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HIV-1 TAT-MEDIATED TRANSCRIPTION AS ATTRACTIVE INTERVENTION SITE FOR SMALL MOLECULES

P. P. P. P. Ac. Ac. Ac. Ac. P. P. HIV-1 TAT-MEDIATED TRANSCRIPTION AS ATTRACTIVE INTERVENTION SITE FOR SMALL MOLECULES. CDK9. CycT1. Hexim1. 7SK RNA. P/CAF. Tat. CDK9. CycT1. Tat. CDK9. CycT1. P300/CBP. ChemMedChem 2010 , 5, 1880-92. TAR. TATA. Tat. NF- kB. SP1.

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HIV-1 TAT-MEDIATED TRANSCRIPTION AS ATTRACTIVE INTERVENTION SITE FOR SMALL MOLECULES

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  1. P P P P Ac Ac Ac Ac P P HIV-1 TAT-MEDIATED TRANSCRIPTION AS ATTRACTIVE INTERVENTION SITE FOR SMALL MOLECULES CDK9 CycT1 Hexim1 7SK RNA P/CAF Tat CDK9 CycT1 Tat CDK9 CycT1 P300/CBP ChemMedChem 2010, 5, 1880-92 TAR TATA Tat NF-kB SP1 RNApII NELF TAR Oriana Tabarrini P P P P DSIF CDK9 CycT1 Ac Department of Chemistry and Technology of Drugs Faculty of Pharmacy, University of Perugia Tat CTD NELF P/CAF RNApII DSIF GRS for Simulation Sciences GmbH, JÜLICH, October, 18, 2011 CTD

  2. 33.3 million [31.4 – 35.3 million]people living with HIV 2.6 million [2.43 – 2.8 million] people newly infected with HIV 1.8 million [1.6 – 2.1 million] AIDS-related deaths http://data.unaids.org/pub/Report/2009/JC1700_Epi_Update_2010_en.pdf AIDS epidemic update 2010 GRS for Simulation Sciences GmbH, JÜLICH, October,18, 2011

  3. TMC 125 RAL MVC TMC 114 26 ENF ATV FAPV FTC 24 TPV 22 20 TDF 18 LPV EFV ABC APV 16 NFV DLV 14 RTV IDV NVP 12 10 SQV3TC 8 d4T 6 ddC ddI 4 AZT 2 0 2003 2004 2005 2006 2007 2008 2002 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 antiretroviral agents fusion inhibitors chemokine receptor inhibitors nucleoside/nucleotide reverse transcriptase inhibitors non-nucleoside reverse transcriptase inhibitors integrase inhibitors protease inhibitors GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  4. HAART (highly active antiretroviral therapy) LIMITS  side-effects  high cost drug resistance development inability to completely eradicate virus infections BENEFITS  reduction of viral load  reductions in morbidity and mortality dramatic increase in life expectancy HAART is no panacea! GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  5. CD4 +T cells dendritic cells sanctuaries central nervous system cells monocytes/ macrophages compartments anatomical sites genital tract gastro-intestinal tract HIV reservoirs such reservoirs are ideally suited to keep hidden copies of the virus, which in turn, can trigger a new systemic infection upon discontinuation of therapy GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  6. AIDS chronic disease “a diamond is forever” ….. can HIV infection ever be cured? GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  7. long-term suppression of the virus eradication of the HIV reservoirs the need to find additional strategies identification of new compounds that act on alternative targets or steps of the viral replicative cycle GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  8. mRNA proviral DNA HIV transcriptional regulation: a valid intervention site the transcription of HIV genome is a crucial step for viral replication amplification of viral genetic information is required to re-initiate the viral replication from post-integration latency after interruption of therapy GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  9. PIC P-TEFb TFIID productive elongation CDK9 CycT1 Tat P-TEFb P/CAF Tat Ac IIH LTR TAFs TAR IIF IIB abortiveelongation P300/CBP P300/CBP RNApII TBP IIE NELF NELF RNApII RNApII IIA MED NELF HAT P P NF-kB SP1 TATA NF-kB SP1 TATA P P P P P P Nuc1 Ac CDK9 TAR transcriptional machinery CDK9 CycT1 P P NFkB SP1 TATA DSIF CTD CTD CycT1 Tat Ac CTD DSIF DSIF HIV Tat-mediated transcription

  10. two new Tat- complexes He N. et al. Mol. Cell2010, 38, 428–438. Sobhian B. et alMol. Cell2010, 38, 439–451. GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  11. control HIV replication not only in acutely infected cells but also in chronically infected ones force the virusto slow down its replication rate DC M/M HIV variants susceptible to drugs HIV variants resistant to drugs why HIV-1 transcription inhibitors? HIV-1 transcription inhibitors decrease the incidence of drug resistance GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  12. Reviewed in: Stevens M. et al. J. Med. Res. Rev.2006, 26, 595-625. Richter SN. et al.Curr. Med. Chem.2006, 13, 1305-1315. Baba M. Curr. Top. Med. Chem.2004, 4, 871-882. Yang M. Curr. Drug Targets Infect. Disord.2005, 5, 433-444. Klebl BM. et al. Future Virol. 2006, 1, 317-330. Wang S. et al.Trends Pharmacol. Sci.2008, 29, 302-313. Tat HIV-1 Tat-mediated transcription inhibitors P-TEFb (CDK9-CycT1) inhibitors  flavopiridol  roscovitine Tat inhibitors  Tat peptidomimetics  siRNAssequesteringTat’s function Tat/TAR complex inhibitors TAR inhibitors  arginine-aminoglycoside coniugate AAA  intercalator  peptoids (TR87)  rbt 550  prochloperazine  acridine CGP40336A  -hairpin cyclic peptide mimetics of Tat CDK9 other transcription inhibitors  Ro 24-7429  K12, K-37, R-762  RD 6-5071  JTK-101 CycT1 TAR RNApII 6-desfluoroquinolones (6-DFQs) GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  13. docking transcription inhibitor 6-DFQs by UniPG: a short overview fluoroquinolones not only exerted their antibacterial action in AIDS patients but also exhibited antiviral activity per se Nozaki-Renard J. et al. AIDS 1990, 4, 1283.1286. RT screening of our in house 6-desfluoroquinolone antibacterial library WM5 lead compound C8166 cells:EC50 = 0.1 µMCC50 = 7 µM SI = 70 Cecchetti V. et al. J. Med. Chem. 2000, 43, 3799-3802. Cruciani G. et al. XII Conv. Naz. Div. Chim. Farm. SCI. Paestum, Italy, 1996. WT5 hit compound C8166 cells:EC50 = 2.0 µM CC50 = 8.1 µM SI = 4 WM5 does not inhibit RT!! GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  14. Tat bulge loop stem TAR RNA WM5: mechanism of action studies  WM5 does not inhibit P, IN or viral entry  WM5 inhibits the Tat-mediated transcription  WM5 binds the viral TAR RNA selectively and efficiently (Kd = 19 ± 0.6 nM)  the bulge is the specific target  WM5 disrupts Tat /TAR complex formation Cecchetti V. et al. J. Med. Chem.2000, 43, 3799-3802. Parolin C. et al.Antimicrob. Agents Chemother.2003, 47, 889-895. Richter S. et al.Antimicrob. Agents Chemother.2004,48, 1895-1899. GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  15. WM5: lead optimization Cecchetti V. et al. J. Med. Chem.2000, 43, 3799-3802. Tabarrini O. et al. J. Med. Chem.2004, 47, 5567-5578. Tabarrini O. et al. J. Med. Chem.2008,5, 5454-5458. Massari S. et al. Bioorg. Med. Chem.2009, 17, 667-674. Massari S. et al. J. Med. Chem.2010, 53, 641-648. Tabarrini O. et al. Future Med. Chem. 2010, 2, 1161-1180. Tabarrini O. et al. ChemMedChem 2010, 5, 1880-1892. GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  16. HP7-13 anti-HIV activity of selected 6-DFQs HM12 MT-4 cells EC50 (HIV-1) = 0.005 ± 0.002 µM EC50 (HIV-2) >0.05 µM CC50 = 0.05  0.003 µM SI (HIV-1) = 10 MT-4 cells EC50 (HIV-1) = 0.16  0.02 µM EC50 (HIV-2) = 0.30  0.04 µM CC50 = 6.04  1.52 µM SI (HIV-1) = 38 SI (HIV-2) = 20 WM14 CEM cells EC50 (HIV-1) = 0.15  0.01 µM EC50 (HIV-2) = 0.20  0.02 µM CC50≥259 µM SI (HIV-1) ≥1726 SI (HIV-2) ≥1295 HM13 M/M cells EC50 (HIV-1) = 0.05  0.01 µM EC90 (HIV-1) = 0.47  0.02 µm no toxic Nevirapine: activity in MT-4 cells. EC50 (HIV-1) = 0.086  0.05 µM, EC50 (HIV-2) > 15 µM, CC50 > 15 µM, SI (HIV-1) > 174 GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  17. HM13N anti-HIV activity of HM13N best antiviral/cytotoxic profile! MT-4 cells EC50 (HIV-1) = 0.06  0.00 µM EC50 (HIV-2) = 0.04  0.00 µM CC50 = 26.33  6.79 µM SI (HIV-1) = 439 SI (HIV-2) = 658 PMA = phorbol 12-myristate 13-acetate TNF- = tumor necrosis factor  Massari S. et al. J. Med. Chem.2010, 53, 641-648. GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  18. structural investigation of the naphthyridone scaffold HM13N NM13 EC50 (HIV-1) ≥0.08 µM CC50 = >296.58 µM SI>3707 EC50 (HIV-1) = 0.06  0.00 µM CC50 = 26.33  6.79 µM SI = 439 Tabarrini O. et al. ChemMedChem 2011, 6, 1249-1257. GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  19. p24 antigen levels (pg/ml) 1600 1400 1200 1000 800 600 400 200 0 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 No TNF (background) With TNF (control) With TNF and HM13 With TNF and HM12 SCID mice number 6-DFQs: antiviral profile showed a pronounced suppressive effect on viral reactivation in an in vivo model of HIV-1 latency Stevens M. et al. Antimicrob. Agents Chemother.2007, 5, 1407-1413 showed no tendency to select for resistant mutations in vitro after 30 weeks of selective pressure Massari S. et al. J. Med. Chem.2010, 53, 641-648. GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  20. control 0 3 10 16 20 30 dextran sulfate AZT 6-DFQs ritonavir 6-DFQs: mechanism of action time-of-addition (TOA) experiment Tat-mediated transcription assay 6-DFQs inhibit the transcription in a dose-dependent manner in the µM range 6-DFQs are inactive in the basal expression 6-DFQs were confirmed to act at a post-integrational level Stevens M. et al. J. Antimicrob. Chemother.2005, 56, 847-855. Massari S. et al. J. Med. Chem.2010, 53, 641-648. Tabarrini O. et al. ChemMedChem 2010, 5, 1880-1892 Tabarrini O. et al. J. Med. Chem.2004, 47, 5567-5578. Tabarrini O. et al. J. Med. Chem.2008,5, 5454-5458. Massari S. et al. J. Med. Chem.2010, 53, 641-648. GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  21. 6-DFQs: mechanism of action  WM5 selectively binds TAR bulge  the same effect was not observed for other potent 6-DFQs hypothesis TARGET: host cellular factor or host cellular factor/viral component complex implicated in the Tat-mediated transcription evidence  different behaviour in various cell lines  inability to select for resistance mutations  ability to inhibit the transactivation process of HIV-unrelated promoters (human CMV IE and human EF1-) Stevens M. et al. J. Antimicrob. Chemother.2005, 56, 847-855. PTEF-b and HAT (p300/CBP) have ruled out as a target Massari S. et al. J. Med. Chem.2010, 53, 641-648. studies are in progress to fish the molecular target GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  22. CDK9 active P-TEFb CycT1 inactive P-TEFb CDK9 Tat CycT1 BRD4 CDK9 CycT1 Hexim1 TAR 7SK RNA P300/CBP NELF RNApII P NF-kB SP1 TATA P P P P CTD DSIF P-TEFb (CDK9/CyclinT1) inhibitors phosphorylates negative elongation factors hyperphosphorylates RNApII increasing the efficacy of transcription elongation GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  23. cyclinT1 CDK9 CycT1 Tat CDK9 flavopiridol P-TEFb inhibitors: SBDD TAR GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  24. P-TEFb inhibitors: SBDD ad hoc library derived from fragments fragment collections docking core selection substructure search • chemical accessibility vendor databases in-house compounds • physicochemical properties • scoring Functions • visual inspection virtual screening • anti-CDK-9 assay • transactivation assay • cytotoxicity • anti-HIV assay • CDK selectivity 16 “Virtual” HITS synthesis biological screening design in-depth biological evaluation • SAR explorations • MM-GB/SA • FEP calculations 5 “Real” HITS GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  25. P-TEFb inhibitors: SBDD CDK9 CycT1 TAR Tat …new opportunities for designing selective compounds GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011

  26. acknowledgments Department of Chemistry and Technology of Drugs (UniPG) University of Leuven, Rega Institute for Medical Research (Belgium) Christophe Pannecouque Dirk Daelemans Violetta Cecchetti Oriana Tabarrini Stefano Sabatini Giuseppe Manfroni M. Letizia Barreca Roberto Bianconi Serena Massari Nunzio Iraci Luca Sancineto University of Padova (Italy) Manlio Palumbo Giorgio Palù Barbara Gatto ICGEB (Trieste, Italy) Alessandro Marcello

  27. is P-TEFb drugable? indirect antiviral agents  host and viral transcription are differently sensitive to CDK9 inhibition  P-TEFb activity is much more heavily involved in pathologic than in normal cellular functions  HIV leads to an increased expression of cyclin T1  prevent the emergence of resistance  combined with the existing direct antiviral drugs, it can result in additive or even synergistic treatment options known P-TEFb Inhibitors evidence Klebl BM. et al. Future Virol. 2006, 1, 317-330. Wang S. et al.Trends Pharmacol. Sci.2008, 29, 302-313. Yes it seems to be DRB, flavopiridol and roscovitine  block HIV-1 replication at concentrations that are not cytotoxic  are also active against various drug-resistant HIV strains and do not lead to resistance formation

  28. NUCLEIC ACID VIRAL factor CELLULAR factor a pharmacophoric model ??

  29. WM5

  30. 6-DFQs do not inhibit integrase Concentration-dependent anti-HIV-1 effects of nevirapine, L-870,810 and compound 3. C8166 T-cells infected with HIV-1 NL4.3 (red triangles) or N/N.Tag.oriT (blue squares) were treated with the indicated compound concentrations. One hundred percent was defined as the level of p24 in the supernatants in control, non-drug-treated samples. Toxicity of the compounds was measured using the MTT-method (green triangles).

  31. elvitegravir GS-9137/JTK-303 6-DFQs do not inhibit integrase Comparison among IFD conformations of L-870,810 (orange), elvitegravir (black) and 3 (white). Molecular surfaces are shown for IN (gray), catalytic loop (residues 140-149; cyan), metal ions (magenta), 3’ DNA strand (green) and 5’ DNA strand (yellow). This Figure was prepared using PyMol.41

  32. D.E.4425647A1(Bayer) fluoroquinolone transcription inhibitors R-71762 – (Sankyo-Ube) EC50 = 0.19 M (CEM cells) CC50 = 14 M (CEM cells) K-12– (Daiichi Pharm) EC50 = 0.085 M PBMCs) CC50 = 16.3 M (PBMCs) Yamashita, M.. et. al. Bioorg. Med. Chem. Lett. 2002, 12, 739-742 Baba, M. et al.Antimicrob. Agents Chemother. 1999, 43,492-497.

  33. Virus reactivation from latent reservoirs: “shock and kill” strategy generalized immune activation “shock phase” “Kill phase” naturally:viral cytopathic effects or by immune effector mechanisms artificially:drugs or antibodies the goal of completely purging latent reservoirs and eradicating HIV in infected patients, is still elusive !!

  34. HP7-13 anti-HIV activity of selected 6-DFQs MT-4 cells EC50 (HIV-1) = 0.16  0.02 µM EC50 (HIV-2) = 0.30  0.04 µM CC50 = 6.04  1.52 µM SI (HIV-1) = 38 SI (HIV-2) = 20 HM12 MT-4 cells EC50 (HIV-1) = 2.30  0.30 µM EC50 (HIV-2) = 2.84  1.82 µM CC50>296 µM SI (HIV-1) >129 SI (HIV-2) >104 H13AM MT-4 cells EC50 (HIV-1) = 0.005 ± 0.002 µM EC50 (HIV-2) >0.05 µM CC50 = 0.05  0.003 µM SI (HIV-1) = 10 SI (HIV-2) <1 complete protection! Nevirapine: activity in MT-4 cells. EC50 (HIV-1) = 0.086  0.05 µM, EC50 (HIV-2) > 15 µM, CC50 > 15 µM, SI (HIV-1) > 174

  35. anti-HIV activity of selected 6-DFQs PBMC cells EC50 (HIV-1) = 0.11  0.00 µM CC50 = 8.75  5.59 µM SI (HIV-1) = 79 WM21 CEM cells EC50 (HIV-1) = 0.15  0.00 µM EC50 (HIV-2) = 0.20  0.00 µM CC50≥259 µM SI (HIV-1) ≥1726 SI (HIV-2) ≥1295 WM14 M/M cells EC50 (HIV-1) = 0.05  0.01 µM EC90 (HIV-1) = 0.47  0.02 µm no toxic HM13 reservoirs!

  36. 6-DFQs: mechanism of action Tat-mediated transcription assay (HeLa cell clone carrying an LTR driven luciferase reporter) 6-DFQs are inactive in the basal expression 6-DFQs inhibits the transcription in a dose-dependent manner in the µM range Stevens M. et al. J. Antimicrob. Chemother. 2005, 56, 847-855. Massari S. et al. J. Med. Chem. 2010, 53, 641-648.

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