Targeting Protein Phosphatase-1 for Activation of Latent HIV-1 Provirus

Sergei Nekhai, PhD

August 09, 2012

Despite the efficient ART treatment, eradication of HIV-1 infection is challenging and requires novel biological insights and therapeutic strategies. Eradication of latent HIV-1 provirus is especially challenging as integrated HIV-1 is not affected by ART drugs until its transcription is activated. Our laboratory identified host protein phosphatase-1 (PP1) as a regulatory factor in HIV-1 transcription. We showed that PP1 dephosphorylates CDK9’s Thr 186 and Ser 175. We recently showed that inhibition of PP1 primarily induces CDK9’s Ser 175 phosphorylation. While PP1 regulatory subunit that mediates CDK9 dephosphorylation is not known, it typically contains the so-called “RVxF” motif that binds to the RVxF –accommodating cavity of PP1. We developed a panel of small molecule compounds targeted to the RVxF-accommodating cavity of PP1 and found that some of these compounds induced HIV-1 transcription and also induced latent HIV-1 provirus. We hypothesize that small molecule compounds targeted to the RVxF cavity in PP1 may transiently disengage PP1 from CDK9, leading to the increased CDK9 Ser 175 phosphorylation and induction of HIV-1 transcription. Thus, development of these reagents and understanding the mechanism of their action might lead to development of novel activators of latent HIV-1 provirus. In this collaborative proposal between Howard University and Petersburg Nuclear Physics Institute (St. Petersburg, Russia) and with Dr. Mudit Tyagi of George Mason University as co-Investigator, we will develop small molecule compounds to induce latent HIV-1 provirus.  In the Aim 1, we will determine the effect of CDK9 Ser 175 phosphorylation on stability of CDK9/cyclin T1/Tat complex. We will analyze the effect of CDK9 Ser 175 phosphorylation on stability and conformational flexibility of CDK9/cyclin T1/Tat complex using in silico molecular modeling and molecular dynamics approaches in collaboration with our Russian co-Investigators. The in silico findings will be validated in the USA PI’s laboratory. We will identify HIV-1 Tat residues that might be involved in the interaction with phosphorylated Ser 175 and mutate these residues to determine experimentally their effect on Tat binding to CDK9/cyclin T1. In the Specific Aim 2, we optimize PP1-targeted small molecule compounds and analyze their effect on the activation of HIV-1 latent provirus and CDK9 Ser 175 phosphorylation.  The existing small molecule compounds will be optimized by Russian collaborators using the crystal structures of PP1 to dock the existing RVxF-targeted compounds in the protein binding sites and optimize their chemical structure to improve the binding constants. In the USA, we will test how these optimized compounds will induce HIV-1 in latently infected T cells and how these compounds affect CDK9 Ser 175 phosphorylation.  Taken together, the proposed studies will determine the role ofCDK9’s Ser 175 phosphorylation in HIV-1 transcriptional activation and yield small molecule compounds that activate HIV-1 transcription. Our proposed study will offer novel therapeutic approaches that will translate our previous basic studies on HIV-1 transcription regulation into novel anti-HIV-1 drugs.