at higher rates than in people aging without HIV or taking ARVs [5]. The additional medications required for treatment of comorbidities increases pill-burden and

7 the potential for drug-drug side effects [5]. New two-drug cART regimens, such as pairing the INSTI Dolutegravir with the NRTI Lamivudine (3TC), have recently been approved for patient use and hold promise in lessening drug-drug interactions and adverse drug events, although the actual long-term benefits or efficacies are not yet known [55][56][57].

Viral resistance to ARVs continues to be a global concern, particularly in areas with restricted access to multiple classes of ARVs [58][59]. Due largely to an error-prone reverse transcription process, HIV features remarkably high mutation rates and genetic diversity, allowing rapid adaptation to selection pressures such as immune response and ARV treatment [60][61][62]. Resistance to ARVs occur from point mutations in the targeted enzyme that render the drug less effective at blocking activity. For example, drug resistant mutations in RT can decrease affinity to NRTIs, or enhance removal of NRTIs after incorporation [37]. Certain mutations in essential enzymes such as RT and Protease generally confer a fitness cost, although continued drug usage can allow the virus to develop secondary mutations that compensate for the primary mutations and restore enzyme function [63]. Furthermore, resistance to newer classes of ARVs such as INSTIs and Fusion inhibitors has also been observed [64][65][66][67]. Additionally, many resistance mutations allow cross-resistance against multiple ARVs within a class, and some resistant strains feature resistance to multiple classes of ARVs. To date, drug- resistant HIV strains have been identified in patients or generated in vitro for all classes of ARVs, including those that target host proteins [66][67][68][69]. Drug-resistant viral strains can lead to regimen failure, complicating treatment. As such, new ARV development is constantly needed in the absence of an effective vaccine or cure.

Even in virally-suppressed individuals on cART, HIV infection is associated with increased risk of chronic inflammation and other non-AIDS comorbidities [70][71]. This is because although current ARVs can inhibit viral replication to below limits of detection, they do not block low levels of viral protein expression from already infected cells. Additionally, there is some evidence for the existence of immune and pharmacologically- privileged sites that may allow persistent, low level viral replication from the reservoir even in patients adhering to cART [72][73][74]; however, the existence of this residual replication is controversial, as several studies have found no evidence of ongoing replication [16][18]. In the absence of a fully sterilizing cure, inhibitors of post-integration processes such as HIV transcription, RNA processing, and protein translation may be

8 useful in blocking viral protein expression or any potential replication from the latent reservoir, and could be used to support existing drug regimens in cases where existing regimens have failed due to antiviral resistance or adverse side-effects. Although several promising candidate Post Integration Inhibitors (PIIs) are currently in pre-clinical development [75][76], there is a likely need for additional, novel PII candidates.

1.5. The HIV Latent Reservoir: Barrier to a Cure

The existence of latent viral reservoirs represents the main barrier to achieving an HIV cure. Latently infected cells feature low or no proviral expression and are thus undetectable by the immune system [77]. Current ARVs only target viral replication and do not affect latently infected cells. Additionally, there is no established cell-surface marker of latently infected cells to facilitate discrimination between infected and healthy cells. Recently, the FCγ receptor CD32 was reported as a reservoir marker [78]; however the original report has been refuted by three studies that suggest CD32 is instead associated with T cell activation [79][80][81]. The ability of HIV to enter a latent state during infection was first observed in cell lines [82][83], and eventually confirmed to occur in patients with undetectable viral loads due to cART [84]. The persistence of the latent reservoir ensures that ARV-treatment interruption will result in viremia rebound within several weeks, necessitating life-long cART [85].

Current detection methods estimate the size of the latent reservoir to be 1-100 in 106 million CD4+ T cells, although the number of cells infected with defective, uninducible proviruses is higher [86][87]. In fact, it is estimated that less than 10% of integrated proviruses are replication-competent, while most feature large internal deletions, hypermutation, or other defects [88][89]. Additionally, initiation of cART quickly after infection correlates with a lower reservoir size [90]. Among CD4+ T cell populations, resting memory T cells make up the largest proportion of the latent reservoir due to long half lives and ability to self renew [91]. Consequently, the half life for clearance of the viral reservoir during sustained cART is approximately 44 months. Full elimination of the virus at this clearance rate has been mathematically modelled to occur after 60-70 years, or essentially the entire life of the patient [92]. Additionally, although the main reservoir is found within memory CD4+ T cells, recent research has shown that HIV-permissive macrophages and other cell types may also contribute to the latent reservoir [93][94].

9 There are two possible mechanisms for persistence of the HIV latent reservoir. First, there is some evidence that latently infected cells compartmentalized in immune- and pharmacologically-privileged sanctuary tissues, such as lymph nodes, testes, and the central nervous system, are exposed to lower drug concentrations and fewer immune responses. This potentially facilitates continuous, low-level viral replication and reservoir reseeding [73][72]. However, several studies have been unable to detect ongoing viral replication in patients on cART [18][16], and its existence is controversial [95]. Secondly, in the absence of active viral replication, latently infected cells can persist and multiply through clonal expansion [19][96].

At a cellular level, many factors control or contribute to HIV latency, including cell activation state [77], cell size and cell cycle progression [97], integration site [98], availability of transcriptional host cofactors such as NFκB and/or P-TEFb, epigenetic silencing [99], and stochastic fluctuations in Tat expression [100]. In general, after infecting a CD4+ T cell, HIV requires cellular activation for optimal integration [101][102]. However, in some cases, reversion to a resting state post-integration causes changes in transcriptional patterns and sequestering of HIV-required cofactors, promoting latency [77]. Indeed, cell activation with stimulating cytokines reverses latency in latently infected cells [103].

The HIV promoter in latently infected cells is associated with repressive epigenetic modifications such as chromatin hypoacetylation and repressive histone methylation [99][104][105]. These modifications are made by recruitment of Class I Histone Deacetylases (HDACs) [106][107][108] and other repressive epigenetic factors [109][110] to the LTR promoter, and drive latency establishment by attenuating transcription factor binding, transcription initiation, and ultimately Tat expression [111]. Inhibition of the epigenetic factors responsible for transcriptional repression at the LTR can lead to reversal of HIV latency [99][109]. For example, HDAC inhibitors are being pursued clinically due to their ability to trigger HIV transcription, potentially leading to identification and elimination of latently infected cells [112][113][114].

Finally, studies of viral transcriptional circuitry have implicated stochastic fluctuations in Tat expression as the key driver of latency establishment and reversal, independent of cell activation state [100][115]. This suggests that latency establishment may be a feature of natural HIV transcriptional mechanisms and potentially selected for

10 during viral evolution, rather than being accidentally dependant on host factors such as cell activation state [116]. For example, latency is never established in cells which express Tat in trans, regardless of activation state, while Tat attenuation by mutation can promote latency establishment in vitro. Additionally, patients with well-suppressed viremia often feature Tat-attenuated viruses in their latent reservoir [111][117].

The latent reservoir is a major barrier in the search for an HIV cure. Strategies to eliminate, silence, or control latent HIV are under development and require the understanding of the multitude of factors involved in latency establishment and reversal. Additionally, the development of novel and innovative therapeutics that target the HIV latent reservoir will be necessary.

1.6. HIV Cure or Remission Strategies

HIV cure or remission strategies must address the latent reservoir. Essentially, there are two broad categories for HIV cure strategies, a sterilizing cure and a functional cure. A sterilizing cure would involve compete eradication of replication-competent HIV from the patient. A functional cure, also called HIV remission, would require long-term control of viral rebound without treatment. Currently, the only known sterilizing cure for HIV involves allogeneic haematopoietic stem-cell transplantation from a donor resistant to HIV infection due to homozygosity for the CCR5-Δ32 mutation, which results in non- functional CCR5 proteins and ablation of viral entry into target cells [118][119]. In this procedure, radiation or chemotherapies eradicates patients’ immune systems, which are then repopulated from donors’ CCR5-Δ32 bone marrows. After a successful procedure, any remaining CCR5-dependant viruses are unable to infect the newly resistant target cells and are eventually eliminated. The Berlin Patient, the first person cured of HIV in this manner, has remained virus-free for over 10 years [118]. Recently, the London patient has reportedly been in long term remission 18 months after cART interruption, with no detection of viremia or HIV DNA, and represents a second potential cure [119]. Unfortunately, this procedure carries a high mortality rate and has not successfully cured HIV infection in all cases, and thus has only been attempted in cases where the patient requires the transplant to treat an aggressive blood cancer [120][121][122][123]. As such, this approach is neither safe nor scalable to the global population of PLWH. However, the success has inspired several gene therapy strategies to create an HIV-

11 resistant immune system without transplant, although more research is required [124][125].

Alternatively, approximately 1% of people infected with HIV are able to spontaneously control the infection without the assistance of cART and despite replication-competent virus [90]. These “Elite Controllers” typically express human leukocyte antigen (HLA) alleles associated with a more robust CD8+ T cell response to HIV. Additionally, some patients who initiate cART very early after infection have been able to control viral rebound after cART interruption and have been termed “post- treatment controllers” [126][127][128][129][130]. High profile examples of this include the Mississippi Baby and the Visconti Cohort, who sustained viral remission for several years without cART. Mechanisms of post treatment control are unclear, but a major contributing factor is that early cART initiation limits the size of the HIV reservoir, helping to control viral rebound for long periods of time [129]. Both elite control and post- treatment control represent functional control of HIV, where the virus is still present but is controlled in the absence of medication. However, low levels of viremia and replication in these populations can result in chronic inflammation and depletion of CD4+ T cells [130][131]. Additionally, post-treatment control is unlikely to represent a true functional cure in the absence of protective host immune factors, and viral rebound is eventually observed in most early cART initiators [129].

Additionally, decades of HIV vaccine development have failed to produce a licensed product [132]. To date, only a single vaccine trial, RV144, has demonstrated any efficacy [133]. The RV144 trial demonstrated a modest 31.2% reduction in HIV acquisition and induced a non-neutralizing binding antibody response, but did not result in viral control within infected individuals [134][135]. Vaccine development has been challenged by the vast genetic diversity of HIV and the heavy glycosylation of Env, which is the only viral antigen present on the surface of infected cells [136][137]. Additionally, vaccine-driven immune responses toward an antigen results in recruitment of CD4+ T cells to infected cells, which can facilitate viral transmission [138]. Finally, an effective HIV vaccine needs to prevent, rather than simply control, HIV infection to preclude establishment of a latent reservoir. Continuation of vaccine research and development is important, and an effective vaccine would revolutionize the global effort against the HIV epidemic.

12 Two strategies are proposed to conclusively deal with the HIV latent reservoir by targeting the reservoir directly: “Shock and Kill” and “Block and Lock”. The Shock and Kill approach involves reactivation of latent viruses by Latency Reversing Agents (LRAs), combined with immune-boosting strategies. Reactivated viruses would then produce viral proteins, allowing the immune system to recognize and destroy infected cells. Additionally, ARVs would be maintained during these treatments to prevent infection of new cells [139].

Compounds with LRA activity act by releasing cell-mediated blocks on HIV expression. To have therapeutic potential, an LRA must also avoid general T cell activation which could cause adverse patient reactions [140]. For example, HDAC inhibitors such as Vorinostat or Panobinostat relieve repressive heterochromatin at the HIV LTR, increasing NFκB and P-TEFb activity [112][114]. Protein Kinase C (PKC) agonists such as Bryostatin or Prostratin activate the PKC signalling pathway, ultimately leading to increases in NFκB activity in the nucleus [141][142]. Other LRAs act by increasing the amount of free P-TEFb available for Tat-driven transcription. For example, the LRA JQ1 competes with P-TEFb for binding to Bromodomain-containing protein 4 (Brd4), a cellular transcription factor responsible for recruiting P-TEFb to host genes. JQ1 removes Brd4 from the HIV promoter and increases the amount of free P-TEFb available to be recruited by Tat [143][144]. Alternatively, Hexamethylene bisacetamide (HMBA) releases P-TEFb from an inhibitory complex formed by HEXIM1 and 7SK small nuclear RNA (snRNA) [145]. In resting T cells, P-TEFb availability is tightly regulated by either sequestering in the inhibitory complex or binding to Brd4 [146]. LRAs that interfere with that regulation increase that amount of free P-TEFb and therefore help Tat coopt P- TEFb for viral transcription.

Despite robust in vitro results, the Shock-and-Kill approach has not yet succeeded in clinical trials [140][147]. For example, although HDAC inhibitors have been shown to transiently increase viral transcription in patients, there has been no resulting decrease in the size of the latent reservoir [113][148][149]. Additionally, determining the optimal dosing regimen for HDACi such as Vorinostat has been complicated, as serial treatments result in inducible proviruses becoming transiently refractory to reactivation [150][151]. Furthermore, HDACi treatment may impair immune responses toward reactivated viruses, complicating the “kill” in the Shock and Kill strategy [152][153]. It is becoming increasingly clear that due to the heterogeneity and dynamism of the latent

13 reservoir, currently identified LRAs are probably not potent or selective enough to reactivate every latent cell [139]. However, cotreatment with LRAs of different classes results in strong synergistic activation of latent HIV, supporting the potential direction of combination LRA therapy [154][155]. Additionally, the discovery of new classes of LRAs or combinations of LRAs with better efficacy is needed. For example, the combination of a TLR7-agonist as the "shock" with a broadly neutralizing antibody as the "kill' has showed promise in early animal trials [156]. Moreover, novel LRA classes such as Second Mitochondrial Activator of Caspases (SMAC) mimetics to activate NFκB without involving the PKC pathway and without activating cells show promise as more specific LRAs [157], but further research is necessary.

In contrast to Shock and Kill, the Block and Lock strategy aims to fully and durably suppress HIV latency reversal, rendering the latent reservoir incapable of reactivation even following suspension of cART. At best, promotion of a lasting deep latent phenotype could result in drug-free HIV remission and a functional cure. Alternatively, even if permanent suppression is not achieved, Block and Lock-mediated delays in viral rebound could support a shift towards long acting antiviral treatment and supplement traditional ARVs while reducing pill burden and the chronic effects of long- term daily medications.

The Block and Lock approach attempts to make use of PIIs or other strategies to durably block expression from integrated proviruses. In this manner, compounds that can increase epigenetic restrictions at the promoter, inhibit Tat function, interfere with HIV mRNA splicing, suppress Rev function, or block viral protein translation are of considerable interest. A leading Block and Lock candidate drug is the direct Tat inhibitor Didihydro Cortistatin A (dCA), which has been shown to promote latency during prolonged treatment of multiple latently infected cell lines as well as in primary cells from individuals on suppressive cART. dCA binds specifically to the basic region of the Tat protein and prevents it from associating with TAR, thus blocking Tat-dependant transcription [158]. Treatment greatly suppresses residual viral transcription compared to ARV treatment alone, an effect which is durable even after dCA is removed from cultures. Additionally, dCA treatment renders cells refractory to stimulation by LRAs, and delays viral rebound in infected humanized mice, compared to ARV-treatment alone [75][159]. Importantly, treatment with dCA increases hypoacetylation and the presence of repressive epigenetic host factors at the HIV promoter, introducing durable blockages

14 to HIV transcription that remain even after the drug is removed, providing a mechanism for durable HIV remission in the absence of ARVs [160]. Furthermore, efforts to induce resistance to dCA resulted in viral mutations that increased basal transcription from the provirus, suggesting that prolonged dCA treatment may select for mutants that are unable to maintain latency and are thus more easily detected and eliminated [161]. However, dCA is still in preclinical development and has not been tested in humans.

Inhibitors of HIV splicing and Rev function are also of interest as PLAs. Efficient HIV replication requires balanced, highly regulated splicing patterns, and compounds that interfere with the host spliceosome have been shown to disrupt HIV replication and prevent latency reversal [162][163]. In particular, ABX464 interferes with viral mRNA splicing by binding to the Cap Binding Complex, a complex that regulates both splicing and nuclear transport of mRNA [76]. ABX464 induces over-splicing of HIV transcripts such that fewer single- and unspliced mRNAs are produced, resulting in fewer translated structural proteins. ABX464 treatment in HIV-infected humanized mice blocked viral replication and delayed viral rebound after treatment was stopped, suggestive of Block and Lock activity [76]. Conversely, it has been suggested that the delay in viral rebound was instead due to viral-mediated depletion of target cells prior to and during treatment, creating a lack of targets and slowing viral spread once treatment was stopped [164]. Regardless, the prevention of viral protein expression could prevent the chronic inflammation suffered by PLWH on long term cART [165]. Further clinical studies of ABX464 are underway [166].

Other preclinical PIIs with Block and Lock potential are also under study. The small molecule ZL0580 specifically binds to BRD4 and increases BRD4-CDK9 binding while reducing Tat-CDK9 interactions [167]. ZL0580 has been demonstrated to delay viral rebound from latently infected primary cells, suggesting Block and Lock potential. Additionally, Sudemycin D6, a specific inhibitor of splicing factor 3B subunit 1 (SF3B1), inhibits Tat-driven viral reactivation and was able to prevent latency reversal even after drug washout [168]. SF3B1 was found to interact directly with both Tat and P-TEFb, although its exact role in HIV transcription is unclear. Furthermore, long noncoding RNAs (lncRNA), such as HEAL, have been shown to play important roles in proviral transcription. Silencing expression of the HEAL lncRNA results in delayed viral rebound, suggesting that HEAL could be a Block and Lock therapy target [169]. Finally, activation

15 of the cellular mTor signalling pathway is required for HIV latency reversal and may be relevant in Block and Lock therapy strategies [169].

1.7. Conclusion

Ending the HIV epidemic will require a cure. Currently, all licensed HIV inhibitors target HIV replication and infection of new cells, but do not target latently infected cells. Aside from an inability to cure HIV infection, current ARVs also feature limitations such as growing antiviral resistance, the need to be taken for life, and an inability to completely suppress low levels of viral protein expression, which can lead to chronic inflammation and other comorbidities in PLWH. PIIs, used in support of traditional ARVs, could potentially alleviate these limitations. Development of PIIs will improve the repertoire of treatment options available in the clinic, in addition to introducing a new class of ARVs that could be used to salvage treatment failure in cases of multidrug antiviral resistance.

Additionally, the Shock and Kill approach to a sterilizing HIV cure has failed in the clinic to date. The Block and Lock approach to durably silence viral expression from the latent reservoir represents an alternative approach to a functional HIV cure, and one that could be used in combination with existing and future therapies. However, there are few candidate Block and Lock agents in development, suggesting a likely need for new candidate drugs.

1.8. Thesis Objectives

The main objective of this thesis is to describe the identification and mechanisms of potential PIIs discovered in high-throughput screens of natural and synthetic compound libraries. In Chapter 2, I describe the methods and results of a screen of natural products from Africa, from which we identified several flavonoid compounds which inhibit replication of wild-type as well as ARV-resistant HIV. I further investigate the mechanisms of these flavonoids, and show that they act at a post integration step of the viral life cycle. While a subset of the flavonoids inhibit Tat-mediated viral transcription, a structural derivative with similar antiviral and PII activity does not. Finally, I explore the Block and Lock potential of the identified flavonoids, and show that a

16 subset promote latency in long term treatment, but that this suppression is reversible after drug washout.

In Chapter 3, I explore potential mechanisms and cellular targets for the antiviral activities of these flavonoids. First, I show that the subset of flavonoids featuring anti-Tat activity are more potent inhibitors of the Tat-required kinase CDK9 than is the flavonoid with no anti-Tat activity, suggesting an explanation for the observed differences in antiviral mechanisms. I then further explore the mechanisms of flavonoid PII activity, including their effects on Casein Kinase II (CK2) activity, HIV alternative splicing, and Rev function. Furthermore, I relate the reversibility of the flavonoids’ Block and Lock activity to concurrent HDAC inhibition, and demonstrate that specific CDK9 inhibition is a potential avenue to establish durable Block and Lock.

In Chapter 4, I describe the results of a high throughput screen of a synthetic compound library to identify specific inhibitors of Tat function, and explore the mechanisms and antiviral activities of the two compound hits. Finally, Chapter 5 is a conclusion that summarizes the thesis and provides future directions and implications of my research.

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30 Chapter 2. Identification of Flavonoids that Suppress HIV-1 Latency Reversal from African Natural Products

2.1. Abstract

The Block and Lock strategy for an HIV functional cure requires durable suppression of HIV-1 latency reversal. However, there are currently no licensed inhibitors of HIV-1 that block expression of viral proteins from integrated proviruses. In an effort to identify novel post integration inhibitors of HIV, we screened 201 compounds from the pan-African Natural Products Library (pANAPL) in a multicycle viral replication assay using CEM-GXR reporter cells, and identified 4 flavonoids with antiviral activity: apigenin, chrysin, luteolin 7-O-β-D-glucoside (L7G), and 3-methyl-6-prenyl-apigenin (3M6PA). We further confirmed antiviral activity in Peripheral Blood Mononuclear Cells (PBMCs) and against ARV-resistant HIV strains, and show that apigenin, chrysin, and L7G act against post integration processes. Interestingly, while apigenin, chrysin, and L7G are capable of inhibiting activation of latent proviruses by latency reversing agents in J-Lat and OM-10.1 cells, apigenin and chrysin act at the Tat-functional level while L7G does not. Furthermore, we show that apigenin and chrysin promote latency during long- term treatment of J-Lat 10.6 cells, suggesting that they may have potential as Block and Lock agents.

2.2. Introduction

Novel HIV PIIs potentially could complement traditional cART, and may have potential in Block and Lock strategies for an HIV functional cure [1]. Natural products are a versatile source of bioactive compounds with a wide variety of activities, including anti- HIV [2][3], and our lab and others have successfully identified novel HIV inhibitors and HIV latency modulators from natural product libraries in the past [4][5][6][7][8]. To identify novel inhibitors of HIV in the present study, we tested 201 compounds from the pan African Natural Product Library (pANAPL) in a multicycle viral replication assay in a reporter CD4+ T cell line. We then characterized compound “hits” for their ability to inhibit HIV latency reversal in cell line models of HIV latency.

31 We identified 4 compounds that inhibited HIV replication, all of which were flavonoids. Flavonoids are a diverse chemical group of plant metabolites that most often take the form of two 6 carbon phenyl rings, the A and B rings, attached by a third 3 carbon linker, the C ring [9]. The flavonoid subtype is determined by the conformation and substitution of the C linker, while A and B ring substitutions determine the chemical diversity within the subgroups. In plants, flavonoids have a variety of functions including pigmentation, cell signalling, UV protection, antioxidation, and antimicrobial activities [10]. Thousands of flavonoid derivatives have been identified from plants, and many feature diverse biological activities such as antioxidative, antibacterial, antiviral, and anticancer properties [9][10]. Due to their abundance in the human diet and the large breadth of biological activities, flavonoids have been researched extensively, and several have been shown to have anti-HIV activity [11][12].

Here, I describe the natural product screen, the identification of several flavonoids that inhibit HIV replication and latency reversal, and the investigation of their mechanisms. I also describe the investigation of these compounds for Block and Lock potential.

2.3. Materials and Methods

2.3.1. Cells and Reagents

CEM and Jurkat T cells (Clone E6-1) were obtained from the American Tissue Culture Collection (ATCC). CEM-derived GXR25 GFP-reporter T cells (CEM-GXR) have been previously described [13]. J-Lat T cells (clones 9.2 and 10.6) were obtained from the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (contributed by Dr. Eric Verdin [14]). OM-10.1 macrophages were also obtained from the NIH AIDS Reagent Program (contributed by Dr. Salvatore Butera [15]). Jurkat Tet-Tat-Dendra+HIV LTR- mCherry (JurTat) cells are described previously [16] and were obtained as a kind gift from Dr. Leor Weinberger (Gladstone Institute, UCSF). Cell lines were cultured in R10+ media [RPMI 1640 with HEPES and L-Glutamine, 10% Fetal Bovine Serum (FBS), 100 U of penicillin/mL, and 100 µg of streptomycin/mL (Sigma)]. HEK-293T cells (Clontech) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L Glucose and L-Glutamine (Lonza), supplemented with FBS and antibiotics as above. PBMCs were obtained from HIV-negative donors following written informed consent under a study

32 protocol approved by the Institutional Review Boards of Simon Fraser University and the University of British Columbia – Providence Health Care Research Institute. PBMCs were cultured in R10+ media supplemented with 100 U/mL human IL2.

All pANAPL compounds were previously confirmed ≥ 95% pure by melting point analysis [17] and were dissolved in DMSO to stock concentrations of 5 mg/mL. TNFα, panobinostat, and prostratin were obtained from Sigma-Aldrich. Luteolin and additional sources of apigenin, chrysin, and luteolin-7-glucoside were purchased as powder from Sigma-Aldritch. Control ARVs Efavirenz and Indinavir were purchased from Sigma, while Raltegravir was provided by Dr. Peter Cheung (BC Centre for Excellence in HIV/AIDS, Vancouver, Canada).

2.3.2. Plasmids

pNL4.3, expressing the HIVNL4.3 subtype B reference strain, was obtained from the NIH AIDS Reagent Program (provided by M. Martin)[18]. pCMV-Tat, an HIV-1 Tat expression vector, and pCMV-ΔTat, its corresponding control lacking Tat, are previously described by Malim et al. [19]. pSelect-GFP expression plasmid was obtained from Invivogen.

2.3.3. Virus Production

HEK-293T cells were transfected with 15 µg pNL4.3 via Lipofectamine LTX (Invitrogen) according to manufacturers instructions. Cell free supernatants containing

HIVNL4.3 virions were collected after 48 hours and stored at -80°C. To estimate viral titres,

CEM-GXR cells were infected with known volumes of HIVNL4.3 stock for 48 hours. CEM- GXR cells feature an integrated Tat-driven GFP reporter construct, and GFP expression in these cells is indicative of active HIV infection. The percentage of GFP expressing cells was quantified by flow cytometry and used to estimate Multiplicities of Infection (MOIs) [20].

ARV-resistant virus strains were generated as described by Tietjen et al [4]. Briefly, patient plasma-HIV-1 RNA sequences were analyzed for resistance mutations using the HIV Drug Resistance Database Genotype Resistance Interpretation Algorithm

[21] and used to generate recombinant HIV-1NL4.3 sequences with resistance mutations

33 to protease and reverse transcriptase inhibitors (HIV-1PR+RT), reverse transcriptase inhibitors (HIV-1RT), and integrase inhibitors (HIV-1INT). Resistance to the PI Indinavir

(IND) and the NNRTI Efavirenz (EFV) by HIV-1PR+RT is primarily conferred by M46L,

I54V, I84V, L90M, K101E, Y181C, and G190A mutations. Resistance to EFV by HIV-1RT is primarily conferred by K103N, V108I, and M230L mutations. Resistance to the INSTI

Raltegravir (RAL) by HIV-1INT was primarily conferred by an N155H mutation. Virus stocks were generated by homologous recombination after co-transfection as reported by Brumme et al [22].

2.3.4. HIV-1 Replication Assays and Compound Screening

CEM cells are an immortalized CD4+ T-lymphocyte line that naturally expresses the HIV-1 coreceptor CXCR4. CEM-GXR cells are a CEM-derived cell line that have been engineered to express high levels of the HIV-1 co-receptor CCR5 and a Tat-driven LTR-GFP expression cassette. Therefore, these cells are susceptible to HIV-1 infection, and viral replication triggers intracellular GFP production. We infected CEM-GXR cells with HIV-1NL4.3 at an MOI of 0.03 for 24 hours. After this, cells were pelleted, re- suspended in R10+ media to 500,000 cells/mL, seeded into 96 well plates at 200 µL/well, and treated with 5 µg/mL of pANAPL compound or 0.1% DMSO vehicle control for a further 72 h. GFP expression was then measured by flow cytometry (Guava EasyCyte 8HT, Luminex). Culture viability was estimated as the relative percentage of compound-treated, infected CEM-GXR cells displaying characteristic forward- and side- scatter parameters compared to infected, vehicle-treated controls. Compounds hits were defined by inhibition of virus-driven GFP production by 50% or greater compared to the vehicle-treated controls. Compounds of interest were then tested in the same infection conditions at multiple concentrations to generate dose-response curves.

Studies involving ARV-resistant virus strains followed the same above procedure, substituting HIV-1NL4.3 with either HIV-1PR+RT, HIV-1RT, or HIV-1INT. Flavonoid anti-viral activity was compared to activity of the licensed ARVs IND, EFV, and/or RAL.

Studies involving PBMCs were performed as described by Mwimanzi et al. [23]. Briefly, PBMCs from 4 healthy, HIV-naïve donors were activated for 3 days with 5 μg/mL phytohemagglutinin, infected with HIV-1NL4.3 for 6 h (MOI = 0.003), then pelleted and resuspended in media supplemented with 100 U IL2 and treated with flavonoids or 0.1%

34 DMSO. Supernatant p24Gag was quantified by ELISA (Xpress Bio, Frederick, MD) on day 6 post-infection. To measure PBMC viability in the presence of flavonoids, the above conditions were replicated without HIV infection. Viable cells were detected by Guava Viacount dye (Luminex, Toronto, ON), which allows detection of live, apoptotic, and dead cells. Proportions of live cells were used to determine culture viability.

2.3.5. Apoptosis Detection Assays

CEM cells were seeded in 96 well plates at 100,000 cells/well then treated for 72 hours with flavonoids, 0.1% DMSO, or 0.1 μM panobinostat, while Jurkat cells were seeded at 200,000 cells/well and treated for 24 hours. 50 µL of culture from each well was stained with Annexin V-APC (Biolegend) as per manufacturers instructions, and apoptosis-positive cells were detected by flow cytometry. Apoptosis in flavonoid-treated cells was normalized to that in DMSO-treated cells and compared to panobinostat- treated cells.

2.3.6. Inhibition of Latency Reversal Assays

J-Lat 9.2 T cells contain an inducible latent provirus with a frameshift mutation in Env and a GFP reporter expressed from the Nef locus. Latency reversal in these cells can be measured by an increase in GFP expression. OM-10.1 macrophage clones contain a full length, latent provirus. p24Gag production is minimal in resting cells, but can be strongly upregulated by LRA treatment.

To test flavonoids for their ability to inhibit latency reversal, J-Lat 9.2 cells or OM- 10.1 cells were seeded in 96 well plates at 200,000 cells/well and co-treated with an LRA and flavonoids or 0.1% DMSO control for 24 hours. J-Lat cells were treated with 10 ng/mL TNFα, 10 μM prostratin, or 0.1 μM panobinostat, while OM-10.1 cells were treated with 1 ng/mL TNFα. J-Lat 9.2 cells were examined for GFP expression by flow cytometry. OM-10.1 cells were fixed and permeabilized using the Cytofix/Cytoperm fixation kit (BD Bioscience), stained for intracellular p24Gag (KC57-RD1 antibody, Beckman Coulter), and examined for p24Gag expression by flow cytometry. J-Lat cells were also examined for p24Gag expression as described above to confirm GFP expression results.

35 2.3.7. GFP Quenching Assay

5x106 CEM cells were resuspended in 200 µL of Opti-MEM media containing 5 μg of pSelect-GFP. Cells were transfected in 0.4 cm cuvettes using a GenePulser MXCell electroporation system (Bio-Rad) using a single 250 V square wave pulse for 25 milliseconds. Cells were rested for 10 minutes at room temperature prior to seeding into 96 well plates at 100,000 cells/well, then treated with flavonoids or 0.1% DMSO control. After 24 hours, GFP expression was detected by flow cytometry.

2.3.8. HIV-1 Tat Inhibition Assays

As described above, CEM-GXR cells feature a Tat-driven LTR-controlled GFP reporter construct. Tat expression in these cells triggers GFP production. 5x106 CEM- GXR cells were transfected with 1.6 µg of pCMV-Tat or pCMV-ΔTat as described above, then seeded into 96 well plates at 100,000 cells/mL and treated with flavonoids or 0.1% DMSO control. After 24 hours, Tat-driven GFP expression was detected by flow cytometry.

JurTat cells feature an integrated Tat-dendra fusion protein expression construct controlled by a Tet-on promoter, in addition to an integrated mCherry reporter under the control of the HIV-1 LTR promoter. Treatment with the tetracycline analogue doxycycline triggers expression of the Tat-dendra fusion protein, which then activates transcription at the HIV-1 LTR and allows expression of the mCherry reporter. To test flavonoids for Tat- inhibiting activity, JurTat cells were stimulated with 500 ng/mL doxycycline and seeded at 200,000 cells/well in 96 well plates in the presence of flavonoids or 0.1% DMSO control. mCherry and Dendra expression levels were analyzed by flow cytometry after 24 hours. The ratio of mCherry expression to Dendra expression was used as a measure of specific inhibition of Tat function.

2.3.9. Block and Lock Assays

J-Lat 10.6 clones feature a proportion of spontaneously GFP-expressing cells that are not fully latent [24][25]. To test the ability of flavonoids to suppress this background latency reversal, J-Lat 10.6 cells were seeded in 24 or 96 well plates at 200,000 cells/mL in 600 or 200 μL, respectively, and treated with flavonoids or DMSO

36 control. Every 3-4 days, samples were taken and either examined directly for GFP expression or fixed via the cytofix/cytoperm protocol and stained for p24Gag expression (KC57-RD1 antibody). GFP and p24Gag expression were analyzed by flow cytometry. Remaining cultures were re-suspended in fresh media and re-seeded to 200,000 cells/mL, with drug replenished to the correct final concentration. Incubation was continued for 21 days, at which point cells were pelleted, washed 1 X with PBS, and re- suspended in fresh, drug free R10+ media or R10+ containing an LRA (10 ng/mL TNFα or 0.1 μM panobinostat). After 24 hours, cells were analyzed for GFP and p24Gag expression.

2.3.10. Data and Statistical Analysis

Flow cytometry data were analyzed using FlowJo v. 10.5.3 (FlowJo LLC, Ashland, OR). Background GFP in uninfected CEM-GXR cultures or unstimulated J-Lat cultures was set to 0.05%. Figures were generated in Prism v. 5.00 (GraphPad, San

Diego, CA). 50% effective and cytotoxic concentrations (EC50s and CC50s, respectively) were calculated from at least 2-3 independent experiments, using the linear regression function in Prism v. 8.2.1. Data are presented as mean ± SD or mean ± SEM. p values of < 0.05 were considered significant, except where corrected for multiple comparisons by Bonferroni Correction.

2.4. Results

2.4.1. Identification of Flavonoids that Inhibit HIV Replication.

We screened 201 compounds from the pan-African Natural Product Library (pANAPL) for anti-viral activity using a multicycle viral replication assay. CEM-GXR cells contain an integrated GFP reporter controlled by the HIV-1 LTR promoter. In these cells, HIV-1 infection and replication drives GFP expression and thus can be quantified by flow cytometry. Cells were infected with HIV-1NL4.3 (MOI = 0.03) for 24 hours, then washed and incubated with compounds (5 μg/mL) or 0.1% DMSO vehicle control. GFP expression was analyzed by flow cytometry after 72 hours.

37

Figure 2.1. Identification of flavonoids that inhibit HIV replication (A) Flavone backbone with numbered carbons, for reference. (B) Flavonoids of interest. Apigenin, 3-methoxy 6-prenyl apigenin (3M6PA), chrysin, and luteolin-7-Glucoside (L7G) were identified from pANAPL screen as compounds that inhibited HIV replication in CEM-GXR cells by ≥50%. Luteolin is the aglycone form of L7G. Naringenin was present in the screened pANAPL but was inactive.

38 We identified 4 compounds that inhibited virally-induced GFP expression by greater than 50%, representing a 2.0% hit rate from the 201 pANAPL compounds screened. The 4 compounds we identified as inhibitors were all flavonoids of the flavone subclass: apigenin, chrysin, luteolin 7-O-β-D-glucoside (L7G), and 3-methoxy-6-prenyl- apigenin (3M6PA). Flavones are a subclass of flavonoids that feature a C4 ketone and a C2-3 double bond on the C ring (Figure 2.1A). Chemical subgroups, commonly hydroxyl, methoxy, and glucosides, can attach to various carbons on the A and B rings and provide the chemical diversity for the flavone class.

Apigenin, chrysin, and L7G have been previously observed to inhibit HIV replication [11][12], while 3M6PA appears to be a novel inhibitor. All 4 compounds share a flavone background but differ in subgroups attached to the A and B rings (Figure 2.1B). On the A ring, all four flavonoids share hydroxyls at C5 and C7 except for L7G, which instead features a glycosyl group at C7. Flavonoid glycosylation is common and associated with differences in biological activity [26]. Additionally, 3M6PA features a prenylation at C6. On the B ring, chrysin features a non-substituted phenyl ring, while apigenin has a C4’ hydroxyl, L7G has hydroxyls at C4’ and C5’, and 3M6PA has a C4’ hydroxyl and a C3’ methoxy group.

The flavanone naringenin was also present in the compound library but was not active at 5 µg/mL, despite its similar structure to apigenin. Flavanones lack the C2-3 double bond and feature chirality around that position. This difference has been observed to change, and in many cases greatly reduce, the biological activity of flavanones compared to flavones, including in regards to anti-HIV activity [12]. We included naringenin in further experiments as an experimental negative control and to explore the structural significance of the C2-3 double bond in the identified flavones.

Finally, the aglycone form of L7G, luteolin, has also been previously described as an HIV inhibitor [11][27]. Although luteolin was not present in pANAPL, we included it in further studies as a comparison to the activities of L7G, as well as to the activity of the structurally-similar aglycone flavones chrysin and apigenin. The three aglycone flavones differ only by the number and position of hydroxyl groups on the B ring.

To characterize the anti-HIV activity of these 6 flavonoids in greater detail, we performed dose response experiments using the same CEM-GXR assay. GFP was

39 again used as a surrogate of HIV replication, while forward and side scatter parameters of treated, infected cells compared to 0.1% DMSO-treated, infected cultures were initially used here to estimate compound toxicity (Figure 2.2A and B). 50% effective (EC50) and cytotoxic (CC50) doses were calculated and used to estimate the selectivity indices

(CC50/EC50, SI) for each flavonoid (Table 2.1). We further confirmed that reductions in GFP expression corresponded with viral inhibition by staining infected and treated cultures for p24Gag expression. p24Gag expression decreased alongside GFP production, confirming viral inhibition (data not shown). Chrysin (EC50 of 3.3 ± 1.2 μM) and apigenin (4.8 ± 0.56 μM) were the most potent HIV-1 inhibitors. Conversely, luteolin (15.5 ± 10.2

μM) was the least potent flavone, as well as the most cytotoxic (CC50 of 23.8 ± 1.2 μM), featuring an SI of only 1.5. In comparison, L7G (EC50 of 7.0 ± 0.67 μM) featured a higher selectivity index than both apigenin and luteolin (12.5 versus 7.2 and 1.5, respectively). Additionally, although apigenin and chrysin were 1.5 to 2-fold more potent inhibitors, they caused 2 to 2.5-fold greater cytotoxicity than L7G (CC50s of 34.6 ± 5.2 μM and 40.8 ± 2.9 μM versus 86.2 ± 8.9 μM). Interestingly, chrysin and luteolin were able to maintain antiviral activity at lower concentrations than apigenin or L7G. 3M6PA featured a selectivity index of only 3.5, the lowest of the four pANAPL hits. Finally, as expected, the flavanone naringenin was much less potent than the flavones, with an estimated EC50 of 113.5 ± 23.0 µM.

40

Figure 2.2. Flavonoids inhibit HIV replication in vitro (A) Effects of flavonoids on infected CEM-GXR cell culture viability, as estimated by the percentage of cells displaying characteristic forward and side scatter profiles of live cells, relative to infected cultures treated with 0.1% DMSO vehicle control. (B) Effects of flavonoids on HIV replication in CEM-GXR cells, relative to infected, 0.1% DMSO-treated cells. (C) Apoptosis in CEM cells treated with flavonoids. Cells were treated with increasing doses of flavonoids or 0.1 μM of the positive control HDACi panobinostat for 72 hours (dotted red line). Data are shown as fold-increase in Apoptosis over 0.1% DMSO-treated controls. (D) Effect of flavonoids on GFP expressed from a constitutive, non-HIV LTR promoter. CEM cells were transfected with pSelect- GFP plasmid and treated for 24 hours with flavonoids. For A-C, data are the mean ± SEM of results from at least 3 independent experiments. For D, data are the mean ± SD of results from 2 independent experiments.

In order to quantify directly the cytotoxicity of the identified flavonoids, we treated uninfected CEM cells with flavonoid, 0.1% DMSO vehicle control, or 0.1 μM of the HDAC

41 inhibitor panobinostat. Panobinostat is known to stimulate apoptosis in Jurkat cells [6] and was included here as a positive control. After 72 hours, we stained cells with Annexin V-APC and detected apoptosis-positive cells via flow cytometry (Figure 2.2C). Compared to 0.1% DMSO-treated controls, L7G-treated cells featured a 3.5 ± 0.40-fold increase (mean ± SEM) in apoptosis at the highest concentration tested (30 μM), while apigenin, luteolin, and chrysin increased apoptosis 5.6 ± 0.31-fold, 7.1 ± 0.56-fold, and 6.7 ± 0.73-fold respectively. In comparison, 0.1 μM panobinostat caused a 7.6 ± 1.7-fold increase in apoptosis. This supports our viability estimations in Figure 2.1B, with L7G being the least cytotoxic of the four flavones.

CEM-GXR cells feature an integrated GFP reporter driven by the HIV LTR promoter. To ensure that our flavonoids were specific for HIV inhibition, and not blocking general transcription or quenching GFP fluorescence, we electroporated CEM cells with a constitutive GFP-expressing plasmid and treated with flavonoids for 24 hours (Figure 2.2D). At the highest tested concentrations (30 µM), apigenin and chrysin reduced GFP expression to 48.6 ± 0.01% and 73.0 ± 0.04% of DMSO-treated controls, respectively, while L7G and luteolin did not affect GFP production. However, in all cases, blockade of constitutive GFP expression occurred at higher concentrations than the concentrations required to block HIV replication. Nevertheless, these nonspecific effects could result in over-estimation of some EC50 values provided in Table 2.1. So, while there are likely off- target effects of flavonoid treatment at higher concentrations, there is also a selectivity towards inhibition of HIV replication, especially in the case of luteolin and L7G.

2.4.2. Flavonoids Inhibit HIV Replication in PBMC

Luteolin has previously been described to inhibit HIV replication in primary cells [27]. In order to compare the antiviral activity of L7G in primary cells to luteolin, apigenin, and chrysin we infected PBMCs from four HIV-uninfected individuals with HIV-1NL4.3 (MOI=0.003), then treated with compounds as previously described [23]. In parallel, we treated uninfected PBMCs in the same assay conditions and measured viability via Guava Viacount. We did not include 3M6PA in this experiment due to its low SI in CEM- GXR cells.

42

Figure 2.3. Flavonoids inhibit HIV replication in PBMCs Effects of flavonoids on viability and HIV replication in PBMCs. (A) Viability of uninfected PBMCs after 6-day treatment with flavonoids, as measured using Guava Viacount dye and relative to cells treated with 0.1% DMSO. (B) Effects of flavonoids on HIV-1NL4.3 replication in PBMCs infected in vitro, as measured by supernatant p24Gag levels on day 6 post-infection, relative to infected cells treated with 0.1% DMSO. For A and B, data are the mean ± SEM of results from 4 independent donors.

We observed that L7G (EC50 of 2.6 ± 0.76 μM) was in fact more potent than apigenin (5.0 ± 1.6 μM), chrysin (9.2 ± 2.9 μM) or luteolin (12.7 ± 3.2 μM) at inhibiting viral replication in infected PBMCs, as measured by supernatant p24Gag levels (Figure 2.3A). L7G, apigenin, and luteolin caused obvious cytotoxic effects at 30 μM, while chrysin was not cytotoxic at the concentrations tested (Figure 2.3B). As before in CEM-

GXR cells, luteolin was the most cytotoxic flavonoid (CC50 of 13.9 ± 5.9 μM), while apigenin (19.8 ± 6.5 μM) and L7G (18.3 ± 2.5) were less toxic. Additionally, the rank order of SIs for these three compounds remained the same in PBMC compared to CEM- GXR, with L7G (SI of 7.0) being a more selective inhibitor than apigenin (4.0) or luteolin (1.5). A SI for chrysin could not be calculated at the concentrations tested, due to its lack of toxicity. EC50s, CC50s, and SIs are included in Table 2.1.

43 Table 2.1. Summary of EC50s, CC50s, and Selectivity Indices of flavonoids in CEM GXR cells and PBMC CEM-GXR

Selectivity Index Compound EC50 (μM) CC50 (μM) (CC50/EC50)

apigenin 4.8 ± 0.56 34.6 ± 5.2 7.2

L7G 7.0 ± 0.67 86.2 ± 8.9 12.3

luteolin 15.5 ± 10.2 23.8 ± 1.2 1.5

chrysin 3.3 ± 1.2 40.8 ± 2.9 12.4

naringenin 113.5 ± 23.0 173.5 ± 15.5 1.5

3M6PA 9.3 ± 0.51 32.2 3.5

PBMC

Selectivity Index Compound EC50 (μM) CC50 (μM) (CC50/EC50)

apigenin 5.0 ± 1.6 19.8 ± 6.5 4.0

L7G 2.6 ± 0.76 18.3 ± 2.5 7.0

luteolin 12.7 ± 3.2 13.9 ± 5.9 1.1

chrysin 9.2 ± 2.9 - -

naringenin >30 >30 -

EC50, 50% effective concentration. CC50, 50% cytotoxic concentration. Calculated from data presented in figure 2.2 and 2.3, shown as mean ± SEM of 3 independent experiments or 4 independent donors.

2.4.3. Flavonoids Inhibit Replication of ARV-Resistant Viruses

Apigenin, L7G, 3M6PA, and luteolin were tested for their ability to inhibit ARV- resistant HIV-1 strains. These recombinant strains were generated by modifying HIV-

1NL4.3 to include protease, reverse transcriptase, and/or integrase sequences of HIV-1 strains from individuals who had developed resistance to the PI Indinavir (IND), the NNRTI Efavirenz (EFV), and/or the INSTI Raltegravir (RAL). Resulting ARV-resistant virus strains are labelled as HIV-1PR+RT, HIV-1RT, and HIV-1INT, respectively.

44 As expected, replication of HIV-1NL4.3 was drastically inhibited in the presence of

EFV (EC50 of 0.0051 ± 0.001 μM), RAL (0.029 ± 0.016 μM), and IND (0.033 ± 0.011 μM) (Figure 2.4A). In comparison, ARV-resistant strains tolerated higher concentrations of ARVs, consistent with their specific mutations (Figure 2.4B-D). For example, EFV had

30-fold and 140-fold higher EC50s against HIV-1PR+RT and HIVRT, respectively, while RAL and IND featured 58-fold and 161-fold higher EC50s against HIV-1PR+RT and HIV-1INT

(Table 2.2). In comparison, apigenin, L7G, and 3M6PA featured similar EC50s against all drug-resistant viruses, with at most a 2.7-fold increase in EC50 against HIVPR+RT.

45

Figure 2.4. Flavonoids inhibit ARV-resistant HIV replication in CEM-GXR cells Effect of flavonoids and ARVs on replication of ARV-resistant HIV-1 strains in CEM-GXR cells. (A) Effects of indinavir (IND, PI), efavirenz (EFV, NNRTI), and/or raltegravir (RAL, INSTI), Apigenin, L7G, and Naringenin on HIVNL4.3 replication in CEM-GXR cells (B-D) Effects of ARVs and flavonoids on replication of ARV-resistant strains. HIV-1PR+RT, resistance to IND and EFV; HIV-1RT, resistance to EFV; HIV-1INT, resistance to IND.

Luteolin had similar activity against HIV-1NL4.3, HIV-1RT, and HIV-1INT, with at most a 1.8 fold decrease in activity (EC50s of 15.1 ± 10.2 μM, 27.3 ± 9.6 μM, 16.1 ± 11.3 μM, respectively). However, it featured a 7-fold higher EC50 against HIV-1PR+RT (109.7 ± 188.8 μM versus 15.1 ± 10.2 μM), suggesting that those particular mutations may be ablating antiviral function. Luteolin has been shown to have modest activity against HIV-1 RT

46 RNAse H activity (EC50 of 12.8 μM) in a cell-free assay [28]; however, Mehla et al. determined that luteolin did not affect HIV-1 reverse transcription in vitro, and instead acted at a post-integration step [27]. HIVPR+RT also tolerated higher concentrations of L7G (2.7-fold), apigenin (2.6-fold), and 3M6PA (2.1-fold) than did the other mutant viral strains, suggesting that inhibition at the protease or reverse transcription levels of HIV replication may be responsible for some of the flavonoids’ antiviral activity. However, the decreases in activity of the flavones are relatively small when compared to those of the tested ARVs, suggesting that flavonoid antiviral activity is mostly maintained against these resistant strains.

Table 2.2. Summary of ARV and flavonoid activities against ARV-resistant HIV strains.

HIV-1NL4.3 HIV-1PR+RT HIV-1RT HIV-1INT Compound Fold- EC Fold- EC Fold- EC (μM) EC (μM) 50 50 50 50 change (μM) change (μM) change 0.033 ± IND 5.4 ± 1.0 163.6 - - - - 0.011 0.0051 ± 0.16 ± 0.73 ± EFV 31.4 141.1 - - 0.001 0.022 0.13 0.029 ± RAL - - - - 1.6 ± 1.5 55 0.016 10.7 ± L7G 7.1 ± 0.67 18.9 ± 3.3 2.7 1.5 9.9 ± 1.3 1.4 1.4 6.1 ± apigenin 4.8 ± 0.56 12.3 ± 4.0 2.6 8.0 ± 1.6 1.7 1.3 0.97 109.7 ± 27.3 ± 16.1 ± luteolin 15.1 ± 10.2 7.3 1.8 1.1 188.8 9.6 11.3 16.2 ± 9.3 ± 3M6PA 9.3 ± 0.51 19.3 ± 1.4 2.1 1.7 1 1.4 0.85

EC50, 50% effective concentration. Fold change is EC50 against ARV-resistant strain/EC50 against HIV-1NL4.3. Data are shown as mean ± SEM of at least 3 independent experiments

2.4.4. Flavonoids Inhibit Latency Reversal in Multiple Cell Lines

Apigenin, luteolin, and chrysin are reported to inhibit HIV at a post-integration step, and have been shown to inhibit reactivation from latency in model cell lines [12][27]. Furthermore, Mehla et al. demonstrated that luteolin inhibits HIV Tat protein function, and is capable of blocking expression from a Tat-driven reporter construct [27]. However, the mechanism of Tat inhibition is unclear. Additionally, although L7G has been observed to inhibit HIV replication [11], a mechanism of action has not been

47 described. Additionally, it is unclear if the glycosyl moiety of L7G modifies its antiviral effects or targets relative to luteolin and other aglycone flavones. Importantly, flavonoids are reported to target multiple host factors simultaneously and have been described as inhibitors of multiple kinases and signalling pathways [29][30]. As such, many biological activities of flavonoids have not been specifically ascribed to individual cellular targets, including in regards to anti-HIV activity.

In order to examine the ability of flavonoids to inhibit expression from an integrated provirus, we first used the J-Lat model of HIV latency [14]. J-Lats are a Jurkat- derived clonal cell line latently infected with a full-length HIV provirus featuring a frameshift mutation in env and a GFP reporter in the place of nef. In the absence of latency reversing stimuli there is minimal GFP production. Latency reversal can be induced by treatment with Latency Reversing Agents (LRAs) such as cell-activating cytokines, PKC agonists, and HDAC inhibitors [6][25][31].

J-Lat cells (clone 9.2) were treated with LRAs TNFα (10 ng/mL), prostratin (10 μM), or panobinostat (0.1 μM), in the presence of flavonoids or 0.1% DMSO vehicle control. After 24-hour incubation, we analyzed cells for GFP expression by flow cytometry, with DMSO-treated J-Lat 9.2 cells set to 0.05% GFP-positive. Representative flow plots are shown in Figure 2.5A, where we also assessed viral protein expression in cells by staining for p24Gag. GFP expression correlated with p24Gag production. Apigenin

(EC50 of 2.6 ± 0.25 μM), luteolin (5.2 ± 0.59 μM), chrysin (2.04 ± 0.33 μM), and L7G (2.6 ± 0.21 μM) antagonized stimulation by the activating cytokine TNFα with similar potencies (Figure 2.5B). Flavonoids were also potent inhibitors of HDACi-mediated latency reversal, with EC50s of 4.06 ± 0.45 μM, 6.8 ± 0.77 μM, 2.8 ± 0.37 μM, and 4.3 ± 0.42, respectively, against panobinostat stimulation (Figure 2.5C). Against latency reversal by the PKC agonist prostratin, luteolin (10.2 ± 3.2 μM) and L7G (9.5 ± 1.7 μM) were less potent than apigenin (2.9 ± 0.73 μM) or chrysin (1.4 ± 0.34 μM), but still active, unlike naringenin (Figure 2.5D). The ability to antagonize latency reversal by multiple classes of LRAs suggests that the flavonoids are acting on targets required for general latency reversal, rather than targets specific to any single LRA pathway. Additionally, and in agreement with our CEM-GXR viral replication assay, chrysin was consistently the most potent latency reversal inhibitor, while luteolin was the least potent. Alternatively, and consistent with previous reports [12], naringenin was much less potent than the related flavones, and was consistently unable to block 50% of latency reversal.

48

49 Figure 2.5. Flavonoids antagonize HIV latency reversal in multiple cell lines (A) J-Lat cell lines contain a latent HIV-1 provirus where nef expression is replaced with GFP. GFP and viral p24Gag production can be stimulated by treatment with an LRA such as TNFα and detected by flow cytometry. (B-D) Effect of flavonoids on latency reversal by different classes of LRAs. Cells were treated with 10 ng/mL TNFα (B), 0.1 uM panobinostat (C), or 10 μM prostratin (D) in the presence of increasing concentrations of flavonoids. Levels of latency reversal were determined by comparison of GFP induced by LRA+flavonoid treatment to treatment with LRA+DMSO vehicle control. Data is shown as mean +/- SEM from at least 3 independent experiments. (E) Effect of flavonoids on Jurkat viability, as measured by increase in apoptosis. Cells were treated with flavonoids or 0.1 µM panobinostat for 24 hours, then measured for increases in apoptosis by Annexin V-APC staining. Results are shown as mean +/- SD from at least 2 independent experiments. Red line represents apoptosis caused by 0.1 μM panobinostat (7.6 ± 1.3-fold increase over DMSO-treated cells) (F) Effect of flavonoids on latency reversal in OM10.1 cells. Cells were stimulated with 1 ng/mL TNFα in the presence of flavonoids. Levels of latency reversal were determined by comparison of intracellular p24Gag expression in TNFα+flavonoid treated cells compared to TNFα+DMSO vehicle control. Results are shown as mean +/- SD from at least 2 independent experiments.

To examine flavonoid cytotoxicity, we measured apoptosis levels in Jurkat cells treated with flavonoids for 24 hours (Figure 2.5E). Consistent with previous experiments, L7G showed lower toxicity than the other flavones. 30 μM of L7G induced a 1.4 ± 0.34-fold increase in apoptosis, compared to apigenin (4.4 ± 0.68-fold), luteolin (3.4 ± 0.61-fold), and chrysin (2.6 ± 0.75-fold). In comparison, none of the flavonoids were as cytotoxic as 0.1 μM Panobinostat, which increased apoptosis 7.6 ± 1.3-fold.

We also used a second model of HIV latency to ensure that latency-promoting activities were not just cell-line or cell-type specific. OM-10.1 macrophage clones are latently infected with a full-length, infectious HIV provirus, and feature minimal viral protein expression at rest [15]. We reversed latency in these cells with 1 ng/mL of TNFα and co-treated with flavonoids for 24 hours, then analyzed intracellular p24Gag levels

(Figure 2.5F). Apigenin (EC50 of 4.1 ± 0.68 μM), chrysin (4.7 ± 0.3 μM), luteolin (10.3 ± 0.55 μM), and L7G (10.3 ± 1.1 μM) were all able to block latency reversal in these cells, although luteolin and L7G were less potent than apigenin and chrysin. Flavonoid EC50s in both J-Lat 9.2 and OM-10.1 cell lines are listed in Table 2.3.

Table 2.3. Summary of EC50s of latency reversal inhibition by flavonoids in J-Lat 9.2 and OM- 10.1 cells J-Lat 9.2 OM-10.1

TNFα Panobinostat Prostratin TNFα Compound EC50 (μM) EC50 (μM) EC50 (μM) EC50 (μM)

L7G 2.6 ± 0.21 4.3 ± 0.42 9.5 ± 1.7 10.3 ± 1.1

50 Apigenin 2.6 ± 0.25 4.06 ± 0.45 2.9 ± 0.73 4.1 ± 0.68

Luteolin 5.2 ± 0.59 6.8 ± 0.77 10.2 ± 3.2 10.3 ± 0.55

Chrysin 2.04 ± 0.33 2.8 ± 0.37 1.4 ± 0.34 4.7 ± 0.3

Naringenin >30 >30 >30 >30

EC50, 50% effective concentration. Calculated from data presented in figure 2.5, shown as mean ± SEM of at least 3 independent experiments

2.4.5. Aglycone Flavones, but not L7G, Inhibit HIV-1 Tat Function

Tat is essential for efficient provirus transcription after integration [32][33][34][35]. In the absence of Tat, HIV transcription initiates, but transcripts fail to elongate due to the presence of host transcription elongation repressors NELF and DSIF. These factors cause pausing of RNAP II and early termination of transcription. HIV Tat recruits the host factor PTEF-b to the HIV LTR, which relieves the transcriptional block by phosphorylating the Ser2 residue of the CTD region of RNAP II [36]. This phosphorylation event causes a conformational change in RNAP II, activating transcriptional elongation. PTEF-b also phosphorylates NELF and DSIF, causing them to dissociate from the LTR and further stimulating transcriptional elongation.

Luteolin has been described as an inhibitor of Tat function [27]. To explore whether related flavonoids also inhibit Tat, we transfected CEM-GXR cells with a Tat expression vector (pCMV-Tat). CEM-GXR cells contain a GFP reporter controlled by the HIV LTR promoter; as such, Tat expression in these cells is sufficient to stimulate GFP expression, while transfection with a Tat-deleted plasmid (pCMV-ΔTat) was not (Figure

2.6A). Treatment of transfected CEM-GXR cells with apigenin (EC50 of 8.0 ± 0.79 μM), chrysin (5.1 ± 0.34 μM), and luteolin (17.5 ± 3.4 μM) caused a dose-dependent decrease in Tat-driven GFP expression, suggestive of Tat inhibition (Figure 2.6B). Conversely, and as expected due to its relative inability to block latency reversal, naringenin did not affect Tat-driven GFP expression. Intriguingly, L7G, which shows comparable antiviral and PII activity to the aglycone flavones, did not decrease Tat-driven GFP expression, suggesting that it is not a Tat inhibitor.

51

Figure 2.6. Aglycone flavones, but not L7G, inhibit HIV Tat-induced gene expression (A) GFP expression in CEM-GXR cells is controlled by a Tat-driven LTR construct. Transfection with pCMV-ΔTat does not stimulate GFP expression, while pCMV-Tat transfection results in GFP expression. (B) Effects of flavonoids on Tat function in CEM-GXR cells. 5x10^6 cells were transfected with 1.6 µg of pCMV-Tat and treated with flavonoids for 24 hours. Data represent

52 mean +/- SD of at least 2 independent experiments (C) JurTat cells contain a Doxycycline (Dox)- driven Tat-Dendra fusion protein, which then drives mCherry production from an HIV-1 LTR. Tat expression and function are thus uncoupled, and can be examined separately. (D) Effect of flavonoids on Tat function in JurTat cells. Cells were activated with 500 ng/mL Dox in the presence of flavonoids. Data is presented as the ratio of mCherry:Dendra, or Tat function:Tat expression, to show selective activity against Tat at a functional level. Results are the mean +/- SEM of at least 3 independent experiments. (E) Representative flow plots show mCherry and Dendra expression in JurTat cells during no treatment, Dox treatment, or Dox + 15 μM Apigenin treatment. 15 μM Apigenin decreased mCherry expression to a greater extent than it did Dendra expression, suggesting specific inhibition of Tat function.

Tat function at the LTR causes a dramatic increase in the number of HIV transcripts produced, which in turn increases the amount of Tat protein expressed [16][32]. This Tat-driven phase of HIV expression results in a transcriptional positive feedback loop, with Tat protein increasing its own transcription. As such, nonspecific inhibition of gene expression that results in decreased Tat expression may mimic specific inhibition of Tat function in our above CEM-GXR assay. In order to confirm that the flavones were specifically inhibiting Tat function, rather than blocking general gene expression leading to lowered GFP expression from the HIV LTR, we used the Jurkat Tet-Tat-Dendra+HIV LTR-mCherry (JurTat) cell line [16]. In JurTat cells, expression of a Tat-Dendra fusion protein is controlled by a Tet-on promoter construct. Furthermore, these cells contain an integrated mCherry reporter under the control of the HIV LTR promoter. Treatment with the tetracycline analogue doxycycline (Dox) triggers expression of the Tat-dendra fusion protein, which is then free to activate transcription at the HIV LTR and allow expression of the mCherry reporter (Figure 2.6C). Unlike in natural HIV transcription, in this system Tat expression is decoupled from Tat function, with expression instead under Dox control. Therefore, treatment with inhibitors that target the Tat functional level, rather than general gene expression, should result in a decrease in mCherry signal without affecting Dendra production. Alternatively, nonspecific inhibitors of gene expression should decrease both Dendra and mCherry signals equally.

In order to test whether the related flavonoids are specific inhibitors of Tat function, we stimulated JurTat cells with Dox, then treated with flavonoids for 24 hours.

We confirmed that apigenin (EC50 of 14.5 ± 1.5 μM), chrysin (12.9 ± 5.8 μM), and luteolin (9.5 ± 1.5 μM) suppressed mCherry signal greater than Dendra signal, as demonstrated by a decrease in the ratio of mCherry fluorescence to that of Dendra and indicative of selective inhibition of Tat function (Figure 2.6D). However, we also observed concurrent

53 decreases in Dendra production with flavonoid treatment. These decreases were likely indicative of off-target effects, and were not as sharp as the decreases in mCherry expression. For example, on average, 15 μM apigenin decreased Dendra expression from 43.6 ± 4.2% to 17.4 ± 2.9% (mean ± SEM), a 2.5-fold decrease. However, 15 μM apigenin concurrently decreased mCherry expression from 13.1 ± 2.4% to 1.8 ± 0.3%, representing a 7.2-fold decrease. The relatively greater fold-decrease suggests selectivity for inhibition of Tat-induced over Dox-induced fluorescence. Figure 2.6E shows representative flow plots of Dendra and mCherry expression levels in the presence and absence of Dox, and when treated with 15 μM apigenin. Chrysin and luteolin also displayed similar effects.

Conversely, naringenin and L7G did not display the same decrease in mCherry:Dendra ratio, corroborating the earlier observation that these are not Tat inhibitors. For example, 15 μM of L7G decreased Dendra expression from 43.6 ± 4.2% to 21.0 ± 3.2%, a 2-fold decrease and similar to that of apigenin. However, 15 μM L7G only decreased mCherry signal from 13.1 ± 2.4% to 8.1 ± 2.6%, a 1.6-fold decrease. Because the mCherry signal decrease was less than that of Dendra (1.6-fold versus 2- fold), it is likely that the mCherry decrease is simply due to lower expression of Tat, rather than actual inhibition of Tat function. EC50s for flavonoid activities in both models of Tat-dependant expression are listed in Table 2.4.

Table 2.4. Summary of flavonoid EC50s against Tat function in JurTat and CEM-GXR cells JurTat CEM-GXR Compound EC50 (μM) EC50 (μM)

L7G - -

Apigenin 14.5 ± 1.5 8.0 ± 0.79

Luteolin 9.5 ± 1.5 17.5 ± 3.4

Chrysin 12.9 ± 5.8 5.1 ± 0.34

Naringenin - -

EC50, 50% effective concentration. Calculated from data presented in figure 2.5, shown as mean ± SEM of at least 3 independent experiments

54 2.4.6. Apigenin, Chrysin, and Luteolin Reversibly Reinforce Latency

Due to the ability of these flavonoids to suppress latency reversal in cell lines, we next explored their potential as Block and Lock agents. Chrysin has been observed to decrease the time needed for LRA-treated, latently infected cells to return to latency [12]. However, despite their activities as latency reinforcers, to our knowledge these flavones have not been examined rigorously for Block and Lock activity. Block and Lock agents should suppress latency reversal, even in the presence of a latency reversing stimuli. Additionally, the effect should be durable, such that it remains even after drug wash out.

The J-Lat 10.6 clonal line, unlike the J-Lat 9.2 clone, feature a proportion of spontaneously GFP-expressing cells that are not fully latent. These cells make up 5-10% of the population and are detectable by flow cytometry (Figure 2.7A). GFP expression is coupled with p24Gag expression, suggesting that these cells are not just expressing GFP at low levels, but also express late viral proteins. We hypothesized that long term treatment with a Block-and-Lock agent would reinforce latency in J-Lat 10.6 cells, decreasing the proportion of GFP-positive cells over time. To test this, we treated J-Lat 10.6 cells with flavonoids for three weeks, testing samples every 3-4 days.

By day 21, treatment with 10 μM of apigenin and chrysin decreased the number of GFP-positive cells by 85.6 ± 8.2% and 91.1 ± 4.4%, compared to cells treated by 0.1% DMSO vehicle control (Figure 2.7B). As shown in a representative flow plot of day 17 of apigenin treatment in Figure 2.7A, p24Gag levels also decreased alongside GFP levels. While 10 μM luteolin was ineffective (data not shown), 15 μM luteolin decreased GFP expression by 55.4 ± 0.8% on day 21. Conversely, long-term treatment with 10 μM naringenin and 10 μM or 15 μM L7G did not reinforce latency. This suggests that the observed latency promotion by aglycone flavones may be driven by a Tat-specific mechanism. Higher concentrations of L7G were associated with extensive cytotoxicity, despite it being the least toxic flavone in previous experiments. As such, we were unable to continue testing 15 μM L7G after day 9.

Furthermore, to explore whether the latency promotion by apigenin, chrysin, and luteolin was durable, we removed flavonoid by washing with PBS, then stimulated with LRAs for 24 hours or left unstimulated. Following flavonoid removal and without stimulation by LRAs, GFP-expression rebounded to control levels within 24 hours,

55 suggesting that the pro-latency activity of these flavonoids is reversible. Further supporting reversibility, none of the flavonoids inhibited latency reversal by TNFα or panobinostat after being removed from culture (Figure 2.7C).

Figure 2.7. Apigenin, chrysin, and luteolin reversibly promote latency J-Lat 10.6 cultures feature a proportion of cells spontaneously expressing GFP and viral protein. J-Lat 10.6 cells were treated with 10 μM of flavonoid for 17 days, with samples taken every 3-4 days. Samples were fixed, then stained for p24Gag and analyzed by flow cytometry. (A) Representative flow plots show that Apigenin reinforces latency by day 17 compared to DMSO- treated controls. (B) Effect of long-term flavonoid treatment on spontaneous latency reversal in J- Lat 10.6 cells. Results are presented as mean +/- SD of 2 independent experiments. (C) Following 50 day treatment with apigenin, chrysin, naringenin, or 0.1% DMSO control, cells were washed once with PBS to remove flavonoid then treated with LRAs or DMSO control. Data are presented as mean +/- SD of 2 independent experiments

56 2.5. Discussion

Our screen of 201 pANAPL compounds identified 4 related flavones that inhibited HIV replication in CEM-GXR T cells by at least 50%, three of which (apigenin, chrysin, and L7G) had SIs above 7 and EC50s at or below 10 μM. These flavonoids, along with luteolin, have been described as having anti-HIV properties in the past. Hu et. al. described apigenin (EC50 of 9 μM), chrysin (5 μM), luteolin (10 μM), and L7G (7 μM) as HIV replication inhibitors by p24Gag antigen capture assay in infected H9 lymphocytes [11]. These results largely agree with our CEM-GXR assay results. Additionally,

Critchfield et. al. described chrysin (EC50 of 2.7 μM) and apigenin (2.5 μM) as inhibitors of HIV latency reversal in OM-10.1 cells, results that are again comparable with those from our OM-10.1 experiments [12]. Furthermore, Mehla et al. observed that 10 μM luteolin was capable of inhibiting viral replication by greater than 50% in TZM-bl reporter cells, Jurkat cells, and primary human lymphocytes [27]. In our CEM-GXR cells and PBMC, luteolin was less potent, but overall comparable. Mehla et al. also demonstrated that 10 μM luteolin was capable of repressing latency reversal in latently infected, TNFα- stimulated THP89 cells, similar to our J-Lat and OM-10.1 results. Finally, Mehla et al. also demonstrated luteolin-mediated inhibition of Tat-driven LTR expression in Tat- transfected TZM-bl reporter cells, which contain an LTR-driven luciferase reporter.

Although these compounds have previously been observed to inhibit HIV, the molecular mechanism of anti-HIV activity of these flavones had not been conclusively reported. Additionally, the flavones had not been investigated for Block and Lock potential. Furthermore, the effect of glycosylation on post integration inhibition of flavonoids has not been explored. We included the aglycone form of L7G, luteolin, and the flavanone naringenin in further experiments to explore the relationship between structure and anti-HIV activity of these compounds.

We show here that the 4 flavones apigenin, chrysin, luteolin, and L7G all feature similar potency in blocking HIV replication, both in vitro and ex vivo. They also suppress latency reversal by LRAs in latently infected cell lines, indicating post integration inhibitory activity. However, while the aglycone flavones (apigenin, chrysin, luteolin) seem to act through suppression of Tat function, L7G did not specifically target Tat. Additionally, apigenin, chrysin, and luteolin promote latency in long-term treatment of J- Lat 10.6 cells, although luteolin is less potent than apigenin and chrysin. Alternatively,

57 L7G did not promote latency in this manner. This suggests that the latency promotion observed in our study is likely due to Tat inhibition. Unexpectedly, higher concentrations of L7G were associated with extensive cytotoxicity over the long-term experiment, despite it being the least toxic flavone in previous experiments. As such, we were unable to continue testing 15 μM L7G after day 9. Alternatively, 15 μM luteolin did not feature significant toxicity during our long-term assay. Furthermore, latency promotion by apigenin, chrysin, and luteolin was reversible within 24 hours of drug removal. This is in contrast to the Block and Lock agent dCA, which has been shown to maintain suppression for several days to weeks after wash out and render latent viruses refractory to latency reversal by LRAs [37].

Flavonoids other than flavones also have anti-HIV activity [38]. For example, flavanols Kaempferol and Myricetin have been described as HIV RT inhibitors [39][40]. Kaempferol has also been shown to have anti-Protease activity. Interestingly, glycosylation of these flavanols generally increases anti-HIV and anti-RT activity [39][41]. Additionally, we found that the antiviral activities of apigenin, L7G, and luteolin were decreased against an HIV strain mutated to resist Protease and RT inhibitors (HIV-

1PR+RT) by 2-fold or greater, in comparison to HIV-1NL4.3. Additionally, apigenin (EC50 of 59.6 μM) and luteolin (12.8 μM) have been described as direct RT inhibitors in a cell free assay [28]. As such, it is possible that a portion of the antiviral activity by these flavonoids is mediated through anti-RT activity. While we cannot rule out that the flavones identified in our study are also affecting pre-integration steps of the HIV life cycle, we have demonstrated that there is clear inhibition of post-integration activities required for HIV replication. Additionally, Mehla et al. found that luteolin did not inhibit integration or reverse transcription [27]. Instead, we have demonstrated here, and others have shown [12][27], that there is a clear inhibition of post integration processes during flavonoid treatment of latently infected cells.

As previously mentioned, flavonoids in general, and including the ones studied here, have multiple reported cellular targets and presumably affect many different cellular pathways. Additionally, many studies have explored the anti-cancer potential of flavonoids due to their tendency to block transcription, potentiate apoptosis, and activate expression of anti-tumour proteins such as p21 and p53 [42][43][44][45][46]. It is therefore not surprising that we observed off-target effects such as suppression of constitutive GFP expression and cytotoxicity at higher flavonoid concentrations.

58 However, these flavones clearly have specific anti-HIV functions, as they inhibit replication and latency reversal at lower concentrations than those that cause significant cytotoxicity or other undesirable off-target effects. Notably, antiviral activities of flavonoids were maintained in PBMCs infected in vitro, indicating that flavonoids continue to selectively inhibit HIV replication in primary cells.

There are currently no licensed ARVs that target HIV transcription or expression from latently infected cells. Tat inhibitors represent a potential class of compounds that could supplement traditional ARV therapy by blocking transcription from latent proviruses. We and others have shown that apigenin, chrysin, and luteolin inhibit HIV transcription by interfering with Tat function. Due to their reputation as kinase inhibitors and ability to target many host factors, these flavones are likely not targeting Tat directly, but instead interfering with a host factor required by Tat. Critchfield et al ascribed the anti-HIV activity of chrysin and related flavones to their ability to inhibit Casein Kinase 2 (CK2) [47]; however, a potential role of CK2 in Tat-mediated HIV transcription is unclear. Alternatively, apigenin, chrysin, and luteolin have been described as PTEF-b inhibitors through interference with CDK9 kinase activity [48]. However, this activity has not been explored in the context of anti-HIV activity. Furthermore, while L7G is not an inhibitor of Tat function, it is still able to block latency reversal by LRAs in multiple latently infected cell lines. This suggests that it is inhibiting an alternative, Tat-independent pathway.

While the flavonoids identified here are not efficient Block and Lock agents on their own, they are potent inhibitors of HIV replication and expression from latency. Additionally, they were effective in blocking replication of ARV-resistant HIV strains. Although flavonoids are non-specific inhibitors of several host targets, there seems to be an overall antiviral effect, even at concentrations lower than those that affect cell viability. This suggests that cells can compensate for the loss of function induced by flavonoid treatment at these concentrations, whereas HIV cannot. Furthermore, by characterizing the mechanisms responsible for post integration activities, we can potentially identify or design more potent or specific inhibitors of those processes, or improve block-and-lock potential. Importantly, we identified that the glycosylated flavone L7G was able to inhibit replication and block latency reversal, but did not act through Tat inhibition, suggesting the presence of a distinct, Tat-independent mechanism.

59 2.6. Contributions

Cole Schonhofer and Dr. Ian Tietjen contributed equally to this study. “We” and “Our” are used throughout this chapter to reflect that several people were involved in this work. My primary role in this study was to collect and analyze much of the data presented in Figures 2.5, 2.6, and 2.7. I also participated in data collection and analysis for Figures 2.2 and 2.3. I also was responsible for training Jennifer Yi, who collected some data used in Figure 2.5 and 2.6. Dr. Ian Tietjen was responsible for the initial compound screen in CEM-GXR cells (Figures 2.1 and 2.2), and the testing of compounds against ARV-resistant viruses (Figure 2.4), and participated in the data collection for Figure 2.3. PBMC used in Figure 2.3 were collected from donor samples by Natalie Kinloch and Aniqa Shahid. Technical support and training was provided by Silven Read, Khumoekae Richard, Steven Jin, and Gursev Anmole. Mentorship and supervision was provided by Dr. Ian Tietjen, Dr. Zabrina Brumme, and Dr. Mark Brockman.

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64 Chapter 3. Mechanistic Investigation of Post Integration Activities of Flavonoids

3.1. Abstract

Flavonoids represent natural product-derived post integration inhibitors of HIV. However, the exact mechanisms of inhibition are not well understood. In Chapter 2, we identified a glycoside flavone, luteolin-7-glucoside (L7G), that suppressed latency reversal in a Tat-independent manner, while its aglycone counterparts (apigenin, luteolin, and chrysin) inhibited Tat-dependant HIV expression. In this chapter, we show that this lack of anti-Tat activity is likely mediated by differences in ability to inhibit CDK9, but not CK2. Additionally, we show that these 4 antiviral flavones interfere with HIV mRNA splicing, resulting in decreased production of unspliced and single spliced mRNAs in J-Lat cells. However, this is not mediated by direct inhibition of HIV Rev function. We also describe the discovery of structurally-related flavonoids with LRA properties, 6-prenyl-apigenin and jaceosidin, from African and Australian natural products. These flavonoids synergistically reactivate HIV from latency during cotreatment with a PKC activator, but not with an HDAC inhibitor. Finally, we demonstrate that jaceosidin, apigenin, and chrysin have HDAC inhibitor activity. This chapter explores a variety of the post-integration mechanisms displayed by flavones, and relates differences in activity and potency to differences in structure. Additionally, we identify CDK9 as a potential target for Block and Lock strategies

3.2. Introduction

Individual flavonoids have multiple effects on treated cells, and have been observed to directly target multiple cellular factors [1][2][3]. Additionally, structural variations between flavonoids contribute to differences in activity, cellular targets, and potency. This functionally promiscuous nature of flavonoids makes it difficult to conclusively establish their antiviral mechanisms. However, by comparing the activities of closely related flavonoids on individual cellular targets, the specific moieties required for activities can be isolated. Additionally, comparison of flavonoid activity to established, specific inhibitors of a factor-of-interest can help determine whether that factor is indeed responsible for flavonoid activity. In this chapter, we attempt to tease apart the multiple

65 activities of the anti-HIV flavonoids identified in Chapter 2, by comparing their activities in assays specific for functions of interest.

The observation that apigenin, luteolin, and chrysin are potent inhibitors of Tat function, while L7G is not, suggested to us that the 7-O-glycosyl moiety was interfering in anti-Tat activity. These aglycone flavones have been extensively described as inhibitors of a variety of protein kinases, usually by ATP-competitive mechanisms due to their small size, planar orientation, and heterocyclic structure. As such, we identified two potential kinase targets that may play a role in flavonoid antiviral activity.

We first investigated flavone-mediated inhibition of P-TEFb, the cellular complex required by Tat to stimulate RNAP II transcriptional elongation. P-TEFb consists of CDK9 and its cyclin partner Cyclin T1, and the kinase activity of CDK9 in particular is well characterized as essential for both Tat function and HIV replication [4]. Apigenin, chrysin, and luteolin have been described as inhibitors of P-TEFb kinase activity, as have structurally-similar flavones such as wogonin and tricin [5][6]. Furthermore, two of the most potent CDK9 inhibitors, Flavopiridol and Vorucilib, are synthetic flavone derivatives [7]. Therefore, flavone-mediated inhibition of CDK9 is an intriguing candidate for antiviral activity.

Importantly, Polier et al. performed an in silico docking analysis of the flavone wogonin binding to CDK9 [8]. Wogonin is structurally similar to chrysin, differing only by the addition of a methoxy group on C8 of the A ring. In this analysis, Polier et al. predicted that Wogonin binding involved docking of its A ring into the ATP-binding pocket of CDK9. We therefore hypothesized that the structurally related apigenin, luteolin, and chrysin bind in a similar orientation, while the glycosyl moiety of L7G, which is present on C7 of the A ring, may force L7G to bind to CDK9 in a different orientation and impact its inhibitory potential. As such, the observed lack of Tat inhibition by L7G may be due to an inability to inhibit CDK9.

An alternative antiviral target is Casein Kinase 2 (CK2). Structurally, CK2 consists of a tetrameric complex of two catalytic α or α’ subunits and two β regulatory subunits. The α and α’ subunits are structurally similar and mostly overlap in function, and are able to function without the β subunits [9]. CK2 has been linked to phosphorylation of over 300 cellular substrates [10] and is involved in many cellular

66 pathways, including several involving RNAP II and cellular transcription [11]. Additionally, CK2 has been described to directly phosphorylate several HIV proteins including Rev [12][13][14][15]. Moreover, HIV Rev has been shown to upregulate CK2 activity, suggesting that CK2 plays a role in HIV replication [16].

Apigenin, chrysin, and luteolin are known inhibitors of CK2 [17]. Additionally, L7G inhibits CK2 in vitro at similar concentrations as luteolin, although it was less potent against the tetrameric holoenzyme than CK2α alone [18]. Relevant to this study, Critchfield et al ascribed the anti-HIV, post integration activity of chrysin and related flavones to their ability to inhibit Casein Kinase 2 (CK2), due to the identification of CK2 subunits as targets of chrysin binding in latently infected OM-10.1 cells [19]. However, a potential role of CK2 in HIV transcription is unclear. CK2 has been linked to RNAP II phosphorylation, suggesting that it may play a direct role in transcriptional elongation [20]; however, a conflicting study suggests that that activity is actually due to CDK11 and does not require CK2 [21].

As L7G does not block Tat-dependent HIV expression, but still prevents latency reversal, it must be acting on another pathway required for latency reversal. Potential targets could include HIV alternative splicing and Rev function. Classically, the HIV genome was shown to be multiply spliced in a regulated manner to produce more than 40 splice forms [22][23]; however, more recent studies have revealed that some HIV strains produce over 100 splice forms from novel splice sites [24][25][26]. Splicing occurs from at least four donor and five acceptor splice sites. Control of splice site selection is regulated by the intrinsic efficiencies of the splice site sequences themselves [27], and the presence of nearby exonic/intronic splicing enhancer or inhibitor sequences [28]. Enhancer sequences are typically bound by proteins containing Serine-Arginine (SR) domains, and binding of these SR proteins promotes splicing at associated splice sites. In contrast, inhibitory sequences are generally recognized and bound by heterogeneous ribonuclear proteins (hnRNPs), which suppress splicing at associated splice sites.

The HIV genome is highly ordered to ensure splicing occurs in the correct order and produces the correct ratios of spliced mRNAs [23]. Initially, the 5’ major splice donor site D1, which is upstream of the Gag/Pol coding region, is spliced to any of the downstream acceptor sites to produce single spliced (SS) mRNAs. As the acceptor sites

67 are downstream of Gag/Pol, the Gag/Pol coding region is excised from SS mRNAs. A second splice event can occur in SS mRNA between HIV’s other major splice donor site, D4, and a downstream acceptor site that results in excision of the Env coding region and production of multiply spliced (MS) mRNA. The production of each HIV protein depends on which acceptor site is spliced to D1 or D4. D1 and D4 are efficiently spliced, while the acceptor sites vary very specifically in their splicing rates due to regulation by the splice site sequence itself, and the activity of nearby SR proteins or hnRNPs. In this way, the prevalence of mRNA produced for each viral protein is tightly regulated, and disruption of this process can greatly affect viral replication [29].

MS mRNAs lack introns and are free to leave the nucleus via normal transport pathways. However, unspliced (US) and SS mRNAs are sequestered in the nucleus due to the presence of introns [30]. The HIV regulatory proteins Tat and Rev are expressed from MS mRNAs, and re-enter the nucleus after translation. While Tat upregulates proviral transcription, Rev binds to the Rev Response Element (RRE), a ~350 nucleotide mRNA element located within the Env coding-region in US and SS mRNAs. Rev then facilitates cytoplasmic transport of these mRNAs via the CRM1 nuclear export pathway [31]. Abolition of Rev expression or function results in a loss of US/SS mRNA translation and suppresses replication [32].

Effects of flavonoids on HIV splicing or Rev function have not been reported. However, apigenin has been described as a direct inhibitor of the splicing factor hnRNPA2 [33][34], and hnRNPA2 seems to have roles in enhancement of HIV transcription and mRNA trafficking [35][36][37]. Additionally, luteolin and apigenin are capable of altering splice site selection in an in vitro splicing reporter construct [38]. Therefore, HIV splicing interference represents a potential mechanism for antiviral activity by L7G.

In contrast to the inhibition of latency reversal that we and others have observed with these flavones, several flavonoids have been shown to have LRA properties. In particular, the epicatechin trimer, procyanidin C1, reverses latency by agonistically activating the MAPK pathway, leading to upregulation of NFκB activity and subsequent HIV expression [39]. Furthermore, at least one flavonoid has been described to have both LRA and antiviral activities: specifically, the flavanol quercetin reverses latency in an NFκB-dependant manner [40], but also inhibits HIV replication [41][42]. These

68 somewhat paradoxical observations suggest that quercetin has different activities at multiple steps of the HIV life cycle.

In Chapter 2, we described the activities of flavonoids isolated from an African natural product library against HIV replication, and described their ability to inhibit latency reversal and Tat-driven transcription. Here, we investigate the mechanisms of these PIIs in detail. Firstly, we compare flavonoid activities against CDK9, and explore a potential role of CK2 in latency reversal. We further explore flavonoid activities against two Tat-independent processes required for HIV latency reversal: alternative splicing and HIV Rev function. Moreover, we describe the discovery of two novel latency reversing flavonoids, 6-prenyl apigenin (6PA) and jaceosidin, and identify HDAC inhibition by flavonoids as both a potential mechanism of jaceosidin’s LRA activity and a limiting factor of apigenin and chrysin as prospective Block and Lock agents. Finally, we demonstrate that selective CDK9 inhibition is a potential method for a more durable Block and Lock than that obtained with apigenin and chrysin.

3.3. Methods

3.3.1. Reagents

ADP-Glo Kinase Assay and HDAC-Glo I/II Assays were obtained from Promega. Purified, activate CDK9/Cyclin T1 was obtained from Sigma. Purified, active CK2α was obtained from Promega. Flavopiridol, quinalizarin, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. Leptomycin B (LMB) was obtained from Cayman Chemical. Jaceosidin from the Davis Open Access Australian Natural Compound Library (Compounds Australia, Griffith University) was kindly provided by Dr. Rohan A. Davis. 6- prenyl-apigenin was identified from the pan-African Natural Products Library [43].

3.3.2. Plasmids

The pSelect-GFP expression plasmid (Invivogen) features a GFP reporter driven by CMV and a second, hEF1-HTLV-driven expression cassette. pSelect-ΔGFP-Rev (pRev) was generated by knockout of the GFP reporter and insertion of the HIV-1 Rev open reading frame into the second expression cassette. A corresponding pΔRev, lacking both GFP and Rev expression, was also generated. pSelect-GFP-p24GagRRE

69 (pGag-RRE) was generated by insertion of the Gag open-reading frame and the RRE sequence into the hEF1-HTLV expression cassette of pSelect-GFP. Transcription from the hEF1-HTLV promoter results in production of transcripts featuring the Gag open reading frame and the RRE. Translation of these transcripts requires co-expression of HIV Rev. A stop codon following the Gag open reading frame prevents the inclusion of the RRE in the final Gag protein. These plasmids were kindly provided by Steven Jin.

3.3.3. Kinase Activity Assays

The Promega ADP-Glo kinase assay was used to measure CDK9 and CK2 kinase activity in the presence of flavonoids by quantifying the levels of ATP converted to ADP. Purified, active CDK9/Cyclin T1 protein was obtained from Sigma, while active, purified CK2α was obtained from Promega. Kinase reactions were performed in white 384 well plates, 10 µL final volumes per well, following manufacturer instructions. Briefly, reagents and flavonoids were diluted in the supplied kinase reaction buffer. For CDK9/Cyclin T1 assays, 30 ng of CDK9/cyclin T1 was incubated with 10 μM ATP, 0.2 µg/mL PDKTide peptide substrate, and flavonoids. For CK2α assays, 10ng of CK2α was incubated with 10 μM ATP, 0.1 μg/mL casein peptide substrate, and flavonoids. No- enzyme and no-inhibitor control conditions were also included. After 40 minutes, kinase reactions were stopped by addition of 10 µl/well ADP-Glo reagent, and mixtures were incubated at room temperature for an additional 40 minutes. Finally, 20 µl/well of Kinase Detection Reagent was added and luminescence intensity was measured on an Infinity M200 multimode plate reader (Tecan Life Sciences) after 30 minutes. Resulting data were normalized to range between 0 and 1, with no-enzyme and no-inhibitor conditions representing 0 and 1, respectively.

3.3.4. Quantitation of Intracellular HIV mRNA Levels

J-Lat 10.6 cells were treated with DMSO, 10 ng/mL TNFα, or TNFα plus 10 μM flavonoids. After 24-hour incubation, cells were pelleted and intracellular viral RNA was extracted and processed as described by Wong et al. [44]. Briefly, RNA was purified by Aurum Total RNA Mini Kits (Bio-Rad), then reverse transcribed using M-MLV (Invitrogen). Resulting cDNA was used to quantify HIV mRNA levels by qRT-PCR. Reaction set-ups, primers, and cycle conditions were as described in [44]. The housekeeping gene β-Actin served as an internal loading control for data normalization.

70 HIV splice form abundance in treated samples was normalized to the levels of MS, SS, and US mRNAs produced by TNFα treatment alone.

3.3.5. Screens for Flavonoids that Synergistically Promote Latency Reversal

J-Lat 9.2 cells were seeded in 96 well plates at 2x105 cells/well and treated with 181 pANAPL compounds at 5 μg/mL and 0.1 μg/mL Phorbol 12-myristate 13-acetate (PMA) for 24 hours. GFP expression was detected by flow cytometry and was an indicator of latency reversal. 512 compounds from the Davis Open Access Australian Natural Compound Library (Compounds Australia, Griffith University) were also screened under the same conditions in J-Lat 9.2 cells. Flavonoid compounds that increased PMA-induced GFP expression were explored further.

To determine whether 6-Prenyl-Apigenin (6PA) synergizes with known LRAs to reverse latency, J-Lat 9.2 cells were treated with 15 μM 6PA alone or in the presence of 10 μM prostratin or 0.1 μM panobinostat. Likewise, J-Lat 9.2 cells were treated with increasing concentrations of Jaceosidin in the presence of prostratin or panobinostat. Presence of synergistic activity in latency reversal was evaluated by Bliss Independence testing of at least 3 independent experiments [45].

3.3.6. HIV Rev Function Assay

5x106 Jurkat cells were co-transfected with 5 μg pRev and 10 μg pGFP-Gag- RRE via electroporation as described in Chapter 2. Transfected cells were resuspended in R10+ media to 1x105 cells/mL, seeded in 96 well plates, then treated with 10 or 20 μM of flavonoids, 5 nM LMB, or DMSO control for 24 hours. Cells were then fixed and stained for p24Gag as described in Chapter 2 and analyzed for p24Gag and GFP expression by flow cytometry. Jurkat cells co-transfected with 10 μg pGFP-Gag-RRE and 5 μg pΔGFP were analyzed alongside experimental cells, as a negative control. Compensation of fluorescence levels was performed in FlowJo v10.5.3. Levels of dual p24Gag and GFP-positive cells were considered indicative of Rev function.

71 3.3.7. HDAC Activity Assay

The Promega HDAC-Glo I/II Assay allows quantification of HDAC I/II activity in live cells via a cell permeable, acetylated, luminogenic peptide substrate. Greater deacetylation of the substrate by cellular HDACs results in greater firefly luciferase production after addition of other kit reagents. Therefore, suppression of HDAC activity with inhibitors results in decreased luminescence.

We examined HDAC activity in the presence of flavonoids as per manufacturer’s instructions. Briefly, HDAC reactions were performed in white 384 well plates, 20 μL final volume/well. Flavonoids were diluted to desired concentrations in the provided buffer and added to wells. Then, Jurkat cells were resuspended in Phenol-Red- and FBS-free RPMI 1640, seeded into wells at 3,000 cells/well, then incubated at 37ºC for 90 minutes. Cells treated with DMSO served as 100% HDAC activity control. Wells containing only media were included to control for signal background. 0.1 μM Panobinostat was included as a positive control for HDAC inhibition.

Following incubation, 20 μL of HDAC-Glo I/II Reagent plus 1% Triton-X100 (prepared as per manufacturer’s instructions) was added to each well. Plates were mixed for 30 seconds, then incubated at room temperature for 30 minutes. Luminescence was detected by an Infinity M200 multimode plate reader (Tecan Life Sciences). Resulting data were normalized to range between 0 and 1, with 0 and 1 representing media-only and no-inhibitor controls, respectively.

3.3.8. Block and Lock Assays

J-Lat 10.6 cells were treated with either 1 or 10 nM Flavopiridol or 0.1% DMSO control for up to 21 days, in experimental conditions as described in Chapter 2. Suppression of spontaneous latency reversal was quantified by comparison of GFP- positive cells between Flavopiridol-treated cultures and DMSO-treated controls, as before. On day 21, cultures were washed once with PBS and either resuspended in drug-free media or stimulated with 10 ng/mL TNFα or 0.1 μM Panobinostat for 24 hours, as before.

72 3.4. Results

3.4.1. Aglycone Flavones are More Potent Inhibitors of CDK9 than L7G

The observed differences in the activities of luteolin and L7G against HIV Tat suggested to us that the glycosyl group of L7G was modifying its anti-HIV activity and likely affecting its ability to inhibit Tat function. We therefore predicted that the ability of L7G to inhibit the cellular target responsible for Tat inhibition would be weaker compared to luteolin, apigenin, or chrysin.

A potential flavonoid target we identified as crucial for Tat function was CDK9. CDK9 and Cyclin T1 form the HIV-required PTEF-B complex. Inhibitors of CDK9 activity, such as Flavopiridol and DRB, have been described as potent inhibitors of HIV replication and latency reversal through abolition of Tat function [4][46]. Additionally, apigenin, luteolin, and chrysin have been described as CDK9 inhibitors [5][8]. Polier et. al demonstrated that treatment with 50 μM of these flavones inhibited RNAPL II phosphorylation in CEM cells, consistent with CDK9 inhibition [8]. They also showed through pull-down experiments that flavones and CDK9 directly interact in CEM cells. However, this activity has not been explored in the context of their anti-HIV activity. We hypothesized that L7G would feature impaired ability to inhibit CDK9 activity in comparison to the aglycone flavonoids.

73

Figure 3.1. Aglycone flavones are more potent inhibitors of CDK9 than L7G (A) CDK9 kinase activity in the presence of no inhibitor or 30 nM Flavopiridol, as measured using the Promega ADP-Glo kinase assay. 30 ng CDK9/Cyclin T1 was incubated with 10 uM ATP and 0.2 μg/μl PDKtide substrate for 45 minutes. The ADP-Glo assay was then preformed to quantify ADP production from ATP during kinase activity, by firefly luminescence. Signal from no-enzyme controls was subtracted as background, then data were normalized to no drug control. (B) CDK9 kinase activity in the presence of flavonoids. Signal from no-enzyme controls was subtracted as background, then data were normalized to no drug control. Data are presented as mean +/- SD from at least two independent experiments. (C) Summary of IC50s against CDK9. Presented as above

We used the Promega ADP-Glo Kinase Assay to measure the activity of purified CDK9-Cyclin T1 in the presence of flavonoids. The ADP-Glo assay uses luminescence to quantify the amount of ATP converted to ADP during a kinase reaction. Kinase inhibition in this assay is detected by a decrease in luminescence relative to untreated controls, as demonstrated by treatment with the potent CDK9 inhibitor Flavopiridol

(Figure 3.1A). Flavopiridol inhibits CDK9 with an IC50 of ~3 nM, and is roughly 10-fold more selective for CDK9 than other CDKs [47]. In our assay, 30 nM flavopiridol inhibited CDK9 activity by 89.6 ± 10.3% (mean ± SD). Inclusion of flavonoids in the kinase assay also resulted in CDK9 inhibition (Figure 3.1B). Apigenin (IC50 of 0.21 ± 0.10 μM), luteolin (0.072 ± 0.033 μM), and chrysin (0.064 ± 0.022 μM) inhibited CDK9 with similar potency, although apigenin was 3 to 4-fold less potent than the other two aglycone flavones. In

74 contrast, inhibition by L7G (6.9 ± 3.9 μM) was 33-fold weaker than apigenin and 96-fold weaker than luteolin, suggesting that 7-O-glycosylation impacts potency. Alternatively, the control flavanone naringenin was observed to have a minimal inhibitory effect on CDK9 activity at the concentrations tested, consistent with its inability to block latency reversal and Tat function. IC50s are summarized in Figure 3.1C.

3.4.2. CK2 Inhibition by Flavones is Not Responsible for Tat Inhibition Activity or Inhibition of Latency Reversal

CK2 has been implicated as a target responsible for flavonoid inhibition of HIV transcription [19]. Critchfield et al. identified that chrysin binds to CK2 subunits in latently infected OM-10.1 cells, and they hypothesized that CK2 may play a role in HIV proviral transcription. However, we and others have demonstrated that the flavonoids in this study are also capable of inhibiting the Tat-required CDK9 kinase [8]. Moreover, a role for CK2 in HIV transcription remains unclear. As such, we explored the activity of these flavones in comparison to the specific CK2 inhibitor quinalizarin (QN) [48]. QN is among the most selective CK2 inhibitors available; in particular, it does not bind to CDK9, unlike other CK2 inhibitors such as apigenin, DRB, and 4,5,6,7-tetrabromo-benzimidazole [49][8][50][51]. As such, QN has been used in several studies to describe the effects of CK2-specific inhibition on human cancer cells lines [52][53]. In particular, QN inhibits CK2 activity in Jurkat cells at concentrations of less than 5 μM [48]. However, the effect of QN on HIV transcription or latency reversal have not been described.

75

Figure 3.2. Activity of a CK2-specific inhibitor compared to flavonoids (A) Effect of flavonoids and QN on CK2 activity, as measured by ADP-Glo assay. 10 ng CK2α was incubated with 10 μM ATP and 0.1 μg/μl Casein peptide substrate for 45 minutes, in the presence or absence of inhibitors. Signal from no-enzyme controls was subtracted as

76 background, then data was normalized to No Drug control. Data are presented as mean ± SD from at least two independent experiments (B) Effect of QN on Tat function in CEM-GXR cells. CEM-GXR cells were transfected with pCMV-Tat as before (Figure 2.6C-D) and treated with compounds. Data are normalized to no-drug control and presented as mean ± SD of two independent experiments (C) Effect of QN on latency reversal in J-Lat 9.2 cells. Cells were treated with LRAs as before (Figure 2.5B-D) in the presence of QN for 24 hours. Results are the shown as the mean +/- SD of at least two independent experiments (D) Comparison of LRA- antagonizing activities of 10 µM QN and L7G in J-Lat 9.2 cells, relative to LRA treatment alone (E) Summary of IC50s against CK2, presented as mean +/- SD

We first used the ADP-Glo kinase assay to compare inhibition of CK2α activity between flavonoids and QN. We found that QN (IC50 of 0.56 ± 0.18 μM) inhibited CK2α activity with similar potency as apigenin (0.23 ± 0.087 μM), chrysin (0.48 ± 0.14 μM), and luteolin (0.24 ± 0.049 μM) (Figure 3.2A). Conversely, L7G was the weakest inhibitor with an IC50 of 0.94 ± 0.44 μM. However, L7G was about 7.3-fold more potent against CK2 than against CDK9, and only 3.9-fold weaker than luteolin. In comparison, luteolin was approximately 96-fold more potent than L7G in inhibiting CDK9 (IC50 6.9 μM versus 0.072 μM), suggesting that the glycosyl moiety interferes less in CK2 inhibition than

CDK9 inhibition. IC50s are summarized in Figure 3.2C.

A potential role of CK2 in Tat activity is unclear. We transfected CEM-GXR cells with pCMV-Tat, as described in Chapter 2, to examine the effect of CK2 inhibition by QN on Tat function. We observed no decrease in Tat-driven GFP from transfected CEM- GXR cells (Figure 3.2B). In comparison, and as described in Chapter 2, L7G also had no effect on Tat signalling, while 10 μM apigenin diminished Tat-driven GFP expression by 60.3 ± 12.6%. This suggests that CK2 inhibition does not significantly affect Tat function, and that Tat inhibition by aglycone flavones is not due to their ability to inhibit CK2.

We next examined the ability of QN to inhibit latency reversal in J-Lat 9.2 cells treated with LRAs (Figure 3.2D). Interestingly, 30 μM of QN was able to block 50.3 ± 7.3% and 50.3 ± 12.0% of latency reversal stimulated by TNFα and panobinostat, respectively, but had little effect on prostratin-driven GFP expression. This suggests that high levels of CK2 inhibition can suppress latency reversal in a Tat-independent manner, similar to what we have observed from L7G. Alternatively, QN could potentially have off- target effects at 30 μM that are responsible for the observed suppression, rather than CK2 inhibition.

77 Regardless, QN was a weaker inhibitor of latency reversal than L7G at 10 μM, despite being a more potent and more selective CK2 inhibitor (Figure 3.2E). Therefore, although L7G may inhibit CK2, this effect is at most partially responsible for its anti- latency reversal activity. There are clearly additional targets that prevent latency reversal in response to LRAs.

3.4.3. Flavonoids Alter HIV Splicing Patterns in J-Lat 9.2 Cells

Efficient gene expression from integrated HIV proviruses requires precise alternative splicing. Unspliced (US), single spliced (SS), and multiply spliced (MS) HIV mRNAs are produced in regulated, precise patterns, and compounds that interfere with the regulation of this process can ablate HIV replication and prevent reactivation from latency [54][55][23]. As such, we examined whether the isolated flavonoids interfere with HIV splicing in J-Lat 10.6 cells. In particular, we were interested in exploring any effects on splicing of L7G, which is able to inhibit replication and latency reversal without suppression of Tat function.

Figure 3.3. Flavonoids alter abundance of HIV mRNA spliceforms (A) The effect of flavonoids (10 µM) on HIV mRNA levels was assessed by qRT-PCR of J-Lat 10.6 cells stimulated with 10 ng/mL TNFα. Abundance of HIV-1 unspliced (US), singly spliced (SS), and multiply spliced (MS) mRNAs are shown relative to TNFα-treated controls. The housekeeping gene, β-actin, served as an internal loading control for data normalization. No TNFα controls featured minimal HIV mRNA production. Data are presented as mean +/- SD from at least 2 independent experiments.

78 J-Lat 10.6 cells were treated with 10 ng/mL TNFα and 10 μM of flavonoids for 24 hours. We then extracted RNA from cell pellets, prepared cDNA via reverse transcription, and examined the prevalence of HIV mRNA splice forms via qRT-PCR. We found that, in comparison to controls treated with TNFα alone, flavonoids reduced the prevalence of SS and US mRNAs, suggesting that they are capable of interfering with post-transcriptional HIV processing. Chrysin was the most potent flavone, inhibiting US prevalence by 86.0 ± 7.2% and SS prevalence by 69.6 ± 18.7%, followed by apigenin (76.9 ± 4.9% and 51.0 ± 20.8%). Luteolin (74.0 ± 8.7% and 30.3 ± 39.0%) and L7G (68.7 ± 7.8% and 15.5 ± 54.6%) were less potent at blocking accumulation of SS and US mRNAs at 10 μM.

Intriguingly, none of the flavonoids decreased MS mRNA prevalence; in fact, L7G and luteolin seemed to increase expression of MS mRNA. This was an unexpected result, as we expected that inhibition of Tat-driven transcription by apigenin, luteolin, and chrysin would decrease the abundance of all three splice forms together. In fact, this result, where MS mRNAs are increased or unaffected while SS and US are decreased, is more indicative of inhibition at the Rev functional level rather than Tat. For example, ABX464 treatment results in an increase in splicing of HIV transcripts, resulting in enrichment of MS mRNAs over US/SS [55], while Digoxin treatment alters splicing patterns such that Rev expression is lost, resulting in nuclear accumulation of US/SS mRNAs and overproduction of MS mRNAs [54].

3.4.4. Flavonoids do not Inhibit HIV Rev Function

As mentioned previously, the result that flavonoids inhibit SS and US mRNA production, but not MS, suggests that they may inhibit Rev at a functional level. HIV Rev is important for the translation of SS and US transcripts, which contain intronic sequences and are not normally translocated out of the nucleus. Rev binds to the RRE present in the introns of these transcripts and facilitates nuclear export via the CRM1 pathway. As such, Rev interference can result in nuclear accumulation and consequential over-splicing and depletion of SS/US transcripts [54][56]. Interference with Rev function, either by suppressing expression of Rev, interfering with Rev-RRE binding, or by prevention of Rev nuclear translocation, inhibits viral protein expression and replication [54][57][58][59].

79

Figure 3.4. Flavonoids do not inhibit HIV Rev function (A) Representational flow plots showing expression of GFP and p24Gag in Jurkat cells during co- transfection with pGag-RRE and pΔRev, p-Gag-RRE and pRev, or pGag-RRE and pΔRev in the presence of 5 nm LMB. p24Gag expression is dependant on pRev co-transfection and can be inhibited by treatment with a Rev inhibitor. Levels of Rev function was quantified by percentage of dual GFP+p24Gag positive cells (upper right quadrant). (B) Effect of apigenin and L7G on Rev function. Jurkat cells were co-transfected as above and treated with DMSO, 5 nM LMB, or 10 or 20 µM of flavonoid. Data were normalized to no drug control and represent mean +/- SD of three independent experiments. Two tail, one sample T Tests were performed, comparing each data set to a theoretical mean of 1. p value significance threshold was Bonferroni corrected for multiple corrections (p<0.05/7<0.0071). Asterix denote significant difference from an expected value of 1.

To examine the effects of flavonoid treatment on Rev function, we modified pSelect-GFP to include the HIV Gag coding region followed by the RRE, driven from the

80 second promoter. This construct, pGAG-RRE, therefore required Rev co-expression to allow nuclear export and translation of p24Gag-mRNA transcripts, while GFP expression was Rev-independent. We co-transfected Jurkat cells with pGAG-RRE and a Rev expression vector, pRev, or its corresponding negative control lacking Rev, pΔRev. We then treated with apigenin, L7G, DMSO, or the control Rev inhibitor Leptomycin B (LMB). LMB is a potent inhibitor of CRM1-mediated nuclear transport via direct binding to CRM1, and abolishes Rev activity by blocking nuclear translocation [58]. We then fixed and stained cells for p24Gag and analyzed p24Gag and GFP expression by flow cytometry. Rev function was assessed by comparing the number of cells double positive for p24Gag and GFP expression in untreated controls to the amount in drug-treated cultures.

As expected, efficient p24Gag expression required co-expression of Rev and was inhibited 88.1 ± 11.2% by 5 nM LMB (Figure 3.4A). However, inclusion of L7G did not affect Rev function in this assay, when compared to untreated controls (Figure 3.4B). Additionally, Apigenin at 20 μM decreased dual p24Gag/GFP-positive cell numbers by 24.6 ± 4.7% compared to untreated controls; however, this effect is within the range expected by the general transcriptional inhibition effects of Apigenin (Chapter 2), and is unlikely to be due to specific inhibition of Rev function. Therefore, we concluded that Apigenin and L7G are unlikely to directly inhibit Rev.

3.4.5. Flavonoids Isolated from Natural Products Synergise with a PKC Agonist, but not an HDAC Inhibitor, to Reverse Latency

Although the flavonoids described to this point are inhibitors of latency reversal, there are several examples of flavonoids in the literature that actually reverse latency, such as procyandin C1 and quercetin [39][40]. During natural products screening for compounds capable of disrupting HIV latency in J-Lat 9.2 cells, we identified two novel latency-reversing flavonoids. In these screens, J-Lat 9.2 cells were treated with 0.1 μg/mL PMA to reverse latency, then treated with compounds from either pANAPL or the Davis Open Access Australian Natural Product library at 5 μg/mL for 24 hours. We identified two flavone compounds, 6-prenyl-apigenin (6PA) and jaceosidin, that increased PMA-induced GFP expression at 5 μg/mL (Figure 3.5A).

81 Both 6PA and jaceosidin are apigenin-derivatives. 6PA is prenylated on C6, while jaceosidin features methoxy substitutions at C6 and C3’. Intriguingly, whereas apigenin inhibited latency reversal in J-Lat 9.2 cells, the structurally similar 6PA and jaceosidin increased the latency-reversing activity of PMA. Additionally, treatment with 15 μM 6PA alone weakly reversed latency, whereas jaceosidin treatment alone did not.

To test whether these latency-reversing effects were consistent during treatment with other LRAs, we treated J-Lat 9.2 cells with either prostratin or panobinostat, and cotreated with either 15 μM of 6PA (Figure 3.5B) or increasing concentrations of jaceosidin (Figure 3.5C). We found that both 6PA and jaceosidin treatment increased activity of the PKC agonist prostratin. In the case of 6PA, 15 μM 6PA alone stimulated 1.5 ± 0.44% GFP expression, while 10 μM prostratin alone stimulated 7.2 ± 0.94% GFP expression. However, in combination they stimulated 23.1 ± 19% GFP expression, representing a 3.2-fold increase over prostratin alone. This was confirmed to be true drug-drug synergy, rather than chance or additive effect, by the Bliss-Independence model [40]. In the case of Jaceosidin, Jaceosidin alone had no observable effect on GFP expression compared to DMSO treatment. However, 10 and 30 μM jaceosidin treatment increased GFP expression stimulated by 10 μM prostratin by 1.8 ± 0.5-fold and 2.3 ± 0.7-fold, respectively. This was also confirmed to be synergistic activity by Bliss- Independence.

Contrastingly, at the concentrations tested, neither drug synergized with the HDAC inhibitor panobinostat. When two classes of LRAs are used in combination, they generally synergize to increase HIV expression to a level greater than would be expected by a simply additive effect [40][60]. Alternatively, co-treatment with two LRAs from the same class, such as two PKC activators or two HDAC inhibitors, does not result in synergism. As such, we hypothesized that 6PA and Jaceosidin were synergizing with PKC agonists, but not an HDAC inhibitor, because they were acting through the HDAC inhibition pathway.

82

Figure 3.5. Discovery of novel flavonoids that synergistically reactivate latency (A) Structures of 6-Prenyl-Apigenin and Jaceosidin. Both flavonoids increased latency reversal by PMA in J-Lat 9.2 cells at 5 µg/mL (B) Activity of 15 µM 6PA alone or in the presence of prostratin or panobinostat. GFP positive cells indicated latency reversal. Data are presented as mean +/- SEM of at least 3 independent experiments. (C) Activity of Jaceosidin in the presence of prostratin or panobinostat. GFP positive cells indicated latency reversal. Data were normalized to effect of LRA alone and is presented as the mean +/- SEM of at least three independent experiments. For B and C, The Bliss independence model was used to determine if flavonoid- LRA interaction was synergistic, additive, or antagonistic. Asterixis denote synergistic activity. For B, significance is p<0.05. For C, p value significance threshold was Bonferroni corrected for multiple comparisons p<0.05/4<0.0125.

3.4.6. Flavonoids Inhibit HDAC Activity in Jurkat Cells

HDAC inhibition by flavonoids has been described by several groups. For example, treatment with quercetin, apigenin, luteolin, and chrysin have all been described to cause global hyperacetylation through anti-HDAC or pro-Histone

83 Acetyltransferase activities [61][62][63][64][65]. However, the activities of 6PA and jaceosidin as HDAC inhibitors have not been described. Additionally, apigenin, chrysin, and luteolin are all inhibitors of latency reversal, despite any additional anti-HDAC activity.

Figure 3.6. Flavonoids inhibit HDAC activity (A) The Promega HDAC-Glo I/II assay uses a luminogenic peptide substrate to detect HDAC activity in living cells. Greater luminescent signal = greater HDAC activity. Treatment of Jurkat cells with 0.1 μM of the potent HDAC inhibitor panobinostat for 90 minutes inhibits HDAC activity. Background from cell-free, media-only wells was subtracted prior to normalization of the data to the no-drug control. Data are presented as mean +/- SD of at least 3 independent experiments (B) Activity of flavonoids against HDAC activity, as measured by HDAC-Glo assay. Assay was performed as per manufacturers instructions. Data were analyzed and is presented as in A. (C) Summary of flavonoid EC50s against HDAC activity in Jurkat cells.

A subset of Class I HDACs are responsible for promoting HIV latency [66]. To examine the ability of jaceosidin and other flavonoids to inhibit Class 1 HDACs, we used the HDAC-Glo I/II assay from Promega. HDAC-Glo allows quantification of HDAC activity in living cell via a cell-permeable, luminogenic, acetylated peptide that serves as a substrate for cellular class I and II HDACs. Deacetylated peptide is then cleaved by

84 addition of a Developer Reagent, which releases aminoluciferin from the peptide. Aminoluciferin release is then quantified in a firefly luciferase reaction. Therefore, greater HDAC activity results in a higher luminescent signal. In comparison to untreated controls, HDAC inhibition by 0.1 μM of the pan-specific HDAC inhibitor panobinostat reduced luminescence by 96.4 ± 0.7% (Figure 3.6A).

We treated Jurkat cells with increasing concentrations of flavonoids for 90 minutes, then examined effects on HDAC function with the HDAC-Glo assay, as per manufacturers instructions. Unfortunately, our supply of 6PA was exhausted prior to this experiment, and thus was not included. As shown in Figure 3.6B and 3.6C, jaceosidin was the most potent HDAC inhibitor, with an IC50 of 13.4 ± 2.1 μM. Chrysin and apigenin were also significant inhibitors of HDAC activity, with IC50s of 19.7 ± 4.8 μM and 46.5 ± 8.9 μM, respectively. Conversely, luteolin, L7G, and naringenin were less potent, featuring IC50s greater than 90 μM.

3.4.7. Specific CDK9 Inhibition by Flavopiridol Promotes Latency

Despite inhibition of CDK9 activity, apigenin, luteolin, and chrysin were only able to transiently promote latency in J-Lat 10.6 cells (Chapter 2). After removal from culture, GFP expression rebounded to control levels, suggesting that the latency promoting effects of these flavonoids was reversible. Intriguingly, concurrent HDAC inhibition could explain the failure of these flavonoids to cause a durable Block-and-Lock state in long term treated cells (Chapter 2). For the latent provirus to remain dormant after PII removal, even when stimulated with an LRA, a build-up of repressive chromatin and epigenetic silencers at the LTR is likely required. However, the HDAC-inhibitory activity of these flavonoids is likely to cause a general hyperacetylation of genome and release of repressive chromatin at the LTR. While treatment is ongoing, the flavones can actively suppress transcription through Tat-inhibition or potentially splicing interference. However, following drug washout, the lack of repressive chromatin could allow for rapid viral rebound.

85

Figure 3.7. The CDK9 inhibitor Flavopiridol promotes latency in J-Lat 10.6 cells (A) J-Lat 10.6 cells were treated with 1 or 10 nM Flavopiridol or 0.1% DMSO control for 21 days. Samples were examined for spontaneous GFP expression every 3-4 days, and media and drug were replenished. Results are presented as mean ± SD of two independent experiments. (B) Following 21-day incubation with Flavopiridol or DMSO, cultures were pelleted, washed once with PBS, and resuspended in either fresh media or media containing 10 ng/mL TNFα or 0.1 μM panobinostat. Cultures were then incubated for 24 hours and examined for GFP expression by flow cytometry. (C) HDAC activity in Jurkat cells treated with increasing concentrations of Flavopiridol, as measured by the HDAC-Glo Assay.

To confirm that CDK9 inhibition is a sufficient to inhibit spontaneous provirus expression, we treated J-Lat 10.6 cells with the potent CDK9 inhibitor Flavopiridol for up to 21 days, in experimental conditions as described in Chapter 2. Because Flavopiridol is associated with cytotoxicity at concentrations above 100 nM, we used subtoxic

86 concentrations of 10 and 1 nM. By day 21, treatment with 10 and 1 nM Flavopiridol suppressed GFP by 85.4 ± 9.4% and 52.2 ± 28.7%, respectively, suggesting that CDK9 blockade is sufficient to reinforce HIV latency in vitro (Figure 3.7A).

Following the 21-day treatment, cells were washed and resuspended in drug-free media or stimulated with LRA, as described in Chapter 2. Prior to washout, cultures treated with 10 nM Flavopiridol for 21 days featured 1.6 ± 1.4% GFP-positive cells, while DMSO-treated cultures featured 9.8 ± 3.2% GFP-positive cells. 24 hours following washout, 3.4 ± 2.2% of 10 nM Flavopiridol treated cultures were GFP-positive, compared to control levels of 10.5 ± 4.9% (Figure 3.7B). These results suggest that, unlike during flavonoid treatment, latency promotion by 10 nM Flavopiridol is durable for at least 24 hours following drug removal. Additionally, the number of GFP-positive cells in cultures treated with 1 nM Flavopiridol remained relatively stable after drug removal, going from 5.1 ± 4.3% GFP-positive cells pre-washout to 6.4 ± 5.7% post-washout. However, these data post-washout are within the margins of error of the DMSO-treated cultures and thus cannot be conclusively characterized as delayed rebound. Finally, treatment with neither 1 nor 10 nM Flavopiridol was able to inhibit latency reversal by TNFα or panobinostat post-washout, suggesting that any delay in latency rebound is overcome during strong proviral stimulation.

3.5. Discussion

In this study, we compared the ability of L7G, apigenin, luteolin, and chrysin to inhibit CDK9 activity, and found that L7G was 96-fold less potent than its aglycone analogue luteolin. We hypothesize that this difference in anti-CDK9 activity is responsible for the lack of Tat inhibition by L7G. Furthermore, although L7G featured inhibitory activity in this assay, we propose that this activity is not potent enough to overcome Tat function in cells, especially in comparison to the three aglycone flavones.

Another potential antiviral target that we identified was CK2, as CK2 has been previously implicated in the anti-HIV activity of these flavonoids [19]. We found that the four flavonoids inhibited CK2 with similar potency to a selective CK2 inhibitor, QN. QN treatment was unable to inhibit Tat-driven GFP expression in CEM-GXR cells, suggesting that CK2 activity is not required for Tat function. However, QN was able to block up to 50% of latency reversal in J-Lat 9.2 cells stimulated with TNFα or

87 Panobinostat. This suggests that CK2 may have a role in a Tat-independent pathway of latency reversal. However, despite similar potency in CK2 inhibition, L7G was a more potent inhibitor of latency reversal, suggesting that CK2 is at most partially responsible for L7G activity.

Critchfield et. al identified CK2 as the putative target of Chrysin and related flavones in PII activity [19]. However, the control CK2 inhibitor used in that study to confirm the anti-HIV effect of CK2 inhibition, 5,6-dichloro-1-β-D-ribofuranosyl-1H- benzimidazole (DRB), is actually more specific for CDK9 than CK2 [49][67][68]. Thus, DRB-induced inhibition of transcriptional elongation may be driven instead by inhibition of CDK9, rather than CK2. Unfortunately, many initial studies in the literature that used compounds such as DRB and apigenin as CK2 inhibitors likely correlated their effects to CK2 inhibition, when in fact those inhibitors are not selective for CK2 [68]. As such, while flavones and DRB likely inhibit both kinases in cells, it is more likely that the anti-HIV, and specifically anti-Tat, effect is predominantly due to CDK9 inhibition rather than CK2 inhibition.

Additionally, Bhargavan and Kanmogne recently reported that CK2 silencing has differing effects on HIV-1 replication and Tat function depending on viral subtype [69]. Importantly, they found that CK2 was not involved in subtype B HIV-1 Tat function or viral replication. This is relevant because all viruses and Tat sources used in our study are subtype B derived, supporting our result that CK2 inhibition was not responsible for the anti-Tat activity we have seen in our assays. In this light, future studies in our lab will involve testing the effects of selective CK2 inhibition by QN in viral replication assays using multiple HIV subtypes, as well as exploring the effect of CK2 inhibition on latency reversal of alternative subtypes.

L7G is a potent inhibitor of latency reversal despite not blocking Tat function. In order to examine Tat-independent mechanisms of action, we examined the prevalence of US, SS, and MS mRNAs in J-Lat 10.6 cells treated with flavonoids. We found that apigenin, luteolin, chrysin, and L7G were able to suppress accumulation of US and SS splice forms, suggesting interference with HIV alternative splicing. Surprisingly, the Tat- inhibiting flavones did not suppress MS mRNA levels. Tat inhibition would be expected to suppress HIV transcription and prevent accumulation of all HIV splice forms. The lack of suppression of MS mRNA suggests that there are multiple pathways being targeted.

88 However, the fact that L7G treatment resulted in an enrichment of MS mRNAs, even in comparison to the aglycone flavones, supports our earlier observations that it is acting through an alternative pathway. An explanation could be that interference in this alternative pathway results in MS mRNA enrichment and US/SS depletion. Additionally, concurrent Tat inhibition by the aglycone flavones could antagonize or overcome the increase in MS mRNA production induced by inhibition of this alternative pathway. In comparison, L7G, which does not inhibit Tat function, does not have a competing secondary activity which may allow full MS mRNA upregulation.

Additionally, treatment of TNFα-stimulated J-Lat 10.6 cells with the 4 flavones results in suppression of both GFP and p24Gag protein expression (Chapter 2). In J-Lat cells, GFP is expressed from the Nef locus, and theoretically represents a MS mRNA. Alternatively, p24Gag protein expression requires production of full-length, US transcripts. It is therefore unsurprising that p24Gag is suppressed during flavonoid treatment, as all four flavonoids greatly decreased the expression levels of US mRNAs. However, none of the flavonoids decreased MS mRNA production, yet greatly suppressed GFP production in response to LRA treatment. This suggests that flavonoids are either acting at a post- transcriptional level to prevent GFP translation, or interfering with splicing such that GFP is mis-expressed. Such effects would not be captured in our assay measuring splice- form abundance.

Additionally, we tested apigenin and L7G for Rev inhibition in a cell-based reporter assay, in which Rev function controlled efficient p24Gag expression. While the CRM1 inhibitor LMB abolished Rev-driven p24Gag expression, apigenin and L7G did not have any significant effect. Apigenin did have an effect (24.6 ± 4.7% inhibition) at 20 μM, but this was within the range of the general transcriptional inhibition we observed in Chapter 2 (Figure 2.2D). As such, this effect is unlikely to represent Rev-specific inhibition. It is possible that our Rev assay would not capture the full range of rev- inhibiting activities. For example, compounds such as Digoxin block Rev function by interfering with alternative splicing and preventing its expression [54]. Such an effect of flavonoids would not be detected in our system, as both Rev and p24Gag were constitutively expressed as full proteins from their plasmids, with no requirement for splicing. As such, we would have expected to detect inhibition of processes such as Rev translocation, as occurs during LMB treatment, or Rev-RRE binding.

89 The discovery of 6PA and jaceosidin as novel latency reversing flavonoids demonstrates that small changes in flavone chemical structure can greatly alter activities. We have shown that the addition of a prenyl group to C6 of apigenin switches it from a PII to an LRA. Interestingly, the same derivative of chrysin, 6-prenyl-chrysin, and 3M6PA were also tested and found to have neither LRA or PII activity (data not shown). Therefore, it can be surmised that the LRA activity of 6PA requires both a C6 prenyl group and a C4’ hydroxyl group, and that the addition of a C3 methoxy reduces activity. Unfortunately, we discovered that our pANAPL stock of 6PA lost activity after a few months of use. As such, we were unable to do more in depth studies with the flavonoid. In the future, we hope to receive a new batch to continue testing.

Conversely, we were able to identify HDAC inhibition as a potential mechanism of jaceosidin’s synergistic LRA activity. Interestingly, the aglycone flavonoids we identified in Chapter 2 as potential block and lock agents - apigenin, luteolin, and chrysin - have all been described as HDAC inhibitors [62][63][64][70]. HDAC inhibitors are a major class of LRA compounds, so it is interesting that these potent inhibitors of latency reversal also have HDACi activity. In particular, apigenin is predicted by in silico modelling to target Class 1 HDACs directly [70], so it is unsurprising that we detected HDAC inhibition in our assay. Surprisingly, luteolin was approximately 2-fold weaker than apigenin in our assay, despite reports showing that both flavones had similar activity against Class I HDACs [64]. In fact, apigenin and luteolin have been reported to inhibit HDAC Class I activity in bovine cardiac tissue with IC50s of 27 μM and 17 μM [64], much more potent than we observed here (46.5 ± 8.9 μM and >90 μM, respectively). It is possible that differences in assays and cell lines used explain the discrepancy.

It is possible that concurrent HDAC inhibition explains the reversibility of latency promotion by apigenin, chrysin, and luteolin following long-term treatment of J-Lat 10.6 cells. In support of this hypothesis, we showed that treatment with 10 nM Flavopiridol, which does not significantly inhibit HDAC activity, suppresses spontaneous latency reversal and maintains that suppression 24 hours following drug removal, in the absence of strong latency reversing stimuli. This suggests that CDK9 is a potential target for Block-and-Lock candidate drugs. Additionally, we were able to demonstrate these antiviral results using Flavopiridol concentrations that were non-cytotoxic. Indeed, HIV is known to be extremely sensitive to P-TEFb inhibition, and is susceptible to lower concentrations of CDK9 inhibitors than those that cause cytotoxicity [4][47]. Even so,

90 future Block-and-Lock studies involving CDK9 will likely require more selective or less cytotoxic inhibitors.

Together, our results demonstrate the variety of activities associated with structurally-related natural flavonoids. It seems that flavones with PII activity have multiple activities that overall result in promotion of latency, despite concurrent activities, such as HDAC inhibition, that generally promote latency reversal. As such, by cataloguing the structural features that result in different activities, more potent or more selective PIIs could potentially be generated. For example, increasing the specificity of apigenin for CDK9 and reducing its specificity toward Class I HDACs could result in a more useful Block-and-Lock candidate. As is, apigenin, chrysin, luteolin, and L7G represent natural compounds with therapeutic potential as post integration inhibitors. Furthermore, we have discovered two novel compounds with LRA potential – 6PA and jaceosidin – that warrant further investigation.

3.6. Contributions

In this chapter, I use “we” and “our” to reflect the contributions of others. My primary role in this chapter was to collect and analyze data for Figures 3.1, 3.2, 3.4, 3.6, and 3.7. Dr. Alan Cochrane (University of Toronto) performed the splicing experiments presented in Figure 3.3 in his lab. Dr. Ian Tietjen and Zahra Haq performed the initial screenings of pANAPL and the Davis Open Access Australian Natural Compound Library (Compounds Australia, Griffith University) to identify 6PA and jaceosidin, respectively, while I was responsible for further experiments with those compounds, resulting in Figure 3.5. Steven Jin kindly generated the pGAG-RRE and pSelect-ΔGFP- Rev expression constructs that I used in Figure 3.4, as well as provided technical support. Technical support and training were also provided by Silven Read and Khumoekae Richard, and mentorship and supervision was provided by Dr. Ian Tietjen and Dr. Zabrina Brumme.

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97 Chapter 4. Discovery and Mechanistic Study of Novel Inhibitors of HIV Tat Function

4.1. Abstract

There are currently no licenced drugs that target Tat-mediated transcription from integrated proviruses. In an effort to identify novel inhibitors of HIV Tat function, we employed the Jurkat Tet-Tat-Dendra+HIV LTR-mCherry (JurTat) cell line. These cells feature inducible Tat-Dendra fusion protein expression that drives production of an mCherry reporter from an integrated LTR; as such, Tat expression and function are decoupled, but can be simultaneously measured. We used these cells in a High Throughput Microscopy Screen of 97,152 compounds from the Rockefeller University High Throughput and Spectroscopy Resource Center compound library, and identified two novel, selective inhibitors of HIV Tat function: A7 (1-[bis(diethylamino)-phenyl-λ5- phosphanylidene]-3-(1-hydroxybutan-2-yl)thiourea) and C11 (2-(1H-pyrazol-1-yl)-N-[6- (thiophen-2-yl)pyrimidin-4-yl]propanamide). Both A7 and C11 inhibited latency reversal in multiple cell lines, and inhibited HIV replication in in vitro infected PBMC. C11 was determined to inhibit the Tat-required kinase activity of CDK9. Additionally, preliminary results suggested that both A7 and C11 were able to block transfer of virus from latently infected PBMCs to a reporter cell line.

4.2. Introduction

In Chapter 2, we identified several natural products capable of inhibiting HIV replication and latency reversal. These flavonoids exerted antiviral effects at several post integration steps, including HIV Tat function and HIV mRNA splicing. However, it is likely that the promiscuity of these compounds limits their ability to induce a lasting, deep- latent state within HIV infected cells. For example, although apigenin and chrysin promoted latency during long-term treatment of J-Lat 10.6 cells, these effects were reversible after drug removal. In Chapter 3, we demonstrated that aside from inhibiting P-TEFb kinase activity and interfering in viral splicing, these two flavonoids also inhibit HDAC activity. HDAC activity at the HIV promoter is an important mechanism in latency establishment [1][2], and the prevention of that cellular process by apigenin and chrysin is a potential reason for their inability to induce a lasting block and lock phenotype. This

98 was further supported by the observation that subtoxic concentrations of Flavopiridol, a more specific CDK9 inhibitor with minimal off-target anti-HDAC activity, induced a more- durable block and lock effect in the same experiment.

Tat inhibition can induce a block and lock phenotype in vitro and ex vivo. The specific Tat inhibitor dCA binds to Tat directly and facilitates development of epigenetic blocks, such as repressive chromatin, at the HIV promoter [3][4]. Targeting of Tat- associating host factors can also induce a lasting “deep latent” state. For example, treatment with Sudemycin D6, an inhibitor of the Tat-associated SF3B1 protein, renders J-Lat 10.6 cells refractory to stimulation with LRAs for at least 72 hours [5]. This is in contrast to the flavonoids we examined, in which latency was reversible by as soon as 24 hours after drug removal.

In Chapter 2 we described the JurTat cell line, which features inducible Tat- Dendra expression that in turn drives mCherry expression from an integrated HIV LTR [6]. We used this cell line to demonstrate that a subset of the identified flavonoids specifically inhibited Tat function, by observing a dose-dependant decrease in the ratio of mCherry to Dendra fluorescence. Now, in an attempt to identify additional compounds that may be capable of inducing a deep latent state, we describe a high throughput screen using the JurTat cell line.

With the help and resources of the laboratory of Dr. Charles M. Rice and the High-Throughput and Spectroscopy Resource Center (HTSRC) at Rockefeller University, we screened 97,152 compounds from the HTSRC compound library by high throughput fluorescent microscopy. Using flow cytometry, we tested hits from that screen at SFU for Tat inhibition in JurTat cells and inhibition of latency reversal in J-Lat 10.6 cells. We identified two specific inhibitors of Tat-dependent HIV latency reversal, A7 and C11, and investigated their mechanisms of action and their activities against HIV replication in primary cells.

4.3. Materials and Methods

4.3.1. Reagents

Compounds for screening were obtained from the Rockefeller University High Throughput and Spectroscopy Resource Center compound library (New York, NY).

99 Doxycycline was obtained from Sigma-Aldrich. MTT reagent (3-(4,5-Dimethylthiazol-2- yl)-2,5-Diphenyltetrazolium Bromide) was purchased from Thermo Fisher. PathDetect pNF-κB-Luc Cis-Reporter plasmid (pNFκB-Luc) was obtained from Agilent. pLTR-RL (an HIV-1 LTR-driven Renilla luciferase reporter) and pLTR-mNFκB-RL (its corresponding control containing inactivating point mutations within its NF-κB sites) are previously described [7][8], as are pCMV-Tat and pCMV-ΔTat [9]. All cell lines were used and cultured as described in Chapters 2 and 3.

4.3.2. High-Throughput Microscopy and Flow Cytometry Screen

97,152 compounds from the Rockefeller University HTSRC compound library were screened in JurTat cells. JurTat cells feature Tet-on inducible Tat-Dendra expression, and an HIV LTR driven mCherry reporter [6]. Doxycycline treatment triggers Tat-Dendra expression, which then drives mCherry expression. Hoescht nuclear staining was used as a measure of viability, and mCherry/Dendra signals were used as a measure of Tat inhibition and specificity. Cells were treated with 500 ng/mL Doxycycline (Dox), seeded in 384 well plates at 3 x 104 cells/well, then treated with 10 µM compound for 24 hours. 10 µM Apigenin was included as a positive control for Tat inhibition, where test assays resulted in a Z’ of 0.7. Z’ values represent the quality and reproducibility of the HTS assay, with Z’ values between 0.5 and 1 are excellent assays [10]. Cells were then fixed with 0.5% paraformaldehyde for 15 minutes, stained with Hoescht dye, diluted to 0.1% paraformaldehyde, and imaged for mCherry, Dendra, and Hoescht by fluorescent microscopy (Molecular Devices Image Xpress Micro X2). Hits were determined as compounds that inhibited ≥ 50% mCherry, < 20% Dendra, and < 25% Hoescht fluorescence, compared to untreated, Dox-stimulated controls. Data were analyzed using internal software developed by the Rockefeller University HTSRC.

96 of 97,152 compounds (0.1%) were identified as Tat functional inhibitors. These hits were then validated by flow cytometry. JurTat cells were activated with Doxycycline as before, seeded into 96 well plates at 106 cells/mL, and treated with 10 μM compound for 24 hours. 32 of 96 (33.3%) compounds inhibited  25% mCherry relative to Dendra fluorescence. These compounds were further tested for their ability to inhibit PMA-induced latency reversal in J-Lat 10.6 cells. Cells were treated with 0.1 μg/mL PMA and 10 μM compound for 24 hours. GFP expression was measured by flow cytometry and used as the measure of latency reversal. 2 of 32 (6.2%) inhibited  20%

100 of GFP expression: A7 (1-[bis(diethylamino)-phenyl-λ5-phosphanylidene]-3-(1- hydroxybutan-2-yl)thiourea) and C11 (2-(1H-pyrazol-1-yl)-N-[6-(thiophen-2-yl)pyrimidin- 4-yl]propanamide). These compounds were then purchased from Enamine (New Jersey, NJ) and dissolved to 80 mM in DMSO.

4.3.3. Tat inhibition Assays

JurTat cells and CEM-GXR cells were treated as before to test for inhibition of Tat function (Chapter 2).

4.3.4. Latency Reversal Assays

J-Lat 9.2 cells and OM 10.1 cells were treated as before to test for inhibition of latency reversal (Chapter 2).

4.3.5. CDK9 inhibition Assay

The Promega ADP-Glo assay was used as before to examine CDK9 activity in the presence of compounds (Chapter 3).

4.3.6. NFκB-reporter Assays

HEK293T cells were seeded at 70% confluency in T25 flasks and allowed to attach overnight. Cells were then transfected with 5.5 μg of pNFκB, using the Lipofectamine 3000 kit and protocol (Thermo Fisher). After 48 hours, cells were seeded into 96 well plates at 104 cells/well and treated with 0.1% DMSO, 10 μM Prostratin, or 10 μM Prostratin + A7 or C11. Following 24-hour incubation, Firefly luciferase was detected by the Dual-Glo Luciferase Assay System (Promega) on an Infinity M200 multimode plate reader (Tecan Life Sciences). Data were normalized to luminescence from 0.1% DMSO-treated cells.

4.3.7. HIV LTR-RL Reporter Assays

5 x 106 Jurkat cells were re-suspended in 200 μL of Opti-MEM media containing 8.4 μg of pLTR-RL (pLTR) or pLTR-mNFκB-RL (pLTR-mNFκB) plus 1.6 μg of either pCMV-Tat or pCMV-ΔTat. Cells were transfected as described previously (Chapter 2).

101 Following 10 min recovery, 96-well plates were seeded with 5 x 104 cells per well and incubated with A7, C11, or DMSO control plus 3 ng/mL TNFα. After 24-hour incubation, Renilla luciferase was detected using the Promega Dual-Glo luciferase procedure. Data were normalized to luminescence of DMSO-treated cells transfected with pLTR or pLTRmNFκB plus pCMV-Tat. Cells transfected with pLTR or pLTRmNFκB plus pCMV- ΔTat served as a negative control to confirm Tat-driven luciferase from the LTR plasmids.

4.3.8. Viability/Apoptosis Assays

A7- and C11-induced apoptosis was measured by Annexin-V-APC staining in Jurkat cells, as before (Chapter 2).

To measure viability in HEK293T cells, cells were seeded into 96 well plates at 104 cells/well in triplicate, and treated with 10 μM Prostratin ± A7 or C11 for 24 hours. Cells were then treated for 4 hours with MTT reagent and cell metabolic activity was measured by absorbance at 570 wave length. Data were normalized to 0.1% DMSO- treated controls.

4.3.9. PBMC Assays

HIVNL4.3 virus was generated as before (Chapter 2). Effects of A7 and C11 on HIV replication and in PBMCs and viability of uninfected PBMCs were tested as before (Chapter 2).

For studies involving HIV-infected PBMCs, PBMCs from 4 donors on stably- suppressive antiretroviral therapy for at least three years were obtained. Study protocols were approved by the Institutional Review Boards of Simon Fraser University and the University of British Columbia – Providence Health Care Research Institute (REB: H15- 03077, approved 8 March 2016). Written informed consent was obtained from all donors. PBMCs were stimulated with 0.1 μg/mL PMA and 1 μg/mL Ionomycin (P.I.) or P.I. + 10 μM A7 or C11 for 48 hours. Viability was then measured by Guava Viacount, while supernatant was diluted 1:8 into 106 CEM-GXR cells and incubated for 24 hours. GFP expression in CEM-GXR cells was measured by flow cytometry after 24 hours, and was used as a measure of infectious virus produced from HIV-positive PBMCs during drug

102 treatments. Data were normalized to CEM-GXR GFP induced by supernatant from untreated HIV-positive PBMCs.

4.4. Results

4.4.1. Discovery of Novel Tat Inhibitors by HTS in JurTat Cells

We used HTS fluorescent microscopy of JurTat cells to screen 97,152 compounds from the Rockefeller University HTSRC compound library at 10 μM. JurTat cells feature doxycycline-driven Tat-Dendra expression and HIV LTR-driven mCherry expression. Thus, mCherry production is dependant on expression of Tat-Dendra, but, unlike in natural HIV transcription, Tat-Dendra expression is decoupled from its LTR activity. By measuring mCherry production relative to Dendra, we were able to discriminate between general inhibitors of transcription and selective Tat-dependant transcription inhibitors. Hoescht nuclear staining was used as a surrogate measure of cell viability during treatment; compounds that decreased Hoescht fluorescence by greater than 75% were excluded from further analysis.

Figure 4.1. Identification of novel Tat inhibitors by high-throughput screening of JurTat cells 97,152 compounds from the Rockefeller University HTSRC compound library were screened in JurTat cells at 10 µM and imaged for mCherry, Dendra, and Hoescht fluorescence by high- throughput microscopy. 96 hits were then validated by flow cytometry, where 32 inhibited  25% mCherry relative to Dendra fluorescence. 2/32 then inhibited  20% of GFP induced by 0.1 µg/mL PMA in J-Lat 10.6 cells. (A) Structures of A7 and C11, the two compound hits.

103 By HTS microscopy we identified 96 (0.1%) compounds that decreased mCherry fluorescence by more than 50% while not affecting Dendra by greater than 20%. These 96 compounds were then validated in JurTat cells by flow cytometry, of which 32 (33.3%) inhibited at least 25% of mCherry fluorescence, relative to Dendra fluorescence, at 10 μM. Of those 32 hits, only 2 (6.2%) were able to supress PMA-induced latency reversal in J-Lat 10.6 cells by greater than 20%. Therefore, we identified 2/97,152 (0.002%) compounds that specifically inhibited Tat function while also suppressing latency reversal. Structures of the two compounds, 1-[bis(diethylamino)-phenyl-λ5- phosphanylidene]-3-(1-hydroxybutan-2-yl)thiourea, or A7, and 2-(1H-pyrazol-1-yl)-N-[6- (thiophen-2-yl)pyrimidin-4-yl]propanamide, or C11, are shown in Figure 4.1A.

4.4.2. A7 and C11 Inhibit Tat Function in JurTat Cells and CEM-GXR Cells

In order to further explore the antiviral and latency-modulating properties of A7 and C11, we first examined the extent to which they were tolerated by cells. We therefore examined treatment-driven apoptosis using annexin-V staining for apoptosis- positive Jurkat cells, as before (Chapter 2). We observed that treatment with 10 μM or less of each compound did not extensively impact apoptosis levels compared to DMSO- treated controls (Figure 4.2A). However, 30 μM A7 stimulated a 6.3 ± 0.3-fold increase in apoptosis over vehicle controls (mean ± SEM). This was similar to apoptosis induced by 0.1 μM Panobinostat (6.0 ± 0.4-fold increase). 30 μM C11 increased apoptosis 3.4 ± 0.3-fold, suggesting that it was more easily tolerated by cells. To ensure that the toxicity of A7 did not interfere with our investigation of its tat-inhibiting mechanisms, we used up to 15 μM A7 in further experiments with Jurkat- and CEM-derived cell lines.

We next explored the ability of A7 and C11 to inhibit Tat-driven reporter expression in JurTat cells and CEM-GXR cells transfected with pCMV-Tat, as before (Chapter 2). We found that C11 inhibited Tat function relatively equally in JurTat cells and transfected CEM-GXR cells, with IC50s of 10.7 ± 1.04 and 7.1 ± 0.40 μM, respectively (Figure4.2B, D). In contrast, A7 was more a more potent inhibitor in CEM-

GXR cells than in JurTat cells, with estimated IC50s of 3.7 ± 0.70 and 25 ± 6.3 μM, respectively.

104

Figure 4.2. A7 and C11 inhibit HIV Tat function (A) Apoptosis in Jurkat T-cells following 24h treatment with A7 or C11, as detected by Annexin-V- APC staining. Apoptosis caused by the HDAC inhibitor Panobinostat (0.1 μM) is shown for comparison (red-dotted line) (B) Effects of A7 and C11 on Tat function in JurTat cells. JurTat cells were stimulated with 500 ng/mL Dox and incubated with A7 or C11 for 24 hours. Decreasing mCherry/Dendra ratios indicate selective inhibition of Tat function. (C) CEM-GXR T-cells contain an HIV-1 LTR driven GFP reporter. GFP expression is Tat-driven. Cells were electroporated with pCMV-Tat and incubated with A7 or C11 for 24 hours, then measured for GFP production by flow cytometry. Decreasing GFP expression indicates block of Tat-dependant transcription (D) Summary of EC50s +/- SD. Data for A and B are presented as mean +/- SEM of 3 or more independent experiments. Data for B are presented as mean +/- SD of 2 independent experiments

105 4.4.3. A7 and C11 Inhibit Latency Reversal in Multiple Cell Lines

We next assessed the abilities of A7 and C11 to inhibit HIV latency reversal in vitro, using the latently-infected J-Lat 9.2 and OM-10.1 cell lines. We stimulated J-Lat 9.2 cells with panobinostat, prostratin, and TNFα as before (Chapter 2), and cotreated with A7 and C11 for 24 hours prior to measuring latency reversal by GFP production (Figure 4.3A-C).

C11 was effective in blocking latency reversal with all 3 LRAs in J-Lat 9.2 cells, with EC50s of 4.9 ± 1.05, 6.2 ± 0.89, and 3.4 ± 0.77 μM, respectively (Table 4.1). In addition, C11 suppressed p24Gag production from OM-10.1 cells stimulated with TNFα with similar potency (4.8 ± 0.68 μM) (Figure 4.3D). Interestingly, while A7 potently inhibited panobinostat-stimulated GFP production (1.7 ± 0.30 μM), it was less potent against prostratin (8.1 ± 2.2 μM) and was completely ineffective against TNFα. In fact, increasing concentrations of A7 increased TNFα-driven GFP expression. In contrast to its effects in J-Lat 9.2 cells, however, A7 treatment of TNFα-stimulated OM-10.1 cells inhibited latency reversal (3.8 ± 0.31 μM). This result suggests that TNFα may affect latency reversal in J-Lat T cells and OM-10.1 macrophages differently, and this difference is likely in a cellular process and/or LTR target with which A7 interferes. Table

4.1 shows a summary of EC50s from each latency-reversal experiment.

Table 4.1. Summary of A7 and C11 EC50s against latency reversal in J-Lat 9.2 and OM-10.1 cells J-Lat 9.2 OM-10.1

TNFα Panobinostat Prostratin TNFα Compound EC50 (μM) EC50 (μM) EC50 (μM) EC50 (μM)

A7 n.d. 1.7 ± 0.30 8.1 ± 2.2 3.8 ± 0.31

C11 3.4 ± 0.77 6.2 ± 0.89 4.9 ± 1.05 4.8 ± 0.68

EC50, 50% effective concentration. Calculated from data presented in Figure 4.3, shown as mean ± SEM of at least 3 independent experiments. n.d. = not determined

106

107 Figure 4.3. A7 and C11 inhibit latency reversal in multiple cell lines (A-C) A7 and C11 were examined for ability to inhibit latency reversal in J-Lat 9.2 cells. Cells were treated with LRAs and inhibitors as before (Chapter 2). Latency reversal inhibition was determined by percent of GFP positive cells in LRA+A7/C11 conditions compared to LRA alone. (D) The effect on A7 and C11 on TNFα-stimulated latency reversal in OM-10.1 cells was examined as before (Chapter 2). Latency reversal was detected by intracellular p24Gag levels. Data are normalized to TNFα-only control. (E) Jurkat cells were transfected with pSelect-GFP and treated with A7 or C11 for 24 hours. GFP is expressed from a non-HIV promoter to check for non-specific inhibition of gene expression or fluorescent-quenching. Data were normalized to no- drug controls. For A-E, data are presented as the mean +/- SEM of at least 3 independent experiments.

We also confirmed the antiviral specificity of A7 and C11 by transfecting Jurkat cells with a constitutively-expressing GFP reporter, pSelect-GFP, as before (Chapter 2). We observed at most a 27.3 ± 15.8% block in GFP expression by 10 μM A7, and a 34.4 ± 0.1% block by 30 μM C11 (Figure 4.3E). While there may be non-specific effects of these compounds on cellular targets and/or transcriptional mechanisms, they occur at higher concentrations than those required to inhibit latency reversal.

4.4.4. C11, but not A7, Inhibits CDK9 Activity

As previously discussed, CDK9 inhibitors are potent suppressors of Tat activity and latency reversal. Additionally, previous screens for Tat-specific inhibitors have identified CDK9 inhibitors as among the most potent [11]. As such, we examined A7 and C11 for ability to inhibit CDK9 activity as before (Chapter 2). We found that C11 inhibited CDK9 activity with an IC50 of 0.016 ± 0.007 μM (Figure 4.4A-B). As such, it is

4-fold more potent than either Luteolin or Chrysin (IC50s 0.072 ± 0.033 and 0.064 ± 0.22 μM, respectively), the most effective flavonoids we tested in this assay (Chapter 3). This result suggests that C11 inhibits Tat function and latency reversal through CDK9 inhibition. Alternatively, A7 only minimally inhibited CDK9; therefore, it must inhibit Tat- driven expression by an alternative mechanism. Neither compound inhibited CK2 activity (data not shown), showing that, unlike the flavonoids described in Chapter 3, dual CK2/CDK9 inhibition is not a feature of C11.

108

Figure 4.4. C11, but not A7, inhibits CDK9 activity (A) The effect of A7 and C11 on CDK9 activity was examined using the ADP-Glo assay procedure, as before (Chapter 2). Data were normalized between the luminescent signal produced by no enzyme (0) and no inhibitor (1) controls, and is presented as the mean +/- SD of at least 2 independent experiments. (B) Summary of activity of A7 and C11 against CDK9. IC50s presented as mean +/- SD. n.d. = not determined

4.4.5. A7 and C11-Mediated Suppression of NFκB Activity is not Essential for Inhibition of Tat-driven Transcription

NFκB signalling is important for effective HIV transcription, independent of Tat [12][13][14]. The HIV LTR contains two conserved NFκB-binding sites, and activation of NFκB signalling by PKC agonists or cytokines enhances transcription at the LTR and reverses latency. After translocation into the nucleus, NFκB binding to the HIV LTR recruits Transcription Factor II H (TFIIH), which, among other factors, enhances transcriptional initiation by phosphorylating Ser5 of the CTD region of RNAP II [15]. Deletion of NFκB sites in the LTR decreases HIV transcription substantially, but does not completely inhibit replication or expression [16][17].

Because of the importance of NFκB in HIV transcription, we examined the effect of A7 and C11 on NFκB signalling. We transfected HEK 293T cells via lipofection with an NFκB-driven Luciferase reporter plasmid (pNFκB-Luc), and treated cells with 0.1% DMSO control or stimulated with 10 μM prostratin ± A7 or C11 for 24 hours. Prostratin treatment upregulated luciferase production by 4.7-fold over untreated controls. This upregulation, however, was inhibited with dose response during cotreatment with A7 or C11 (Figure 4.5A). We found that both A7 and C11 inhibited NFκB-driven Luciferase

109 production with IC50s of 8.04 ± 1.5 and 8.5 ± 5.6 μM, respectively, without causing major toxicity when assessed by MTT reagent (Figure 4.5B). MTT staining is a measure of cellular metabolic activity; we observed significant cytotoxicity only when treating HEK 293T cells with 30 μM of A7, where viability was reduced to 30.0 ± 21.5% of controls.

To examine NFκB inhibition in the context of the HIV LTR, we employed two LTR-driven Luciferase constructs, pLTR and pLTR-mNFκB [7][8]. These constructs both feature Renilla luciferase induced by activation of transcription at an HIV LTR promoter. However, pLTR-mNFκB has been mutated to delete the two NFκB binding sites present in the promoter. Therefore, luciferase production from this plasmid is independent of direct NFκB binding. Luciferase production from both of these plasmids can be stimulated by Tat co-expression.

We co-transfected Jurkat cells with pCMV-Tat and either pLTR or pLTR-mNFκB, via electroporation as discussed previously (Chapter 2). Then, we treated with either A7 or C11, in the presence of 3 ng/mL TNFα, for 24 hours prior to measuring luciferase production. As a control, cells co-transfected with either LTR construct and the Tat- deleted plasmid, pΔTat, featured minimal luciferase production, confirming that even in the presence of TNFα, expression from both LTRs is Tat-dependant. Consistent with previous assays that confirmed that A7 and C11 were Tat inhibitors, we observed a decrease in luciferase production in Jurkat cells co-transfected with pLTR and pCMV-Tat following treatment with either compound (Figure 4.5C). Interestingly, A7 was the more potent inhibitor in this assay (EC50 of 4.6 ± 1.1 μM), while C11 was less effective, particularly in comparison to previous experiments in CEM-GXR cells and JurTat cells

(EC50 of 22.4 ± 19.4 μM versus 7.1 ± 0.40 and 10.7 ± 1.04 μM, respectively). Nevertheless, C11 still exhibited a clear trend toward Tat inhibition.

110

Figure 4.5. A7 and C11-mediated suppression of NFκB signalling is not required for Tat inhibition (A) HEK293T cells were transfected with pNFκB-Luc, then cotreated with 10 µM prostratin and increasing concentrations of A7 or C11 for 24 hours. Cells were then examined for NFκB-driven firefly luciferase production via Promega Dual-Glo. Data were normalized to DMSO control (dotted line at 1), and prostratin treatment increased NFκB-driven luciferase by 4.6-fold, on average (dotted line at 4.6). Data are presented as mean +/- SEM of 3 independent experiments. (B) Viability of HEK 293T cells treated with 10 µM prostratin and increasing concentrations of A7 and C11, measured by MTT assay. Data are normalized to 0.1% DMSO control and presented as mean +/- SEM of 3 independent experiments. (C-D) Effect of A7 and C11 on Tat-driven expression from a wildtype HIV LTR (C) or an LTR lacking NFκB binding sites (D). Jurkat cells

111 were co-transfected with pLTR or pLTR-mNFkB and pΔTat or pTat, then treated for 24 hours with 3 ng/mL TNFa ± A7 or C11. Tat-driven luciferase production was measured via Promega Dual- Glo procedure. Data were normalized to untreated, Tat-expressing controls, and are presented as mean ± SEM of three independent experiments (C) or mean ± SD of two independent experiments (D).

Interestingly, both compounds were also able to inhibit Tat-driven luciferase production from pLTR-mNFκB (Figure 4.5D). While A7 featured similar potency against both constructs (EC50s of 4.6 ± 1.1 and 5.8 ± 5.9 μM, respectively), C11 seemed to be slightly more potent in the absence of productive NFκB signalling (EC50 of 10.7 ± 5.2 versus 22.4 ± 19.4 μM). This result suggests that although A7 and C11 are able to inhibit NFκB signalling, this activity is insufficient to inhibit Tat-driven reporter expression, and additional antiviral mechanisms remain likely.

Table 4.2. Summary of EC50s of A7 and C11 against NFκB signaling in HEK 293T cells and Tat- driven luciferase production in Jurkat cells HEK 293T cells Jurkat cells

NFκB Activity LTR Activity LTR-mNFκB Activity Compound EC50 (μM) EC50 (μM) EC50 (μM)

A7 8.04 ± 1.5 4.6 ± 1.1 5.8 ± 5.9

C11 8.5 ± 5.6 22.4 ± 19.4 10.7 ± 5.2

EC50, 50% effective concentration. Calculated from data presented in Figure 4.5, shown as mean ± SEM of at least 3 independent experiments. LTR-mNFκB data are shown as mean ± SD of 2 independent experiments.

4.4.6. A7 and C11 Inhibit HIV Replication in PBMCs

Having observed that A7 and C11 inhibit Tat, we next explored their effects on viral replication. We infected PBMCs from 4 HIV-negative donors with HIVNL4.3, as before (Chapter 2), and treated with A7 and C11 for 6 days before measuring supernatant p24Gag (Figure 4.6A). We also tested the effects of the compounds on cell viability by treating uninfected PBMCs in parallel, then detecting live cells with Guava Viacount dye

(Figure 4.6B). A7 (EC50 of 1.6 ± 0.33 μM) was slightly more potent than the most effective flavonoid we tested in PBMCs, L7G (EC50 = 2.6 ± 0.76 μM, Chapter 2). The high potency of A7 yielded an SI of 5, despite a relatively low CC50 of 8.01 ± 3.5 μM.

Comparatively, the SI of L7G in PBMC infection was 7, due to a higher CC50 of 18.3 ±

2.5 μM. C11 also limited HIV infection in PBMCs, with an EC50 of 9.2 ± 1.4 μM. Despite

112 being 5.8-fold less potent than A7, it was also much less cytotoxic, featuring a predicted

CC50 of 107.0 ± 2.9 μM and an SI of 11.6.

Figure 4.6. A7 and C11 inhibit HIV replication in PBMCs Effects of A7 and C11 on viability and HIV infection of PBMC. PBMC were infected and treated as before (Chapter 2) (A) Effects of A7 and C11 on HIV-1NL4.3 replication in PBMC, as measured by supernatant p24Gag levels on day 6 post-infection, relative to infected cells treated with DMSO. (B) Viability of uninfected PBMCs after 6 day treatment with A7 or C11, as measured using Guava Viacount and relative to cells treated with DMSO. For A and B, data are the mean ± SEM of results from 4 independent donors. (C) Summary of CC50, EC50, and SI of A7 and C11 in virus replication inhibition.

4.4.7. A7 and C11 Prevent Viral Transfer from HIV-Infected Cells Reactivated from Latency

After confirming that A7 and C11 inhibited HIV replication in PBMCs, we next tested their ability to prevent viral transfer from stimulated PBMCs from HIV-positive donors. We treated PBMCs from 4 HIV-positive donors with suppressed viral loads with either 0.1% DMSO control or 0.1 μg/mL PMA and 1 μg/mL Ionomycin. PMA is a potent PKC agonist and stimulates NFκB activity, while Ionomycin is calcium ionophore that

113 increases intracellular calcium concentrations and thereby stimulates NFAT activation [18]. This combination mimics cellular activation by T cell receptor engagement and has been shown to be among the most potent in stimulating HIV latency reversal in primary cells [19][20]. We also treated stimulated PBMCs with 10 μM A7 or C11. After 48 hours, we measured cell viability by Guava Viacount dye, as before (Chapter 2) and did not observe any toxicity by any of the treatments (Figure 4.7A). We then diluted virus- containing culture supernatants 1:8 and added it to cultures of CEM-GXR cells. After 24 hours, we observed GFP expression in CEM-GXR cells, suggesting productive infection by viruses induced from the HIV-positive PBMCs.

Figure 4.7. A7 and C11 treatment reduces viral transfer from stimulated HIV+ PBMCs PBMCs from 4 HIV-positive donors on suppressive cART were treated with PMA+Ionomycin (P.I.) ± 10 μM A7 or C11 for 48 hours. Cell viability was measured by Guava Viacount (A) and supernatant was diluted 1:8 into CEM-GXR cells and incubated for 24 hours. Resulting GFP expression in CEM-GXR cells indicates infection (B). p-values were obtained from one-tailed, paired t-test analysis, and p values < 0.05 were considered significant.

Supernatant from PMA/Ionomycin-stimulated PBMCs resulted in an average 4.6 ± 1.2-fold increase in GFP production in CEM-GXR cells, compared to that from non- stimulated PBMC (Figure 4.7B). In contrast, concurrent treatment with A7 or C11 decreased GFP expression to 3.3 ± 1.2-fold (p=0.004, pairwise T-test) or 3.5 ± 1.1-fold

114 (p=0.033, pairwise T-test) of unstimulated controls. This suggests that A7 and C11 limited the amount of infectious virus transferred to CEM-GXR cells from latently infected PBMCs and subsequently decreasing GFP expression.

4.5. Discussion

A high throughput fluorescence microscopy screen of over 97,000 synthetic compounds identified 96 potential Tat-functional inhibitors, 33 of which were validated by flow cytometry. Of these, two also inhibited latency reversal in J-Lat 10.6 cells. Those two compounds, A7 and C11, have not been described as inhibitors of HIV replication or latency reversal previously. Additionally, they act through distinct mechanisms, with C11 being a potent inhibitor of the Tat-required kinase CDK9. A7 is not a CDK9 inhibitor, and at this time its mechanism of Tat inhibition is unclear. While both A7 and C11 inhibit NFκB signalling in vitro, this activity was not responsible for inhibition of Tat-driven transcription, as both blocked Tat function at a NFκB-deleted LTR. Furthermore, both compounds inhibited HIV replication in primary PBMCs, and suppressed infectious virus transfer from HIV-infected PBMCs reactivated from latency.

It is interesting that out of 97,152 compounds, only two consistently inhibited both Tat function and latency reversal. This represented an ultimate hit rate of only 0.002%. In comparison, the pANAPL screen described in Chapter 2 had a hit rate of 2% (4/201), and so was a 1000-fold more successful screen. Screening for specific Tat inhibitors, rather than general inhibitors of HIV replication, likely explains this discrepancy. For example, of the four hits we identified from pANAPL in the CEM-GXR screen against HIV replication, two were not Tat inhibitors and would not have been hits in this JurTat screen (L7G and 3M6PA). Alternatively, it is possible that natural product libraries are comprised of more biological-relevant molecules than libraries comprised of randomly- synthesized compounds. Many chemicals isolated from plants and other natural sources have functions in cellular signalling, as well as evolved antiviral and antimicrobial responses; ; it is unsurprising that many natural products have been observed to have HIV-relevant activities [21][22].

The activities of each compound varied in three assays used to assess Tat inhibition. A7 was quite potent in CEM-GXR cells transfected with pCMV-Tat (IC50 = 3.7

± 0.7 μM) and Jurkat cells transfected with pCMV-Tat and pLTR (IC50 = 4.6 ± 1.1 μM),

115 but was less potent when measuring mCherry suppression relative to Dendra expression in JurTat cells (IC50 = 25 ± 6.3 μM). In contrast, C11 was effective in JurTats and transfected CEM-GXR cells (IC50s = 10.7 ± 1.0 μM and 7.1 ± 0.4 μM, respectively), but less effective in transfected Jurkats (IC50 = 22.4 ± 19.4 μM). We attribute these variabilities to potential cell line differences in expression of Tat-required host factors that in turn impact drug function, or potentially to differences between expression from a non-integrated plasmid construct compared to integrated LTRs with associated epigenetic factors.

A7 was unable to block TNFα-stimulated GFP expression in J-Lat 9.2 cells, despite potent inhibition of TNFα-stimulated p24Gag expression in OM-10.1 cells. While we are unable to completely explain this at this time, we hypothesize that it may be related to potential differences in cell signalling responses to TNFα between the T cell derived J-Lat 9.2 cell line and the macrophage-derived OM-10.1 cell line. In particular, we treated J-Lat cells with 10 ng/mL TNFα, while OM-10.1 cells were treated with only 1 ng/mL TNFα to obtain maximal reactivation. It is possible that this difference in TNFα concentration, combined with different cell lineages, is associated with the observed differences in A7 activity. Alternatively, there may be differences in the promoters of the integrated proviruses of each cell line, leading to one being susceptible to inhibition by A7 while the other is not. Regardless, identifying the cause of this phenomenon is a goal of future investigations, and will help to elucidate the mechanism and target of Tat inhibition by A7.

Interestingly, both A7 and C11 inhibited NFκB signalling from a luciferase reporter. Pharmacological inhibition or RNAi silencing of CDK9 has been reported to suppress NFκB activity [23][24][25][26][27], most likely through blocking transcriptional elongation of at least a subset of NFκB-responsive genes. Therefore, it is unsurprising that we observed dose-dependent inhibition of NFκB activity during C11 treatment. Additionally, although it was not the primary cause of Tat inhibition, the ability of A7 to inhibit NFκB signalling provides direction for future studies. The observation that A7 inhibited NFκB signalling, but failed to inhibit TNFα-stimulated GFP in J-Lat 9.2 cells, is particularly perplexing because TNFα-stimulated activation from latency in J-Lat cells is mediated by NFκB activation and translocation into the nucleus to initiate proviral transcription [28][29]. Clearly, further investigation of A7 is warranted.

116 Both A7 and C11 were able to decrease transfer of viruses from latently infected PBMCs, when supernatant from PMA/Ionomycin-stimulated and A7 or C11 treated PBMCs was used to infect CEM-GXR cells. However, it is possible that this activity is not due solely to lower levels of stimulated virus produced during A7/C11 treatment. Despite dilution of supernatant before infection of CEM-GXR cells, the presence of A7 and C11 in the supernatant could be partially responsible for the observed inhibition in GFP expression. GFP production from CEM-GXR cells is Tat-driven, so the transfer of A7 and C11 along with induced virus could suppress activity at the reporter LTR. However, the 1:8 supernatant dilution prior to transferring should decrease A7 and C11 concentrations from 10 μM to approximately 1.25 μM, which is below their respective IC50 values in CEM-GXR cells transfected with pCMV-Tat.

Alternatively, it is also possible that aside from Tat inhibition, A7 and C11 act at additional steps of HIV replication, including pre-integration steps. This would also serve to limit GFP production without being necessarily due to the promotion of latency in stimulated PBMCs. However, we believe that by diluting the supernatant before transfer to CEM-GXR cells, we limited those potential effects. A third potential limitation of this assay could be non-specific activation of the CEM-GXR GFP reporter by PMA/Ionomycin and/or stimulating cytokines present in the transferred supernatant. A7 and/or C11 could block production of stimulating cytokines from PMA/Ionomycin- activated PBMCs, and thus limit future cytokine-stimulated GFP expression. However, if this is the case, A7 and C11 would still be demonstrating potentially relevant immune- dampening activity. An alternative measure of viral reactivation from induced PBMCs would be useful in corroborating these results, and is a required future experiment. For example, more sensitive experiments to measure viral reactivation ex vivo in isolated, resting CD4 cells could include quantitative Viral Outgrowth Assay (qVOA) or Tat/Rev Induced Limiting Dilution Assay (TILDA) [30][31].

In sum, A7 and C11 are novel inhibitors of HIV Tat function. Both compounds are capable of selectively inhibiting Tat in multiple in vitro assays, blocking latency reversal in latently infected cell lines, and inhibiting HIV replication in primary PBMCs. C11 is a potent inhibitor of Tat-required CDK9, and both compounds inhibit NFκB signalling, although this mechanism is not primarily responsible for their anti-Tat activities. These two compounds represent intriguing candidates for future study, including as post integration inhibitors of HIV and as potential block and lock candidate drugs.

117 4.6. Contributions

Myself and Jennifer Yi contributed equally to this study. I use “we” and “our” throughout to acknowledge the contributions of everyone involved in this project. My role was in training and supervising Jennifer Yi, who collected and analyzed much of the data presented in Figures 4.2 and 4.3. Additionally, I conducted the flow cytometry validation of the 96 hits from the HTS in JurTat cells, eventually leading to the identification of A7 and C11 (Figure 4.1). Jennifer Yi also contributed to the flow cytometry validations. I further contributed to data collection and analysis in Figures 4.2, 4.3, 4.5, and 4.6. I was fully responsible for the experiments shown in Figure 4.4. Maya Naidu produced much of the data presented in Figure 4.5, while Dr Ian Tietjen performed the experiments presented in Figure 4.7 and contributed to data collection for Figure 4.6. Natalie Kinloch and Aniqa Shahid collected HIV-negative and HIV-positive PBMCs from blood donors under the clinical supervision of Dr. Marianne Harris. Additionally, Dr. Ian Tietjen performed the original JurTat HTS of the Rockefeller University HTSRC compound library with the assistance and resources of Dr Charles Rice, Dr. Brandon Razooky, Jeanne Chiaravalli, Brittiny Dhital, Marianne Harris, and J. Fraser Glickman.

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121 Chapter 5. Conclusion

5.1. Summary

Despite effective antiretroviral therapies, there is currently no safe, scalable cure for HIV infection. This is because current licensed ARVs only target actively replicating virus. The persistence of a latent HIV reservoir during long-term cART, from which infectious virus re-emerges in the absence of treatment, complicates treatment and represents a major barrier in cure efforts [1][2]. Currently, no licensed ARV targets transcription or expression from HIV reservoirs. This results in low levels of residual viral protein expression in patients, leading to chronic inflammation and other associated conditions [3]. Additionally, the presence of the reservoir ensures that cART must be maintained for life, leading to long-term issues such as drug toxicities, induced comorbities, and emerging viral resistance [3][4][5].

The Block and Lock strategy for a functional HIV cure involves durably promoting latency of integrated HIV proviruses and preventing them from re-activating [2][6]. Theoretically, permanent or long-term latency could allow for drug-free HIV remission, wherein individuals could discontinue cART without viral rebound, while also preventing low levels of viral protein expression. At the very least, an effective inhibitor of HIV protein expression would represent a new class of ARV and could supplement current cART strategies. Of particular interest pharmaceutically are two potential viral drug targets that, if targeted, can prevent expression of viral proteins from integrated proviruses. The HIV regulatory proteins Tat and Rev are required for viral reactivation from latency: Tat greatly upregulates productive transcription from the integrated genome, while Rev allows translation of the full complement of HIV proteins, by exporting intron-containing mRNAs before they are fully spliced [7][8]. Pharmaceutical targeting of these important proteins and their functions has been shown to delay viral rebound from latency in the absence of ARVs, in cell lines, primary cells, and humanized mice, thus providing proof-of-concept for Block-and-Lock [6][9][10].

However, there are currently no licensed therapies that target either Tat or Rev. Novel compounds that can target these proteins, and thereby induce a deep latent state, are of particular interest. In this thesis, I have described efforts to discover novel inhibitors of HIV replication that act a post-integration step of the viral life cycle. I further

122 described the investigation into the mechanism of several natural flavonoids and characterized their potential as Block-and-Lock agents. Furthermore, I have described the identification of novel synthetic Tat inhibitors.

In Chapter 2, I described the results of a multicycle HIV replication assay in CEM-GXR cells, which identified four structurally related antiviral flavonoids from African natural products: apigenin, chrysin, luteolin-7-O-β-glucoside, and 3-methoxy-6-prenyl- apigenin. In our investigation of these flavonoids, we characterized their antiviral activities in experimentally-infected PBMCs and against ARV-resistant HIV strains. We further investigated their activities as post integration inhibitors of latency reversal in J- Lat 9.2 T cells and OM-10.1 macrophages. We discovered that while apigenin and chrysin were potent inhibitors of HIV Tat, L7G was not. Interestingly, apigenin, chrysin, and luteolin were also capable of promoting latency in long-term treated J-Lat 10.6 cells, while L7G was not.

In Chapter 3, I described the continuing investigation into the mechanisms of action of these flavonoids. Using a luminescence-based kinase assay, we discovered that apigenin, chrysin, and luteolin were more potent inhibitors of the Tat-required kinase CDK9 than L7G, suggesting that the glycosyl-moiety was interfering in CDK9 inhibition and providing an explanation for the inability of L7G to inhibit Tat. Furthermore, we showed that CK2 inhibition was not likely responsible for the flavonoids’ antiviral activity, as a selective CK2 inhibitor was unable to block Tat function and was less potent at blocking latency reversal, despite similar activity against CK2 in vitro. Additionally, we showed that the flavonoids interfered with HIV splicing, preventing accumulation of single-spliced and unspliced transcripts. However, this activity was not due to Rev inhibition. I further described the identification of two structurally-similar flavonoids that, in contrast to the previously-described flavonoids, synergised with a PKC activator, but not an HDAC inhibitor, to reverse latency. We showed that one of these flavonoids, jaceosidin, had HDAC inhibiting activity; additionally, apigenin and chrysin showed HDAC inhibiting activity as well, providing an explanation for their reversibility in our Block and Lock assay described in the previous chapter. Finally, we confirmed that CDK9 is a potential target for Block-and-Lock strategies by demonstrating that long-term treatment with subtoxic concentrations of the CDK9 inhibitor Flavopiridol durably promoted latency in J-Lat 10.6 cells.

123 In Chapter 4, I described the discovery of A7 and C11, two novel inhibitors of HIV Tat function, from a high throughput microscopy screen of more than 90,000 synthetic compounds. After confirming their activities as latency reversal inhibitors, we identified C11 as a potent CDK9 inhibitor, while showing that the activity of A7 is not due to inhibition of NFκB signalling. Finally, we demonstrated that A7 and C11 could inhibit HIV replication in PBMCs, and block viral transfer from PBMCs from HIV-positive donors.

My thesis research has increased our knowledge of the anti-HIV activities and probable mechanisms of several natural flavonoids. Apigenin, luteolin, and chrysin have been described as HIV post integration inhibitors; however, the source of their anti-HIV activity is not fully understood. Our work investigating their mechanisms of action has increased our understanding of how these compounds interfere with multiple steps in HIV latency reversal and underscored their potential as antivirals. In addition, I have highlighted the potential of these compounds as Block-and-Lock agents, and identified CDK9 inhibition as a potential avenue for the development of future Block-and-Lock compounds. Furthermore, I have described several structural variations that increase, decrease, or completely change the function of flavones; understanding which structural variations are responsible for which activities can be important in the identification of new drug targets and the design of more specific or more potent inhibitors. Finally, A7 and C11 represent the success of a high throughput screen for novel Tat inhibitors; the JurTat cell line had not previously been used in this manner and represents a potentially useful tool for identifying new drug candidates. Moving forward, investigating the mechanism of action of A7, in particular, will hopefully increase our understanding of the factors involved in HIV Tat function and identify potential targets for pharmaceutical interventions. Taken together, we anticipate this work will contribute to ongoing efforts to achieve drug-free HIV remission in people living with HIV/AIDS in Canada and worldwide.

5.2. References

[1] R. F. Siliciano and W. C. Greene, “HIV latency.,” Cold Spring Harb. Perspect. Med., vol. 1, no. 1, p. a007096, Sep. 2011.

[2] G. Darcis, B. Van Driessche, and C. Van Lint, “HIV Latency: Should We Shock or Lock?,” Trends Immunol., vol. 38, no. 3, pp. 217–228, 2017.

124 [3] S. Zicari et al., “Immune Activation, Inflammation, and Non-AIDS Co-Morbidities in HIV-Infected Patients under Long-Term ART.,” Viruses, vol. 11, no. 3, 2019.

[4] M. Massanella, R. Fromentin, and N. Chomont, “Residual inflammation and viral reservoirs: alliance against an HIV cure.,” Curr. Opin. HIV AIDS, vol. 11, no. 2, pp. 234–41, Mar. 2016.

[5] A. Chawla et al., “A Review of Long-Term Toxicity of Antiretroviral Treatment Regimens and Implications for an Aging Population,” Infectious Diseases and Therapy, vol. 7, no. 2. Springer, pp. 183–195, Jun-2018.

[6] C. F. Kessing et al., “In Vivo Suppression of HIV Rebound by Didehydro- Cortistatin A, a ‘Block-and-Lock’ Strategy for HIV-1 Treatment,” Cell Rep., vol. 21, no. 3, pp. 600–611, 2017.

[7] C. A. Rosen, “Tat and Rev: positive modulators of human immunodeficiency virus gene expression.,” Gene Expr., vol. 1, no. 2, pp. 85–90, May 1991.

[8] J. Karn and C. M. Stoltzfus, “Transcriptional and posttranscriptional regulation of HIV-1 gene expression,” Cold Spring Harb. Perspect. Med., vol. 2, no. 2, p. a006916, Feb. 2012.

[9] G. Mousseau, C. F. Kessing, R. Fromentin, L. Trautmann, N. Chomont, and S. T. Valente, “The tat inhibitor didehydro-cortistatin a prevents HIV-1 reactivation from latency,” MBio, vol. 6, no. 4, pp. 1–14, 2015.

[10] N. Campos et al., “Long lasting control of viral rebound with a new drug ABX464 targeting Rev - mediated viral RNA biogenesis,” Retrovirology, vol. 12, no. 1, p. 30, Dec. 2015.

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