Small Molecule Modulators of Histone Acetyltransferase p300

Histone acetyltransferases (HATs) are a group of enzymes that play a significant role in the regulation of gene expression. These enzymes covalently modify the N-terminal lysine residues of histones by the addition of acetyl groups from acetyl-CoA. Dysfunction of these enzymes is often associated with the manifestation of several diseases, predominantly cancer. Here we report that anacardic acid from cashew nut shell liquid is a potent inhibitor of p300 and p300/CBP-associated factor histone acetyltranferase activities. Although it does not affect DNA transcription, HAT-dependent transcription from a chromatin template was strongly inhibited by anacardic acid. Furthermore, we describe the design and synthesis of an amide derivative N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) using anacardic acid as a synthon, which remarkably activates p300 HAT activity but not that of p300/CBP-associated factor. Although CTPB does not affect DNA transcription, it enhances the p300 HAT-dependent transcriptional activation from in vitro assembled chromatin template. However, it has no effect on histone deacetylase activity. These compounds would be useful as biological switching molecules for probing into the role of p300 in transcriptional studies and may also be useful as new chemical entities for the development of anticancer drugs. Histone acetyltransferases (HATs) are a group of enzymes that play a significant role in the regulation of gene expression. These enzymes covalently modify the N-terminal lysine residues of histones by the addition of acetyl groups from acetyl-CoA. Dysfunction of these enzymes is often associated with the manifestation of several diseases, predominantly cancer. Here we report that anacardic acid from cashew nut shell liquid is a potent inhibitor of p300 and p300/CBP-associated factor histone acetyltranferase activities. Although it does not affect DNA transcription, HAT-dependent transcription from a chromatin template was strongly inhibited by anacardic acid. Furthermore, we describe the design and synthesis of an amide derivative N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) using anacardic acid as a synthon, which remarkably activates p300 HAT activity but not that of p300/CBP-associated factor. Although CTPB does not affect DNA transcription, it enhances the p300 HAT-dependent transcriptional activation from in vitro assembled chromatin template. However, it has no effect on histone deacetylase activity. These compounds would be useful as biological switching molecules for probing into the role of p300 in transcriptional studies and may also be useful as new chemical entities for the development of anticancer drugs. The eukaryotic genome is organized as a highly complex nucleo-protein structure called chromatin, the unit of which is the nucleosome. The nucleosome is composed of two copies each of four different histones, H3, H2B, H2A, and H4, which is wrapped around by 146 base pairs of DNA. Therefore, for any process that requires access to the DNA (e.g. transcription, replication, recombination, and repair), the chromatin needs to be opened by the remodeling systems. There are two different biochemical processes to modify chromatin structure, namely the covalent modifications of histone tails and the ATP-dependent chromatin remodeling. Among the several covalent modifications of histones known, the reversible acetylation of key lysine residues in histones holds a pivotal position in transcriptional regulation (1Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1624) Google Scholar, 2Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64, 2: 435-459Crossref Scopus (1412) Google Scholar). Acetylation of histones is a distinctive feature of the transcriptionally active genes, whereas deacetylation indicates the repressed state of a gene (1Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1624) Google Scholar, 2Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64, 2: 435-459Crossref Scopus (1412) Google Scholar). A balance between the acetylation and deacetylation states of histones regulates transcription. Dysfunction of the enzymes involved in these events, the histone acetyltransferases (HATs) 1The abbreviations used are: HAT, histone acetyltransferase; HDAC, histone deacetylase; PCAF, p300/CBP-associated factor; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; AA, anacardic acid; CTPB, N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide. and histone deacetylases (HDACs), is often associated with the manifestation of cancer (3Redner R.L. Wang J. Liu J.M. Blood. 1999; 94, 2: 417-428Crossref Google Scholar). These enzymes thus become potential new targets for antineoplastic therapy (4Marks P.A. Rifkind R.A. Richon V.M. Breslow R. Miller T. Kelly W.K. Nat. Rev. Cancer. 2001; 1: 194-202Crossref PubMed Scopus (1693) Google Scholar). A wide repertoire of transcriptional co-activator proteins is now recognized to possess histone acetyltransferase activity (1Roth S.Y. Denu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1624) Google Scholar, 2Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64, 2: 435-459Crossref Scopus (1412) Google Scholar). These include p300/CBP-associated factor (PCAF), which is similar to GCN5, nuclear hormone receptor cofactors such as steroid receptor cofactor 1 (SRC1) and activator of thyroid and retinoid receptor, and the multifunctional p300/CBP. The p300/CBP is a global transcriptional coactivator, which plays a critical role in a variety of cellular process including cell cycle control, differentiation, and apoptosis. Mutations in p300/CBP are associated with different human cancers and other human diseases (5Giles R.H. Peters D.J. Breuning M.H. Trends Genet. 1998; 14: 178-183Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 6Murata T. Kurokawa R. Krones A. Tatsumi K. Ishii M. Taki T. Masuno M. Ohashi H. Yanagisawa M. Rosenfeld M.G. Glass C.K. Hayashi Y. Hum. Mol. Genet. 2001; 10: 1071-1076Crossref PubMed Scopus (136) Google Scholar). It is one of the most potent histone acetyltransferases, which can acetylate all four-core histones within nucleosomes as well as free histone forms. The HAT activity of p300 is regulated by several other factors. For example, the viral oncoprotein E1A binds to p300 and inhibits its activity, whereas phosphorylation of CBP by cyclin E/Cdk2 kinase activates its HAT activity (7Ait-Si-Ali S. Ramirez S. Barre F.X. Dkhissi F. Magnaghi-Jaulin L Girault J.A. Robin P. Knibiehler M. Pritchard L.L. Ducommun B. Trouche D. Harel-Bellan A. Nature. 1998; 396: 184-186Crossref PubMed Scopus (270) Google Scholar). During the process of transcription, p300 is recruited on to the chromatin template through the direct interaction with the activator and enhances the transcription by acetylation of promoter proximal nucleosomal histones (8Kundu T.K. Palhan V. Wang Z. An W. Cole P.A. Roeder R.G. Mol. Cell. 2000; 6: 551-561Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Although significant progress has been made in the field of histone deacetylase inhibitors as antineoplastic therapeutics, and some of the compounds are already in human trials (4Marks P.A. Rifkind R.A. Richon V.M. Breslow R. Miller T. Kelly W.K. Nat. Rev. Cancer. 2001; 1: 194-202Crossref PubMed Scopus (1693) Google Scholar), the reports of HAT inhibitors/activators are scanty. Prior to the molecular characterization of HAT enzymes, several polyamine-CoA conjugates were found to block HAT activity in cell extracts. However, the target enzyme(s) for these conjugates was not known (9Cullis P.M. Wolfeuden R. Cousens L. Alberts B.M. J. Biol. Chem. 1982; 257: 12165-12169Abstract Full Text PDF PubMed Google Scholar). Recently, two peptide-CoA conjugates, namely Lysyl CoA (Lys-CoA) and H3-CoA-20, were synthesized that specifically inhibit the HAT activity of p300 and PCAF, respectively (10Lau O.D. Kundu T.K. Soccio R.E. Ait-Si-Ali S. Khalil E.M. Vassilev A. Wolfe A.P. Nakatani Y. Roeder R.G. Cole P.A. Mol. Cell. 2000; : 589-595Abstract Full Text Full Text PDF PubMed Google Scholar, 11Costanzo A. Merlo P. Pediconi N. Fulco M. Sartorelli V. Cole P.A. Fontemaggi G. Fanciulli M. Schiltz L. Blandino G. Balsano C. Levrero M. Mol. Cell. 2002; 9: 175-186Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Here we report that a small molecule compound anacardic acid from cashew nut shell liquid, known to have antitumor activity (12Kubo I. Ochi M. Vieira P.C. Komatsu S. J. Agric. Food Chem. 1993; 41: 1012-1015Crossref Scopus (81) Google Scholar), inhibits HAT activity of p300 and PCAF. Surprisingly, the amide derivative of the same compound shows an enhancement of p300 HAT activity with human core histones and positive transcriptional coactivator, PC4, as substrates. Furthermore, since this compound does not induce the HAT activity of PCAF, it is p300-specific. It had no effect in HDAC1 activity. As expected, it enhanced the HAT-dependent transcriptional activation from chromatin template but did not show any effect on in vitro DNA transcription. This selective activator should be a very useful biological tool to understand the mechanisms of p300 function and may also introduce a novel group of compounds for antineoplastic therapeutics. Purification of Human Core Histones and Recombinant Proteins— Human core histones were purified from HeLa nuclear pellet as described previously (13Kundu T.K. Wang Z. Roeder R.G. Mol. Cell. Biol. 1999; 19: 1605-1615Crossref PubMed Scopus (107) Google Scholar). The FLAG epitope-tagged human topoisomerase I, histone deacetylase 1 (HDAC1), and PCAF were purified from the recombinant baculovirus-infected insect cell line, Sf21, by immunoaffinity purification using M2-agarose (Sigma) (14Wang Z. Roeder R.G. Genes Dev. 1997; 11: 1315-1326Crossref PubMed Scopus (129) Google Scholar). Full-length p300 was also purified from the recombinant baculovirus-infected Sf21 cells as a His6-tagged protein through the nickel-nitrilotriacetic acid affinity column (Qiagen) as described previously (13Kundu T.K. Wang Z. Roeder R.G. Mol. Cell. Biol. 1999; 19: 1605-1615Crossref PubMed Scopus (107) Google Scholar). The His6-tagged nucleosome assembly protein 1 (NAP1) used for the in vitro chromatin assembly was purified from Escherichia coli cells as reported previously (13Kundu T.K. Wang Z. Roeder R.G. Mol. Cell. Biol. 1999; 19: 1605-1615Crossref PubMed Scopus (107) Google Scholar), and the FLAG-tagged chimeric activator Gal4-VP16 was expressed in E. coli and purified by immunoaffinity purification with M2 agarose. Human positive transcriptional coactivator, PC4, was expressed in E. coli and purified as described earlier (15Prashanth Kumar B.R. Swaminathan V Banerjee S. Kundu T.K. J. Biol. Chem. 2001; 276: 16804-16809Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The peptide substrate, a 45-residue core histone H3 N-terminal peptide (N-CARTKQTARKSTGGKAPRQLASKAARKSAPSTGGVKKPHRYKPG-C), was synthesized and purified (by high performance liquid chromatography) in the Peptide Research Laboratory, Cancer Research, London, UK. Synthesis of N-(4-Chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) (6Murata T. Kurokawa R. Krones A. Tatsumi K. Ishii M. Taki T. Masuno M. Ohashi H. Yanagisawa M. Rosenfeld M.G. Glass C.K. Hayashi Y. Hum. Mol. Genet. 2001; 10: 1071-1076Crossref PubMed Scopus (136) Google Scholar) from Anacardic Acid (2Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64, 2: 435-459Crossref Scopus (1412) Google Scholar)—Anacardic acid 2 (molecular weight, 348) was treated with diethyl sulfate (16Kiong L.S. Tyman J.H.P. J. Chem. Soc. Perkin Trans. 1. 1981; : 1942-1952Crossref Google Scholar) in the presence of K2CO3 in acetone, which yielded diethyl ester 3. Diethyl ester on treatment with potassium t-butoxide (17Chang F.C. Wood N.F. Tetrahedron Lett. 1964; : 2969-2973Crossref Scopus (85) Google Scholar) in Me2SO yielded O-ethyl anacardic acid 4. The compound 4 on treatment with thionyl chloride in the presence of a catalytic amount of N,N-dimethylformamide yielded the corresponding acid chloride 5. The resultant acid chloride was taken for a condensation step without further purification. Compound 5 was condensed with 5-amino-2-chloro benzenetriflouride in dichloromethane in the presence of triethylamine as acid scavenger to yield compound (CTPB) 6 (molecular weight, 554) (see Fig. 3A). The formation of the derivatives was confirmed by IR and NMR spectroscopy (data not shown). X-ray Structure Determination of CTPB—Good quality, needle-shaped crystals of the CTPB (0.1 mm, 0.055 g), grown from a mixture of hexane and dichloromethane (2:1) by slow evaporation at room temperature, were chosen after examination under an optical microscope and coated with epoxy before mounting. X-ray diffraction intensities were measured by ω scans using a Siemens three-circle diffractometer attached with a CCD area detector and a graphite monochromator for the MoKα radiation (50 kV, 40 mA). A hemisphere of reciprocal space was collected using the SMART software with 2θ setting of the detector at 28°. Data reduction was performed using the SAINT program (Siemens). The phase problem was solved by direct methods, and the non-hydrogen atoms were refined anisotropically, by means of the full matrix least-squares procedure using the SHELXTL program (Siemens). All the hydrogen atoms were located using the difference Fourier method. The absolute structure of CTPB is shown in Fig. 3B. HAT Assay—HAT assays were performed as described elsewhere (13Kundu T.K. Wang Z. Roeder R.G. Mol. Cell. Biol. 1999; 19: 1605-1615Crossref PubMed Scopus (107) Google Scholar). Briefly, indicated amounts of proteins/peptide (see the figure legends) were incubated in HAT assay buffer containing 50 mm Tris-HCl, pH 8.0, 10% (v/v) glycerol, 1 mm dithiothreitol, 1 mm phenylmethyl sulfonyl fluoride, 0.1 mm EDTA, pH 8.0, 10 mm sodium butyrate at 30 °C for 10 min in the presence or absence of compound followed by the addition of 1 μl of 6.2 Ci/mmol [3H]acetyl coenzyme A (acetyl-CoA) and were further incubated for another 10 min. The final reaction volume was 30 μl. The reaction mixture was then blotted onto P-81 (Whatman) filter papers, and radioactive counts were recorded on a Wallac 1409 liquid scintillation counter. To characterize the inhibition kinetics of anacardic acid, filter binding assays were done using a constant amount of HeLa core histones in the presence or absence of AA with increasing concentrations of [3H]acetyl-CoA (see the legend for Fig. 2). To visualize the radiolabeled acetylated histones, the reaction mixtures were resolved on 15% SDS-PAGE and processed for fluorography as described elsewhere (15Prashanth Kumar B.R. Swaminathan V Banerjee S. Kundu T.K. J. Biol. Chem. 2001; 276: 16804-16809Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Histone Deacetylase Assay—Deacetylation assays were performed in the HAT assay buffer without sodium butyrate. 2 μg of core histones was incubated with 20 ng of p300 and 1 μl of 6.2 Ci/mmol [3H]acetyl-CoA for 15 min at 30 °C. The activity of p300 was inhibited by incubating the reaction mixture with 10 nm p300-HAT-specific inhibitor, Lysyl-CoA (10Lau O.D. Kundu T.K. Soccio R.E. Ait-Si-Ali S. Khalil E.M. Vassilev A. Wolfe A.P. Nakatani Y. Roeder R.G. Cole P.A. Mol. Cell. 2000; : 589-595Abstract Full Text Full Text PDF PubMed Google Scholar), for 10 min, after which 50 ng of HDAC1 was added, in the presence or absence of the compound, and incubated further for 45 min. The samples were analyzed by fluorography. In Vitro Chromatin Assembly—A chromatin template for in vitro transcription experiments was assembled and characterized as described earlier (8Kundu T.K. Palhan V. Wang Z. An W. Cole P.A. Roeder R.G. Mol. Cell. 2000; 6: 551-561Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) In Vitro Transcription Assay—Transcription assays were essentially carried out as described elsewhere (8Kundu T.K. Palhan V. Wang Z. An W. Cole P.A. Roeder R.G. Mol. Cell. 2000; 6: 551-561Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) with minor modifications. The scheme of transcription is enumerated in Fig. 5A. Briefly, 30 ng of DNA/equivalent amount of chromatin template was incubated with 30 ng of activator (Gal4-VP16) in a buffer containing 4 mm HEPES (pH 7.8), 20 mm KCl, 2 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, 10 mm sodium butyrate, 0.1 mg/ml bovine serum albumin, 2% glycerol (8Kundu T.K. Palhan V. Wang Z. An W. Cole P.A. Roeder R.G. Mol. Cell. 2000; 6: 551-561Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The compound CTPB was added to the acetylation reaction along with p300 and acetyl-CoA and incubated for 30 min at 30 °C. This was followed by addition of the p300-specific inhibitor Lysyl CoA (5 μm) to quench the acetylation reaction (see "Results and Discussion"). For AA, the HAT p300 was preincubated with indicated amounts of AA on ice for 20 min, following which it was added to the acetylation reaction in the transcription assay (see Fig. 5A, scheme). For the DNA transcription assays and chromatin transcription inhibition assays, the Lysyl CoA step was omitted. After acetylation, HeLa nuclear extract (5 μl, which contains ∼8 mg/ml protein) was added to initiate the preinitiation complex formation. Transcription reaction was started by the addition of NTP-mix and α-[32P]UTP after the preinitiation complex formation. The incubation was continued for 40 min at 30 °C. Transcription was terminated by the addition of 250 μl of stop buffer (20 mm Tris-HCl pH 8.0, 1 mm EDTA, 100 mm NaCl, 1% SDS, and 0.025 ng/μl tRNA). The 32P-radiolabeled transcript was extracted with phenol-chloroform, ethanol-precipitated, dried pellet-dissolved in loading dye (8 m urea, 0.005% bromphenol blue, and xylene cyanol), and analyzed on 5% urea-polyacrylamide gel. Gels were then dried and subjected to autoradiography at -70 °C. Quantification of transcription was done by Fuji BAS system. Quantitation of DNA and chromatin transcription data represents three independent experiments. There are several reports on the inhibitors of histone deacetylases (HDAC), whereas those regarding HAT inhibitors are scanty. Recently, two enzyme-specific HAT inhibitors have been synthesized that are peptide conjugates of acetyl-CoA, Lys-CoA for p300 and H3-CoA-20 for PCAF (10Lau O.D. Kundu T.K. Soccio R.E. Ait-Si-Ali S. Khalil E.M. Vassilev A. Wolfe A.P. Nakatani Y. Roeder R.G. Cole P.A. Mol. Cell. 2000; : 589-595Abstract Full Text Full Text PDF PubMed Google Scholar). However, there is no report yet regarding naturally occurring inhibitors of any of the HATs. We have screened the plant extracts known to possess anticancer properties. The extracts (both polar and nonpolar) were tested for their HAT inhibitory activity in filter binding assays (see "Experimental Procedures") using highly purified HeLa core histones (Fig. 1A) as a substrate- and baculovirus-expressed PCAF or p300, as the histone acetyltransferases (Fig. 1, C and D). This led us to find out that cashew nut shell liquid possessed inhibitory activity toward p300. The systematic bioactivity-guided fractionation of cashew nut shell liquid yielded an unsaturated anacardic acid mixture, namely the 8′Z-monoene, the 8′Z, 11′Z-diene, and the 8′Z, 11′Z, 14′Z-triene, which are the chief constituents (∼75%) of cashew nut-shell liquid (18Shobha S.V. Ravindranath B. J. Agric. Food Chem. 1991; 39: 2214-2217Crossref Scopus (47) Google Scholar), having maximum HAT inhibitory activity (data not shown). The hydrogenation of the unsaturated anacardic acids mixture yielded a single compound, anacardic acid (2-hydroxy-6-pentadecylbenzoic acid), showing an equally potent inhibitory activity toward p300 (Fig. 2A). These data indicated that the absence of unsaturation in anacardic acid did not alter its HAT inhibitory property. To further test the specificity and concentration dependence of inhibition, we compared the effect of a range of concentrations of anacardic acid on the HAT activities of p300 and PCAF. Since the compound was in Me2SO, we added an appropriate control in the HAT assays. The solvent does not produce any appreciable change in the HAT activities of either p300 or PCAF (Fig. 2A, bar 3 versus bar 2). Although at 5 μm concentration of anacardic acid, p300 showed a minimal enhancement in HAT activity, the same concentration of anacardic acid with PCAF showed an inhibition of 50% (Fig. 2A, bar 4 versus bar 3, see inset). Further increase in the concentration of anacardic acid produced a rapid inhibition of HAT activities of both p300 and PCAF, with 15 μm anacardic acid quenching HAT activity >90% (Fig. 2A, bar 6 versus bar 3). To visualize the anacardic acid-mediated inhibition of p300 HAT activity on different core histones, we analyzed the radiolabeled histones on SDS-PAGE followed by fluorography. The results were similar to the observation made in the filter binding assays. In the presence of 5 μm anacardic acid, there is an increase in the acetylation of histone H4 (Fig. 2B, lane 4). However, the overall acetylation remains the same as compared with acetylation in absence of the inhibitor. Further increase in the concentration of anacardic acid completely quenches the HAT activity (Fig. 2B, lane 7). The gel assay for PCAF shows a correlation with the filter binding assay (Fig. 2C), with higher concentrations of anacardic acid completely shutting off HAT activity (Fig. 2C, lane 7). The IC50 values of anacardic acid for p300 and PCAF were found to be ∼8.5 μm (data not shown) and ∼5 μm (Fig. 2A, bar 4), respectively. The filter binding assays were also repeated, using the human histone H3 N-terminal peptide as the substrate for p300 HAT to confirm the specificity of AA toward the HAT. Addition of AA to the reaction produced the characteristic inhibition of HAT activity (Fig. 2D), and the IC50 value for H3 peptide was found to be ∼500 nm (Fig. 2D, bar 7 versus bar 3). We went on to characterize the nature of inhibition of anacardic acid on the p300 HAT activity. The rate of the acetylation reaction at different concentrations of the inhibitors (and in its absence) was recorded with increasing concentrations of [3H]acetyl-CoA and a constant amount of core histones. The double reciprocal plot for each inhibitor concentration and in its absence (1/cpm versus 1/[acetyl-CoA]) was plotted as shown in Fig. 2E. The results suggest that AA is a non-competitive type of p300-HAT inhibitor. Due to the apparent lack of specificity toward HATs, we were interested in altering the various functional groups of anacardic acid, keeping the parent structure intact, to end up with a molecule with a better inhibitory effect or even selectivity. Since the side chain of the compound had already been negated from having any effect (no change in the HAT inhibition of unsaturated versus saturated anacardic acid), we modified the other functional groups on the phenolic ring in anacardic acid. The acidic group on the anacardic acid was modified to different amide derivatives using substituted anilides ("Experimental Procedures"). One of these compounds, with a 5-amino-2-chlorobenzotrifluoride moiety substituted on anacardic acid, CTPB (Fig. 3, A and B), when tested in an in vitro HAT assay (filter binding), surprisingly showed an enhancement in the p300 HAT activity while keeping the PCAF HAT activity mostly unperturbed (Fig. 4A). The concentration-dependent HAT activity profile revealed a maximum for p300 HAT activity at 275 μm CTPB (Fig. 4A, inset, bar 7c), an ∼4-fold increase over the Me2SO control (Fig. 4A, inset, lane 7c versus lane 3). Further increase in the concentration of CTPB to 300 μm resulted in a drop in the activation levels. These results were confirmed using the H3 peptide as a substrate in the HAT assays with p300 (data not shown). To analyze the effect of the compound on the acetylation of the individual core histones, the radiolabeled products of the HAT assays were separated on a 15% SDS-PAGE and visualized by fluorography. Addition of CTPB in increasing amounts in the presence of p300 resulted in the gradual increase in the acetylation of both histones H3 and H4, peaking at 200 μm (Fig. 4B, lane 7). In contrast to the filter binding assays, the enhancement in the gel assays is ∼5-fold more than the Me2SO control (Fig. 4B, lane 7 versus lane 3). Further increase in the concentration of CTPB brings down the HAT activity. A drop in the enhancement of HAT activity at higher concentrations of the activator, CTPB, a nonpolar compound, could be due to an alteration in the reaction condition affecting the efficiency of the HAT. In case of PCAF, there is a slight increase in the levels of H4 acetylation, whereas the H3 acetylation levels remain constant (Fig. 4C). This reflects the congruence between the filter binding and gel assay data. Despite using highly purified p300 and PCAF for our HAT assays, we went on to check the effect of CTPB on histone deacetylases, enzymes that catalyze the reverse reaction of HATs. This was done to ensure that CTPB does not affect other enzymes or the substrate (histones) in a nonspecific manner. The HDAC assays protocol was modified (as elaborated under "Experimental Procedures") to include acetylation of histones followed by the quenching of the HAT activity by the p300-selective HAT inhibitor, Lys-CoA, and the incubation with the purified recombinant human HDAC1 (Fig. 1B), either in the presence or in the absence of CTPB. Deacetylation of the core histones in the presence or absence of the compound, CTPB at 100 or 500 μm, shows no difference whatsoever (Fig. 4D, lanes 5 and 6 versus lane 3). Preincubation of HDAC1 with CTPB does not affect its deacetylation activity (data not shown). Interestingly, addition of CTPB, after quenching the p300 HAT activity by Lys-CoA, does not show the characteristic enhancement (Fig. 4D, lane 7 versus lane 2). Addition of the solvent, Me2SO, has a slight inhibitory effect on the deacetylase activity of HDAC1 (Fig. 4D, lane 4 versus lane 3). These results indicate the specific nature of CTPB toward p300. To confirm that the target of CTPB is p300, we have used a nonhistone substrate, human transcription coactivator PC4, for p300 acetylation. PC4 is acetylated specifically by p300 (16Kiong L.S. Tyman J.H.P. J. Chem. Soc. Perkin Trans. 1. 1981; : 1942-1952Crossref Google Scholar). The addition of CTPB (100 μm) enhances PC4 acetylation by p300 (Fig. 4E, lane 5 versus lane 3) substantially. A further increase in the concentration of CTPB produces a drop in the enhancement of PC4 acetylation (data not shown), as observed in the case of the histones. Taken together, these results indicate that the probable target of CTPB is the enzyme p300. p300, along with its homologue CBP, is known to be a prominent transcription coactivator, capable of interacting with a large number of transcriptional activators possessing HAT activity. Acetylation of histones is a distinctive feature of active genes (19Allfrey V. Faulkner R.M. Mirsky. A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Scopus (1787) Google Scholar). It has been conclusively proved that the acetylation of the promoter proximal histones by p300 is necessary and sufficient for the initiation of transcription. Thus, modulators of the HAT activity of p300 can be applied in the study of transcriptional regulation. To address the effect of CTPB on transcription from a chromatin template, we used the in vitro chromatin-based transcription system (8Kundu T.K. Palhan V. Wang Z. An W. Cole P.A. Roeder R.G. Mol. Cell. 2000; 6: 551-561Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). This system requires the HAT activity of p300 for the initiation of transcription (Fig. 5A). Such a system would be ideal for testing CTPB. To establish the HAT-specific nature of our compound, we first tested its effect on transcription from a histone-free DNA template. This system does not require p300 HAT activity for transcriptional initiation (Fig. 5B). Addition of the solvent, Me2SO, to this assay produces a drop in transcript levels (Fig. 5B, lane 3 versus lane 2). However, the addition of increasing concentration of either AA or CTPB does not produce any variation in the transcript levels as compared with the Me2SO control (Fig. 5B, lanes 4–7 for AA and lanes 8–10 for CTPB versus lane 3, and see also the legend for Fig. 5). This suggests that the compounds do not affect any component of the basal transcription machinery. The drop in transcript levels upon addition of Me2SO may be due to the disruption of certain key protein-protein interactions. We went on to test the effect of the compound CTPB on HAT-dependent transcription from a chromatin template. The template pG5ML-array (8Kundu T.K. Palhan V. Wang Z. An W. Cole P.A. Roeder R.G. Mol. Cell. 2000; 6: 551-561Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) was assembled into chromatin by using the NAP1-mediated assembly method ("Experimental Procedures"). Addition of CTPB to the HAT-dependent transcription reaction along with the p300 and acetyl-CoA (without addition of Lys-CoA) did not produce a significant variation in the transcript levels with or without the compound (data not shown). A close scrutiny of the transcription assay scheme revealed that the HAT activity of p300 remains active throughout the assay period, within which time the promoter proximal histones could be completely acetylated. To characterize a HAT activator, it would be necessary to limit the period of acetylation to a small window. We achieved this by adding the p300-specific inhibitor Lys-CoA (10Lau O.D. Kundu T.K. Soccio R.E. Ait-Si-Ali S. Khalil E.M. Vassilev A. Wolfe A.P. Nakatani Y. Roeder R.G. Cole P.A. Mol. Cell. 2000; : 589-595Abstract Full Text Full Text PDF PubMed Google Scholar) after allowing for 30 min of acetylation either in the presence or in the absence of the compound. Under these conditions, we found that the addition of Me2SO produced a slight drop in the transcript levels (Fig. 5C, lane 5 versus lane 4), whereas the addition of CTPB enhanced the levels of transcription 1.6-fold over the Me2SO control (Fig. 5C, lane 6 versus lane 5). Thus, this result indicates that CTPB specifically enhances the HAT activity of p300, a function that is reflected even at the transcriptional level. To explain the 1.6-fold increase in transcription levels, in contrast to the ∼5-fold increase in the histone acetylation levels, we carried out a time course experiment to analyze the effect of CTPB on histone acetylation over a 30-min time period. We used Me2SO-treated p300 acetylation reactions as the control on which the activation levels were calculated (Fig. 5D). Since the histone concentration remains constant, the difference in the levels of histone acetylation drops over time. After a 30-min incubation, the difference stands at 1.6-fold (Fig. 5D, bar 4), the same as what we observe in the transcription assay. Anacardic acid did not affect the transcription from the DNA template, but the HAT-dependent transcription from chromatin template was inhibited by anacardic acid even at 10 μm concentration (Fig. 5C, lane 10 versus lane 9). We have identified a natural compound that can broadly inhibit the HAT activity but not any other enzymatic activity as revealed by the DNA transcription. Although anacardic acid is not specific for any particular group of HATs, it may serve as a lead compound to synthesize other non-peptide-based specific HAT activity modulators. The most significant finding of this study is the synthesis of a specific activator of p300 HAT activity, CTPB, using anacardic acid as a synthon. The enhancement of p300 HAT activity by CTPB is also reflected at the transcriptional level, where acetylation of histones in the promoter proximal region dictates transcription initiation. Further investigation of the effect of CTPB on in vivo histone acetylation and the functions thereof should be studied to understand the mechanism of action of the compound. This information would be very useful to design a novel group of antineoplastic drugs targeted toward histone acetyltransferases. We thank Drs. Robert G. Roeder, Philip A. Cole, James T. Kadonaga, and Alain Verreault for providing invaluable reagents. We thank Profs. C. N. R. Rao and G. U. Kulkarni for providing facilities for synthesis and x-ray crystal analysis of the compound. We acknowledge John Thomas for help during the synthesis.