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Targeting Protein Phosphatase-1 for the Inhibition of HIV-1. Sergei Nekhai, Ph.D. Center for Sickle Cell Disease, Howard University. NIGMS, NIH RCMI –NCRR NHLBI, NIH Civilian Research and Development Foundation. HIV-1 Life Cycle. DENDRITIC CELL. ENTRY. UNCOATING. CYTOPLASM. - PowerPoint PPT Presentation
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Targeting Protein Phosphatase-1 for the Inhibition of HIV-1
Sergei Nekhai, Ph.D.
NIGMS, NIH RCMI –NCRR
NHLBI, NIH Civilian Research and Development Foundation
Center for Sickle Cell Disease, Howard University
CYTOPLASMREVERSE TRANSCRIPTION
NUCLEUS
INTEGRATION
TRANSCRIPTIONTRANSLATION
BUDDING ASSEMBLY
MATURATION
UNCOATING
VIRAL PROTEINS
DENDRITIC CELL
CELL FACTORS
RNA SPLICING, TRANSPORT
T CELL
ENTRY
HIV-1 Life Cycle
Nekhai and Jerebtsova, Curr.Opin.Mol. Therapy, 2006
Karn, J. (1999) J. Mol. Biol. 293: 235-254
HIV-1 Tat Regulates Phosphorylation of RNA Polymerase II C-terminal Domain
CTDo
RNA Pol II
PPPCTDa
RNA Pol II
Tat: CDK9/cyclin T1,
RPII CTD = (YSPTSPS)52
Tat : FCP1,
PPP
PP1
CDK2/cyclin E
TatCTD
RPII
CTD
PRPII
Tat interaction network
Gautier VW et al. Retrovirology. 2009 May 19;6:47
How a small viral protein can be involved in enormous amount of protein- protein
interactions?
• Tat modulates activity of a key enzyme that can regulate different nuclear processes by protein modification (phosphorylation)
• A fundamental difference in the substrate recognition by protein kinases and phosphatases:
• Each kinase recognizes its distinct substrate • Protein phosphatases consist of a constant catalytic
subunit and a variable regulatory subunit that determines the localization, activity and substrate-specificity of the phosphatase
Hypothesis HIV-1 Tat interacts with PP1
Tat-PP1 interaction serves to dephosphorylate multiple proteins (CDK9, Sp1 or RNAPII CTD during HIV-1 transcription)
Disruption of Tat-PP1 interaction inhibits HIV-1
Regulation of HIV-1 transcription by Protein Phosphatase-1
• PP1 supports Tat-mediated transcription in vitro (Bharucha et al., Virology 2002; Nekhai et al., Biochem. J. 2002)
• PP1 serves as RNA polymerase II phosphatase (Washington et al., J. Biol. Chem. 2002)
• NIPP1 expression inhibits Tat-dependent HIV-1 transcription (Ammosova et al., J. Biol. Chem. 2003)
• Tat interacts with PP1 and reallocates it to the nucelus (Ammosova et al., 2005, J. Biol. Chem. )
Design of PP1 InhibitorsDesign of PP1 Inhibitors
Candidate
Target: PP1Target: PP1
Compounds SelectionCompounds Selection
Macromolecular Modeling
Organic ChemistryScreening
Biochemistry ScreeningScreening
Howard U.Howard U. Enamine, UkraineEnamine, Ukraine
Active site
Surface of the PP1 colored by hydrophobicity: Blue – hydrophilic residuesOrange – hyprophobic residues
RVSF peptide from GmKVKF peptide from MYPT1
The Crystal Structure of PP1 Bound to an RVxF-containing Peptide
Plate well Inhibition of HIV-1 Transcription in CEM-GFP cells (IC50, M)
Toxicity in CEM cells, (IC50, M)
Inhibition of HIV-1 transcription in 293T cells (IC50, M)
Plate 01 H04 12.5 >25 5
Plate 01 C07 10 6 >10
Plate 01 G10 20 >25
Plate 01 D12 20 7.5
Plate 02 D02 20 >25
Plate 02 C03 15 10 >10
Plate 02 B05 10 15
Plate 02 C06 15 15 >10
Plate 02 B07 5 5
Plate 02 E09 15 20 >10
Plate 02 G10 20 20
Plate 03 E01 20 20
Plate 03 G01 15 15
Plate 03 C02 20 >25
Plate 03 A06 20 >25 >10
Plate 03 A08 25 >25 >10
Plate 03 C08 25 25 >10
Selected Compounds that Inhibited HIV-1 Transcription
B
Inhibitor, M
Tra
nscr
ipat
ion,
% o
f con
trol
0
25
50
75
100
0 10 20 30 40 50
1H41G3
HIV-1 Transcription
C
0
20
40
60
80
100
0 20 40 60 80 100 120
1H41G3A02
Inhibitor, M
Via
bilit
y, %
of c
ontr
ol
Toxicity
0
10
20
30
40
50
60
0 2 4 6 8 10
Viral infection, Days
RT
, a
rbitr
ary
co
un
ts
1G3 2M1G3 10 M1G3 25 M
1H4 2 M1H4 10 M1H4 25 M
DMSO D HIV-1 replication
1H4 Inhibits HIV-1 Transcription and Replication
A
RVTF1H4
Inhibition Tat-induced transcription
in CEM cells, IC50
Inhibition Tat-induced transcription
in 293T cells, IC50
50% Inhibition of HIV-1
replication
Toxicity in CEM cells, uptake of
PI
Toxicity in 293T cells, LDH assay
1H04 10 M 5 M 10 M Not toxic Not toxic
1E07 2 M 3 M 1 M Not toxic Not toxic
1B03 1M NO Not toxic Not toxic
Optimization of the 1H4 Compound
Conclusions• HIV-1 can be inhibited by small molecule
compounds that mimics the PP1-binding RVXF peptide
• 1H4 inhibits dephosphorylation of hybrid pRb-Tat substrate by PP1 and disrupt the interaction of HIV-1 Tat with PP1 (not shown here
• 1H4 is the first example of a small molecule non-competitive inhibitor of PP1 that affects HIV-1
• Our study opens PP1 as a new avenue for the design of novel antiretroviral therapeutics
Acknowledgements Victor Gordeuk Howard University,
Tatiana Ammosova Center for Sickle Xiaomei Niu Cell Disease Sharroya Charles
Zufan Debebe Altreisha Foster
Mathieu Bollen Catholic University, Leuven, Belgium
Kuan-Teh Jeang NIAID, NIH
Marina Jerebtsova Children’s National Medical Center Patricio Ray
Dmytro Kovalskyy Enamine, UkraineMaxim Platonov