Hiv 1 tat mediated transcription as attractive intervention site for small molecules
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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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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

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

AIDS

chronic disease

“a diamond is forever”

….. can HIV infection ever be cured?

GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

cyclinT1

CDK9

CycT1

Tat

CDK9

flavopiridol

P-TEFb inhibitors: SBDD

TAR

GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

P-TEFb inhibitors: SBDD

CDK9

CycT1

TAR

Tat

…new opportunities for designing selective compounds

GRS for Simulation Sciences GmbH, JÜLICH October,18, 2011


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

NUCLEIC ACID

VIRAL factor

CELLULAR factor

a pharmacophoric model ??


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

WM5


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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).


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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.


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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 !!


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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!


Hiv 1 tat mediated transcription as attractive intervention site for small molecules

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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