From the
Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore 560064, India,
Chemistry and Physics of Materials Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore 560064, India
Received for publication, February 13, 2003
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ABSTRACT |
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INTRODUCTION |
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A wide repertoire of transcriptional co-activator proteins is now recognized to possess histone acetyltransferase activity (1, 2). 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 (5, 6). 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 (7). 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 (8).
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 (4), 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 (9). 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 (10, 11).
Here we report that a small molecule compound anacardic acid from cashew nut shell liquid, known to have antitumor activity (12), 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.
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EXPERIMENTAL PROCEDURES |
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Synthesis of N-(4-Chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) (6) from Anacardic Acid (2)Anacardic acid 2 (molecular weight, 348) was treated with diethyl sulfate (16) in the presence of K2CO3 in acetone, which yielded diethyl ester 3. Diethyl ester on treatment with potassium t-butoxide (17) 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).
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X-ray Structure Determination of CTPBGood 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 scans using a Siemens three-circle diffractometer attached with a CCD area detector and a graphite monochromator for the MoK
radiation (50 kV, 40 mA). A hemisphere of reciprocal space was collected using the SMART software with 2
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 AssayHAT assays were performed as described elsewhere (13). 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 (15).
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Histone Deacetylase AssayDeacetylation 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 (10), 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 AssemblyA chromatin template for in vitro transcription experiments was assembled and characterized as described earlier (8)
In Vitro Transcription AssayTranscription assays were essentially carried out as described elsewhere (8) 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 (8). 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
-[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.
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RESULTS AND DISCUSSION |
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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).
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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 (16). 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 (19). 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 (8). 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 47 for AA and lanes 810 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 (8) 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 (10) 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.
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FOOTNOTES |
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* This work was supported by the Jawaharlal Nehru Center for Advanced Scientific Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
¶ To whom correspondence should be addressed: Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore 560064, India. Tel.: 0091-80-8462750 (ext. 2257); Fax: 0091-80-8462766; E-mail: tapas{at}jncasr.ac.in.
1 The 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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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