The transcriptional coactivator p300/CBP (CREBBP) is a histone acetyltransferase (HAT) that regulates gene expression by acetylating histones and other transcription factors. Dysregulation of p300/CBP HAT activity contributes to various diseases including cancer. Sequence alignments, enzymology experiments and inhibitor studies on p300/CBP have led to contradictory results about its catalytic mechanism and its structural relation to the Gcn5/PCAF and MYST HATs. Here we describe a high-resolution X-ray crystal structure of a semi-synthetic heterodimeric p300 HAT domain in complex with a bi-substrate inhibitor, Lys-CoA. This structure shows that p300/CBP is a distant cousin of other structurally characterized HATs, but reveals several novel features that explain the broad substrate specificity and preference for nearby basic residues. Based on this structure and accompanying biochemical data, we propose that p300/CBP uses an unusual 'hit-and-run' (Theorell-Chance) catalytic mechanism that is distinct from other characterized HATs. Several disease-associated mutations can also be readily accounted for by the p300 HAT structure. These studies pave the way for new epigenetic therapies involving modulation of p300/CBP HAT activity.

Ghrelin is a gastric peptide hormone that stimulates weight gain in vertebrates. The biological activities of ghrelin require octanoylation of the peptide on Ser3, an unusual post-translational modification that is catalyzed by the enzyme ghrelin O-acyltransferase (GOAT). Here, we describe the design, synthesis, and characterization of GO-CoA-Tat, a peptide-based bisubstrate analog that antagonizes GOAT. GO-CoA-Tat potently inhibits GOAT in vitro, in cultured cells, and in mice. Intraperitoneal administration of GO-CoA-Tat improves glucose tolerance and reduces weight gain in wild-type mice but not in ghrelin-deficient mice, supporting the concept that its beneficial metabolic effects are due specifically to GOAT inhibition. In addition to serving as a research tool for mapping ghrelin actions, GO-CoA-Tat may help pave the way for clinical targeting of GOAT in metabolic diseases.The persistent rise in the proportion of overweight individuals in Western society over the past 30 years has been associated with substantial excess morbidity and is widely recognized as a major public health concern. To address this problem, intensive efforts are underway to ‡ To whom correspondence should be addressed. pcole@jhmi.edu. * These authors contributed equally to this work. † These authors contributed equally to this work. clarify nutrient-hormone interactions contributing to weight gain. Starting with the isolation of leptin (1), a series of hormones acting centrally and peripherally to influence body mass have been discovered. Among these, the gastric peptide hormone acyl ghrelin has generated considerable interest as an important stimulus for weight gain (2-5) and modulator of glucose homeostasis (6-8). Various strategies in therapeutic development have been devised to antagonize acyl ghrelin (9,10), although none has yet emerged as clinically beneficial. Acyl ghrelin has an unusual Ser3 octanoylation; only acylated ghrelin can bind and activate the growth hormone secretagogue receptor (GHSR-1a). The cDNA for the enzyme responsible for this esterification, GOAT, has recently been cloned (11,12). GOAT has been suggested as a potential therapeutic target for modulating weight gain and glucose control, but thishas not yet been directly tested (9,13). An acyl ghrelin product analog Dap-ghrelin blocks GOAT activity in a microsomal assay (14).We designed bisubstrate analog GO-CoA-Tat based on the theory that if GOAT uses a ternary complex mechanism which templates octanoyl-CoA and ghrelin peptide, then linking the two substrates with a non-cleavable bridge could combine the binding energies of the individual ligands without the entropic loss associated with forming the ternary complex (Fig. 1A). A related strategy has been used for other peptide modifying enzymes including histone acetyltransferases (HAT) and protein kinases (15,16). Since we were uncertain about the ghrelin peptide length needed for recognition by GOAT, we selected amino acids 1-10 for coupling to octanoyl-CoA, to maximize inclusion of highly conserved...

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)butane], a naturally occurring isothiocyanate derived from cruciferous vegetables, is a highly potent inducer of phase 2 cytoprotective enzymes and can protect against electrophiles including carcinogens, oxidative stress, and inflammation. The mechanism of action of sulforaphane is believed to involve modifications of critical cysteine residues of Keap1, which lead to stabilization of Nrf2 to activate the antioxidant response element of phase 2 enzymes. However, the dithiocarbamate functional group formed by a reversible reaction between isothiocyanate of sulforaphane and sulfhydryl nucleophiles of Keap1 is kinetically labile, and such modification in intact cells has not yet been demonstrated. Here we designed sulforaphane analogs with replacement of the reactive isothiocyanate by the more gentle electrophilic sulfoxythiocarbamate group that also selectively targets cysteine residues in proteins but forms stable thiocarbamate adducts. Twenty-four sulfoxythiocarbamate analogs were synthesized that retain the structural features important for high potency in sulforaphane analogs: the sulfoxide or keto group and its appropriate distance to electrophilic functional group. Evaluation in various cell lines including hepatoma cells, retinal pigment epithelial cells, and keratinocytes as well as in mouse skin shows that these analogs maintain high potency and efficacy for phase 2 enzyme induction as well as the inhibitory effect on lipopolysaccharide-induced nitric oxide formation like sulforaphane. We further show in living cells that a sulfoxythiocarbamate analog can label Keap1 on several key cysteine residues as well as other cellular proteins offering new insights into the mechanism of chemoprotection.