KATching-Up on Small Molecule Modulators of Lysine Acetyltransferases

Perspective pubs.acs.org/jmc KATching-Up on Small Molecule Modulators of Lysine Acetyltransferases Roman P. Simon,† Dina Robaa,‡ Zayan Alhalabi,‡ Wolfgang Sippl,‡ and Manfred Jung*,† † Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, Freiburg 79104, Germany Department of Pharmaceutical Chemistry, University Halle-Wittenberg, Halle/Saale 06120, Germany ‡ ABSTRACT: The reversible acetylation of lysines is one of the best characterized epigenetic modifications. Its involvement in many key physiological and pathological processes has been documented in numerous studies. Lysine deacetylases (KDACs) and acetyltransferases (KATs) maintain the acetylation equilibrium at histones but also many other proteins. Besides acetylation, also other acyl groups are reversibly installed at the side chain of lysines in proteins. Because of their involvement in disease, KDACs and KATs were proposed to be promising drug targets, and for KDACs, indeed, five inhibitors are now approved for human use. While there is a similar level of evidence for the potential of KATs as drug targets, no inhibitor is in clinical trials. Here, we review the evidence for the diverse roles of KATs in disease pathology, provide an overview of structural features and the available modulators, including those targeting the bromodomains of KATs, and present an outlook. regulating tool to control transcriptional activity of specific chromatin loci.2 Upon acetylation, local affinity of the modified histone protein to negatively charged DNA is decreased, resulting in a less condensed chromatin structure and in exposure of promoter sites. As a consequence of the increased accessibility, the DNA globally becomes more prone to access of the transcriptional machinery.3 In addition, transcription factors and other regulatory elements are recruited in a modification-specific manner to the relaxed promoter locus by interaction of specialized reader domains with acetyl lysine moieties.1,4 Thus, HATs and histone acetylation are functionally linked with the control of transcription activation, replication, and DNA damage repair.5 Mass spectrometrybased proteomics targeting the acetylome revealed that this kind of PTM occurs not just on histones but on thousands of other acetylation sites in more than 1500 proteins. Affected proteins play vital roles in fundamental cellular processes like cell division, signaling, apoptosis, and metabolism.6,7 Because of the broad acceptance of substrates, histone acetyltransferases and deacetylases are hence often referred to as lysine INTRODUCTION For more than 50 years, the enzymatic acetylation of histones and other proteins and the functional consequences have been topics of scientific interest. A wide range of experimental efforts identified protein acetylation to be one of the key mechanisms in the regulation of biological functions. The transfer or removal of acetyl groups to ε-amino group of lysine residues is mediated by two classes of enzymes. Histone acetyltransferases (HATs) catalyze the covalent attachment of acetyl groups to lysine residues of histones and other proteins by using acetyl-coenzyme A (acetyl-CoA) as a cofactor. Histone deacetylases (HDACs) conversely catalyze the amide hydrolysis of acetylated lysine. The attachment of acetyl groups to lysine residues goes along with two functional consequences. First, the positive charge of physiologically protonated ε-amino groups is abolished, resulting in altered electrostatic as well as steric properties of the affected protein region. Second, acetylation serves as a mark for distinct “reader” domains, which comprise specialized tertiary structures (e.g., bromodomains) in proteins that undergo a selective interaction with acetylated lysines.1 The most studied targets of HATs are histone proteins. Together with other epigenetic posttranslational modifications (PTMs) (e.g., phosphorylation, methylation, ubiquitinylation, sumoylation, ADP ribosylation), acetylation serves as a ■ © 2015 American Chemical Society Special Issue: Epigenetics Received: September 28, 2015 Published: December 23, 2015 1249 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective Table 1. Type A Lysine Acetyltransferases (KATs) KAT synonym organism physiological histone substrates associated complexes/proteins GNAT Family Gcn5 hGcn5 PCAF Elp3 Hpa2 Hpa3 Nut1 KAT2 KAT2A KAT2B KAT9 KAT10 dCBP CBP p300 KAT3 KAT3A KAT3B Tip60 Esa1 Sas3 MOZ/MYST3 MORF/MYST4 HBO1/MYST2 MOF/MYST1 Sas2 KAT5 KAT5 KAT6 KAT6A KAT6B KAT7 KAT8 KAT8 TAF1/TBP TFIIIC90 KAT4 KAT12 SRC1 AIB1/ACTR/SRC3 p160 CLOCK KAT13A KAT13B KAT13C KAT13D Rtt109 CMLO3 KAT11 S. cerevisiae, D. melanogaster H3K9,14,18,23,36/H2B H. sapiens H3K9,14,18/H2B H. sapiens H3K9,14,18/H2B H. sapiens, S. cerevisiae, D. melanogaster H3 S. cerevisiae H3K14/H4K5,12 S. cerevisiae H4K8 S. cerevisiae H3 > H4 p300/CBP Family D. melanogaster H4K5,8/H3K14,18 H. sapiens H2AK5/H2BK12,15 H. sapiens H2AK5/H2BK12,15 MYST Family H. sapiens, D. melanogaster H4K5,8,12,16 S. cerevisiae H4K5,8,12,16/H2BK4,7 S. cerevisiae H3K14,23 H. sapiens H3K14 H. sapiens H3K14 H. sapiens H4K5,8,12 > H3 H. sapiens, D. melanogaster H4K16 S. cerevisiae H4K16 Transcription Factors Containing KAT Activity H. sapiens, S. cerevisiae, D. melanogaster H3 > H4 H. sapiens H3K9,14,18 Nuclear Receptor Coactivators H. sapiens H3/H4 H. sapiens H3/H4 H. sapiens H3/H4 H. sapiens H3K9,14 Other S. cerevisiae H3K9,27,56 X. levis H4 mediator TIP60 NuA4, Pic. NuA4 NuA3 MOZ/MORF MOZ/MORF HBO1 MSL SAS TFIID TFIIIC transcription process, as they were still detected in nuclei pretreated with the transcription inhibitor puromycin. Moreover, the modifications decreased the histone-mediated repression of RNA synthesis.15 In consecutive work, they showed that the ε-amino group of lysine residues is the target of acetylation.16 During the following years, histone acetylation was linked to gene activation by a number of different studies. In 1978, sodium butyrate was described as a histone deacetylase inhibitor by Davie and co-workers.17 This was a milestone, as for the first time it was shown that epigenetic regulators could be targeted by small molecules. It took about 20 years until the first KAT enzyme Gcn5 (general control nonderepressible 5, p55) was identified by Brownell and Allis in 1995. In their studies, an acetyltransferase activity gel assay was employed, which detects the incorporation of 3H-acetate into histone substrates after electrophoretic separation of nuclear extracts in a SDS/polyacrylamide gel matrix.18 One year later, the Gottschling group reported the isolation and cloning of Hat1 from Saccharomyces cerevisiae lysates as the first KAT exhibiting cytosolic localization.19 The identification of distinct enzymes, which catalyze the transfer of acetyl groups from acetyl-CoA to histone protein substrates, formed the starting point for extensive studies about the precise mechanism and functional consequence of this PTM. New insights into structural aspects of chromatin organization and histone function were provided when Luger et al. solved the nucleosome particle crystal structure in 1997.20 It was the same year that Grant et al. acetyltransferases (KATs) and lysine deacetylases (KDACs), respectively.8 In concert, these enzymes dynamically regulate the lysine acetylation levels within the cellular proteome. This acetylation equilibrium is adjusted in response to cellular stimuli like autoacetylation, protein−protein interactions, phosphorylation, or the cellular acetyl-CoA level, leading to altered gene transcription and subsequently to phenotype adaption.9 Aberrant acetylation levels have been connected with a diversity of disease phenotypes including cancer, neurological disorders, and cardiovascular and metabolic malignancies.10−14 Hence, KAT enzymes seem to be deeply involved in the manifestation and progression of such diseases and therefore the elucidation of their precise mechanism is required to assess their potential as possible drug targets and small molecule modulators are of high interest for probing these pathways and as potential drugs. ■ SAGA, SLIK, ADA, HAT-A2, ATAC STAGA, TFTC PCAF elongator Hpa2 FIFTY YEARS OF LYSINE ACETYLATION In 1964, Allfrey and colleagues proposed an innovative hypothesis of reversible posttranslational histone modifications being a dynamic regulatory mechanism for RNA synthesis after they reported the incorporation of acetyl and methyl groups into histone proteins as a result of treating isolated nuclei with radiolabeled acetate (acetate-2-C14) and methionine (methionine-methyl-C14), respectively. The incorporation of these modifications was found to happen independently of the 1250 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective enzymes differ in their N- and C-terminal regions, which are responsible for recognition, positioning, and binding of substrates. Structural motifs within these regions, like bromoand chromodomains, zinc binding moieties, and cysteine/ histidine rich modules (PHD, TAZ, ZZ) promote the target structure diversity.33 One example for substrate discrimination by such structural motifs is Gcn5. Human Gcn5 can be distinguished from its yeast homologue by a 400-residue Nterminal region. Under in vitro conditions, recombinant human Gcn5 acetylates free histone H3 on lysine 14 and, to a minor degree, histone H4 on lysine 8 and 16. It is remarkable that in contrast to yeast Gcn5, which targets the same substrate residues, the human homologue is capable of acetylating histones in a nucleosomal context, indicating the role of the Nterminal region in substrate recognition.5 PCAF and human Gcn5 share about 80% sequence similarity and both exhibit site preference for in vitro H3K14 acetylation within free or nucleosomal histones. On the cellular level, the substrate pattern is shifted toward multiple H3 and H2B acetylation sites, stating that recombinant enzymes may differ in substrate specificity and turnover comparing in vitro and in vivo conditions.34 Like the most KAT enzymes, Gcn5 and PCAF natively occur as part of multiprotein complexes. Functional interaction between subunits, as well as different PTMs on substrate or protein structures, direct affinity pattern to the more physiological substrates. Together, GNAT family enzymes and their multiprotein complexes are generally involved in transcriptional activation, elongation and DNA damage repair.35 The ubiquitously expressed and metazoan specific KATs p300 and CBP (CREB-binding protein) form the p300/CBP family. Both enzymes share close structural and functional homology and are competent to acetylate all four histone proteins under in vitro conditions. The p300/CBP-mediated transfer of acetyl groups comprises a Theorell−Chance mechanism, which is characterized by stable binding of the cofactor acetyl-CoA followed by transient and rather weak association of the lysine substrate to the enzyme. This catalytic mechanism is distinct from the GNAT and MYST KAT families and may contribute to the broad substrate acceptance of p300/CBP. On molecular level, p300 and CBP interact with a variety of transcription factors and coactivators to form regulatory complexes at promoter regions. In this way, they stimulate transcription of specific genes and serve as inciting regulatory elements. About 100 protein substrates have been described for p300/CBP so far.36 Interestingly, while being intensively studied in mammals, plant orthologues of p300/ CBP have also been found and characterized, suggesting a fundamental functional implication of this enzyme family in all metazoan organisms.37 The MYST family is named after the initially identified members (MOZ (monocytic leukemia zinc finger protein), Ybf2 (Sas3), Sas2 (something about silencing), and Tip60 (Tat-interactive protein)).38 These enzymes show sequence similarities, which reaches a remarkable high degree in a particular MYST homology region within the catalytic domain. Like in the GNAT enzymes discussed above, the highly conserved cofactor-binding motif A is also present in the structure of MYST family members.39 The acetyltransferase MOZ targets histone H3 lysine residue 14 and is correlated with transcription activation.8 Sas2 and Sas3 are involved in transcriptional silencing processes in S. cerevisiae.40 Tip60 was the first reported human member of the MYST family with identified and partially characterized the first KAT-containing protein complex SAGA (Spt-Ada-Gcn5-acetyltransferase) from Saccharomyces cerevisiae with its catalytic subunit Gcn5.21 Their findings added another level of complexity to the field of acetyltransferases because it demonstrated that their regulatory activity in a cellular context depends on multidomain protein complexes with a specific composition. The starting point of KAT inhibitor discovery was set two years later when Marmorstein and Allis published the crystal structure of Gcn5 as a nascent acetyltransferase as well as in a context with the cofactor acetyl-CoA and the physiologic ligand histone H3.22 Following these discoveries, Cole and colleagues synthesized the first KAT inhibitors. Inspired by earlier observations on serotonin N-acetyltransferases, they covalently linked lysine residues to CoA to generate pseudo bisubstrates of the addressed enzymes.23 In 2006, Kim et al. performed the first proteomic screen targeting lysine acetylation and identified 388 acetylation sites in 195 proteins.7 Over the past few years, advances in assay technology and in computational methods have led to an increased understanding of acetylation biology and pathology.6,24 Furthermore, a growing number of other acyl groups are identified to be the subject of reversible attachment and cleavage on lysines in proteins. Examples are formyl, propionyl, butyryl, crotonyl, malonyl, succinyl, or long chain fatty acids.25 There is no doubt that expanding on these observations will elucidate the full potential of the field of lysine acylation. ENZYMES AND ENZYME FAMILIES After the initial discovery of Hat1 and Gcn5, biological experiments led to the identification of additional KAT activity-containing enzymes that are classified according to their preferred cellular localization. Type B KATs share cytoplasmic localization and acetylate nascent histones to facilitate translocation into the nucleus, where the histone proteins are deacetylated and subsequently incorporated into chromatin fibers. Hat1 (KAT1) is one of two members of type B KATs. It is highly conserved through evolution in eukaryotes and acetylates free (not in a nucleosome) H4 protein on lysine residues 5 and 12, in humans also on H2A lysine residue 5. Together with the WD40 protein Hat2, Hat1 forms the Hat1/2 complex, which exhibits 10-fold increased activity compared to native Hat1. The intriguing observation that Hat1 is not solely found in the cytoplasm but also in the nucleus suggests that the enzyme activity shuttles between different cell compartments.26 The second member of type B KATs is Hat4, which is located in the Golgi apparatus where it catalyzes the acetylation of H4 protein on position 79 and 91.27 Type A KATs comprise a number of heterogenic enzymes that share nuclear localization. On the basis of their structural homology and catalytic mechanism, these enzymes are grouped into distinct families (Table 1). The GNAT (Gcn5 related N-acetyltransferase) family includes the enzymes Gcn5,18 PCAF (p300/CBP associated factor),28 Elp3,29 Hpa2/3,30 and Nut1.31 A characteristic feature of this enzyme family is the presence of up to four sequence motifs (A−D) with motif C being almost exclusively found in GNAT family enzymes. The A motif is the most highly conserved region, and it contains an acetyl-CoA binding domain that is defined by an Arg/Gln-X-X-Gly-X-Gly/Ala segment.32 This segment is not limited to GNAT enzymes, but also found in other KAT families. While the catalytic domains of KATs show high structural homology within a certain family, ■ 1251 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective preference for H4 acetylation. Transcriptional activation and DNA damage response are mediated by the acetylase activity of this enzyme.41 Following the founding members, more MYST family enzymes were identified and characterized. The yeast enzyme Esa1 is a homologue of human Tip60 and the catalytic subunit of the NuA4 complex. This complex promotes H4 acetylation and subsequently stimulates transcription of genes that are essential for cell cycle progression.42 The in vivo activity and specificity of almost all MYST family enzymes is highly determined by the composition of their protein complexes. HBO1 (HAT bound to ORC1) interacts with subunits of the origin of replication complex, consequently playing a functional role in DNA replication.43 The isolated HBO1 complex acetylates histones H4 at position 5, 8, and 12 and less effectively H3, whereas recombinant HBO1 shows no significant acetylase activity, implicating protein−protein interactions and in vivo modifications in efficient substrate recognition and turn over.8,43 A close homologue to the MYST family founding member MOZ is the transcription activator MORF (MOZ related factor). While recombinant and fulllength MORF catalyzes acetylation of H4 and H3 protein, under physiological condition solely histone H3 is targeted.8,44 The product of the mof (males absent on the first) gene represents one more member of this KAT family. The enzyme was identified in the course of studies targeting dosagecompensation of the X-chromosome in D. melanogaster.45 MOF is related to the yeast enzyme Sas2 but targets different acetylation sides on H4, H3, and H2A proteins in vitro. In the native MSL complex, MOF catalyzes the acetylation of nucleosomal histone H4 exclusively on lysine residue 16, emphasizing the influence of the associated MSL proteins on MOF activity and substrate specificity. KAT activity was also found in protein components of transcription factor complexes like TAF1/TBP and TFIIIC90. These protein complexes affect transcription directly and form a separate KAT family.46,47 There are a few coactivators of nuclear hormone receptors, which exhibit ligand-dependent KAT activity. They serve as regulatory elements in hormone related transcriptional processes and are grouped in a unique KAT family.48,49 The global regulator of circadian gene expression CLOCK was shown to be a member of this family.50 The fungal specific lysine acetyltransferase Rtt109 shows very little sequence homology to any of the other KAT family members, but its tertiary fold adopts a structure that is surprisingly similar to p300. However, the catalytic mechanism of Rtt109 involves a ternary intermediate complex and therefore differs significantly from the one of p300. Because of these differences, the enzyme is not assigned to any of the known KAT families. Isolated Rtt109 is not competent to efficiently acetylate lysine substrates, but its activity is stimulated by association with either of two different histone chaperones, Asf1 and Vps75. Upon formation of the enzyme− chaperone complex, the activity of the catalytic domain is dramatically increased and the substrate specificity is directed toward distinct lysine residues (H3K56 for Asf1 and H3K9,23 for Vps75).51 Bioinformatic analysis of the zebrafish genome led to the identification of the Camello protein family member CMLO3, for which lysine acetyltransferase activity on histone protein H4 was demonstrated. Their structural divergence and their perinuclear localization distinguish Camello proteins from other KATs and place them outside of any of the other KAT families.52 Considering the vast number of identified nonhistone acetylation sites on a constantly growing number of proteins, covering a wide functional spectrum, it should be noted that for many of these acetylation reactions the responsible enzymes have not been identified yet. 6 While several histone acetyltransferases were shown to also accept nonhistone substrates, the implications of specific nonhistone acetyltransferases, such as the α-tubulin acetyltransferase αTAT153 and the cohesin acetyltransferase Eco1/ESCO1,54 still need to be determined. KATS IN DISEASES Lysine acetylation of histone and nonhistone proteins is generally linked to activation of transcriptional activity and therefore affects pivotal physiological processes within an organism. As a consequence of misregulated acetylase activity, the manifestation and progression of certain malignancy phenotypes correlates with pathological aberrations of the acetylation equilibrium. This could be either due to altered activity of the responsible enzymes or because of changes in their expression levels. The role of distinct KAT subtypes in diseases like cancer, neurodegenerative disorders, viral and parasitic infections, inflammation, and metabolic and cardiovascular malignancies have been extensively investigated.55,56 p300 and CBP are global coactivators of gene transcription and involved in multiple cellular processes. Point mutation and microdeletion of the CBP gene result in Rubinstein−Taybi syndrome (RTS), which is characterized by physical abnormalities and mental retardation. 55 Elevated p300 expression levels have been associated with several types of cancer, including esophageal squamous cell carcinoma (ESCC),57 hepatocellular carcinoma (HCC),58 and melanoma. In the latter case, downregulation of p300 activity retards cell cycle progression in human melanocytes by activating replicative senescence.59 It is reported that p300/CBP interaction with c-Myb facilitates myeloid differentiation block and is required for acute myeloid leukemia (AML) induction.60 A characteristic feature of this hematologic malignancy is the presence of KAT activity-containing fusion proteins in consequence of chromosomal translocations. Fusion products of p300/CBP with MOZ, MORF, or MLL exhibit aberrant KAT activity and substrate specificity and hence lead to abnormal transcription activation.11 The AML1-ETO oncogene is acetylated by and colocalized with p300 at specific promoter regions, which was found to be pivotal for leukemogenesis.61 After infection of a cell with the human immunodeficiency virus (HIV), p300-mediated acetylation of the viral protein integrase is crucial for incorporation of virus DNA into the cells’ genome.62 Upon integration, p300/CBP interaction with the HIV-1-Tat protein promotes transcription of the HI provirus.63 In patients with elevated glucose levels, binding of p300 to promoter sites is increased, which leads to upregulation of vasoactive factors and extracellular matrix proteins, suggesting a possible role of p300 in chronic diabetes related complications.64 p300 and CBP stimulate cardiac growth and p300 activity is increased in agonist induced hypertrophy of cardiomyocytes. Recruitment and acetylation of specific transcription factors, such as GATA4 and MEF2, mediate elevated transcription levels of hypertrophy related effector proteins.13 The acetylation of NF-κB by p300/CBP is associated with a loss of affinity for IκB, leading to enhanced expression of proinflammatory downstream genes products. In neurodegenerative diseases, reduced CBP activity is associated ■ 1252 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective the reader is referred to more specific reviews regarding this topic.11,36,55,81 with loss of neuronal plasticity and destabilization of short-term memory, implicating CBP as an antitarget in this context.12 Like p300 and CBP, the GNAT family KATs serve as regulators of transcriptional activity and are similarly implicated in fundamental physiological processes. Their activity and specificity highly depend on protein−protein interactions, as these enzymes are usually part of multienzyme complexes. It was shown that Gcn5 activity is crucial for cell cycle progression.65 While Gcn5 and PCAF play important roles in the activation and stabilization of the tumor suppressor p53, PCAF-mediated acetylation of the cyclin-dependent kinase inhibitor p27 facilitates its degradation and further leads to uncontrolled cell cycle progression.66 The invasive potential and growth rate of urothelial cancer cells is hampered by PCAF knockout.67 Drug resistant cancer cells exhibit elevated levels of PCAF and Gcn5-mediated H3K9 acetylation in promoter regions of the MDR1 (multidrug resistant protein 1) gene.68 Alongside p300, Gcn5 is competent to acetylate and thus activate HIV integrase and Tat proteins with essential impact on the HIV replication cycle.69 A Plasmodium falciparum homologue of Gcn5 (PfGcn5) was found to play a key role in antigenic switching and expression of plasmodial proteins.70 Interestingly, despite some evidence on an antitarget role of p300 in neurodegenerative diseases, knockout of PCAF promotes insensitivity to β-amyloid peptide toxicity in mice, suggesting PCAF to be a possible target for the treatment of Alzheimer disease.71 It was also shown that PCAF is involved in the extinction process of conditioned fear and that PCAFmediated acetylation of connexin 43 is implicated in cardiac dystrophy.72,73 In type 2 diabetes, Gcn5 and PCAF were found to acetylate PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), a key coactivators in energy metabolism, thereby regulating its transcriptional activity.74 MYST family KATs are more diverse in domain organization and complex formation than p300/CBP and GNAT family proteins. Aberrant activity of this KAT family has predominantly been implicated in manifestation and progression of cancer. The aforementioned fusion proteins of MOZ and MORF with other KAT family members in AML inductions are complemented by the MOZ-TIF2 fusion protein, which is yielded after chromosomal inversion. In AML, MOZ-TIF2 interacts with CBP and disrupts normal CBP-dependent transcriptional activation.75 In addition, mutation in MOZ was associated with esophageal adenocarcinoma.76 Altered activity of the MYST family member MORF has been linked to breast cancer, prostate cancer, and leiomyoma. Furthermore, these KATs are involved in developmental processes and mutation in their encoding genes have been found in several developmental disorders.77 The acetyltransferase Tip60 plays an important role in hormone receptor signaling and DNA damage repair. The androgen receptor is activated in an androgen-independent manner upon Tip60-mediated acetylation. Related to this, the proliferation of prostate cancer has been correlated to aberrant Tip60 activity.78 Moreover, resistance to apoptotic signaling cascades in cancer cells after DNA damage was associated with Tip60 mutations.79 HBO1 is a key regulator of DNA replication and proliferation. Overexpression of HBO1 has been reported in a specific subset of human primary cancers.80 Together, this emphasizes the role of mistargeted acetylation in oncogenesis and other malignancy phenotypes. The field of lysine acetylation in cell function and its involvement in diseases goes beyond the examples listed in this article. For more detailed information, STRUCTURAL ASPECTS OF KATS KAT modules, which typically occur alongside other conserved protein modules as a part of much larger proteins, show different sizes between the various KAT families. A high sequence similarity is shared between members of the same family, whereas poor to no sequence homology is found between the families.82 Despite the poor sequence homology, all KATs adopt a globular α/β fold where the central core is structurally conserved. This central core is associated with the binding and catalysis of the cofactor acetyl-CoA. Meanwhile, the N- and C-terminal protein regions flanking the central core are structurally divergent and are believed to contribute to the substrate specificity of these enzymes. It is however questionable if substrate specificity of KATs can be solely attributed to the divergence of the N- and C-terminal domains. As previously mentioned, KATs are often found as subunits in large protein complexes, and their interacting protein partners were found to play a role in regulating their substrate selectivity as well as enhance their acetylase activity.83 It should hence be noted that further biochemical and structural investigations are necessary to decipher the exact role of protein partners on the activity and substrate specificity of KATs. The GNAT, p300/ CBP, and MYST families are the most extensively studied KATs, and crystal structures of several members of these families are available in apo form and/or in various ligandbound forms. The overall structures of representative members for each family are shown in Figure 1. Crystal structures of the KAT domain of Gcn5 (human, yeast and Tetrahymena) and PCAF have provided valuable information on the mechanism of catalysis as well as cofactor and substrate-binding of these enzymes. Acetyl-CoA is bound to the central core cleft via numerous hydrogen-bond interactions, mostly between the pantothenic and pyrophosphate moieties of the cofactor and the neighboring amino acid residues.84 Meanwhile, the adenosine base shows no contacts with the protein, and it was found to adopt different orientations in various crystal structures.85,86 An insight into the binding of the peptide substrates to GNAT family KATs was attained by determining the crystal structures of Tetrahymena Gcn5 (tGcn5) with H3, H4, and p53 peptides (PDB IDs: 1q2c, 1q2d, 1pu9, 1pua, and 1qsn). The central core and the flanking N- and C-terminal residues create an L-shaped binding cleft, which accommodates acetyl-CoA and the peptide substrate. The active lysine residue is in contact with the conserved core region, showing van-der-Waals (vdW) interactions with Val123, Leu126, and Phe169 and a hydrogen-bond interaction between its backbone-NH and the backbone carbonyl group of Ala124. The rest of the histone tail occupies a large cleft formed by the flanking, less conserved N- and Cterminal regions. Interestingly, most of the interactions are mediated through hydrogen bonds between the backbone of the histone peptide and the protein, in addition to vdW contacts between the peptide side chains and some protein residues in the binding cleft.22,86 It was hypothesized that residues C-terminal to the reactive lysine of the peptide substrates play an important role in substrate selectivity.86 Of particular importance are residues at the +2 and +4 position, which show strong vdW interactions with the protein. The Nterminal residues of the peptide seem to mainly contribute to the substrate affinity; only substrates with high affinities (H3 ■ 1253 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective surrounded by the hydrophobic residues Tyr1397, Trp1436, Cys1438 (L1 loop), and Tyr1446 (L1 loop). The ε-NH of the lysine moiety undergoes H-bond interactions with the backbone carbonyl of Trp1436. A common feature of all p300/CBP substrates is the presence of a basic residue (lysine or arginine) at the −3 or +4 position of the active lysine residue.89,91 This is consistent with the highly electronegative surface area of the crystallized p300 KAT domain (Figure 2), Figure 2. (a) Electrostatic surface of p300 (PDB ID 3biy) in complex with Lys-CoA (white sticks). Red color indicates electronegative regions, blue electropositive, and white regions having a neutral potential. The arrow points to the electronegative subpocket, which accommodates the Lys/Arg residue at −3 or +4 position of the peptidic substrate. Lys-CoA can be partly seen as white sticks. (b) the structure of p300 protein containing the bromo- (cyan), PHD (orange), RING (yellow), and KAT (green) domains (4bhw.pdb). The L1 loop is shown as red ribbon, and the zinc ions as violet spheres. which distinguishes this family from other KATs. In particular, a highly electronegative pocket, formed of Thr1357, Glu1505, Asp1625, and Asp1628 and located 10 Å away from the active lysine pocket, is responsible for accommodating the basic residue at the −3 or +4 position. Mutations of these residues showed a significant decrease in p300 KAT activity.89 In addition, the p300 KAT activity is regulated by a lysine-rich loop (aa 1520−1560; activation loop) that is hyperacetylated in the active form.92 It was suggested that the activation loop, in its deacetylated form, folds into the highly electronegative surface, thereby occluding the substrate binding pocket. Upon acetylation, this loop flips outward and allows substrate binding. Owing to its high proteolytic sensitivity, all crystal structures of p300 have been determined without the activation loop. Recently, a crystal structure of the KAT, RING, PHD, and BRD domains of the p300 protein has been determined.88 These domains form a compact module, where the RING domain is in direct contact with the L1 loop and hence in close proximity of the KAT substrate pocket (Figure 2). The RING domain was found to have an inhibitory function on the acetylase activity of the KAT domain. Indeed, mutations which disrupt the integrity of the RING domain or the attachment between the RING and KAT domains resulted in an increase in the autoacetylation of the p300 activation loop and the p300 acetylase activity. Similar mutations were correlated with the pathology of some diseases; C1204R mutation is found in B-cell lymphomas, while deletion of the RING domain (1198−1234) occurs in breast cancer tissues.88 The overall structure of the yeast MYST member Esa1 (PDB ID 1fy7)93 and the later disclosed human homologue hMOF94−97 reveal, as expected, structural homology of the central core with other KATs. Meanwhile, the flanking N- and C-terminal segments show a structural divergence to other Nacetyltransferases. A distinctive feature of the MYST family is Figure 1. Overall structure of nuclear KATs. Acetyl-CoA (cyan sticks) is shown in complex with (a) hGcn5 (PDB ID 1zr4), (b) p300 (PDB ID 4pzr) with the L1 loop depicted as green ribbon, and (c) hMOF (PDB ID 2giv) as a representative of the MYST family. peptide) display interactions between their N-terminal residues and the protein.86,87 It is noteworthy, that a loop region (aa 184−188 tGcn5), which constitutes a part of the substrate binding pocket, shows a considerable flexibility and a 9 Å outward movement is observed upon peptide binding.22,86 A similar conformational change is not observed in the crystal structure of Gcn5 in complex with the bisubstrate inhibitor LysCoA (PDB ID 1m1d).85 As previously noted, p300 and CBP share a structural conservation of the central core with members of the Gcn5/ PCAF and MYST family KATs and a structural divergence of the N- and C-terminal regions.88−90 However, p300/CBP show a unique feature among KATs, namely a ∼25 residues long loop (L1 loop), which constitutes a part of the cofactor and lysine-binding pockets.89 A salt bridge between Asp1399 with His1415 is essential for holding the L1 loop in the right conformation to allow for the acetyl-CoA binding. The crystal structure of p300 in complex with the bisubstrate inhibitor LysCoA (3biy.pdb) demonstrates that the lysine moiety is 1254 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective the regulation of its acetylase activity by intramolecular autoacetylation of a conserved lysine residue proximal to the substrate binding site (K274 in hMOF).96,97 K274 autoacetylation was demonstrated to be essential for the in vitro and in vivo KAT catalytic activity.97 The acetylated lysine residue (K274, hMOF) is buried into a side pocket of the enzyme active site, where it is stabilized by H-bond interactions with the conserved residues Tyr3012 and Ser303. The terminal methyl group displays vdW contacts with the conserved residues Phe283 and Phe285 (Figure 3).96 In the deacetylated form, the KAT MODULATORS The multifold implication of lysine acetylation in physiologic pathways as well as manifestation and progression of diseases emphasizes the potential of KAT modulators as therapeutic strategy or versatile mechanistic tools. However, the identification of such compounds has proven to be challenging and the development of potent and selective KAT inhibitors lags far behind modulators of other epigenetic enzymes, like KDACs. Despite different approaches to find small molecule inhibitors of KAT enzymes, only few potent substances have been obtained so far. With the aid of computational methods and advanced assay technologies, recently progress was made toward compounds with improved KAT modulatory properties and in vivo characterization. According to their origin and mode of action, the known KAT modulators can be assigned to four distinct classes: bisubstrate inhibitors, natural compounds and their analogues and derivatives, synthetic small molecules, and bromodomain inhibitors. Bisubstrate Inhibitors. The first published KAT inhibitors were bisubstrate analogues mimicking the ternary complex of cofactor acetyl-CoA and lysine substrate in spatial proximity during the catalytic process (Figure 4). Cole and colleagues ■ Figure 3. (a) Comparison of the interactions of the autoacetylated lysine (K274) in hMOF (green ribbons and sticks, PDB ID 3qah), with those of the deacetylated form as seen in the K274R mutant (yellow ribbons and sticks, PDB ID 2pq8). Only side chains of the involved residues are shown for clarity. Acetyl-CoA is shown as magenta sticks. (b) Structure of hMOF KAT domain (surface depiction in cyan) in complex with MSL1 segment (surface depiction in red); 4dnc.pdb. Lys-CoA is shown as white spheres. lysine residue flips out of the side pocket and consequently blocks the substrate binding site, as observed in the crystal structure with the K274R mutant (Figure 3) (PDB ID 2pq8). The flipped-out form is stabilized by salt bridge interactions with the catalytically important Glu350, thus quenching its proton abstraction ability. The activity and substrate specificity of hMOF is regulated by its association with other proteins to form larger protein complexes.98,99 hMOF association with MSL1 and MSL3 in the MSL (male-specific lethal) complex leads to a pronounced enhancement of its enzymatic activity and a refinement of its substrate specificity to selectively target the nucleosomal H4K16.99 Meanwhile, the complex of hMOF with NSL1, as found in the NSL (nonspecific lethal) complex, is more efficient in specifically acetylating K20 on p53 than hMOF alone. 98 The crystal structure of hMOF with MSL1470−540 has been recently revealed (PDB IDs 4dnc and 2y0m). The MSL1 segment interacts extensively with the N-terminal part of hMOF, mainly through H-bond and saltbridge interactions. A depiction of the MOF−MSL1 complex is shown in Figure 3. The MSL1 segment is believed to act as a tether between MOF and MSL3, which is another essential member in the MSL complex.94,95 Although the determined crystal structures provide some basic structural insights into the nature of the interplay between the subunits in these complexes, further structural investigations are necessary to fully understand how the activity and substrate specificity of MOF is regulated by the complex subunits. Despite the availability of structural information on numerous KATs, so far only very few studies have reported on the use of in silico screening approaches for the identification of KAT inhibitors. Figure 4. Structures of bisubstrate KAT inhibitors. covalently linked CoA to the lysine residue of a substrate peptide of various chain lengths. The bisubstrates concept was adopted by several other groups to generate specific KAT inhibitors. Compound 1 (Lys-CoA) is yielded by connecting CoA and a single lysine residue via a methylene linker.23 The compound is a potent inhibitor of p300 with an IC50 value of 0.5 μM and pronounced selectivity toward p300 compared to PCAF. The particular potency of 1 results from the Theorell− Chance kinetic mechanism of p300/CBP catalysis, which is characterized by a strong cofactor-binding and a weak transient interaction with the histone substrate.23 The selectivity pattern of bisubstrate inhibitors can efficiently be altered by modifying 1255 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective Figure 5. Structures of natural KAT modulators, synthetic analogues, and derivatives. Figure 6. Structures of selected natural KAT inhibitors, their derivatives, and structural analogues. the length and composition of the peptide chain that comprises the lysine-CoA construct. Derivative 1.1 (H3-CoA-20) mimics the native substrate H3K14 of PCAF and therefore inhibits the enzyme selectively with an IC50 value of 0.3 μM.23 To address MYST family enzymes, a series of H4 peptide-containing bisubstrates analogues was designed and structure 1.2 (H4K16CoA) was reported as an acetyl-CoA competitive and potent Tip60 inhibitor with an IC50 value in the low micromolar range.100 It should be noted that the mentioned bisubstrate analogues also inhibit the Tip60 yeast homologue Esa1 at low micromolar concentrations and therefore special caution in yeast studies is advised. Deduced from the crystal structure of p300, compound 2 (Boc-C5-CoA) was reported as p300 inhibitor (IC50 0.07 μM) with an optimized linker length that is capable to occupy two binding pockets (P1, P2) within the enzyme active site.101 Attributed to the polar phosphate moieties and the partially peptidic structure, bisubstrate inhibitors generally suffer from poor cell permeability and metabolic instability, which limits their use to in vitro applications or requires membrane penetrating techniques like micro injection or lipid permeabilization. Different approaches have been applied to circumvent this limitation. Truncation of the CoA moiety in 3′ position led to a significant reduction of inhibitory activity.102 Coupling of the inhibitors’ amino acid backbone to the Tat protein transduction domain or to arginine rich peptides succeeded in facilitating cellular uptake and activity.103,104 However, the application of such inhibitor/peptide constructs is rather limited due to their 1256 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 results from biochemical testing compd 1 name/code Lys-CoA 1.1 H3-CoA20 1.2 H4K16-CoA 2 Boc-C5-CoA 4 anacardic acid approach lead structurebased lead structurebased lead structurebased lead structurebased fucussed library screen p300/CBP Gcn5 PCAF 0.5 μM 200 μM 0.98 μM 0.05 μM 30 μM 0.0032 μM [GST-p300C] 200 μM 108.3 μM 6.62 μM 58.47 μM Tip60 other 29.8 μM 7 μM [Esa1] 17.59 μM 5.51 μM [Esa1] 0.3 μM 0.07 μM 8.5 μM 5 μM >1000 μM >200 μM 667.1 μM >200 μM 347.6 μM 64 μM 297.2 μM [Esa1] 43 μM [MOF] Ki = 64 μM [MOF] 1257 1−2 mM 33.9 μM 4.15 μM, max 60% lead structurebased 4.1 662 μM n.i. (200 μM) 4.2 CTPB 4.4 LoCAM lead structurebased lead structurebased 100% (200 μM) n.i. ∼400% (275 μM) 74% (50 μM) n.i. 81.2% (100 μM) 5 EGCG 6 curcumin CTK7A 7 garcinol 7.2 LTK-14 synthetic optimization fucussed library screen synthetic optimization ∼40% (50 μM) 37.5% (100 μM) 60 μM 30 μM/50 μM 25 μM 70 μM n.i. (100 μM) ref 23 radiometric radiometric radiometric radiometric radiometric filter-binding gel gel filter-binding gel 100 104 121 132 23 radiometric filter-binding 100 radiometric 101 radiometric filter-binding 107 radiometric filter-binding radiometric filter-binding colorimetric CoA-scavenging immunosorbent ELISA-like radiometric filter-binding colorimetric CoA-scavenging immunosorbent ELISA-like 100 108 112 111 115 148 111 radiometric filter-binding 108 radiometric filter-binding 107 immunosorbent ELISA-like 114 radiometric filter-binding 115 colorimetric 116 radiometric filter-binding 118 radiometric filter-binding radiometric filter-binding radiometric gel AlphaLisa proximity immunoassay radiometric filter-binding 115 100 121 127 122 6.5 μM >40 μM >400 μM 6.5 μM >50% (50 μM) >50% (50 μM) 7 μM 5 μM radiometric filter-binding 124 n.i. (50 μM) radiometric filter-binding 126 n.i. (80 μM) radiometric gel 125 5−7 μM Ki = 5.1 μM >200 μM n.i. (50 μM) n.i. (100 μM) >200 μM [Esa1] Perspective DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 6.3 fucussed library screen fucussed library screen 65% (200 μM) assay system radiometric gel Journal of Medicinal Chemistry Table 2. KAT Modulators Listed with Their Results from Biochemical Evaluation: IC50 Values or Percentage Enzyme Inhibition at a Specified Concentration (n.i. = No Inhibition) results from biochemical testing compd name/code approach 8 EML425 9 plumbagin 10 Embelin lead structurebased fucused library screen HTS 11 11.1 12.2 NK13650A NK13650B PU139 HTS HTS virtual screen p300/CBP Gcn5 PCAF 2.9 μM [p300]/1.1 μM [CBP] n.i. n.i. 20−25 μM [full length]; 2 μM [cat. domain] 75% (25 μM) PU141 1258 MC1823 14.2 CPTH6 15 MB-3 phenotypic screen phenotypic screen rational design 16 C646 virtual screen 17 L002 18 19 20 NU9056 TH1834 phenotypic screen virtual screen HTS rational design HTS 75% (25 μM) n.i. (9 μM) n.i. (9 μM) 1.64 μM 8.39 μM 9.74 μM 53% (5 μM) [MOF] 130 μM 5.92 μM [p300]/2.85 μM [CBP] 21 7.2 μM virtual screen 13 other 50 μM 0.011 μM [GST-p300C] 0.022 μM [GST-p300C] 5.35 μM [p300]/2.49 μM [CBP] 12.3 Tip60 87.36 μM 130 μM 60% (5 μM) [MOF] 24% (25 μM); 30% (50 μM) [U937 nuclear extracts] 500 μM CBP 55% (50 μM) [CBP] 1.6 μM, Ki = 0.4 μM, 86% (10 μM) 0.32 μM 1.98 μM, 128 μM [p300]/32% (100 μM) [CBP] 150 μM ∼58 μM n.i. ∼40% (800 μM) 100 μM <10% (10 μM) 33.9 μM ∼40% (800 μM) <10% (10 μM) 34.7 μM >100 μM ∼35 μM n.i. < 10% (10 μM) [Rtt109, Sas2, MOZ] 2% (100 μM) 149 μM 2 μM 60% (500 μM) 1% (100 μM) [HBO1]; −28% (100 μM) [MORF] 190 μM [Esa1] 0.56 μM [Rtt109] assay system ref AlphaLisa proximity immunoassay 127 radiometric filter-binding 129 radiometric filter-binding 131 radiometric filter-binding radiometric filter-binding time-resolved fluorescence immunosorbent time-resolved fluorescence immunosorbent time-resolved fluorescence immunosorbent time-resolved fluorescence immunosorbent immunosorbent ELISA-like 132 132 136 140 radiometric filter-binding 142 radiometric gel immunosorbent ELISA-like radiometric gel 144 114 145 colorimetric CoA-scavenging colorimetric CoA-scavenging/ radiometric filter-binding radiometric filter-binding radiometric filter-binding immunosorbent gel 112 148 149 150 151 colorimetric CoA-scavenging 51 Journal of Medicinal Chemistry Table 2. continued 137 136 137 Perspective DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective inhibitory potency superior to 4 in PCAF inhibition.112 Interestingly, in a subsequent publication, compound 4.1 was identified as an activator of PCAF with no effect on p300 and inhibitory activity on Tip60.108 The activation of KAT enzymes by small molecules is consistent with observations of other groups. In an inhibitor screen of substituted benzamide analogues of 4, derivative 4.2 (CTPB) promoted activation of p300 but not of PCAF and increased p300-dependent transcription activation.107 An interesting attempt was followed by Chatterjee et al., which included binding of the small molecule p300/CBP activator 4.3 (TTK21) to glucose-based carbon nanospheres.113 The resulting particles readily penetrated the cell membrane to increase histone acetylation without causing apparent toxicity. Treatment with 4.3-loaded nanospheres was further correlated with beneficial neurologic effects in a mouse xenograft model. Sbardella, Mai, and colleagues published the long chain alkylidenmalonate 4.4 (LoCAM) as a selective activator of PCAF while inhibiting p300/CBP in a comparable degree to the parent structure 4.114 Structure−activity relationship studies on the LoCAM scaffold revealed that the replacement of one or both of the ester moieties with keto- or carboxylic acid groups greatly alters the modulation profile of the resulting analogues, ranging from selective activation to unselective inhibition. In terms of the aliphatic chain, it was shown that the introduction of a heteroatom is detrimental for binding efficiency and variations in alkyl chain length result in abrogated modulatory activity.115 The precise underlying mechanism of this activator/inhibitor ambivalence is still under investigation. Similar to compound 4, disruption of NF-κB signaling due to repression of KAT activity was demonstrated for some polyphenols like 5 (epigallocatechin-3-gallate (EGCG)).116 However, these compounds interact with a wide range of other protein targets and their structural optimization to achieve selectivity seems to be challenging.117 Compound 6 (curcumin) is a major component of Curcuma longa rhizome that is commonly used in Indian and Chinese traditional medicine.118 The compound was reported in 2004 as a p300 inhibitor (IC50 25 μM) with no effect on PCAF. Treatment with 6 represses p300-dependent H3, H4, and p53 acetylation in different cell lines. Kinetic studies revealed a covalent mode of action at a binding site apart from the substrate and cofactor-binding pocket. The double bonds in the cinnamoyl structure serve potentially as Michael reaction acceptors, and their presence is crucial for binding with p300.119 Although certain selectivity between different KAT enzymes was demonstrated, the compounds’ inhibitory activity is rather promiscuous as it also inhibits other epigenetic targets (e.g., KDACs, DNMT1, LSD1) as well as a multitude of nonepigenetic related proteins.120 Still, 6 is under clinical investigation for several indications. Open-chain and cyclic cinnamoyl analogues (6.1−6.2) have been shown to inhibit p300 activity with similar potency as the lead structure.121 The sodium salt of the hydrazinocurcumin 6.3 (CTK7A) is more water-soluble while maintaining inhibitory potency.122 It should be noted that, like the parent structure, the described analogues interact with multiple target proteins aside from p300.117 Such promiscuous effects generally complicate the correlation of in vitro with in vivo observations. In addition, curcumin is known to be a membrane disruptor and therefore it is likely that some of its activities could be attributed to nondrug-like modes of action.123 complex handling and elaborate production. Bandyopadhyay et al. fused CoA to the polyamine spermidine to generate compound 3 (Spd(N1)-CoA), which is internalized into cells via polyamine transporter uptake.105,106 They further truncated the CoA moiety to the cysteamine-β-alanine core structure to achieve structure 3.1 and reported cellular activity comparable to 3. 105 Both polyamine conjugates impeded histone acetylation-dependent repair and synthesis of DNA and consequently led to radio- and chemosensitization. Natural Products and Synthetic Analogues and Derivatives. Screening of plant or microbial extracts has proven to be one of the most successful strategies in the discovery of KAT modulators. Several structures have been identified comprising micromolar activity (Figures 5 and 6 and Table 2). Common structural scaffolds of natural compounds, such as Michael reaction acceptors and polyphenols, are reflected in their frequent lack of selectivity and hence often pleiotropic effects in cellular systems. In addition, natural compounds usually comprise unfavorable physicochemical properties, which limit their further development. Synthetic and semisynthetic approaches have been carried out in order to determine structure−activity relationships and to optimize compound properties. Interestingly, in the course of biological evaluation, alongside with KAT inhibitors, compounds with enzyme activating capacity and mixed activities have been found. In this way, natural compounds are useful templates for further development into modulators of KAT activity, yet more druglike structures are still desirable. The natural product 4 (anacardic acid), rich in the liquid of cashew nut shells, emerged from a screening of plant extracts with anticancer activity.107 The substance was described in 2003 as a nonselective, noncompetitive inhibitor of p300/CBP and PCAF, but following studies also reported activity in Tip60 inhibition under similar experimental conditions.108 The inhibitory potency of 4 varies between different studies and IC50 values between 5 μM and 1000 μM were obtained for p300 inhibition, depending on assay conditions and enzyme source. On the cellular level, repression of the NF-κB signaling pathway by KAT-dependent acetylation of the p65 subunit has been found. However, the application of compound 4 in numerous pharmacological studies is attributed to its pleiotropic affinity that affects multiple enzyme targets.109 A limiting factor for further development of this natural product as a therapeutic tool is its unfavorable physicochemical properties, especially its high lipophilicity. The 6-alkyl salicylic acid structure has extensively been mined in order to generate compounds with increased solubility, cell permeability, and inhibitory efficacy. Our group probed a set of phenoxyacetic acid analogues (structures not shown) for their inhibitory capacity against KAT activity in nuclear extracts. We demonstrated that the position and length of the alkyl chain are pivotal for enzyme inhibition and that substitution of the phenolic hydroxyl group is well tolerated.110 Deduced from the co-crystal structure of PCAF with acetyl-CoA, Ghizzoni et al. proposed a binding model for 4 and synthesized a set of compounds with variations in the alkyl chain and the salicylic acid residue. Esterification of the carboxyl group diminished inhibitory activity, whereas modulation of the alkyl chain or hydroxylation in C4 position caused a shift of specificity toward MYST family KATs. This structure−activity relationship is supported by a recent study on alkyl salicylic acid derivatives in MOF inhibition, with 4 as a reference inhibitor.111 Among the compounds tested by Ghizzoni et al., derivative 4.1 showed 1259 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective Figure 7. Structures of synthetic KAT inhibitors. proposed high affinity for the same alternative binging site as for 7.2. The suggested noncovalent manner of this binding mode is intriguing, as the benzylidene barbituric acid scaffold is potentially prone to Michael addition reactions and has been exploited in targeting enzyme structures apart from p300/ CBP.128 The hydroxynaphthoquinone 9 (plumbagin), isolated from Plumbago rosea, has been shown to attenuate p300-dependent acetylation of histones H3, H4, and p53 in HepG2 liver cancer cells without causing any effect on PCAF activity.129 Structure− activity studies suggested the hydroxyl group in C5 position to be pivotal for forming hydrogen-bond interactions with Lys1358 in the active site of the enzyme. In consequence, derivatization of the naphthochinonein this position diminishes the inhibitory effect. Methylation in C3 position abrogated the thiol reactivity and therefore cytotoxicity of 9 while maintaining its function as a p300 inhibitor.130 The 3-alkyl dihydroxybenzoquinone 10 (Embelin) is isolated from Embelia ribes and displays a similar scaffold.131 Compound 10 has been shown to inhibit KAT activity of recombinant PCAF and to promote H3K9 hypoacetylation in treated mice. The compound also attenuated PCAF-mediated MyoD acetylation in HEK239T cells, which was correlated to a block of differentiation in C2C12 cells. Chemical variation revealed that the 11-carbon alkyl chain in structure 10 is crucial for the inhibitory capacity as an analogue with one carbon less was reported to be completely inactive. The polyisoprenylated benzophenone 7 (garcinol) was isolated from Garcinia indica and identified as a micromolar inhibitor of p300 (IC50 7 μM) and PCAF (IC50 5 μM) KAT activity.124 Repression of histone acetylation and induction of apoptosis were found in human cancer cell lines upon treatment with this natural product. Isothermal calorimetric titration data proposed a two centered binding mode with the catechol hydroxyl groups interacting with the acetyl-CoA binding pocket, while the isoprenoid moieties are placed into the substrate binding domain of the enzyme.125 Monomethylation of the intramolecular cyclization product 7.1 (isogarcinol) in C14-position led to derivative 7.2 (LTK-14), which provides a shifted inhibition pattern toward p300 activity and an attenuated T-cell cytotoxicity.126 Human HeLa cells that are treated with 7.1 or one of its analogues develop concentrationdependent histone hypoacetylation and repression of gene transcription. The binding mode of 7.2 was proposed to be different from the parent compound, with a single unique binding side within the enzymes KAT domain.125 Sbardella and colleagues recently published the benzylidene barbituric acid derivative 8 (EML425) as a selective and reversible inhibitor of p300/CBP with an inhibitory potency in the low micromolar range (IC50 2.9 μM for p300 and 1.1 μM for CBP).127 The compound resulted from a molecular pruning approach of structure 7 with isosteric replacement of the benzophenone core. Compound 8 was found to promote cell cycle arrest in G0/G1 phase accompanied by H3K9 and H4K5 hypoacetylation in U937 cells. Molecular modeling studies of this derivative 1260 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective inhibition as novel strategy in the control of schistosomiasis. In a recent publication, Gajer et al. demonstrated two selected pyridoisothiazolones (12.2 and 12.3 (PU141)) to trigger growth inhibition and histone hypoacetylation in multiple cancer cell lines and to block neuroblastoma cell growth in a SK-N-SH xenograft model in vivo.139 The compounds were previously screened against a cysteine protease panel to assess their off-target selectivity, and no significant in vitro activity on these enzymes was observed. Thus, the inhibitors do not have a general reactivity toward all cysteine dependent enyzmes. For 12.2, the authors also reported reduction of histone acetylation in healthy mice and synergistic effects with the DNAintercalating drug doxorubicin in the xenografts. This is one of very few examples for the demonstration of hypoacetylation in vivo after inhibitor treatment. Yeast phenotypic screenings have yielded quinoline and hydrazone derivatives with KAT inhibiting activity. Structural optimization of the quinolone structure resulted in compound 13 (MC1823), which comprises a scaffold similar to that of 4.140 The reduction in cell viability of S. cerevisiae was correlated to a histone H3 and α-tubulin hypoacetylation as a result of Gcn5 inhibition. The same effect has been observed for the hydrazones 14−14.2.141−143 In addition, p300 and PCAF activities are repressed by 14 (BF1) and 14.2 (CPTH6), respectively, and both compounds are competent to cause histone protein hypoacetylation in different human cancer cell lines. In 2004, the α-methylene-γ-butyrolactone 15 (MB-3) was published as an inhibitor of Gcn5, selective over CBP.144 Biel et al. followed a rational design strategy based on the electrostatic interaction fields within the active site of Gcn5. The assessed IC50 value of 100 μM is comparable with the KD value of the H3 substrate and despite the apparent Michael reaction acceptor scaffold, the binding mode of MB-3 was reported to follow a noncovalent fashion. The pyrazolone-containing inhibitor 16 (C646), was identified as a potent, selective, and reversible p300/CBP KAT inhibitor by means of a virtual screening approach.145 Bowers et al. docked a database of ∼500000 commercially available compounds into the same binding pocket as occupied by the bisubstrate inhibitor 1 in the p300 KAT enzyme and selected 194 compounds for biological testing. Three compounds were found to inhibit p300 KAT activity in the micromolar and submicromolar range, where 16 showed the highest inhibitory activity (Ki = 460 nM). The compound was predicted to be a bisubstrate inhibitor of p300, and the proposed binding mode shows numerous H-bond and salt bridge interactions of 16, with the side chains of the binding pocket similar to those observed with the CoA moiety of the cocrystallized inhibitor 1. Chemical modifications of functional groups indicated that the free carboxylic acid, as well as certain interactions mediated by the nitro group, are essential for inhibitory activity, although replacement of the nitro group with more metabolically favorable functionalities resulted in only minor loss of potency. Further, a reduced derivative demonstrated that the conjugated pyrazolone structure is pivotal for binding to the p300 active site, indicating that a certain level of planarity of the molecule is required for efficient active-site targeting. However, this structural entity is potentially prone to nucleophilic attack. Despite it being shown in the original publication that binding of 16 to p300 happens in a nontime dependent manner and is not abrogated by DTT or β-mercaptoethanol, the formation of covalent Two compounds have been identified during a microorganism broth library screen. 11 (NK13650A) and 11.1 (NK13650B) are fungal metabolites of a Penicillium strain with a peptidic structure that contains a citric acid moiety.132 The evaluation of their inhibitory capacity revealed high selectivity for p300 KAT activity over Tip60, and IC50 values were determined to be 11 and 22 nM, respectively. It was further demonstrated that these compounds were competent to repress androgen- and estrogen receptor-dependent activation of gene transcription and to be cytotoxic to different cancer cell lines. Owed to their peptidic nature, these compounds suffer from poor cell permeability and metabolic instability, which requires structural optimization. Nevertheless, the identification of secondary metabolites comprising KAT inhibitory activity supplies a promising strategy in lead structure discovery as such approaches were successfully applied on other biological targets, like KDACs.133 Synthetic Compounds. Different approaches, such as high-throughput-strategies, rational design, and in silico screenings have been applied in order to find new potent small molecule KAT modulators. Derivatization of initial hits and investigations on the structure−activity relationships gained new insights into the characteristics of KAT enzymes. With the aid of computational methods, it was possible to identify and develop new compounds with pronounced inhibitory activity (Figure 7 and Table 2). Only few of these modulators are fully characterized in vitro and in vivo so far. The inhibition of p300 and PCAF enzymes in biochemical and cell-based assays with concomitant repression of growth and histone acetylation by compounds comprising an isothiazolone structure (12) was first reported in 2005 by Aherne and colleagues.134 An irreversible binding mode was suggested that involves disulfide bond formation between the isothiazolone sulfur and a cysteine residue in the enzyme. Consistent with this, addition of DTT to the reaction abolished inhibitory activity. Because the general reactivity of these substances is a major drawback, several efforts have been dedicated to developing derivatives with reduced promiscuity while maintaining inhibitory potency. Furdas et al. identified pyridoisothiazolones as novel PCAF inhibitors by applying a computational screening approach.135 The NCI database was screened for compounds which contain an isothiazolone or isothiazolidinone substructure as found in the PCAF inhibitor 12.1.134 A subsequently carried out similarity search retrieved 51 related compounds from commercial databases, which were docked into the substrate binding site observed in the crystal structure of PCAF. Only compounds, which showed a reactive S−N moiety in close proximity to PCAF Cys574 in the docking results, were considered for further biological testing. By this means, different pyridoisothiazolones were discovered as novel KAT inhibitors with low micromolar IC50 values and reduced general bioreactivity.136 Structure−activity studies revealed a crucial role of the substituent in 2-position for inhibitory activity. N-Aryl substituted compounds were shown to cause pan-KAT inhibition on a series of enzymes (PCAF, Gcn5, p300, CBP, and MOF), while N-benzyl or N-alkyl substituents led to defined subtype selectivity patterns.137 The Fantappie group incubated Schistosoma mansoni parasites with the compound 12.2 (PU139) and reported impaired promoter activity of the egg shell protein Smp14, probably evidently as a result of diminished SmGCN5 and SmCBP1 activity.138 The repression of Smp14 controlled gene products led to production of abnormal and defective eggs, implying KAT 1261 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective inhibition of enzyme-mediated H3K56 acetylation in presence of histone chaperone cofactor protein Vps75 or Asf1, respectively. Compound 21 did not show any apparent effect on p300 and Gcn5 activity but inhibited Rtt109 potently (IC50 0.56 μM) in a sulfhydryl scavenging assay. Kinetic measurements proposed a tight or irreversible binding mode as inhibitory activity increased over time and was not diminished by dialysis of the inhibitor from the reaction mixture. Inhibitors of the fungal specific acetyltransferase Rtt109 are proposed to be promising drug candidates against pathogenic fungal species, such as Candida albicans. The further development of compound 21 is limited by its inability to induce cellular effects, presumably due to drug efflux or rapid metabolism. Structure−activity studies need to be conducted to assess the overall potential of this scaffold. Bromodomain Inhibitors. In addition to modulators targeting the catalytic domain of KATs, a more recent approach concentrates on the identification and development of small molecule inhibitors of KAT protein−protein interaction domains.153,154 One of the most thoroughly investigated interaction domains is the bromodomain, which recognizes and binds to acetylated lysine residues. The disruption of acetylation-dependent protein−protein interactions by small molecule inhibitors abrogates the assembly of transcriptional regulator complexes and subsequently represses the phenotypic consequences of acetylation signaling. The successful implementation of BET bromodomain ligands has evoked a growing interest in the identification of potent and selective small molecule inhibitors of non-BET bromodomains, as they are structural entities of several KATs. Mostly structure-guided approaches have yielded a set of KAT bromodomain ligands whose structures are depicted in Figure8. The N-aryl-propane-1,3-diamine 22 has been identified as an inhibitor of the PCAF bromodomain.155 Binding of this bromodomain to the acetylated K50 moiety of the HIV-1-Tat protein is required to induce transcription of the integrated HIV-1 provirus. 22 successfully inhibited the transactivation of the HIV-1 promoter by blocking the PCAF bromodomain in C1866 cells with an IC 50 value of 0.6 μM. Higher concentrations of the inhibitor were shown to be cytotoxic, which is a drawback for further development of this scaffold. Acetylation of the effector protein p53 at lysine residue 382 facilitates interaction with the CBP bromodomain. Upon binding, a coactivator complex is formed, which is recruited to p53 controlled promoter sites. Zhou et al. performed a cellbased compound screen against the p53-dependent expression of p21 in response to doxorubicin-mediated DNA damage. Structural optimization of initial hits yielded the azobenzene 23 (ischemin), which was demonstrated to inhibit the p53-CBP bromodomain interaction with an IC50 value of 5 μM and a 5fold selectivity over other bromodomains (PCAF, BRD4, BAZ2B).156 Compound 23 was shown to be competent in protecting rat cardiomyocytes from p53-induced apoptosis in consequence of doxorubicin treatment. Rooney et al. employed a fragment-based approach combined with chemical expansion of the initial hits to develop a series of dihydroquinoxalinones as the first submicromolar ligands for the CBP bromodomain. The most potent compound 24 was shown to bind to the bromodomain via an induced fit pocket, which is occupied by the tetrahydroquinoline side chain and stabilized by a cation−π interaction of this residue with R1173 of the protein.157 The KD of 24 was assessed in an isothermal titration calorimetry assay to be 390 conjugates with a number of different cellular cysteine containing proteins was observed in a recent publication.146 The large conjugated system in structure 16 causes intrinsic fluorescence of the compound, which is an obstacle for its use in fluorescent-based assay methods. Cole and colleagues circumvented this hurdle by replacing the furan group with a phenyl ring.147 The resultant derivative was absent of intrinsic fluorescence while mostly maintaining its inhibitory capacity (IC50 9 μM (p300)). In melanoma and lung cancer cell lines, treatment with 16 led to inhibition of cell growth and concomitant histone H3 and H4 hypoacetylation. Cell cycle arrest and induction of apoptosis upon administration was also observed in AML1-ETO positive leukemia cells.148 The small molecule inhibitor 17 (L002) has been reported as a result of a large high-throughput screening approach of more than 600000 substances for their cytotoxic activity against the triple-negative breast cancer cell line MDA-MB-231.149 Radioactive filter binding assays revealed inhibitory activity against p300 (IC50 128 μM) and GNAT family KATs (IC50 34.7 μM for PCAF and 33.9 μM for Gcn5) but no inhibition of MYST family members. The IC50 value of 17 against p300 was determined to be 1.98 μM in an orthogonal fluorescence assay. The sulfonyl-coupled iminoquinone structure was placed in silico into the acetyl-CoA domain of the p300 enzyme. In cellular experiments, the compound has been found to promote histone and p53 hypoacetylation and to suppress STAT3 activation in lymphoma, leukemia, and breast cancer cell lines. Reduction of tumor growth and decreased histone acetylation were correlated to in vivo administration in a MDA-MB-468 xenograft model. Different scaffolds have been identified and tested in order to find inhibitors for the Tip60 histone acetyltransferase as a potential therapeutic strategy in correlated malignancies. The Zheng group published the phthalimide analogue 18 in 2011 as the result of a virtual screening campaign based on the Tip60 yeast homologue Esa1 crystal structure.150 KAT inhibition efficacy was assessed in a radiometric in vitro assay for different KAT family members (Tip60, Esa1, p300, PCAF) and IC50 values between 100 and 190 μM were determined, stating an unselective inhibitory activity. Computational docking and kinetic studies suggested that 18 targets the acetyl-CoA binding site of Tip60. One year later, Coffey et al. performed a highthroughput screening for Tip60 inhibitors of ∼80000 substances. Structure 19 (NU9056), a derivative of an initial hit, bearing an isothiazole scaffold, was demonstrated to specifically inhibit Tip60 activity (IC50 2 μM).151 It was further shown that the derivative blocks acetylation of histone proteins in a concentration-dependent manner and induces apoptosis via caspase activation in prostate cancer cell lines. In 2014, 20 (TH1834) was developed during a rational design approach in accordance with the electrostatic surface potential of a Tip60 active site model.152 Under physiological conditions, the structure comprises two oppositely charged ends, which were predicted to interact with amino acid side chains on different sides of the Tip60 binding pocket. The ethylbenzene side chain was introduced as an isosteric replacement of acetyl-CoAs’ adenine residue. Throughout the biological evaluation, compound 20 was tested at high concentration (500 μM), promoting induction of apoptosis and radiosensitization in MCF7, DU-145, and PC-3 cancer cell lines. An inhibitor (21) of the fungal KAT Rtt109 was published in 2013 as the result of a high-throughput screening campaign.51 The authors reported specific and noncompetitive in vitro 1262 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective ■ DISCUSSION AND CONCLUSIONS Altogether, epigenetic modifiers have emerged in the last 20 years as one of the most promising class of new targets for a variety of diseases. The majority of findings points to a pivotal role in cancer, but also for metabolic diseases, CNS disorders, and infectious pathologies promising data is available.160 The evidence for the roles of different KAT enzymes in these indications was mostly gathered with the aid of molecular biology methods like gene knockdown or gene knockout. To distinguish whether the resultant phenotypic changes upon enzyme depletion can be assigned to the repression of either the acetyltransferase activity or protein−protein interactions, small molecule inhibitors are needed that specifically target either of these features. There is no doubt that new approaches in molecular biology will help to further elucidate the field of lysine acetylation to give a more rounded picture of their specific implication in pathological and physiological processes.161 Among the histone modifiers, inhibitors of histone deacetylases have gained approval for use in patients and five drugs are now on the market.162 For some epigenetic regulators, e.g., the bromodomains, the time span from first reports as druggable targets163 to first clinical trials was about four to five years only.164 KATs were among the first epigenetic modifiers to be discovered,18 yet, strangely, almost 20 years later, there is still no drug in advanced development. This is true despite numerous studies showing the involvement of KATs in disease and rich structural data. Structural reasons for the rather difficult development of potent and selective inhibitors are the structurally conserved cofactor binding site, which makes it difficult to achieve pronounced selectivity for a specific enzyme subtype and the relatively shallow substrate binding cleft where the histone substrate is placed.160 Another problem in the identification of active compounds seems to be the variation in the different assay formats. Various assay strategies have been employed in the biochemical characterization of KAT enzymes (Table 2). Radiometric measurements have emerged as one of the standard procedures, allowing robust and sensitive readouts, but their application requires special hazard precautions and waste disposal. Colorimetric assays are susceptible for quenching effects and interference of the tested compound with the assay components. The need for time-consuming washing steps is the biggest disadvantage of heterogeneous immunosorbent methods, which complicates their application in high-throughput approaches. In comparison, the data generated by different biochemical assays for published compounds is very inconsistent. For example, for compound 4, the measured potencies in p300 inhibition vary between IC50 values of 8.5 and >1000 μM in three radiometric filter-binding assays. The huge discrepancy may be explained by differences in enzyme source, type of substrate, substrate and cofactor concentration, buffer composition, and incubation time. Therefore, direct comparison of IC50 values, even for similar assay types, can be misleading. Standardized assay protocols and reliable reference compounds would be valuable tools to achieve comparability of biochemical data. The implementation of new assay methods, like thermal shift165 or microscale thermophoresis,166 could prove beneficial for the identification and characterization of new substances and detection of promiscuous inhibition mechanisms. To avoid misinterpretation of compound hits from initial screens, it is important to confirm such observations in orthogonal assays that rely on a different type of readout. In addition, a panel of Figure 8. Structures of PCAF and CBP/p300 bromodomain inhibitors. nM, and the selectivity for the CBP bromodomain over BRD4 (KD 1.4 μM) was stated to be modest. Mining of the 3,5-dimethylisoxazole scaffold resulted in the identification of 25 (SGC−CBP30).158 The aryl substituent of the isoxazole core forms a cation−π interaction with R1173 in the bromodomain binding site. The compounds binds to the CBP bromodomain with a KD value of 21 nM, and the achieved inhibition is highly selective over BRD4. In a luciferase-based reporter assay, the authors observed diminished expression of CBP-dependent p53 downstream genes as a result of SGC− CBP30 incubation. SGC and GSK developed the CBP bromodomain inhibitor 26 (I-CBP112) on the basis of a benzoxazepine scaffold.159 The acyl group in this structure mimics the acetyl residue of the native recognition motive, thereby forming hydrogen bonds to the binding site, while the aryl substituent interacts with the arginine moiety in position 1173 of the CBP bromodomain. The KD values of 26 for binding to p300 and CBP bromodomains were determined to be 167 and 151 nM, respectively, with selectivity over a number of other bromodomains (e.g., BRD2, BRD4, PCAF, TIF1α). The compound proved cellular activity in a FRAP assay on U2OS cells. Further studies revealed that 26 is capable to impair p53 interaction, which results in reduced p21 expression. Treatment of leukemia cell lines with the bromodomain inhibitor led to cell cycle arrest in G1-phase and morphological differentiation. 1263 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective treatment of mantle cell lymphoma and is pursued with high intensity. Thus, starting out from covalent modifiers of cysteines in KATs, affinity and selectivity could be built in subsequently, resulting potentially in highly potent and selective KAT inhibitors. For inhibitors that rely on the covalent targeting of cysteine residues, as it is the case with compounds 12.2 and 12.3, further structural development of these lead structures is needed to direct their general reactivity further toward specific targets only. Because even for well characterized compounds, like 16, covalent interaction with offtarget protein structures has been demonstrated. Hence, such molecules are placed at the intersection of PAINS and covalent modifiers. Therefore, at the moment, maybe not a single KAT inhibitor does satisfy the rigid rules demanded for high quality chemical probes.169 As most of the known KAT modulators were identified due to screenings of large compound libraries or in the course of chemical optimization or derivatization of such initial hits, it becomes more and more evident that this might not be the ideal way to find highly potent and specific ligands for KAT enzymes. Approaches like rational-design, fragmentbased design, or virtual ligand screening could provide better suitable methods to deal with the KAT specific challenges concerning druggability. On the other hand, for therapeutic endeavors compounds with a more pleiotropic profile may still be useful, as a drug needs to be safe and effective and knowledge on the mode of action is not a prerequisite for successful treatment. The problem is of course that optimization toward an assumed target may then not be successful in terms of efficacy and needs to be performed with phenotypic cellular and animal models. A big problem is the widespread use of published KAT inhibitors in mechanistic studies with a danger of overinterpretation of the link of histone acetyltransferase inhibition and the studied mechanism in question, but on the other hand, it may still be informative to use these inhibitors to potentially gain insight in KAT biology. Therefore, for both drug discovery but especially chemical epigenetics, there is clearly still a big demand for high quality KAT inhibitors. Many points need to be addressed such as the relevance of nonhistone acetylation,170−172 the question of the relevance of acyl groups other than acetyl,173 or the role of nonenzymatic versus enzymatic acetylation.174,175 The big question is whether after 20 years a new way can be discovered to reach that goal. As bromodomains have emerged as druggable and promising targets, one strategy is to target those domains that are part of KATs rather than the enzymatic activity. As there is no crystal structure of any KAT catalytic domain in context with a small molecule inhibitor aside from 1, more structural data of KAT enzymes in complex with small molecule ligands and in context of their native multidomain protein complexes is needed to guide further development. Combined with new approaches in modulator discovery and optimization, computational methods using such information will largely improve the chances of developing potent and selective modulators of KAT enzymes. These will be highly useful as chemical probes and will show whether we can KATch up in terms of drug development with this difficult, yet still promising class of epigenetic targets. counter screens against possible off-targets should be carried out to ensure target selectivity. The field of modulator development is further complicated by the fact that activity and substrate specificity of enzymes in biochemical assays can significantly differ from a cellular environment. This holds especially true for KATs as they natively occur in multiprotein complexes. Therefore, profound evaluation of cellular activity is needed to complement biochemical data. A number of inhibitors have been discovered by various approaches but many of them suffer from several drawbacks, such as low in vivo potency, metabolic instability, or poor selectivity (Table 2). Among the different subtypes of KAT enzymes, p300/CBP seems to be the preferred target of most modulators. This is presumably due to the structural features of these isoenzymes that distinguish them from the other families. Although bisubstrate inhibitors of KAT activity excel in potency and selectivity, their further development into more druglike small molecules is largely prevented by their complex structure. The simple rationale of covalently linking ligands of both the cofactor and the substrate binding site is contradicted in terms of selectivity by the high structural conservation of KAT isoforms in these regions. The incorporation of large peptidic groups into the inhibitor structure is needed to achieve subtype selectivity, and the resulting compounds suffer from poor in vivo efficacy. However, analogues and derivatives of 1 might serve as a starting point for fragment-based approaches in future inhibitor discovery. Natural products have been investigated deeply as a class of KAT modulators. Extensive structure−activity studies have been carried out to assess their full potential, and a multifold of derivatives have been generated with the aim to improve physicochemical and modulatory properties. A lot of them feature structural motifs that are suspicious in terms of promiscuous behavior, such as Michael acceptors, phenolic or quinone moieties.167 While for some of them selectivity and target engagement among the KATs has been shown, clearly data for the analysis on other targets is missing for most of these compounds. Or, like in the case of curcumin, inhibition of many other targets has been shown already. Despite large efforts that have been put into the characterization of natural KAT modulators and the development of improved derivatives, no compound with pronounced potency and especially high selectivity, for KAT enzymes and distinct KAT isoforms, has been obtained. The identification of structural analogues of known inhibitors with reversed inhibitory potency, actually increasing enzyme activity, as well as the isolation of highly potent peptidic natural products, are interesting findings. Crystal structures of these modulators in complex with the KAT enzyme would provide valuable information for further development. The class of synthetic compounds comprises a structurally heterogeneous set of molecules, which were identified by different approaches. Although some of these compounds were shown to be potent KAT inhibitors, only few of them feature subtype selectivity and have been characterized profoundly in a cellular setting. Screening and designing of small molecules with the aid of computational methods has proven to be useful in the targeting of specific interaction fields. In this way, it becomes much more likely to achieve subtype selectivity, like in the case of compound 16. Another possibility for direct enzyme inhibition is targeting cysteines involved in the catalysis or present at the active site of the enzyme. The use of covalent inhibitors has emerged as a highly promising strategy in kinase inhibitors that has already culminated in the approval of ibrutinib168 for the ■ AUTHOR INFORMATION Corresponding Author * Phone: +497612034896. Fax: +497612036321. E-mail: manfred.jung@pharmazie.uni-freiburg.de. 1264 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective specific lethal; PTM, posttranslational modification; vdW, vander-Waals Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ■ REFERENCES (1) Zeng, L.; Zhou, M. M. Bromodomain: an acetyl-lysine binding domain. FEBS Lett. 2002, 513, 124−128. (2) Kouzarides, T. Chromatin modifications and their function. Cell 2007, 128, 693−705. (3) Smith, B. C.; Denu, J. M. Chemical mechanisms of histone lysine and arginine modifications. Biochim. Biophys. Acta, Gene Regul. Mech. 2009, 1789, 45−57. (4) Marmorstein, R.; Zhou, M. M. Writers and readers of histone acetylation: structure, mechanism, and inhibition. Cold Spring Harbor Perspect. Biol. 2014, 6, a018762. (5) Sterner, D. E.; Berger, S. L. Acetylation of histones and transcription-related factors. Microbiol. Mol. Biol. Rev. 2000, 64, 435− 459. (6) Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M. L.; Rehman, M.; Walther, T. C.; Olsen, J. V.; Mann, M. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009, 325, 834−840. (7) Kim, S. C.; Sprung, R.; Chen, Y.; Xu, Y.; Ball, H.; Pei, J.; Cheng, T.; Kho, Y.; Xiao, H.; Xiao, L.; Grishin, N. V.; White, M.; Yang, X. J.; Zhao, Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol. Cell 2006, 23, 607−618. (8) Allis, C. D.; Berger, S. L.; Cote, J.; Dent, S.; Jenuwein, T.; Kouzarides, T.; Pillus, L.; Reinberg, D.; Shi, Y.; Shiekhattar, R.; Shilatifard, A.; Workman, J.; Zhang, Y. New nomenclature for chromatin-modifying enzymes. Cell 2007, 131, 633−636. (9) Mellert, H. S.; McMahon, S. B. Biochemical pathways that regulate acetyltransferase and deacetylase activity in mammalian cells. Trends Biochem. Sci. 2009, 34, 571−578. (10) Feinberg, A. P. Phenotypic plasticity and the epigenetics of human disease. Nature 2007, 447, 433−440. (11) Sun, X. J.; Man, N.; Tan, Y.; Nimer, S. D.; Wang, L. The role of histone acetyltransferases in normal and malignant hematopoiesis. Front. Oncol. 2015, 5, 108. (12) Schneider, A.; Chatterjee, S.; Bousiges, O.; Selvi, B. R.; Swaminathan, A.; Cassel, R.; Blanc, F.; Kundu, T. K.; Boutillier, A. L. Acetyltransferases (HATs) as targets for neurological therapeutics. Neurotherapeutics 2013, 10, 568−588. (13) Wang, Y.; Miao, X.; Liu, Y.; Li, F.; Liu, Q.; Sun, J.; Cai, L. Dysregulation of histone acetyltransferases and deacetylases in cardiovascular diseases. Oxid. Med. Cell. Longevity 2014, 2014, 641979. (14) Caton, P. W.; Nayuni, N. K.; Kieswich, J.; Khan, N. Q.; Yaqoob, M. M.; Corder, R. Metformin suppresses hepatic gluconeogenesis through induction of SIRT1 and GCN5. J. Endocrinol. 2010, 205, 97− 106. (15) Allfrey, V. G.; Faulkner, R.; Mirsky, A. E. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc. Natl. Acad. Sci. U. S. A. 1964, 51, 786−794. (16) Gershey, E. L.; Vidali, G.; Allfrey, V. G. Chemical studies of histone acetylation. The occurrence of epsilon-N-acetyllysine in the f2a1 histone. J. Biol. Chem. 1968, 243, 5018−5022. (17) Candido, E. P.; Reeves, R.; Davie, J. R. Sodium butyrate inhibits histone deacetylation in cultured cells. Cell 1978, 14, 105−113. (18) Brownell, J. E.; Allis, C. D. An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 6364−6368. (19) Parthun, M. R.; Widom, J.; Gottschling, D. E. The major cytoplasmic histone acetyltransferase in yeast: links to chromatin replication and histone metabolism. Cell 1996, 87, 85−94. (20) Luger, K.; Mader, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997, 389, 251−260. (21) Grant, P. A.; Duggan, L.; Cote, J.; Roberts, S. M.; Brownell, J. E.; Candau, R.; Ohba, R.; Owen-Hughes, T.; Allis, C. D.; Winston, F.; Berger, S. L.; Workman, J. L. Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Notes The authors declare no competing financial interest. Biographies Roman P. Simon holds a diploma degree in Pharmaceutical Sciences at the Institute of Pharmaceutical Sciences of the Albert-Ludwigs University Freiburg and works as a Ph.D. candidate in the group of Prof. M. Jung. He studied Pharmacy (state examination) at the University of Freiburg from 2008 to 2013. After a six months research stay in the group of Prof. Ganesan at the University of East Anglia in 2014, he returned to Freiburg. Since the beginning of 2015, he is a doctoral student, with his research focusing on development and characterization of novel small molecule KAT inhibitors. Dina Robaa studied Pharmacy at the University of Alexandria in Egypt. She obtained her Ph.D. in Pharmaceutical Chemistry at the University of Jena in the group of Jochen Lehmann. Since 2011, she has been working as a postdoctoral fellow in the research group of Wolfgang Sippl. Her research focuses on structure-based drug design of several epigenetic modulators. Zayan Alhalabi studied Pharmacy at the University of Damascus in Syria. She finished her diploma in Pharmaceutical Science. She obtained her master’s degree in Pharmaceutical Chemistry at the University of Damascus 2012. In 2013, she started her Ph.D. at the Department of Medicinal Chemistry in the research group of Prof. Wolfgang Sippl. Her research focuses on structure-based drug design for epigenetic targets (Sirtuins). Wolfgang Sippl is Professor for Medicinal Chemistry and Director of the Institute of Pharmacy at the Martin-Luther-University of HalleWittenberg (Germany). He obtained a Ph.D. in Pharmaceutical Chemistry at the University of Düsseldorf in the group of Hans-Dieter Höltje and was a postdoctoral fellow at the Université Louis-Pasteur in Strasbourg (France) where he worked with Camille G. Wermuth. Since 2003, he is Full Professor at the Institute of Pharmacy in Halle. His main interests are focussed on computational chemistry and structure-based drug design of novel epigenetic modulators for the therapy of cancer and parasitic diseases. Manfred Jung did his Ph.D. with W. Hanefeld on the synthesis of aromatic retinoids at the University of Marburg. In 1993/94, he did a postdoc with T. Durst (University of Ottawa, Canada). From 1994 to 2003 he was a group leader at the University of Münster and obtained his habilitation in Pharmaceutical Chemistry (2000). Since 2003, he is a Professor of Pharmaceutical Chemistry at the University of Freiburg. In 2010, he declined an offer for a full professorship to the University of Mainz and is a Full Professor in Freiburg since 2011. The topic of his research is Chemical Epigenetics. His group is working on inhibitor synthesis, assay development, and screening for inhibitors of reversible histone acetylation and methylation but also histone readers. ACKNOWLEDGMENTS R.P.S and M.J. thank the Deutsche Forschungsgemeinschaft for funding (Ju 295/9-2 within SPP1463). ■ ABBREVIATIONS USED BRD, bromodomain; H3K9, histone H3 lysine residue 9; HAT, histone acetyltransferase; KAT, lysine acetyltransferase; KD, dissociation constant; KDAC, lysine desacetylase; MSL, male ■ 1265 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective acetyltransferase that is essential for growth in yeast. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 3561−3565. (43) Iizuka, M.; Stillman, B. Histone acetyltransferase HBO1 interacts with the ORC1 subunit of the human initiator protein. J. Biol. Chem. 1999, 274, 23027−23034. (44) Champagne, N.; Bertos, N. R.; Pelletier, N.; Wang, A. H.; Vezmar, M.; Yang, Y.; Heng, H. H.; Yang, X. J. Identification of a human histone acetyltransferase related to monocytic leukemia zinc finger protein. J. Biol. Chem. 1999, 274, 28528−28536. (45) Smith, E. R.; Pannuti, A.; Gu, W.; Steurnagel, A.; Cook, R. G.; Allis, C. D.; Lucchesi, J. C. The drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation. Mol. Cell. Biol. 2000, 20, 312−318. (46) Mizzen, C. A.; Yang, X. J.; Kokubo, T.; Brownell, J. E.; Bannister, A. J.; Owen-Hughes, T.; Workman, J.; Wang, L.; Berger, S. L.; Kouzarides, T.; Nakatani, Y.; Allis, C. D. The TAF(II)250 subunit of TFIID has histone acetyltransferase activity. Cell 1996, 87, 1261− 1270. (47) Hsieh, Y. J.; Kundu, T. K.; Wang, Z.; Kovelman, R.; Roeder, R. G. The TFIIIC90 subunit of TFIIIC interacts with multiple components of the RNA polymerase III machinery and contains a histone-specific acetyltransferase activity. Mol. Cell. Biol. 1999, 19, 7697−7704. (48) York, B.; O’Malley, B. W. Steroid receptor coactivator (SRC) family: masters of systems biology. J. Biol. Chem. 2010, 285, 38743− 38750. (49) Spencer, T. E.; Jenster, G.; Burcin, M. M.; Allis, C. D.; Zhou, J.; Mizzen, C. A.; McKenna, N. J.; Onate, S. A.; Tsai, S. Y.; Tsai, M. J.; O’Malley, B. W. Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 1997, 389, 194−198. (50) Doi, M.; Hirayama, J.; Sassone-Corsi, P. Circadian regulator CLOCK is a histone acetyltransferase. Cell 2006, 125, 497−508. (51) Lopes da Rosa, J.; Bajaj, V.; Spoonamore, J.; Kaufman, P. D. A small molecule inhibitor of fungal histone acetyltransferase Rtt109. Bioorg. Med. Chem. Lett. 2013, 23, 2853−2859. (52) Karmodiya, K.; Anamika, K.; Muley, V.; Pradhan, S. J.; Bhide, Y.; Galande, S. Camello, a novel family of Histone Acetyltransferases that acetylate histone H4 and is essential for zebrafish development. Sci. Rep. 2014, 4, 6076. (53) Friedmann, D. R.; Aguilar, A.; Fan, J.; Nachury, M. V.; Marmorstein, R. Structure of the alpha-tubulin acetyltransferase, alphaTAT1, and implications for tubulin-specific acetylation. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 19655−19660. (54) Hou, F.; Zou, H. Two human orthologues of Eco1/Ctf7 acetyltransferases are both required for proper sister-chromatid cohesion. Mol. Biol. Cell 2005, 16, 3908−3918. (55) Portela, A.; Esteller, M. Epigenetic modifications and human disease. Nat. Biotechnol. 2010, 28, 1057−1068. (56) Selvi, B. R.; Chatterjee, S.; Modak, R.; Eswaramoorthy, M.; Kundu, T. K. Histone acetylation as a therapeutic target. Subcell. Biochem. 2013, 61, 567−596. (57) Li, Y.; Yang, H. X.; Luo, R. Z.; Zhang, Y.; Li, M.; Wang, X.; Jia, W. H. High expression of p300 has an unfavorable impact on survival in resectable esophageal squamous cell carcinoma. Ann. Thorac. Surg. 2011, 91, 1531−1538. (58) Li, M.; Luo, R. Z.; Chen, J. W.; Cao, Y.; Lu, J. B.; He, J. H.; Wu, Q. L.; Cai, M. Y. High expression of transcriptional coactivator p300 correlates with aggressive features and poor prognosis of hepatocellular carcinoma. J. Transl. Med. 2011, 9, 5. (59) Bandyopadhyay, D.; Okan, N. A.; Bales, E.; Nascimento, L.; Cole, P. A.; Medrano, E. E. Down-regulation of p300/CBP histone acetyltransferase activates a senescence checkpoint in human melanocytes. Cancer Res. 2002, 62, 6231−6239. (60) Pattabiraman, D. R.; McGirr, C.; Shakhbazov, K.; Barbier, V.; Krishnan, K.; Mukhopadhyay, P.; Hawthorne, P.; Trezise, A.; Ding, J.; Grimmond, S. M.; Papathanasiou, P.; Alexander, W. S.; Perkins, A. C.; Levesque, J. P.; Winkler, I. G.; Gonda, T. J. Interaction of c-Myb with p300 is required for the induction of acute myeloid leukemia (AML) by human AML oncogenes. Blood 2014, 123, 2682−2690. Ada complex and the SAGA (Spt/Ada) complex. Genes Dev. 1997, 11, 1640−1650. (22) Rojas, J. R.; Trievel, R. C.; Zhou, J.; Mo, Y.; Li, X.; Berger, S. L.; Allis, C. D.; Marmorstein, R. Structure of Tetrahymena GCN5 bound to coenzyme A and a histone H3 peptide. Nature 1999, 401, 93−98. (23) Lau, O. D.; Kundu, T. K.; Soccio, R. E.; Ait-Si-Ali, S.; Khalil, E. M.; Vassilev, A.; Wolffe, A. P.; Nakatani, Y.; Roeder, R. G.; Cole, P. A. HATs off: selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF. Mol. Cell 2000, 5, 589−595. (24) Choudhary, C.; Weinert, B. T.; Nishida, Y.; Verdin, E.; Mann, M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat. Rev. Mol. Cell Biol. 2014, 15, 536−550. (25) Lin, H.; Su, X.; He, B. Protein lysine acylation and cysteine succination by intermediates of energy metabolism. ACS Chem. Biol. 2012, 7, 947−960. (26) Parthun, M. R. Hat1: the emerging cellular roles of a type B histone acetyltransferase. Oncogene 2007, 26, 5319−5328. (27) Yang, X.; Yu, W.; Shi, L.; Sun, L.; Liang, J.; Yi, X.; Li, Q.; Zhang, Y.; Yang, F.; Han, X.; Zhang, D.; Yang, J.; Yao, Z.; Shang, Y. HAT4, a Golgi apparatus-anchored B-type histone acetyltransferase, acetylates free histone H4 and facilitates chromatin assembly. Mol. Cell 2011, 44, 39−50. (28) Yang, X. J.; Ogryzko, V. V.; Nishikawa, J.; Howard, B. H.; Nakatani, Y. A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 1996, 382, 319−324. (29) Wittschieben, B. O.; Otero, G.; de Bizemont, T.; Fellows, J.; Erdjument-Bromage, H.; Ohba, R.; Li, Y.; Allis, C. D.; Tempst, P.; Svejstrup, J. Q. A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme. Mol. Cell 1999, 4, 123− 128. (30) Sampath, V.; Liu, B.; Tafrov, S.; Srinivasan, M.; Rieger, R.; Chen, E. I.; Sternglanz, R. Biochemical characterization of Hpa2 and Hpa3, two small closely related acetyltransferases from Saccharomyces cerevisiae. J. Biol. Chem. 2013, 288, 21506−21513. (31) Lorch, Y.; Beve, J.; Gustafsson, C. M.; Myers, L. C.; Kornberg, R. D. Mediator-nucleosome interaction. Mol. Cell 2000, 6, 197−201. (32) Dyda, F.; Klein, D. C.; Hickman, A. B. GCN5-related Nacetyltransferases: a structural overview. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 81−103. (33) Marmorstein, R. Protein modules that manipulate histone tails for chromatin regulation. Nat. Rev. Mol. Cell Biol. 2001, 2, 422−432. (34) Roth, S. Y.; Denu, J. M.; Allis, C. D. Histone acetyltransferases. Annu. Rev. Biochem. 2001, 70, 81−120. (35) Kimura, A.; Matsubara, K.; Horikoshi, M. A decade of histone acetylation: marking eukaryotic chromosomes with specific codes. J. Biochem. 2005, 138, 647−662. (36) Dancy, B. M.; Cole, P. A. Protein lysine acetylation by p300/ CBP. Chem. Rev. 2015, 115, 2419−2452. (37) Bordoli, L.; Netsch, M.; Luthi, U.; Lutz, W.; Eckner, R. Plant orthologs of p300/CBP: conservation of a core domain in metazoan p300/CBP acetyltransferase-related proteins. Nucleic Acids Res. 2001, 29, 589−597. (38) Borrow, J.; Stanton, V. P., Jr.; Andresen, J. M.; Becher, R.; Behm, F. G.; Chaganti, R. S.; Civin, C. I.; Disteche, C.; Dube, I.; Frischauf, A. M.; Horsman, D.; Mitelman, F.; Volinia, S.; Watmore, A. E.; Housman, D. E. The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREBbinding protein. Nat. Genet. 1996, 14, 33−41. (39) Neuwald, A. F.; Landsman, D. GCN5-related histone Nacetyltransferases belong to a diverse superfamily that includes the yeast SPT10 protein. Trends Biochem. Sci. 1997, 22, 154−155. (40) Reifnyder, C.; Lowell, J.; Clarke, A.; Pillus, L. Yeast SAS silencing genes and human genes associated with AML and HIV-1 Tat interactions are homologous with acetyltransferases. Nat. Genet. 1997, 16, 109. (41) Sapountzi, V.; Logan, I. R.; Robson, C. N. Cellular functions of TIP60. Int. J. Biochem. Cell Biol. 2006, 38, 1496−1509. (42) Smith, E. R.; Eisen, A.; Gu, W.; Sattah, M.; Pannuti, A.; Zhou, J.; Cook, R. G.; Lucchesi, J. C.; Allis, C. D. ESA1 is a histone 1266 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective (61) Sun, X. J.; Wang, Z.; Wang, L.; Jiang, Y.; Kost, N.; Soong, T. D.; Chen, W. Y.; Tang, Z.; Nakadai, T.; Elemento, O.; Fischle, W.; Melnick, A.; Patel, D. J.; Nimer, S. D.; Roeder, R. G. A stable transcription factor complex nucleated by oligomeric AML1-ETO controls leukaemogenesis. Nature 2013, 500, 93−97. (62) Cereseto, A.; Manganaro, L.; Gutierrez, M. I.; Terreni, M.; Fittipaldi, A.; Lusic, M.; Marcello, A.; Giacca, M. Acetylation of HIV-1 integrase by p300 regulates viral integration. EMBO J. 2005, 24, 3070− 3081. (63) Deng, L.; de la Fuente, C.; Fu, P.; Wang, L.; Donnelly, R.; Wade, J. D.; Lambert, P.; Li, H.; Lee, C. G.; Kashanchi, F. Acetylation of HIV-1 Tat by CBP/P300 increases transcription of integrated HIV1 genome and enhances binding to core histones. Virology 2000, 277, 278−295. (64) Chen, S.; Feng, B.; George, B.; Chakrabarti, R.; Chen, M.; Chakrabarti, S. Transcriptional coactivator p300 regulates glucoseinduced gene expression in endothelial cells. Am. J. Physiol. Endocrinol. Metab. 2010, 298, E127−E137. (65) Kikuchi, H.; Takami, Y.; Nakayama, T. GCN5: a supervisor in all-inclusive control of vertebrate cell cycle progression through transcription regulation of various cell cycle-related genes. Gene 2005, 347, 83−97. (66) Perez-Luna, M.; Aguasca, M.; Perearnau, A.; Serratosa, J.; Martinez-Balbas, M.; Jesus Pujol, M.; Bachs, O. PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27 Kip1. Nucleic Acids Res. 2012, 40, 6520−6533. (67) Shiota, M.; Yokomizo, A.; Tada, Y.; Uchiumi, T.; Inokuchi, J.; Tatsugami, K.; Kuroiwa, K.; Yamamoto, K.; Seki, N.; Naito, S. P300/ CBP-associated factor regulates Y-box binding protein-1 expression and promotes cancer cell growth, cancer invasion and drug resistance. Cancer Sci. 2010, 101, 1797−1806. (68) Toth, M.; Boros, I. M.; Balint, E. Elevated level of lysine 9acetylated histone H3 at the MDR1 promoter in multidrug-resistant cells. Cancer Sci. 2012, 103, 659−669. (69) Terreni, M.; Valentini, P.; Liverani, V.; Gutierrez, M. I.; Di Primio, C.; Di Fenza, A.; Tozzini, V.; Allouch, A.; Albanese, A.; Giacca, M.; Cereseto, A. GCN5-dependent acetylation of HIV-1 integrase enhances viral integration. Retrovirology 2010, 7, 18. (70) Miao, J.; Fan, Q.; Cui, L.; Li, J.; Li, J.; Cui, L. The malaria parasite Plasmodium falciparum histones: organization, expression, and acetylation. Gene 2006, 369, 53−65. (71) Duclot, F.; Meffre, J.; Jacquet, C.; Gongora, C.; Maurice, T. Mice knock out for the histone acetyltransferase p300/CREB binding protein-associated factor develop a resistance to amyloid toxicity. Neuroscience 2010, 167, 850−863. (72) Wei, W.; Coelho, C. M.; Li, X.; Marek, R.; Yan, S.; Anderson, S.; Meyers, D.; Mukherjee, C.; Sbardella, G.; Castellano, S.; Milite, C.; Rotili, D.; Mai, A.; Cole, P. A.; Sah, P.; Kobor, M. S.; Bredy, T. W. p300/CBP-associated factor selectively regulates the extinction of conditioned fear. J. Neurosci. 2012, 32, 11930−11941. (73) Colussi, C.; Rosati, J.; Straino, S.; Spallotta, F.; Berni, R.; Stilli, D.; Rossi, S.; Musso, E.; Macchi, E.; Mai, A.; Sbardella, G.; Castellano, S.; Chimenti, C.; Frustaci, A.; Nebbioso, A.; Altucci, L.; Capogrossi, M. C.; Gaetano, C. Nepsilon-lysine acetylation determines dissociation from GAP junctions and lateralization of connexin 43 in normal and dystrophic heart. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2795−2800. (74) Sun, C.; Wang, M.; Liu, X.; Luo, L.; Li, K.; Zhang, S.; Wang, Y.; Yang, Y.; Ding, F.; Gu, X. PCAF improves glucose homeostasis by suppressing the gluconeogenic activity of PGC-1alpha. Cell Rep. 2014, 9, 2250−2262. (75) Kindle, K. B.; Troke, P. J.; Collins, H. M.; Matsuda, S.; Bossi, D.; Bellodi, C.; Kalkhoven, E.; Salomoni, P.; Pelicci, P. G.; Minucci, S.; Heery, D. M. MOZ-TIF2 inhibits transcription by nuclear receptors and p53 by impairment of CBP function. Mol. Cell. Biol. 2005, 25, 988−1002. (76) Dulak, A. M.; Stojanov, P.; Peng, S.; Lawrence, M. S.; Fox, C.; Stewart, C.; Bandla, S.; Imamura, Y.; Schumacher, S. E.; Shefler, E.; McKenna, A.; Carter, S. L.; Cibulskis, K.; Sivachenko, A.; Saksena, G.; Voet, D.; Ramos, A. H.; Auclair, D.; Thompson, K.; Sougnez, C.; Onofrio, R. C.; Guiducci, C.; Beroukhim, R.; Zhou, Z.; Lin, L.; Lin, J.; Reddy, R.; Chang, A.; Landrenau, R.; Pennathur, A.; Ogino, S.; Luketich, J. D.; Golub, T. R.; Gabriel, S. B.; Lander, E. S.; Beer, D. G.; Godfrey, T. E.; Getz, G.; Bass, A. J. Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity. Nat. Genet. 2013, 45, 478−486. (77) Yang, X. J. MOZ and MORF acetyltransferases: molecular interaction, animal development and human disease. Biochim. Biophys. Acta, Mol. Cell Res. 2015, 1853, 1818−1826. (78) Shiota, M.; Yokomizo, A.; Masubuchi, D.; Tada, Y.; Inokuchi, J.; Eto, M.; Uchiumi, T.; Fujimoto, N.; Naito, S. Tip60 promotes prostate cancer cell proliferation by translocation of androgen receptor into the nucleus. Prostate 2010, 70, 540−554. (79) Ikura, T.; Ogryzko, V. V.; Grigoriev, M.; Groisman, R.; Wang, J.; Horikoshi, M.; Scully, R.; Qin, J.; Nakatani, Y. Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis. Cell 2000, 102, 463−473. (80) Iizuka, M.; Takahashi, Y.; Mizzen, C. A.; Cook, R. G.; Fujita, M.; Allis, C. D.; Frierson, H. F., Jr.; Fukusato, T.; Smith, M. M. Histone acetyltransferase Hbo1: catalytic activity, cellular abundance, and links to primary cancers. Gene 2009, 436, 108−114. (81) Dekker, F. J.; van den Bosch, T.; Martin, N. I. Small molecule inhibitors of histone acetyltransferases and deacetylases are potential drugs for inflammatory diseases. Drug Discovery Today 2014, 19, 654− 660. (82) Kuo, M. H.; Allis, C. D. Roles of histone acetyltransferases and deacetylases in gene regulation. BioEssays 1998, 20, 615−626. (83) Lee, K. K.; Workman, J. L. Histone acetyltransferase complexes: one size doesn’t fit all. Nat. Rev. Mol. Cell Biol. 2007, 8, 284−295. (84) Schuetz, A.; Bernstein, G.; Dong, A.; Antoshenko, T.; Wu, H.; Loppnau, P.; Bochkarev, A.; Plotnikov, A. N. Crystal structure of a binary complex between human GCN5 histone acetyltransferase domain and acetyl coenzyme A. Proteins: Struct., Funct., Genet. 2007, 68, 403−407. (85) Poux, A. N.; Cebrat, M.; Kim, C. M.; Cole, P. A.; Marmorstein, R. Structure of the GCN5 histone acetyltransferase bound to a bisubstrate inhibitor. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 14065− 14070. (86) Poux, A. N.; Marmorstein, R. Molecular basis for Gcn5/PCAF histone acetyltransferase selectivity for histone and nonhistone substrates. Biochemistry 2003, 42, 14366−14374. (87) Clements, A.; Poux, A. N.; Lo, W. S.; Pillus, L.; Berger, S. L.; Marmorstein, R. Structural basis for histone and phosphohistone binding by the GCN5 histone acetyltransferase. Mol. Cell 2003, 12, 461−473. (88) Delvecchio, M.; Gaucher, J.; Aguilar-Gurrieri, C.; Ortega, E.; Panne, D. Structure of the p300 catalytic core and implications for chromatin targeting and HAT regulation. Nat. Struct. Mol. Biol. 2013, 20, 1040−1046. (89) Liu, X.; Wang, L.; Zhao, K.; Thompson, P. R.; Hwang, Y.; Marmorstein, R.; Cole, P. A. The structural basis of protein acetylation by the p300/CBP transcriptional coactivator. Nature 2008, 451, 846− 850. (90) Maksimoska, J.; Segura-Pena, D.; Cole, P. A.; Marmorstein, R. Structure of the p300 histone acetyltransferase bound to acetylcoenzyme A and its analogues. Biochemistry 2014, 53, 3415−3422. (91) Thompson, P. R.; Kurooka, H.; Nakatani, Y.; Cole, P. A. Transcriptional coactivator protein p300. Kinetic characterization of its histone acetyltransferase activity. J. Biol. Chem. 2001, 276, 33721− 33729. (92) Thompson, P. R.; Wang, D.; Wang, L.; Fulco, M.; Pediconi, N.; Zhang, D.; An, W.; Ge, Q.; Roeder, R. G.; Wong, J.; Levrero, M.; Sartorelli, V.; Cotter, R. J.; Cole, P. A. Regulation of the p300 HAT domain via a novel activation loop. Nat. Struct. Mol. Biol. 2004, 11, 308−315. (93) Yan, Y.; Barlev, N. A.; Haley, R. H.; Berger, S. L.; Marmorstein, R. Crystal structure of yeast Esa1 suggests a unified mechanism for catalysis and substrate binding by histone acetyltransferases. Mol. Cell 2000, 6, 1195−1205. 1267 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective (94) Huang, J.; Wan, B.; Wu, L.; Yang, Y.; Dou, Y.; Lei, M. Structural insight into the regulation of MOF in the male-specific lethal complex and the non-specific lethal complex. Cell Res. 2012, 22, 1078−1081. (95) Kadlec, J.; Hallacli, E.; Lipp, M.; Holz, H.; Sanchez-Weatherby, J.; Cusack, S.; Akhtar, A. Structural basis for MOF and MSL3 recruitment into the dosage compensation complex by MSL1. Nat. Struct. Mol. Biol. 2011, 18, 142−149. (96) Sun, B.; Guo, S.; Tang, Q.; Li, C.; Zeng, R.; Xiong, Z.; Zhong, C.; Ding, J. Regulation of the histone acetyltransferase activity of hMOF via autoacetylation of Lys274. Cell Res. 2011, 21, 1262−1266. (97) Yuan, H.; Rossetto, D.; Mellert, H.; Dang, W.; Srinivasan, M.; Johnson, J.; Hodawadekar, S.; Ding, E. C.; Speicher, K.; Abshiru, N.; Perry, R.; Wu, J.; Yang, C.; Zheng, Y. G.; Speicher, D. W.; Thibault, P.; Verreault, A.; Johnson, F. B.; Berger, S. L.; Sternglanz, R.; McMahon, S. B.; Cote, J.; Marmorstein, R. MYST protein acetyltransferase activity requires active site lysine autoacetylation. EMBO J. 2012, 31, 58−70. (98) Li, X.; Wu, L.; Corsa, C. A.; Kunkel, S.; Dou, Y. Two mammalian MOF complexes regulate transcription activation by distinct mechanisms. Mol. Cell 2009, 36, 290−301. (99) Morales, V.; Straub, T.; Neumann, M. F.; Mengus, G.; Akhtar, A.; Becker, P. B. Functional integration of the histone acetyltransferase MOF into the dosage compensation complex. EMBO J. 2004, 23, 2258−2268. (100) Wu, J.; Xie, N.; Wu, Z.; Zhang, Y.; Zheng, Y. G. Bisubstrate inhibitors of the MYST HATs Esa1 and Tip60. Bioorg. Med. Chem. 2009, 17, 1381−1386. (101) Kwie, F. H.; Briet, M.; Soupaya, D.; Hoffmann, P.; Maturano, M.; Rodriguez, F.; Blonski, C.; Lherbet, C.; Baudoin-Dehoux, C. New potent bisubstrate inhibitors of histone acetyltransferase p300: design, synthesis and biological evaluation. Chem. Biol. Drug Des. 2011, 77, 86−92. (102) Cebrat, M.; Kim, C. M.; Thompson, P. R.; Daugherty, M.; Cole, P. A. Synthesis and analysis of potential prodrugs of coenzyme A analogues for the inhibition of the histone acetyltransferase p300. Bioorg. Med. Chem. 2003, 11, 3307−3313. (103) Wadia, J. S.; Dowdy, S. F. Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Adv. Drug Delivery Rev. 2005, 57, 579−596. (104) Zheng, Y.; Balasubramanyam, K.; Cebrat, M.; Buck, D.; Guidez, F.; Zelent, A.; Alani, R. M.; Cole, P. A. Synthesis and evaluation of a potent and selective cell-permeable p300 histone acetyltransferase inhibitor. J. Am. Chem. Soc. 2005, 127, 17182−17183. (105) Bandyopadhyay, K.; Baneres, J. L.; Martin, A.; Blonski, C.; Parello, J.; Gjerset, R. A. Spermidinyl-CoA-based HAT inhibitors block DNA repair and provide cancer-specific chemo- and radiosensitization. Cell Cycle 2009, 8, 2779−2788. (106) Cullis, P. M.; Wolfenden, R.; Cousens, L. S.; Alberts, B. M. Inhibition of histone acetylation by N-[2-(S-coenzyme A)acetyl] spermidine amide, a multisubstrate analog. J. Biol. Chem. 1982, 257, 12165−12169. (107) Balasubramanyam, K.; Swaminathan, V.; Ranganathan, A.; Kundu, T. K. Small molecule modulators of histone acetyltransferase p300. J. Biol. Chem. 2003, 278, 19134−19140. (108) Ghizzoni, M.; Wu, J.; Gao, T.; Haisma, H. J.; Dekker, F. J.; George Zheng, Y. 6-alkylsalicylates are selective Tip60 inhibitors and target the acetyl-CoA binding site. Eur. J. Med. Chem. 2012, 47, 337− 344. (109) Hemshekhar, M.; Sebastin Santhosh, M.; Kemparaju, K.; Girish, K. S. Emerging roles of anacardic acid and its derivatives: a pharmacological overview. Basic Clin. Pharmacol. Toxicol. 2012, 110, 122−132. (110) Eliseeva, E. D.; Valkov, V.; Jung, M.; Jung, M. O. Characterization of novel inhibitors of histone acetyltransferases. Mol. Cancer Ther. 2007, 6, 2391−2398. (111) Wapenaar, H.; van der Wouden, P. E.; Groves, M. R.; Rotili, D.; Mai, A.; Dekker, F. J. Enzyme kinetics and inhibition of histone acetyltransferase KAT8. Eur. J. Med. Chem. 2015, 105, 289−296. (112) Ghizzoni, M.; Boltjes, A.; Graaf, C.; Haisma, H. J.; Dekker, F. J. Improved inhibition of the histone acetyltransferase PCAF by an anacardic acid derivative. Bioorg. Med. Chem. 2010, 18, 5826−5834. (113) Chatterjee, S.; Mizar, P.; Cassel, R.; Neidl, R.; Selvi, B. R.; Mohankrishna, D. V.; Vedamurthy, B. M.; Schneider, A.; Bousiges, O.; Mathis, C.; Cassel, J. C.; Eswaramoorthy, M.; Kundu, T. K.; Boutillier, A. L. A novel activator of CBP/p300 acetyltransferases promotes neurogenesis and extends memory duration in adult mice. J. Neurosci. 2013, 33, 10698−10712. (114) Sbardella, G.; Castellano, S.; Vicidomini, C.; Rotili, D.; Nebbioso, A.; Miceli, M.; Altucci, L.; Mai, A. Identification of long chain alkylidenemalonates as novel small molecule modulators of histone acetyltransferases. Bioorg. Med. Chem. Lett. 2008, 18, 2788− 2792. (115) Castellano, S.; Milite, C.; Feoli, A.; Viviano, M.; Mai, A.; Novellino, E.; Tosco, A.; Sbardella, G. Identification of structural features of 2-alkylidene-1,3-dicarbonyl derivatives that induce inhibition and/or activation of histone acetyltransferases KAT3B/p300 and KAT2B/PCAF. ChemMedChem 2015, 10, 144−157. (116) Choi, K. C.; Jung, M. G.; Lee, Y. H.; Yoon, J. C.; Kwon, S. H.; Kang, H. B.; Kim, M. J.; Cha, J. H.; Kim, Y. J.; Jun, W. J.; Lee, J. M.; Yoon, H. G. Epigallocatechin-3-gallate, a histone acetyltransferase inhibitor, inhibits EBV-induced B lymphocyte transformation via suppression of RelA acetylation. Cancer Res. 2009, 69, 583−592. (117) Mai, A.; Cheng, D.; Bedford, M. T.; Valente, S.; Nebbioso, A.; Perrone, A.; Brosch, G.; Sbardella, G.; De Bellis, F.; Miceli, M.; Altucci, L. Epigenetic multiple ligands: mixed histone/protein methyltransferase, acetyltransferase, and class III deacetylase (sirtuin) inhibitors. J. Med. Chem. 2008, 51, 2279−2290. (118) Balasubramanyam, K.; Varier, R. A.; Altaf, M.; Swaminathan, V.; Siddappa, N. B.; Ranga, U.; Kundu, T. K. Curcumin, a novel p300/ CREB-binding protein-specific inhibitor of acetyltransferase, represses the acetylation of histone/nonhistone proteins and histone acetyltransferase-dependent chromatin transcription. J. Biol. Chem. 2004, 279, 51163−51171. (119) Neckers, L.; Trepel, J.; Lee, S.; Chung, E. J.; Lee, M.-J.; Jung, Y.-J.; Marcu, M. G. Curcumin is an inhibitor of p300 histone acetylatransferase. Med. Chem. 2006, 2, 169−174. (120) Fu, S.; Kurzrock, R. Development of curcumin as an epigenetic agent. Cancer 2010, 116, 4670−4676. (121) Costi, R.; Di Santo, R.; Artico, M.; Miele, G.; Valentini, P.; Novellino, E.; Cereseto, A. Cinnamoyl compounds as simple molecules that inhibit p300 histone acetyltransferase. J. Med. Chem. 2007, 50, 1973−1977. (122) Arif, M.; Vedamurthy, B. M.; Choudhari, R.; Ostwal, Y. B.; Mantelingu, K.; Kodaganur, G. S.; Kundu, T. K. Nitric oxide-mediated histone hyperacetylation in oral cancer: target for a water-soluble HAT inhibitor, CTK7A. Chem. Biol. 2010, 17, 903−913. (123) Ingolfsson, H. I.; Thakur, P.; Herold, K. F.; Hobart, E. A.; Ramsey, N. B.; Periole, X.; de Jong, D. H.; Zwama, M.; Yilmaz, D.; Hall, K.; Maretzky, T.; Hemmings, H. C., Jr.; Blobel, C.; Marrink, S. J.; Kocer, A.; Sack, J. T.; Andersen, O. S. Phytochemicals perturb membranes and promiscuously alter protein function. ACS Chem. Biol. 2014, 9, 1788−1798. (124) Balasubramanyam, K.; Altaf, M.; Varier, R. A.; Swaminathan, V.; Ravindran, A.; Sadhale, P. P.; Kundu, T. K. Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression. J. Biol. Chem. 2004, 279, 33716−33726. (125) Arif, M.; Pradhan, S. K.; G R, T.; Vedamurthy, B. M.; Agrawal, S.; Dasgupta, D.; Kundu, T. K. Mechanism of p300 specific histone acetyltransferase inhibition by small molecules. J. Med. Chem. 2009, 52, 267−277. (126) Mantelingu, K.; Reddy, B. A.; Swaminathan, V.; Kishore, A. H.; Siddappa, N. B.; Kumar, G. V.; Nagashankar, G.; Natesh, N.; Roy, S.; Sadhale, P. P.; Ranga, U.; Narayana, C.; Kundu, T. K. Specific inhibition of p300-HAT alters global gene expression and represses HIV replication. Chem. Biol. 2007, 14, 645−657. 1268 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective (127) Milite, C.; Feoli, A.; Sasaki, K.; La Pietra, V.; Balzano, A. L.; Marinelli, L.; Mai, A.; Novellino, E.; Castellano, S.; Tosco, A.; Sbardella, G. A novel cell-permeable, selective, and noncompetitive inhibitor of KAT3 histone acetyltransferases from a combined molecular pruning/classical isosterism approach. J. Med. Chem. 2015, 58, 2779−2798. (128) Maurer, B.; Rumpf, T.; Scharfe, M.; Stolfa, D. A.; Schmitt, M. L.; He, W.; Verdin, E.; Sippl, W.; Jung, M. Inhibitors of the NAD(+)dependent protein desuccinylase and demalonylase Sirt5. ACS Med. Chem. Lett. 2012, 3, 1050−1053. (129) Ravindra, K. C.; Selvi, B. R.; Arif, M.; Reddy, B. A.; Thanuja, G. R.; Agrawal, S.; Pradhan, S. K.; Nagashayana, N.; Dasgupta, D.; Kundu, T. K. Inhibition of lysine acetyltransferase KAT3B/p300 activity by a naturally occurring hydroxynaphthoquinone, plumbagin. J. Biol. Chem. 2009, 284, 24453−24464. (130) Vasudevarao, M. D.; Mizar, P.; Kumari, S.; Mandal, S.; Siddhanta, S.; Swamy, M. M.; Kaypee, S.; Kodihalli, R. C.; Banerjee, A.; Naryana, C.; Dasgupta, D.; Kundu, T. K. Naphthoquinonemediated inhibition of lysine acetyltransferase KAT3B/p300, basis for non-toxic inhibitor synthesis. J. Biol. Chem. 2014, 289, 7702−7717. (131) Modak, R.; Basha, J.; Bharathy, N.; Maity, K.; Mizar, P.; Bhat, A. V.; Vasudevan, M.; Rao, V. K.; Kok, W. K.; Natesh, N.; Taneja, R.; Kundu, T. K. Probing p300/CBP associated factor (PCAF)-dependent pathways with a small molecule inhibitor. ACS Chem. Biol. 2013, 8, 1311−1323. (132) Tohyama, S.; Tomura, A.; Ikeda, N.; Hatano, M.; Odanaka, J.; Kubota, Y.; Umekita, M.; Igarashi, M.; Sawa, R.; Morino, T. Discovery and characterization of NK13650s, naturally occurring p300-selective histone acetyltransferase inhibitors. J. Org. Chem. 2012, 77, 9044− 9052. (133) Furumai, R.; Komatsu, Y.; Nishino, N.; Khochbin, S.; Yoshida, M.; Horinouchi, S. Potent histone deacetylase inhibitors built from trichostatin A and cyclic tetrapeptide antibiotics including trapoxin. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 87−92. (134) Stimson, L.; Rowlands, M. G.; Newbatt, Y. M.; Smith, N. F.; Raynaud, F. I.; Rogers, P.; Bavetsias, V.; Gorsuch, S.; Jarman, M.; Bannister, A.; Kouzarides, T.; McDonald, E.; Workman, P.; Aherne, G. W. Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity. Mol. Cancer Ther. 2005, 4, 1521−1532. (135) Furdas, S. D.; Kannan, S.; Sippl, W.; Jung, M. Small molecule inhibitors of histone acetyltransferases as epigenetic tools and drug candidates. Arch. Pharm. (Weinheim, Ger.) 2012, 345, 7−21. (136) Furdas, S. D.; Shekfeh, S.; Bissinger, E. M.; Wagner, J. M.; Schlimme, S.; Valkov, V.; Hendzel, M.; Jung, M.; Sippl, W. Synthesis and biological testing of novel pyridoisothiazolones as histone acetyltransferase inhibitors. Bioorg. Med. Chem. 2011, 19, 3678−3689. (137) Furdas, S. D.; Hoffmann, I.; Robaa, D.; Herquel, B.; Malinka, ́ W.; Swiątek, P.; Akhtar, A.; Sippl, W.; Jung, M. Pyrido- and benzisothiazolones as inhibitors of histone acetyltransferases (HATs). MedChemComm 2014, 5, 1856−1862. (138) Carneiro, V. C.; de Abreu da Silva, I. C.; Torres, E. J.; Caby, S.; Lancelot, J.; Vanderstraete, M.; Furdas, S. D.; Jung, M.; Pierce, R. J.; Fantappie, M. R. Epigenetic changes modulate schistosome egg formation and are a novel target for reducing transmission of schistosomiasis. PLoS Pathog. 2014, 10, e1004116. (139) Gajer, J. M.; Furdas, S. D.; Grunder, A.; Gothwal, M.; Heinicke, U.; Keller, K.; Colland, F.; Fulda, S.; Pahl, H. L.; Fichtner, I.; Sippl, W.; Jung, M. Histone acetyltransferase inhibitors block neuroblastoma cell growth in vivo. Oncogenesis 2015, 4, e137. (140) Mai, A.; Rotili, D.; Tarantino, D.; Ornaghi, P.; Tosi, F.; Vicidomini, C.; Sbardella, G.; Nebbioso, A.; Miceli, M.; Altucci, L.; Filetici, P. Small-molecule inhibitors of histone acetyltransferase activity: identification and biological properties. J. Med. Chem. 2006, 49, 6897−6907. (141) Secci, D.; Carradori, S.; Bizzarri, B.; Bolasco, A.; Ballario, P.; Patramani, Z.; Fragapane, P.; Vernarecci, S.; Canzonetta, C.; Filetici, P. Synthesis of a novel series of thiazole-based histone acetyltransferase inhibitors. Bioorg. Med. Chem. 2014, 22, 1680−1689. (142) Trisciuoglio, D.; Ragazzoni, Y.; Pelosi, A.; Desideri, M.; Carradori, S.; Gabellini, C.; Maresca, G.; Nescatelli, R.; Secci, D.; Bolasco, A.; Bizzarri, B.; Cavaliere, C.; D’Agnano, I.; Filetici, P.; RicciVitiani, L.; Rizzo, M. G.; Del Bufalo, D. CPTH6, a thiazole derivative, induces histone hypoacetylation and apoptosis in human leukemia cells. Clin. Cancer Res. 2012, 18, 475−486. (143) Chimenti, F.; Bizzarri, B.; Maccioni, E.; Secci, D.; Bolasco, A.; Chimenti, P.; Fioravanti, R.; Granese, A.; Carradori, S.; Tosi, F.; Ballario, P.; Vernarecci, S.; Filetici, P. A novel histone acetyltransferase inhibitor modulating Gcn5 network: cyclopentylidene-[4-(4′chlorophenyl)thiazol-2-yl)hydrazone. J. Med. Chem. 2009, 52, 530− 536. (144) Biel, M.; Kretsovali, A.; Karatzali, E.; Papamatheakis, J.; Giannis, A. Design, synthesis, and biological evaluation of a smallmolecule inhibitor of the histone acetyltransferase Gcn5. Angew. Chem., Int. Ed. 2004, 43, 3974−3976. (145) Bowers, E. M.; Yan, G.; Mukherjee, C.; Orry, A.; Wang, L.; Holbert, M. A.; Crump, N. T.; Hazzalin, C. A.; Liszczak, G.; Yuan, H.; Larocca, C.; Saldanha, S. A.; Abagyan, R.; Sun, Y.; Meyers, D. J.; Marmorstein, R.; Mahadevan, L. C.; Alani, R. M.; Cole, P. A. Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor. Chem. Biol. 2010, 17, 471−482. (146) Shrimp, J. H.; Sorum, A. W.; Garlick, J. M.; Guasch, L.; Nicklaus, M. C.; Meier, J. L. Characterizing the Covalent Targets of a Small Molecule Inhibitor of the Lysine Acetyltransferase P300. ACS Med. Chem. Lett. 2015, DOI: 10.1021/acsmedchemlett.5b00385. (147) Dancy, B. M.; Crump, N. T.; Peterson, D. J.; Mukherjee, C.; Bowers, E. M.; Ahn, Y. H.; Yoshida, M.; Zhang, J.; Mahadevan, L. C.; Meyers, D. J.; Boeke, J. D.; Cole, P. A. Live-cell studies of p300/CBP histone acetyltransferase activity and inhibition. ChemBioChem 2012, 13, 2113−2121. (148) Gao, X. N.; Lin, J.; Ning, Q. Y.; Gao, L.; Yao, Y. S.; Zhou, J. H.; Li, Y. H.; Wang, L. L.; Yu, L. A histone acetyltransferase p300 inhibitor C646 induces cell cycle arrest and apoptosis selectively in AML1ETO-positive AML cells. PLoS One 2013, 8, e55481. (149) Yang, H.; Pinello, C. E.; Luo, J.; Li, D.; Wang, Y.; Zhao, L. Y.; Jahn, S. C.; Saldanha, S. A.; Planck, J.; Geary, K. R.; Ma, H.; Law, B. K.; Roush, W. R.; Hodder, P.; Liao, D. Small-molecule inhibitors of acetyltransferase p300 identified by high-throughput screening are potent anticancer agents. Mol. Cancer Ther. 2013, 12, 610−620. (150) Wu, J.; Wang, J.; Li, M.; Yang, Y.; Wang, B.; Zheng, Y. G. Small molecule inhibitors of histone acetyltransferase Tip60. Bioorg. Chem. 2011, 39, 53−58. (151) Coffey, K.; Blackburn, T. J.; Cook, S.; Golding, B. T.; Griffin, R. J.; Hardcastle, I. R.; Hewitt, L.; Huberman, K.; McNeill, H. V.; Newell, D. R.; Roche, C.; Ryan-Munden, C. A.; Watson, A.; Robson, C. N. Characterisation of a Tip60 specific inhibitor, NU9056, in prostate cancer. PLoS One 2012, 7, e45539. (152) Gao, C.; Bourke, E.; Scobie, M.; Famme, M. A.; Koolmeister, T.; Helleday, T.; Eriksson, L. A.; Lowndes, N. F.; Brown, J. A. Rational design and validation of a Tip60 histone acetyltransferase inhibitor. Sci. Rep. 2014, 4, 5372. (153) Majmudar, C. Y.; Hojfeldt, J. W.; Arevang, C. J.; Pomerantz, W. C.; Gagnon, J. K.; Schultz, P. J.; Cesa, L. C.; Doss, C. H.; Rowe, S. P.; Vasquez, V.; Tamayo-Castillo, G.; Cierpicki, T.; Brooks, C. L., 3rd; Sherman, D. H.; Mapp, A. K. Sekikaic acid and lobaric acid target a dynamic interface of the coactivator CBP/p300. Angew. Chem., Int. Ed. 2012, 51, 11258−11262. (154) Sachchidanand; Resnick-Silverman, L.; Yan, S.; Mutjaba, S.; Liu, W. J.; Zeng, L.; Manfredi, J. J.; Zhou, M. M. Target structurebased discovery of small molecules that block human p53 and CREB binding protein association. Chem. Biol. 2006, 13, 81−90. (155) Wang, Q.; Wang, R.; Zhang, B.; Zhang, S.; Zheng, Y.; Wang, Z. Small organic molecules targeting PCAF bromodomain as potent inhibitors of HIV-1 replication. MedChemComm 2013, 4, 737−740. (156) Borah, J. C.; Mujtaba, S.; Karakikes, I.; Zeng, L.; Muller, M.; Patel, J.; Moshkina, N.; Morohashi, K.; Zhang, W.; Gerona-Navarro, G.; Hajjar, R. J.; Zhou, M. M. A small molecule binding to the 1269 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270 Journal of Medicinal Chemistry Perspective coactivator CREB-binding protein blocks apoptosis in cardiomyocytes. Chem. Biol. 2011, 18, 531−541. (157) Rooney, T. P.; Filippakopoulos, P.; Fedorov, O.; Picaud, S.; Cortopassi, W. A.; Hay, D. A.; Martin, S.; Tumber, A.; Rogers, C. M.; Philpott, M.; Wang, M.; Thompson, A. L.; Heightman, T. D.; Pryde, D. C.; Cook, A.; Paton, R. S.; Muller, S.; Knapp, S.; Brennan, P. E.; Conway, S. J. A series of potent CREBBP bromodomain ligands reveals an induced-fit pocket stabilized by a cation-pi interaction. Angew. Chem., Int. Ed. 2014, 53, 6126−6130. (158) Hay, D. A.; Fedorov, O.; Martin, S.; Singleton, D. C.; Tallant, C.; Wells, C.; Picaud, S.; Philpott, M.; Monteiro, O. P.; Rogers, C. M.; Conway, S. J.; Rooney, T. P.; Tumber, A.; Yapp, C.; Filippakopoulos, P.; Bunnage, M. E.; Muller, S.; Knapp, S.; Schofield, C. J.; Brennan, P. E. Discovery and optimization of small-molecule ligands for the CBP/ p300 bromodomains. J. Am. Chem. Soc. 2014, 136, 9308−9319. (159) Picaud, S.; Fedorov, O.; Thanasopoulou, A.; Leonards, K.; Jones, K.; Meier, J.; Olzscha, H.; Monteiro, O.; Martin, S.; Philpott, M.; Tumber, A.; Filippakopoulos, P.; Yapp, C.; Wells, C.; Che, K. H.; Bannister, A.; Robson, S.; Kumar, U.; Parr, N.; Lee, K.; Lugo, D.; Jeffrey, P.; Taylor, S.; Vecellio, M. L.; Bountra, C.; Brennan, P. E.; O'Mahony, A.; Velichko, S.; Muller, S.; Hay, D.; Daniels, D. L.; Urh, M.; La Thangue, N. B.; Kouzarides, T.; Prinjha, R.; Schwaller, J.; Knapp, S. Generation of a selective small molecule inhibitor of the CBP/p300 bromodomain for leukemia therapy. Cancer Res. 2015, 75, 5106−5119. (160) Arrowsmith, C. H.; Bountra, C.; Fish, P. V.; Lee, K.; Schapira, M. Epigenetic protein families: a new frontier for drug discovery. Nat. Rev. Drug Discovery 2012, 11, 384−400. (161) Hilton, I. B.; D’Ippolito, A. M.; Vockley, C. M.; Thakore, P. I.; Crawford, G. E.; Reddy, T. E.; Gersbach, C. A. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 2015, 33, 510−517. (162) Mottamal, M.; Zheng, S.; Huang, T. L.; Wang, G. Histone deacetylase inhibitors in clinical studies as templates for new anticancer agents. Molecules 2015, 20, 3898−3941. (163) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.; Fedorov, O.; Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.; Philpott, M.; Munro, S.; McKeown, M. R.; Wang, Y.; Christie, A. L.; West, N.; Cameron, M. J.; Schwartz, B.; Heightman, T. D.; La Thangue, N.; French, C. A.; Wiest, O.; Kung, A. L.; Knapp, S.; Bradner, J. E. Selective inhibition of BET bromodomains. Nature 2010, 468, 1067−1073. (164) Brand, M.; Measures, A. M.; Wilson, B. G.; Cortopassi, W. A.; Alexander, R.; Hoss, M.; Hewings, D. S.; Rooney, T. P.; Paton, R. S.; Conway, S. J. Small molecule inhibitors of bromodomain-acetyl-lysine interactions. ACS Chem. Biol. 2015, 10, 22−39. (165) Andreotti, G.; Monticelli, M.; Cubellis, M. V. Looking for protein stabilizing drugs with thermal shift assay. Drug Test. Anal. 2015, 7, 831−834. (166) Jerabek-Willemsen, M.; Andre, T.; Wanner, R.; Roth, H. M.; Duhr, S.; Baaske, P.; Breitsprecher, D. MicroScale Thermophoresis: Interaction analysis and beyond. J. Mol. Struct. 2014, 1077, 101−113. (167) Baell, J. B.; Holloway, G. A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 2010, 53, 2719−2740. (168) Pan, Z.; Scheerens, H.; Li, S. J.; Schultz, B. E.; Sprengeler, P. A.; Burrill, L. C.; Mendonca, R. V.; Sweeney, M. D.; Scott, K. C.; Grothaus, P. G.; Jeffery, D. A.; Spoerke, J. M.; Honigberg, L. A.; Young, P. R.; Dalrymple, S. A.; Palmer, J. T. Discovery of selective irreversible inhibitors for Bruton’s tyrosine kinase. ChemMedChem 2007, 2, 58−61. (169) Arrowsmith, C. H.; Audia, J. E.; Austin, C.; Baell, J.; Bennett, J.; Blagg, J.; Bountra, C.; Brennan, P. E.; Brown, P. J.; Bunnage, M. E.; Buser-Doepner, C.; Campbell, R. M.; Carter, A. J.; Cohen, P.; Copeland, R. A.; Cravatt, B.; Dahlin, J. L.; Dhanak, D.; Edwards, A. M.; Frederiksen, M.; Frye, S. V.; Gray, N.; Grimshaw, C. E.; Hepworth, D.; Howe, T.; Huber, K. V.; Jin, J.; Knapp, S.; Kotz, J. D.; Kruger, R. G.; Lowe, D.; Mader, M. M.; Marsden, B.; Mueller-Fahrnow, A.; Muller, S.; O'Hagan, R. C.; Overington, J. P.; Owen, D. R.; Rosenberg, S. H.; Roth, B.; Ross, R.; Schapira, M.; Schreiber, S. L.; Shoichet, B.; Sundstrom, M.; Superti-Furga, G.; Taunton, J.; Toledo-Sherman, L.; Walpole, C.; Walters, M. A.; Willson, T. M.; Workman, P.; Young, R. N.; Zuercher, W. J. The promise and peril of chemical probes. Nat. Chem. Biol. 2015, 11, 536−541. (170) Scholz, C.; Weinert, B. T.; Wagner, S. A.; Beli, P.; Miyake, Y.; Qi, J.; Jensen, L. J.; Streicher, W.; McCarthy, A. R.; Westwood, N. J.; Lain, S.; Cox, J.; Matthias, P.; Mann, M.; Bradner, J. E.; Choudhary, C. Acetylation site specificities of lysine deacetylase inhibitors in human cells. Nat. Biotechnol. 2015, 33, 415−423. (171) Glozak, M. A.; Sengupta, N.; Zhang, X.; Seto, E. Acetylation and deacetylation of non-histone proteins. Gene 2005, 363, 15−23. (172) Singh, B. N.; Zhang, G. H.; Hwa, Y. L.; Li, J. P.; Dowdy, S. C.; Jiang, S. W. Nonhistone protein acetylation as cancer therapy targets. Expert Rev. Anticancer Ther. 2010, 10, 935−954. (173) Weinert, B. T.; Scholz, C.; Wagner, S. A.; Iesmantavicius, V.; Su, D.; Daniel, J. A.; Choudhary, C. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation. Cell Rep. 2013, 4, 842−851. (174) Baeza, J.; Smallegan, M. J.; Denu, J. M. Site-specific reactivity of nonenzymatic lysine acetylation. ACS Chem. Biol. 2015, 10, 122− 128. (175) Weinert, B. T.; Moustafa, T.; Iesmantavicius, V.; Zechner, R.; Choudhary, C. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions. EMBO J. 2015, 34, 2620−2632. 1270 DOI: 10.1021/acs.jmedchem.5b01502 J. Med. Chem. 2016, 59, 1249−1270