Targeting of Histone Acetyltransferase p300 by Cyclopentenone Prostaglandin Δ12-PGJ2 through Covalent Binding to Cys1438

Abstract

Inhibitors of histone acetyltransferases (HATs) are perceived to treat diseases like cancer, neurodegeneration, and AIDS. On the basis of previous studies, we hypothesized that Cys1438 in the substrate binding site could be targeted by Δ12-prostaglandin J2 (Δ12-PGJ2), a cyclopentenone prostaglandin (CyPG) derived from PGD2. 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 Δ12-PGJ2 was crucial for the inhibitory activity, while the 9,10-dihydro-15-deoxy- Δ12,14-PGJ2, which lacks the electrophilic carbon (at carbon 9), was ineffective. Molecular docking studies suggested that Δ12-PGJ2 places the electrophilic carbon in the cyclopentenone ring well within the vicinity of Cys1438 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 Δ12-PGJ2 with Cys1438. Using biotinylated derivatives of Δ12-PGJ2 and 9,10-dihydro-15-deoxy- Δ12,14-PGJ2, we demonstrate the covalent interaction of Δ12-PGJ2 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, Δ12-PGJ2 also inhibited the acetylation of the HIV-1 Tat by recombinant p300 in in vitro assays. This study demonstrates, for the first time, that Δ12-PGJ2 inhibits p300 through Michael addition, where α,β-unsaturated carbonyl function is absolutely required for the inhibitory activity.