Inhibitors of human histone deacetylase: Synthesis and enzyme assay of hydroxamates with piperazine linker

Article abstract of DOI:10.1002/ardp.200900117

The histone deacetylase (HDAC) enzyme plays an important role in gene transcription. Inhibitors of histone deacetylases induce cell differentiation and suppress cell proliferation in tumor cells. Hydroxamates with rigid linker have displayed better inhibi

Full text of DOI:10.1002/ardp.200900117

Arch. Pharm. Chem. Life Sci. 2010, 343, 167–172
S. Chakrabarty et al.
167
Full Paper
Inhibitors of Human Histone Deacetylase: Synthesis and
Enzyme Assay of Hydroxamates with Piperazine Linker
Shubhashis Chakrabarty1, Jasmine1, Chetan Bhadaliya1, Barij Nayan Sinha1, Aruna Mahesh2,
He Bai2, Sylvie Y. Blond2, 3, and Venkatesan Jayaprakash1
1 Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India
2 Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, USA
3 UMRS 940, Hopital Saint Louis, Institut de Gꢀnꢀtique molꢀculaire, Paris, France
The histone deacetylase (HDAC) enzyme plays an important role in gene transcription. Inhibitors
of histone deacetylases induce cell differentiation and suppress cell proliferation in tumor cells.
Hydroxamates with rigid linker have displayed better inhibition profiles than those with linear
and flexible aliphatic linkers. We have designed and synthesized several potential histone deace-
tylase inhibitors with a piperazine moiety in the linker region to test the effect of reduced linker
flexibility. Inhibitors were evaluated for their inhibitory action on human HDAC3/NCoR2 and
HDAC8. N-Hydroxycarboxamide derivatives (compounds 4a–d) were found to be better than N-
hydroxyacetamide derivatives (compounds 6a–d) against HDAC8. Amongst the synthesized com-
pounds, 4a (HDAC8, IC50: 3.15 lM) with no substitution in the aryl cap was the most active and
promising lead for further investigations.
Keywords: Docking / Histone deacetylase inhibitors / Piperazine hydroxamates /
Received: May 31, 2009; Accepted: October 14, 2009
DOI 10.1002/ardp.200900117
ylation, acetylation, ubiquitination, and sumoylation are
the known post-translation modifications. To date, acety-
lation is the most thoroughly studied of these modifica-
tions [1–3, 4]. While the acetylation state of chromatin
protein is unquestionably very dynamic, it seems to
depend on the net local balance between histone acetyl-
transferase (HAT) and histone deacetylase (HDAC) activ-
ities [5]. Over-expression of class 1 HDACs in human can-
cer tissues has been nicely reviewed by M. Nakagawa et al.
and the effectiveness of HDAC inhibitors in the control of
cancer progression has been very well documented in
reviews by different groups [6–8].
HDAC inhibition has recently been clinically validated
as a new therapeutic strategy for cancer treatment with
the FDA approval of suberoylanilide hydroxamic acid
(SAHA) for the treatment of cutaneous T cell lymphoma
[9]. Many other small molecules that inhibit HDAC pro-
teins are currently in clinical trials for cancer treatment
e.g., MS-275 and NVP-LAQ824 (Fig. 1).
Introduction
The packaging of DNA and the dynamic structure of chro-
matin provide a vital control for the regulation of gene
expression and the binding of transcription factors to
DNA. Chromatin is composed of multiple repeating units
termed nucleosomes, which are comprised of 146 base
pairs of DNA wrapped around a core of eight histone pro-
teins composed of two copies each of H2A, H2B, H3, and
H4 [1, 2]. Regulation of gene expression is mediated by
several mechanisms such as DNA methylation, ATP-
dependent chromatin remodeling, and post-transla-
tional modifications of histones. Phosphorylation, meth-
Correspondence: Venkatesan Jayprakash, Department of Pharmaceuti-
cal Sciences, Birla Institute of Technology, Mesra, Ranchi, Jharkhand
835 215, India.
E-mail: venkatesanj@bitmesra.ac.in
Fax: +91 651 227-5290
The classic pharmacophore for HDAC inhibitors con-
sists of three distinct structural motifs: the zinc-binding
group (ZBG), a hydrophobic linker, and a recognition cap
Abbreviations: histone acetyltransferase (HAT); histone deacetylase
(HDAC); suberoylanilide hydroxamic acid (SAHA); zinc-binding group
(ZBG)
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Arch. Pharm. Chem. Life Sci. 2010, 343, 167–172
Figure 1. Structure and pharmacophoric model of representa-
tive HDAC inhibitors.
group [10]. The X-ray crystal structures of a bacterial
HDAC homolog, histone deacetylase-like protein (HDLP)
bound to SAHA and Trichostatin A (TSA), and more
recently of human HDAC8 confirmed that the ZBG inter-
acts with a Zn2+ ion at the base of a channel-like active
site [6, 10]. The common ZBG of HDAC inhibitors is the
hydroxamate moiety. The structural modification of the
hydroxamate ZBG has been modestly successful; yielding
isosteres such as benzamide, a-ketoesters, electrophilic
ketones, mercaptoamide, and phosphonates [6, 11].
Hence, the linker and cap group present an alternative
opportunity to discover potent and more selective HDAC
inhibitors [12].
Reagents and conditions: a) Piperazine hexahydrate, acetonitrile, potassium carbo-
nate, reflux, 2–16 h at 808C; b) ethyl chloroformate (ClCOOC2H5), ethanol, sodium
carbonate, stirring 1–3
h at room temperature; c) hydroxylamine hydrochloride
(NH2OH . HCl), methanol, stirring 1–2 h at room temperature; d) 2-(piperazin-1-yl)
acetic acid, acetonitrile, reflux 2–16 h at 808C; e) ethyl chloroformate (ClCOOC2H5, 0–
58C stirring for 10 min; f) hydroxylamine hydrochloride (NH2OH N HCl), methanol, stir-
ring15–30 min at room temperature.
Scheme 1. Synthesis of compounds 4a–d and 6a–d.
evaluate our molecules against recombinant human
The available literature suggests that a bulky hydro- HDAC3/NCoR2 and HDAC8, two class-I HDAC isoforms
phobic cap group contributes towards class-I selectivity [6–8]. Three compounds inactivated HDAC8 preferen-
while an aromatic linker and a metal-binding group tially and were further analyzed with the molecular-
together contribute towards isoform selectivity within docking program (GLIDE) based on the X-ray structure of
class-I HDAC [13]. The residues in the hydrophobic active- the HDAC8-SAHA complex.
site channel that accommodate the linker region of the
HDAC inhibitors are highly conserved among HDAC iso-
forms. Due to this HDAC inhibitors designed to exploit
structural differences in the linker region to achieve class
Results and discussion
and isoform selectivity have been very few. It was clearly Chemistry
demonstrated that a rigid and bulky linker region that All the compounds were synthesized according to
increases the hydrophobic interaction with tunnel resi- Scheme 1. Substituted chloroacetanilides [18], 2-(pipera-
dues increases the potency and selectivity towards zin-1-yl) acetic acid [19], and hydroxylamine stock solu-
HDAC1 and HDAC3 [14]. Alicyclic rigid analogues of tion were prepared by reported procedures [20]. For com-
SAHA and Trichostatin were reported to have mamma- pounds 4a–d: N-aryl-2-(piperazine-1-yl) acetamides 2a–d
lian HDAC inhibition and antiproliferative activities [15– were prepared by the simple addition reaction of substi-
17]. But reports on human HDAC inhibition, class, and tuted chloro acetanilides with piperazine hydrate, which
isoform selectivity for these analogues are not available. was then made to react with ethyl chloroformate fol-
With this in mind, we have designed and synthesized lowed by hydroxylamine hydrochloride to yield com-
novel, structurally simple HDAC inhibitors with a pounds 4a–d. In both the reactions, either sodium carbo-
hydroxamic acid as the ZBG attached to a piperazine nate or potassium carbonate was used to neutralize the
linker domain and an aromatic cap. Over-expression of acid released. For compounds 6a–d the 2-[4-(2-(aryl
class-I HDAC in certain cancer cells and the anticancer amino)-2-oxoethyl) piperazine-1-yl] acetic acid 5a–d were
property of inhibitors of class-I HDACs prompted us to prepared similar to 2a–d but using 2-(piperazin-1-yl) ace-
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Arch. Pharm. Chem. Life Sci. 2010, 343, 167–172
Hydroxamates with Piperazine Linker
169
Table 1. Physico-chemical characteristics of the synthesized
Table 3. Docking scores of synthesized compounds.
compounds.
Compound
Glide Score (Kcal/mol)
1T69
1T69 Mutated
(MET274fiLEU274)
4a
4b
4c
4d
6a
6b
6c
6d
–7.33
–7.12
–5.49
–6.87
–4.45
–5.49
–5.35
–5.34
–6.06
–6.51
–4.79
–5.99
–5.55
–7.18
–6.95
–6.07
Com-
pound
R
MF
MW
Yield
(%)
M.p.
(8C)
4a
4b
4c
4d
6a
6b
6c
6d
-H
-Cl
-CH3
-OCH3
-H
-Cl
-CH3
-OCH3
C13H18N4O3
C13H17ClN4O3 312
C14H20N4O3
C14H20N4O4
C14H20N4O3
C14H19ClN4O3 326
C15H22N4O3
C15H22N4O4
278
75
59
84
63
40
56
42
47
230
250
150
190
78
92
152
62
292
308
292
306
322
most potent one in this series with an IC50 of 1.5 € 0.4 lM,
followed by compound 4c with an IC50 of 17 € 4 lM. Com-
pound 6a was the only active N-hydroxyacetamide deriv-
ative with an IC50 of 36 € 15 lM. The present study evalu-
ates the successful suitability of piperazine as a linker
moiety. Within this small library of eight compounds, a
SAR discussion may not be reliable but a few guiding
points may be considered for further design. N-Hydroxy-
carbamides were better than N-hydroxyacetamides, and
para substitution reduced potency. Rational modification
of the aryl cap of the N-hydroxycarbamide derivative 4a
will be carried out to optimize this potential candidate.
Table 2. HDAC8 inhibitory activity of the synthesized com-
pounds.
Compound
IC50
(lM)*
Ki
(lM)
4a
4b
4c
4d
6a
1.5 € 0.4
>100
17 € 4.0
>100
36 € 15
>100
2.8
–
19.3
–
29
–
6b
6c
>100
–
6d
SAHA
>100
0.36 € 0.036
–
0.33
Docking studies
Interaction of compound 4a with human HDAC8 was
studied by the molecular docking program GLIDE (Schrꢀ-
dinger, LLC) using the SP docking protocol. Docking was
performed for all eight molecules and the results are pre-
* Experiments were performed in triplicates.
tic acid in place of piperazine (without using a base), sented in Table 3. Compound 4a shows hydrogen-bond
which was then made to react with ethyl chloroformate interaction with TYR306 and ASP101 (Fig. 2a).
followed hydroxylamine to yield the compounds 6a–d.
The structures and physicochemical characteristics of
the synthesized compounds are presented in Table 1.
Hydroxamate’s carbonyl O and hydroxyl O of com-
pound 4a were at 2.315 ꢁ and 2.246 ꢁ (SAHA, 2.332 and
2.136 ꢁ) distance from Zn2+ (Fig. 2a). This makes com-
pound 4a to exactly superimpose on crystal structure of
SAHA with top docking score of –7.33 amongst all the
HDAC8 enzyme-inhibition studies
All synthesized compounds were screened against eight. All the 4 N-hydroxycarboxamide derivatives estab-
HDAC3/NCoR2 and HDAC8 using the fluorogenic sub- lish hydrogen bonds with TYR306 and ASP101 and are
strate FLUOR de LysTM and commercially available found to be oriented in a similar direction to the aryl cap
of SAHA. The hydrogen-bond interaction with ASP101
recombinant proteins. None of them were found to inacti-
vate HDAC3/NCoR2 when tested at concentrations equal keeps the head group near the TYR100 in the entry point.
to 60 lM, but inhibition was observed against HDAC8 for Any substitution in para-position of the cap aryl group
compounds 4a, 4c, and 6a tested at 20 and 100 lM. Those did not favor interaction with TYR100. Possibly a func-
tional group with a hydrogen-bonding capability with
compounds were further evaluated using the HDAC8 flu-
orescent activity assay kit (Biomol Research Laboratories, TYR100 in the cap aryl region may increase the interac-
Plymouth Meeting, PA, USA) at a wider range of inhibitor tion and, thereby, increase the potency. The observation
concentrations. IC50 values of compounds are presented is consistent with the recent report of the importance of
in Table 2. Compounds 4a, 4c, and 6a were found to have ASP101 of HDAC8 in establishing hydrogen-bond interac-
IC50 values in the lower lM range. Compound 4a was the
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170
S. Chakrabarty et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 167–172
LUE274 and conformation of 4a in the mutated protein are in green.
Figure 3. Interaction of 4a with 1T64 and 1T64 (MET274fi
LEU274).
less, and none established a hydrogen bond with ASP101.
Conformation of 4a in 1T69 and mutated 1T69
(MET274fiLEU274) are presented in Fig. 3. Compounds
6a–d displayed better scoring in mutated 1T69, but
experimentally failed to show better inhibition profile
against HDAC3/NCoR2 compared with HDAC8 in prelimi-
nary screening against recombinant proteins at the con-
centration of 60 lM. It seems that MET274 in the tunnel
region influences the conformation of the piperazine
ring to orient amide -NH near the cap group to establish
a hydrogen bond with ASP101. This confers selectivity
towards HDAC8 with respect to the isoforms used in the
study. Further testing of compound 4a with other iso-
forms is required to establish the merit of a piperazine
linker in the design of selective HDAC8 inhibitors. Ana-
logues of 4a with ortho- and meta-substitution in the aryl
cap region are currently under investigation.
Hydrogen-bonding interactions are shown in yellow broken lines; distances between
Zn and the hydroxamate group are shown in orange lines.
Figure 2. Interaction of compounds 4a–4d (2a) and 6a–6d (2b)
with human HDAC8.
tion with the backbone NH of the substrate and different
classes of HDAC inhibitors [21].
Interestingly, none of the N-hydroxyacetamide deriv-
atives was found to establish a hydrogen-bonding interac-
tion with ASP101, and, owing to this, the cap aryl group
pointed away from TYR100 (Fig. 2b). Apparently, the
extra carbon of the acetamide causes the molecule to ori-
ent differently inside the active-site channel. Docking
studies with these series of molecules reveal the follow-
ing: the hydrogen-bonding interactions with TYR306 and
ASP101 are crucial and interaction of the aryl cap with
TYR100 appears to be significant. Functional groups with
hydrogen-bonding capability in the cap aryl region
should be explored.
The hydrophobic tunnel region of HDAC8 differs from
other class-1 isoforms by having MET274 in place of
LUE274. We mutated MET274fiLEU274 of 1T69 using the
script mutate.py from Schrꢀdinger, LLC to mimic the tun-
nel region of other class-1 HDACs. The results of docking
with this mutated protein are presented in Table 3. It was
found that the docking scores for compounds 4a–d were
Experimental
General
Unless otherwise noted, all solvents and reagents were of
reagent grade and purified as per standard procedures. The melt-
ing points were determined using a Thermonic melting point
apparatus (Campbell electronics, Bombay, India), by capillary
method and are uncorrected. The IR spectra of the compounds
synthesized were taken on IR-Prestige 21, Shimatzu Corpora-
tion, Japan from 4000–400 cm–1 using KBr discs. 1H-NMR spectra
were recorded at 300 MHz in DMSO-d6 solvent on Bruker DRX-
300, Bruker Instruments Inc., USA. The chemical shifts were
measured at d units (ppm) relative to tetramethyl silane (TMS).
Elemental analysis was performed on Vario EL III, Elementar,
Germany, using sulfanilamide as standard. All chemicals were
purchased from Merck, Spectrochem, or Central Drug House,
Lucknow, India. Reactions were monitored by TLC on silica gel
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Arch. Pharm. Chem. Life Sci. 2010, 343, 167–172
Hydroxamates with Piperazine Linker
171
plates in either iodine chamber or in UV chamber. Intermediates
were characterized by IR and CHN analysis and the final com-
pounds were analyzed by1H-NMR and FAB-MS.
Substituted chloroacetanilides [18], 2-(piperazin-1-yl) acetic
acid [19], and the hydroxylamine stock solution were prepared
by the reported procedure [20].
4-(2-(4-Methoxyphenylamino)-2-oxoethyl)-N-
hydroxypiperazine-1-carboxamide 4d
Prepared from 3b following the procedure of 4a, recrystallized
1
from methanol. Yield: 63%, m.p.: 1908C; H-NMR (DMSO-d6) d:
9.51 (s, 1H, OH), 6.85–7.53 (m, 4H, ArH), 3.09 (s, 2H, methylene),
2.57 (m, 8H, piperazine), 3.71 (s, 3H, Ar-OCH3); FAB-MS (m/z): 308
[M+].
Chemistry
General procedure for the synthesis of 2-[4-(2-(aryl
General procedure for the synthesis of N-aryl-2-
amino)-2-oxoethyl)piperazine-1-yl]acetic acid 5a–d
2-(Piperazin-1-yl)acetic acid (0.013 mol) in 5 mL acetonitrile was
added dropwise to substituted chloroacetanilide (0.01 mol) in 25
mL acetonitrile with stirring. The reaction mixture was then
refluxed for 2 to 16 h at 808C. Then, the reaction mixture was
cooled to room temperature and dissolved in water and
extracted with dichloromethane. The organic layer was evapo-
rated to yield 2-[4-(2-(aryl amino)-2-oxoethyl)piperazine-1-yl]ace-
tic acids 5a–d.
(piperazine-1-yl)-acetamide 2a–d
Piperazine hexahydrate (0.013 mol) in 5 mL acetonitrile was
added dropwise to substituted chloroacetanilide (0.01 mol) in 25
mL acetonitrile with stirring. To this mixture potassium carbo-
nate (0.12 mol) was added and was refluxed for 2 to 16 h at 808C.
Then, the reaction mixture was cooled to room temperature, dis-
solved in water, and extracted with dichloromethane. The
organic layer was evaporated to yield N-aryl-2-(piperazine-1-yl)
acetamides 2a–d.
2-[4-(2-(Phenylamino)-2-oxoethyl)piperazine-1-yl]
General procedure for the synthesis of ethyl-4-(2-oxo-2-
N-hydroxyacetamide 6a
Ethyl chloroformate (0.0023 mol) and triethylamine (0.0025
mol) were added to solution 5a (0.0018 mol) in tetrahydrofuran
(20 mL) at 0–58C with stirring. Stirring was continued for 10 to
15 min and the precipitate obtained was filtered off. To the fil-
trate, a freshly prepared solution of hydroxylamine (0.003 mol
equivalent from hydroxylamine stock solution) in methanol was
added. The resulting mixture was stirred at room temperature
for another 15 to 30 min and the organic layer was evaporated
(aryl amino)-ethyl)piperazine-1-carboxylate 3a–d
Ethyl chloroformate (0.00327 mol) was added dropwise with stir-
ring to a solution of N-aryl-2-(piperazine-1-yl) acetamide in etha-
nol (25 mL). Sodium carbonate (0.006 mol) was then added to the
above mixture and stirring was continued for about 1 to 3 h. A
precipitate of ethyl 4-(2-oxo-2-(aryl amino)ethyl)piperazine-1-car-
boxylates 3a–d was obtained and washed with cold ethanol and
recrystallized from hot ethanol.
to
yield
2-[4-(2-(phenylamino)-2-oxoethyl)piperazine-1-yl]N-
hydroxyacetamide. It was washed with cold methanol and
recrystallized from hot methanol. Yield: 40%, m.p.: 788C; 1H-
NMR (DMSO-d6) d: 10.37 (s, 1H, OH), 7.30–7.61 (m, 4H, ArH), 4.26
(s, 2H, methylene), 1.21–3.06 (m, 8H, piperazine); FAB-MS (m/z):
292 [M+].
4-(2-(Phenylamino)-2-oxoethyl)-N-hydroxypiperazine-1-
carboxamide 4a
Hydroxylamine (0.002 M equivalent from hydroxylamine stock
solution) in methanol was added to 3a (0.000623 M) in methanol
(25 mL), dropwise with stirring for about 2 h. The precipitate
obtained was washed with ether and recrystallized from metha-
nol. Yield: 75%, m.p.: 2308C; 1H-NMR (DMSO-d6) d: 9.60 (s, 1H, OH),
8.22 (s, 1H, NH), 7.19–7.85 (m, 5H, ArH), 3.90 (s, 2H, methylene),
1.15–3.29 (m, 8H, piperazine); FAB-MS (m/z): 278 [M+].
2-[4-(2-(4-Chlorophenylamino)-2-oxoethyl)piperazine-1-
yl]N-hydroxyacetamide 6b
Prepared from 5b following the procedure of 6a, recrystallized
from methanol. Yield: 56%, m.p.: 928C; 1H-NMR (DMSO-d6) d:
10.60 (s, 1H, OH), 9.92 (s, 1H, NH), 7.36–7.65 (m, 4H, ArH), 4.27 (s,
2H, methylene), 1.18–3.04 (m, 8H, piperazine); FAB-MS (m/z): 326
[M+], 327 [M + 1], 328 [M + 2].
4-(2-(4-Chlorophenylamino)-2-oxoethyl)-N-
hydroxypiperazine-1-carboxamide 4b
Prepared from 3b following the procedure of 4a, recrystallized
from methanol. Yield: 59%, m.p.: 2508C; 1H-NMR (DMSO-d6) d:
10.91 (s, 1H, OH), 7.40–7.70 (m, 4H, ArH), 3.44 (s, 2H, methylene),
1.23–4.04 (m, 8H, piperazine); FAB-MS (m/z): 312 [M+], 313 [M + 1],
314 [M + 2].
2-[4-(2-(4-Methylphenylamino)-2-oxoethyl)piperazine-1-
yl]N-hydroxyacetamide 6c
Prepared from 5c following the procedure of 6a, recrystallized
1
from methanol. Yield: 42%, m.p.: 1528C; H-NMR (DMSO-d6) d:
10.21 (s, 1H, OH), 7.11–7.47 (m, 4H, ArH), 4.21 (s, 2H, methylene),
1.17–3.07 (m, 8H, piperazine); FAB-MS (m/z): 306 [M+].
4-(2-(p-Methylphenylamino)-2-oxoethyl)-N-
hydroxypiperazine-1-carboxamide 4c
Prepared from 3c following the procedure of 4a, recrystallized
from methanol. Yield: 84%, m.p.: 1508C; 1H-NMR (DMSO-d6) d:
10.7 (s, 1H, OH), 7.13–7.51 (m, 4H, ArH), 3.42 (s, 2H, methylene),
4.20 (m, 8H, piperazine), 2.26 (s, 3H, Ar-CH3); FAB-MS (m/z): 292
[M+].
2-[4-(2-(4-Methoxyphenylamino)-2-oxoethyl)piperazine-
1-yl]N-hydroxyacetamide 6d
Prepared from 5d following the procedure of 6a, recrystallized
from methanol. Yield: 47%, m.p.: 628C; 1H-NMR (DMSO-d6) d:
10.26 (s, 1H, OH), 6.88–7.51 (m, 4H, ArH), 3.71 (s, 2H, methylene),
1.18–3.06 (m, 8H, Piperazine); FAB-MS (m/z): 322 [M+].
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S. Chakrabarty et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 167–172
Biology
characterization of the compounds. The author also acknowl-
edges Prof. Alan P. Kozikowski for his suggestions.
In-vitro enzyme-inhibition assay against human HDAC3/
NCoR2 and HDAC8 – IC50 and Ki determination
The in-vitro HDAC enzymatic assays were all performed in tripli-
cate using 96-well white microtitration plate from Costar and 50
lL of HDAC assay buffer (25 mM Tris, pH 8.0, 137 mM NaCl, 2.7
mM KCl, 1 mM MgCl2) supplemented with 0.5 mg/mL BSA, 0.375
units of recombinant human HDAC8 (BioMol, Plymouth Meet-
ing, PA, USA) or 0.1 lg of HDAC3/NCoR2 purified active complex
(BPS, San Diego, CA, USA). This mixture was pre-incubated for
5 min at 258C with increasing concentrations of HDAC inhibitor
prior to the addition of 25 lM final concentration of the fluoro-
genic acetylated HDAC substrate Fluor de LysTM (BioMol KI178)
for HDAC8 assays or 5 lM final concentration of fluorogenic
HDAC substrate 3 (BPS, 50037) for HDAC3 assays. After 30 min
incubation at 258C, the enzymatic reaction was stopped by addi-
tion of 50 lL of developer (BioMol KI176), supplemented with
5 lM Trichostatin. The fluorescence intensity was measured at
kexcitation = 360 nm and kemission = 460 nm using a POLARStar
Optima microtitration plate reader (BMG Labtech, Durham, NC,
USA). Data were plotted as the percentage of HDAC8 inhibition
as a function of the log of the inhibitor concentration. The IC50
values were determined by fitting the experimental data to a
four-parameter logistical model using the Graphpad prism5
software version 5.01 for Windows (GraphPad Software, San
Diego, CA, USA). The Ki values were calculated using a KM for the
substrate (S) equal to 222 lM (determined experimentally under
the same conditions) and the simplified Cheng-Prusoff equation:
Ki = IC50/1 + (S/KM) [22].
The authors have declared no conflict of interest.
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Docking studies
Docking studies were performed using the SP docking protocol
of GLIDE (Schrꢀdinger, LLC) in HP system with Intel core2 duo
CPU E8300 @ 2.83 GHz processor, 1 GB RAM and with the operat-
ing system Red hat linux enterprises (version 4.0). The crystal
structure of the ligand SAHA bound to human HDAC8 (PDB
code: IT69) was retrieved from the RCSB Protein Data Bank. Pro-
tein for docking was prepared with the Maestro interface of
Schrꢀdinger using the Protein Preparation Wizard. Minimiza-
tion of macromolecule was carried out with the OPLS2001 Force
Field. The docking grid was generated with the GLID Grid gener-
ation module. Ligand molecules were sketched using the built
panel as part of the Maestro interface and were prepared for
docking using the Ligand Preparation Wizard. Minimization of
ligand molecules was carried out using MMFF Force Field. Dock-
ing was performed with the GLIDE ligand docking module using
the SP docking protocol. The pose viewer file generated was then
analyzed by Maestro.
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The author acknowledges the University Grants Commission,
India, for receiving the fellowship and the Sophisticated Instru-
mentation Facility (SAIF) of the Central Drug Research Insti-
tute, Lucknow, for being able to use the facility for spectral
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