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 Center for Sickle Cell Disease, Howard University

2. CYTOPLASM REVERSE TRANSCRIPTION NUCLEUS INTEGRATION TRANSCRIPTION TRANSLATION 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

3. Karn, J. (1999) J. Mol. Biol. 293: 235-254

4. HIV-1 Tat Regulates Phosphorylation of RNA Polymerase II C-terminal Domain CTDo RNA Pol II PP P CTDa RNA Pol II Tat: CDK9/cyclin T1, RPII CTD = (YSPTSPS) 52 Tat : FCP1, PPP PP1 CDK2/cyclin E Tat CTD RPII CTD P RPII

5. Tat interaction network Gautier VW et al. Retrovirology. 2009 May 19;6:47

6. 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

7. 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

9. 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. )

10. Candidate Macromolecular Modeling Organic Chemistry Screening Biochemistry

11. Active site Surface of the PP1 colored by hydrophobicity: Blue – hydrophilic residues Orange – hyprophobic residues RVSF peptide from Gm KVKF peptide from MYPT1 The Crystal Structure of PP1 Bound to an RVxF-containing Peptide

12. PlatewellInhibition of HIV-1 Transcription in CEM-GFP cells (IC 50,  M) Toxicity in CEM cells, (IC 50,  M) Inhibition of HIV-1 transcription in 293T cells (IC 50,  M) Plate 01H0412.5>25 5 Plate 01C07106 >10 Plate 01G1020>25 Plate 01D12207.5 Plate 02D0220>25 Plate 02C031510 >10 Plate 02B051015 Plate 02C0615 >10 Plate 02B0755 Plate 02E091520 >10 Plate 02G1020 Plate 03E0120 Plate 03G0115 Plate 03C0220>25 Plate 03A0620>25 >10 Plate 03A0825>25 >10 Plate 03C0825 >10 Selected Compounds that Inhibited HIV-1 Transcription

13. B Inhibitor,  M Transcripation, % of control 0 25 50 75 100 01020304050 1H4 1G3 HIV-1 Transcription 1H4 Inhibits HIV-1 Transcription and Replication A RVTF 1H4

14. Inhibition Tat-induced transcription in CEM cells, IC 50 Inhibition Tat-induced transcription in 293T cells, IC 50 50% Inhibition of HIV-1 replication Toxicity in CEM cells, uptake of PI Toxicity in 293T cells, LDH assay 1H04 10  M5  M 10  M Not toxic 1E07 2  M3  M 1  M Not toxic 1B03 1M1M NO Not toxic Optimization of the 1H4 Compound

15. 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

16. Acknowledgements Victor GordeukHoward University, Tatiana Ammosova Center for Sickle Xiaomei Niu Cell Disease Sharroya Charles Zufan Debebe Altreisha Foster Mathieu BollenCatholic University, Leuven, Belgium Kuan-Teh Jeang NIAID, NIH Marina Jerebtsova Children’s National Medical Center Patricio Ray Dmytro Kovalskyy Enamine, Ukraine Maxim Platonov