Sirtuins, New Potential Targets for Hiv-Therapy

SIRTUINS, NEW POTENTIAL TARGETS FOR HIV-THERAPY Di Rosa Michelino1,2,Gnemmi Ilaria1, Riva Beatrice1, Galli Ubaldina1, Genazzani Armando A1, Canonico Pier Luigi 1, Marilia Rita Pinzone2, Condorelli Fabrizio1, Nunnari Giuseppe2,3 1 Dept. of Pharmaceutical Sciences, School of Pharmacy, University of Piemonte Orientale “A. Avogadro”, Novara, Italy 2 Dept. of Clinical and Experimental Medicine, Division of Infectious Diseases, University of Catania, Italy 3 Dept. of Clinical and Experimental Medicine, Division of Infectious Diseases, University of Messina, Italy

ABSTRACT EVALUATION OF INTRINSIC CYTOTOXICITY OF DRUGS AFFECTING SIRTUIN ENZYMES

BACKGROUND: Highly active antiretroviral therapy (HAART) has shown great efficacy in increasing the survival of Human Immunodeficiency Virus (HIV)-infected individuals, although this protocol, which is mainly based on targeting viral encoded molecules, cannot eradicate the virus. T6 C2 At the molecular level, HIV life-cycle critically relies not only on the action of viral proteins since many host genes have to interact with them in order to allow infection. In particular, after retrotranscription of the viral genomic 150 150 RNA, integration of the proviral DNA into the host genome is allowed by the interaction between viral integrase and broken-DNA fixing proteins, provided by the host cell, in a process called “post-integration repair”. 100 100

50 50 AIM: Based on these assumptions, we investigated the potential impact on HIV integration of those enzymes that catalyse the removal of acetyl groups from chromatin proteins. In particular, we studied the sirtuin class of 0 T6 24h 0 C2 24h proteins, since this family of NAD+-dependent de-acetylases is recruited to the sites of DNA damage to establish functional interactions with “repairing factors”, such as Ku70 or the ATM/Nsb1 complex. -50 T6 48h -50 C2 48h T6 72h C2 72h

METHODS: To quantitatively assess the integration of viral DNA into the host genome, we challenged HeLa cells with an HIV-based, replication-defective, lentivirus that carries the “green fluorescent protein” (GFP) reporter -100 -100

Abs [570nm-b] % of ctrl Abs [570nm-b] % of ctrl gene. Indeed, by infecting this human cell line in a range of 0,1-0,2 MOI (in order to avoid multiple infections in the same cell), we were able to measure “integrational” events by discriminating, through flow cytometry, -150 -150 between population of cells expressing lower levels of the GFP protein (as consequent to “episomal” localization of the viral genome), from cells characterized by a brighter green fluorescence enabled by the integration of the 0,01 0,1 1 10 100 0,01 0,1 1 10 100

GFP gene into the host genome. conc. (µM) conc. (µM)

RESULTS: By this approach we identified, from a library of commercially available and newly synthesized sirtuins inhibitors, three molecules, T6 (Sirt1 and 2), B2 (Sirt2) and compound-2 (Sirt3), which were capable to inhibit HIV B2 integration with a good concentration/toxicity profile. 150 Importantly, data emerging after flow cytometry were calibrated on the detection and quantification of the Alu-gag sequences, which represent hybrid regions resulting from the integration of viral genes into the host genome, via nested PCR on the DNA purified from infected cells. Also supporting the involvement of sirtuins in the molecular steps leading to viral integration, treatments with resveratrol, which activates the whole family of de- 100 Fig. 4. Effect of sirtuin inhibitors on HeLa cells acetylases by increasing intracellular NAD+ levels, enhanced HIV integration, as determined by assessment of GFP-fluorescence intensity. 50 viability. HeLa cells were treated for 24, 48 and

0 B2 24h 72 h with tenovin-6 (T6, 0,1-1-3-10-30-100 µM), CONCLUSION: Taken together, our observations provide the first evidence that sirtuins may be considered as a new potential class of targets for HIV-therapy. Importantly, this paves the way to the development of drugs able to -50 B2 48h compound-2 (C2, 0,1-1-3-10-30-100 µM) or B2 inhibit HIV life-cycle on the cellular side in order to avoid resistance to therapies consequent to the tendency of viral proteins to modify their structures over replication rounds.

-100 B2 72h (1-5-25 µM). Then, cell viability was assessed by Abs [570nm-b] % of ctrl -150 MTT assay. 0,1 1 10 100 HIV LIFE-CYCLE conc. (µM) Fusion inhibitors enfuvirtide EVALUATION OF VIRAL INTEGRATION AS MODIFIED BY PHARMACOLOGICAL TREATMENTS gp120 inhibitors

Reverse Transcriptase RESVERATROL CD4 inhibitors CTRL Inhibitors NRTIs-abacavir, didanosine Emtricitabine, lamivudine Co-receptors inhibitors NNRTIs-atevirdine, delavirdine, maraviroc Efavirenz, nevirapine, pyrdinones

Integrase inhibitors Raltegravir

HeLa HeLa Protease inhibitors HeLa infected HeLa infected Atazanavir, darunavir, HeLa infected+RESV 40 µM Fosamprenavir, indinavir, Ritonavir, saquinavir Hela % G F P tot 0,59 Modified by http://www.niaid.nih.gov and Pirrone V. et al. % E pi G F P 0,5 Antimicrob. Agents Chemother. 2011;55:1831-1842. HeLa infected+RESV % G F P tot 34,25 % Int G F P 0,09 % E pi G F P 19,76 Fig. 1. Schematic representation of the HIV life-cycle and the molecular targets of the HAART protocol. Virus enters target cells by binding to CD4 and a coreceptor (CCR5 or CXCR4), thus allowing fusion Hela infected % G F P tot 10,6 % Int G F P 14,49 % E pi G F P 7,03 between the cellular and viral membranes. After entry, the viral nucleoprotein core, containing the genomic RNA, is released into cytoplasm, where reverse transcription takes place. The originating viral % Int G F P 3,57 DNA is then integrated into the host genome, where the so-called pro-virus serves as a transcription template for the synthesis of both viral mRNAs and genomic RNA. Following to their synthesis, viral proteins are assembled to generate new virions; such virus particles need to go through a maturation step in order to generate infectious HIV. Steps in the HIV life-cycle that are blocked by different classes T6 B2 C2 of antiretroviral drugs are indicated in the figure. WORKING HYPOTHESIS

HeLa HeLa HeLa HeLa infected HeLa infected HeLa infected HeLa infected+T6 7.5 µM HeLa infected+B2 25 µM HeLa infected+C2 1 µM

HeLa infected+T6 % G F P tot 10,22 HeLa infected+B2 % G F P tot 5,94 HeLa infected+C2 % G F P tot 7,41 % E pi G F P 7,79 % E pi G F P 4,76 % E pi G F P 6,18 % Int G F P 2,43 % Int G F P 1,18 % Int G F P 1,23

Modified by Van Maele B. et al. Trends Biochem Sci. 2006 Feb;31(2):98-105. Fig. 2. HIV genomic integration and host cell contribution. HIV integration occurs in two steps: 3’-processing and strand transfer. The 3’-processing occurs in the cytoplasm of host cells in the context of a pre-integration complex (PIC), containing viral DNA and proteins (including integrase) as well as cellular proteins. Following to nuclear import of the PIC, the strand transfer step occurs. Finally, the integrase- mediated integration of viral DNA is accomplished by means of post-integration repair (PIR) events. PIR probably relies on DNA double-strand breaks (DSB) repairing enzymes of the host (for example Nsb1, ATM, Ku70), likely to include sirtuin class of de-acetylases. DISCRIMINATION BETWEEN “EPISOMAL” AND “INTEGRATED” EXPRESSION OF VIRUS-CODED PROTEINS

Fig. 5. Effects of sirtuin-targeting drugs on viral integration. HeLa cells, exposed to 0,2 MOI of viral particles were treated with the reverse transcriptase inhibitor efavirenz (EFV, 42,5 nM) or the integrase inhibitor raltegravir (RAL, 250 nM) in order to set thresholds for the discrimination of cells expressing “episomal” GFP from those with integrated copies of viral DNA. Alternatively, sirtuin inhibitors tenovin-6 (T6, 7,5 µM), B2 (25 µM) and compound-2 (C2, 1 µM), or the sirtuin activator resveratrol (RESV, 40 µM), were given contextually to viral particles. Total (tot), episomal (Epi) and integrated (Int) GFP expression levels were assessed, after 72 h, by flow cytometry. Treatments with an inhibitor of the nicotinamide phosphoribosyltransferase (NAMPT), FK866 (2,5 nM) were performed in order to evaluate the impact of NAD+ depletion on viral integration.

CONCLUSIONS

Sirtuin inhibitors decrease HIV integration into host cell genome, representing new potential therapeutics targets

• C2 (Sirt3 specific inhibitor) and B2 (Sirt2 specific inhibitor) cause a shift of GFP expression to the “episomal” mode as efficiently as the integrase inhibitor raltegravir;

• Resveratrol, which activates the whole family of de-acetylases by increasing intracellular NAD+ levels, enhanced HIV integration, as determined by assessment of GFP-fluorescence intensity.

FUTURE PERSPECTIVES Modified by Van Loock M. et al. J Virol Methods 2013 Feb;187(2):238–247.

Fig. 3. Schematic representation of the flow cytometric assessment of HIV integration. HeLa cells were exposed for 72 h to HIV1-based, replication-defective, lentiviruses that carry the “green fluorescent • To dissect the molecular mechanisms involved in C2 and B2 antiretroviral effects through a genetic manipulation of sirtuins subtypes expression. protein” (GFP) reporter gene and treated, contextually, with different drugs. Total, “episomal” and “integrated” expression of GFP was assessed through flow cytometry. A) Integration of the viral DNA into the host genome hesitates into the expression of high levels of GFP while its episomal localization leads to a lower efficiency in GFP production (reduced intensity of the green fluorescence). B) Inhibition of • To evaluate C2 or B2 synergisms with other drugs included in the HAART protocol. the reverse transcriptase by efavirenz (EFV) results in the absence of a GFP signal as no viral DNA is synthesized. C) Administration of the integrase inhibitor raltegravir (RAL) avoids integration thus shifting GFP expression to the “episomal” mode (lower brightness). • Synthesize and characterize C2 and B2 analogues with a better pharmacological profile.