Novel histone deacetylase 8 ligands without a zinc chelating group: Exploring an 'upside-down' binding pose

Article abstract of DOI:10.1016/j.bmcl.2012.08.104

A novel series of HDAC8 inhibitors without a zinc-chelating hydroxamic acid moiety is reported. Photoaffinity labeling and molecular modeling studies suggest that these ligands are likely to bind in an 'upside-down' fashion in a secondary binding site pro

Full text of DOI:10.1016/j.bmcl.2012.08.104

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6621–6627
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel histone deacetylase 8 ligands without a zinc chelating group: Exploring
an ‘upside-down’ binding pose
Aditya Sudheer Vaidya a, , Raghupathi Neelarapu a, , Antonett Madriaga a, He Bai a, Emma Mendonca b,
Hazem Abdelkarim a, Richard B. van Breemen a, Sylvie Y. Blond c, Pavel A. Petukhov a,
⇑
a Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, United States
b Department of Bioengineering, University of Illinois at Chicago, 851 South Morgan Street, Chicago, IL 60607, United States
c Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, IL 60612, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of HDAC8 inhibitors without a zinc-chelating hydroxamic acid moiety is reported. Photo-
affinity labeling and molecular modeling studies suggest that these ligands are likely to bind in an
‘upside-down’ fashion in a secondary binding site proximal to the main catalytic site. The most potent
Received 16 July 2012
Revised 23 August 2012
Accepted 28 August 2012
Available online 7 September 2012
ligand in the series exhibits an IC50 of 28
H4 but not -tubulin in SH-SY5Y cell line.
lM for HDAC8 and is found to inhibit the deacetylation of
a
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
HDAC8
Upside down
Photolabeling
Zinc chelating group
Secondary binding site
Histone deacetylases (HDAC) are considered as promising
therapeutic targets.1,2 The vast majority of the HDAC inhibitors
contains a zinc chelating group (ZCG) as an essential part respon-
sible for their binding affinity. The most commonly used ZCG in
HDAC inhibitors is a hydroxamic acid group that shows poor
metabolic stability in vivo and is rapidly hydrolyzed to carboxylic
acid, a poor ZCG, and hydroxylamine.3,4 Compounds containing
hydroxamic acid moieties are poorly absorbed in vivo and show
extensive glucuronidation and sulfation.5 The sulfate conjugates
of hydroxamic acids may lead to formation of electrophiles such
as isocyanates which may further react with nucleophiles in pro-
teins and other biological macromolecules.6–8 Non-hydroxamate
ZCGs typically include carboxylic acids (sodium valproate), epoxy
ketones (trapoxin), o-amino anilides (MS 275), electrophilic
ketones, and thiols.9–12 These non-hydroxamate ZCGs often suffer
from poor potency,13 redox activity,14 general reactivity,10 meta-
bolic instability,15 and poor aqueous solubility.15 For life-long
applications, for example as a potential treatment of Alzheimer’s
disease,16,17 these types of properties of HDAC inhibitors are highly
undesirable. Clearly, there is an urgent need for the development of
HDAC inhibitors that may utilize novel binding modes that do not
require a ZCG. A series of non-selective tetrapeptides likely not
chelating to the active site Zn2+ of HDAC1-3 is the only example
of the non-ZCG HDAC ligands we could find.18
Recently, we have published the Binding Ensemble Profiling
with Photoaffinity Labeling (BEProFL) approach that was used to
map experimentally the multiple binding poses of the surface
binding groups (SBG) of inhibitors of HDAC8,19 a novel therapeutic
target for neuroblastoma,20–24 leukemia,25 and cognitive disor-
ders.26 In our paper19 we noted that some of the covalent modifi-
cations of HDAC8 may correspond to a novel ‘upside-down’ pose
of the diazide probe that was strikingly similar to that found in
HDAC8 (PDB:1T64)27 for one of the two molecules of Trichostatin
A bound to the secondary site proximal to the main catalytic site.
Our main objective in this study was to design and synthesize a
series of diazide-based probes/ligands lacking the hydroxamic acid
moiety, test HDAC8 inhibition by these probes, and explore using
photoaffinity labeling studies if they bind in an ‘upside-down’ fash-
ion in the secondary binding site in HDAC8.
The synthesis of the ligands/probes is outlined in Scheme 1.
Commercially available 4-benzyloxyaniline hydrochloride 4 was
coupled with suberic acid monomethyl ester 5 or octanoic acid 6
using a facile carbodiimide based coupling reaction. The benzylhy-
doxy group of 1e and 7 was deprotected via catalytic hydrogena-
tion and later coupled with bromide 11 or tosylates 10 and 15.
Esters 1b, 1f, and 16 were subjected to basic hydrolysis and
re-esterified with appropriate alcohols resulting in probes 1c, 1g,
1h, and 1i, respectively, in 20–65% overall yields. The structures
⇑
Corresponding author. Tel.: +1 312 996 4174; fax: +1 312 996 7107.
E-mail address: pap4@uic.edu (P.A. Petukhov).
These authors contributed equally to the work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.104

6622
A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6621–6627
MeO
X
a
Y
OTs
N3
OTs
Br
10
11
N3
12, X = OMe, Y = COOH, Z = NH2
N3
Z
13,
X = OMe, Y = CH2OH, Z = NH2
14, X = OMe, Y = CH2OH, Z = N3
15
b
c
, X = OMe, Y = CH2OTs, Z = N3
H
N
R1
6
H
R3
O
N
NH2.HCl
R1
6
O
f
HO
d
R1
6
O
RO
RO
O
R2
4
, R = Bn, R1 = COOMe
, R = Bn, R = CH3
5, R1 = COOMe
1e
7
1b, R1= COOMe, R2 = N3, R3 = CH2N3
1d, R1, R2, R3 = CH3
1
, R1 = CH3
6
e
, R = H, R1 = COOMe
1f, R1 = COOMe, R2 = CH3, R3 = CH3
8
9
, R =H, R1 = CH3
1
, R = COOMe, R2 = N3, R3 = OMe
16
H
N
R1
6
R3
O
O
g, h or i
1f or 1b or 16
R2
1c, R1= COO-t-Bu, R2 = N3, R3 = CH2N3
1g, R1 = COO-t-Bu, R2 = CH3, R3 = CH3
1h, R1= COO(CH2)2N3, R2 = N3, R3 = OMe
1
, R = COOCH2-p-Ph-CH2N3, R2 = N3, R3 = OMe
1i
Scheme 1. Reagents and conditions (a) LAH, THF, 0 °C to rt; (b) NaNO2, HCl(aq), NaN3, 0 °C to rt; (c) TsCl, Et3N, CH2Cl2, 0 °C; (d) suberic acid monomethyl ester or octanoic
acid, EDCI, HOBt, DIPEA, CH2Cl2, 0 °C to rt; (e) Pd/C, MeOH, rt; (f) 10 or 11 or 15, K2CO3, acetone, reflux; (g) NaOH(aq) or KOH(aq), rt (h) (COCl)2, t-BuOH, DMF, Et3N, CH2Cl2; (i)
[4-(azidomethyl)phenyl]methanol or 2-azidoethanol, EDCI, HOBt, CH2Cl2, 0 °C to rt.
of the final compounds were elucidated by NMR and confirmed by
HRMS.28 Synthetic procedures and data for compounds 1a, 1b, 1e,
and 8 and 2a, 2b, 3a, 3b, and 10 were previously reported by
us.19,29 [4-(Azidomethyl)phenyl]methanol and 2-azidoethanol
were synthesized according to the reported methods.30,31
The inhibition by the probes/ligands was measured using com-
mercially available recombinant human HDAC1, 2, 3, and HDAC8
using fluorogenic substrates Boc-Lys(Ac)-AMC for HDAC1, 2, and
3 and Fluor de Lys for HDAC8, respectively, using previously
reported procedures.19,29 The resulting IC50 values are given in
Table 1. The analysis of the SAR was facilitated by docking of the
ligands to HDAC8 (PDB:1T64)27 using GOLD v.5.1.32 and the dock-
ing procedure reported by us previously.33
At first, we replaced the hydroxamic acid moiety in 1a with a
methyl ester and found that ligand 1b is able to maintain its
HDAC8 inhibitory properties (IC50 of 1b is 37.4 lM) despite the
lack of a chemical moiety that can serve as a bidentate chelator
to Zn2+ (Table 1). Although encouraging, this finding needed fur-
ther proof that the ester group of ligand 1b does not serve as a
monodentate ZCG. Our preliminary modeling studies have shown
that a t-butyl ester moiety instead of the methyl ester in 1b would
not be able to fit the binding site of HDAC8 because it is too bulky.
The corresponding t-butyl ester 1c was found to inhibit HDAC8
To explore if the presence of the two lipophilic azide groups is
required, we synthesized compound 1e where hydrogen atoms
were placed in both the meta positions in the terminal phenyl ring.
Compound 1e was found to be inactive up to 66 lM. The analysis
of the binding site available to the ligands in those HDAC8 X-ray
structures where the second copy or an empty binding site adja-
cent to the catalytic site is present showed that the second pocket
is relatively large and there are two additional lipophilic sub-pock-
ets at the bottom. Our docking experiments suggested that in the
case of ligands 1a–c their meta-azido and meta-methylene azido
moieties can occupy these two lipophilic sub-pockets and form
extensive hydrophobic interactions with the binding site. In the
case of 1e such interactions were missing. Next, to further verify
if the presence of meta substituents is required, we synthesized
compound 1f that has two meta-methyl substituents in the termi-
nal phenyl ring. The HDAC8 inhibitory potency of 1f was restored
to the level of ligands 1a–d. The strong dependence on the pres-
ence of additional substituents in the terminal phenyl ring is con-
sistent with the ‘upside-down’ pose where extended interactions
with the binding site would be necessary to compensate for the
missing interactions between the ZCG and the active site Zn2+. In
agreement with the proposed ‘upside-down’ binding pose and
the data for esters 1b and 1c, potency of the substantially bulkier
t-butyl ester 1g is only marginally lower than that of the corre-
sponding methyl ester 1f. Comparable potency of diazides 1b
and 1c with dimethyl-substituted 1g and 1f indicates that the
azide moieties do not form an R-N3Á Á ÁZn2+ complex if the ligands
bind ‘upside-down’.
with an IC50 of 42.6 lM, which is similar to that of ligand 1b. Dock-
ing showed that ligand 1c can only fit the binding site by adopting
an ‘upside-down’ pose shown in Figure 1.
Next, we removed the ester group in 1b. The resulting ligand 1d
with a rather flexible unsubstituted alkyl chain was found to be
inactive. If the binding mode of 1d is similar to that of 1c, its long
alkyl chain substituent would be solvent exposed and may not con-
tribute to the binding. Thus, lack of potency comparable to that of
1b appears to be in line in the proposed binding mode.
To confirm that the scaffold containing the terminal phenyl ring
with two lipophilic meta substituents is, indeed, a universal fea-
ture responsible for activity, we synthesized and tested methyl es-
ter derivatives of the diazide probes 2a and 3a.7 Both 2a and 3a are

A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6621–6627
6623
Table 1
HDAC8 IC50 values for compounds 1a–h, 2a–b, and 3a–b
R2
H
N
H
N
O
R1
R1
R1
6
N
N
6
HN
O
N
H
6
R2
O
O
R2
N
O
O
N
R3
R3
R3
1
2
3
O
O
O
HOHN
H
NHOH
N
NHOH
O
N
OMe
O
SAHA
TSA
PCI34051
Compd #
R1
R2
R3
HDAC8 IC50 (lM)
1aa
1b
1c
1d
1e
1f
1g
1h
CONHOH
COOCH3
COO-t-Bu
CH3
COOCH3
COOCH3
COO-t-Bu
COOCH2CH2N3
N3
N3
N3
CH3
H
CH3
CH3
N3
N3
N3
N3
N3
N3
—
CH2N3
CH2N3
CH2N3
CH3
H
CH3
7.34 0.22
37.4 2.34
42.6 1.81
NA
NAd
47.8 2.35
53.2 6.49
67.5 4.79e
28.0 2.65f
0.017 0.003
36.0 2.20
0.651 0.12
47.1 1.76
0.44 0.0217
0.39 0.018
0.0125
CH3
OCH3
OCH3
CH2N3
CH2N3
CH2N3
CH2N3
—
1i
COOCH2-p-Ph-CH2N3
2ab
2b
CONHOH
COOCH3
CONHOH
COOCH3
—
3ac
3b
SAHA
TSA
PCI34051
—
—
—
—
—
—
a
b
c
d
e
f
HDAC3 IC50 1.48
HDAC3 IC50 128 nM, Ki 0.064
HDAC3 IC50 45 nM, Ki 0.023
The residual activity is more than 50% at 66
HDAC1 IC50 111 14.8 M, HDAC2 IC50 72 2.2
HDAC1 IC50 86.0 7.1 M, HDAC2 IC50 79 3.0
l
M, Ki 0.74
l
l
M.
M.
M.
l
l
M, IC50 was not determined.
M.
M.
l
l
l
l
were found to inhibit HDAC8 with IC50 of 36.0 and 47.1 lM,
respectively, which is comparable to the potency of compounds
in series 1. Relatively a small difference in the potency of 2b and
3b and similar compounds in series 1 is consistent with the bind-
ing pose of the ligands without ZCG (Fig. 1). The terminal phenyl
ring of 2b and 3b is completely immersed in the binding site
whereas the pyrazole and the isoxazole portions of 2b and 3b,
respectively, are only partially enclosed in the binding site but
otherwise are solvent exposed.
Next, we performed a series of photolabeling experiments with
purified recombinant HDAC8 to further delineate the putative
binding modes of the probes.34 In all the photolabeling experi-
ments, the concentration of purified HDAC8 used was 0.5 lM,
and all the experiments were performed in non-denaturing
conditions unless otherwise stated. The probe or probe/competitor
mixtures were preincubated with purified recombinant HDAC8
from E. coli,35 in photolabeling buffer for 3 h at room temperature,
exposed to 254 nm UV light for 3 Â 1 min with 1 min resting. In
case of denaturation experiments, after photoirradiation a solution
of sodium dodecyl sulfate was added to attain a final concentration
of 2% w/v and the mixture incubated at 37 °C for 30 min. In all
photolabeling experiments an alkyne containing biotin tag19 was
attached to the HDAC8-probe adduct using Cu(I) catalyzed (3+2)
cycloaddition reaction carried out at room temperature for 1 h.
The biotinylated HDAC8 was visualized by streptavidin–HRP and
western blot and the loading was confirmed by using anti-HDAC8
antibody. The control wells contained purified HDAC8, a mixture of
HDAC8 and the biotin tag, or a mixture of HDAC8 and the probes
Figure 1. ‘Upside-down’ binding pose proposed for HDAC8 inhibitors 1c (magenta),
1f (yellow), 1h (cyan), and 3b (green). TSA bound to the main catalytic site (PDB:
1T64) is rendered brown. The binding site is rendered by solvent accessible surface,
green—lipophilic, magenta—hydrophilic.
nanomolar inhibitors of HDAC8. In these ligands the SBG contains a
pyrazole or an isoxazole moiety, respectively, as well as the meta-
azido and meta-methyleneazido substituted phenyl ring. Consis-
tently with the ‘upside-down’ hypothesis, both esters 2b and 3b

6624
A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6621–6627
Figure 2. Characterization of biotinylated HDAC8 protein and total HDAC8 protein. (A) Western blot analysis of probe 1i binding to HDAC8 using Strep–HRP and anti-HDAC8
antibodies, control 1-HDAC8 (0.5 M), click reagents, control 2-HDAC8 (0.5 M), biotin tag (50 M), click reagents, all other lanes contain different concentrations (indicated)
of 1i, biotin tag (50 M), click reagents. (B) Western blot analysis of probes 1a, 1b, and 1h binding to HDAC8 using Strep–HRP and anti-HDAC8 antibodies, control-HDAC8
(0.5 M), biotin tag (50 M), click reagents, all other lanes contain different probes at concentrations indicated, biotin tag (50 M), click reagents. (C) Western blot analysis of
l
l
l
l
l
l
l
probe 1b binding to HDAC8 in the presence and the absence of equimolar concentration of SAHA. (D) Western blot analysis of probe 1b binding to HDAC8 under denaturing
conditions and non-denaturing conditions. (E) Western blot analysis of probe 1b binding to HDAC8 in the presence and the absence of an excess of HDAC8 inhibitors SAHA,
TSA, PCI34051, and ligand 1f. (F) Western blot analysis of probe 1i binding to HDAC8 in the presence and the absence of an excess of HDAC8 inhibitors SAHA, TSA, and
PCI34051, control 1-HDAC8 (0.5
all other lanes contain different inhibitors of HDAC8 at concentrations indicated, probe 1i (1.5
probe 1a in the presence of varying concentrations of SAHA. (H) Western blot analysis of binding of probe 1i in the presence and the absence of excess of ligand 1f. Click
l
M), click reagents, control 2-HDAC8 (0.5
lM), biotin tag (50
lM), click reagents, control 3-HDAC8 (0.5
lM), probe (1.5 lM), click reagents,
l
M), biotin tag (50 M), click reagents. (G) Western blot analysis of binding of
l
reagents in all experiments are TCEP (500
lM), TBTA (100
l
M), and CuSO4 (1000
lM). Shown are representative blots of two or more independent experiments.
used in the photolabeling experiments with click reagents. The
click reagents in all the experiments are TCEP (500 M), TBTA
(100 M), and CuSO4 (1000
M).19
of SAHA did not result in a decrease in the biotinylation signal pro-
duced by 1b (Fig. 2C), suggesting 1b does not bind to the main
catalytic site and produces only non-specific biotinylation under
non-denaturing conditions. A comparison of the density of the bio-
tinylated bands corresponding to the HDAC8 adducts with 1a and
1h provided further evidence for a novel binding mode of our non-
ZCG probes. Specifically, probe 1h, which is at least ninefold less
potent than probe 1a, gave much higher biotinylation than 1a at
the same concentrations of both these probes (Fig. 2B). Although
the efficiency of the (3+2) cycloaddition and the biotinylation re-
sponse may depend on the geometry of the moiety bearing the azi-
do group and its accessibility, this observation clearly indicates
that the preferable binding mode of 1h is such that its azido group
is exposed to the solvent, is accessible for the (3+2) cycloaddition
reaction, and the resulting biotin-containing adduct is available
for recognition by the Strep–HRP.
We envisioned that denaturing the protein prior to initiating
the (3+2) cycloaddition reaction in case of probe 1b may expose
the alkyl azide, which was previously buried inside the secondary
binding site, thus making it more accessible to the (3+2) cycloaddi-
tion reaction. Photolabeling of HDAC8 with probe 1b followed by
reaction with the biotin tag resulted in a much higher biotinylation
under denaturing conditions as compared to non-denaturing con-
ditions as shown in Figure 2D. To further confirm that the biotinyl-
ation produced 1b under denaturing conditions is primarily due to
interaction of the probe with the binding site of HDAC8, we per-
formed a series of competition experiments between probe 1b
and potent inhibitors SAHA, TSA, PCI34051, and ligand 1f
(Fig. 2E). All of the competitors exhibited a pronounced decrease
in biotinylation of HDAC8 by 1b. Interestingly, despite 100–1000
l
l
l
Our photolabeling studies required probes able to differentiate
between the ‘normal’ and the hypothesized ‘upside-down’ poses
leading us to design and synthesis of two additional probes 1h
and 1i. We envisioned that after crosslinking both the probes 1h
and 1i would still be detectable by the Strep–HRP antibody if they
bind ‘upside-down’ and their alkyl azide groups point toward the
solvent and can react with the alkyne-containing biotin tag via
(3+2) cycloaddition. If 1h and 1i bind with their ester group near
the zinc atom of HDAC8 then the alkyl–azido ester group would
be hidden in the binding site, and these ligands are not expected
to react with the alkyne moiety of the biotin tag and the resulting
adducts should not be recognized by the Strep–HRP antibody. In
agreement with the already established SAR, the effect of different
ester groups on potency was marginal, and 1h and 1i inhibited
HDAC8 with IC50 of 67.4 and 28.9 lM, respectively. We found that
similar to probe 1a,19 probes 1h (not shown) and 1i produce a
dose-dependent increase in biotinylation of HDAC8 (Fig. 2A).
Next, we compared the dose-dependent increase in biotinyla-
tion for ligands 1a, 1b, and 1h (Fig. 2B). It was expected that li-
gands 1a and 1h would produce a pronounced dose-dependent
biotinylation increase, whereas 1b would not. Consistently with
the binding mode proposed, probe 1b gave only a very low non-
specific biotinylation, whereas 1a and 1h gave the pronounced
dose-dependent biotinylation of HDAC8. To rule out the possibility
of probe 1b binding to the main catalytic site, a competition exper-
iment was carried out at an equimolar concentration of 85-fold
more potent SAHA. The presence of an equimolar concentration

A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6621–6627
6625
fold difference in potency (Table 1), the competing ligands com-
pletely decreased the biotinylation of HDAC8 only at very high
concentrations (low concentrations are not shown), suggesting
that the binding of SAHA, TSA, and PCI34051 to the main catalytic
site only partially obstructs the binding of probe 1b to the second-
ary binding site. We observed that at 50 fold molar excess 1f also
decreased the biotinylation of HDAC8 produced by 1b in a manner
comparable to competition with SAHA, TSA, and PCI34051. Consid-
ering the remarkable difference in potency between 1f and the lat-
ter HDAC inhibitors, the outcome of this photolabeling experiment
appears to strongly indicate that the binding mode of 1b and 1f dif-
fers from that of the ligands that have ZCG.
Next, we investigated if probe 1i can be competed by SAHA,
TSA, PCI34051, and 1f under non denaturing conditions. We ob-
served that similar to probe 1b, probe 1i could be competed out
by SAHA, TSA, or PCI34051 at 25 to 50-fold molar excess concen-
trations as indicated in Figure 2F. The decrease in biotinylation of
status of histone H4 after a 24 h treatment (Fig. 3A). Unlike the
HDAC6 selective compound 18,37 probe 1i did not inhibit the
deacetylation of
class I HDACs.
a-tubulin, suggesting that 1i targets only nuclear
In summary, we demonstrated that HDAC8 enzymatic activity
can be inhibited by compounds that lack an active site Zn2+ biden-
tate chelating group. Although the structure and activity require
further optimization, multiple lines of evidence presented in the
Letter indicate that the ligands 1b, 1c, 1f–1i, 2b, and 3b have bind-
ing modes different from those of the parent compounds 1a, 2a,
and 3a and are likely to be positioned ‘upside-down’ in the second-
ary binding site proximal to the main catalytic site. One of the most
potent ‘upside-down’ inhibitors 1i was shown to increase acetyla-
tion of H4 but not
a tubulin in SH-SY5Y cells. Studies to expand the
SAR presented in the Letter, to elucidate the key pharmacophore
and auxophore portions, to improve the HDAC8 inhibition and
selectivity, and to co-crystallize HDAC8 with the ‘upside-down’ li-
gands are now in progress.
HDAC8 observed with 1a and SAHA at 0.25 and 2.5
is strikingly more pronounced than that observed for 1i: SAHA/
TSA/PCI34051 for both 37.5 M and 75 M of these ligands. These
lM (Fig. 2G)
l
l
Acknowledgments
observations suggest that unlike the hydroxamate containing
probe 1a, which probably binds primarily to the main catalytic site,
probe 1i is likely to bind to a secondary binding site where its bind-
ing is only partially obstructed by the ligands binding to the main
catalytic site such as SAHA, TSA, or PCI34051. This is consistent
with the experiments conducted with 1b under denaturing condi-
tions. We also observed that ligand 1f is able to compete out probe
1i (Fig. 2H). Due to relatively low potency of 1f and low solubility
the competition is less pronounced than in the case of SAHA, TSA,
and PCI34051. These results further support our hypothesis that
the binding of 1i and other non-hydroxamate inhibitors in Table 1
occurs to the secondary binding site as shown in Figure 1. With 1i
and other probes in this series, we could only conduct the photola-
beling studies since the kinetic experiments would require concen-
trations of the inhibitors at which they were not soluble.
This study was funded by the National Cancer Institute/NIH
grant R01 CA131970 and ADDF grant #20101103. Molecular mod-
eling was performed using the UCSF Chimera package from the Re-
source for Biocomputing, Visualization, and Informatics at the
University of California, San Francisco (supported by NIH P41 RR-
01081) and OpenEye Scientific Software, Santa Fe, NM. We thank
Dr. Carol Fierke, University of Michigan, MI, for generously provid-
ing us with the plasmid for in-house expression and purification of
HDAC8. We also thank Mr. Furong Sun of the University of Illinois
at Urbana-Champaign for high-resolution mass spectrometry data.
The Q-Tof Ultima mass spectrometer used for HRMS data was pur-
chased in part with a grant from the National Science Foundation,
Division of Biological Infrastructure (DBI-0100085).
To obtain a preliminary selectivity profile for other HDAC class I
isoform, we measured the inhibition of recombinant HDAC1, 2, and
3 by probes 1h and 1i.19,29 We also assessed the inhibitory effect of
probe 1i on cytoplasmic HDAC6, a representative class II HDAC iso-
form, vs nuclear class I HDACs by monitoring the acetylation status
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ther 1h nor 1i inhibited HDAC3 at concentrations up to 66
whereas HDAC1 and HDAC2 were inhibited by these ligands with
IC50 of 111 and 72 M and 86 and 79 M, respectively. The binding
a
-tubulin and histone H4 in SH-SY5Y cells, respectively.36 Nei-
lM,
l
l
modes of these ligands in HDAC1 and 2 are currently being inves-
tigated. Probe 1i showed a dose dependent increase in acetylation
9. Suzuki, T.; Miyata, N. Mini. Rev. Med. Chem. 2006, 6, 515.
10. Suzuki, T.; Miyata, N. Curr. Med. Chem. 2005, 12, 2867.
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Bioorg. Med. Chem. 2005, 13, 4332.
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Mills, E.; Berghs, S. C.; Carey, N.; Finn, P. W.; Collins, L. S.; Tumber, A.; Ritchie, J.
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Pease, L. J.; Glaser, K. B.; Marcotte, P. A.; Bouska, J. J.; Murphy, S. S.; Davidsen, S.
K. Bioorg. Med. Chem. Lett. 2002, 12, 3443.
16. Guan, J. S.; Haggarty, S. J.; Giacometti, E.; Dannenberg, J. H.; Joseph, N.; Gao, J.;
Nieland, T. J.; Zhou, Y.; Wang, X.; Mazitschek, R.; Bradner, J. E.; DePinho, R. A.;
Jaenisch, R.; Tsai, L. H. Nature 2009, 459, 55.
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Figure 3. (A) Western blot detection of acetylation of histone H4 following a 24 h
treatment with probe 1i in SH-SY5Y cells. (B) Western blot detection of acetylation
21. Muhlethaler-Mottet, A.; Meier, R.; Flahaut, M.; Bourloud, K. B.; Nardou, K.;
Joseph, J. M.; Gross, N. Mol. Cancer. 2008, 7, 55.
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a tubulin following a 24 h treatment with probe 1i and ligand 18 in SH-SY5Y
cells. Shown is a representative blot of two or more independent experiments.

6626
A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6621–6627
22. Witt, O.; Deubzer, H. E.; Lodrini, M.; Milde, T.; Oehme, I. Curr. Pharm. Des. 2009,
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25. Balasubramanian, S.; Ramos, J.; Luo, W.; Sirisawad, M.; Verner, E.; Buggy, J. J.
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7-[4-(3-Azido-5-azidomethylbenzyloxy)phenylcarbamoyl]heptanoic acid tert-
butyl ester (1c). Compound 1c (18 mg, 64% overall) was prepared from 1b
(50 mg, 0.107 mmol) following the same procedure described for 1g. 1H NMR
(400 MHz, CDCl3): d 7.45–7.41 (d, J = 8.4 Hz, 2H), 7.16–7.09 (m, 3H), 6.94–6.92
(m, 2H), 5.05 (s, 2H), 4.38 (s, 2H), 2.36–2.34 (s, 2H), 2.23 (m, 2H), 1.75 (m, 2H),
1.61 (m, 2H), 1.46 (s, 9H), 1.40 (m, 4H). 13C NMR (100 MHz, CDCl3): d 173.2,
171.1, 155.0, 141.1, 139.8, 137.8, 131.6, 123.1, 121.6 (2C), 118.0, 117.5, 115.2
(2C), 80.0, 69.4, 54.2, 37.4, 35.4, 28.7, 28.6, 28.1 (3C), 24.9, 24.8. HRMS (ESI) m/z
[M+H]+ calcd for C26H34N7O4 508.2672, found 508.2673.
26. Fass, D. M.; Reis, S. A.; Ghosh, B.; Hennig, K. M.; Joseph, N. F.; Zhao, W. N.;
Nieland, T. J.; Guan, J. S.; Groves Kuhnle, C. E.; Tang, W.; Barker, D. D.;
Mazitschek, R.; Schreiber, S. L.; Tsai, L. H.; Haggarty, S. J. Neuropharmacology
2012.
27. Somoza, J. R.; Skene, R. J.; Katz, B. A.; Mol, C.; Ho, J. D.; Jennings, A. J.; Luong, C.;
Arvai, A.; Buggy, J. J.; Chi, E.; Tang, J.; Sang, B. C.; Verner, E.; Wynands, R.; Leahy,
E. M.; Dougan, D. R.; Snell, G.; Navre, M.; Knuth, M. W.; Swanson, R. V.; McRee,
D. E.; Tari, L. W. Structure 2004, 12, 1325.
28. Octanoic acid (4-benzyloxyphenyl)amide (7). To an ice cooled solution of 6
(0.61 g, 4.2 mmol) in anhydrous dichloromethane was added EDCI (0.9 g,
4.7 mmol) followed by HOBt (0.63 g, 4.7 mmol) and stirred for 1 h. To this, was
then added a solution of 4 (0.023 g, 0.14 mmol) and DIPEA (1.5 mL, 8.5 mmol)
in anhydrous dichloromethane at 0 °C and stirred for 8 h. The reaction mixture
was treated with 2 N aqueous HCl solution and extracted with EtOAc. The
organic layer was washed with saturated aqueous NaHCO3 solution followed
by brine. The organic extracts were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was further
purified by flash chromatography (90:30 ? 50:50, hexanes:EtOAc) to afford
7 as a light brown solid (0.97 g, 70%). 1H NMR (400 MHz, CDCl3): d 7.44–7.32
(m, 7H), 6.93 (d, J = 8.8 Hz, 2H), 5.05 (s, 2H), 2.34 (t, J = 7.6 Hz, 2H), 1.76–1.69
(m, 2H), 1.34–1.31 (m, 8H), 0.91 (t, J = 6.6 Hz, 3H). 13C NMR (100 MHz, CDCl3): d
171.4, 155.5, 136.9, 131.4, 128.6 (2C), 127.9, 127.4 (2C), 121.7, 115.1, 70.3,
37.6, 31.7, 29.3, 29.0, 25.7, 22.6, 14.1. HRMS (ESI) m/z [M+H]+ calcd for
(3-Amino-5-methoxyphenyl)methanol (13). To a solution of 12 (5 g, 0.03 mol) in
anhydrous THF (100 mL) was added LiALH4 (2.3 g, 0.06 mol) in portions at 0 °C.
The reaction mixture was stirred overnight, cooled to 0 °C, and quenched by
slow addition of aqueous KOH solution (20%, 5 mL) followed by ethyl acetate
(50 mL). The mixture was filtered through a pad of Celite and rinsed with ethyl
acetate. The filtrate was dried over Na2SO4, filtered, and concentrated under
reduced pressure. The crude was purified by flash chromatography
(60:40 ? 40:60, hexanes:EtOAc) to give compound 13 as a pale yellow solid
(2.75 g, 60%). 1H NMR (400 MHz, CDCl3): d 6.38 (s, 1H), 6.34 (s, 1H), 6.31 (s, 1H),
4.39 (s, 2H), 3.79 (s, 3H), 3.77 (s, 2H). 13C NMR (100 MHz, CDCl3): d 160.61,
147.49, 143.13, 105.90, 102.04, 99.87, 64.98, 54.78.
(3-Azido-5-methoxyphenyl)methanol (14). To
a solution of 13 (1.75 g,
11.46 mmol) in glacial acetic acid AcOH/H2SO4 (98% w/v)/water (4:1:5)
(15 mL) was added NaNO2 (1.58 g, 22.93 mmol) in portions at 0 °C and
stirred for 10 min. NaN3 (2.24 g, 34.4 mmol) was then added portion wise at
0 °C and the resulting mixture was stirred for 5 h at rt. The reaction was
quenched with saturated aqueous NaHCO3 solution and extracted with EtOAc
(3 Â 30 mL). The combined organic extracts were washed with brine, dried
(Na2SO4), filtered, and concentrated under reduced pressure. The crude was
purified by flash chromatography (70:30 ? 50:50, hexanes:EtOAc) to give
compound 14 as a pale yellow oil (1.88 g, 90%). 1H NMR (400 MHz, CDCl3): d
6.69 (s, 1H), 6.65 (s, 1H), 6.47 (s, 1H), 4.64 (s, 2H), 3.81 (s, 3H), 2.26 (bs, 1H). 13
C
NMR (100 MHz, CDCl3): d 160.93, 144.02, 141.43, 109.46, 108.85, 104.02,
64.68, 55.45.
C21H28NO2 326.2120, found 326.2119.
Octanoic acid [4-(3,5-dimethylbenzyloxy)phenyl]amide (1d). A suspension of 7
(0.95 g, 2.92 mmol) and Pd/C (150 mg, 0.14 mmol) in MeOH was allowed to
react under H2 atmosphere (balloon pressure) at rt for 5 h. The catalyst was
removed by filtration through a pad of celite. The filtrate was evaporated and
the white crystalline phenol 9 (0.63 g, 92%) obtained almost in pure form was
directly used for next reaction.
Toluene-4-sulfonic acid 3-azido-5-methoxybenzyl ester (15). To a solution of 14
(1.88 g, 10.49 mmol) in anhydrous dichloromethane was added triethylamine
(2.92 mL, 20.98 mmol) followed by tosyl chloride (2.20 g, 11.54 mmol) at 0 °C
portion wise. The reaction mixture was stirred for 30 min at the same
temperature. After completion of the reaction, the mixture was concentrated
under reduced pressure and the residue was immediately purified by flash
chromatography (90:10 ? 80:20, hexanes:EtOAc) to give 15 as a white solid
(1.75 g, 70%). 1H NMR (400 MHz, CDCl3): d 7.82(d, J = 8.2 Hz, 2H), 7.36 (d,
J = 8.2 Hz, 2H), 6.58 (s, 1H), 6.50 (s, 1H), 6.47 (s, 1H), 5.01 (s, 2H), 3.78 (s, 3H),
2.47 (s, 3H). 13C NMR (100 MHz, CDCl3): d 160.91, 145.04, 141.68, 136.24,
133.09, 129.90, 127.99, 110.95, 110.39, 105.40, 71.05, 55.52, 21.66.
7-[4-(3-Azido-5-methoxybenzyloxy)phenylcarbamoyl]heptanoic acid methyl ester
(16). To a solution of 15 (0.72 g, 2.15 mmol) and 8 (0.50 g, 1.79 mmol) in dry
acetone (8 mL) was added K2CO3 (1.48 g, 10.74 mmol) at ambient temperature,
and the reaction mixture was refluxed for 12 h. The mixture was poured into
water and extracted with EtOAc (3 Â 30 mL). The organic layer was washed
with water, dried over Na2SO4, and evaporated under reduced pressure. The
residue was purified by flash chromatography (90:10 ? 70:30, hexanes:EtOAc)
to afford 5 as a white solid (0.56 g, 75%). 1H NMR (400 MHz, CDCl3): d (ppm)
7.43 (d, J = 8.8 Hz, 2H), 7.24 (s, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.76 (s, 1H), 6.72 (s,
1H), 6.51 (s, 1H), 5.01 (s, 2H), 3.82 (s, 3H), 3.68 (s, 3H), 2.32 (m, 4H) ,1.69 (m,
4H), 1.35 (m, 4H). 13C NMR (100 MHz, CDCl3): d 174.25, 171.11, 160.99, 155.15,
141.56, 140.18, 131.57, 121.65, 115.18, 110.01, 109.51, 104.32, 69.69, 55.49,
51.51, 37.42, 33.95, 28.77, 28.31, 25.39, 24.69.
A mixture of 9 (100 mg, 0.42 mmol), 11 (110 mg, 0.55 mmol), and powdered
K2CO3 (90 mg, 0.64 mmol) in acetone (10 mL) was refluxed for 6 h. The mixture
was cooled to rt and concentrated leaving a residue which was dissolved in
ethyl acetate and washed with water followed by brine. The organic fractions
were dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was purified by flash chromatography (95:05 ? 80:20,
hexanes:EtOAc) to furnish 1d as
a
white solid (110 mg, 73%). 1H NMR
(400 MHz, CDCl3): d 7.49–7.41 (m, 2H), 7.06 (s, 1H), 6.98–6.92 (m, 4H), 4.97
(s, 2H), 2.35 (s, 6H), 2.34–2.31 (m, 2H), 1.75–1.69 (m, 2H), 1.37–1.31 (m, 8H),
0.91 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, CDCl3): d 171.8, 156.0, 138.6 (2C),
137.2, 131.7, 130.0, 125.8 (2C), 122.2 (2C), 115.5 (2C), 70.8, 38.0, 32.1, 29.7,
29.5, 26.2, 23.0, 21.7 (2C), 14.5. HRMS (ESI) m/z [M + H]+ calcd for C23H32NO2
354.2433, found 354.2426.
7-[4-(3,5-Dimethyl-benzyloxy)phenylcarbamoyl]heptanoic acid methyl ester (1f).
A mixture of 8 (100 mg, 0.36 mmol), 11 (107 mg, 0.54 mmol), and powdered
K2CO3 (100 mg, 0.72 mmol) in acetone (10 mL) was refluxed for 6 h. The
mixture was cooled to rt and concentrated leaving a residue which was
dissolved in ethylacetate and washed with water followed by brine. The
organic fractions were dried over Na2SO4, filtered, and concentrated under
reduced pressure. The crude product was purified by flash chromatography
(90:10 ? 80:20, hexanes:EtOAc) to furnish 1f as a white solid (107 mg, 75%).
1H NMR (300 MHz, CDCl3): d 7.42–6.92 (m, 7H), 4.97 (s, 2H), 3.67 (s, 3H), 3.33
(s, 6H), 3.32 (m, 4H), 1.73–1.62 (m, 4H), 1.40 (m, 4H). 13C NMR (75 MHz,
CDCl3): d 174.2, 171.2, 155.5, 138.1 (2C), 136.7, 131.3, 129.5, 125.3 (2C), 121.6
(2C), 115.0 (2C), 70.3, 51.4, 37.2, 33.9, 28.7, 28.6, 25.4, 24.6, 21.2 (2C). HRMS
(ESI) m/z [M+H]+ calcd for C24H32NO4 398.2331, found 398.2337.
7-[4-(3-Azido-5-methoxybenzyloxy)phenylcarbamoyl]heptanoic acid (17). To
a
solution of 16 (0.26 g, 0.59 mmol) in THF/MeOH (1:1, 5 mL) was added 4 N
KOH (0.43 mL, 1.78 mmol) at rt, and stirred for 24 h. The mixture was
neutralized with 2 N HCl and extracted with EtOAc. The combined organic
extracts were washed with brine, dried (Na2SO4), filtered, and concentrated
under reduced pressure. The crude 17 obtained as a yellow solid (0.24 mg, 98%)
was used for next reaction without any further purification.
7-[4-(3-Azido-5-methoxybenzyloxy)phenylcarbamoyl]heptanoicacid-4-
7-[4-(3,5-Dimethylbenzyloxy)phenylcarbamoyl]heptanoic acid tert-butyl ester
(1g). An ice cooled solution of 1f (50 mg, 0.126 mmol) in THF–MeOH (5 mL,
1:1) was treated with 2 N NaOH solution (130 lL, 0.25 mmol) and the reaction
mixture was allowed to stir at rt for 1 h. The reaction was diluted with ethyl
acetate (10 mL) and acidified with 2 N HCl (5 mL). The organic layer was
separated, dried over Na2SO4, filtered, and evaporated under reduced pressure.
The crude acid obtained as white solid (40 mg, 85%) was directly used for next
reaction without further purification.
azidomethylbenzyl ester (1i). To an ice cooled solution of 17 (0.05 g, 0.18 mmol)
in anhydrous dichloromethane was added EDCI (0.038 g, 0.18 mmol) followed
by HOBt (0.024 g, 0.18 mmol) and stirred for 1 h. To this, was then added a
solution of [4-(azidomethyl)phenyl]methanol (0.023 g, 0.14 mmol) and DIPEA
(61.3
lL, 0.35 mmol) in anhydrous dichloromethane at 0 °C and stirred for
12 h. Saturated aqueous NaHCO3 solution was added to the reaction mixture
and extracted with EtOAc. The organic layer was washed with water, dried
over Na2SO4, and evaporated under reduced pressure. The residue was purified
by flash chromatography (90:10 ? 70:30, hexanes:EtOAc) to afford 1i as a
white solid (0.02 g, 68%). 1H NMR (400 MHz, CDCl3): d 7.43 (d, J = 8.8 Hz, 2H),
7.38 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.15 (s, 1H), 6.93 (d, J = 8.0 Hz,
2H), 6.77 (s, 1H), 6.72 (s, 1H), 6.51 (s, 1H), 5.14 (s, 2H), 5.01 (s, 2H), 4.36 (s, 2H),
3.83 (s, 3H), 2.38 (t, J = 7.6 Hz ,2H), 2.31 (t, J = 7.6 Hz ,2H), 1.69 (m, 4H), 1.38 (m
,4H). 13C NMR (100 MHz, CDCl3) d 173.53, 171.03, 160.99, 155.16, 141.57,
140.18, 136.25, 135.38, 131.54, 128.65, 128.40, 121.63, 115.19, 110.01, 109.51,
104.32, 69.69, 65.66, 55.49, 55.46, 37.41, 34.15, 28.69 (2C), 25.38, 24.27. HRMS
(ESI) m/z [M+H]+ calcd for C30H34N7O5 572.2621, found 572.2625.
To a solution of acid (40 mg, 0.1 mmol) in CH2Cl2 (5 mL) were added oxalyl
chloride (50
was stirred at rt for 8 h and a solution of tert-butanol (135
CH2Cl2 (2 mL) and triethylamine (45 L, 0.31 mmol) were added. The reaction
l
L, 0.53 mmol) and one drop of dry DMF. The reaction mixture
l
L, 1.25 mmol) in
l
mixture was stirred at rt for 12 h and concentrated under reduced pressure.
The residue was purified by flash chromatography (90:10 ? 70:30,
hexanes:EtOAc) to give 1g as a semi solid (20 mg, 44%). 1H NMR (400 MHz,
CDCl3): d 7.43–7.41 (d, J = 8.8 Hz, 2H), 7.30–7.28 (m, 1H), 7.05 (bs, 1H), 6.97–
6.93 (m, 4H), 4.97 (s, 2H), 2.36 (s, 6H), 2.35–2.20 (m, 4H), 1.67–1.61 (m, 4H),
1.45 (s, 9H), 1.43–1.27 (m, 4H). 13C NMR (100 MHz, CDCl3): d 173.3, 171.2,
155.6, 138.1 (2C), 136.7, 131.2, 129.6, 125.3 (2C), 121.6 (2C), 115.1 (2C), 80.0,
70.4, 37.4, 35.4, 33.8, 28.7, 28.1 (3C), 25.4, 24.8, 21.2 (2C). HRMS (ESI) m/z
[M+H]+ calcd for C27H38NO4 440.2801, found 440.2802.
7-[4-(3-Azido-5-methoxybenzyloxy)phenylcarbamoyl]heptanoicacid-2-azidoethyl
ester (1h). Compound 1h was prepared by a coupling reaction between acid 17
and 2-azido ethanol following the same procedure described for probe 1i.
White solid, 65% yield. 1H NMR (400 MHz, CDCl3) d 7.43 (d, J = 8.8 Hz, 2H), 6.93

A. S. Vaidya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6621–6627
6627
(d, J = 8.8 Hz, 2H), 6.75 (s, 1H), 6.71 (s, 1H), 6.50 (s, 1H), 5.00 (s, 2H), 4.25 (t,
J = 4.8 Hz, 2H), 3.82 (s, 3H), 3.48 (t, J = 4.8 Hz, 2H), 2.38 (m, 4H), 1.69 (m, 4H),
1.40 (m, 4H). 13C NMR (100 MHz, CDCl3): d 173.40, 171.11, 160.99, 155.15,
141.55, 140.18, 131.57, 121.67, 115.17, 110.01, 109.51, 104.31, 69.69, 62.83,
55.48, 49.80, 37.39, 33.95, 28.69, 28.69, 25.38, 24.57. HRMS (ESI) m/z [M+H]+
calcd for C24H30N7O5 496.2308, found 496.2318.
membrane was incubated for 1 h with 3% albumin fraction V (USB, OH) and
washed three times with 1x phosphate buffer saline supplemented with 0.1%
of Tween-20 (PBS-T). The membrane was then incubated with an anti-HDAC8
antibody (1:3000) for 1 h under rt with slight agitation. After three washes in
PBS-T, the membranes were incubated with a secondary antibody antirabbit-
HRP for 1 h at rt. The signals were detected using the enhanced
chemiluminescence (ECL) kit from Pierce (Pierce Biotechnology, Rockford, IL).
35. Dowling, D. P.; Gattis, S. G.; Fierke, C. A.; Christianson, D. W. Biochemistry 2010,
49, 5048.
29. Neelarapu, R.; Holzle, D. L.; Velaparthi, S.; Bai, H.; Brunsteiner, M.; Blond, S. Y.;
Petukhov, P. A. J. Med. Chem. 2011, 54, 4350.
30. Zhang, C. J.; Li, L.; Chen, G. Y.; Xu, Q. H.; Yao, S. Q. Org. Lett. 2011, 13, 4160.
31. Zhao, Y. L.; Dichtel, W. R.; Trabolsi, A.; Saha, S.; Aprahamian, I.; Stoddart, J. F. J.
Am. Chem. Soc. 2008, 130, 11294.
32. Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D. Proteins
2003, 52, 609.
33. Brunsteiner, M.; Petukhov, P. A. J. Mol. Model. 2012.
34. Photoaffinity labeling protocol: The probes or probe/competitor mixture was
incubated with human recombinant HDAC8 (purified from E. coli) for 3 h in
photolabeling buffer (25 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl,
1 mM MgCl2, 0.1% Triton-X), exposed to 254 nm UV light 3 Â 1 min with 1 min
resting. In case of denaturation experiments, post photocrosslinking, a solution
of sodium dodecyl sulfate in water was added to a attain final concentration of
2% w/v. Biotin tag BT was attached to HDAC8-probe adduct using the (3 + 2)
cycloaddition reaction catalyzed by TBTA and Cu(I) produced in situ from
CuSO4 and TCEP. The final concentrations of BT, TCEP, TBTA, and CuSO4 are
36. Cell Culture and western blot protocol: SH-SY5Y cells seeded at 1.0 x 105 cells/
well in 6 well plates and grown to 80% confluence in 1:1 DMEM:OPTI-MEM
(Gibco) supplemented with 10% FBS (Gibco), 1% Antibiotic–Antimyctotic
(Gibco), and 1% penicillin–streptomycin (Mediatech). Cells were treated with
either vehicle, increasing concentrations of 1i, or HDAC6-selective compound
18. Cells were maintained at 37 °C and 5% CO2 for 24 h after treatment. Cells
were lysed using 1Â RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) containing 1:100 dilutions of
protease inhibitor cocktail (Sigma) and 1:100 dilution of phosphatase inhibitor
(Sigma) with shearing. Lysates were clarified through centrifugation at
13,000 rpm at 4 °C. Total protein concentration was determined via the BCA
Protein Assay Kit (Pierce). Lysates were stored at À20 °C until later use. Sample
vials were prepared by aliquoting 20
lg of total protein and adding 4Â Sample
Buffer with 10% b-mercaptoethanol. Sample vials were boiled for 5 min, cooled
to RT, and separated by gel electrophoresis at 80 V. Proteins in the gel were
transferred to PVDF membrane in 4 min using the Invitrogen iBlot system.
Membranes were blocked using 5% Milk in PBS-T and probed using anti-
50 lM, 0.5 mM, 0.1 mM, and 1 mM respectively. After 60 min of incubation at
rt, protein samples were analyzed by SDS-PAGE and western blot using an
antibiotin primary antibody, streptavidin conjugated to horseradish
peroxidase (Pierce, Rockford, IL), and ECL (Pierce, Rockford, IL). The
membranes were then stripped in 0.2 M glycine, pH 2.6 for 10 min, and then
in 0.2 M glycine, pH 2.3 for another 10 min, before being reblocked and
redecorated with an anti-HDAC8 primary antibody and an antirabbit-HRP
GAPDH (1:5000), anti-acetyl
a-tubulin, or anti-acetyl histone H4 (1:1000)
overnight at 4 °C. Membranes were incubated with either anti-mouse (1:5000)
or anti-rabbit (1:5000) in 5% milk in PBS-T. Results were visualized using
Femto chemiluminescent substrate (Pierce) in CCD camera. HRP-conjugated
secondary antibody. Western blotting was done with 0.5
lg of purified protein
anti-rabbit IgG (H+L) was purchased from GE (Piscataway, NJ). Anti-acetyl a-
with 5Â loading buffer containing 10% SDS, 0.05% bromophenol blue, 50%
tubulin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All
other antibodies (anti-GAPDH, anti-acetyl histone H4, and HRP-conjugated
anti-mouse IgG (H+L)) were purchased from Millipore.
glycerol, and b-mercaptoethanol. Protein samples were boiled for 5 min and
allowed to cool before loading on
a denaturing 10% polyacrylamide gel
electrophoresis (SDS PAGE). After electrophoresis, protein was transferred to a
polyvinylidiene difluoride membrane (Imobilon-Millipore, Bedford, MA). The
37. Butler, K. V.; Kalin, J.; Brochier, C.; Vistoli, G.; Langley, B.; Kozikowski, A. P. J.
Am. Chem. Soc. 2010, 132, 10842.