Small molecule modulators of histone acetyltransferase p300

The activity of inhibited by incubating the reaction mixture with 10 nM p300-HAT specific inhibitor, Lysyl-CoA 10 min after which 50 ng of added, in the presence or absence of the compound, and incubated further 45 min. The samples were analysed by flourography.


Introduction
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 histones 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 (1,2). Acetylation of histones is a distinctive feature of the transcriptionally active genes, whereas deacetylation indicates the repressed state of a gene (1,2). A balance between the acetylation and deacetylation states of histones regulates transcription. Dysfunction of the enzymes involved in these events, the histone acetyltransferases (HATs) and histone deacetylases (HDACs) is often associated with the manifestation of cancer (3). These enzymes thus become potential new targets for antineoplastic therapy (4).
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 (ACTR) 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 acetyltransferase, 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).
Though a significant progress has been made in the field of histone deacetylase inhibitors as antineoplastic therapeutics, some of the compounds are already in human trials (4); the reports by guest on March 22, 2020 http://www.jbc.org/ Downloaded from mounting. X-ray diffraction intensities were measured by ω scans using a Siemens threecircle 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, USA, 1995). The phase problem was solved by direct methods and the non-hydrogen atoms were refined anisotropically, by means of the fullmatrix least-squares procedure using the SHELXTL program (Siemens, USA, 1995). All the hydrogen atoms were located using the difference Fourier method. The absolute structure of CTPB is shown in Figure 3B.
HAT Assay: HAT assays were performed as described elsewhere (13). Briefly, indicated amounts of proteins/peptide (see 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 presence or absence of compound followed by the addition of 1 µl of 6.2 Ci/mmol [ 3 H]acetyl Coenzyme A (acetyl-CoA) and were further incubated for another 10 min. The final reaction volume was 30µl. The reaction mixture was then blotted onto P-81 (Whatman) filter papers and radioactive counts were recorded on a Wallac 1409 liquid scintillation counter. In order to characterize the inhibition kinetics of anacardic acid, filter-binding assays were done using constant amount of HeLa core histones in the presence or absence of AA with increasing concentrations of [ 3 H]-acetyl CoA (see Figure Legends, 2E). To visualize the radiolabeled acetylated histones, the reaction mixtures were resolved on 15% SDS-PAGE and processed for fluorography as described elsewhere (15). 7 In Vitro Chromatin assembly: Chromatin template for in vitro transcription experiments was assembled and characterized as described earlier (8) In Vitro Transcription Assay: Transcription assays were essentially carried out as described elsewhere (8), with minor modifications. The scheme of transcription is enumerated in Figure   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 DTT, 0.2 mM PMSF, 10 mM sodium butyrate, 0.1 mg/ml BSA, 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 o 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 scheme, Figure 5A). 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 pre-initiation complex formation.

RESULTS AND DISSCUSSION
There are several reports on the inhibitors of histone deacetylases (HDAC), while that regarding HAT inhibitors are scanty. Recently, two enzyme specific HAT inhibitors have been synthesized, which are peptide conjugates of acetyl-CoA, Lys-CoA for p300 and H3-CoA-20 for PCAF (10).
However there is no report yet regarding naturally occurring inhibitors of any of the HATs. We have screened the plant extracts known to possess anti-cancer 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 (Figure 1 A)  This led us to find out that cashew nut shell liquid (CNSL) possessed inhibitory activity towards p300. The systematic bio-activity guided fractionation of CNSL yielded unsaturated anacardic acids 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 nutshell liquid (18), having maximum HAT inhibitory activity (data not shown). The hydrogenation of unsaturated anacardic acids mixture yielded a single compound, Anacardic acid (2-hydroxy-6-pentadecylbenzoic acid) showing an equally potent inhibitory activity towards p300 ( Figure 2A). This data indicated that 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 DMSO, 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 ( . The IC 50 values of anacardic acid for p300 and PCAF was found to be ~8.5 µM (data not shown) and ~5 µM (Figure 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 towards the HAT. Addition of AA to the reaction produced the characteristic inhibition of HAT activity ( Figure 2D) and IC 50 value for H3 peptide was found to be ~500 nM ( Figure 2D was plotted as shown in Figure 2E. The results suggest that AA is a non-competitive type of p300-HAT inhibitor. Due to the apparent lack of specificity towards HATs, we were interested to alter the various functional groups of anacardic acid, keeping the parent structure intact, to end up in 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, ( Figure 3A and B) when tested in vitro HAT assay (filter binding), surprisingly showed an enhancement in the p300 HAT activity, while keeping the PCAF HAT activity mostly unperturbed ( Figure 4A). The concentration dependent HAT activity profile revealed a maxima for p300 HAT activity at 275 µM of CTPB ( Figure 4A, inset, bar 7c); an ~4 fold increase over the DMSO control ( Figure 4A inset, lane 7c versus lane 3). Further increase in the concentration of CTPB to 300µM 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).  Figure 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 in order to ensure that CTPB does not affect other enzymes or the substrate (histones) in a non-specific manner. The HDAC assays protocol was modified (as elaborated in These results indicate the specific nature of CTPB towards p300. In order to confirm that the target of CTPB is p300, we have used a non-histone substrate, human transcription coactivator PC4, for p300 acetylation. PC4 is acetylated specifically by p300 (16).
The addition of CTPB (100µM) enhances PC4 acetylation by p300 ( Figure 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. In order 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 ( Figure 5A). Such a system would be ideal for testing CTPB. In order 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 ( Figure 5B). Addition of the solvent, DMSO, to this assay, produces a drop in transcript levels  Figure   Legends). This suggests that the compounds do not affect any component of the basal transcription machinery. The drop in transcript levels upon addition of DMSO 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 pG 5 ML-array (8) was assembled into chromatin 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. In order 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)  this result indicates that CTPB specifically enhances the HAT activity of p300, a function that is reflected even at the transcriptional level. In order 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 DMSO treated p300 acetylation reactions as the control on which the activation levels were calculated ( Figure 5D). Since the histone concentration remains constant, the difference in the levels of histone acetylation drops over time. After a 30-minute incubation, the difference stands at 1.6-fold ( Figure 5D, bar 4), 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 10µM concentration ( Figure 5C, lane 10 versus lane 9).
We have identified a natural compound, which can broadly inhibit the HAT activity but not any other enzymatic activity as revealed by the DNA transcription. Though 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 in in vivo histone acetylation and the functions thereof should be studied in order to understand the mechanism of action of the compound. This information would be very useful in order to design a novel group of antineoplastic drugs targeted towards histone acetyltranferases.    Histone acetyltransferase assays were performed in the presence and absence of CTPB using highly purified HeLa core histones (800 ng) and either with p300 (5 ng) or PCAF (15 ng) and