Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the dynamic and reversible acetylation of proteins, an epigenetic regulatory mechanism associated with multiple cancers. Indeed, HDAC inhibitors are already approved in the clinic. The HAT paralogs p300 and CREB-binding protein (CBP) have been implicated in human pathological conditions including several hematological malignancies and androgen receptor-positive prostate cancer. Others have reported CoA-competitive inhibitors of p300 and CBP with cell-based activity. Here, we describe 2 compounds, CPI-076 and CPI-090, discovered through p300-HAT high throughput screening screening, which inhibit p300-HAT via binding at an allosteric site. We present the high resolution (1.7 and 2.3 Å) co-crystal structures of these molecules bound to a previously undescribed allosteric site of p300-HAT. Derivatization yielded actionable structure-activity relationships, but the full-length enzymatic assay demonstrated that this allosteric HAT inhibitor series was artifactual, inhibiting only the HAT domain of p300 with no effect on the full-length enzyme.

Epigenetics refers to a broad regulatory system that controls gene expression by modifying chromatin. Chromatin is DNA wrapped around an assembly of proteins called histones. Whether a specific set of genes is turned on or off depends on the action of epigenetic regulators. Epigenetic regulators change the architecture of chromatin, allowing it to adopt an open configuration to facilitate gene expression or, conversely, a closed configuration to suppress gene expression. An open chromatin configuration turns on a gene by allowing access and “readout” of the genetic information stored in DNA. A closed chromatin configuration turns off a gene by preventing access to DNA and results in silencing of gene expression. Various chromatin regulatory mechanisms (multiple classes of reader, writer, and eraser proteins) impact the chromatin structure to alter the transcriptional machinery's access to DNA (Jenuwein and Allis, 2001).

Most epigenetic drug targets fall into three main classes: First, epigenetic writers, which are enzymes that add chemical modifications onto chromatin (e.g., histone acetyltransferases such as p300 and histone methyltransferases such as EZH2); second, epigenetic readers, which are protein families that recognize chemical modifications on chromatin and bind to these modifications using specialized protein domains (e.g., bromodomains); third, epigenetic erasers, which are enzymes that remove chemical modifications from chromatin (e.g., lysine demethylases such as LSD1) (Biswas and Rao, 2018). The action of each class of epigenetic regulators alters the structure of chromatin and ultimately controls transcription.

As illustrated in Fig. 1, epigenetic regulators within the writer, reader, and eraser classes modify chromatin and affect gene expression by adding, binding to, or removing chemical tags, which are indicated by dots, on chromatin. In normal cells, these epigenetic mechanisms are tightly regulated so that genes are “turned on” or “turned off” as appropriate.

FIG. 1.

Three classes of epigenetic regulators. Writers add chemical modifications onto chromatin. Readers recognize chemical modifications on chromatin and bind to them. Erasers remove chemical modifications from chromatin. The action of each class controls the expression of certain genes in different ways. Reproduced with permission from Constellation's website.

FIG. 1.

Three classes of epigenetic regulators. Writers add chemical modifications onto chromatin. Readers recognize chemical modifications on chromatin and bind to them. Erasers remove chemical modifications from chromatin. The action of each class controls the expression of certain genes in different ways. Reproduced with permission from Constellation's website.

Close modal

Abnormal cells, such as proliferating cancer cells, can dysregulate these epigenetic mechanisms, ultimately leading to disease (Yoo and Jones, 2006). Epigenetic inhibition is particularly attractive as a therapeutic approach for several reasons. (1) Small-molecule inhibitors can block the abnormal function of epigenetic regulators that cancer cells depend on for growth and potentially restore normal gene expression. (2) Cells use hundreds of epigenetic regulators to control gene expression, which provides a large number of potential drug targets.

One epigenetic writer/eraser pair is HAT/KAT and HDAC. Histone or lysine acetyltransferases (HATs/KATs) and histone deacetylases (HDACs) catalyze the dynamic and reversible acetylation of histone proteins, an epigenetic regulatory mechanism associated with multiple cancers. While several HDAC inhibitors are already approved, relatively little is known about HAT inhibitors (Struhl, 1998).

The HAT paralogs p300 and CREB-binding protein (CBP) have been implicated in human pathological conditions (Dutta et al., 2016) including several hematological malignancies and androgen receptor-positive prostate cancer. The focus of this work is the inhibition of histone acetyltransferase p300, a master transcriptional regulator. P300 is an ∼2400AA multidomain protein whose catalytic core consists of bromo-, RING, PHD, and histone acetyltransferase (HAT) domains. By acetylating histones, p300 promotes gene expression. It can also acetylate itself, as well as other proteins, and recruit a larger complex of proteins including transcription factors. The opportunity to target these multiple functions offers broad therapeutic potential in oncology (Lasko et al., 2017).

To identify starting points for modulating p300/CBP activity, high throughput screening was carried out using the WT P300 HAT domain (WT HAT construct = K1287-F1666 including the L1520-L1580 regulatory loop, expressed as HisTev fusion) and a peptide substrate in a catalytic assay. After extensive triage, several validated inhibitor types were identified, one of which will be the focus of this report. A more detailed description of the HTS efforts will be the subject of a forthcoming publication by Huhn et al. At this stage of the project, only the HAT domain of p300 was used, as it was not yet feasible to obtain sufficient quantities of full-length P300 (FL-P300) to conduct a high-throughput screen of >150,000 compounds.

Two different chemotypes, CPI-076 and CPI-090 (Table I), showed a common mechanism of inhibition, with both displaying weaker inhibition at high concentrations of either a peptide substrate or AcCoA. This mechanism initially appeared to be mixed inhibition because a shift in IC50 was seen for both excess substrate MOI tests. On further examination, neither the 10× AcCoA nor the 30× histone peptide met the IC50 shift threshold necessary to be considered truly competitive. In both situations, CPI-076 and CPI-090 are AcCoA- and peptide-modulating compounds but not wholly competitive compounds.

TABLE I.

Allosteric P300 HAT inhibitor HTS hits.

CPI-numberCPI-076CPI-090
Primary assays 
p300 HAT SPA IC50
CBP HAT SPA IC50 
20 μM
39 μ
0.31 μM
0.74 μ
DSF
ΔTm @ 250 μ
+1.3 °C +5.8 °C 
CPI-numberCPI-076CPI-090
Primary assays 
p300 HAT SPA IC50
CBP HAT SPA IC50 
20 μM
39 μ
0.31 μM
0.74 μ
DSF
ΔTm @ 250 μ
+1.3 °C +5.8 °C 

Additionally, given their small size, it seemed unlikely that CPI-076 and CPI-090 blocked both sites. Followup with dynamic scanning fluorimetry (DSF) of the P300-HAT domain indicated strong stabilization, especially for CPI-090. As detailed below, crystallography was key to unraveling these observations by revealing that these inhibitors occupied a previously undescribed allosteric pocket.

To improve homogeneity, a construct omitting the autoacetylation-prone autoinhibitory loop was inactivated through mutation of residue Y1467 to F: p300‐HAT‐YF‐Δloop [K1287‐Q1663/Y1467F/Δ(L1520‐L1580)] and overexpressed in E. coli. The enzyme was purified by Nickel Sepharose and ion-exchange chromatography. Initial crystals were obtained with CoA by vapor diffusion crystallization. From these, stocks of microseeds were prepared.

Blade-form ternary crystals of p300‐HAT‐YF‐Δloop with CoA and CPI-076 were obtained at 4° C from 200 + 150 nl microseeded sitting drops with a 60 μl reservoir consisting of 20% MPD, 0.1M Tris pH 8, and 7.5% PEG 5000 MME. Ternary crystals of p300‐HAT‐YF‐Δloop with CoA and CPI-090 (Fig. 2) were obtained at 4° C from 200 + 150 nl sitting drops with a 60 μL reservoir consisting of 0.1 M CHES pH 9.5 and 30% w/v PEG 3000 [MCSG1 (MCSG) (purchased from Anatrace), condition A2]. As these were small crystals grown in MPD and PEG conditions, low-viscosity perfluoropolyether oil (purchased as “LVCO” from MiTeGen) was sufficient for cryoprotection (Riboldi‐Tunnicliffe and Hilgenfeld, 1999).

FIG. 2.

Crystals of p300‐HAT‐YF‐Δloop with CoA and CPI-090.

FIG. 2.

Crystals of p300‐HAT‐YF‐Δloop with CoA and CPI-090.

Close modal

Following synchrotron data collection and reduction with XDS (Kabsch, 2010), the structures were solved by molecular replacement with Phaser (McCoy et al., 2007), with 2 chains in the ASU, iterative cycles of rebuilding in Coot (Emsley and Cowtan, 2004) and refining in Refmac5 (Murshudov et al., 1997). Coordinates and structure factors were deposited in the PDB with codes 6PGU (CPI-076) and 6PF1 (CPI-090). Data collection and refinement parameters are presented in Table II.

TABLE II.

Crystallographic data collection and refinement statistics.

CPI-076CPI-090
Deposition code 6PGU 6PF1 
Resolution range (Å) 71.78, 1.72 70.69, 2.32 
Resolution range last shell (Å) 1.76, 1.72 2.44, 2.32 
Space group P212121 P212121 
Unit cell (Å) 66.253, 97.599, 105.932 65.062, 90.634, 112.950 
Unique reflections 69876 29312 
Overall and last-shell completeness (%) 99.75 93.55, 90.32 
Overall and last shell Rmeas 0.067 0.126, 0.726 
Overall and last shell σ(I) 28.7 2.0 10.8, 2.3 
Solution method Molecular replacement Molecular replacement 
Refinement software Refmac5 Refmac5 
Rwork, Rfree 0.16727, 0.20627 0.18355, 0.24728 
Number of atoms 5754 5315 
Average of B factors 23.28 34.247 
RMSD bond lengths 0.019 0.013 
RMSD angles 2.033 1.645 
Molprobity score and percentile rank 1.214, 98 1.154, 100 
CPI-076CPI-090
Deposition code 6PGU 6PF1 
Resolution range (Å) 71.78, 1.72 70.69, 2.32 
Resolution range last shell (Å) 1.76, 1.72 2.44, 2.32 
Space group P212121 P212121 
Unit cell (Å) 66.253, 97.599, 105.932 65.062, 90.634, 112.950 
Unique reflections 69876 29312 
Overall and last-shell completeness (%) 99.75 93.55, 90.32 
Overall and last shell Rmeas 0.067 0.126, 0.726 
Overall and last shell σ(I) 28.7 2.0 10.8, 2.3 
Solution method Molecular replacement Molecular replacement 
Refinement software Refmac5 Refmac5 
Rwork, Rfree 0.16727, 0.20627 0.18355, 0.24728 
Number of atoms 5754 5315 
Average of B factors 23.28 34.247 
RMSD bond lengths 0.019 0.013 
RMSD angles 2.033 1.645 
Molprobity score and percentile rank 1.214, 98 1.154, 100 

The overall structure is as expected from previous p300-HAT structures: a central β-sheet is composed of seven β-strands and surrounded by nine α-helices (Fig. 3). Despite their chemical differences, these new allosteric compounds bind in the same location (Fig. 4), ∼8 Å distant from the active site, between the α5 and α6 helices. While these compounds' MOA is formally modulated by the substrate and AcCoA, the structures show that the binding mode is distal from the substrate and AcCoA sites, which starts to rationalize the difference.

FIG. 3.

Lys-CoA dual substrate analog P300-HAT structure 3BIY superposed with the CPI-090 P300-HAT structure (yellow C atoms for CPI-090 and CoA). Lys's closest approach to CPI-090 is ∼8 Å.

FIG. 3.

Lys-CoA dual substrate analog P300-HAT structure 3BIY superposed with the CPI-090 P300-HAT structure (yellow C atoms for CPI-090 and CoA). Lys's closest approach to CPI-090 is ∼8 Å.

Close modal
FIG. 4.

Comparing the binding modes of CPI-090 (yellow C-atoms) with CPI-076 (salmon C-atoms).

FIG. 4.

Comparing the binding modes of CPI-090 (yellow C-atoms) with CPI-076 (salmon C-atoms).

Close modal

It is worth noting that in both cases, CoA was carried through purification and appeared fortuitously in the AcCoA binding site. So though these compounds are AcCoA modulated, there is obviously no direct competition for the AcCoA binding site.

There are few structural differences between these ternary compound/CoA/HAT structures and CoA/HAT binary structures. Comparing the HAT/CoA/CPI-090 structure with HAT/CoA (4PZR), the RMSD value was found to be 0.27 Å over 270 CA atoms. Essentially, the allosteric compounds displace water molecules (Fig. 5). The grounds for mechanistic competition with the substrate and AcCoA are not readily apparent. CPI-076 is secured in p300-HAT-CoA by hydrophobic interactions and a hydrogen bond with Asp1507 and also creates a water network out to Arg1392/Ile1486.

FIG. 5.

CPI-076-p300-HAT-CoA (light blue C atoms for the compound, green protein, and CoA) aligned with p300-HAT-CoA (pink protein and CoA) shows minimal perturbations in the protein backbone, as compound binding acts to displace five water molecules found in the CoA-only structure. Water molecules colored to match protein color.

FIG. 5.

CPI-076-p300-HAT-CoA (light blue C atoms for the compound, green protein, and CoA) aligned with p300-HAT-CoA (pink protein and CoA) shows minimal perturbations in the protein backbone, as compound binding acts to displace five water molecules found in the CoA-only structure. Water molecules colored to match protein color.

Close modal

With CPI-076's measured HAT-domain IC50 20 μM, SBDD efforts focused on driving the biochemical activity. CPI-076's structure offered 2 potential vectors for compound elaboration [Fig. 6(a)]. However, derivatization failed to improve the compound activity from this starting point (data not shown). The indole compound CPI-090, a stronger HAT inhibitor at 0.31 μM, offered vectors for optimization from the 4, 5, and 6 positions of its pyridine ring [Fig. 6(b)]. Derivatization yielded actionable SAR and improvement in the biochemical activity (data not shown), and so this series was selected for cell-based assays.

FIG. 6.

(a) CPI-076 offered two vectors for structure-based elaboration. (b) The indole compound CPI-090, a stronger HAT inhibitor, offered vectors for optimization from the 4, 5, and 6 positions of the pyridine ring.

FIG. 6.

(a) CPI-076 offered two vectors for structure-based elaboration. (b) The indole compound CPI-090, a stronger HAT inhibitor, offered vectors for optimization from the 4, 5, and 6 positions of the pyridine ring.

Close modal

The sequence of 2414-residue full-length human p300 (FL-P300) was codon-optimized for insect cell expression and synthesized in three parts (Genscript). After assembly of the full-length sequence, the complete ORF was subcloned into a modified pTriEx vector (Novagen/EMD Millipore) to express FL-P300 with both an N-terminal, cleavable polyhistidine tag and C-terminal StrepII tag (HisTev-p300-Strep). Baculovirus was produced by co-transfection of the plasmid into Sf9 cells with linearized baculovirus BestBac2.0 (Expression Systems). The protein was expressed in Sf9 cells and purified with Talon Metal Affinity Resin (Clontech/Takara). The purified protein was concentrated to 1.6 mg/ml in storage buffer (50 mM Tris pH 8, 100 mM NaCl, 10% glycerol, and 0.5 mM TCEP).

Moving the allosteric series compounds into cell-based assays assessing the ability of the compound to inhibit H3K18 acetylation in HCT116 cells delivered a setback to this allosteric series, as these compounds displayed such a large shift in cell-potency to be inactive. At this point in the program, quantities of highly purified FL P300 were more readily available (preparation described above), and so HAT inhibitors were retested against FL P300 under optimized conditions. A large shift relative to the P300-HAT catalytic domain assay was observed. CPI-090 does not inhibit the FL enzyme in our experiments (Fig. 7). The related compound CPI-076 had no measurable FL activity (Table III).

FIG. 7.

Inhibition of P300-HAT and P300-FL by CPI-090.

FIG. 7.

Inhibition of P300-HAT and P300-FL by CPI-090.

Close modal
TABLE III.

Comparison of allosteric inhibitor activities in various assays.

CPI-numberCPI-076CPI-090
Primary assays   
P300 HAT SPA IC50 20 μ0.31 μ
CBP HAT SPA IC50 39 μ0.74 μ
H3K18Ac EC50  >30 μ
FL p300 SPA IC50 Undetectable >160 μ
CPI-numberCPI-076CPI-090
Primary assays   
P300 HAT SPA IC50 20 μ0.31 μ
CBP HAT SPA IC50 39 μ0.74 μ
H3K18Ac EC50  >30 μ
FL p300 SPA IC50 Undetectable >160 μ

It became clear from the optimized FL assay that the (sub)micromolar inhibition observed against the catalytic HAT domain had been an artifact of using the HAT domain, which had been used because of initial limitations of quantity and purity in obtaining full-length P300.

The grounds for mechanistic modulation with the substrate and AcCoA in the HAT-only construct are not readily apparent. The structure of the p300-p53 complex (Ghosh et al., 2019) (5xzc, at 10.7 Å) appeared at first to hint at a hypothesis, but further investigation ruled it out. The p300-p53 structure is noteworthy in that it contains a model of the p300 regulatory loop (residues ∼1520–1560), which has not to our knowledge been visualized before. While nothing in the p300-p53 complex structure appears to directly block access to the CPI-090/CPI-076 binding site, the regulatory loop packs against helix 7; in our HAT structures, helix 7 comes into contact with CPI-090/CPI-076 and is mobile (which lacks the loop). The activation loop was intact in our screening construct, though, which would seem to rule out the hypothesis of regulatory loop interference/mimicry.

The cellular and FL-P300 biochemical assays demonstrated that the allosteric HAT inhibitor series, which had been co-crystallized with a truncated form of the target enzyme and enhanced by SBDD, was artifactual. This series was ultimately abandoned, and series of compounds with alternative mechanisms of action were prioritized for our medicinal chemistry campaign. The results of this case study demonstrate that hits from partial enzymes may be artifactual, not translating into cellular efficacy, despite compelling support from DSF and crystallographic experiments. The lesson learned is to prioritize the use of full-length enzymes when possible in biochemical assays early in compound screening or follow-up and to carefully examine any anomalies or discrepancies between assay results. The challenges in obtaining FL-P300 led us to rely on a truncated form of the target enzyme and a focus on this series, which ultimately lacked pharmacological relevance.

The work of Robert Campbell, Rishi Vaswani, and Venita Watson is gratefully acknowledged. The authors acknowledge the support and screening deck provided by Genentech. Joshua Carter and Ardian Soca Wibowo (current affiliation: Helix BioStructures) performed the data collection and processing. The authors also thank Steve Bellon, Julian Levell, and Robert Sims for helpful discussions. This work was performed at Constellation, while the authors received compensation as Constellation employees.

1.
Biswas
,
S.
and
Rao
,
C. M.
, “
Epigenetic tools (The Writers, The Readers and The Erasers) and their implications in cancer therapy
,”
Eur. J. Pharmacol.
837
,
8
24
(
2018
).
2.
Dutta
,
R.
,
Tiu
,
B.
, and
Sakamoto
,
K. M.
, “
CBP/p300 acetyltransferase activity in hematologic malignancies
,”
Mol. Genet. Metab.
119
,
37
43
(
2016
).
3.
Emsley
,
P.
and
Cowtan
,
K.
, “
Coot: Model-building tools for molecular graphics
,”
Acta Crystallogr., Sect. D: Biol. Crystallogr.
60
,
2126
2132
(
2004
).
4.
Ghosh
,
R.
,
Kaypee
,
S.
,
Shasmal
,
M.
,
Kundu
,
T. K.
,
Roy
,
S.
, and
Sengupta
,
J.
, “
Tumor Suppressor p53-mediated structural reorganization of the transcriptional coactivator p300
,”
Biochemistry
58
,
3434
3443
(
2019
).
5.
Jenuwein
,
T.
and
Allis
,
C. D.
, “
Translating the histone code
,”
Science
293
,
1074
1080
(
2001
).
6.
Kabsch
,
W.
, “
Xds
,”
Acta Crystallogr., Sect. D: Biol. Crystallogr.
66
,
125
132
(
2010
).
7.
Lasko
,
L. M.
,
Jakob
,
C. G.
,
Edalji
,
R. P.
,
Qiu
,
W.
,
Montgomery
,
D.
,
Digiammarino
,
E. L.
,
Hansen
,
T. M.
,
Risi
,
R. M.
,
Frey
,
R.
,
Manaves
,
V.
,
Shaw
,
B.
,
Algire
,
M.
,
Hessler
,
P.
,
Lam
,
L. T.
,
Uziel
,
T.
,
Faivre
,
E.
,
Ferguson
,
D.
,
Buchanan
,
F. G.
,
Martin
,
R. L.
,
Torrent
,
M.
,
Chiang
,
G. G.
,
Karukurichi
,
K.
,
Langston
,
J. W.
,
Weinert
,
B. T.
,
Choudhary
,
C.
,
de Vries
,
P.
,
Van Drie
,
J. H.
,
McElligott
,
D.
,
Kesicki
,
E.
,
Marmorstein
,
R.
,
Sun
,
C.
,
Cole
,
P. A.
,
Rosenberg
,
S. H.
,
Michaelides
,
M. R.
,
Lai
,
A.
, and
Bromberg
,
K. D.
, “
Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours
,”
Nature
550
,
128
132
(
2017
).
8.
McCoy
,
A. J.
,
Grosse-Kunstleve
,
R. W.
,
Adams
,
P. D.
,
Winn
,
M. D.
,
Storoni
,
L. C.
, and
Read
,
R. J.
, “
Phaser crystallographic software
,”
J. Appl. Crystallogr.
40
,
658
674
(
2007
).
9.
Murshudov
,
G. N.
,
Vagin
,
A. A.
, and
Dodson
,
E. J.
, “
Refinement of macromolecular structures by the maximum-likelihood method
,”
Acta Crystallogr., Sect. D: Biol. Crystallogr.
53
,
240
255
(
1997
).
10.
MCSG Crystal Production, see http://bioinformatics.anl.gov/mcsg/technologies/crystalproduction.html for details of the MCSG crystallization screens.
11.
Riboldi‐Tunnicliffe
,
A.
and
Hilgenfeld
,
R.
, “
Cryocrystallography with oil – an old idea revived
,”
J. Appl. Crystallogr.
32
,
1003
1005
(
1999
).
12.
Struhl
,
K.
, “
Histone acetylation and transcriptional regulatory mechanisms
,”
Genes Dev.
12
,
599
606
(
1998
).
13.
Yoo
,
C. B.
and
Jones
,
P. A.
, “
Epigenetic therapy of cancer: Past, present and future
,”
Nat. Rev. Drug Discovery
5
,
37
50
(
2006
).