Author Correction: Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

In the originally published version of this Letter, the authors Arthur F. Kluge, Michael A. Patane and Ce Wang were inadvertently omitted from the author list. Their affiliations are: I-to-D, Inc., PO Box 6177, Lincoln, Massachusetts 01773, USA (A.F.K.); Mitobridge, Inc. 1030 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (M.A.P.); and China Novartis Institutes for BioMedical Research, No. 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong District, Shanghai 201203, China (C.W.). These authors contributed to the interpretation of results and design of compounds. In addition, author 'Edward A. Kesicki' was misspelled as 'Ed Kesicki'. These errors have been corrected online.

Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.

Regulation of S-adenosylhomocysteine hydrolase by lysine acetylation.

S-Adenosylhomocysteine hydrolase (SAHH) is an NAD(+)-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys(401) and Lys(408) but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys(401) and Lys(408) and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD(+) binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns.

Epinephrine inhibits PI3Kα via the Hippo kinases.

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.

Probing the reaction coordinate of the p300/CBP histone acetyltransferase with bisubstrate analogs.

Histone and protein acetylation catalyzed by p300/CBP transcriptional coactivator regulates a variety of key biological pathways. This study investigates the proposed Theorell-Chance or "hit-and-run" catalytic mechanism of p300/CBP histone acetyltransferase (HAT) using bisubstrate analogs. A range of histone peptide tail peptide-CoA conjugates with different length linkers were synthesized and evaluated as inhibitors of p300 HAT. We show that longer linkers between the histone tail peptide and the CoA substrate moieties appear to allow for dual engagement of the two binding surfaces. Results with D1625R/D1628R double mutant p300 HAT further confirm the requirement for a negatively charged surface on the enzyme to interact with the histone tail.

Analysis of p300/CBP histone acetyltransferase regulation using circular permutation and semisynthesis.

The histone acetyltransferase (HAT) p300/CBP has been shown to undergo autoacetylation on lysines in an apparent regulatory loop that stimulates HAT activity. Here we have developed a strategy to introduce acetyl-Lys at up to six known modification sites in p300/CBP HAT using a combination of circular permutation and expressed protein ligation. We show that these semisynthetic, circularly permuted acetylated proteins retain high affinity for an acetyl-CoA substrate analogue and that HAT activity correlates positively with degree of acetylation. This study provides novel evidence for control of p300/CBP HAT activity by site-specific autoacetylation and outlines a potentially general strategy for using expressed protein ligation and circular permutation to chemically interrogate internal regions of proteins.

Use of Fluorinated Functionality in Enzyme Inhibitor Development: Mechanistic and Analytical Advantages.

On the one hand, owing to its electronegativity, relatively small size, and notable leaving group ability from anionic intermediates, fluorine offers unique opportunities for mechanism-based enzyme inhibitor design. On the other, the "bio-orthogonal" and NMR-active 19-fluorine nucleus allows the bioorganic chemist to follow the mechanistic fate of fluorinated substrate analogues or inhibitors as they are enzymatically processed. This article takes an overview of the field, highlighting key developments along these lines. It begins by highlighting new screening methodologies for drug discovery that involve appropriate tagging of either substrate or the target protein itself with (19)F-markers, that then report back on turnover and binding, respectively, via an the NMR screen. Taking this one step further, substrate-tagging with fluorine can be done is such a manner as to provide stereochemical information on enzyme mechanism. For example, substitution of one of the terminal hydrogens in phosphoenolpyruvate, provides insight into the, otherwise latent, facial selectivity of C-C bond formation in KDO synthase. Perhaps, most importantly, from the point of view of this discussion, appropriately tailored fluorinated functionality can be used to form to stabilized "transition state analogue" complexes with a target enzymes. Thus, 5-fluorinated pyrimidines, alpha-fluorinated ketones, and 2-fluoro-2-deoxysugars each lead to covalent adduction of catalytic active site residues in thymidylate synthase, serine protease and glycosidase enzymes, respectively. In all such cases, (19)F NMR allows the bioorganic chemist to spectrally follow "transition state analogue" formation. Finally, the use of specific fluorinated functionality to engineer "suicide substrates" is highlighted in a discussion of the development of the alpha-(2'Z-fluoro)vinyl trigger for amino acid decarboxylase inactivation. Here (19)F NMR allows the bioorganic chemist to glean useful partition ratio data directly out of the NMR tube.

Discovery of Spiro Oxazolidinediones as Selective, Orally Bioavailable Inhibitors of p300/CBP Histone Acetyltransferases.

p300 and its paralog CBP can acetylate histones and other proteins and have been implicated in a number of diseases characterized by aberrant gene activation, such as cancer. A novel, highly selective, orally bioavailable histone acetyltransferase (HAT) domain inhibitor has been identified through virtual ligand screening and subsequent optimization of a unique hydantoin screening hit. Conformational restraint in the form of a spirocyclization followed by substitution with a urea led to a significant improvement in potency. Replacement of the hydantoin moiety with an oxazolidinedione followed by fluoro substitution led to A-485, which exhibits potent cell activity, low clearance, and high oral bioavailability.

Mini-ISES identifies promising carbafructopyranose-based salens for asymmetric catalysis: Tuning ligand shape via the anomeric effect.

This study introduces new methods of screening for and tuning chiral space and in so doing identifies a promising set of chiral ligands for asymmetric synthesis. The carbafructopyranosyl-1,2-diamine(s) and salens constructed therefrom are particularly compelling. It is shown that by removing the native anomeric effect in this ligand family, one can tune chiral ligand shape and improve chiral bias. This concept is demonstrated by a combination of (i) x-ray crystallographic structure determination, (ii) assessment of catalytic performance, and (iii) consideration of the anomeric effect and its underlying dipolar basis. The title ligands were identified by a new mini version of the in situ enzymatic screening (ISES) procedure through which catalyst-ligand combinations are screened in parallel, and information on relative rate and enantioselectivity is obtained in real time, without the need to quench reactions or draw aliquots. Mini-ISES brings the technique into the nanomole regime (200 to 350 nmol catalyst/20 µml organic volume) commensurate with emerging trends in reaction development/process chemistry. The best-performing ß-d-carbafructopyranosyl-1,2-diamine-derived salen ligand discovered here outperforms the best known organometallic and enzymatic catalysts for the hydrolytic kinetic resolution of 3-phenylpropylene oxide, one of several substrates examined for which the ligand is "matched." This ligand scaffold defines a new swath of chiral space, and anomeric effect tunability defines a new concept in shaping that chiral space. Both this ligand set and the anomeric shape-tuning concept are expected to find broad application, given the value of chiral 1,2-diamines and salens constructed from these in asymmetric catalysis.

α-Vinylic Amino Acids: Occurrence, Asymmetric Synthesis and Biochemical Mechanisms.

This report presents an overview of the family of naturally occurring 'vinylic' amino acids, namely those that feature a C-C double bond directly attached to the α-carbon, along the side chain. Strategies that have been brought to bear on the stereocontrolled synthesis of these olefinic amino acids are surveyed. The mechanistic diversity by which such 'vinylic triggers' can be actuated in a PLP (pyridoxal phosphate) enzyme active site is then highlighted by discussions of vinylglycine (VG), its substituted congeners, particularly AVG [4E-(2'-aminoethoxy)vinylglycine], and a naturally occurring VG-progenitor, SMM (S-methylmethionine).

Double-cuvette ISES: in situ estimation of enantioselectivity and relative rate for catalyst screening.

Described is a new method for the screening of an array of catalysts, in situ, to estimate enantioselectivity and relative rates. We term this approach "double-cuvette ISES (in situ enzymatic screening)". The Co(III)-salen mediated hydrolytic kinetic resolution (HKR) of (+/-)-propylene oxide is used as a model reaction to demonstrate proof of principle. In two parallel cuvettes, a lower CHCl3-based organic layer is loaded with the epoxide and the chiral salen catalyst. Aqueous reporting layers, containing distinct "reporting enzymes" and their nicotinamide cofactors, are layered above the organic layers. The 1,2-propanediol enantiomers formed by the chiral catalyst diffuse into the aqueous layer and are oxidized there by the reporting enzymes at rates dependent upon the diol concentration, the R:S ratio of the diol, and the enantioselectivity of the reporting enzymes. A focused chiral salen library was constructed from seven chiral 1,2-diamines, derived from amino acid, terpenoid, and carbohydrates skeletons, and seven salicylaldehyde derivatives. Double-cuvette ISES identified a couple of interesting combinatorial hits in this salen array, wherein either the sense or magnitude of enantioselection for a given chiral diamine depends significantly upon the choice of "salicylaldehyde" partner. A comparison of predicted ee's and relative rates using this new screening tool with those independently measured is provided.