The crystal structure of p300 HAT in complex with bisubstrate analog Lys-CoA (pdb: 3BIY) was prepared for virtual screening using the Internal Coordinate Mechanics (ICM-Pro) software version 3.5 (MolSoft LLC, San Diego CA) (Abagyan et al., 2005; Abagyan et al., 1994
). The ligand and heteroatom molecules were removed from the structure and a continuous dielectric was used in place of the water molecules. Missing hydrogen and heavy atoms were added and atom types and partial charges were assigned. The protein model was adjusted so that the optimal positions of polar hydrogens were identified, the most isomeric form of each histidine was assigned and the correct orientation of the side-chains of glutamine and asparagine were found. Steric clashes were removed by an energy minimization procedure.
Virtual screening was undertaken with the ICM-VLS software version 3.5 (MolSoft LLC, San Diego CA) using dockScan version 4.4. This method uses an extension of the Empirical Conformational Energy Program for Peptides 3 (ECEPP/3) (Nemethy et al., 1992
) force field parameters for proteins and the Merck Molecular Force Field (MMFF) (Halgren and Nachbar, 1996
) for small molecules. Five continuously differentiable potential grid maps were calculated for the receptor using spline interpolation for efficient gradient minimization (Totrov and Abagyan, 1997
). These maps include energy terms for electrostatics, directional hydrogen bond, hydrophobic interactions, and two for Van der Waals interactions for steric repulsion and dispersion attraction including a soft potential to limit the effect of minor steric clashes. A collection of 492,793 compounds from the ChemBridge small molecule database (ChemBridge Corp, San Diego CA) was screened to the p300 HAT bisubstrate inhibitor binding site. Each ligand was docked into the binding pocket three times. During docking the ligand is flexible and its position and internal torsions are sampled using the ICM biased probability Monte Carlo procedure which includes a local minimization after each random move. Each docked ligand is assigned a score according to the weighted components of the ICM-VLS scoring function (Totrov and Abagyan, 1999
Purification of semisynthetic p300 HAT domain
A variant of the HAT domain of p300 (residues 1287 to 1652) was expressed as a fusion with the VMA intein chitin binding domain as described previously (Thompson et al., 2004
). Residues 1529-1560, comprising the regulatory autoacetylation loop, were deleted in the construct, rendering the enzyme constitutively active. The construct also contained an M1652G mutation. E. coli
BL21(RIL)-DE3 cells containing the construct were grown to OD600
of 0.6 at 37°. The incubator was cooled to 16°C, and expression was induced by addition of 500 μM IPTG. Following overnight expression, the cells were centrifuged and resuspended in lysis buffer prior to lysis via
three passes through a French pressure cell. Lysates were clarified through centrifugation and incubated with chitin resin for 30 min at 4°C. The resin was washed thoroughly before addition of 200 mM MESNA and a C-terminal peptide corresponding to residues 1653-1666 of p300. The expressed protein ligation reaction was allowed to proceed for 16 h followed by purification over a MonoS 5/50 GL strong cation exchange column (GE Healthcare) using linear gradients of NaCl (50 to 1000 mM). Purified semisynthetic p300 was concentrated and dialyzed against 20 mM HEPES, pH 7.9, 50 mM NaCl, 1 mM DTT, and 10% glycerol (v/v) prior to flash-freezing in liquid N2
and storage at -80°C. Protein concentration was determined by gel and by Bradford assay using BSA as a standard.
Initial screen of VLS hits
The top 194 p300 HAT inhibitor candidates identified by VLS were screened using a coupled spectrophotometric assay. In this assay, CoASH produced by the p300 reaction is used by α-ketoglutarate dehydrogenase (α-KGDH) to produce NADH, which can be monitored spectrophotometrically at 340 nm (Kim et al., 2000
). Reactions were performed at 30°C in 1 M HEPES, pH 7.9, and contained 200 μM H4-15, 200 μM TPP, 5 mM MgCl2
, 1 mM DTT, 50 μg/mL BSA, 200 μM NAD, 2.4 mM α-ketoglutarate, 200 μM acetyl-CoA, 0.1 units α-KGDH, and 100 nM p300. DMSO was kept at a constant 3.3%, and candidate compounds were screened at 100 μM. Reaction mixtures were incubated at 30°C for 10 min prior to initiation. Reactions were initiated with addition of H4-15 and followed over the linear portion of the progress curve, which provides the initial velocity via
linear regression. Percent inhibition was determined by comparison with velocity without candidate added. Compounds that exhibited over 40% inhibition were subjected to further screening steps. To ensure that compounds were not inhibiting α-KGDH instead of p300, compounds were assayed with 0.2 units of α-KGDH, two times the amount used in the initial screen. To ensure that compounds were not inhibiting through nonspecific aggregation, compounds were assayed in the presence of 0.01% Triton X-100. Compounds whose inhibition was greatly decreased either by raising the α-KGDH concentration or by preventing nonspecific aggregation were removed from further consideration.
Compounds that passed the iterative verification process in the initial screen were then tested in a direct radioactive assay. In this assay, production of 14
C-labeled Ac-H4-15 is monitored electrophoretically (Thompson et al., 2001
). Reactions were performed in 20 mM HEPES, pH 7.9, and contained 5 mM DTT, 80 μM EDTA, 40 μg/mL BSA, 100 μM H4-15, and 5 nM p300. DMSO was kept constant at 2.5%, and inhibitors were screened at 25 μM. Reactions were incubated at 30°C for 10 minutes, initiated with addition of a 1:1 mixture of 12
C-acetyl-CoA and 14
C-acetyl-CoA to a final concentration of 20 μM, and allowed to run for 10 min at 30°C. Reactions are then quenched with addition of 14% SDS (w/v). Turnover was kept below 10%. All compounds were screened in duplicate. Samples were then loaded onto a 16% Tris-Tricine gel along with a BSA standard and run at 140 V for 90 minutes. Gels were washed and dried, and exposed in a PhosphorImager cassette for ~2 days. Bands corresponding to Ac-H4-15 were then quantified using ImageQuant. Compounds exhibiting over 40% inhibition compared to control were then kinetically characterized.
Kinetic characterization of verified inhibitors and analogs
IC50 values for the putative p300 HAT inhibitors identified through the initial screen were determined using the direct radioactive assay described above. Reactions were performed in 20 mM HEPES, pH 7.9, and contained 5 mM DTT, 80 μM EDTA, 40 μg/mL BSA, 100 μM H4-15, and 5 nM p300. Putative inhibitors were added over a range of concentrations, with DMSO concentration kept constant (<5%). Reactions were incubated at 30° for 10 minutes, then initiated with addition of a 1:1 mixture of 12C-acetyl-CoA and 14C-acetyl-CoA to 20 μM. After 10 min at 30°C, reactions were quenched with 14% SDS (w/v). All concentrations were screened in duplicate. Gels were run, washed, dried, and exposed to a PhosphorImager plate as described above, and production of Ac-H4-15 quantified to obtain IC50s.
Patterns of inhibition of putative p300 HAT inhibitors were determined in a similar fashion. One substrate was held constant while the other was varied over a range of three inhibitor concentrations (0, 0.5xIC50
, and IC50
). AcCoA was varied from 5-120 μM while holding H4-15 constant at 100 μM, and H4-15 was varied from 25-500 μM while holding acetyl-CoA constant at 10 μM. Reactions were performed in duplicate as described above; enzyme concentration and reaction time were varied to keep turnover below 10%. Following quantification, data were globally fit to equations for competitive or noncompetitive inhibition to determine the optimal pattern of inhibition (Copeland, 2000
Determining inhibitor specificity for p300 HAT
C646, C375, and C146 were screened spectrophotometrically against PCAF (p300/CBP-associated factor, a histone acetyltransferase) and AANAT (arylalkylamine N
-acetyltransferase, a non-histone acetyltransferase) using a similar coupled assay as described above. PCAF reactions were performed in 100 mM HEPES, pH 7.9, and contained 200 μM TPP, 5 mM MgCl2
, 1 mM DTT, 50 mM NaCl, 0.05 mg/mL BSA, 200 μM NAD, 2.4 mM α-KG, 30 μM acetyl-CoA, 0.037 units α-KGDH, 3.3% DMSO, and 10 μM inhibitor. Reactions took place at 30°C. Reactions were initiated by addition of 10 nM PCAF and were followed at 340 nm over the linear portion of the curve below 10% turnover. AANAT reactions were performed in 100 mM NH4
OAc, pH 6.8, and contained 200 μM TPP, 5 mM MgCl2
, 1 mM DTT, 50 mM NaCl, 0.05 mg/mL BSA, 200 μM NAD, 2.4 mM α-KG, 200 μM AcCoA, 0.1 units α-KGDH, 3.3% DMSO, and 10 μM inhibitor. Reactions took place at 25°. Reactions were initiated with addition of 65 nM AANAT, and followed at 340 nm, as above. Percent inhibition values were compared to those with p300, which were repeated using the protocol given above. C646 was further analyzed as a potential HAT inhibitor with yeast GCN5, the Sas2/4/5 complex, MOZ, and Rtt109. Yeast GCN5, MOZ, and the Rtt109/Vps75 complex, were purified as described elsewhere (Poux et al., 2002
; Tang et al., 2008
; Holbert et al., 2007
). The SAS complex was expressed and purified in E. coli.
as detailed elsewhere (Shia et al., 2005
; Sutton et al., 2003
). Briefly, the SAS2, SAS4 and SAS5 proteins were co-expressed using the Duet system (Novagen) in BL21-CodonPlus(DE3)-RIL cells (Strategene). The complex was purified using a combination of nickel affinity, ion exchange (HisTrap SP) and gel filtration (Superdex 200) chromatography.
HAT assays with yeast GCN5, SAS complex, MOZ, and Rtt109/Vps75 complex used the direct radioactive assay described above. Reactions were carried out at 30 °C for times varying from 2 to 4 min under the following reaction conditions: 50 mM HEPES, pH 7.9, 50 mM NaCl, 0.05 mg/ml BSA, 5 mM DTT, 0.05 mM EDTA, 0.25% DMSO, 10 μM of X. laevis histone H3, and varying concentrations of C646 (0, 3, 10 μM). The reactions contained either 70 nanograms of Rtt109/Vps75, 15 nanograms of yGcn5, 300 nanograms of the SAS complex or 1 microgram of hMOZ. The amount of enzyme used in each assay was estimated by comparing Coomassie Blue staining of samples with bovine serum albumin standards, analyzed by SDS-PAGE. The mixture was allowed to equilibrate at 30°C for 10 min before the reaction was initialed with addition of a 1:1 mixture of 12C-AcCoA and 14C-AcCoA to a final concentration of 20 μM. After the appropriate time the reaction was quenched with 6 X Tris-Tricine gel loading buffer which contained 0.2 M Tris-Cl pH 6.8, 40% v/v glycerol, 14% w/v SDS, 0.3 M DTT, and 0.06% w/v Coomassie Blue. The 14C-labeled histone substrates were separated from reactants on a 16.5% Tris-Tricine SDS-PAGE gel. The rate of 14C-incorporation into histone H3 was quantified by autoradiography. We performed all assays in duplicate, and these generally agreed within 20%.
Time course studies
Time courses of p300 HAT with C646 were determined using the radioactive assay described above. Reactions were performed using the conditions detailed above with 1.5 μM C646. Reactions were quenched at particular time intervals, then run on a 16% Tris-Tricine gel and quantified as described above. Similar studies were performed varying the time of p300 HAT pre-incubation with C646. Assays contained the conditions detailed above, with 1.5 μM C646 added at various times prior to initiation with 10 μM acetyl-CoA. Reactions were quenched after 5 min, then run on a 16% Tris-Tricine gel and quantified as described above. All time points were screened in duplicate.
Inhibition with p300 mutants
Sites for mutation were chosen by examination of the C646 binding model generated during VLS. T1411A, W1466F, Y1467F, and R1410A mutations were installed using QuikChange protocols. p300 variants were expressed in E. coli BL21(RIL)-DE3 cells and purified using expressed protein ligation as described above. IC50 values for C646 with all mutants were obtained using the methods described above. All assays contained 10 μM of a 1:1 mixture of 12C-acetyl-CoA and 14C-acetyl-CoA and 400 μM H4-15. Reaction time was varied between 5 and 10 min to keep turnover below 10%. Enzyme concentrations were altered for each mutant, as active site mutations affected enzyme activity. In a similar fashion, kinetic parameters for each mutant vs. AcCoA were determined. [H4-15] was 400 μM, and DMSO was held constant at 2.5%. Reactions proceeded for 6 minutes before being quenched and run on a 16% Tris-Tricine gel as described above. Data were quantified and fit to the Michaelis-Menten equation.
The 2D 1H-1H correlation spectra were acquired at 30 °C on an 11.7 T Varian INOVA spectrometer using a pentaprobe equipped with z-axis pulse-field gradient coils. Data were processed and analyzed using NMRPipe . The sample contained 600 μL of 10 mM C646 DMSO-d6 solution.
Histone acetylation assays in mouse cells
C3H 10T1/2 mouse fibroblasts were grown in DMEM with 10 % FCS at 37 °C with 6 % CO2
. Confluent cultures were rendered quiescent in DMEM with 0.5 % FCS for 18-20 h prior to treatment. Cells were treated with the following compounds: TSA (10 ng/ml [33 nM]; Sigma), C646 (25 μM), C37 (25 μM). Antibodies were used at the following concentrations: total H3 (1:10000; ab7834; Abcam); H4K12ac (1:2500; 06-761; Upstate). Rabbit anti-H3K9ac (1:10000) antibodies were generated in-house (Edmunds et al., 2008
). Histones were isolated from cells by acid extraction, separated by SDS and acid-urea polyacrylamide gel electrophoresis and analyzed by Western blotting as described previously (Thomson et al., 1999
; Clayton et al., 2000
Cancer cell studies
Melanoma cell lines WM35, WM983A, and 1205Lu were generous gifts from Meenhard Herlyn's lab at the Wistar Institute (Philadelphia, PA). Non-small cell lung cancer (NSCLC) cell lines H23 and H838 were obtained from Dr. Charles Rudin's lab, and H1395 from Dr. Craig Peacock's lab, at Johns Hopkins. Melanoma cells were maintained in Dulbecco's Modified Eagle Medium. NSCLCs were maintained in RPMI Medium 1640. Both types of media were supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin, and L-glutamine. Media, pen-strep, and L-glutamine were purchased from Invitrogen. FBS was purchased from Gemini Bio-Products (#100106).
Before treating cells with p300 inhibitors (C646, Lys-CoA-Tat [Zheng et al., 2005
; Guidez et al., 2005
]) or control compounds (C37, Ac-Asp-Asp-Asp-Asp-Tat [also known as Ac-DDDD-Tat]), cells were plated at sub-confluent concentration (~60%) and incubated at 37°C until attached to the plating surface. Compound stocks (10-20 mM in anhydrous DMSO) were directly added to culture media at desired concentrations. DMSO concentration was kept constant at 0.2% between different treatment conditions. Cells were seeded in 96-well plates at ~5000 cells per well on average, depending on each cell line's doubling time. After attachment, cells were treated with p300 inhibitors, control compounds, or DMSO for 24 h. After treatment, 3
H-thymidine (1 mCi/ml stock) was added to media at 10 μCi/ml final concentration. Cells were incubated for an additional 5 h, then trypsinized and collected onto a filter mat using a Cell Harvester (PerkinElmer). Radioactivity was measured with a MicroBeta plate reader (PerkinElmer). Each sample was tested in triplicate.
Cell Cycle Analysis
Cells treated with C646 or DMSO were stained with propidium iodide (PI) according to a published protocol (Robinson, 2009
). Briefly, equal numbers of cells (greater than 106
) in 0.5 ml PBS were fixed in 70% ethanol for at least 2 h at 4°C. A stock staining solution containing 10 ml of 0.1% Triton X-100 in PBS, 400 μl of RNase cocktail (equivalent of ~200 units of RNase A) (Ambion), and 200 μl of 1 mg/ml PI was prepared. Fixed cells were spun at 200 g for 5 min (Beckman Coulter Allegra® X-12R, with an SX4750A rotor), re-hydrated in 5 ml PBS, and spun again to remove all traces of ethanol. Cells were stained with 1 ml staining solution for 20 min at 37°C, then immediately analyzed on a FACSCalibur flow cytometer (BD Biosciences) at the Johns Hopkins Flow Cytometry Core Facility. Data acquisition and analysis were performed with the CellQuest software (BD). WinMDI 2.9 (http://facs.scripps.edu.software.html
) was also used for data presentation.