Small Molecules Targeted to a Non-Catalytic “RVxF” Binding Site of Protein Phosphatase-1 Inhibit HIV-1

Tatiana Ammosova,Dmytro Kovalskyy,Maxim Platonov,Yuri Obukhov,Sergei Nekhai,Victor R Gordeuk,Venkat R K Yedavalli,Kuan-Teh Jeang,Kylene Kehn-Hall

doi:10.1371/journal.pone.0039481

Abstract

HIV-1 Tat protein recruits host cell factors including CDK9/cyclin T1 to HIV-1 TAR RNA and thereby induces HIV-1 transcription. An interaction with host Ser/Thr protein phosphatase-1 (PP1) is critical for this function of Tat. PP1 binds to a Tat sequence, Q35VCF38, which resembles the PP1-binding “RVxF” motif present on PP1-binding regulatory subunits. We showed that expression of PP1 binding peptide, a central domain of Nuclear Inhibitor of PP1, disrupted the interaction of HIV-1 Tat with PP1 and inhibited HIV-1 transcription and replication. Here, we report small molecule compounds that target the “RVxF”-binding cavity of PP1 to disrupt the interaction of PP1 with Tat and inhibit HIV-1 replication. Using the crystal structure of PP1, we virtually screened 300,000 compounds and identified 262 small molecules that were predicted to bind the “RVxF”-accommodating cavity of PP1. These compounds were then assayed for inhibition of HIV-1 transcription in CEM T cells. One of the compounds, 1H4, inhibited HIV-1 transcription and replication at non-cytotoxic concentrations. 1H4 prevented PP1-mediated dephosphorylation of a substrate peptide containing an RVxF sequence in vitro. 1H4 also disrupted the association of PP1 with Tat in cultured cells without having an effect on the interaction of PP1 with the cellular regulators, NIPP1 and PNUTS, or on the cellular proteome. Finally, 1H4 prevented the translocation of PP1 to the nucleus. Taken together, our study shows that HIV- inhibition can be achieved through using small molecules to target a non-catalytic site of PP1. This proof-of-principle study can serve as a starting point for the development of novel antiviral drugs that target the interface of HIV-1 viral proteins with their host partners.

Highlights

The emergence of drug-resistant HIV-1 presents a challenge for the design of new therapeutics
Transcription of HIV-1 is dependent on the viral Tat protein, which binds a nascent transactivation responsive (TAR) RNA [1] and recruits host cell transcription factors including CDK9/cyclin T1 to the viral LTR
Our previous studies showed that PP1 is important for the activation of HIV-1 transcription and that disruption of the HIV-1 Tat/PP1 interaction is inhibitory for HIV-1. These findings suggest the possibility of identifying small molecules that can act in a manner similar to the expression of central domain of NIPP1 (cdNIPP1) to disrupt the interaction of Tat and PP1 and inhibit HIV-1 transcription

Summary

Introduction

The emergence of drug-resistant HIV-1 presents a challenge for the design of new therapeutics. Targeting host cell factors employed by HIV-1 for replication could be an approach to address HIV-1 drug resistance. Transcription of HIV-1 is dependent on the viral Tat protein, which binds a nascent transactivation responsive (TAR) RNA [1] and recruits host cell transcription factors including CDK9/cyclin T1 to the viral LTR (reviewed in [2]). CTD-dephosphorylation mediated through protein phosphatase-1 (PP1) is essential for HIV-1 transcription [3,4]. The PP1 holoenzyme consists of a constant catalytic subunit (PP1a, PP1b/d or PP1c) and a variable regulatory subunit that determines the localization, activity and substrate-specificity of the phosphatase [7]. Major PP1 regulators, such as NIPP1 (Nuclear Inhibitor of PP1) or PNUTS (Phosphatase Nuclear Targeting Subunit), bind PP1 with nanomolar affinity and modulate the dephosphorylation of a wide range of PP1 substrates [7]. We found that Tat binds PP1 in a similar manner to how NIPP1 binds PP1, except that the Tat-PP1 interaction is weaker and occurs with micromolar affinity [6]

Methods

Results

Conclusion