Histone deacetylase activators: N-acetylthioureas serve as highly potent and isozyme selective activators for human histone deacetylase-8 on a fluorescent substrate

Article abstract of DOI:10.1016/j.bmcl.2011.07.080

We report, for the first time, that certain N-acetylthiourea derivatives serve as highly potent and isozyme selective activators for the recombinant form of human histone deacetylase-8 in the assay system containing Fluor-de-Lys as a fluorescent substrate. The experimental data reveals that such activating feature is manifested via decrease in the K_m value of the enzyme's substrate and increase in the catalytic turnover rate of the enzyme.

Full text of DOI:10.1016/j.bmcl.2011.07.080

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5920–5923
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Histone deacetylase activators: N-acetylthioureas serve as highly potent
and isozyme selective activators for human histone deacetylase-8
on a fluorescent substrate
Raushan K. Singh a, Tanmay Mandal b, Narayanaganesh Balsubramanian a, Tajae Viaene a, Travis Leedahl a,
a,
Nitesh Sule a, Gregory Cook a, , D.K. Srivastava
⇑
⇑
a Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108, USA
b Department of Organic Chemistry, University of Leipzig, 04103 Leipzig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
We report, for the first time, that certain N-acetylthiourea derivatives serve as highly potent and isozyme
selective activators for the recombinant form of human histone deacetylase-8 in the assay system con-
taining Fluor-de-Lys as a fluorescent substrate. The experimental data reveals that such activating feature
is manifested via decrease in the Km value of the enzyme’s substrate and increase in the catalytic turnover
rate of the enzyme.
Received 9 June 2011
Revised 20 July 2011
Accepted 22 July 2011
Available online 4 August 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Enzyme activation
Epigenetic regulation
Histone deacetylase
Isozyme selectivity
Thiourea derivative
Of different modes of epigenetic regulation of gene expression,
acetylation and deacetylation of histore core proteins have gained
considerable interest in the recent years. This has been particularly
important from the point of view of developing therapeutic agents
for the treatment of various human diseases.1 The acetylation of
the lysine groups of histone is accomplished by the action of his-
tone acyltransferase (HAT), and their deacetylation reaction is cat-
alyzed by various isoforms of histone deacetylases (HDACs). The
dynamic (catalytic) actions of both these enzymes dictate the ex-
tent of acetylated form of histone within the chromatin structure.2
Since the acetylation eliminates the positive charges from the his-
tone’s lysine residues, their electrostatic interactions with nega-
tively charged phosphate groups of DNA is impaired, resulting in
loosening of the nucleosomal assembly and subsequently facilitat-
ing the transcriptional activation of various genes. The reversal of
the overall process (i.e., the suppression of the gene expression)
is manifested due to the diminution in the extent of acetyl func-
tionality within the histone core, which ‘compacts’ the nucleo-
somal assembly due to favorable electrostatic interaction
between the positively charged histone and negatively charged
phosphate groups of DNA. In contrast to this simplistic model, it
has been observed that the transcriptional activation and suppres-
sion of genes is not only controlled by the extent of histone acety-
lation/deacetylation but also on the type of histone molecules
which are acetylated.3 Since the extent and/or the type of histone
acetylation within the nucleosome is maintained by the catalytic
efficiencies as well as selectivity of HAT and HDAC isozymes, it is
possible to control the expression of various genes simply by mod-
ulating the activities of these enzymes by using isozyme selective
activators and inhibitors. With realization that small molecular
weight HDAC inhibitors have anticancer effect, there has been a
growing interest (by major Pharmaceutical and Biotech compa-
nies) in designing novel inhibitors against the above enzyme not
only for the treatment of cancers but various other diseases.4,5
Presently, two HDAC inhibitors, namely SAHA and romidepsin,
have been approved by FDA for the treatment of cutaneous lym-
phoma, and there are about a dozen of other inhibitors which are
at various stages of clinical trials.6,7
To date, 14 different types of HDACs are known to be involved
in promoting deacetylase reaction of different histones as well as
selected non-histone proteins.8 Based on the phylogenetic relation-
ship, HDACs have been grouped in four different classes: (1) Class I
(HDAC 1, 2, 3, and 8), (2) Class II (HDAC-4, -5, -6, -7, -9 and -10), (3)
Class III (Sirtuins), and (4) class IV (HDAC 11). Of these, whereas
Class I, II and IV HDACs catalyze metal dependent deacetylation
reaction, Class III HDACs/Sirtuins utilize NAD+ as the coenzyme
⇑
Corresponding authors. Tel.: +1 701 231 7413; fax: +1 701 231 8831 (G.C.);
tel.: +1 701 231 7831; fax: +1 701 231 8342 (D.K.S.).
E-mail addresses: gregory.cook@ndsu.edu (G. Cook), DK.Srivastava@ndsu.edu
(D.K. Srivastava).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.080

R. K. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5920–5923
5921
Table 1
and its ADP-ribose moiety serves as the acetyl group acceptor dur-
ing the catalytic cycle.9
Fold-activation of HDAC-8 by 10
derivatives
l
M
concentration of selected N-acetylthiourea
Of these, except for Class III HDACs (Sirtuins), there has been
limited interest in delineating the roles of HDAC activators in gene
expression, leading to the modulation of different physiological/
pathological processes.10 The activation of selected isoforms of
HDACs has been found to be desirable in treating chronic obstruc-
tive pulmonary disease (COPD), which is caused by increased
expressions of pro-inflammatory factors due to reduced HDAC
activity, and theophyllin has been found to relieve the symptoms
of COPD by indirectly activating HDACs (especially HDAC-2).11,12
Aside from the histone mediated regulation at the transcriptional
level, circumstantial evidence suggest the potential role of activa-
tion of specific isoforms of HDACs in promoting physiologically
desirable metabolic processes.13 Hence, not only HDAC inhibitors
but also the small molecular weight HDAC-activators are expected
to find applications in human health and diseases. However, to the
best of our knowledge, except for a U.S. patent application13, there
has been no report (in any scientific journal) of small molecular
weight compounds serving as the direct activators of metallo-
HDACs (i.e., HDAC class I, II, and IV).
In pursuit of investigating the structural–functional and inhibi-
tory features of the recombinant form of human HDAC-8, we have
been synthesizing a variety of structurally diverse small molecular
weight compounds, which would interact at the active site pocket
of the enzyme to unravel their influence on the enzyme catalysis.
With precedent of selected benzamide and thiourea derivatives
serving as the inhibitors of different class of HDACs,14 we
synthesized N(phenylcarbothiol)benzamide(TM-2-51) using ben-
zoylchloride and aniline as the precursors as outlined in Scheme 1.
We evaluated the effectiveness of TM-2-51(Scheme 1) on the
HDAC-8 catalyzed reaction using Fluor-de-LysÒ as the commercial
fluorogenic substrate as described previously.15 To our surprise, we
observed that the initial rate of the HDAC-8 catalyzed reaction was
significantly enhanced in the presence of TM-2-51, suggesting that
the latter compound served as an ‘activator’ rather than inhibitor
of HDAC-8. This preliminary finding prompted us to synthesize dif-
ferently substituted N-acetylthiourea derivatives (see the Supple-
Entry
Structure
Compound ID
TM-2-51
Fold-activationa
12
O
O
S
1
N
H
N
H
S
2
3
TM-2-87
TM-2-88
2
N
H
N
H
O
O
OMe
O
S
8.4
N
H
N
H
OMe
F
O
S
4
5
6
7
TM-2-138
TM-2-97
TM-2-105
TM-2-90
3.5
5.6
2.0
3.3
N
N
H
H
O
O
O
O
S
S
S
S
N
N
H
H
N
H
N
H
N
H
N
H
N
H
N
H
8
TM-2-101
1.1
Ph
O
O
O
S
S
S
CH
(s)
3
9
TM-2-104
TM-2-130
4.5
1.5
N
H
N
H
CH
3
10
N
H
N
H
(R)
N
H
N
H
11
12
TM-2-131
TM-2-126
3.0
1.2
F
O
O
S
S
N
H
N
H
CF
3
mentary information) and evaluated their efficacies (at 10 lM;
CF
3
N
H
N
H
the concentration of compounds customarily used in our lab for
initial screening of inhibitors/activators against HDAC-8 as well
as other pathogenic enzymes (Table 1).
A casual perusal of the data of Table 1 reveals that most of N-
acetylthioureas, with a few exceptions, serve as activators of
HDAC-8. Depending on the structure of the activator, the activity
13
TM-2-125
1.1
CF
3
F
O
S
14
15
TM-2-107
TM-2-139
3.0
1.0
N
H
N
H
S
F
O
S
of HDAC-8 is increased up to 12-fold by TM-2-51 (at 10 lM con-
N
N
H
H
centration) and there appears to be a strong structure–activity
relationship among N-acetylthiourea derivatives. As long as one
of thiourea’s nitrogen forms the amide linkage with benzoic acid
and the other nitrogen is directly attached to a six member aro-
matic (TM-2-51, TM-2-88) or cyclic (TM-2-97) ring, the activator
exhibits maximal potency. Several factors, for example, the pres-
ence of methylene group(s) between thiourea nitrogen and aro-
matic ring (TM-2-105, TM-2-90, TM-2-101, TM-2-125, TM-2-107,
TM-2-139), the presence of fluoro group(s) at either of the aro-
matic rings (TM-2-138, TM-2-126, TM-2-125, TM-2-139) and/or
a
Defined as the ratio of the initial rate of the HDAC-8 catalyzed reaction in the
absence and presence and of 10 M activator.
l
bulky substituents (TM-2-87 and TM-2-101) all appear to impair
the activation potencies of N-acetylthioureas.
Given that TM-2-51 serves as the most potent activator of
HDAC-8 at 10 lM concentration, it was of interest to determine
its apparent binding affinity for the enzyme and its maximal
NH2
O
O
O
S
NH4NCS
(1:1 eq)
Acetone /reflux
Benzoyl isocyanate
Aniline
Cl
NCS
N
H
N
H
Acetone/
reflux
Benzoyl chloride
N
-(phenylcarbamothioyl)-benzamide
Scheme 1. Synthetic scheme of TM-2-51.

5922
R. K. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5920–5923
Table 2
activation potency at saturating concentration. Toward this goal,
we measured the initial rate of the HDAC-8 catalyzed reaction in
the presence of increasing concentration of the activator. Figure 1
shows the increase in the fold activation of the enzyme (given by
the ratio of the enzyme catalyzed rate in the presence, v, and ab-
sence, v0, of the activator) as a function of the activator concentra-
tion. The solid smooth line is the best fit of the data for the
hyperbolic dependence of v/v0 on the activator concentration for
Steady-state kinetic parameters of HDAC-8 catalyzed reaction in the absence and
presence of TM-2-51
Condition
Km
(l
M)
kcat (sÀ1
)
kcat/Km (MÀ1 sÀ1
)
Control (no activator)
+Activator
744
230
0.007
0.036
9.4
150
an apparent activation constant (Ka) of 12.4 1.2
l
M and the max-
Clearly, the activators stabilize both the ground as well as the tran-
sition state (albeit by slightly different magnitudes) of the HDAC-8
catalyzed reaction. This can be achieved either by binding of acti-
vators within or in the vicinity of the enzyme’s active site pocket
leading to the stabilization of enzyme bound substrate as well as
facilitating the deacetylation reaction, or due to their binding at
some putative allosteric site and eliciting their influence via long
range conformational changes in the enzyme structure. However,
to further ascertain whether the activators indeed interacted with
HDAC-8, we performed isothermal microcalorimetric studies for
the binding of TM2-51 to the enzyme. The experimental data re-
vealed that the binding affinity of TM2-51 to HDAC-8 was similar
to the activation constant (see Fig. 1) of the compound, suggesting
that the observed activation phenomenon was not due to some
unforeseen kinetic complexity (data not shown).
imum fold activation as being equal to 26.8 1.1. Hence, at saturat-
ing concentration of TM-2-51, HDAC-8 catalyzed reaction is
enhanced by about 27-fold.
To further probe the kinetic mechanism of activation of the
HDAC-8 catalyzed reaction by TM-2-51, we determined the Km va-
lue of the enzyme’s substrate and kcat of HDAC-8 in the absence
and presence of saturating concentration of the activator. As appar-
ent from the data of Table 2, the activator decreased the Km value
of the substrate by about three-fold and it increased the kcat value
of the enzyme by five-fold. Hence, the specificity constant (kcat/Km
value) of the enzyme is enhanced by about 16-fold in the presence
of saturating concentration of the activator. It should be noted that
the latter value is somewhat lower than that obtained from the
data of Figure 1. We believe the origin of this discrepancy lies in
the mechanistic complexity of the enzyme activation at different
concentrations of the substrate. We are currently investigating this
feature and we will report our finding subsequently.
To determine the putative binding site(s) of activators on the
enzyme’s surface, we performed modeling studies by docking
TM2-51 to the known crystal structure of HDAC-8-substrate com-
plex via AutoDock Vina18 (as described in the Supplementary infor-
mation). A blind docking approach (i.e., without specifying the
putative binding regions on the structural coordinates of the en-
zyme) revealed that most of the activator molecules clustered in
the vicinity of the active site pocket of HDAC-8 (see Fig. S1 of Sup-
plementary information), and one of aromatic rings of the activator
was stacked between Phe306 and the coumarin moiety of the sub-
strate. Such stacking is believed to increase the binding affinity of
the substrate in the presence of the activator (as observed experi-
mentally; Table 2). Since Tyr306 (the corresponding amino acid
present in the native enzyme) has been known to be involved in
polarization of the carbonyl group of the acetylated lysine moiety
of the substrate and in stabilization of the oxyanion intermediate
during catalysis,19 it is conjectured that the enzyme bound activa-
tor would increase the kcat value of the enzyme (as also observed
experimentally; see Table 2). Aside from our docking results, the
structural data of HDAC-8 suggests that the enzyme’s surface in
the vicinity of the active site is extremely malleable which
facilitates the binding of a variety of ligands.19 Hence, it is not sur-
prising that TM-2-51 as well as its derivatives selectively activates
HDAC-8 (via binding to the enzyme site) as compared to other
HDAC isozymes (see Table S1 of Supplementary information).
However, we realize that our demonstration of N-acetylthiou-
rea-mediated activation of HDAC-8 primarily relies on the employ-
ment of Fluor-de-Lys as the fluorogenic substrate, and thus one can
argue that the observed activation is due to potential interaction
between the fluorescent moiety of the substrate and the aromatic
rings of the activators as observed in the case of sirtuin-1.16,17 In
case of sirtuin-1, it has been demonstrated that the putative activa-
tors bind to the enzyme only in the presence of fluorogenic sub-
strates17,20 and the activation is manifested via lowering the Km
value of the substrate. This is in marked contrast to our observation
that N-acetylthioureas directly bind to HDAC-8 (in the absence of
substrate or any other ligand) and their binding results in the de-
crease in the Km value of the substrate as well as increase in the kcat
value of the enzyme. These coupled with the fact that the overall
activation profile (at sub-saturating substrate concentrations) con-
forms to the marked co-operativity (our unpublished results)
prompt us to surmise that N-acetylthiourea-mediated activation
of HDAC-8 is real, and it is not an artifact of the employment of
To ascertain whether TM-2-51 as well as other N-acetylthioure-
as of Table 1 functions as selective activator for HDAC-8, or they
serve as non-specific ‘pan’ activators for other HDAC isozymes,
we determined the initial rates of the selected HDAC (viz., HDAC-
1, HDAC-2, HDAC-3, HDAC-4, HDAC-6, HDAC-10 and HDAC-11)
catalyzed reaction in the presence of 10 and 100 lM concentra-
tions of different activators. We observed that none of the N-acet-
ylthioureas of Table 1 activated other HDAC isozymes at either of
the above concentrations (see the Supplementary information).
In fact, we observed that at 100
N-acetylthioureas inhibited several HDACs by 4 to 15%, and the
compound TM-2-107 (at 100 M concentration) inhibited the
lM concentration, some of the
l
HDAC-10 catalyzed reaction by 47%. Such weak inhibitory feature
of some of thiourea derivatives is similar to that observed with sir-
tuins.14 In view of these results, we propose that depending on the
structural variants, some of N-acetylthioureas serve as the highly
potent and isozyme selective activators for the recombinant form
of HDAC-8 particularly in the Fluoro-de-Lys Assay system.
The question arose as to how some of N-acetylthiourea deriva-
tives selectively activated HDAC-8 but not other HDAC isozymes,
and the activation was manifested via increasing the binding affin-
ity of the enzyme’s substrate (i.e., decrease in the Km value) as well
as increase in the catalytic turnover rate (kcat value) of the enzyme.
20
15
10
5
0
0
12
24
36
48
µM)
[Activator; TM-2-51] (
Figure 1. Fold activation of HDAC8 catalyzed reaction is dependent on activator
concentration with apparent activation constant of 12.4 1.2 M.
l

R. K. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5920–5923
5923
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ating the mode of binding our activators to HDAC-8 and their over-
all mechanism of activation, and we will report these findings
subsequently.
11. Ito, K.; Ito, M.; Elliott, W. M.; Cosio, B.; Caramori, G.; Kon, O. M.; Barczyk, A.;
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This research was supported by the NIH grants CA113746 and
CA132034 to D.K.S., and the NIH COBRE grant NCRR-P20-
RR15566 to G.C.
14. Lain, S.; Hollick, J. J.; Campbell, J.; Staples, O. D.; Higgins, M.; Aoubala, M.;
McCarthy, A.; Appleyard, V.; Murray, K. E.; Baker, L.; Thompson, A.; Mathers, J.;
Holland, S. J.; Stark, M. J. R.; Pass, G.; Woods, J.; Lane, D. P.; Westwood, N. J.
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Supplementary data
15. Singh, R. K.; Mandal, T.; Balasubramanian, N.; Cook, G.; Srivastava, D. K. Anal.
Biochem. 2011, 408, 309.
Supplementary data (synthetic details of different activators,
experimental protocol for the activation of the enzyme, and docking
of an activator to the enzyme site) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2011.07.080.
16. Dittenhafer-Reed, K. E.; Feldman, J. L.; Denu, J. M. Chembiochem 2011, 12, 281.
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A.; Lavu, S.; Medvedik, O.; Sinclair, D. A.; Olefsky, J. M.; Jirousek, M. R.; Elliott, P.
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