Background:  Our recent studies indicate that CDK2 and protein phosphatase-1 (PP1) might regulate HIV-1 transcription. Treatment of the cells with iron chelators inhibited HIV-1 transcription by reducing the activity of CDK2; preventing association of CDK9 with cyclin T1, and inhibiting phosphorylation of RNAPII. We also previously showed that PP1 is required for the Tat-induced HIV-1 transcription and that Tat binds to PP1 through the Tat's Q³⁵VCF³⁸ sequence and translocates it to the nucleus. In this study, we show that both CDK2 and PP1 affect CDK9 phosphorylation. Furthermore, we design small molecular inhibitors to disrupt the interaction of Tat with PP1 to inhibit HIV-1 transcription and viral replication.

Results:  We show here that CDK2 directly phosphorylates CDK9 in vitro and that PP1 dephosphorylates the phosphorylated CDK9. Inhibition of PP1 resulted in the inhibition of CDK9 activity and increased association of CDK9 with the inhibitory 7SK RNA. Virtual screening of PP1 inhibitors was performed against PP1 crystal structure. The selected compounds were analyzed for the inhibition of HIV-1 transcription in CEM cells infected with Adeno-Tat. Of the compounds, 4 showed inhibition with IC₅₀ of <10 µM. Only 1 compound, 1H4, was inhibitory in the transiently transfected 293T cells. 1H4 was not toxic for 293T cells. Interestingly, 1H4 competed for the binding of Tat to PP1 in vitro, but did not directly affect PP1 enzymatic activity. The 1H4 compound inhibited HIV-1 replication at 10-µM or 25-µM concentrations.

Conclusions:  Our results presented here and our previous observations indicate that CDK2 and PP1 are involved in the activation of CDK9 and HIV-1 transcription. Dephosphorylation of CDK9 by PP1 allows dissociation of inhibitory 7SK RNA and HEXIM1 protein; then phosphorylation by CDK2 might help CDK9 to associate with cyclin T1 and regain the kinase activity. Our studies also provide evidence that HIV-1 transcription is inhibited when interaction of HIV-1 Tat with PP1 is disrupted by a small molecular compound. Our study is the first example of a development of small molecular inhibitors of PP1 designed to inhibit HIV-1 transcription. Thus our studies open a future avenue for the design of small molecular compounds that may be selected from the promising scaffold that we described.