15-Deoxy-Δ^12,14-prostaglandin J₂ inhibits HIV-1 transactivating protein, Tat, through covalent modification

Controlling the HIV/AIDS epidemic remains a major challenge, with approximately 5 million new HIV infections annually. Cyclopentenone prostaglandins (CyPG), such as 15-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂), are arachidonic acid-derived endogenous electrophiles that possess anti-HIV activity by an unknown mechanism. Given that the reactive α,β-unsaturated ketone in the cyclopentenone ring of 15d-PGJ ₂ covalently modifies key Cys thiols in select proteins, we hypothesized that 15d-PGJ₂ inhibits HIV transcription and replication by targeting Cys thiols in HIV-1 Tat. Tat is a potent transactivator of viral gene expression required for HIV transcriptional elongation and replication. Our studies indicate that 15d-PGJ₂ treatment of cells inhibits Tat-dependent transcription and replication of HIV-1, while 9,10-dihydro-15d- PGJ₂, PGE₂, PGF_2α, or PGD₂ that lack the reactive α,β-unsaturated ketone were ineffective. The inhibition of Tat activity by 15d-PGJ₂ was dose-dependent, with an IC₅₀ of 1.2 μM and independent of NF-κB pathway. Furthermore, using a biotinylated derivative of 15d-PGJ₂, we demonstrate that 15d-PGJ₂ modifies free Cys-thiols in Tat to form covalent Michael adducts and that the interaction was further increased on reduction of Tat. 15d-PGJ₂-modified Tat was unable to transactivate the HIV long terminal repeat in U937 human macrophages. These data demonstrate that Tat acts as a molecular target of CyPG leading to the inhibition of transcription and also suggest a novel therapeutic approach to complement current antiretroviral strategies for HIV/AIDS.