Summary The dynamic and reversible acetylation of proteins catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) is a major epigenetic regulatory mechanism of gene transcription 1 associated with multiple diseases. While HDAC inhibitors are approved to treat certain cancers, progress on the development of drug-like HAT inhibitors has lagged 2. The HAT paralogs p300 and CBP (p300/CBP) are key transcriptional co-activators essential for a multitude of cellular processes and also implicated in human pathological conditions, including cancer 3. Current p300/CBP HAT domain inhibitors including natural products, 4 bi-substrate analogs (Lys-CoA) 5 and the widely utilized C646 6, 7 lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like p300/CBP catalytic inhibitor. We show the first high resolution (1.95Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 is acetyl-CoA competitive. A-485 selectively inhibited proliferation across lineage-specific tumor types, including several hematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen sensitive and castrate resistant prostate cancer and inhibited tumor growth in a castration resistant xenograft model. These results demonstrate the feasibility of selectively targeting the catalytic activity of histone acetyltransferases.

The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30 min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities.

Benzene is a human carcinogen that induces hematopoietic malignancies. It is believed that benzene does not initiate leukemias directly, but rather generates DNA damage through a series of phenolic metabolites, especially 1,4-benzoquinone. The cellular consequences of 1,4-benzoquinone are consistent with those of topoisomerase II-targeted drugs. Therefore, it has been proposed that the compound initiates specific leukemias by acting as a topoisomerase II poison. This hypothesis, however, has not been supported by in vitro studies. While 1,4-benzoquinone has been shown to inhibit topoisomerase II catalysis, increases in enzyme-mediated DNA cleavage have not been reported. Because of the potential involvement of topoisomerase II in benzene-induced leukemias, we re-examined the effects of the compound on DNA cleavage mediated by human topoisomerase IIalpha. In contrast to previous reports, we found that 1,4-benzoquinone was a strong topoisomerase II poison and was more potent in vitro than the anticancer drug etoposide. DNA cleavage enhancement probably was unseen in previous studies due to the presence of reducing agents in reaction buffers and the incubation of 1,4-benzoquinone with the enzyme prior to the addition of DNA. 1,4-Benzoquinone increased topoisomerase II-mediated DNA cleavage primarily by enhancing the forward rate of scission. In vitro, the compound induced cleavage at DNA sites proximal to a defined leukemic chromosomal breakpoint and displayed a sequence specificity that differed from that of etoposide. Finally, 1,4-benzoquinone stimulated DNA cleavage by topoisomerase IIalpha in cultured human cells. The present findings are consistent with the hypothesis that topoisomerase IIalpha plays a role in the initiation of specific leukemias induced by benzene and its metabolites.