Structure–Activity Relationships on Cinnamoyl Derivatives as Inhibitors of p300 Histone Acetyltransferase

Article abstract of DOI:10.1002/cmdc.201700040

Human p300 is a polyhedric transcriptional coactivator that plays a crucial role in acetylating histones on specific lysine residues. A great deal of evidence shows that p300 is involved in several diseases, including leukemia, tumors, and viral infection. Its involvement in pleiotropic biological roles and connections to diseases provide the rationale to determine how its modulation could represent an amenable drug target. Several p300 inhibitors (i.e., histone acetyltransferase inhibitors, HATis) have been described so far, but they all suffer from low potency, lack of specificity, or low cell permeability, which thus highlights the need to find more effective inhibitors. Our cinnamoyl derivative, 2,6-bis(3-bromo-4-hydroxybenzylidene)cyclohexanone (RC56), was identified as an active and selective p300 inhibitor and was proven to be a good hit candidate to investigate the structure–activity relationship toward p300. Herein, we describe the design, synthesis, and biological evaluation of new HATis structurally related to our hit; moreover, we investigate the interactions between p300 and the best-emerged hits by means of induced-fit docking and molecular-dynamics simulations, which provided insight into the peculiar chemical features that influence their activity toward the targeted enzyme.

Full text of DOI:10.1002/cmdc.201700040

Accepted Article
Title: Structure-Activity Relationships on Cynnamoyl Derivatives as
Inhibitors of p300 Histone Acetyltransferase
Authors: Valentina Noemi Madia, Rosaria Benedetti, Maria Letizia
Barreca, Liza Ngo, Luca Pescatori, Antonella Messore,
Giovanni Pupo, Francesco Saccoliti, Sergio Valente,
Antonello Mai, Luigi Scipione, Yujun George Zheng, Cristina
Tintori, Maurizio Botta, Violetta Cecchetti, Lucia Altucci,
Roberto Di Santo, and Roberta Costi
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201700040
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700040
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10.1002/cmdc.201700040
ChemMedChem
Structure-Activity Relationships on Cynnamoyl Derivatives
as Inhibitors of p300 Histone Acetyltransferase
Valentina Noemi Madia,[a] Rosaria Benedetti,[b] Maria Letizia Barreca,[c] Liza Ngo,[d] Luca
Pescatori,[a] Antonella Messore,[a] Giovanni Pupo,[a] Francesco Saccoliti,[a] Sergio
Valente,[a]Antonello Mai,[a] Luigi Scipione,[a] Yujun George Zheng,[d] Cristina Tintori,[e] Maurizio
Botta,[e] Violetta Cecchetti,[c] Lucia Altucci,[b],[f] Roberto Di Santo,[a] and Roberta Costi *[a]
[a]
Dr. V. N. Madia 0000-0002-5724-612X, Dr. L. Pescatori 0000-0003-4734-7856, Dr. A. Messore 0000-0003-0158-5816, Dr. G. Pupo 0000-0002-
6011-0223, Dr.F. Saccoliti 0000-0002-9175-9199, Dr. S. Valente 0000-0002-2241-607X, Prof. A. Mai 0000-0001-9176-2382, Dr. L. Scipione
0000-0002-2006-7005, Prof. R. Di Santo 0000-0002-4279-7666, Prof. R. Costi 0000-0002-1314-9029
Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti
“Sapienza” Università di Roma
P.le Aldo Moro 5, I-00185, Roma, Italy
E-mail: roberta.costi@uniroma1.it
[b]
[c]
[d]
[e]
[f]
Dr. R. Benedetti, Prof. L. Altucci 0000-0002-7312-5387
Dipartimento di Biochimica, Biofisica e Patologia generale
Università degli Studi della Campania Luigi Vanvitelli
Vico L. De Crecchio 7, I-80138, Napoli, Italy
Prof. M. L. Barreca 0000-0003-3530-5042, Prof. V. Cecchetti 0000-0001-8558-7004
Department of Pharmaceutical Sciences
University of Perugia
Via A. Fabretti 48, I-06123 Perugia, Italy
Dr. L. Ngo, Prof. Y. G. Zheng 0000-0001-7116-3067
Department of Pharmaceutical and Biochemical Sciences
University of Georgia
Athens, Georgia, 30602, U.S.A.
Dr. C. Tintori 0000-0002-6002-0332, Prof. M. Botta 0000-0003-0456-6995
Dipartimento di Biotecnologie, Chimica e Farmacia
Università degli Studi di Siena
Via Aldo Moro 2, I-53100, Siena, Italy
Prof. L. Altucci 0000-0002-7312-5387
Istituto di Genetica e Biofisica, IGB ‘Adriano Buzzati Traverso’
Via P. Castellino 111 I-80131, Napoli, Italy
Supporting information for this article is given via a link at the end of the document.
1
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Abstract: Human p300 is a polyhedric transcriptional coactivator, playing a crucial role by acetylating histones on specific lysine
residues. A great deal of evidences shows that p300 is involved in several diseases as leukemia, tumors and viral infection. Its
involvement in pleiotropic biological roles and connections to diseases provide the rationale as to how its modulation could
represent an amenable drug target. Several p300 inhibitors (HATi) have been described so far, but all suffer from low potency, lack
of specificity or low cell-permeability, highlighting the need to find more effective inhibitors. Our cinnamoyl derivative, RC 56, was
identified as active and selective p300 inhibitor, proving to be a good hit candidate to investigate the structure-activity relationship
towards p300. Herein we describe the design, synthesis and biological evaluation of new HATi structurally related to our hit,
investigating, moreover, the interactions between p300 and the best-emerged hits by means of induced fit docking and molecular
dynamics simulations, gaining insight on the peculiar chemical features that influenced their activity toward the targeted enzyme.
Introduction
Described for the first time as adenoviral E1A-binding protein,[1] p300/CBP is a HAT (histone acetyltransferase) enzyme
family together with other four families, including GNAT, MYST, nuclear receptor coactivators, and TBP-associated factor
TAFII.[2] p300 acts primarily as histone acetyltransferase, with the ability to transfer an acetyl group from acetyl-coenzyme A
(acetyl-CoA) to the histone ε-NH2 of lysine side chain, for remodeling chromatin to a relaxed superstructure. This
modification results in the change of histone-DNA and histone-protein interactions. Histone acetylation can occur in
promoter regions, where p300 acts as a transcription co-factor for a variety of nuclear proteins (including oncoproteins,[3]
viral proteins[4] and tumor suppressor proteins[5]) or over large regions of chromatin, affecting global gene expression
levels.[6] p300 substrates are not limited to histone proteins, but include more than 75 different proteins.[7] Noteworthy, p300
acetylation activity can be also addressed to viral proteins such as HIV-1 integrase, contributing significantly to viral
replication.[8] Moreover, an extensive autoacetylation process occurs in an intermolecular fashion, regulating the activation
state and the mediated transcriptional regulation of p300.[9]
By acetylating different substrates, p300 is implicated in a wide array of cellular processes, such as cell-cycle regulation,[10]
differentiation[11] and DNA damage response,[12] and can promote opposite cellular outcomes such as proliferation and
apoptosis.[13] Because of the biological importance of p300, the alteration in the gene sequence (mutation, chromosomal
translocation and dysregulation) is correlated to many disease conditions, including cancer,[14] chronic neuropathic pain[15]
and cognitive and neurodegenerative disorder.[16] Most recently, implication of p300 was also described in ventricular
remodeling after myocardial infarction.[17]
p300 is characterized by highly conserved regions, which is possible to distinguish by four different functional domains: (i) a
catalytic HAT domain, where the histone and proteins acetylation occurs; (ii) four recognized transactivation domains, that
mediates protein-protein interactions with DNA-binding transcription factors and transcription machinery; (iii) the
bromodomain that recognize histone acetylated tails and (iv) the cysteine-histidine-rich region. Recently, mutagenesis
studies and resolution of structure of semi-synthetic heterodimeric p300 HAT domain in complex with Lys-CoA revealed the
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10.1002/cmdc.201700040
ChemMedChem
catalytic mechanism of p300, where Tyr1467 and Trp1436 residues play a significant role in the acetylation reaction.[18]
Tyr1467 guides and protonates the sulfur atom of acetyl-CoA, while Trp1436 orients the lysine side chain for the nucleophilic
attack of the acetyl-CoA cofactor. More recently, the resolution of crystal structure of the p300 HAT domain bound to acetyl-
CoA confirmed this hypothesis and gave insight for the design of p300 inhibitors.[19]
Due to the relevant role in different pathological conditions, HAT p300 is considered an amenable target and its modulation
holds promise for future therapeutic strategies.[20] p300 inhibitors (HATi) so far described comprise natural compounds,
some of them supplied from dietary plants, (i.e. anacardic acid,[21] curcumin,[22] garcinol,[23] epigallocatechin-3-gallate[24] and
plumbagin[25]) and synthetic small molecules (i.e., Lys-CoA,[26] C646,[27] isothiazolones[28] and cinnamoyl compounds[29]
)
(Chart 1).
O
OH
O
OH
O
CH3
H3CO
HO
OCH3
OH
OH
OH
O
Plumbagin
Anacardic acid
Curcumin
OH
OH
OH
HO
O
HO
O
O
O
O
N X
O
O
S
OH
OH
OH
Cl
OH
OH
Epigallocatechin-3-gallate
Isothiazolones
Garcinol
O2N
O
O
HN
O
N
O
O
N
O
SCoA
N
H
Br
Br
H
NH2
HO
OH
COOH
Lys-CoA
C646
RC56
Chart 1. Chemical structures of known natural products and synthetic small molecules p300 inhibitors.
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Nevertheless, in the field of HATi a huge investigation margin is left due to the necessity to find further inhibitors endowed
with good pharmacokinetic and pharmacodynamic profiles, and to establish the exact inhibition mode towards p300. In the
last decade our research group was involved in the identification of HATi[29] and among them RC 56 [2,6-bis-(3-bromo-4-
hydroxybenzylidene)cyclohexanone] (Figure 1) was identified as the most active and selective p300 inhibitor, showing
cellular activity displayed by the down regulation of histone H3 acetylation level. Most recently RC 56 was used as
investigation tool to evaluate the acetylation level in H3K4 by p300 and the expression of multidrug resistance (MDR1) gene
in drug-resistant and drug-sensitive breast carcinoma cell lines.[30] We considered RC 56 a good hit candidate to investigate
the structure-activity relationship towards p300. Herein we report the design of new HATi (1a-s and 2a) addressing six
modifications to the RC 56 structure, which involved both the benzylidene moiety and the central cyclohexanone portion, so
as to evaluate the role of the arylidene substituents, the central ring and the carbonyl group in inhibiting the enzymatic
target. In particular, 1) the bromine atom in 3-position of the benzylidene portion was substituted with other atoms or groups:
F (1b), Cl (1c), I (1d), methyl group (1g), hydroxyl group (1e), hydrogen atom (1a) and phenyl ring (1f); 2) the hydroxyl group
in 4-position of the benzylidene portion was substituted with a bromine atom (1h); 3) a third substituent in 5-position of the
benzylidene portion (Br, F, Cl) was introduced (1i-j); 4) the cyclohexanone was reduced to cyclohexanol (2a); 5) a carboxylic
acid function was introduced in 4-position of the cyclohexanone ring (1k-l); 6) the cyclohexanone was substituted with 5-
member ring (1m) or heterocycles: tetrahydro thiopyranone (1n) or piperidinone (1o-s). Thus, we report the synthesis and
biological evaluation of the HATi 1a-s and 2a (Table 1). All the synthesized compounds were tested on recombinant p300
using an in vitro radiometric assay. Cell-based assays were performed with the most promising compounds to corroborate
and strengthen the activities. Moreover, theoretical investigation of the interactions between p300 HAT and the best-
emerged hits (i.e., 1d and 1i) was carried out by means of induced molecular modeling experiments.
Results and discussion
Chemistry. Bis-arylidene derivatives 1a-s were obtained by microwave-assisted (1a-j,m-n) or acid catalyzed (1k-l,o-s)
condensation of the appropriate five- or six-member cycle (see Table 1) with the appropriate benzaldehyde following a
previously reported synthetic approach (Scheme 1).[29],[31] Derivative 2a was obtained by reduction of the carbonyl group of
RC 56 to hydroxyl group in presence of lithium aluminum hydride (Scheme 2). All the benzaldehydes used as starting
materials are commercially available, except 3d and 3f. Benzaldehyde 3d was obtained as reported in literature,[32] while 3f
was synthesized by a Suzuki coupling reaction (Scheme 3).
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O
O
O
R1
R2
a
R1
R2
R1
R2
H
Cycle
Cycle
R3
3a-n
R3
R3
4a-g
1a-s
a Reagents and conditions: a) 1a-j,m-n: montmorillonite K-10, 100 W, 100 °C, 5 min; 1k-l,o-s: HClg,
CH3COOH, room temp, 48 h.
Scheme 1.a Key synthetic step for the synthesis of the bis-arylidene derivatives 1a-s.
O
OH
a
Br
Br
Br
Br
HO
OH
HO
OH
2a
RC 56
a Reagents and conditions: a) THF, LiAlH4, 0 °C to room temp, 2h, 32%.
Scheme 2.a Synthesis of derivative 2a.
O
O
H
a
H
HO
HO
Br
3k
3f
a Reagents and conditions: a) phenylboronic acid, tetrakis(triphenylphosphine)palladium,
Ba(OH)2·8H2O, DME/H2O, reflux, 4h, 32%.
Scheme 3.a Preparation of benzaldehyde 3f.
Biological evaluation. The newly synthesized compounds 1a-s and 2a could be categorized according to the nature of the
central cycle (cyclohexanone derivatives 1a-l; cyclopentanone derivative 1m; tetrahydrothiopyranone derivative 1n;
piperidinone derivatives 1o-s; cyclohexanol derivative 2a). All the compounds were tested against recombinant p300 (0.025
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ChemMedChem
µM) using the standard filter binding assay. Conditions for this assay included 100 µM H3-20 peptide and 1 µM [14C] Ac-CoA
with a six-minute incubation at 30°C (Table 1). The activity of p300 was measured in the presence of individual compounds
each at fixed concentration of 100 µM. The positive control contained no inhibitor. The activity of each inhibitor solution was
compared with the positive control to calculate the percentage of p300 retaining activity (%), which was the quantitative
representation of the potency of each inhibitor. IC50 values were further determined for those compounds showing less than
10% of p300 retaining activity at 100 µM (1b-d,g,i,n). RC 56 was found previously by us to be a potent p300 inhibitor and
thus was used as the reference compound. In this experiment RC 56 revealed an IC50 value of 30.9 µM, six times greater
than the one already reported.[29] The difference in the IC50 value of RC 56 was attributed to altered assay conditions. In the
previous study, GST-p300 HAT domain was used to acetylate a histone protein mixture with [14C] Ac-CoA as cofactor. The
acetylated products were visualized with phosphorimaging after SDS-PAGE separation. In the present study, a tag-free
p300 was used as enzyme source and a high concentration of H3 peptide was used as substrate. The reaction yields were
controlled under the initial reaction condition (product conversion typically is less than 20%).
Among the tested compounds, the cyclohexanone derivative 1i was the most active compound showing complete inhibition
to p300 activity at 100 µM. Its IC50 value was measured to be 2.3 ± 0.5 µM, fifteen times lower than RC 56 (30.9 ± 8.2 µM).
Of note, we also measured the IC50 of C646 at the same condition to be 3.6 ± 0.6 µM. C646 was regarded one of the most
potent p300 inhibitor.[27] Therefore, the potency of 1i is at the same level as C646.
Among the cyclohexanone derivatives 1a-l, we discuss preliminary structure-activity relationship according to the
substituents on the benzylidene portion. The substitution of the bromine atom of RC 56 with hydrogen (1a), hydroxyl group
(1e), phenyl ring (1f) resulted in the decrease of the inhibitory activity (% of residual acetylation of 28.6, 89.0, 92.2,
respectively). The substitution of the bromine atom of RC 56 with other halogen atoms (1b-d) as well as a methyl group (1g)
led to compounds with good inhibitory activity. In fact, compounds 1b, 1c and 1g showed IC50 values comparable with that
of RC 56 (IC50 values of 38.9 µM, 45.5 µM, 26.8 µM, respectively). Noteworthy, the substitution of bromine of RC 56 with an
iodine atom (1d) led to a five times more active compound (IC50 value of 8.1 µM). The introduction of a second bromine
atom on the benzylidene portion (1i) generated the most potent compound (IC50 value of 2.3 µM), conversely the substitution
of the two bromine atoms of 1i with a fluorine and a chlorine atom (1j) decreased the activity (% of residual acetylation of
41.1). The biological data also suggested a key role of the hydroxyl groups on the benzene rings as highlighted by the
comparison between RC 56 (IC50 value of 30.9 µM) and derivative 1h (% of residual acetylation of 85.1 at 100 mM).
The cyclohexanone and tetrahydrothiopyranone derivatives showed comparable activity (RC 56 and 1n IC50 values of 30.9
µM and 35.0 µM, respectively), while the substitution of the cyclohexanone ring with cyclopentanone (1m), piperidinone (1o-
s) and cyclohexanol (2a) led to a decreasing in the inhibition potency (% of residual acetylation in the range of 12.5 and
128).
Among the piperidinone series featuring a 3,4-dihydroxybenzylidene substitution pattern, 1o-p,r, the most active compound
was the N-benzyl substituted piperidinone 1r. In this series, the introduction of a benzyl group in 4-position of the
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piperidinone ring (1r) increased the inhibition potency if compared with the unsubstituted counterpart (1o) (% of residual
acetylation of 19.4 and 114, respectively). Moreover, comparing derivative 1r with its analog 1s, characterized by the
substitution of the hydroxyl in 3-position of the benzylidene ring with a bromine atom, an activity decrease was observed (%
of residual acetylation of 19.4 and 81.6, respectively). Interestingly, the replacement of the cyclohexanone of RC 56 with a
N-benzyl substituted piperidinone ring (1s), led to a decrease of inhibitory activity (% of residual acetylation of 81.6).
Interestingly, three compounds (1l,o,q) showed slight activation of p300 activity. This phenomenon is not unusual. In our
previous study of anacardic acid analogs for HAT inhibition, we had similar observation that certain HAT inhibitors showed
activating effect on p300 and PCAF activities.[33] The exact mechanism is not clear. Possibly, some inhibitor analogs can
bind to HAT enzyme at a different conformation, thus working as an agonist instead of antagonist.
Table 1. p300 inhibitory activity of compounds 1a-s and 2a in enzyme assays.
R1
R2
Y
X
R1
R2
R3
R3
% retaining
activity (100
µM)[a]
Cpd
1a
1b
1c
1d
1e
1f
X
Y
R1
H
R2
R3
IC50[b] (µM)
CH2
CH2
CH2
CH2
CH2
CH2
CH2
C=O
C=O
C=O
C=O
C=O
C=O
C=O
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
28.6
6.3
F
38.9 ± 10.1
45.5 ± 9.8
8.1 ± 2.1
Cl
7.7
I
5.8
OH
Ph[c]
CH3
89.0
92.2
3.0
1g
26.8 ± 11.2
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1h
1i
CH2
CH2
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
CH-OH
C=O
Br
Br
Br
H
Br
Cl
H
H
H
H
H
H
H
H
H
H
H
85.1
0
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
2.3 ± 0.5
1j
CH2
F
41.1
70.1
128
41.2
7.13
114
82.4
128
19.4
81.6
12.5
0
1k
CH2COOH
CH2CH2COOH
0
COOH
OH
Br
1l
1m
1n
S
Br
35.0 ± 12.3
1o[d]
1p[d]
1q[d]
1r[d]
1s[d]
2a
NH
OH
OH
NO2
OH
Br
NHCH3
NHCH2CH3
NHCH2Ph
NHCH2Ph
CH2
Br
RC56
CH2
Br
30.9 ± 8.2
C646
3.6 ± 0.6
[a]
[b]
The relative activity of p300 compared to the positive control which has no inhibitor present in the presence of 100 µM inhibitor;
IC50
[c]
[d]
(µM) determined from dose-response curves. The data represents the mean value of at least duplicates; phenyl; compounds tested as
HCl salt.
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Influence on the cell-cycle regulation.
Some of the compounds displaying activity against p300 were also tested in “cell-based assay” to verify their capability to
modify cell cycle progression and induce cell death. Leukemic monoblast U937 cells were treated with 50 µM of each
compound (as reported in Figure 1) for 30 hours. Next, cell cycle distribution and percentage of cell death were both
analyzed. As shown in Figure 1, compound 1d induced a weak G2 phase accumulation, whereas compounds 1a, 1b, 1f, 1g
and 1n caused an increase of cell number percentage in S phase.
Compounds 1b and 1g also showed a G1 block, coupled with induction of cell death (Figure1, panel B) greater than RC56.
Compound 1i, although did not change cell cycle distribution, compared to the ctr-untreated point, induced a weak cellular
death (around 8.5%). It is interesting to note that for compounds 1b, 1d, 1g and 1i a strong correlation between the
percentage of p300 inhibition (shown in Table 1) and the biological effect, reported here as inhibition of proliferation and
induction of cell death (Figure 1, panel A and B), exists. The effect of compounds 1b and 1g seems to be very similar to
RC56, showing a S phase accumulation and a value of p300 IC50 ranging from 38.9 ± 10.1 and 8.1 ± 2.1. However for both
compound the percentage of cell death induction is greater than the one obtained with RC56.
Figure 1. Cell cycle distribution; (A) % of cells in cell cycle phases and (B) apoptosis induction. SAHA was used as reference compound.
Experiments were carried out as independent biological triplicates. Error bars show the standard deviation of triplicates.
Molecular modeling study. An in-depth theoretical investigation of the interactions between p300 HAT and the best-
emerged hits (i.e., 1i and 1d) was carried out by means of induced fit docking (IFD), molecular mechanics-generalized
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Born/Poisson Boltzmann surface area (MM-GBSA) experiments and Molecular Dynamics (MD) simulations. RC 56 was
analyzed as well as a reference compound.
Induced-fit docking experiments
Prior to docking, the compounds were built using the Schrödinger Maestro interface[34] and then submitted to the LigPrep
utility,[35] which rapidly produces low energy 3D structures, taking into account ionization states, tautomerism,
stereochemistry, and ring conformations at the desired pH. For our study, the default pH range of 6−8 was kept. This
analysis suggested that three molecules could exist in a neutral (N), monoanionic (M) or dianionic (D) form. Given that, nine
different structures were obtained: RC 56-N, RC 56-M and RC 56-D; 1i-N, 1i-M and 1i-D; 1d-N, 1d-M and 1d-D.
Then, docking experiments were carried out using the available crystal structure of p300 HAT–Lys-CoA complex (PDB ID
3BIY) and the IFD procedure,[36],[37],[38],[39],[40] which takes into account receptor flexibility upon ligand binding in an attempt to
describe the inhibitor binding mode.
IFD of the nine structures into the Lys-CoA binding domain generated a number of protein/ligand complexes, and the best
ten scoring poses for each ionization state were retained and further analyzed. A comparison between the IFD
conformations for each ligand in the different N, M and D forms highlighted that all the docked positions could be clustered
into two main distinct binding modes A and B (Figure 2), independently of the ionization state of the inhibitor.
B
A
C
Figure 2. Alternative binding modes obtained for the cinnamoyl-based inhibitors at the p300 HAT domain. Figure 2A and 2B show a
schematic representation of the interactions between the protein and 1i, as representative inhibitor, for the binding mode A and B,
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respectively. The superimposition of the two binding modes (A: yellow, B: green) and Lys-CoA (violet) substrate is represented as well
(Figure 2C).
It is worth noting that a binding mode similar to A has been also predicted for compound RC 56 by Devipriaya and
Kumaradhas using a different docking protocol.[41]
In an attempt to gain a more accurate ranking of the ligand docking poses, the ligand-protein complexes were rescored
using the MM-GBSA approach implemented in the Schrödinger’s Maestro suite,[41] and the results are summarized in Table
2.
The binding pose A was predicted as the most favorable for both RC 56 and 1d inhibitors, although the lowest energy
orientations were obtained for two different ionization states. Comparable MM-GBSA-dG values emerged for binding modes
A and B of compound 1i-M, suggesting that both poses could be reliable. It is also worth noting that for both binding modes
the predicted binding energies reproduced the trend observed for the p300 HAT inhibitory activity, i.e. the most interesting
compounds were in the order 1i, 1d and RC 56.
Table 2. MM-GBSA-DG values obtained for ligand-protein complexes selected by the IFD procedure.
Binding mode A
Binding mode B
Ligand in
complex with
HAT p300
MM-GBSA-ΔG (Kcal/mol)
MM-GBSA-ΔG (Kcal/mol)
RC56-N
RC56-M
RC56-D
1d-N
-135.53
-126.98
-113.05
-136.46
-147.15
-120.63
-145.42
-154.93
-136.29
-127.95
-120.15
-117.82
-131.57
-128.09
-112.70
-137.21
-154.47
-111.96
1d-M
1d-D
1i-N
1i-M
1i-D
Molecular dynamics simulations.
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In order to assess the stability of binding modes A and B previously identified for compound 1i-M and to identify the most
energetically favorable structure, the two ligand-p300 HAT complexes were submitted to 28 ns MD simulations in explicit
solvent.
The superimposition between the final structures and the initial modeled structures showed that there were structural
rearrangements within the binding site of both complexes (see Figure S2, Supporting Information). Analyzing the dynamical
behavior of the complex between p300 HAT and the ligand with the binding mode A, we observed that the ligand did tend to
move more internally into the pocket to maximize the interaction with the Arg1410 (more than 110% of occupancy), while the
hydrogen bond with the Tyr1467 was lost and the interactions with this residue were later mainly of hydrophobic character.
In its final pose the ligand established hydrophobic interactions with Ile1457, Leu1463, Leu1398, Trp1466, Tyr1414, Ile1395
and Ile1435 and made a weak hydrogen bond in turn with the carbonyl backbone of Ser1396 or Trp1436 (Figure 3A and
3C). On the other hand, a marked rearrangement of the residues of the binding site was observed for the complex B. In its
final position the ligand made polar interactions with Arg1410 (over 70% of occupancy), with Tyr1467 (67% of occupancy),
and with the carbonyl backbone of Trp1436 (occupancy around 22%) (Figure 3B and 3D). Hydrophobic contacts were also
detected between 1i-M and Trp1466, Tyr1414, Leu1398, Trp1436, Tyr1446, Ile1435 and Tyr1467. In this final complex,
polar interactions as well as hydrophobic contacts were maximized. Accordingly, complex B resulted to be the most
energetically favorable one in terms of free energy of binding between ligand and protein, being substantial the difference
between the DG values of the two complexes (DDG of 9.47 Kcal/mol in favor of complex B that A).
The outputs of this study provide some useful information to aid the rational design of new cinnamoyl inhibitors of p300 HAT
in the future.
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B
A
C
D
Figure 3. Minimized structures resulting from MD simulations of 1i-M/p300 complexes A and B. Panels A and B show a 2D schematic
representation of the interactions between the protein and 1i for the binding mode A and B, respectively. Panels C and D provide the same
information in a 3D perspective For the sake of clarity, only a few key residues are labeled and hydrogen-bonding interactions are
represented by black dashed lines.
Conclusions
Herein we report a new series of cell-permeable cynnamoyl derivatives as inhibitors of p300 HAT addressing various
structural modifications to our hit RC 56, involving both the benzylidene moiety and the central cyclohexanone portion.
Among the tested derivatives, substituting the cyclohexanone with other central ring highlighted a decreasing trend in the
inhibition potency (1m,s, 2a), with the sole exception of the tetrahydrothiopyranone derivative (1n), characterized by a
comparable activity in respect of RC 56. As regards the cyclohexanone derivatives, replacing the bromine atom of RC 56
with other halogen atoms (1b-d) as well as a methyl group (1g) led to derivatives with IC50 values comparable with that of
our hit. In particular, among them 1d showed good inhibitory activity, proving to be five times more active (IC50 value of 8.1
µM) than reference compound, though data arising from cell-based assay showed higher percentage of cell death induction
in leukemic monoblast cells for compounds 1b and 1g, even greater than the one obtained with RC 56. Noteworthy, the
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cyclohexanone derivative 1i, characterized by the introduction of a second bromine atom on the benzylidene portion, proved
to be the most active compound with complete inhibition of p300 activity at 100 mM and IC50 value (2.3 ± 0.5 µM) fifteen
times lower than RC 56 (30.9 ± 8.2 µM) and, notably, very similar to that of C646 (3.6 ± 0.6 µM), regarded one of the most
potent p300 inhibitor. Furthermore, molecular modeling studies highlighted the main structural features involved in ligand-
protein complex formation as for the key role played by the phenolate moieties in establishing a salt bridge with amino acidic
residues at the active site, as stated by biological data (indeed, the substitution of hydroxyl groups with bromine atoms in
derivative 1h led to a remarkable decrease of the inhibition potency). Interestingly, two distinct binding modes within the
p300 HAT domain, one for RC 56 and 1d and another for 1i, were identified, suggesting that the introduction of a second
bromine atom may cause a rearrangement of the inhibitor within the enzymatic binding site.
In conclusion, this study provided useful insights for future development of novel small molecules as p300 HAT inhibitors. In
particular, the low micromolar inhibitor 1i represents a very interesting compound whose further rational modifications might
represent a line of inquiry for antitumor and antiviral vanguard chemotherapies.
Experimental section
Chemistry. General. Melting points were determined with a Büchi 530 capillary apparatus and are uncorrected.
Compounds purity were always > 95% determined by high-pressure liquid chromatography (HPLC). HPLC analyses were
carried out with Shimadzu LC-10AD VP CTO-10AC VP, using Discovery Bio Wide Pore C18 (10 cm x 4.6 mm, 3 mm)
1
column. Infrared (IR) spectra were recorded on a Perkin-Elmer Spectrum-one spectrophotometer. H NMR spectra were
recorded on a Bruker AC 400 spectrometer. Merk silica gel 60 F254 plates were used for analytical TLC (thin layer
chromatography). Developed plates were visualized by UV light. Column chromatographies were performed on silica gel
(Merck; 70-230 mesh). Concentration of solution after reactions and extractions involved the use of a rotary evaporator
operating at reduced pressure. Analytical results agreed to within ± 0.40% of the theoretical values. Dimethylsulfoxide-d6
99.9% (code 44,139-2) and deuterochloroform 98.8% (code 41,675-4) of isotopic purity (Aldrich) were used. Solvents were
reagent grade and, when necessary, were purified and dried by standard methods. Organic solutions were dried over
anhydrous sodium sulfate (Merck).
Microwave irradiation experiments. Microwave reactions were conduced using a CEM Discover system unit (CEM. Corp.,
Matthews, NC). The machine consists of a continuous focused microwave-power delivery system with operator selectable
power output from 0 to 300 W. The temperature of the contents of the vessel was monitored using a calibrated infrared
temperature control mounted under the reaction vessel. All experiments were performed using a stirring option whereby the
contents of the vessel are stirred by means of a rotating magnetic plate located below the floor of the microwave cavity and
a Teflon-coated magnetic stir bar in the vessel.
All the final compounds 1a-s and 2a were synthesized according to the procedure reported in literature[29],[31] and described
in Scheme 1. Spectroscopic, chemical and physical data of compounds 1a-b,e,g,i,l,n-r are already described in
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literature.[29].[31],[42] Spectroscopic, chemical and physical data of derivatives 1c-d,f,h,j-k,m,s are reported below.
1
Experimental procedure, yield (%), melting point (°C), recrystallization solvent, IR, H NMR of derivatives 2a and 3f are
reported below.
6-Hydroxy-[1,1’-biphenyl]-3-carboxaldehyde (3f). To a solution of 3k (13.15 mmol) in DME (180 mL) phenylboronic acid
(15.76 mmol), tetrakis(triphenylphosphine)palladium (0) (0.77 mmol), Ba(OH)2 8H2O (19.73 mmol) and H2O (22.6 mL) were
added in turn. The reaction was stirred at reflux under argon atmosphere for 4 h. The reaction was quenched with water,
extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, filtered and
evaporated under reduced pressure obtaining 6 g of crude product as yellow oil. Purification of crude product was performed
by column chromatography on silica gel (chloroform/ethyl acetate 3:1 as eluent) obtaining 0.88 g of pure product as yellow
oil (880 mg, 32% yield). Spectroscopic data as by reference.[43]
1
2,6-Bis(3-chloro-4-hydroxybenzylidene)cyclohexan-1-one (1c). Yield: 79 mg (40%); oil; H NMR (400 MHz, [D6]DMSO):
δ=1.72-1.75 (m, 2H, CH2), 2.85 (t, JCH2 = 8 Hz, 4H, CH2), 7.04 (d, Jo = 8.4 Hz, 2H), 7.41 (d, Jo = 8.4, Jm = 1.8 Hz, 2H), 7.50 (s,
2H, =CH-), 7.54 (d, Jm = 1.8 Hz, 2H), 10.4 ppm (s br, 2H, OH); IR (KBr): ν= 3401 (OH), 1639 (C=O) cm-1; Anal. Calcd for
C20H16Cl2O3: C 64.02, H 4.30, Cl 18.89, found C 63.70, H 4.02, Cl 18.55.
1
2,6-Bis(4-hydroxy-3-iodobenzylidene)cyclohexan-1-one (1d). Yield: 98 mg (22%); mp: 183 °C; methanol; H NMR (400
MHz, [D6]DMSO): δ=1.71-1.74 (m, 2H, CH2), 2.84 (t, JCH2 = 8 Hz, 4H, CH2), 6.95 (d, Jo = 8.4 Hz, 2H), 7.43 (d, Jo = 8.4, Jm =
2.1 Hz, 2H), 7.48 (s, 2H, =CH-), 7.86 (d, Jm = 2.1 Hz, 2H), 11 ppm (s br, 2H, OH); IR (KBr): ν=3223 (OH), 1651 (C=O) cm-1;
Anal. Calcd for C20H16I2O3: C 43.04, H 2.89, I 45.47, found C 42.68, H 2.58, I 45.21.
1
2,6-Bis((6-hydroxy-[1,1'-biphenyl]-3-yl)methylene)cyclohexan-1-one (1f). Yield: 125 mg (42%); oil; H NMR (400 MHz,
[D6]DMSO): δ=1.73-1.76 (m, 2H, CH2), 2.91 (t, JCH2 = 8 Hz, 4H, CH2), 7.05 (d, Jo = 8 Hz, 2H), 7.31 (d, Jo = 8 Hz, 2H), 7.42-
7.46 (m, 8H), 7.86-7.89 (m, 6H), 10.1 ppm (s br, 2H, OH); IR (KBr): ν=3223 s br, 1664 (C=O) cm-1; Anal. Calcd for C32H26O3:
C 83.82, H 5.72, found C 83.60, H 6.08.
2,6-Bis(3,4-dibromobenzylidene)cyclohexan-1-one (1h). Yield: 111 mg (36%); mp: 165 °C; benzene; 1H NMR (400 MHz,
[D6]DMSO): δ=1.72-1.75 (m, 2H, CH2), 2.86 (t, JCH2 = 8 Hz, 4H, CH2), 7.43 (d, Jo = 8.3, Jm = 1.9 Hz, 2H), 7.54 (s, 2H, =CH-),
7.83 (d, Jo = 8.3 Hz, 2H), 7.91 ppm (d, Jm = 1.8 Hz, 2H); IR (KBr): ν=1661 (C=O) cm-1; Anal. Calcd for C20H14Br4O: C 40.72,
H 2.39, Br 54.18, found C 40.37, H 1.99, Br 53.89.
2,6-Bis(3-chloro-5-fluoro-4-hydroxybenzylidene)cyclohexan-1-one (1j). Yield: 90 mg (22%); mp: 160-161 °C; toluene;
1H NMR (400 MHz, [D6]DMSO): δ=1.73-1.76 (m, 2H, CH2), 2.86 (t, JCH2 = 8 Hz, 4H, CH2), 7.42-7.47 (m, 6H), 10.97 ppm (s
br, 2H, OH); IR (KBr): ν=1664 (C=O) cm-1; Anal. Calcd for C20H14Cl2F2O3: C 58.42, H 3.43, Cl 17.24, F 9.24, found C 58.71,
H 3.22, Cl 17.61, F 9.49.
5,5'-((5-carboxy-2-oxocyclohexane-1,3-diylidene)bis(methanylylidene))bis(2-hydroxybenzoic acid) (1k). Yield: 131 mg
(40%); mp: 170-173 °C; toluene; 1H NMR (400 MHz, [D6]DMSO): δ=2.80-2.82 (m, 2H, CH2), 3.06-3.09 (m, 2H, CH2), 7.06 (d,
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Jo = 8.6 Hz, 2H), 7.61 (s, 2H, =CH-), 7.72 (d, Jo = 8.6, Jm = 1.2 Hz, 2H), 7.98 (d, Jm = 1.2 Hz, 2H), 12-13 ppm (s br, 2H, OH);
IR (KBr): ν=3500-2500 (OH), 1650 (C=O) cm-1; Anal. Calcd for C23H18O9: C 63.02, H 4.14, found C 63.34, H 3.89.
1
2,5-bis((E)-3-bromo-4-hydroxybenzylidene)cyclopentan-1-one (1m). Yield: 110 mg (33%); mp: 154-156 °C; toluene; H
NMR (400 MHz, [D6]DMSO): δ=2.99 (s, 4H, CH2), 6.89 (d, Jo = 8.6 Hz, 2H), 7.42 (s, 2H, =CH-), 7.58 (d, Jo = 8.6, Jm = 1.2 Hz,
2H), 7.91 (d, Jm = 1.2 Hz, 2H), 10.2 ppm (s br, 2H, OH); IR (KBr): ν=3229 (OH), 1671 (C=O) cm-1; Anal. Calcd for
C19H14Br2O3: C 50.70, H 3.14, Br 35.50, found C 50.98, H 3.00, Br 35.29.
1-benzyl-3,5-bis((E)-3-bromo-4-hydroxybenzylidene)piperidin-4-one (1s). Yield: 94 mg (38%); mp: 180 °C;
1
dioxane/diethyl ether; H NMR (400 MHz, [D6]DMSO): δ=3.42 (s, 4H, CH2 piperidinone), 3.57 (s, 2H, CH2Bz), 6.69 (d, Jo =
7.5 Hz, Jm = 1.2 Hz, 2H), 7.22-7.31 (m, 5H, Ph), 7.43 (d, Jo = 7.5, Jm = 1.2 Hz, 2H), 7.59 (s, 2H, =CH-), 8.26 (d, Jm = 1.2 Hz,
2H), 9.98 ppm (s br, 2H, OH); IR (KBr): ν=3225 (OH), 1674 (C=O) cm-1; Anal. Calcd for C21H19Br2NO3: C 51.14, H 3.88, Br
32.40, N 2.84, found C 51.41, H 3.70, Br 32.79, N 2.99.
4,4'-(2-Hydroxycyclohexane-1,3-diylidene)bis(methanylylidene)bis(2-bromophenol) (2a). To a well stirred suspension
of lithium aluminum hydride (50 mmol) in anhydrous THF (494 mL) cooled to 0 °C, a solution of RC 56 (0.75 g, 1.63 mmol)
in anhydrous THF (53 mL) was added drop wise. The reaction was then stirred at room temperature for 4 h. Crushed ice
was added to the reaction mixture until the formation of hydrogen vapors was stopped. The produced aluminum hydroxide
was filtered off by means a Buchner filter and the resulting solution was evaporated at reduced pressure obtaining 0.74 g of
crude product. Purification of crude product was performed by column chromatography on silica gel (chloroform as eluent)
1
obtaining 0.25 g of pure product as yellow oil (250 mg, 32% yield). H NMR (400 MHz, [D6]DMSO): δ=1.73-1.76 (m, 2H,
CH2), 2.84-2.87 (m, 4H, CH2), 7.02 (d, Jo = 8.44 Hz, 2H), 7.41 (d, Jo = 8.50 Hz, 2H), 7.49 (s, 1H, =CH-), 7.68 (s, 1H=CH-),
10.79 ppm (s br, 1H, OH); IR (KBr): ν=3468 (OH), 3234 (OH) cm-1; Anal. Calcd for C20H18Br2O3: C 51.53, H 3.89, Br 34.28,
found C 51.69, H 4.13, 33.92.
Biological methods
Enzyme assays. The expression and purification of the tag-free recombinant human p300 HAT domain enzyme (residues
1287-1666) was done using the method developed by Cole’s laboratory.[44] The standard filter binding assay was used to
measure the acetyltransferase activity of p300 in the presence of various inhibitors. A histone H3 peptide containing the
amino-terminal 20 residues (H3-20) was used as the substrate, and the concentration was set at 100 µM. 1 µM [14C]-Ac-
CoA as the acetyl donor. The reaction buffer contained 50 mM HEPES (pH 8), 1 mM EDTA, and 0.5 mM dithiothreitol (DTT).
The reaction time was 6 minutes at 30°C. A 30-µL reaction volume comprised of inhibitor, peptide, and cofactor incubated
for 5 minutes at 30°C followed by the addition of p300 (final at 0.025 µM). After 6 minutes of incubation the reaction was
quenched by spreading 20 µL of the reaction mixture over a Whatman P81 filter disc. Once the filter discs were dried, they
were washed three times with 50 mM NaHCO3 (pH 9.0) and re-dried. Acetylated products were quantified using the
MicroBeta2 (Perkin Elmer) after the addition of scintillation cocktail. The enzymatic reaction was controlled in the initial linear
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phase, with a typical conversion yield of less than 20%. Each measurement was done at least in duplicate with errors less
than 20%.
Cell-based assays. Drugs. SAHA (MERCK, Readington, NJ, USA) was dissolved in dimethylsulphoxide and used at 5 µM
concentration.
Cell lines. U937 cells (ATCC) were cultured using standard procedures, in RPMI (Euroclone) supplemented with 10% FBS
(Sigma) and 50 µg/ml penicillin-streptomycin and 2 mM glutamine.
Cell cycle Analysis. The cells were plated (2x105 cells/mL) and stimulated for 30 hours with compounds at 50 µM. Treated
and untreated U937 cells were harvested and resuspended in staining solution containing RNaseA, PI (50 µg/ml), sodium
citrate (0.1%), NP40 (0.1%) in cold PBS for 30 minutes in the darkness. Cell cycle distribution was assessed with a
FACScalibur flow cytometer using the Cell Quest software (Becton Dickinson, Milan, Italy). ModFit LT version 3 Software
(Verity, Topsham, ME, USA) was used for the analysis.
Cell death analysis. After stimulation with selected compounds for 30 hours at 50 µM, treated an untreated U937 cells were
collected in 0.1% sodium citrate and 50 µg/ml PI. After 30 min incubation, the percentage of cells with sub-G1 DNA was
analyzed with FACS (FACScalibur; BD Biosciences, San Jose, CA).
Molecular Modeling study. Protein and Ligand Preparation: The crystal structure of p300 HAT domain in complex
with a bisubstrate inhibitor, i.e., Lys-CoA, was retrieved from the RCSB Protein Data Bank (PDB ID 3BIY)[45] and used as
a target for the modeling studies. The inhibitor and the water molecules were deleted, and the Schrödinger Protein
Preparation Wizard[46] was then used to obtain a satisfactory starting structure for docking studies. This facility is designed to
ensure chemical correctness and to optimize a protein structure for further analysis. In particular, hydrogen atoms were
added, and bond orders and charges were assigned; the orientation of hydroxyl groups on Ser, Thr and Tyr, the side chains
of Asn and Gln residues, and the protonation state of His residues were optimized. Steric clashes were relieved by
performing a small number of minimization steps, not intended to minimize the system completely. In our study, the
minimization (OPLS force field) was stopped when the RMSD of the non-hydrogen atoms reached 0.30 Å. The analyzed
compounds RC56, RC172 and RC175 were constructed using Maestro 9.9[34] and then submitted to the LigPrep module[35]
using the default parameters.
Docking Studies: The IFD protocol,[36],[37],[38],[39] developed by Schrödinger was employed for prediction of ligand binding
modes and concomitant structural changes in the p300 HAT receptor. The prepared protein structure was used to generate
the receptor grid, which was centered on the crystallographic position of Lys-CoA substrate; afterwards, the simulations
were run setting the “Extended Sampling” protocol and refining residues within 6 Å of all ligand poses. All the other
parameters were left at the default values. Binding-free energy estimates were evaluated for the top docked complexes
using MM–GBSA calculations.
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Molecular Dynamics. Molecular Dynamic (MD) simulations have been performed through the use of AMBER 12 suite of
programs (http://ambermd.org/)[47] and the ff03.r1 force field. An appropriate number of counter-ions were added to
neutralize the system, and complexes were placed in an octagonal box of TIP3P water molecules. The distance between the
box walls and the protein was set to 10 Å. MD runs were carried out with a protocol previously validated.[48],[49],[50] Before MD
simulation, two stage of energy minimization were performed to remove bad contacts. In the first stage, we kept the protein
fixed with a constraint of 500 kcal/mol and we minimized the positions of the water molecules. Then, in the second stage, we
minimize the entire system, applying a constraint of 10 kcal/mol on the α carbons. MD trajectories were run using the
minimized structure as starting input. Constant volume simulations were performed for 50 ps, during which time temperature
was raised from 0 to 300 K using the Langevin dynamics method. Then, 150 ps of constant-pressure MD simulations were
performed at 300 K in three steps of 50 ps each. During the three periods of this second stage, the α carbons were blocked
with harmonic force constants of 10, 5, and 1 kcal/mol·Å, respectively. Finally a 28 ns MD simulation without restraint was
run at a constant temperature of 300 K and a constant pressure of 1 atm. The root mean square deviations (RMSDs) of the
α carbons were then calculated throughout the simulations with respect to the starting structure for both systems. After the
first ns, both 1i-M/p300 HAT complexes reached an equilibrium state, and stable RMSDs were detected during the
remaining time of MD simulations (see Figure S1, Supporting Information).
Acknowledgments:
We wish to thank FIRB RBFR10ZJQT_003, EU (Blueprint High Impact project, n° 28251) and MIUR (PRIN 2012ZHN9YH;
20152TE5PK), EPIGEN (MIUR-CNR), AIRC (17217), POR Campania FESR 2007-2013-MOVIE (B25C13000240007) and
COST EPICHEMBIO CM1406. Y. G. Z. was supported by AHA grant 12GRNT12070056 and NIH grant R01GM086717.
Keywords: Antitumor agents • Transferases • Medicinal chemistry • Drug discovery • Structure-Activity Relationships.
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Inhibiting p300 HAT in cancer therapy
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This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700040
ChemMedChem
HAT is an attractive anticancer target. Several HAT inhibitors have been identified, but all exhibit low potency or
pharmacodynamics limits. We report the design and synthesis, along with biological evaluation and theoretical investigation,
of potent and selective cinnamoyl compounds, highlighting the peculiar features required to develop effective HATi.
20
This article is protected by copyright. All rights reserved.