Enzyme kinetics and inhibition of histone acetyltransferase KAT8

Article abstract of DOI:10.1016/j.ejmech.2015.10.016

Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of anacardic acid (AA) was proposed. This enabled the calculation of the inhibition constant K_i of anacardic acid derivatives using an adaptation of the Cheng-Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT.

Full text of DOI:10.1016/j.ejmech.2015.10.016

European Journal of Medicinal Chemistry 105 (2015) 289e296
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Enzyme kinetics and inhibition of histone acetyltransferase KAT8
a
a
b
c
Hannah Wapenaar , Petra E. van der Wouden , Matthew R. Groves , Dante Rotili ,
c
,
d
a, *
Antonello Mai , Frank J. Dekker
Department of Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands
Department of Drug Design, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands
Department of Drug Chemistry and Technologies, ‘Sapienza’ University, Rome, Italy
a
b
c
d
Pasteur Institute, Cenci Bolognetti Foundation, ‘Sapienza’ University, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16
on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor
suppressor protein p53. Further studies on its biological function and drug discovery initiatives will
benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this
aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a
ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone
substrate. This mechanism is supported by affinity measurements of both substrates using isothermal
titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection
around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with ana-
cardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory
Received 10 August 2015
Accepted 7 October 2015
Available online 22 October 2015
Keywords:
Epigenetics
Histone acetylation
KAT8
Enzyme kinetics
Catalytic mechanism
i
action of anacardic acid (AA) was proposed. This enabled the calculation of the inhibition constant K of
anacardic acid derivatives using an adaptation of the ChengePrusoff equation. The results described in
this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized
small-molecule inhibitors for this HAT.
©
2015 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-
ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
particular the HAT lysine acetyltransferase 8 (KAT8) is marginally
explored in drug discovery projects.
Epigenetic modifications of histones, such a lysine acetylation,
The HATs are a disparate group of enzymes that can be divided
into different families based on their structural homology. The
three main families are GNAT (Gcn5-related N-acetyltransferases),
MYST (MOZ, YBF2/SAS3, SAS2 and TIP60) and p300/CBP (CREB
binding protein). The HAT of our interest, KAT8, is a member of the
MYST family. This enzyme was originally discovered in Drosophila,
where it is involved in dose-compensation of the X-chromosome
gene transcription in male flies. KAT8 functions in two protein
complexes, MSL and MSL1v1, that are conserved throughout the
eukaryotic kingdom, including humans [11]. Both KAT8 complexes
have been described to be responsible for acetylating lysine 16 on
histone H4 (H4K16) and were shown to play a role in cell cycle
progression [12]. However, only the MSL1v1 complex seems to be
involved in acetylation of the tumor suppressor protein p53 [13].
KAT8 has also been shown to play a role in embryonic stem cell
renewal. Embryonic stem cells lacking KAT8 lose differentiation
potential and show changes in morphology and gene expression of
essential transcription factors [14]. Thus, it is clear that KAT8 plays a
play a key role in the regulation of gene transcription. Histone
acetyltransferases (HATs) are a class of acetyltransferases that
catalyze the acetylation of ε-amino groups on lysine residues in
both histone and non-histone proteins. Through histone acetyla-
tion, they play a regulatory role in the chromatin structure, thereby
influencing gene transcription. The acetylation of non-histone
proteins, for example transcription factors, is involved in the
regulation of many processes, such as cell growth and inflamma-
tory signaling [1]. This enzyme class has been linked to the pa-
thology of various diseases, including cancer [2e4], inflammatory
diseases [5e7], viral infections [8] and neurological diseases [9,10].
However, knowledge on their role in specific diseases and drug
discovery efforts towards this class of enzymes are still limited. In
*
Corresponding author. Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands.
E-mail address: f.j.dekker@rug.nl (F.J. Dekker).
http://dx.doi.org/10.1016/j.ejmech.2015.10.016
223-5234/© 2015 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).
0

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H. Wapenaar et al. / European Journal of Medicinal Chemistry 105 (2015) 289e296
very important role in normal physiology and disease.
(Table 1), which is characteristic for a ping-pong mechanism. In a
ping-pong mechanism the donor substrate binds first to the
enzyme. In case of KAT8, Ac-CoA acts as an acetyl donor. Subse-
quently the acetyl group is transferred temporarily to a residue on
the enzyme and CoA leaves the binding pocket. Then the histone
substrate binds and the acetyl group is transferred onto its lysine
residue, upon which the second product is formed.
From the Vmax and the enzyme concentration, the turnover
number of the enzyme (kcat) can be calculated. This is the
maximum number of substrate molecules that the enzyme can
convert per catalytic site per unit of time. The kcat ranged between
0.2 and 1.1 molecules per minute, which is lower than the kcat
observed by Yang et al. [19]. This difference can be explained by the
concentration dependency of Vmax and kcat on the concentration of
both substrates (Table 1).
KAT8 is a bi-substrate enzyme that binds two substrates; acetyl
coenzyme A (Ac-CoA) and histone H4 containing free lysine
ε-amino groups. Development of inhibitors for bi-substrate en-
zymes requires knowledge of the catalytic mechanism. It is
important to understand if the substrates, acetyl coenzyme A and
histone H4, bind simultaneously or consequently and if the indi-
vidual binding events are inter-dependent. In addition, knowledge
on the catalytic mechanism combined with inhibitor kinetics, en-
ables the calculation of the assay-independent inhibition constant
i
(K ) from the assay-dependent inhibitory concentration (IC50) as
described by Cheng and Prusoff [15]. Therefore, we investigated the
catalytic mechanism of KAT8 using enzyme kinetic studies based
on models described by Copeland [16]. We demonstrated that the
non-selective HAT inhibitor anacardic acid (AA) [17] also inhibits
KAT8 and performed kinetic studies to further investigate this in-
hibitor. Based on the results, we proposed a model comprising the
catalytic activity of KAT8 and the inhibitory action of AA. We
employed this knowledge to study the inhibitory potency of a small
collection of anacardic acid derived inhibitors and to calculate their
As described by Copeland, in case of a ping-pong mechanism the
histone peptide should have little or no affinity for the enzyme in
absence of Ac-CoA. To find further evidence for this mechanism
isothermal titration calorimetry (ITC) experiments were performed
(Fig. 2). Titration of Ac-CoA to KAT8 indicated binding with an
respective binding constants (K
not reveal selectivity between both enzymes for the compound
collection that was investigated.
i
). Inhibition studies on p300 did
equilibrium dissociation binding constant (K
This is close to the K determined for GNAT family HATs [20] and
the yeast HAT ESA1 in the picNuA4 complex [21]. The stoichiometry
N) of the interaction was close to 1, showing that one molecule of
Ac-CoA binds to one molecule of enzyme. Subsequent titration of
histone substrate to the KAT8/Ac-CoA complex demonstrated a K
of 6.7 M for the histone substrate. In contrast, titration of the
d
) of 8.7 mM (Table 2).
d
(
2
. Results and discussion
d
2.1. Catalytic mechanism
m
histone substrate solution to the KAT8 enzyme in absence of Ac-
CoA showed no measurable binding. These observations are
consistent with the ping-pong mechanism for the KAT8 acetyl-
transferase activity as postulated based on the enzyme kinetic ex-
periments, providing further proof for this mechanism.
It is of interest to mention that binding of both substrates has an
unfavorable enthalpy component that is compensated for by a
strongly favorable entropy component (Table 2). Strongly favorable
entropy components in equilibrium binding kinetics are often
linked to the release of bound water molecules upon substrate
As described by Copeland16, steady state kinetic experiments
can be employed to determine by which catalytic mechanism the
enzyme operates. In our studies we adapted an enzyme activity
assay based on fluorescence detection of the HAT reaction product
CoA, as described by Gao et al. [18] for use with KAT8. In this assay
the CoA thiolate is detected by the thiol sensitive fluorescent dye 7-
0
diethylamino-3-(4 -maleimidylphenyl)-4-methylcoumarin (CPM).
As histone substrate, a synthetic peptide corresponding to the first
0 amino acids of the histone H4 N-terminal (histone H4 peptide)
2
was used. First, the K of the histone substrate and kcat of the
m
enzyme were determined using increasing concentrations of his-
tone substrate at constant concentration of Ac-CoA (Fig. 1A). Then
the catalytic mechanism was determined according to procedures
described by Copeland. The velocity of recombinantly expressed
KAT8 was determined at different concentrations of Ac-CoA in the
presence of varying concentrations of histone substrate (Fig. 1B).
Table 1
K
m
, Vmax and kcat of Ac-CoA at varying concentrations of histone H4 peptide (Fig. 1).
cat (min 1
ꢀ
)
Histone H4 peptide
V
max (pmol/min)
K
m
(
m
M)
k
15
30
60
90
m
m
m
m
M
M
M
M
3.4 ± 0.2
4.5 ± 0.3
9.9 ± 0.3
14.7 ± 0.6
1.1 ± 0.3
1.2 ± 0.3
2.1 ± 0.3
2.8 ± 0.5
0.2 ± 0.02
0.3 ± 0.02
0.7 ± 0.04
1.1 ± 0.06
m m
The K and Vmax of Ac-CoA were determined. Both K and Vmax of
Ac-CoA increased at increasing concentrations of histone substrate
Fig. 1. Kinetics and catalytic mechanism of the bi-substrate enzyme KAT8. A) Determination of Vmax, K
velocity was determined of recombinantly expressed KAT8 using 0e400 M histone substrate and 4
the non-linear MichaeliseMenten regression. B) KAT8 operates via a ping-pong mechanism. The steady-state velocity was determined of KAT8 at 0e20
presence of 15, 30, 60 and 90
pong mechanism.
m
and kcat of the histone substrate (histone H4 peptide). The steady-state
M Ac-CoA. Vmax, K and kcat of the histone substrate were derived from
M of Ac-CoA in the
m
mM of histone substrate. Both K and Vmax of Ac-CoA increased at increasing concentrations of histone substrate (H4peptide), which suggests a ping-
m
m
m
m

H. Wapenaar et al. / European Journal of Medicinal Chemistry 105 (2015) 289e296
291
Fig. 2. ITC experiments support a ping-pong mechanism. ITC experiments were done to determine the affinity of the substrates for KAT8. A) Titration of Ac-CoA to KAT8
demonstrated a K of 8.7 M. B) Titration of the histone substrate (histone H4peptide) to the KAT8/Ac-CoA complex demonstrated a K of 6.7 M. However, this peptide showed no
affinity for KAT8 in absence of Ac-CoA. Based on these data the binding energies were calculated as shown in Table 2.
d
m
d
m
Table 2
CoA showed bound CoA rather than Ac-CoA and acetylation of
cysteine-304 (PDB: 1MJA, Fig. 3B) [22]. Taken together, the postu-
lated ping-pong mechanism as observed in the enzyme kinetic
experiments can be rationalized by structural data for KAT8 and
closely related HATs thus further supporting the evidence for this
mechanism.
Parameters for Ac-CoA and the histone substrate as derived from ITC experiments
(
Fig. 2).
Ac-CoA vs KAT8 Histone H4 peptide vs Ac-CoA/KAT8
Kd (
N (stoichiometry)
H (cal/mol)
m
M)
8.7 ± 1.2
0.95
6.7 ± 2.1
1.03
D
10,470 ± 470
3732 ± 300
ꢀ10665
ꢀ6933
Despite this evidence we should note that enzyme kinetics for
HATs frequently seem to depend on the assay conditions. For
example, in contrast to the structural data described before, a ki-
netic study on the yeast analog ESA1 indicated the formation of a
ternary complex between the enzyme, Ac-CoA and the substrate in
catalysis [21]. Comparable complications were encountered in the
analysis of the catalytic mechanism of p300. Based on enzyme ki-
netics a ping-pong mechanism was proposed [23]. However, it was
demonstrated that an electrophilic acetyl-CoA affinity labelling-
based probe did not target a residue that is critical for catalysis,
arguing against a ping-pong mechanism [24], but a ternary com-
plex mechanism could not be confirmed either. Despite the fact
that p300 contains a cysteine in the binding pocket no mutagenesis
studies have yet been performed to investigate whether this
cysteine is important for HAT activity. Based on structural and
biochemical data, it was proposed that p300/CBP uses a modified
mechanism denoted as the TheorelleChance (‘hit-and-run’) cata-
lytic mechanism. In the TheorelleChance mechanism, there is no
stable ternary complex as formed in a standard ternary complex
mechanism. After acetyl-CoA binds, the histone substrate associ-
ates weakly with the p300 surface, allowing the lysine to react with
the acetyl group, but kinetically only the interaction with acetyl-
CoA is important [25]. Therefore, we do not exclude the possibil-
ity that a more refined enzyme kinetic model will be assigned to
KAT8 in the future. Nevertheless, the steady state kinetic study here
clearly indicates a ping-pong mechanism in which Ac-CoA binds
first followed by binding of the histone H4 peptide. Therefore, we
ꢀ
T
G
D
S (cal/mol/degK) ꢀ17228
D
ꢀ6758
binding, which may also be the case in KAT8.
A requirement for a ping-pong mechanism is that the binding
pocket has a residue that can temporarily accept the acetyl group
before transfer to the substrate. This role is usually taken by a
cysteine residue because acetylation of a cysteine residue results in
a thioester, which is prone to aminolysis by the lysine ε-amine
functionalities of the histone substrates. Previously published KAT8
crystal structures (PDB: 2GIV, 3TOA [19]) indicate that cysteine-143
is close to the binding site of Ac-CoA (Fig. 3A). It seems reasonable
to presume that this cysteine residue plays a role as an initial
acceptor of the acetyl group in the postulated ping-pong mecha-
nism, although none of these published crystals structures contain
an acetylated cysteine residue in this position. Surprisingly, close to
this cysteine, both crystal structures show an acetylated lysine
residue (Ac-Lys-101) that is auto-acetylated by KAT8 itself. As
shown by mutagenesis studies, both the cysteine and lysine resi-
dues were essential for HAT activity [19]. The observation that the
cysteine residue is essential for catalysis also supports the postu-
lated ping-pong mechanism.
Further support for the ping-pong mechanism in KAT8 catalysis
comes from the crystal structure of the yeast HAT ESA1, which has a
high sequence and structural similarity with KAT8. Importantly, the
Ac-CoA binding pocket is almost identical and the aforementioned
cysteine and lysine residues are conserved (Cys-304 and Lys-262).
Interestingly, a crystal structure of ESA1 co-crystallized with Ac-
applied this mechanism for calculation of the K
i
values of inhibitors
of this enzyme using an adaptation of the ChengePrusoff equation

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H. Wapenaar et al. / European Journal of Medicinal Chemistry 105 (2015) 289e296
Fig. 3. Structural support for a ping-pong mechanism. A) A crystal structure of KAT8 (PDB:2GIV) shows Ac-CoA bound to the enzyme. Close to the binding pocket, a cysteine is
19
situated, which could facilitate a ping-pong mechanism. Additionally, an acetylated lysine is shown, which is essential for activity of KAT8 . B) A crystal structure of yeast ESA1, a
MYST family HAT and homologue to KAT5, shows an almost identical binding pocket with an acetylated cysteine. It is possible that either the cysteine or the lysine, or both,
contribute to a ping-pong mechanism.
ꢁ
as described by Copeland [16].
the published procedures of 5 N KOH in THF/water at 55 C required
5
days giving 8a in 24% yield. Therefore 7b was deprotected by acid
using 11 M HCl in dioxane/water. An improved yield of 46% was
observed under these conditions. The methyl protecting group of
2.2. Inhibitors e chemistry
10c was removed with boron tribromide in 53% yield. The removal
Anacardic acid is a known natural product HAT inhibitor, which
of the benzyl protective group could be done simultaneously with
the hydrogenation and was therefore used for the other com-
pounds. Hydrogenation and hydrogenolysis of 10d-e was done very
efficiently, giving 11d-e in 88e97% yield.
also shows activity on KAT8. A focused compound collection
inspired by AA was assembled from newly synthesized compounds
8
a-b, 11c-e and 13, previously synthesized compounds 14 and 15
[26] and the known p300 inhibitor C646. The compound collection
To investigate whether the salicylic acid could be replaced by a
was designed to vary the position of alkylation of the salicylate core
and the length and polarity of the aliphatic substituent. The com-
pounds were synthesized, using a convenient and flexible synthetic
route employing Sonogashira coupling as a key step as published
previously [27]. Using this strategy, different salicylate triflates or
halides were linked to various alkynes.
2
-hydroxy acetophenone group, product 13 was synthesized
0
(Scheme 1D). A substitution reaction using 2-bromo-2 -hydrox-
yacetophenone 12 and 1-heptane thiol provided 13 in one step in
5% yield.
8
6
-alkyl substitution of the salicylate core was achieved using
2.3. Inhibitor kinetics-KAT8
Sonogashira coupling of terminal alkynes to triflate 5. Triflate 5 was
synthesized from 2,6-dihydroxybenzoic acid using a previously
published two-step synthesis [28]. 5-alkyl substitution of the sa-
licylate core was achieved with aryl halides 9 as starting materials,
which were prepared using known procedures [29,30].
Commercially available terminal alkynes were used, except for
terminal alkynes 4a and 4b. Alkyne 4a was synthesized by benzy-
lation of 3a using previously published procedures [31]
To investigate the binding kinetics of the inhibitors, we con-
ducted enzyme kinetic studies on KAT8 with AA. The velocity of
KAT8 was determined at different concentrations of Ac-CoA and
constant concentration of the histone substrate in the presence of
varying concentrations of AA (Fig. 4A). A clear decrease in Vmax and
K
m
with increasing concentrations of AA was observed (Table 3).
The decrease in Vmax indicates that AA binds a site allosteric from
the Ac-CoA binding pocket. The decrease in K suggests that the
(Scheme 1A). Alkyne 4b was synthesized from methyl undec-10-
m
ynoate (1) by hydrolysis of the ester (2), reduction of the acid (3b)
and subsequent benzylation in an overall yield of 44% over 3 steps.
Sonogashira couplings in the salicylate 6-position (Scheme 1B)
and the 5-position (Scheme 1C) were performed with moderate to
good yields (46e98%). The purification was in some cases
demanding due to side products formed in the reaction. The
resulting alkynes were reduced by hydrogenation, which simulta-
neously resulted in the removal of the benzyl moiety from the
aliphatic alcohols. Intermediate 6b could not be completely puri-
fied after the Sonogashira coupling due to presence of a side
product with similar polarity. After an initial hydrogenation step,
which proved to reduce the triple bond, the impurity could be
removed. Removal of the benzyl required a separate hydrogenolysis
step giving 7b in 60% yield. Hydrolysis of the acetonide of 7a using
binding of AA stabilizes the binding of Ac-CoA. This is a charac-
teristic of uncompetitive inhibition, where the substrate must be
present for the inhibitor to bind. The same experiment was done
using different concentrations of the histone substrate and a con-
stant concentration of Ac-CoA (Fig. 4B). Strikingly, the curves do not
follow MichaeliseMenten kinetics when AA is present, but have a
sigmoidal appearance. This shows that there is cooperativity
resulting in a Hill coefficient, a measure for the slope of the curve,
that is not equal to
deWymaneChangeux (MWC) model [32]. It is not possible to
derive a true K from a sigmoidal curve and therefore the con-
centration of peptide that gives half-maximal velocity (khalf) is
determined, which resembles the K , but is dependent on the hill
slope (for calculation of khalf see SI). At increasing concentrations of
1
as described in the Mono-
m
m

H. Wapenaar et al. / European Journal of Medicinal Chemistry 105 (2015) 289e296
293
ꢁ
ꢁ
Scheme 1. Synthesis of anacardic acid derivatives. a) lithium hydroxide, THF/water, RT; b) LiAlH
4
, THF, 0 C - RT; c) Benzyl bromide, NaH, THF, 0 C - RT; d) PdCl
2
(PPh
3
)
2
, CuI, Et
(3 atm), Pd/C,
in DCM,
2
NH,
ꢁ
CH
3
CN, respective aliphatic alkynes or alkyne 4a-b, 100 C, MW; e) Hydrogenation triple bond and hydrogenolysis of benzyl protected salicylic acids or alkynes: H
MeOH/ethyl acetate, 40 C, PARR apparatus; f) 5 N KOH, THF, 55 C or HCl (37%), dioxane, RT; g) deprotection of methyl protected salicylic acids: 1 M BBr
2
ꢁ
ꢁ
3
acetonitrile, ꢀ78 ꢁC to RT; h) 1-heptanethiol, KOH, MeOH, 0 C to RT.
ꢁ
Fig. 4. Inhibitor kinetics of AA on KAT8. The velocity of KAT8 was determined at 0e20
decrease at increasing concentrations of AA. The velocity of KAT8 was determined at 0e200
40 M AA respectively.
m
M Ac-CoA in the presence of 0, 80, 160 and 240
m
mM AA respectively. Both K and Vmax
mM of the histone substrate (histone H4 peptide) in the presence of 0, 80, 160 and
2
m
Table 3
max and K
V
m
for Ac-CoA at different concentrations of inhibitor AA. Vmax, hill slope and khalf for histone H4 peptide at different concentrations of inhibitor AA (Fig. 4).
Ac-CoA Histone H4 peptide
half
max (pmol/min) max (pmol/min) (mM)
[AA] (mM)
V
K
m
(
mM)
V
Hill slope
k
0
8
1
2
17 ± 0.7
12 ± 0.3
9.2 ± 0.3
6.1 ± 0.2
4.8 ± 0.5
2.4 ± 0.2
1.9 ± 0.2
1.0 ± 0.1
13 ± 0.7
12 ± 0.5
12 ± 0.5
12 ± 0.4
1.5 ± 0.2
2.3 ± 0.3
3.4 ± 0.6
3.4 ± 0.3
36
47
54
0
60
40
514
AA, the Vmax is constant, but an increase is observed in khalf. This
suggests that the binding of AA opposes the binding of the histone
substrate.
The cooperativity may be explained using a model based on the
existence of two conformations of the enzyme, E and E* (Fig. 5). Ac-
CoA binds the free enzyme (E), which has low catalytic activity. The

294
H. Wapenaar et al. / European Journal of Medicinal Chemistry 105 (2015) 289e296
m
catalytic mechanism, the K of one substrate depends on the
concentration of the other substrate. The observed IC50 values are
therefore dependent on the concentrations of both substrates in the
assay and their respective K values. This will give large variations
m
in the IC50 values observed in different assays and makes direct
comparison of inhibitors published in literature impossible. Cor-
recting for the assay conditions by calculating the K
comparison between assays and assay conditions. The K
i
enables
values
i
were calculated from the IC50 values using a model that takes into
account that the enzyme operates via a ping-pong mechanism as
observed in the enzyme kinetic study. It also takes into account that
the inhibitors like AA affect only one form of the enzyme, EX, as
shown by the kinetic model (Fig. 5). According to Cheng and Prusoff
[
15] if these requirements are met, Equation (1) can be used, which
includes the K values of both substrates and their respective
concentrations used in the assay. The K values of the inhibitors
were calculated (example calculation in SI) using Equation (1) in
which K is the K of Ac-CoA at the concentration of histone sub-
strate used in the assay (2.1 M), K is the K of the histone sub-
strate at the concentration of Ac-CoA used (71 M) and A and B the
m
i
a
m
Fig. 5. Proposed model for the inhibitory and catalytic activity of AA and KAT8. Ac-CoA
binds to the free enzyme (E), which has low catalytic activity. The histone substrate is
not able to bind this conformation. Following the ping-pong mechanism, the acetyl
group (X) is transferred to the enzyme (EX). Binding of the histone substrate (S) in-
duces a conformational change of the enzyme (E*XS), which is catalytically active. Ac-
CoA has no or lower affinity for this conformation. The histone substrate is acetylated
and leaves the enzyme as product (Ac-histone substrate) upon which the free enzyme
conformation (E) is regenerated. The inhibitor AA binds to EX and stabilizes the
catalytically inactive conformation, therefore inhibiting the catalytic activity of the
enzyme, but increasing the affinity of Ac-CoA.
m
b
m
m
concentrations of either substrate in the assay (4 and 60
respectively).
mM
ꢀ
ꢁ
IC50 ¼ Ki 1 þ K þ K
a
B
KbA
A
(1)
a
The results show that the inhibitors 15 and 11d with the 10
carbon atom aliphatic tail bind slightly better than the inhibitors
with the 15 carbon atom tail AA and 11e. The inhibitors with the 5
carbon atom tail 14 and 11c completely lose their affinity within the
investigated range of concentrations as was observed in a previous
study on KAT5 [26]. This indicates that hydrophobic interactions
with the aliphatic tail play a major role in the inhibitory potency of
this type of compound for KAT8. The importance of hydrophobic
interactions of the aliphatic tail is further confirmed by the loss of
potency of compound 8b that includes an aliphatic alcohol in the
aliphatic tail. The substitution position of the salicylate makes a
small difference in the observed potencies with the best potency
observed in the salicylate 6-position. We also note that replacing of
the salicylate moiety by a 2-hydroxy acetophenone moiety (13)
completely removed activity, stressing the importance of the sa-
licylate functionality.
histone substrate is not able to bind this conformation, which is
shown by the ITC data (Fig. 2). Upon binding of Ac-CoA, the acetyl
group (X) is transferred onto the enzyme (EX) and the histone
substrate (S) can bind. This is shown by the ITC experiments as well
as the mechanistic studies. Binding of the histone substrate induces
a conformational change (E*XS), which is catalytically active. Ac-
CoA has lower or no affinity for this conformation, which is
shown by the increase in K
m
of Ac-CoA induced by higher con-
centrations of histone H4 peptide (Fig. 1). The histone substrate is
acetylated, leaves the enzyme as product (Ac-histone substrate)
and free enzyme (E) is regenerated. The inhibitor AA binds to EX,
thereby stabilizing the catalytically inactive conformation of the
enzyme (E). This is shown by the increase in K
m
of the histone
substrate and especially by the decrease in K of Ac-CoA. Ac-CoA
m
regains affinity for the enzyme, even though the enzyme activity is
inhibited. Additionally, an increasing concentration of histone
substrate, which induces conformation E* for which AA has little or
no affinity, will eventually be able to restore activity of the enzyme.
Cooperativity between an active and an inactive conformation can
cause the sigmoidal behavior observed in the inhibitor kinetics
with the histone substrate. This was also observed in case of the bi-
substrate enzyme phosphofructokinase [33]. MichaeliseMenten
kinetics can be observed when the enzyme has maximal activity. A
shift towards sigmoidal kinetics is therefore only observed in case
of the histone substrate where the khalf decreases due to the
presence of an inhibitor. Based on mechanistic and inhibitor kinetic
studies, we propose a model comprising the catalytic activity of
KAT8 and the inhibitory action of AA, which can be used to further
investigate the inhibitor properties.
2.5. Inhibitors e P300
In order to assess the selectivity of the KAT8 inhibition by the
compounds of this focused collection, we tested them for inhibitory
potency on p300. Towards this aim we employed an assay based on
radiolabeling of the histone substrate (Table 4). The reference
compound C646 had an IC50 of 0.32
literature [34]. AA showed 97% inhibition at a concentration of
50 M. The salicylic acid derivatives inhibited p300 as well and
mM, which is consistent with
m
showed comparable SAR as observed for KAT8. This suggests a
similar binding mode and interactions, although KAT8 and p300
are structurally very different. It is not possible to align either the
amino acid sequence or the 3D structures of KAT8 and p300 by
conventional means. It is however possible that either the Ac-CoA
or histone substrate pockets, due to the similarity of the ligands,
show a certain resemblance thus resulting in a comparable SAR for
these inhibitors.
i
2.4. Inhibitors e K calculation
The inhibitory potency for the HAT enzyme KAT8 was deter-
mined using the same fluorescence-based assay as used for the
3. Conclusions
kinetic studies. The inhibitory concentrations 50% (IC50) were
determined if more than 50% inhibition was observed at 400
inhibitor concentration. As shown in the kinetic studies for the
m
M
In this study, the catalytic mechanism of KAT8 histone acetyl-
transferase has been investigated. Enzyme kinetic experiments

H. Wapenaar et al. / European Journal of Medicinal Chemistry 105 (2015) 289e296
295
Table 4
Inhibitory potency of anacardic acid (AA) derivatives on KAT8 and p300. The inhibitory potency for KAT8 was measured using an assay based on fluorescent detection of CoA.
The K values were calculated using an adaptation of the ChengePrusoff equation based on the postulated ping-pong mechanism and inhibitor kinetics (Equation (1)). The
inhibition of p300 was measured using an assay based on radiolabeling of the histone substrate.
i
Compound
AA
Structure
KAT8 K
i
(m
M)
P300 inhibition at 50
97%
mM
64 ± 8.9
1
1
1
5
37 ± 7.0
88%
4
No inhibitiona
No inhibitionb
85%
1e
79 ± 11
1
1
1d
1c
57 ± 6.6
95%
No inhibitiona
No inhibitionb
1
8
3
b
No inhibitiona
No inhibitionb
No inhibition
157 ± 7.2
8
a
No inhibitiona
No inhibitiona
No inhibition
C646
IC50 ¼ 0.32
mM
a
No Inhibition at 400
No Inhibition at 200
m
m
M.
M.
b
indicate that this bi-substrate enzyme operates by a ping-pong
mechanism. This mechanism is supported by the observation that
binding of the first substrate Ac-CoA is required for binding of the
second substrate histone H4 as determined by ITC measurements.
The presence of cysteine 143 in the KAT8 active site combined with
the previous evidence that this residue is essential for catalysis
further supports the evidence for a ping-pong mechanism for
acetyltransferase activity of KAT8. We employed this model for
SAR as on KAT8, suggesting a similar binding mode even though the
two enzymes are structurally different.
This study gives insight in KAT8 through the catalytic mecha-
nism and presents a series of small-molecule inhibitors for this
HAT. Based on kinetic studies of AA and the catalytic mechanism, a
model has been proposed comprising the catalytic activity of KAT8
and the inhibitory action of AA, which includes an active and an
inactive conformation of the enzyme. This provides a basis for
development of inhibitors and the interpretation of the enzyme
inhibition studies, which will ultimately enable the exploitation of
KAT8 as a novel drug target in disease.
i
calculation of the K values of inhibitors of this enzyme. In order to
generate small molecule inhibitors for KAT8 we assembled a
focused compound collection around the known non-selective HAT
inhibitor AA. This compound collection was tested for inhibition of
KAT8. Kinetic studies were performed with the reference com-
pound AA and based on both inhibitor kinetics and mechanistic
studies, a catalytic model was proposed involving two different
Acknowledgments
We thank Prof. Y. G. Zheng for kindly providing the KAT8
plasmid. We acknowledge the European Research Council for
providing an ERC starting grant (309782) to F. J. Dekker. RF-2010-
2318330 grant (A.M.), IIT-Sapienza Project (A.M.), FP7 Projects
BLUEPRINT/282510 and A-PARADDISE/602080 (A.M.), Sapienza
Ateneo Award Project 2014 (D.R.), PRIN 2012 (prot.2012CTAYSY)
(D.R.).
conformations of the enzyme. The equilibrium binding constant K
i
was calculated using an adaptation of the ChengePrusoff equation
based on the catalytic mechanism and the proposed model. AA and
its derivatives inhibited KAT8 and both the aliphatic tail and the
salicylate functionality proved to be important for binding. The
inhibitors were tested for activity on p300 and showed a similar

2
96
H. Wapenaar et al. / European Journal of Medicinal Chemistry 105 (2015) 289e296
Practical Introduction to Structure, Mechanism, and Data Analysis, second ed.,
Appendix A. Supplementary data
John Wiley & Sons, 2004, pp. 350e366.
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Supplementary data related to this article can be found at http://
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