Binding ensemble profiling with photoaffinity labeling (BEProFL) approach: Mapping the binding poses of HDAC8 inhibitors

Article abstract of DOI:10.1021/jm9005077

A binding ensemble profiling with (f)photoaffinity labeling (BEProFL) approach that utilizes photolabeling of HDAC8 with a probe containing a UV-activated aromatic azide, mapping of the covalent modifications by liquid chromatography-tandem mass spectrome

Full text of DOI:10.1021/jm9005077

J. Med. Chem. 2009, 52, 7003–7013 7003
DOI: 10.1021/jm9005077
Binding Ensemble Profiling with Photoaffinity Labeling (BEProFL) Approach: Mapping the Binding
Poses of HDAC8 Inhibitors
Bai He,† Subash Velaparthi,† Gilles Pieffet,† Chris Pennington,†,§ Aruna Mahesh,‡ Denise L. Holzle,‡ Michael Brunsteiner,†
Richard van Breemen,† Sylvie Y. Blond,‡, and Pavel A. Petukhov*,†
†Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago,
Illinois 60612, and ‡Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 South Ashland, Chicago, Illinois 60612. §Present
address:AbbottLaboratories GPRD, Process Analytical Chemistry, 1401 Sheridan Road, NorthChicago, Illinois 60064. Alternative address: Institut
de Geꢀneꢀtique Moleꢀculaire, INSERM/University Denis Diderot Paris 7, UMRS-940, 27 Rue Juliette Dodu, 75010 Paris, France.
Received April 21, 2009
A binding ensemble profiling with (f)photoaffinity labeling (BEProFL) approach that utilizes photo-
labeling of HDAC8 with a probe containing a UV-activated aromatic azide, mapping of the covalent
modifications by liquid chromatography-tandem mass spectrometry, and a computational method to
characterize the multiple binding poses of the probe is described. By use of the BEProFL approach, two
distinct binding poses of the HDAC8 probe were identified. The data also suggest that an “upside-
down” pose with the surface binding group of the probe bound in an alternative pocket near the catalytic
site may contribute to the binding.
Introduction
Histone deacetylases (HDACsa) comprise a family of
enzymes that regulate chromatin remodeling, gene transcrip-
tion, and activity of partner proteins. They control critical
cellular processes, including cell growth, cell cycle regulation,
DNA repair, differentiation, proliferation, and apoptosis.1
Chemical inhibitors of HDACs (Figure 1) have been shown to
inhibit tumor cell growth and induce differentiation and cell
death.2 SAHA (1, Figure 1) has been recently approved by
FDA for use against cutaneous T-cell lymphoma. The
HDACs can be divided into four classes based on structure,
sequence homology, and domain organization.3 It is hypothe-
sized thatdepending on isoform selectivity HDAC ligands can
be either cytotoxic or neuroprotective.4 The development of
isoform-selectiveHDAC inhibitorswould bea significant step
in reducing off-target effects of HDAC-based therapeutics.
One of the key challenges in designing isoform-selective
HDAC inhibitors is a poor understanding of the binding
modes (poses) available to the highly solvent exposed surface
binding group (SBG) of HDAC inhibitors targeting the
grooves and ridges on the protein surface directly adjacent
to the catalytic well of HDACs (Figure 2). It has been
hypothesized by us in our preliminary experiments and a
recent publication by Wiest and colleagues5 that the SBG
groups may have more than one preferred position on the
surface and each of them contributes to the overall binding
affinity. Although the available HDAC X-ray data provide
Figure 1. General structure of HDAC inhibitors.
*To whom correspondence should be addressed. Phone: 312-996-
4174. Fax: 312-996-7107. E-mail: pap4@uic.edu.
Figure 2. Description of the grooves (G1-G3) and ridges
(R1-R3) formed by the protein surface of HDAC8 (PDB 1T69).
a Abbreviations: HDAC, histone deacetylase; BEProFL, binding
ensemble profiling with (f)photoaffinity labeling; BT, biotin tag; ArN3,
aromatic azide group; AlkN3, aliphatic azide group; ZBG, zinc binding
group; SBG, surface binding group; linker, a portion of the molecule
connecting the zinc binding group and surface binding group; SAHA,
suberoylanilide hydroxamic acid; TSA, trichostatin A.
information on structure of proteins and binding of ligands,
its use is limited because of high solvent exposure of the SBG
of the ligands and additional copies of the same protein in the
r
2009 American Chemical Society
Published on Web 11/03/2009
pubs.acs.org/jmc

7004 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
He et al.
Figure 3. Application of photoaffinity probes for detection of binding poses of an HDAC ligand.
Figure 4. Chemical structures of the probes 2 and 3 and the biotin tag 4.
crystallographic cell that interfere with the binding of the
cocrystallized ligands.
simulations to map the ensemble of the poses of HDAC8
inhibitors upon binding (Figure 3) or BEProFL (binding
ensemble profiling with (f)photoaffinity labeling).
Photoaffinity labeling is a strategy offering methods for the
identification of drug target proteins of biologically active
compounds and mapping their binding sites.6-8 Among
several photosensitive groups, arylazide is perhaps one of
the most widely used because of its universality, reactivity,
and relative simplicity in implementation.6 One of the recent
advances in the photoaffinity labeling probe development is
application of “click-chemistry” and bioorthogonal probes
for activity-based proteomics profiling by Cravatt et al.9-15
and its recent modification by Suzuki et al.16-18 This latter
study is based on a concept in which a bifunctional ligand is
connected to a target protein by activation of a photoreactive
group, such as an aromatic azido or 3-trifluoromethyl-3H-
diazirin-3-yl group, and identification of the ligand product is
achieved by anchoring a detectable tag to an alkylazido
moiety, which survives photolysis, using the Staudinger-
Bertozzi ligation.19-24 A similar approach was recently
utilized to discover a new binding site in HIV-1 integrase.25
In this case a different photoactivated moiety, benzophenone,
facilitated by mass spectrometry and docking analysis was
used to determine the exact ligand binding position. The
recent study by Cravatt et al.15 presents a novel proteomics
probe for histone deacetylases and is intended to discover new
proteins interacting with the histone deacetylases. It does not
address, however, the problem of multiple binding poses of
HDAC inhibitors.
Chemistry and Analyses
Synthesis. The synthesis of probes 2 and 3 (Figure 4) is
outlined in Schemes 1 and 2. Commercially available sub-
ericacid monomethyl ester 5 was coupled with 4-(3-nitro-
phenyl)thiazol-2-ylamine in the presence of pyridine and
POCl3 to give ester 6.26 The nitro group of ester 6 was
reduced resulting in amine 7 that was converted to azide 8
by diazotization and nucleophilic substitution with sodium
azide. Further treatment of 8 with hydroxylamine resulted in
hydroxamic acid 2. The condensation of 5 with an equivalent
amount of 4-benzyloxyaniline in the presence of POCl3 and
pyridine gave amide 9. The benzylhydroxy group of amide 9
was deprotected by catalytic hydrogenation and coupled
with mesylate 12,27 resulting in diazide 11 that was then
converted to the corresponding hydroxamic acid 3 in a 40%
yield. BT 4 was synthesized following the procedure de-
scribed earlier.28
HDAC Activity Assay. The inhibition of HDAC8 was
measured as recommended by the supplier BIOMOL Inter-
national using the fluorescent acetylated HDAC substrate
Fluor de Lys (BIOMOL, KI178) and commercially available
recombinant human HDAC8 (BIOMOL). The activity data
are summarized in Table 1.
Photolabeling and Visualization of Probes. The probes 2
and 3 or probe 3-SAHA mixture (in the competition
experiment) were incubated with the HDAC8 protein, ex-
posed to 254 nM UV light 3 ꢀ 1 min with 1 min resting. In the
Here we introduced for the first time the use of diazide-
based photoaffinity labeling probes, liquid chromatography-
tandem mass spectrometry, and molecular dynamics

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7005
Scheme 1. Synthesis of Probe 2a
a Reagents and conditions: (a) POCl3, pyridine, 12 h, room temp, 76%; (b) H2, Pd-C, EtOH, AcOH, 50 °C, 2 h, 80%; (c) NaNO2, AcOH/H2O (9:1),
0 °C, 3 min, then NaN3, 0 °C, 30 min, 73%; (d) NH2OH HCl, KOH, MeOH, 4 h, 0 °C to room temp, 28%.
3
Scheme 2. Synthesis of Probe 3a
a Reagents and conditions: (a) POCl3, pyridine, 12 h, room temp, 61%; (b) H2, Pd-C, MeOH, room temp, 5 h, 81%; (c) K2CO3, acetone, 12 h, 66%;
(d) NH2OH HCl, KOH, MeOH, 4 h, 0 °C to room temp, 40%.
3
Table 1. IC50 and KI Values of Inhibition of HDAC8 with SAHA and
Probes 2 and 3
out followed by mass spectrometric liquid chromatography-
tandem mass spectrometry (LC-MS/MS) and proteomics
database analyses using SEQUEST proteomics software.
Two proteomics data sets were generated; one used an
accurate mass peptide mass tolerance of 5 ppm between the
theoretical and measured peptide masses, and the second
data set used a mass tolerance of 10 ppm. It should be noted
that both 5 and 10 ppm are considered accurate mass
measurements. The results are provided in Table 3 and
Figures 6-8.
Molecular Dynamics Simulations. Molecular Dynamics
Simulations of SAHA and ligand 3 in complex with HDAC8
were performed using the GROMACS software package
(version 3.3.1)29-32 and the Amber03 force field33 as ported
to GROMACS.34 Ligands were parametrized using the
general Amber force field (GAFF)35,36 augmented with
custom azide parameters37and AM1BCC charges.38,39 The
structure of HDAC8 and the starting conformation of
SAHA were taken from PDB 1T69. The starting structures
for 3 were obtained from the docking with Gold 3.2.40 Root
mean square deviation (rmsd) matrices of the ligands were
built for each simulation trajectory by calculating the rmsd
of each structure of the ligand with all other structures of the
same ligand in the trajectory. All protein structures in the
trajectory were aligned using the least-squares fitting of the
protein backbone. The final rmsd calculations of the ligands
HDAC8
compd
IC50 (μM)
Ki (μM)
SAHA
0.44 ( 0.021
2.71 ( 0.14
7.34 ( 0.22
0.396
2.43
6.59
2
3
case of 3, BT 4 was attached to HDAC8-3 adduct using
[3 þ 2]-cycloligation catalyzed by TBTA and Cu(I) produced
in situ from CuSO4 with TCEP. The tagged HDAC8 protein
was visualized with Western blotting. The procedures were in
general similar to those recently described by Cravatt et al.15
and Suzuki et al.16-18 The results are shown in Figure 5.
MALDI-ToF MS Analysis of Intact HDAC8 Protein. The
four HDAC8 samples (three of them were treated with 2 at
different concentrations) were purified and subjected to
MALDI-ToF MS positive ion analysis. The results are
shown in Table 2.
Tryptic Digestion and 1-D LC-MS/MS Analysis.
HDAC8 protein was incubated with the photoaffinity probe
3, exposed to UV light to form HDAC8-3 covalent adduct,
and tagged with BT 4. Next, the modified protein was
purified using avidin-agarose chromatography. Unmodi-
fied HDAC8 was used as a negative control. To facilitate the
analysis of the modified protein, tryptic digestion was carried

7006 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
He et al.
Figure 5. Characterization of biotinylated HDAC proteins and total HDAC proteins. (a) Western blot analyses of probe 2 (labeled as #) and
probe 3 (labeled as /) binding to HDAC8 probed by strep-HRP and anti-HDAC8 antibodies. All the numbers stand for the concentration in
μM. (b) Same as for part a. Competition of probe 3 with SAHA.
Table 2. MALDI MS of Intact HDAC8 and Treated with 2
nitrene moiety known to be very reactive and nonspecific,
which is expected to be essential for capturing a “snapshot” of
the bound probe close to where this reactive moiety was
generated. In addition there are several critical requirements
for photoaffinity probes in general. First, the probes should
exhibit a respectable level of HDAC binding to maximize the
yield of the modified HDAC protein. Second, the procedures
for UV-activated cross-linking should be efficient enough so
that the UV damage of the protein is minimized while the yield
of the cross-linked adduct is maximized. Third, the attachment
procedure of the biotin probe should be fast and efficient to
minimize possible aggregation and precipitation of the protein.
The probes used in this study were designed to mimic the
scaffold of SAHA (1) and a phenylthiazole-based HDAC
inhibitor.41 The synthesis of probes 2 and 3 is shown in
Schemes 1 and 2. It was found that probe 2 is 6.1-fold and
probe 3 is 16.6-fold less active in inhibiting HDAC8 compared
to SAHA (Table 1). The moderate HDAC8 activity of the
ligands with this scaffold is consistent with other reports.42
The decrease in activity of the probes is not surprising, since
introduction of the azide moieties in probes 2 and 3 increased
the lipophilic nature of the solvent exposed SBGs.
av MW (Da)
[M þ H]þ
mass shift,
Δ CTRL
sample
N
std dev
HDAC8 CTRL
HDAC8 (1:1)
HDAC8 (1:5)
HDAC8 (1:10)
6.0
6.0
6.0
4.0
45 620
45 624
45 733
45 877
57.5
41.3
37.1
37.1
0.0
4.0
113.5
256.8
were performed on their heavy atoms using the atoms
corresponding to the scaffold of SAHA. The results of the
rmsd calculations are given in Supporting Information to-
gether with the analysis of the distances between the SBGs of
SAHA and ligand 3 and representative amino acids in
G1-G3. The most representative binding poses are shown
in Figure 9.
Results and Discussion
A general idea for capturing the binding poses of the HDAC
probe using BEProFL approach is outlined in Figure 3.
Among other reactive groups typically used for covalent
modification of the protein we selected an aromatic azide
because (1) it is hydrophobic and therefore is likely to stay
near the protein surface, minimizing the reaction with the
solvent and maximizing the amount of the covalently modified
protein, and (2) upon UV photoactivation it generates a
The success of the procedure shown in Figure 3 depends on
the ability of the probes to modify the protein rather than the
solvent surrounding the ligand and the protein. Since the SBG

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7007
Table 3. Summary of Identified HDAC8 Peptides Modified with Probe 3-BT 4 Adduct
charge
state (z) mods
no. of mass tolerance
(ppm)
entry no.
peptidea
sequence (AA nos.)
[M þ H]þ
3330.72571 1109.57524
m/z
1
IPKRASM*VHSLIEAYALHKQM*R
RASMVHSLIEAYALHKQM*RIVKPK
ASM*VHSLIEAYALHKQMRIVKPK
ASM*VHSLIEAYALHKQMRIVKPK
QM*RIVKPK.
034-055
037-060
038-060
038-060
053-060
133-145
147-171
222-239
240-258
259-286
259-286
3
4
3
3
2
2
6
3
2
4
5
1
1
2
2
1
2
1
1
1
2
2
5
10
5
2
3541.88969 886.22242
4105.10938 1367.70313
4105.10938 1367.70313
3
4
5
5
1734.93338 867.96669
2901.37544 1450.18772
10
5
6
VAINWSGGWHHAKK
7
DEASGFCYLNDAVLGILRLRRKFER
GRYYSVNVPIQDGIQDEK
YYQICESVLKEVYQAFNPK
3660.86731
2800.35061
609.31122
934.11687
5
8
10
10
5
9
3041.46828 1521.23414
4244.03997 1060.25999
10
11
AVVLQLGADTIAGDPMCSFNMTPVGIGK
AVVLQLGADTIAGDPMCSFNMTPVGIGK
4244.03997
849.60799
10
a The asterisk (/) indicates an oxidized methionine. The underlined residues correspond to the region of the HDAC8 protein where modification by
probe 3 occurred.
of HDAC inhibitors is highly solvent exposed, there was a
chance that the aromatic azide moiety would not be able to
reach the protein surface and instead would prefer to react
with the solvent.
protein showed negligible levels of nonspecific biotinyla-
tion with BT 4 (data not shown). In the competition
experiment shown in Figure 5b, the level of the covalent
modifications with probe 3 drops by 50% when SAHA is
present during the photolabeling, indicating that the cross-
linking of the probe 3 to HDAC8 is likely to happen when 3
is bound at the catalytic site.
A total of 11 modified HDAC8 peptides were identified
from the avidin-agarose purified HDAC8-3,4 adduct based
on peptide mapping using high resolution accurate mass
measurement (within 10 ppm) and peptide sequencing using
MS/MS (Table 3). No modified peptides were detected in the
negative control using unmodified HDAC8 (data not shown).
The amino acids underlined in Table 3 correspond to the
residues modified by 3 based on MS/MS analysis. Because of
occasional missed trypsin cleavage sites, which are common in
trypsinization reactions, there are some amino acid sequence
overlaps among several peptides. For example, entries 1 and 2
correspond to overlapping amino acid sequences 35-55 and
37-60, respectively. Also, because of the high reactivity of the
photoaffinity ligand, several amino acid residues became
modified in entries 3, 4, 6, 10, and 11 (Table 3).
As the first step, we confirmed that an aromatic azide group
can indeed be used to covalently attach the probes to the
HDAC8 protein by detecting a mass shift in a MALDI MS
experiment. The results of the MALDI MS analysis are
summarized in Table 2. The average mass of the untreated
HDAC8 protein was 45620 Da. After incubation with ligand
2, the mass of the HDAC8 protein increased in dose-depen-
dent manner. A maximum mass shift of 256.8 Da was
observed at an enzyme to reagent ratio of 1:10. As the amount
of protein modified approaches 100%, the observed average
mass shift would be expected to approach the mass of the
reagent (360 Da). We attribute the relatively high concentra-
tion of probe 2 needed to its relatively poor potency against
HDAC8 and high solvent exposure of the SBG. The results of
the MALDI-MS analysis of the intact protein supported the
conclusion that the protein was successfully modified by the
UV activated nitrene group generated from the probe 2, and
we moved to the next stage, cross-linking followed by attach-
ment of BT 4 to the alkyl azide moiety of probe 3.
Toevaluatethe selectivityofphotolabeling in vitro, purified
recombinant HDAC8 was incubated with various concentra-
tions of ligands 2 and 3. The conditions for UV cross-linking
were optimized to increase the signal-to-noise ratio. We found
that in case of HDAC8 3 ꢀ 1 min exposure to UV light with
1 min rest gives the best signal-to-noise ratio. The “click-
chemistry” conditions with catalysis by Cu(I) generated in situ
were successfully used to attach BT 4 to the adduct between
HDAC8 and probe 3. In our preliminary studies we also tried
the Staudinger-Bertozzi ligation19-24 as an attachment pro-
cedure of the biotin tag and determined that the “click-
chemistry” approach is much faster and gives better yield as
determined by Western blotting. This is consistent with the
observation made by Cravatt et al.9
Among the sites modified by probe 3 (Table 3, Figure 8a),
the two nearest the binding site amino acids located on the
surface of the protein were Asp233 and Asp272. Asp272 maps to
the R2/G1 region of HDAC8, and Asp233 neighbors with the
R2/G2 (Figure 2). The two other areas near the binding site
corresponding to His142-His143 (entry 6, Table 3) and Ile135
-
Asn136-Trp137-Ser138 (entry 6, Table 3) are not on the surface
of the protein. Photolabeling of these residues may be ex-
plained if the binding site of HDAC8 adopts a conformation
similar tothat foundin1T64 X-ray structure wherethe second
molecule of TSA is bound upside down.43 The access to these
residues in other crystal structures is blocked by loop L1
(Figure 8b) that was shown to shift its position to accommo-
date the second molecule of TSA. If loop L1 swings far
enough, it may expose the residues in entries 1-6 for mod-
ification by the SBG of probe 3. The other labeled peptides
correspond to sites on the outer periphery of HDAC8 and
were likely modified by excess unbound 3 upon activation by
UV light. An analysis of these areas shows that they are either
hydrophobic or close to hydrophobic areas and thus may
participate in aggregation with probe 3.
As expected, the amount of biotinylated HDAC increases
ina concentration-dependentmanner in thepresenceofligand
3 but not with ligand 2 (Figure 5a) that lacks the alkyl azide
group designed to bind the biotin tag; only nonspecific,
background level signals are observed. A comparison of the
background levels observed in lanes 3 and 9-11 shows that
the background levels of biotinylation are due to nonspecific
modification of the HDAC8 protein with BT 4 and do not
depend on the presence of probes 2 and 3. It appears that the
nonspecific biotinylation with BT 4 may be specific to the
HDAC8 protein because in a similar experiment HDAC3
Modified Asp233 was contained in peptide GRYYSVNV-
PIQDGIQDEK (222-239), and the MS/MS spectrum of this
modified peptide is shown in Figure 6. On the basis of Sequest
proteomics analysis, the standard peptide fragment ions such
asy61þ and a152þ were identified, andmanual inspection of the

7008 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
He et al.
Figure 6. MS/MS spectrum of HDAC8 tryptic peptide 222-239,
GRYYSVNVPIQDGIQDEK (see Table 3, entry 8), showing alky-
lation at Asp233. The peaks are annotated using the conventional
proteomics MS/MS nomenclature (e.g., y61þ). Upper case letters
denote the additional side chain cleavages of ligand 3 modified by
biotin tag 4 (the structures of possible fragmentation of the resulting
construct are shown in Figure 7).
Figure 8. (a) Location of the peptides (red) modified with probe 3
mapped on HDAC8 PDB 1T69 (blue model). The numbers corre-
spond to the entries in Table 3. The residues rendered as “sticks”
correspond to the residues in the immediate proximity to SAHA in
1T69. (b) Same as for panel a. Overlay of HDAC8 1T69 (blue) and
1T64 (magenta). SAHA and TSA are rendered as “stick” models
colored by atom types (SAHA) or green color (TSA).
ligands 1 and 3 bound to the HDAC8 and compared them with
the outcomes of the photolabeling experiments described above
for ligand 3. The binding modes of the ligands are characterized
by the conformation of the ligand inside the binding site and the
resulting interactions between the SBG of the ligands and the
various structural elements of the HDAC8 surface, i.e., R1-R3
and G1-G3 (Figure 2). To determine the binding modes
available to the ligands, identical conformations sampled dur-
ing the course of an MD simulation were identified using the
matrix rmsd and confirmed with an analysis of the distances
between the SBGs of SAHA and ligand 3 and representative
amino acids in G1-G3 (Supporting Information).
In ligand 1 the binding mode 1 is represented by the areas in
the rmsd matrix at 1, 5, 8.5-12, and 19.5-20 ns, the binding
mode 2 at 2-4.5, 5.5, 13, 14-17, and 17.5-19.5 ns, and the
binding mode 3 at 2, 5.5-8.5, 12, 13.5, and 17 ns. The rmsd
matrix (Supporting Information) shows that ligand 1 changed
from one binding mode to another multiple times during the
courseof the simulation, suggesting thatthey arethe onlyones
available to the ligand. The fact that no new conformations
were found after 2 ns in the remaining 18 ns of the simulation
suggests that the conformational space of the bound ligand
was fully sampled.
Figure 7. Structure and 10 possible bond cleavages of ligand 3
modified with biotin tag 4. Bond cleavages are labeled A-J, and R
denotes the amino acid side chain.
data was used to identify additional fragment ions formed by
side chain fragmentation of 3. By accounting for these side
chain fragments, it was possible to identify and assign the
other peaks present in the MS/MS spectra.
During MS/MS analysis of peptides from HDAC8 that had
been modified by 3, fragmentation of probe 3 was observed at
the sites indicated in Figure 7. These types of cleavages (e.g.,
y152þ-A) are indicated in the tandem mass spectra of mod-
ified HDAC8 peptides. There were a total of three fragment
ions, m/z 689.1 (y61þ), 885.4 (b173þ), and 1190.4 (a152þ), that
could be directly assigned to peptide backbone bond clea-
vages. Of those three, only the y61þ fragment corresponds to a
region of the peptide not containing probe 3. The remaining
fragment ions were assigned to peptide backbone cleavages
plus fragmentation of 3. For three of the fragment ions, more
than one type of fragment ion could be assigned. The overall
HDAC8 sequence analyses ranged between 61% and 68% for
the control HDAC8 samples and between 52% and 58% for
modified HDAC8 protein samples.
Characteristic interactions of each binding mode are deter-
mined by visual inspection of the conformation of the ligand
To explore if there are more than one binding mode for
HDAC ligands, we performed extensive MD simulations of

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7009
the protonation state of Asp101 remains a matter of debate.45
Interestingly, with very few exceptions,45 the majority of the
HDAC X-rays contain an additional copy of the HDAC
protein that not only shields the ligand from the solvent but
also directly affects the binding of the ligand. It is therefore
unclear how relevant the binding pose observed in the X-ray is
to the actual pose of the cocrystallized ligands in the solution.
Similar exploration of all conformational space available to
probe 3 would require running simulations for an unrealisti-
cally long time because of the increased number of degrees of
freedom. To avoid it, we decided to use multiple binding poses
of ligand 3 as starting conformations and perform several
simulations simultaneously, a well accepted approach for
exploring large conformational spaces. Probe 3 was docked
to HDAC8, and 10 diverse binding poses were selected for
further MD simulations. The total cumulated simulation time
was more than 160 ns. The rmsd matrix of ligand 3
(Supporting Information) shows that, similar to ligand 1,
ligand 3 has three main binding modes. The binding mode 1
corresponds to 0-3, 28-32, 60-65, and 135-160 ns, the
binding mode 2 to 20-28, 32-40, 50-60, 85-90, 120-135,
and 165-168 ns, and the binding mode 3 to 3-20, 40-50,
65-85, 90-120, 120-135, 160-165, and 168-180 ns. As
expected for a larger andmoreflexiblemolecule, the transition
time from one binding mode to another is now longer
compared to that observed for the binding modes of SAHA
(SupportingInformation). Thebindingmodes1, 2, and3 have
a weight of approximately 19%, 12%, and 46%, respectively,
with the binding mode 3 now being predominant. Whereas
these binding modes are not present in the same proportion as
in ligand 1, more than one major binding mode is available for
ligand 3 as well.
Figure 9b shows the spatial distribution of the aromatic
azide group throughout the simulations. The cyan dots
represent all the locations occupied by the last nitrogen atom
of the azide group of ligand 3. The coordinates of this atom
were taken from every frame of the simulation trajectory. The
main density areas are located in the grooves and ridges
G1/R2, R2, and G2/R2. The spatial distribution of the
aromatic azide group during the simulation is consistent with
the modification of the residues Asp233 and Asp272 observed
by LC-MS/MS analysis, as it is mostly located in the vicinity
of these residues. Importantly, the modification of these
residues cannot be accomplished if the ligand exists only in a
single binding pose, as the residues modified are located too
far away from each other to be reacting with any single
binding pose. The absence of modification corresponding to
the azido group oriented toward G3 in pose 1 of probe 3 is
consistent with its predominant solvent exposure and the fact
that G3 is relatively deeper compared to G1 and G2. The
sharper curvature of the surface near G3 prevents the remo-
tely located aromatic azide moiety of probe 3 from making
contact with the surface. These observations suggest that to
improve the surface contact with G3, the introduction of a
more flexible moiety between the linker and the SBG portions
of the HDAC ligands may be necessary.
Figure 9. (a) Binding modes 1, 2, and 3 of SAHA indicated in white,
green, and red, respectively. The surface is colored according to
atom types using standard atom color convention. (b) The residues
Asp233 and Asp272 modified by photoactivation of the probe 3 are
marked by a cyan surface. The locations of the aromatic azide
throughout the simulations are projected on the surface and are
marked by cyan dots. Probe 3 is represented in a binding mode
similar to the binding mode 2 observed for SAHA. G1-G3 and
R1-R3 are grooves and ridges on the protein surface, respectively.
inside the binding site of HDAC8. In the first binding mode,
the phenyl ring interacts with the area G2/R3 of the protein,
whereas in the second binding mode the phenyl ring interacts
with G2/R2 (Figure 9a). The third binding mode is similar to
the binding mode of 1 observed in X-ray, as they are both
characterized by strong electrostatic interaction between the
amide group of 1 and Asp101 and weak interaction between
the phenyl ring of 1 and the protein surface.
The analysis indicates that the second binding mode dom-
inates, contributing approximately 35% of the total conforma-
tions. The first and third binding modes follow with an
approximately equal weight, 22% and 21%, respectively. It
is therefore expected that all the three major binding modes of
SAHA collectively contribute to inhibition of HDAC8. Only
the binding mode 3 is observed in the crystal structure PDB
1T69. The only difference between 1T69 and the binding mode
3 is the conformation of the amide group of SAHA. In 1T69
the carbonyl points toward the carboxylate of Asp101, one of
the residues conserved in class I and II HDAC isoforms,
whereas in the binding mode 3 the amide nitrogen of SAHA
forms hydrogen bond with Asp101. Although recent crystal
structures44 of HDAC8 indicate the presence of a hydrogen
bond between Asp101 and the amide nitrogen of the inhibitors,
Conclusions
This is the first study where the binding poses available to
the ligands in HDAC8 were determined experimentally by the
BEProFL approach. The tagging of the probe 3 attached to
HDAC8 with BT 4 resulted in a concentration dependent
increase in biotinylation of the protein with the increase of the

7010 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
He et al.
concentration of probe 3 but not with probe 2 which lacks the
second azido group. The selectivity of probe 3 for the active
site of HDAC8 was demonstrated by blocking it with SAHA.
Using the photoaffinity experiments, we show that probe 3
modifies the HDAC8 binding site at or near Asp233 and
Asp272, the interface areas between G1 and R2 and R2 and
G2 that can only be reached if probe 3 adopts at least two
different binding poses. The results of MD simulations are
consistent with these observations. Sharper curvature of the
HDAC8 protein surface in G3 explains the lack of covalent
7-[4-(3-Azidophenyl)thiazol-2-ylcarbamoyl]heptanoic Acid Methyl
Ester (8). To a solution of 7 (0.350 g, 0.96 mmol) in a 9:1 mixture
of acetic acid and water (15 mL) was added NaNO2 (0.100 g,
1.45 mmol) at 0 °C, and the mixture was stirred for 5 min. To this
was added NaN3 (0.094 g, 1.45 mmol) at the same temperature,
and stirring was continued for 30 min. To this was added
saturated aqueous NaHCO3 solution followed by NaHCO3
powder until the mixture reached pH 7. The mixture was
extracted with EtOAc and the combined organic extracts were
washed with brine, dried (Na2SO4), filtered, and concentrated
1
under reduced pressure to afford 8 (0.273 g, 73%). H NMR
modifications in it. The modifications observed at His142
-
(400 MHz, CD3OD) δ 7.70 (d, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.44
(s, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.00 (dd, J = 7.6, 1.6 Hz, 1H),
3.65 (3H, s), 2.49 (t, J = 7.2 Hz, 2H), 2.34 (t, J = 7.6 Hz, 2H),
1.73 (t, J = 7.2 Hz, 2H), 1.64 (t, J = 6.8 Hz, 2H), 1.41(brs, 4H);
13C NMR (100 MHz, DMSO-d6) δ (ppm) 174.1, 170.2, 158.5,
148.5, 146.7, 136.0, 130.2, 122.5, 122.5, 117.5, 116.7, 108.7, 51.5,
36.2, 33.9, 28.6, 24.6, 24.5.
His143 and Ile135-Asn136-Trp137-Ser138 are likely attributed to
the “upside-down” binding pose of probe 3 analogous to that
of TSA. The other few modifications of the HDAC8 protein
on the side opposite the binding site appear to be a result of
aggregation with probe 3. Although the three major poses
available to SAHA and probe 3 are similar, they are not
equally populated as determined by MD analysis. Overall, the
findings demonstrated that multiple binding poses of the
HDAC ligands are likely to contribute to the binding. Future
studies are needed to learn how this observation can be used in
HDAC drug design to improve the activity and selectivity of
the ligands.
Octanedioic Acid [4-(3-Azidophenyl)thiazol-2-yl]amide Hydro-
xyamide (2). KOH (0.607 g, 10.85 mmol) was added at 40 °C for
10 min to a solution of hydroxylamine hydrochloride (0.753 g,
10.85 mmol) in MeOH. The reaction mixture was cooled to 0 °C
and filtered. Compound 8 (0.210 g, 0.54 mmol) was added to the
filtrate followed by KOH (36 mg, 0.65 mmol) at room tempera-
ture for 30 min and stirred for 4 h. The reaction mixture was
extracted with EtOAc, and the organic layer was washed with a
saturated NH4Cl aqueous solution and brine, dried over Na2SO4,
filtered, and concentrated. The crude solid was purified by
preparative HPLC to give compound 2 (0.058 g, 28%). 1H
NMR (400 MHz, DMSO-d6) δ (ppm) 12.25 (s, 1H), 10.32 (s,
1H), 8.64 (s, 1H), 7.74-7.62 (m, 3H), 7.05 (brs, 1H), 6.67 (brs,
1H), 2.49 (m, 2H), 1.93 (m. 2H), 1.26 (m, 2H), 1.10 (brs, 4H); 13C
NMR (100 MHz, DMSO-d6) δ (ppm) 172.0, 169.5, 158.4, 148.0,
140.3, 136.5, 130.8, 122.8, 118.8, 116.3, 109.5, 35.2, 32.6, 25.4,
25.0; MS (ESI) m/z 389.1 [M þ H]þ; HRMS (ESI) calculated for
[C17H20N6O3S1 þ H]þ 389.1390, found 389.1383.
The study also highlights further directions in refinement of
the BEProFL approach. Minimization of the fragmentation
of the probe is highly desirable to facilitate the analysis of MS/
MS data. The source of nonspecific tagging of the HDAC8
protein upon incubation with biotin tag 4 remains unclear,
warranting further investigation.
The methodology supplies a unique power in analysis of the
binding of ligands to their macromolecular targets by expanding
the data typically obtained in the protein X-ray crystallography
or providing an alternative tool when cocrystallization of the
protein with the ligand of interest has failed. Most importantly,
the binding poses determined by BEProFL are determined in
solution and, thus, the captured “snapshots” reflect the dynamic
nature of the ligand and its macromolecular target conforma-
tions. Extension of the BEProFL approach to study the binding
poses of the ligands in cells is in progress in our laboratory. The
approach has the potential not only to guide ligand optimization
but also to become a new tool in disciplines such as molecular
modeling, development, validation, and application of compu-
ter-aided drug design methods, especially those for rapid pre-
diction of the protein-ligand interactions such as docking and
scoring.
7-(4-Benzyloxyphenylcarbamoyl)heptanoic Acid Methyl Ester
(9). A stirring solution of 4-benzyloxyaniline hydrochloride
(2.01 g, 10.10 mmol) and suberic acid monomethyl ester 5
(1.90 g, 10.10 mmol) in dry pyridine (20 mL) was cooled to
-15 °C, and POCl3 (1.2 mL, 13.13 mmol) was added dropwise
over 30 min and stirred for 12 h at room temperature. The
reaction mixture was diluted with EtOAc and washed thor-
oughly with saturated KHSO4 solution and brine. The organic
phase was dried over Na2SO4. The solvent was removed by
rotary evaporation. The crude solid was washed with EtOAc to
give compound 9 (2.27 g, 61%). 1H NMR (400 MHz, CD3OD):
δ 7.42-7.26 (m, 7H), 7.11 (s, 1H), 6.93 (d, J = 7.8 Hz, 2H), 5.22
(s, 2H), 3.66 (s, 3H), 2.34-2.31 (m, 4H), 1.77-1.71 (m, 2H),
1.70-1.62 (m, 2H), 1.40-1.36 (m, 4H); 13C NMR (100 MHz,
CD3OD) δ 174.2, 171.0, 155.5, 136.9, 131.3, 128.5, 127.9, 127.4,
121.6, 115.2, 70.3, 51.5, 37.4, 33.9, 28.7, 28.7, 25.4, 24.6.
7-(4-Hydroxyphenylcarbamoyl)heptanoic Acid Methyl Ester
(10). A suspension of compound 9 (1.20 g, 3.24 mmol) and Pd/C
(10 wt %, 0.120 g) in MeOH (20 mL) was reacted under
hydrogen atmosphere at room temperature for 5 h. The catalyst
was removed by filtration through a pad of Celite. The solvent
was evaporated. The crude material was purified by flash
chromatography (EtOAc/hexane, 2:1) to give compound 10
Experimental Procedures
General Synthetic Procedures. 1H NMR and 13C NMR
spectra were recorded on Bruker spectrometer at 300/400 and
75/100 MHz, respectively, with TMS as an internal standard.
Standard abbreviations indicating multiplicity were used as
follows: s = singlet, bs = broad singlet, d = doublet, t =
triplet, q = quadruplet, and m = multiplet. HRMS experiments
were performed on Q-TOF-2TM (Micromass). TLC was per-
formed with Merck 60F254 silica gel plates. Column chroma-
tography was performed using Merck silica gel (40-60 mesh).
Analytical HPLC was carried out on an Ace 5AQ column (100
mm ꢀ 4.6 mm) with a Shimadzu 10 VP series HPLC with a diode
array detector with detection at λ = 254 nm. Method A
consisted of the following: H2O/MeCN (0.05% TFA), 70/30
to 0/100 in 25 min, flow rate of 1.3 mL/min. Method B consisted
of the following: H2O/MeCN (0.05% TFA), 90/10 to 0/100 in
25 min, flow rate of 1.3 mL/min. The purity of compounds
determined by HPLC (methods A and B) was g95% for all the
synthesized compounds.
1
(0.742 g, 81%). H NMR (400 MHz, CD3OD) δ 7.31 (d, J =
8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H), 3.65 (s, 3H), 2.34-2.31
(m, 4H), 1.71-1.61 (m, 4H), 1.39-1.34 (m, 4H); 13C NMR
(100 MHz, CD3OD) δ 174.5, 172.8, 153.9, 130.2, 121.9, 114.7,
50.5, 36.2, 33.2, 28.4, 25.4, 24.2.
7-[4-(3-Azido-5-azidomethylbenzyloxy)phenylcarbamoyl]hep-
tanoic Acid Methyl Ester (11). To a solution of 12 (0.250 g, 0.886
mmol) and7-(4-hydroxyphenylcarbamoyl)heptanoicacid methyl
ester 10 (0.247 g, 0.886 mmol) in dry acetone (8 mL) was added
K2CO3 (0.367 g, 2.65 mmol) at ambient temperature, and the
reactionmixture was stirred at the same temperature for 12 h. The

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7011
mixture was poured into water and extracted with EtOAc. The
organic layer was washed with water three times, dried over
Na2SO4, and evaporated in vacuo. The residue was purified by
flash chromatography (EtOAc/hexane, 2:1) to afford 11 (0.272 g,
66%). 1H NMR (400 MHz, CDCl3): δ 7.43 (d, J = 8.8 Hz, 2H),
7.15 (s, 1H), 7.11 (s, 1H), 7.08 (s, 1H), 6.92 (d, J = 9.2 Hz, 2H),
5.04 (s, 1H), 4.37 (s, 1H), 3.69 (s, 3H), 2.36-2.31 (m, 4H), 1.74 (t,
J = 8.0 Hz, 2H), 1.65 (t, J = 7.2 Hz, 2H), 1.40 (brs, 4H); 13C
NMR (100 MHz, CDCl3) δ174.2, 171.0155.0, 141.0, 139.8, 137.8,
131.6, 124.2, 123.1, 121.6, 119.1, 118.7, 117.9, 117.5, 115.1, 69.3,
54.1, 51.4, 37.4, 33.9, 28.6, 25.3, 24.6.
covalently to the protein. The biotin tag 4 was added to all the
tubes at a final concentration of 50 μM, and the chemical
reaction was initiated by addition of TCEP (0.5 mM final
concentration), TBTA (0.1 mM), and CuSO4 (1 mM). After
90 min of incubation at room temperature, protein samples were
analyzed by SDS-PAGE and Western blot using an antibiotin
primary antibody, streptavidin conjugated to horseradish per-
oxidase (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-HDAC
primary antibody and an antirabbit-HRP secondary antibody.
Western Blotting. Western blot was performed using 10 μg of
total protein from cell lysate or 0.5 μg of purified protein with
5ꢀ loading buffer containing 10% SDS, 0.05% bromophenol
blue, 50% glycerol, and β-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 membrane was incubated for 1 h with 1%
albumin fraction V (USB, OH) and washed three times with 1ꢀ
phosphate buffer saline supplemented with 0.05% of Tween-20
(PBS-T). The membrane was then incubated with an anti-
HDAC8 antibody (1:3000) for 2 h under room temperature
with slight agitation. After three washes in PBS-T, the mem-
branes were incubated with a secondary antibody antirabbit-
HRP for 1 h at room temperature. The signals were detected
using the enhanced chemiluminescence (ECL) kit from Pierce
(Pierce Biotechnology, Rockford, IL). Densitometry scanning
of the films was performed using the ImageJ software provided
by NIH.47,48 Data obtained from at least three independent
experiments are presented as the mean of the relative intensity of
the biotinylated bands over the total immunoreactive HDAC
bands ( standard deviation (SD).
MALDI-ToF MS Analysis of Intact HDAC8 Protein. The
samples were purified and concentrated using Centricon 10 000
molecular weight cutoff filter filters (Millipore) according to the
manufacturer’s instructions. Prior to use, the filters were washed
three times with 400 μL of 50 mM ammonium bicarbonate, pH
7.8. Aliquots of 25 μL of each HDAC8 sample were then diluted
to a total volume of 250 μL with the ammonium bicarbonate
buffer and applied to the prerinsed filters. The samples were
centrifuged at 13500g at 4 °C for 60 min. After centrifugation, the
filter was transferred to a second 1.5 mL microcentrifuge tube
inverted and centrifuged at 1000g for 6 min. The recovered
protein (approximately 3-5 μL) was mixed with 3 μL of MALDI
matrix and spotted on a MALDI plate for analysis. The MALDI
matrix was saturated with sinapinic acid in 50:50 (v/v) H2O/ACN
þ 0.1% TFA. Positive ion MALDI mass spectra were acquired
using an Applied Biosystems Voyager DE Pro MALDI-ToF MS.
Tryptic Digestion of HDAC8 and Modified HDAC8. HDAC8
(∼10 μg) was incubated with 5 equiv of probe 3, irradiated with
UV light to activate the probe, and tagged with BT 4. Next, the
modified protein was purified using avidin-agarose chroma-
tography. Unmodified HDAC8 was used as a negative control.
Each sample of modified or unmodified HDAC8 was diluted to
a final volume of 100 μL with an aqueous buffer containing
50 mM ammonium bicarbonate (AMBIC) and 1 mM TCEP (pH
7.8). Prior to digestion, all samples were tested to ensure that their
pH was between 7.0 and 8.0. Control samples containing 10 μg of
HDAC8 were treated with 200 ng of sequence grade modified
trypsin (Promega), while the modified HDAC8 samples (e10 μg
each) were treated with 100 ng of trypsin. Digestions were carried
out for 15 h and then analyzed using LC-MS/MS.
Octanedioic Acid [4-(3-Azido-5-azidomethylbenzyloxy)phenyl]-
amide Hydroxyamide (3). KOH (0.481 g, 8.60 mmol) was added
at 40 °C for 10 min to a solution of hydroxylamine hydrochloride
(0.597 g, 8.60 mmol) in MeOH. The reaction mixture was cooled
to 0 °C and filtered. Compound 11 (0.200 g, 0.43 mmol) was
added to the filtrate followed by KOH (28 mg, 0.51 mmol) at
room temperature for 30 min and stirred for 4 h. The reaction
mixture was extracted with EtOAc, and the organic layer was
washed with a saturated NH4Cl aqueous solution and brine,
dried over Na2SO4, filtered, and concentrated. The crude solid
was purified by preparative HPLC to give compound 3 (0.080 g,
1
40%). H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.72
(s, 1H), 8.65 (s, 1H), 7.48 (d, J = 7.3 Hz, 2H), 7.26 (s, 1H), 7.16 (s,
1H), 7.09 (s, 1H), 6.94 (d, J = 8.6 Hz, 2H), 5.09 (s, 1H), 4.50 (s,
1H), 2.24 (t, J = 7.2 Hz, 2H), 1.93 (t, J = 7.0 Hz, 2H), 1.55-1.46
(m, 4H), 1.26 (brs, 4H); 13C NMR (100 MHz, DMSO-d6) δ 171.1,
169.52, 154.19, 140.48, 140.38, 138.56, 133.37, 124.36, 120.96,
118.61, 118.0, 115.2, 68.9, 53.3, 36.6, 32.6, 28.8, 25.52, 25.47; MS
(ESI) m/z 467.2 [M þ H]þ; HRMS (ESI) calculated for
[C22H26N8O4 þ H]þ 467.2140, found 467.2149.
Biological Materials. The HDAC8 fluorescent assay kit and
the human recombinant HDAC8 protein were purchased from
Biomol International (Plymouth Meeting, PA). Cell culture
media MEM/EBSS, Ham’s F12, fetal bovine serum (FBS),
and nonessential amino acids (NEAA) were purchased from
Hyclone (Logan, UT). Cell culture dishes were purchased from
BD Falcon (BD Biosciences, San Jose, CA). The primary anti-
HDAC8 antibody was from Santa Cruz (Santa Cruz, CA)
respectively. The antirabbit secondary antibody conjugated to
horseradish peroxidase (HRP) was from GE (Piscataway, NJ).
Streptavidin and the colorimetric HRP substrate o-phenylene-
diamine (OPD) were from Pierce (Rockford, IL) and the 96-well
plates from Nunc (Rochester, NY). Tris(2-carboxyethyl)phos-
phine) (TCEP) was from (Alfa Aesar chemicals, Ward Hill,
MA). CuSO4, dimethyl sulfoxide (DMSO), tris((1-benzyl-1H-
1,2,3-triazol-4-yl)methyl)amine (TBTA), and all other chemi-
cals were purchased from Sigma (St. Louis, MO) if not stated
otherwise.
HDAC Activity Assay. The assay proceeded through a simple
two-step procedure that can be carried out in half-volume micro-
titration plates. The in vitro fluoro HDAC assay was performed
in 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.
After 30 min, an amount of50 μL ofdeveloper supplementedwith
5 μM trichostatin A to inactivate the enzyme was added. The
fluorescence experiments were conducted at λexcit = 360 nm and
λemis = 460 nm using a POLARStar Optima microtitration plate
reader (BMG Labtech, Durham, NC). The IC50 values were
determined using the GraphPad Prism 5 software. KI values were
calculated using the Cheng-Prusoff equation46 and a Km for the
substrate equal to 222 μM for HDAC8.
Visualization of Probe 3 Attached to HDAC 8 Using Biotiny-
lated Tag 4. Purified recombinant human HDAC8 (0.5 μg in
5 μL of assay buffer) was incubated with various concentrations
of 2, 3, and SAHA for 3 h in the dark, after which the formation
of a covalent bond between the azide group present in the ligand
and reactive side chains of the HDAC was initiated by UV
irradiation (λ = 254 nm) for 3 ꢀ 1 min with 1 min rest. Then
“click chemistry” was used to attach BT 4 to probe 3 attached
1-D LC-MS/MS Analysis of Tryptic Digests. Tryptic digests
were analyzed using an Ultimate 3000 capillary HPLC system
(Dionex) interfacedwitha LTQ-FThybridlinear iontrapFTICR
mass spectrometer (Thermo-Fisher Scientific) that was operated
using positive ion nanoelectrospray with data-dependent MS/MS.

7012 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
He et al.
The resolving power of the FT ICR mass spectrometer was
∼100 000. Charge state screening was used to exclude singly charged
ions. Samples were diluted to a concentration of ∼0.1 pmol/μL of
total protein using 50 mM AMBIC. The LC-MS/MS injection
volume was 5 μL. The mobile phases consisted of A (95/5 (v/v)
H2O/CH3CN þ 0.1% formic acid) and B (5/95 (v/v) H2O/CH3CN
þ 0.1% formic acid). The trapping column (0.3 mm i.d. ꢀ 5 mm
Dionex C18 PepMap column) was operated at a flow rate of 15 μL/
min using 100% solvent A during sample loading. The analytical
column (75 μm ꢀ 150 mm Agilent Zorbax 300-SB C18 column) was
used to separate the peptide digests of HDAC, and modified
HDAC was operated at a flow rate of 190 nL/min and was equipped
with a PEEK sleeve fitting containing a 75 μm i.d. pulled fused silica
nanoelectrospray emitter (New Objective) with a tip i.d. of 15 μm.
An 85 min linear gradient was used from 3% to 95% solvent B
followed by a 10 min re-equilibration prior to the next analysis.
Following LC-MS/MS analysis, the data were searched using
SEQUEST (Bio Works 3.2) proteomics software.
The binding modes of 1 and 3 determined using the rmsd
matrix were confirmed by monitoring characteristic distances
between the interacting groups. The residues were selected in
such a way that a distance less than 5 A between the center of
mass of the phenyl ring of 1 and Phe208 would indicate that
˚
ligand 1 is in G2/R3. Similarly a distance less than 5 A between
the center of mass of the phenyl ring of 1 and the center of mass
of Met274 would be indicative of ligand 1 in G2/R2, a distance
˚
less than 5 A between the amide NH of the SBG and the center of
mass of Asp101 would be indicative of ligand 1 near R1. The
same distance characteristics of the three binding modes of 1
have been monitored for 3. The results of this analysis are shown
in the Supporting Information.
˚
Acknowledgment. This study was in part funded by the
Breast Cancer CongressionallyDirected Research Programof
Department of Defense Idea Award BC051554 and by the
National Cancer Institute/NIH Grant 1R01 CA131970-
01A1. We also thank Chicago Biomedical Consortium and
Searle family for the support of FT ICR MS and the National
Center for Research Resources/NIH Grant 1S10 RR14686
for the MALDI ToF mass spectrometer. Molecular graphics
images were produced using the UCSF Chimera package
from the Resource for Biocomputing, Visualization, and
Informatics at the University of California, San Francisco
(supported by NIH Grant P41 RR-01081) and OpenEye
Scientific Software, Santa Fe, NM.
Graphics. The protein surface in Figure 2 is rendered in
Vida3.0.49 The images in Figures 8 and 9 were rendered in
Chimera.50
Molecular Dynamics Simulations. Molecular dynamics simu-
lations were performed using the GROMACS software package
(version 3.3.1),29-32 the Amber03 force field33 and the general
Amber force field (GAFF)35,36 together with AM1BCC
charges.38,39 The structure of HDAC8 and the starting confor-
mation of SAHA were taken from the X-ray structure of
HDAC8 forming a complex with SAHA (PDB structure
1T69). Since no crystal structure is available for ligand 3, the
starting structures were obtained from the docking of ligand 3 to
HDAC8 using the docking software Gold 3.2.40 TIP3P explicit
solvent was used.51 All simulations were run in a dodecahedral
box containing approximately 9100 water molecules. The bonds
of the protein and ligands were constrained using the LINCS
algorithm,52 while the bonds and angle of the water molecules
were constrained using the SETTLE algorithm.53 A time step of
2 fs was used for integrating the equations of motion. The system
was simulated in an NTP ensemble at a fixed temperature of
300 K and a fixed pressure of 1 bar. Temperature was kept
constant using a Berendsen thermostat54 with a coupling time of
0.1 ps, and pressure was controlled by a weak coupling to a
reference pressure55 with a coupling time of 1 ps and an
isothermal compressibility of 4.6 ꢀ 10-5 bar-1. Long-range
Coulombic interactions were evaluated using the particle mesh
Ewald (PME) algorithm56 with an interpolation order of 4 and
Supporting Information Available: The rmsd matrixes and
distance analysis figures and synthetic procedures for BT 4. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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sented by a white point on the matrix, whereas different
conformations were represented by a black point. As a conse-
quence, a white block on the matrix diagonal represents a group
of identical conformations and a white block off diagonal
indicates that the corresponding two groups on the diagonal
contain identical conformations. By use of this technique,
different sets of identical conformations corresponding to the
different binding modes of the ligand can be readily identified.
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