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Transcript of Targeting Protein Phosphatase-1 for the Inhibition of HIV-1 Sergei Nekhai, Ph.D. NIGMS, NIH RCMI...
![Page 1: 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.](https://reader035.fdocuments.us/reader035/viewer/2022062222/5697bff31a28abf838cbc748/html5/thumbnails/1.jpg)
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
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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
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Karn, J. (1999) J. Mol. Biol. 293: 235-254
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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
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Tat interaction network
Gautier VW et al. Retrovirology. 2009 May 19;6:47
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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
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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
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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. )
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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
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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
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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
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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
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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
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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
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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