1 / 16

Targeting Protein Phosphatase-1 for the Inhibition of HIV-1

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.

brandy
Download Presentation

Targeting Protein Phosphatase-1 for the Inhibition of HIV-1

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

  2. HIV-1 Life Cycle DENDRITIC CELL ENTRY UNCOATING CYTOPLASM REVERSE TRANSCRIPTION VIRAL PROTEINS MATURATION T CELL ASSEMBLY BUDDING INTEGRATION CELL FACTORS TRANSLATION TRANSCRIPTION RNA SPLICING, TRANSPORT NUCLEUS 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 Tat: CDK9/cyclin T1, CTDo CTDa P P P Tat : FCP1, RNA Pol II RNA Pol II P RPII RPII CTD CTD Tat (YSPTSPS)52 CTD = RPII CDK2/cyclin E PP1 PPP

  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 fundamentaldifference 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

  8. 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. )

  9. Design of PP1 Inhibitors Target: PP1 Compounds Selection Howard U. Enamine, Ukraine Macromolecular Modeling Organic Chemistry Screening Biochemistry Screening Candidate

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

  11. Plate well Inhibition of HIV-1 Transcription in CEM-GFP cells (IC50, mM) Toxicity in CEM cells, (IC50, mM) Inhibition of HIV-1 transcription in 293T cells (IC50, mM) 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

  12. 1H4 Inhibits HIV-1 Transcription and Replication A RVTF 1H4 C Toxicity 1H4 100 1G3 A02 80 Viability, % of control 60 40 20 0 0 20 40 60 80 100 120 Inhibitor, mM D HIV-1 replication DMSO 1G3 2mM 60 1G3 10 mM 1G3 25 mM 50 1H4 2 mM 40 1H4 10 mM 30 1H4 25 mM RT, arbitrary counts 20 10 0 0 2 4 6 8 10 Viral infection, Days B HIV-1 Transcription 100 1H4 75 1G3 50 Transcripation, % of control 25 0 0 10 20 30 40 50 Inhibitor, mM

  13. 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 mM 5 mM 10 mM Not toxic Not toxic 1E07 2 mM 3 mM 1 mM Not toxic Not toxic 1B03 1mM NO Not toxic Not toxic Optimization of the 1H4 Compound

  14. 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

  15. 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, Ukraine Maxim Platonov

More Related