Targeting of histone acetyltransferase p300 by cyclopentenone prostaglandin Δ ¹²-PGJ ₂ through covalent binding to Cys ¹⁴³⁸

Inhibitors of histone acetyltransferases (HATs) are perceived to treat diseases like cancer, neurodegeneration, and AIDS. On the basis of previous studies, we hypothesized that Cys ¹⁴³⁸ in the substrate binding site could be targeted by Δ ¹²-prostaglandin J ₂ (Δ ¹²-PGJ ₂), a cyclopentenone prostaglandin (CyPG) derived from PGD ₂. We demonstrate here the ability of CyPGs to inhibit p300 HAT-dependent acetylation of histone H3. A cell-based assay system clearly showed that the α,β-unsaturation in the cyclopentenone ring of Δ ¹²-PGJ ₂ was crucial for the inhibitory activity, while the 9,10-dihydro-15-deoxy-Δ ^12,14-PGJ ₂, which lacks the electrophilic carbon (at carbon 9), was ineffective. Molecular docking studies suggested that Δ ¹²-PGJ ₂ places the electrophilic carbon in the cyclopentenone ring well within the vicinity of Cys ¹⁴³⁸ of p300 to form a covalent Michael adduct. Site-directed mutagenesis of the p300 HAT domain, peptide competition assay involving p300 wild type and mutant peptides, followed by mass spectrometric analysis confirmed the covalent interaction of Δ ¹²-PGJ ₂ with Cys ¹⁴³⁸. (Figure presented) Using biotinylated derivatives of Δ ¹²-PGJ ₂ and 9,10-dihydro-15-deoxy-Δ ^12,14-PGJ ₂, we demonstrate the covalent interaction of Δ ¹²-PGJ ₂ with the p300 HAT domain, but not the latter. In agreement with the in vitro filter binding assay, CyPGs were also found to inhibit H3 histone acetylation in cell-based assays. In addition, Δ ¹²-PGJ ₂ also inhibited the acetylation of the HIV-1 Tat by recombinant p300 in in vitro assays. This study demonstrates, for the first time, that Δ ¹²-PGJ ₂ inhibits p300 through Michael addition, where α,β-unsaturated carbonyl function is absolutely required for the inhibitory activity.