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