A Dual-Mode Fluorescence Strategy for Screening HAT Modulators

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A Dual-Mode Fluorescence Strategy for Screening HAT Modulators Nan Xie, Emilia N. Elangwe, Sabrina Asher, and Yujun George Zheng* Department of Chemistry, Georgia State University, PO Box 4098, Atlanta, Georgia 30302. Received October 27, 2008

Histone acetyltransferases (HATs) are an important class of epigenetic enzymes involved in chromatin restructuring and transcriptional regulation. We describe in this paper a novel approach for the identification and characterization of HAT inhibitors using both fluorescence resonance energy transfer (FRET) and fluorescence polarization. Expressed protein ligation (EPL) was used to label HATs PCAF and p300 with Dabcyl (Dab) as FRET acceptors. Methoxycoumarin (Mca) is conjugated to HAT substrate analogues to function as fluorescent donors, namely, H3CoA20Mca for interacting with PCAF and LysCoAMca for p300. When a ligand-protein interaction occurs, the fluorescent intensity of the donor fluorophore decreases due to FRET quenching by the Dab acceptor. Meanwhile, the formation of ligand-protein complexes causes reduction of the molecular mobility of the donor fluorophore, resulting in increased fluorescence anisotropy. Thus, dual modes of fluorescence measurement, FRET and anisotropy, are integrated in the same assay system. In particular, we demonstrated that both FRET and anisotropy measurements can be used to effectively detect and characterize HAT inhibitors. The developed strategy should be useful in the search of new anticancer drugs that target the substrate interfaces of the HAT targets, as well as find values in mechanistic study of HATs.

INTRODUCTION The genomes of eukaryotic organisms are tightly packed into chromatin complexes in the nucleus, which not only serve as structural scaffolds of the DNA duplexes, but also function as a dynamic regulator of various DNA-involved nuclear processes such as DNA transcription, replication, recombination, and damage repair (1, 2). The core histones in chromatin are subject to a diverse post-translational modification including acetylation, methylation, and phosphorylation, which has been determined to be an important molecular process that regulates the chromatin structure and gene expression (3, 4). As one of the key types of histone modifications, lysine acetylation is catalyzed by histone acetyltransferases (HATs), which elicit chromatin structural relaxation and gene transcriptional activation either by altering DNA-histoneinteractionsorbyattractingdownstreameffectors(5,6). HATs are grouped into several distinct families based on sequence and structural homology (7-11), which include Gcn5/ PCAF, p300/CBP, and the MYST family (named after its founding members, MOZ, YBF2/SAS3, SAS2, and Tip60) (12). These HAT proteins not only are essential to regulate gene expression in normal cellular processes (e.g., growth, differentiation, apoptosis), but expression and/or activity deregulation of many HATs have also been observed in a range of disease states, especially cancer (8, 13-17). It has increasingly become apparent that dysfunctional regulation of histone acetylation constitutes a significant mechanism for malignant diseases (8, 14, 15). For example, in prostate carcinoma, several HATs including PCAF, p300/CBP, and Tip60 are up-regulated and participate in the acetylation and activation of the androgen receptor and contribute to the pathway of hormone resistance (18, 19). Prostate cancer cells expressing acetylation mimic AR mutants were more resistant to androgen-antagonist flutamide (20). Thus, inhibition of deregulated HATs represents a promising approach to new cancer therapy. Although HDAC inhibitors have been extensively studied and several are in clinical trials (21-23), progress in developing low-molecular-weight inhibitors of HATs has been relatively * [email protected].

slow, partly because of the lack of effective experimental tools for inhibitor screening. Most of the reported small molecule HAT inhibitors to date, such as anacardic acid, garcinol, isothiazolone, curcumin, and cinnamoyl compounds, exhibit nonspecific inhibition and have limited usage in pharmacological settings (24). Therefore, new endeavors are needed to develop small molecule inhibitors of HATs. Currently, only limited approaches exist for HAT inhibitory study in a high-throughput format (25-29). The classical radioisotope-labeled assay is useful for enzymatic characterization of HATs but is difficult to be implemented to automation and high-throughput screening (HTS) (30). Spectrophotometric methods that rely on quantification of the product coenzyme A (CoA) have been reported (31-33). These coupled reactions contain additional components and are much more complex than a pure HAT assay, which greatly compromises their application. Immunoblotting protocols (e.g., TRF-Cellisas) that rely on antibodies recognizing acetyl-lysines are widely used to study histone acetylation and deacetylation and have also been proposed for inhibitor screening use (28, 29, 34, 35). Although the applicability of this type of assay to natural product screening with medium throughput has been demonstrated (36, 37), it suffers from several aspects of shortcomings: antibodies are expensive, the procedure involves extensive washing and is timeconsuming, and achieving quantification results is technically problematic. Also, a robust deconvolution scheme is additionally required to validate the action of hits identified in a cell-based screen. Therefore, certain reservation exists concerning their practical utility in a real HTS context (38). We recently developed a simple, single-step assay for direct readout of acetylation products via fluorescent intensity changes (39). To gain fluorescence signals adequate for use in inhibitor screening contexts, herein we present a new fluorescence strategy for the study of HAT-ligand interactions. Uniquely, we integrated two principles of photophysical measurement, namely, fluorescence resonance energy transfer (FRET) and fluorescence polarization/anisotropy, within one single system. In this approach, the HAT protein is expressed and chemoselectively labeled with an acceptor chromophore (quencher). On the other hand, a HAT-binding ligand (e.g., a modified substrate analogue)

10.1021/bc800467a CCC: $40.75  2009 American Chemical Society Published on Web 01/15/2009

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Scheme 1. Schematic Diagram of the Fluorescent Reporter System

Figure 1. Preparation of PCAF-Dab acceptor with expressed protein ligation strategy. (a) Scheme of ligation. (b) 15% SDS-PAGE of PCAF expression and ligation with peptide CSSK(Dabcyl)G. Lane 1, MW marker; 2, chitin beads before ligation; 3, chitin beads after ligation; 4-5, eluting fractions of the ligated product.

is chemically synthesized and labeled with a donor fluorophore. When the donor-labeled ligand is mixed with the acceptorlabeled HAT protein to form a complex, a FRET interaction occurs between the donor and the acceptor (e.g., coumarin and dabcyl), and thus, the fluorescence of the donor is quenched (Scheme 1). Along this process, the molecular mobility of the small-molecular-weight donor increases greatly due to complexation with the protein target. This allows for simultaneous fluorescence polarization and anisotropy readout. HAT inhibitors, on the other hand, have the potential to competitively displace the donor molecule from the enzyme, thus inversing the FRET and fluorescence anisotropy responses.

EXPERIMENTAL PROCEDURES Materials. Fmoc-protected amino acids, Boc-protected amino acids, Rink amide resin, and preloaded Wang resin were purchased from NovaBiochem. All the other chemical reagents were purchased from Sigma-Aldrich or Fisher and used without further purification. E. coli strains XL1-Blue, BL-21(DE3), and BL-21(DE3) Codonplus RIPL competent cells were obtained from Stratagene. Chitin beads were purchased from New England Biolab. Synthesis of Compounds. Solid-phase peptide synthesis (SPPS) was performed on a PS3 peptide synthesizer (Protein Technologies) using the Fmoc (N-(9-fluorenyl) methoxycarbonyl) strategy (40). All the reactions were performed at room temperature unless indicated otherwise. Removal of Fmoc was performed with 20% v/v piperidine/DMF. For the coupling of each amino acid (AA), 4 equiv of AA/HBTU/HOBt (Nhydroxybenzotriazole) were used. N-Methylmorpholine (NMM) was used as a base catalyst. The N-terminal amino group was acetylated with acetic anhydride unless indicated otherwise. After solid-phase synthesis, the resins were subsequently washed with DMF and dichloromethane and then dried in vacuum for at least 2 h before cleavage. Peptides were cleaved from resin by treatment with 95% trifluoroacetic acid (TFA), 2.5% H2O, and 2.5% triisopropylsilane, or with 94% TFA, 2.5% H2O, 2.5% ethanedithiol, and 1% triisopropylsilane if the sequences of peptides contain Cysteine(Trt), or with Reagent K (82.5% trifluoroacetic acid, 5% phenol, 5% H2O, 5% thioanisole, 2.5% ethanedithiol) if the sequences of peptides contain Arginine(Mtr) (handling of TFA must be performed in a secure hood) for 4 h. Cold diethyl ether was used to precipitate the products. Crude products were collected by centrifugation and were washed twice with cold diethyl ether. After lyophilization, the compounds were redissolved in water and purified with reverse-phase (RP) HPLC (C18, Varian) on a Varian Prostar HPLC system using linear gradients of H2O/0.05% TFA (solvent A) vs acetonitrile/0.05% TFA (solvent B). Analytical HPLC and MALDI-MS were used for characterization.

Cys-Ser-Ser-Lys(Dabcyl)-Gly (1). Fmoc-Lys(Dde)-OH, FmocSer-OH, Boc-Cys(Trt)-OH, and Fmoc-Gly-Wang resin were used in SPPS. The Dde (dimethyldioxocyclohexylidene) group was removed with a solution of 2% hydrazine in DMF for 2 h (41). For fluorescent labeling with the Dabcyl group, the resin was treated with 4 equiv of Dabcyl-OSu and 10 equiv of N-methylmorpholine in DMF for 4 h. The final compound was purified with preparative RP-HPLC (gradient 5-50% B over 60 min; flow 10 mL/min; absorption detection at 460 nm), and the structure was confirmed using mass spectrometry. MALDIMS: calcd. for C32H44N9O9S [M + 1] 731.3, found 732.3. H3(Br)20Mca (2) [Ac-K(Mca)RTKQTARKSTGGK(Br)APRKQL]. The protected sequence Ac-K(Mca)RTKQTARKSTGGK(Dde)APRKQL was synthesized on preloaded FmocLeu-Wang resin using SPPS protocol. After Dde removal, the resin was treated with 10 equiv of bromoacetic acid and 10 equiv diisopropylcarbodiimide (DIC) in a minimal volume of DMF for 4 h. The resins were subsequently washed with DMF and dichloromethane and then dried in vacuum for 2 h before cleavage. The compound was purified with preparative RPHPLC (gradient 5-35% B over 60 min; flow 10 mL/min; UV detection at 214 nm), and the structure was confirmed using mass spectrometry. MALDI-MS: calcd. for C110H185BrN36O33 [M + 1] 2620.3, found 2620.5. H3CoA20Mca (3). A mixture of 5.4 mg of H3(Br)20Mca and 4.7 mg of CoASH was dissolved in 100 µL of sodium phosphate buffer (100 mM, pH 8). The mixture was allowed to stand in the dark for 16 h and then purified with RP-HPLC (gradient 15-20% B over 60 min; flow 10 mL/min; UV detection at 214 nm), and the structure was confirmed using mass spectrometry. MALDI-MS: calcd. for C131H217N43O49P3S [M + Li+] 3309.5, found 3309.2. H3(Br)20 (4)[Ac-ARTKQTARKSTGGK(Br)APRKQL]. It was synthesized following the same procedure as for H3(Br)20Mca, but Fmoc-ALa-OH was employed in the place of FmocLys(Mca)-OH. The compound was purified with preparative RPHPLC (gradient 5-30% B over 60 min; flow 10 mL/min; UV detection at 214 nm), and the structure was confirmed using mass spectrometry. MALDI-MS: calcd. for C95H170BrN35O29 [M + 1] 2347.2, found 2347.1. H3CoA20 (5). 6.8 mg of H3(Br)20 and 6.4 mg of CoASH were dissolved in 100 µL of sodium phosphate buffer (100 mM, pH 8). The mixture was allowed to stand in the dark for 16 h and then purified with RP-HPLC (gradient 10-25% B over 60 min; flow 10 mL/min; UV detection at 214 nm), and the structure was confirmed using mass spectrometry. MALDI-MS: calcd. for C116H202N42O45P3S [M + Li+] 3036.4, found 3034.4. CMLVELHTQSQDRFK(Dabcyl)-NH2 (6). The sequence was synthesized on Rink amide resin using the general SPPS protocol. Following Dde removal, then, the resin was treated with 4 equiv of Dabcyl-OSu and 10 equiv of N-methylmorpholine in DMF for 4 h. It should be noted that the N-terminal cysteine needs to be protected with Boc prior to the Dde removal

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Figure 2. Chemical synthesis of the H3CoAMca donor.

Figure 3. FRET changes of H3CoAMca. (a) The emission spectral changes of H3CoAMca (0.2 µM) at different concentrations of PCAFDab (λex ) 326 nm). (b) The titration curve of H3CoAMca emission at 396 nm (0.2 µM) vs the concentrations of PCAFDab. (c) The spectral changes of H3CoAMca (0.2 µM) at increasing concentrations of the PCAF inhibitor H3CoA20 while fixing the concentration of PCAFDab at 12 µM (λex ) 326 nm). (d) The fluorescent intensity changes of H3CoAMca (0.2 µM) at 396 nm vs the concentrations of H3CoA20 (the concentration of PCAFDab is 12 µM).

with 2% hydrazine. The compound was purified with preparative RP-HPLC (gradient 10-60% B over 70 min; flow 10 mL/min; UV-vis detection at 460 nm), and the structure was confirmed using mass spectrometry. MALDI-MS: calcd. for C93H141N27O24S2 [M + 1] 2085.0, found 2085.1. Ac-Lys(Br)-Ahx-Lys(Mca)-NH2 (7). Fmoc-Lys(Mca)-OH, Fmoc-Ahx-OH, Fmoc-Lys(Dde)-OH, and acetic anhydride were sequentially coupled to Rink amide resin using the SPPS protocol. Then, the Dde group was removed, followed by bromoacetylation of the ε-amino group of the lysine. The resins were subsequently washed and then subjected to TFA cleavage. The compound was purified with preparative RP-HPLC (gradient 5-30% B over 60 min; flow 10 mL/min; UV detection at 214 nm), and the structure was confirmed using MALDI-MS: calcd. for C34H49BrN6O9 [M + Na+] 789.26, found 789.3.

Ac-Lys-CoA-Ahx-Lys(Mca)-NH2 (LysCoAMca) (8). A mixture of 2 mg of Ac-Lys(Br)-Ahx-Lys(Mca)-NH2 and 4.1 mg of CoASH was dissolved in 200 µL of sodium phosphate buffer (100 mM, pH 8) and 200 µL of acetonitrile. The mixture was allowed to stand in the dark for 16 h and then purified with RP-HPLC (gradient 15-35% B over 60 min; flow 10 mL/min; UV detection at 214 nm), and the structure was confirmed by MALDI-MS: calcd. for C55H81N13O25P3S [M + Li+] 1455.5, found 1458.3. Ac-Lys(Br)-NH2 (9). Fmoc-Lys(Dde)-OH and acetic anhydride were sequentially coupled to Rink amide resin using SPPS protocol. Following Dde removal with 2% hydrazine in DMF, bromoacetylation of the ε-amino group of the lysine was achieved by treating the resin with 10 equiv of bromoacetic acid and 10 equiv diisopropylcarbodiimide (DIC) in a minimal

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Figure 4. Use of fluorescence anisotropy for measurement of PCAF-ligand interaction. (a) Fluorescence anisotropy of H3CoAMca (0.2 µM) at 396 nm at different concentrations of PCAF-Dab. (b) Fluorescence anisotropy of the binary mixture (0.2 µM of H3CoAMca and 12 µM of PCAFDab) at 396 nm at different concentrations of the HAT inhibitor H3CoA20.

Figure 5. FRET changes of LysCoAMca. (a) The emission spectral changes of LysCoAMca (0.2 µM) at different concentrations of p300-Dabcyl (λex ) 326 nm). (b) The titration curve of LysCoAMca (0.2 µM) emission at 396 nm vs the concentrations of p300-Dabcyl. Kd is calculated to be 0.40 ( 0.06 µM. (c) The spectral changes of LysCoAMca (0.2 µM) at increasing concentrations of the p300 inhibitor LysCoA while fixing the concentration of p300Dab at 2.2 µM (λex ) 326 nm). (d) The fluorescent intensity changes of LysCoAMca (0.2 µM) at 396 nm vs the concentrations of LysCoA (the concentration of p300Dab is 2.2 µM). Ki of LysCoA is calculated to be 0.57 ( 0.07 µM.

Figure 6. Fluorescence anisotropy changes of LysCoAMca. (a) Titration of LysCoAMca (0.2 µM) with p300-Dab by fluorescence anisotropy (λex ) 326 nm, λem ) 396 nm). Kd is calculated to be 0.73 ( 0.15 µM. (b) Titration of the binary mixture (0.2 µM of LysCoAMca and 2.2 µM of p300Dab) with HAT inhibitor LysCoA by fluorescence anisotropy (λex ) 326 nm, λem ) 396 nm). Ki of LysCoA is calculated to be 1.92 ( 0.29 µM.

volume of DMF for 4 h. The resins were subsequently washed with DMF and dichloromethane and then dried in vacuum for 2 h before cleavage. The compound was purified with preparative RP-HPLC (gradient 5-15% B over 60 min; flow 10 mL/

min; UV detection at 214 nm). MALDI-MS: calcd. for C10H18BrN3O3 [M + Na+] 332.04, found 332.0. Ac-Lys-CoA-NH2 (LysCoA) (10). A mixture of 2 mg of AcLys(Br)-NH2 and 4.1 mg of CoASH was dissolved in 50 µL of

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sodium phosphate buffer (100 mM, pH 8) and 50 µL of methanol. The mixture was allowed to stand in the dark for 16 h and then purified with RP-HPLC (gradient 4-5% B over 60 min; flow 10 mL/min; UV detection at 214 nm), and the structure was confirmed using mass spectrometry. MALDI-MS: calcd. for C31H50N10O19P3S [M + Li+] 998.2, found 995.3. Expression of Dabcyl-Labeled PCAF Protein. The DNA construct encoding for the PCAF HAT domain (aa 493-658) in the PTYB2 vector was used for protein expression. In a typical procedure, to 50 µL of BL-21(DE3) competent cells was added 1 µL of the PCAF-pTYB2 plasmid and incubated on ice for 30 min. The tube was then heat shocked at 42 °C for 30 s and put on ice for 2 min. 0.5 mL of NZY solution was added to the cells and allowed to incubate while rotating at 37 °C, 225 rpm, for 1 h. The cells were spread to agar plates and distributed with glass-ball beads. The plates were then incubated overnight at 37 °C. One colony was selected for inoculation of 8 mL of LB media containing ampicillin. The solution was then allowed to incubate at 37 °C overnight with shaking at 225 rpm. The 8 mL cell culture was added to 1 L of LB media and cultured until the OD595 reached 0.6-0.8. Then, the temperature of the incubator was decreased to 16 °C at which point protein expression was induced with 0.3 mM of IPTG. The culture was kept in the incubator for 16-20 h. The cells were harvested by centrifugation at 5000 rpm, 4 °C, for 10 min. The cells were then resuspended in 25 mL of lysis buffer (Hepes 25 mM, pH 8.0, NaCl 500 mM, glycerol 10%, MgSO4 1 mM, PMSF 1 mM). The cells were lysed with a French Press twice, and the lysates immediately were centrifuged at 4 °C, 14,000 rpm for 30 min. The supernatant was collected and loaded to 3.0 mL of chitin beads. The beads were washed with 1× chitin column buffer (25 mM HEPES pH 8.0, 500 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, glycerol 10%, and 1 mM PMSF). After washing, 2 mM of the ligation peptide (CSSK(Dabcyl)G, 1) in 3 mL of ligation buffer (25 mM HEPES pH 8.0, 500 mM NaCl, 1 mM EDTA, glycerol 10%, 1 mM PMSF, and 200 mM MESNA) was added to the beads. The ligation reaction proceeded at room temperature for 40 h. After that, the ligated product was eluted, combined, and dialyzed against a storage buffer (25 mM HEPES pH 7.0, 250 mM NaCl, 1 mM EDTA, glycerol 10%, and 5 mM DTT) and then concentrated in a Millipore centrifuge filter tube. The protein concentration was determined by Bradford assay. The final labeled protein (PCAFDab) was aliquoted, flash-frozen with liquid N2, and stored at -80 °C until use. Expression of Dabcyl-Labeled p300 Protein. Labeling of p300 protein with Dacyl was achieved using the EPL protocol essentially the same as in the literature (33). First, the DNA sequence encoding for the p300 HAT domain (aa 1287-1652) was subcloned into PTYB2 vector (a gift from Prof. Phil Cole). BL-21(DE3) Codonplus RIL competent cells were used for protein expression. The expressed p300-intein-CBP fusion protein was loaded to chitin beads. After extensive washing, 6 mg of the ligation peptide (2. CMLVELHTQSQDRFK(Dabcyl)NH2) in 3 mL of ligation buffer (25 mM HEPES pH8.0, 250 mM NaCl, 1 mM EDTA, and 200 mM MESNA solution) was added to the beads. The ligation reaction was kept at room temperature for 48 h. The protein product was confirmed with 12% SDS-PAGE. The combined eluents were dialyzed against the storage buffer and then concentrated in a centrifuge filter tube. The concentration of labeled p300 protein (p300Dab) was determined by Bradford assay. Fluorescence Measurement. The fluorescence experiments were performed on a Fluoromax-4 instrument (Horiba Jobin Yvon, Edison, NJ) at 25 °C using a 0.5 mL cuvette in a buffer solution containing 50 mM Hepes (pH 8.0), 0.1 mM EDTA, 150 mM NaCl, and 1 mM DTT. The dissociation constants (Kd)

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were determined by measuring the fluorescence signals of Mcalabeled ligands (at fixed concentration) with increasing Dablabeled protein concentrations. fraction bound ) (F - Fo) ⁄ (Ff - Fo) ) {(Ff - Fo) ⁄ 2[Pr]tot}{b(b2 - 4[L]tot[Pr]tot)1⁄2} + Fo (1)

b ) Kd + [L]tot + [PCAF]tot Where F0 and Ff are the initial and final fluorescence intensities, respectively, [Pr]tot is the total protein concentration, and [L]tot is the total concentration of Mca-labeled ligand. The inhibition constants (Ki) were determined by measuring the fluorescence signals of the binary mixture containing Mcalabeled ligand and Dab-labeled protein (at fixed concentrations) with increasing concentrations of respective inhibitors. Equation 2 was used for Ki calculation (42). Ki ) [I]50 ⁄ ([L]50 ⁄ Kd + [Pr]o ⁄ Kd + 1)

(2)

where [I]50 denotes the concentration of the free inhibitor at 50% inhibition, [L]50 is the concentration of the free Mca-labeled ligand at 50% inhibition, [Pr]0 is the concentration of the free protein at 0% inhibition, and Kd is the dissociation constant of the protein-ligand complex.

RESULTS AND DISCUSSION PCAF Protein Expression and Ligation. To design the donor-acceptor reporter for HATs, it would be ideal if the HAT enzyme can be regioselectively labeled so that its active site will not be affected by the chemical labeling reaction. Mixing a protein sample with common labeling reagents (e.g., fluorescein succinimidyl ester) has a high chance of leading to random placement of introduced tags. To achieve the regioselectivity, we took advantage of expressed protein ligation (EPL), a technique widely used for site-specific protein engineering (43, 44). As a proof of principle, we first conducted fluorescent labeling of the HAT PCAF (Figure 1). The gene sequence encoding for the human PCAF (493-658) was subcloned into a PTYB2 expression vector. From this system, the recombinant protein was expressed in E. coli in the fusion form of PCAFintein-CBD (chitin binding domain). The CBD motif allows for purifying the fusion protein on chitin beads. After washing out impurities, the beads were treated with a ligation buffer containing peptide Cys-Ser-Ser-Lys(Dabcyl)-Gly, abbreviated as CSSK(Dab)G, and 200 mM of 2-mercaptoethanesulfonic acid (MESNA). The thiol treatment produced the PCAF protein with a thioester motif at its carboxyl terminus. In the course, PCAFthioester reacted with CSSK(Dabcyl)G via native chemical ligation (45) and formed the labeled PCAF-CSSK(Dabcyl)G product (PCAFDab). Design and Synthesis of the Fluorescent Donor Ligand H3CoAMca. PCAF was reported to acetylate the N-terminal tail of histone H3 (46). The bisubstrate analogue H3CoA20 in which a CoA motif is linked to the H3 N-terminal sequence at the acetylation lysine site (K14) has been shown as a potent inhibitor of PCAF/GCN5 (47). We modified the bisubstrate analogue H3CoA20 with a fluorophore, methoxycoumarin (Mca), to produce a fluorescent donor ligand (Figure 2). The Mca group is attached at the N-terminal end via a lysine residue, distant from the CoA-attaching site to avoid potential interference with the binding ability of the ligand to PCAF. FRET Response of the PCAF Reporter H3CoAMca. The Mca-labeled H3CoA20 conjugate (H3CoAMca) was characterized for its fluorescence properties. The compound was excited at 326 nm (extinction coefficient ε ) 11 820 M-1 cm-1) and exhibited a maximum emission at 396 nm (Figure 3). On the other hand, PCAFDab showed a broad absorption across

Screening HAT Modulators

360-500 nm (not shown). Therefore, excited-state energy of Mca can be effectively transferred to the Dab acceptor when the two chromophores are within the Fo¨rster distance. We tested fluorescence emission of H3CoAMca in the presence of PCAFDab. As expected, the fluorescent intensity of H3CoAMca (0.2 µM) gradually decreased as a function of PCAFDab concentration (Figure 3a,b). The maximum quenching was achieved when the concentration of PCAFDab reached 12 µM or higher. From the titration data, the apparent Kd was calculated to be 0.57 ( 0.06 µM. Next, we evaluated the performance of the donor-acceptor reporter for use in the detection of HAT inhibitors by monitoring FRET-mediated fluorescence intensity changes. A solution of a binary mixture was prepared containing 0.2 µM of H3CoAMca and 12 µM of PCAFDab. Then, an increasing amount of the nonfluorescent HAT inhibitor, H3CoA20, was added to the solution. As shown in Figure 3c,d, the fluorescent intensity increased in response to increasing amounts of H3CoA20. This matches well with our concept that the presence of a HAT inhibitor expels the H3CoAMca donor out of the binding pocket in PCAFDab, which will release the FRET interaction between the donor and the acceptor, thus causing fluorescence enhancement of the H3CoAMca donor. The Ki of H3CoA20 can be conveniently calculated from the competitive binding curve as 0.33 ( 0.03 µM, which is in close range to the value determined from the radioactive assay (0.14 µM) (48). Anisotropy Response of the PCAF Reporter. Compared to its free form, the H3CoAMca ligand in the PCAF complex possesses a larger molecular mass and more restricted molecular mobility, which will correspond to a larger fluorescence polarization and anisotropy. Therefore, the HAT binding assay can also be possibly realized using the mode of fluorescence polarization or anisotropy measurement. As shown in Figure 4a, the fluorescence anisotropy of H3CoAMca gradually increased when increasing amounts of PCAFDab were added to the solution. Kd was calculated to be 1.58 ( 0.22 µM from the titration curve. The value is slightly higher than the value obtained from the FRET measurement, which most likely reflects that not all proteins are labeled by Dab chromophore: FRET measurement is dependent on the Dab labeling, while anisotropy is not. We also tested the use of fluorescence anisotropy in the measurement of inhibitor binding (Figure 4b). A solution of binary mixture containing 0.2 µM of H3CoAMca and 12 µM of PCAF-Dab was prepared. When an increasing amount of the PCAF inhibitor H3CoA20 was added, the fluorescence anisotropy of H3CoAMca at 396 nm decreased concurrently. From the anisotropy assay, Ki of H3CoA20 was determined to be 0.83 ( 0.10 µM. These experiments substantiate that, when H3CoAMca is competitively displaced from binding to the large HAT protein by an inhibitor, its fluorescence anisotropy will decrease due to reduction in molecular mass and rigidity. Fluorescent Reporter of p300. To demonstrate whether the dual-mode strategy can be applied as a general approach to the study of other HAT proteins, we constructed a similar donor-acceptor assay and evaluated its performance for p300, another important HAT enzyme. Like the site-specific labeling of PCAF, p300 HAT domain (1287-1652) was expressed using a pTYB2 vector and then ligated to a C-terminal peptide CMLVELHTQSQDRFK(Dabcyl) (6), thus generating a protein fluorescent acceptor, p300Dab. To make a corresponding fluorescent donor ligand, an Mca-labeled compound, LysCoAMa, was designed on the basis of the reported p300 inhibitor, LysCoA (47). As anticipated, upon addition of p300Dab, the fluorescent intensity of LysCoAMca declined in a dose-dependent manner owing to FRET quenching interaction between Dab and Mca (Figure 5a,b). Again, the fluorescence intensity was recovered when the nonfluorescent ligand LysCoA was added to the solution, competitively displacing LysCoAMca

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out of the binding pocket in p300 (Figure 5c,d). We also performed fluorescence anisotropy measurements of LysCoAMca (Figure 6). In this regard, the performance of LysCoAMca was analogous to that of H3CoA20Mca; the anisotropy value increased upon binding to p300Dab protein, and the change was reversed by increasing concentrations of the competitive inhibitor LysCoA. Therefore, both FRET and anisotropy signals were effective to probe and characterize the p300-inhibitor interactions. Taken together, we demonstrated a displacement strategy for the study of HAT-inhibitor interactions. The assay is built on the binding between an acceptor-labeled HAT protein and a fluorescent donor-labeled ligand. The HAT protein (PCAF or p300) was chemoselectively labeled using the technique of EPL. FRET and fluorescence anisotropy were utilized as readout to identify and quantify ligand binding. This approach is fast, homogeneous, non-radioactive, and friendly to automation, thus holding promise as a platform for the discovery and characterization of HAT modulators. It should be noted that current screening equipments often harness capabilities to collect data in multiple modes, such as absorption, fluorescence intensity, polarization, FRET, and chemiluminescence, and so forth. Since FRET and polarization measurements are based on two totally different photophysical principles (the measurement of Mca polarization is dispensable to the Dab), availability of dual signals in a primary assay will allow for the generation of two independent data sets from one screening experiment, thus having a high efficacy in substantiating genuine hits and eliminating false positives. Apart from inhibitor discovery, this fluorescence strategy can also be used as a valuable tool to investigate binding kinetics of HAT substrate analogues as well as the enzymatic mechanisms of HAT catalysis.

ACKNOWLEDGMENT This work is supported by Georgia Cancer Coalition Distinguished Cancer Scholar Award and a Georgia State University Research Initiation Grant.

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