6-alkylsalicylates are selective Tip60 inhibitors and target the acetyl-CoA binding site

Article abstract of DOI:10.1016/j.ejmech.2011.11.001

Histone acetyltransferases are important enzymes that regulate various cellular functions, such as epigenetic control of DNA transcription. Development of HAT inhibitors with high selectivity and potency will provide powerful mechanistic tools for the elu

Full text of DOI:10.1016/j.ejmech.2011.11.001

European Journal of Medicinal Chemistry 47 (2012) 337e344
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
6-alkylsalicylates are selective Tip60 inhibitors and target the acetyl-CoA binding
site
,
,
,
Massimo Ghizzoni a 1, Jiang Wu b 1, Tielong Gao b, Hidde J. Haisma a, Frank J. Dekker a
**
*
,
,
Y. George Zheng b
a Department of Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of Groningen A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands
b Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone acetyltransferases are important enzymes that regulate various cellular functions, such as
epigenetic control of DNA transcription. Development of HAT inhibitors with high selectivity and potency
will provide powerful mechanistic tools for the elucidation of the biological functions of HATs and may
also have pharmacological value for potential new therapies. In this work, analogs of the known HAT
inhibitor anacardic acid were synthesized and evaluated for inhibition of HAT activity. Biochemical assays
revealed novel anacardic acid analogs that inhibited the human recombinant enzyme Tip60 selectively
compared to PCAF and p300. Enzyme kinetics studies demonstrated that inhibition of Tip60 by one such
novel anacardic acid derive, 20, was essentially competitive with Ac-CoA and non-competitive with the
histone substrate. In addition, these HAT inhibitors effectively inhibited acetyltransferase activity of
nuclear extracts on the histone H3 and H4 at micromolar concentrations.
Received 29 June 2011
Received in revised form
27 October 2011
Accepted 1 November 2011
Available online 10 November 2011
Keywords:
Histone acetylation
Epigenetics
Anacardic acid
Ó 2011 Elsevier Masson SAS. All rights reserved.
Histone acetyltransferase (HAT)
Inhibitors
1. Introduction
stability, [17] enzyme activity, [18] proteineprotein interaction, [19]
metabolism, [20,21] etc. On the chromatin template, generally,
The plant natural product anacardic acid (AA, 6-
pentadecylsalicylic acid) is isolated from cashew nut shell liquid
and is used in traditional medicine. [1] It exerts beneficial biological
effects such as antitumor activity [2] and antioxidant activity. [3]
AA was also shown to inhibit the activity of oxidative enzymes,
such as tyrosinase, [4] cyclooxygenase, [5] and lipoxygenase. [6]
Interestingly, AA and its derivatives were reported to modulate
the enzymatic activity of histone acetyltransferases (HATs), such as
p300 and p300/CBP-associated factor (PCAF), and to affect HAT-
dependent gene transcription [7e13].
HATs are grouped into distinct families based on their sequence
and structural homology [14,15]. The best studied families are the
GNAT (GCN5-related N-acetyltransferase) family that includes
PCAF and GCN5, the p300/CBP family that includes p300, and the
MYST family that includes Tip60 (TAT-interacting protein 60) and
MOF (maleless on the first). Acetylation of histones and other
proteins by HATs regulates chromatin restructuring, [16] protein
histone acetylation is connected to activation of gene transcription,
whereas acetylation of transcription factors can either activate or
inactive gene transcription [22]. Combinations of acetylation and
other posttranslational modifications regulate gene transcription in
response to extra- and intracellular stimuli. Moreover, accumulating
evidence reveals that HAT activities are deregulated in many
diseases, which highlights the pharmacologic importance of HATs as
potential drug targets [23,24]. Acetylations of the nuclear factor
kB
(NF- B) transcription factor as well as the histones play a crucial role
k
in activation of this pathway [25]. Recent years have seen great
efforts for designing and screening chemical HAT modulators
[26e28]. The known HAT inhibitor AA has been shown to inhibit NF-
kB mediated gene transcription, presumably by inhibition of the
HAT p300 [29]. Furthermore, it has been shown that AA inhibits
Tip60, which is one key member of the MYST family. AA blocks the
Tip60-dependent activation of the ataxia telangiectasia mutated
(ATM) protein kinase and the DNA-dependent protein kinase,
catalytic subunit (DNAePKcs) by DNA damage and sensitizes human
tumor cells to the cytotoxic effects of ionizing radiation [8]. This
indicates that HAT inhibition by small molecules provides a potential
novel therapeutic approach for cancer and inflammatory diseases.
The molecular mechanism by which AA and its analogs inhibit
HAT activity remains, however, poorly understood. It has been
* Corresponding author. Tel.: þ31 503638030; fax: þ31 50 3637953.
** Corresponding author. Tel.: þ1 4044135491; fax: þ1 4044135505.
E-mail addresses: f.j.dekker@rug.nl (F.J. Dekker), yzheng@gsu.edu (Y. George
Zheng).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.001

338
M. Ghizzoni et al. / European Journal of Medicinal Chemistry 47 (2012) 337e344
corresponding trimethylsilylalkynes in yields between 36 and 76%.
The trimethylsilyl protective group was cleaved to give the corre-
sponding alkynes 3ae3e. Alkynes 3ae3e were coupled to triflate 4
using Sonogashira couplings to provide compounds 5ae5e in yields
between 49 and 82% (Scheme 2). Hydrogenation of the alkynes
using Pd/C under H2 atmosphere and hydrolysis of the acetonide
using KOH in THF provided compounds 6a-6c and 6f. Hydrogena-
tion of 5c using higher catalyst loading resulted in partial or
complete reduction of the acetyl group to give either the ethyl (6e)
or hydroxyethyl (6d) functionality. Furthermore, hydrogenation of
alkyne 5e resulted in reduction of the pentylpyridin-2(1H)-one
functionality (5e) to give a pentylpiperidin-2-one functionality
(6g). Compounds 9, 12 and 15 (Scheme 2) present variations to the
6-alkyl substitution pattern of AA. These compounds were
synthesized by Sonogashira couplings of alkynes on building blocks
7, 10 and 13. Products 8, 11 and 14 were obtained in yields around
60%. These alkynes were hydrogenated using Pd/C under H2
atmosphere. The methyl ester and methoxy functionality in 8 were
simultaneously deprotected using BBr3 and the methyl ester in 11
was hydrolyzed using KOH in THF. Hydrogenation of 14 provided
the final product 15 in one step. Compound 16 (AA) to 26 have been
published previously [12].
Scheme 1. Synthesis of acetylene building blocks for Sonogashira coupling. a)
R-halide, K2CO3, DMF, b) TMS-acetylene, PdCl2(PPh3)2, Et2NH, CuI, PPh3, CH3CN, c)
TBAF, THF.
suggested that the salicylate functionality of anacardic acid binds to
the same pocket as the Ac-CoA pyrophosphate, [12] which suggest
competitive binding in respect to Ac-CoA. In contrast, some studies
suggested non-competitive inhibition for AA against Ac-CoA in
p300 and PCAF assays, [7,30] which implicates a different inhibitory
mechanism for p300 and PCAF.
3. Pharmacology
Herein, we describe the design and synthesis of a series of AA
analogs and the evaluation of their regulatory activity on the
human recombinant HATs p300, PCAF, and Tip60 as well as cellular
HAT activity. We aim to develop potent and selective inhibitors for
recombinant HATs that also inhibit cellular HATs. Furthermore, we
aim to gain a better understanding of the HAT inhibitory mecha-
nism of AA analogs.
3.1. Inhibitory potency of the AA analogs on three major HAT
proteins
The potencies of 16 (AA) and its analogs to modulate the ace-
tyltransferase activities of recombinant HATs were evaluated using
human recombinant Tip60, PCAF and p300, which represent the
three major HAT families in mammalian cells. Standard
radioisotope-labeled histone acetyltransferase assays were carried
out with 14C-labeled Ac-CoA as the acetyl donor. Histone N-
terminal peptides were used as the acetyl acceptor of the HAT
reaction: H4-20 for p300 and Tip60 catalysis, and H3-20 for PCAF
catalysis. The compounds shown in Scheme 1, 2 and in Table 1 were
screened for inhibition of HAT activity. The compounds in Table 1
were published previously [12] The activities of the three HATs in
2. Chemistry
A compound collection was designed based on a versatile
synthetic strategy, which includes two sonogashira couplings as
key steps. This resulted in salicylate derivatives with a phenethyl
substituent in the 6-position. This strategy allows including
substituents with different polarities and sizes. The sonogashira
couplings were performed under microwave irradiation, which
provided fast conversion with moderate to high yields. Aryl-
bromides were alkylated using alkylhalides to give the products
2ae2d in high yields (>90%) (Scheme 1). Alkylation of 1c provided
the N-alkylated product 2e as the main product in 55% yield. The O-
alkylated product was isolated in minor amounts. The N-alkylated
product 2e showed a characteristic amide carbonyl signal in the IR
spectrum, whereas this signal was absent in the corresponding O-
alkylated product. Subsequently, the arylbromides were coupled to
trimethylsilylacetylene by a Sonogashira coupling to yield the
the presence of 200 mM of each compound are collectively shown in
Fig. 1. Clearly, the effect of the AA analogs on the enzymatic activity
of the three HAT enzymes varies significantly from one to another,
which offers the opportunity to derive structureeactivity rela-
tionship (SAR).
The overall observation is that the 16 (AA) analogs show
stronger inhibitory potency for Tip60 compared to p300 and PCAF,
suggesting that the studied AA analogs have a general tendency for
specific inhibition for the MYST HATs. In particular, compounds 6a,
6c, 16 (AA), 17, 18, 19 and 20 inhibited more than 76% of the Tip60
Scheme 2. Sonogashira coupling and subsequent hydrogenation and deprotection to give anacardic acid derivatives 6ae6h. a) PdCl2(PPh3)2, Et2NH, CuI, PPh3, CH3CN, b) H2, Pd/C,
MeOH, c) KOH, THF.

M. Ghizzoni et al. / European Journal of Medicinal Chemistry 47 (2012) 337e344
339
Table 1
hydrophobic substitution in the salicylate 6-position is indispens-
able for Tip60 inhibition. Compounds 16 (AA), 17 and 6h show that
aliphatic side chains with 10e15 carbon atoms (16, 17) in the
salicylate 6-position provide good inhibitory potency in contrast to
aliphatic chains with 5 carbon atoms (6h). These data indicate that
side chains that correspond to the length of a linear aliphatic chain
with 10e15 carbon atoms provide good inhibitory potency. Also, it
seems plausible that the first atom of the side chain adjacent to the
salicylic ring needs to be a hydrophobic carbon, as replacement of
this carbon by a hydrophilic oxygen greatly diminished inhibitory
activity of the compounds. This is exemplified in compound 25 that
contains an alpha-oxygen and shows only modest inhibitory
activity.
Previously published anacardic acid devatives that were applied in this study [12].
R1
R2
R3
16 (AA)
17
18
H
H
H
H
H
H
OH
OH
e(CH2)14CH3
e(CH2)9CH3
e(CH2)14CH3
e(CH2)9CH3
19
20
H
H
In this study the potency of 16 (AA) deviates from studies
previously published by others. For example, the IC50 of 16 (AA) was
21
22
H
H
H
estimated be 8.5
mM for p300 and 5 mM for PCAF inhibition by
CH3
e(CH2)14CH3
Kundu et al. [7] In contrast, Souto et al. reported an IC50 between 50
and 100 uM for p300 [11]. In our previous characterization with
a different assay, the IC50 of 16 (AA) is about 1000
The last finding is well in line with our current finding that 16 (AA)
inhibits about 20% of the HAT activity of p300 and PCAF at 200 M.
The reported differences demonstrate that the inhibitory potency
of 16 (AA) depends strongly on the assay conditions and the
enzyme source.
mM for PCAF [12].
23
24
H
H
H
H
m
Of interest, some of the 16 (AA) analogs showed activation of the
acetyltransferase activity of p300 and PCAF in contrast with the
observed inhibitory effect on Tip60. Under the applied assay
conditions, 21 and 24 enhance the PCAF activity significantly at
25
26
H
H
H
H
eO(CH2)13CH3
eOCH2Ph
a concentration of 200
mM. Our observations are not alone: the
activation of HAT activity by several AA analogs was also observed
previously [7,31,32,33]. However, the exact mechanism, the
concentration dependence and time dependence of the activation
remain to be investigated in order to validate small molecule HAT
activation as a relevant phenomenon.
Among the effective Tip60 inhibitors, there are three salicylates
with a phenethyl substitution in the 6-position, 6a, 6c and 20.
These inhibitors have calculated logD values at pH 7.4 of respec-
tively 4.53, 3.07 and 4.19, which are lower than the calculated value
5.21 for 16 (AA) (calculated using MarvinSketch 5.4). The high logD
value for AA is generally considered to be a disadvantage for its
bioactivity [34] and therefore the inhibitors with the 6-phenethyl
activity at 200 mM, whereas their inhibitory effect on the activity of
p300 and PCAF was little or very modest (ꢀ35%). All these
compounds have the conserved 6-substituted salicylate function-
ality, which highlights that this motif is critical for Tip60 inhibition.
In contrast to compound 16 (AA) that inhibits the Tip60 activity
almost completely, compound 22, in which the carboxylate is
replaced for a methyl ester, completely lost its inhibitory potency.
Moreover, all the compounds with more than 70% inhibitory
activity at 200
position. In contrast, compounds with hydrophilic substituents, e.g.
6b and 6d, very weakly inhibit Tip60 at 200 M. This indicates that
mM have an extended hydrophobic substituent in 6-
m
Fig. 1. The effect of 16 (AA) and its analogs on the HAT activity of the enzymes Tip60, p300, and PCAF. The inhibitors were applied at a concentration of 200
the reaction mixture contained 100 nM Tip60, 10 M Ac-CoA, 100 M H4-20. For the p300 assay, the reaction mixture contained 5 nM p300, 10 M Ac-CoA, and 100
the PCAF assay, the reaction mixture contained 5 nM PCAF, 10 M Ac-CoA, and 100 M H3-20.
m
M. For the Tip60 assay,
m
m
m
m
M H4-20. For
m
m

340
M. Ghizzoni et al. / European Journal of Medicinal Chemistry 47 (2012) 337e344
Table 2
explained by the 67% sequence similarity in their catalytic region
(Blast sequence alignment) [35].
IC50 data of AA and 20 for the inhibition of HATs.
IC50 of 16 (AA)
IC50 of 20
Tip60
PCAF
p300
MOF
64 ꢂ 15
m
M
74 ꢂ 20
mM
3.2. Kinetic pattern of Tip60 inhibition by compound 20
>200
>200
m
m
M (40% inhibition)
M (40% inhibition)
M
>200
>200
m
m
M (3% inhibition)
M (30% inhibition)
mM
We carried out steady-state kinetic characterization of 20 for
inhibition of Tip60 with respect to Ac-CoA and the H4 peptide to
understand the mechanism by which the AA analogs inhibit the
HAT activity of Tip60. The inhibition pattern was analyzed by
measuring initial velocities of Tip60 at different concentrations of
one substrate, a fixed concentration of the second substrate, and
43 ꢂ 2
m
47 ꢂ 14
salicylate type can be considered improved leads for development
of Tip60 inhibitors. Inhibitor 20 inhibited the Tip60 activity by 88%
at 200
PCAF, in contrast to AA that showed about 20% inhibition of these
enzymes at 200 M. We therefore selected this inhibitor for further
evaluation in order to understand the potency and selectivity of this
AA analog for HAT inhibition. We determined the IC50 value for
inhibition of the HAT activity of Tip60 and MOF for 20 and
compared that with 16 (AA). MOF is another MYST family HAT that
is relevant in eukaryotic gene transcription. The IC50 was deter-
mined as the concentration of the inhibitor at which half of the
enzyme activity was inhibited. The results are shown in Table 2.
Under these experimental conditions, the IC50’s of 16 (AA) and 20
mM and had no observable inhibitory effect on p300 and
several selected concentrations of 20 (i.e. 0, 80, 120
mM). The
MichaeliseMenten plots and the LineweavereBurk double recip-
rocal plots are shown in Fig. 2. To determine the inhibition pattern
of compound 20, the MichaeliseMenten data were fitted to the
nonlinear non-competitive inhibition equation (1) [10]. In this
m
equation,
n is the measured initial reaction velocity, V is the
maximal velocity (in the absence of inhibitor), [S] is the concen-
tration of the varied substrate, Km is the corresponding apparent
MichaeliseMenten constant, [I] is the concentration of the inhib-
itor, and Kis and Kii are the slope and intercept inhibition constants,
respectively.
are 64
m
M and 74
mM for Tip60, and 43
m
M and 47
mM for MOF. In
contrast, IC50’s of both 16 (AA) and 20 are higher than 200
m
M for
v ¼ V½Sꢁ=½Kmð1 þ ½Iꢁ=KisÞ þ ½Sꢁð1 þ ½Iꢁ=KiiÞꢁ
(1)
p300 and PCAF (the exact IC50 values cannot be determined due to
compound insolubility at high concentrations). These data indicate
that 16 (AA) and the other tested analogs tend to be specific
inhibitors for the MYST family HATs. This is exemplified by 20,
which at 200 mM inhibited about 90% of Tip60 activity but had no
inhibitory impact on p300 and PCAF (Fig. 1). The comparable
inhibitory potency for the MYST family HATs Tip60 and MOF can be
The MichaeliseMenten data were fit to equation (1) and yielded
Kis of 54 M and Kii of 572 M. Because Kis is 10-fold smaller than Kii,
m
m
it is concluded that the inhibition of Tip60 by 20 is essentially
competitive with respect to Ac-CoA, binding to the same enzyme
form. In the primary double reciprocal plot of E/V versus 1/[Ac-CoA]
a series of straight lines intersect at a point very close to the
Fig. 2. Steady-state kinetic characterization of Tip60 inhibition by 20. (a) and (b) MichaeliseMenten and LineweavereBurk plots showing relation of Tip60 activity versus Ac-CoA
concentration at three selected concentrations of 20 (0 M, red; 80 M, blue; 120 M, green). The reaction contained 50 nM Tip60 and 100 M H4-20. (c) and (d) MichaeliseMenten
and LineweavereBurk plots showing function of Tip60 activity versus H4-20 concentration at three selected concentrations of 20 (0 M, red; 80 M, blue; 120 M, green). The
reaction contained 25 nM Tip60 and 2 M Ac-CoA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
m
m
m
m
m
m
m
m

M. Ghizzoni et al. / European Journal of Medicinal Chemistry 47 (2012) 337e344
341
ordinate, which further supports the competitive inhibition mode.
The measurable Kii may be an indication that 20 has additional
weak binding site(s) in Tip60. On the other hand, the intersecting
point in the double reciprocal plot of E/V versus 1/[H4-20] is clearly
positioned to the left of the ordinate. The kinetic data fitting yielded
at four different regions in the Tip60 structure. In particular, half of
the generated poses are located in the Ac-CoA binding pocket of
Tip60 (i.e. region I). A further zoom-in examination reveals that in
this site 20 or the adenine group of Ac-CoA interacts with several
common amino acid residues of the Tip60, such as Ser364, Lys331,
and Arg326. It is also interesting that the hydrophobic tail in
molecule 20 shows a similar orientation as the long chain of Ac-
CoA. These docking results suggest that 20 targets the active site
of Tip60, which coincides well with the aforementioned steady-
state kinetic analysis showing that inhibition by 20 is predomi-
nantly competitive with respect to Ac-CoA. The docking model
provides a structural basis for future optimization to obtain more
potent and selective AA analog inhibitors.
Kis of >660 mM and Kii of 78 mM. Therefore, 20 is non-competitive
against the peptide substrate, preferentially binding to the enzyme
form which is different from that the peptide binds to. The inhib-
itory pattern of 20 also offers insight into the kinetic mechanism of
Tip60 catalysis. It suggests a likely ordered substrate binding
pathway: Ac-CoA binds to Tip60 first and the peptide binds at
a point downstream of Ac-CoA binding.
3.3. Docking study
3.4. Inhibition of cellular HAT activity
To elucidate the structural basis of Tip60 inhibition by the AA
analogs, we performed a docking study of 20 with the crystal
structure of Tip60 HAT domain (PDB ID 2OU2). As displayed in
Fig. 3, the 50 structural poses of the ligand were found to be located
The inhibition of HAT activity in nuclear extracts from HeLa cells
or tissue samples from different brain regions from rats was studied
using an ELISA assay. In this assay either the biotinylated histone H3
Fig. 3. Docking of 20 in the crystal structure (2OU2) of Tip60 HAT domain. (a) The spatial distribution of the 50 docking poses is clustered into four regions. (b) Ac-CoA and one pose
in region I interact with some common residues of Tip60.

342
M. Ghizzoni et al. / European Journal of Medicinal Chemistry 47 (2012) 337e344
Fig. 4. Concentration dependent inhibition of HAT activity in HeLa nuclear extracts by AA analogs. Averages and standard deviations of triplicates are reported relative to the
positive control in which no inhibitor was present and the negative control where no enzyme was present.
(aa 1e21) or histone H4 (aa 2e24) peptides were linked to a 96-
well plate via streptavidin. The histone peptides were subjected
to acetylation by using nuclear extracts as enzyme source and
histone acetylation was detected using an anti-acetyl Lysine anti-
body. Most of the studied 6-alkylsalicylates show concentration
dependent inhibition of histone H4 acetylation (Fig. 4). Compounds
with a polar chain in the salicylate 6-position have a strongly
reduced HAT inhibitory potency (6b, 6g, 6h, 23, 26). Also methyl-
ation of the salicylate carboxylate (22) is detrimental for the HAT
inhibitory activity. An aliphatic substituent in the salicylate 6-
position is beneficial for inhibition of HAT activity (16 (AA), 17, 18,
19). Interestingly, compound 21 that has been described previously
to be a more potent inhibitor of histone acetylation than 16 (AA) in
HEP G2 cells also showed high inhibitory activity on HeLa nuclear
extracts [12].
(Supplementary data e Figure S1). Interestingly, a change in
inhibitory potency was observed between histone H3 and histone
H4. For example 16 (AA) and 17 show almost equal potency for both
histones, whereas for example, 6a, 6f, 15, 18 and 20, are more
potent for histone H3 compared to histone H4. The opposite is true
for compounds like 21, 24 and 25. Given the complexity of HAT
enzyme composition, distribution, and catalytic turnover rates of
each HAT complex in the nucleus, it would be very difficult to
interpret the differential impact of the AA analogs on H3 acetyla-
tion and H4 acetylation. However, the data shown here indicate
that selective inhibitors towards histone H3 or H4 acetylation could
be developed.
An assay on the HAT activity of nuclear extracts of brain tissue
samples from rats showed no significant difference between HAT
activity in brain tissue in different brain regions (n ¼ 3, p > 0.05)
(Fig. 5). Studies on two different animals showed no significant
deviations between the animals (n ¼ 2, p > 0.05). HAT inhibition
studies with the inhibitors 16 (AA) and 20 demonstrated that both
compounds inhibited the HAT activity of the nuclear extracts of
different regions significantly (p < 0.05). Compound 20 showed
slightly impaired inhibition compared to 16 (AA), which is statis-
tically significant for the hippocampus (p < 0.05).
It is of note that, the inhibition profile of the AA analogs on
nuclear extract acetyltransferase activity correlates well with the
assay on recombinant Tip60. For instance, the compounds that
inhibited the cellular HAT activity more then 50% at 200 mM, e.g. 6a,
6c, 6f, 11, 12, 16 (AA), 17, 18, 19, 20, 21, 24, and 25, also inhibited the
Tip60 HAT activity. On the other hand, most of the compounds that
showed poor inhibition of HeLa nuclear extract activity, 6b, 6g, 6h,
22, 23, and 26, were also very poor inhibitors for the recombinant
enzyme. The compounds 6d, 6e and 15 are exceptions because they
seem to be more potent on HeLa nuclear extracts than for the
recombinant Tip60. These observations could be explained by the
presence of multiple HATs in nuclear extracts.
4. Conclusion
AA (16) analogs with various 2-phenylethane substituents in the
salicylate 6-position were synthesized and tested for modulating
enzymatic activities of three major types of mammalian HAT
proteins. The recombinant enzyme inhibition assays demonstrated
that inhibitors 18, 19, 20 and 21 are selective for Tip60 in compar-
ison with PCAF and p300. Compounds that inhibited Tip60 showed
in most cases also inhibition of HAT activity in HeLa nuclear
extracts, which suggests that MYST type HATs play an important
role in the HAT activity of the studied extracts. Inhibitor 20 was
subjected to further investigation because its logD value is lower
than AA, whereas it inhibitory potency on recombinant Tip60 as
well as HeLa nuclear extracts is maintained. Enzyme kinetics
showed that inhibition of Tip60 by 20 is competitive to Ac-CoA and
non-competitive to histone H4, which supports the hypothesis that
AA derivatives bind to the Ac-CoA binding pocket. Inhibitor 20
showed also equal potency to AA in nuclear extracts of tissue
samples from different brain regions of rats, which indicates that 20
inhibits HAT activity from various sources.
The inhibitory potency of the compound collection on histone
H3 acetylation by HeLa nuclear extracts was investigated at 50 mM
concentrations and compared to histone H4 acetylation
Fig. 5. Acetyltransferase activity of nuclear extract from tissue samples from different
brain regions in mice. The acetyltransferase activity was determined using an ELISA
assay with histone H3 as acetyl acceptor. The inhibitors 16 (AA) and 20 were applied in
5. Experimental section
5.1. Synthesis
a concentration of 30 mM. Experiments were performed in triplicate for two different
animals. The averages and standard deviations for the two animals are shown and
blank values were subtracted. *p < 0.05.
See supplementary data.

M. Ghizzoni et al. / European Journal of Medicinal Chemistry 47 (2012) 337e344
343
5.2. Protein expression
40 mg/mL. The reaction time for the enzymatic reaction was 15 min.
The brain tissue samples were obtained from Prof. Dr. B. Roo-
zendaal, Neurosciences, UMCG, Groningen, The Netherlands. The
samples were obtained from animal experiments that were
approved by the animal experiment committee (Dier Exper-
imenten Commissie, DEC) from this institute according to national
regulations described in the ‘law for animal experiments’.
His-tagged PCAF HAT domain (493-658) was expressed with the
pET28a vector. His-tagged full length Tip60 was expressed with the
pET21a(þ) vector. Recombinant p300 HAT domain was a gift from
Dr. Philip Cole. Human MOF (125-458) was expressed with the
pET19 vector. All the protein expression was carried out with E. coli
BL21(DE3). The His-tagged proteins were purified on Ni-NTA beads.
Protein concentrations were determined using the Bradford assay.
Proteins were flash frozen in a storage buffer containing 25 mM
HEPES pH ¼ 7.0, 500 mM NaCl, 10 mM DTT, 1 mM EDTA and 10%
Glycerol, and stored at ꢃ80 ꢄC.
5.5. Modeling
The PDB file of Tip60 structure (2OU2) was modified with
Maestro 9.0.211 software to add hydrogens and delete the water
and Ac-CoA molecules. The 3-dimensional structure of 20 was also
constructed with Maestro 9.0.211. AutoDock Version 4.2 was then
employed to generate PDBQT files for both the ligand and the
macromolecule for docking. AutoGrid was used to generate a grid
box covering the whole protein unit for docking processing. The
Genetic Algorithm with 2,500,000 maximum numbers of evalua-
tions and 50 generations for picking individuals was selected as the
docking parameters. Structural analysis with PyMOL was per-
formed following the docking process.
5.3. Biochemical inhibition assays
Radioisotope-labeled acetyltransferase assays were carried out
at 30 ꢄC in a reaction volume of 30
mL. The reaction buffer contained
50 mM HEPES at pH 8.0, 0.1 mM EDTA, 50 g/mL BSA, 1 mM
m
dithiothreitol, 0.1% Triton-X100, and 2% DMSO. 14C-labeled Ac-CoA
(Perkin Elmer) was used as the acetyl donor. The peptide containing
the N-terminal 20-amino acid sequence of histone H4 (i.e. H4-20)
was used as substrate for p300 and Tip60, and the peptide con-
taining the N-terminal 20-amino acid sequence of histone H3 (i.e.
H3-20) was employed as substrate for PCAF. The HAT reaction was
initiated with the HAT enzyme after the other components (Ac-CoA,
peptide substrate, and the inhibitor) were equilibrated at 30 ꢄC for
5 min. Rate measurements were based on initial conditions
(generally less than 15% consumption of the limiting substrate).
After the reaction, the mixture was loaded onto a Waterman P81
filter paper and then washed with 50 mM sodium bicarbonate (pH
9.0) for three times. The paper was air dried and the amount of
radioactivity incorporated into the peptide substrate was quanti-
fied by liquid scintillation counting. In all the cases, background
acetylation (in the absence of enzyme) was subtracted from the
total signals. The IC50 was determined as the concentration of an
inhibitor at which half of the enzyme activity was inhibited. For IC50
determination, a range of at least seven inhibitor concentrations
varied at least 20-fold around the IC50 were tested. All the assays
were performed at least twice, and duplicates generally agreed
within 20%. The conditions for the IC50 measurement were; for the
Acknowledgements
YGZ would like to acknowledge the partial financial support
from AHA grant 09BGIA2220207 and NIH grant R01GM086717. We
acknowledge Prof. Dr. B. Roozendaal, Neurosciences, University
Medical Centre Groningen, The Netherlands for providing samples
from rat brain tissue. We acknowledge A. Boltjes for his contribu-
tion to the synthesis of the anacardic acid analogs.
Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.11.001.
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