Human HDAC isoform selectivity achieved via exploitation of the acetate release channel with structurally unique small molecule inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.06.030

Herein we report the discovery of a family of novel yet simple, amino-acid derived class I HDAC inhibitors that demonstrate isoform selectivity via access to the internal acetate release channel. Isoform selectivity criteria is discussed on the basis of X

Full text of DOI:10.1016/j.bmc.2011.06.030

Bioorganic & Medicinal Chemistry 19 (2011) 4626–4634
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Human HDAC isoform selectivity achieved via exploitation of the acetate
release channel with structurally unique small molecule inhibitors
⇑
Lewis Whitehead , Markus R. Dobler, Branko Radetich, Yanyi Zhu, Peter W. Atadja, Tavina Claiborne,
Jonathan E. Grob, Andrew McRiner, Margaret R. Pancost, Anup Patnaik, Wenlin Shao, Michael Shultz,
Ritesh Tichkule, Ruben A. Tommasi, Brian Vash, Ping Wang, Travis Stams
Novartis Institutes for Biomedical Research, 100 Technology Square & 250 Massachusetts Avenue, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report the discovery of a family of novel yet simple, amino-acid derived class I HDAC inhibitors
that demonstrate isoform selectivity via access to the internal acetate release channel. Isoform selectivity
criteria is discussed on the basis of X-ray crystallography and molecular modeling of these novel inhib-
itors bound to HDAC8, potentially revealing insights into the mechanism of enzymatic function through
novel structural features revealed at the atomic level.
Received 1 April 2011
Revised 29 May 2011
Accepted 2 June 2011
Available online 15 June 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Histone deacetylase
X-ray crystallography
Selectivity
Acetate release hypothesis
Metal binding
1. Introduction
studies indicate, enzyme selectivity between HDAC1, 3 and 8 may be
obtained by exploring the opening of the active site, however, differ-
Histone deacetylases (HDACs) are an important class of enzymes
involved in regulating chromatin unfolding, gene expression and
DNA repair via histone acetylation and deacetylation processes. As
such HDACs represent important therapeutic targets in anti-
cancer1,2 as well as anti-viral, anti-parasitic and anti-inflammatory
indications.3 HDACs are divided into three classes comprising Zn2+
cofactor dependent class I (HDAC1–3 and 8), class II (HDAC4–7, 9,
10), and the NADPH cofactor dependent class III (Sirtuins 1–7)
whose primary function is deacetylation of lysine residues at the
N-terminal of Histone domains 3 and 4. The majority of inhibitors
currently being tested in the clinic were developed by academic
and industrial laboratories targeting class I and II HDACs with
non-selective, generally zinc chelating small molecules with liabili-
ties including cross-reactivity and toxicity.4,5 There is evidence
emerging that suggests that HDAC inhibitors, which specifically in-
hibit class I enzymes, offer distinct therapeutic advantage compared
to the classical broad-spectrum inhibitors.6–13 However the high
homology of the class I HDACs in the active site region and the
11 Å vertical channel promises to challenge any attempt to achieve
isoform selectivity, when designing and developing novel therapeu-
tic small molecule HDAC inhibitors. As previous molecular modeling
entiation between HDAC1 and 2 will be very difficult.14 We report
the discovery of a new family of non-hydroxamate based, isoform
selective, class I HDAC inhibitors capable of reaching into the acetate
release channel deep within the enzyme and binding with novel zinc
chelation.
In general HDAC inhibitors such as Trichostatin A (TSA, 1) closely
resemble the aliphatic acetyl-lysine substrate shown in Figure 1,
and deliver a hydroxamic acid or another zinc binding group to
the catalytic zinc cation. The metal cofactor is deeply buried in
the narrow active site pocket, marking the intersection of the
11 Å vertical channel (acetyl-lysine binding tunnel) and the 14 Å
horizontal channel (acetate release channel) as seen in co-crystal
structures HDLP (HDAC-like protein),5 HDAH (HDAC-like amidohy-
drolase)15 and human HDAC8.16 All inhibitors consist of a zinc bind-
ing group and a polar capping group connected through a linker,
preferably containing a vinylic or aryl group. In accordance with
modeling predictions, previous medicinal chemistry attempts to
generate the isozyme-specific HDAC inhibitors generally focused
on varying the capping functionality or in a few cases the linker
group.8,17
Our investigation of a high throughput compound screening as-
say led to the discovery of a novel family (2) of non-hydroxamic
acid containing class I HDAC inhibitors.18 These compounds are
represented generically in structure 2 and exemplified as 3, 4 in
Figure 2. They have been found to exhibit a remarkable selectivity
⇑
Corresponding author.
E-mail addresses: lewis.whitehead@novartis.com, lewlou1@verizon.net
(L. Whitehead).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.030

L. Whitehead et al. / Bioorg. Med. Chem. 19 (2011) 4626–4634
4627
Figure 1. Schematic display of the HDAC enzymatic process. (A) Acetylated lysine binds into HDAC’s binding tunnel coordinating with zinc, aspartic acid and tyrosine side-
chains. R groups represent histone amino-acid chain (B) As lysine departs one would expect bulk-solvent water molecules to begin entering the tunnel, acetic acid is released
into the acetate release channel.
Figure 2. Histone deacetylase inhibitors; Trichostatin A (1); generic representation of the (R)-a-amino-ketone moiety 2; class I HDAC binding ligands 3, 4.
Table 1
In the clinic, hydroxamic acid containing therapeutics are proving
to be effective in saving lives, for instance vorinostat20 is FDA ap-
proved for use in T-cell lymphoma, with panobinostat21 and beli-
nostat22 close to market approval. However, due to the metal
chelating strength of the hydroxamic moiety, it is believed to be
responsible for clinically observed side-effects such as nausea,
thrombocytopenia, anemia and other metabolic abnormalities that
are detrimental to the health of patients in contrast to their life-
saving profile.23 As such, the search for alternative small molecules
containing weaker metal binding features has been of great inter-
est to the HDAC community, as well as for the broader medicinal
chemistry audience where metal binding chemical features are
common in a variety of therapeutic siderophores,24 protein,25
RNA26,27 and DNA27 targets in drug discovery.
Catalytic IC50 values of the in vitro activity of 1, 3 and 4 in HDAC isoforms 1, 2, 6 and 8
Molecule
HDAC1
3 nM
HDAC2
4 nM
HDAC6
51 nM
HDAC8 (nM)
1
3
4
960
200
90
>30
1.7
l
M
M
>30
3.9
l
lM
M
>30
>30
l
l
M
M
l
profile against class I HDAC isoforms, data that is shown in Table 1,
which may be used to aid in understanding and modifying the
molecular mechanisms connecting HDAC activity to cancer
formation.
2. Results and discussion
In this manuscript thediscovery of a novel chiral a-amino-ketone
HDAC binding moiety is disclosed. The discussion will initially detail
the nature of the chemical novelty, and then follow on with the de-
HDAC8 is a member of the class I histone deacetylase enzyme
family and is predominantly expressed in smooth muscle tissue.
It has been implicated as a key player in the progression of can-
cers,19 and is therefore an enzymatic target of tremendous interest
to laboratories around the world concerned with discovering a
cure for these life threatening conditions. The most commonly rec-
ognized chemical class for inhibition of HDACi are the hydroxamic
acids. These acids bind to the catalytic zinc cation at the base of the
acetyl-lysine tunnel via an electrostatically favorable bi-dentate
binding mode with two oxygen atoms, leaving the amide NH to
form an additional hydrogen bonding interaction with a histidine.
tails of co-crystallization of two closely related
a-amino-ketone
containing ligands within HDAC8. It will continue with the rational-
ization of isoform selectivity data and conclude with a speculative
assessment of the mechanism of acetate release.
2.1. Small molecule structural novelty
The novelty of chelation to the catalytic zinc in HDAC8 with an
-amino-ketone motif is shown in Figure 3, the R chirality of the
a

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L. Whitehead et al. / Bioorg. Med. Chem. 19 (2011) 4626–4634
The difluoro-benzyl-piperazine and the dihydro-isoindole moie-
ties of 3 and 4 perform van der Waals interactions with His-
143, Gly-151, Phe-152, His-180 and Phe-208 of the acetyl-lysine
substrate tunnel. A
p-stack between the difluoro-benzyl-pipera-
zine moiety of 3 and Phe-208 and T-stacking interactions of
the dihydro-isoindole in 4 with Phe-152 and Phe-208 are also
noted. Novel inhibitor HDAC8 interactions are observed between
the 3-chlorophenyl and the 2,4-dichlorophenyl moiety of com-
pound 3 and 4, respectively, with side-chains of Ile-34, Trp-
141, Gly-303, Gly-304 and Tyr-306 of the acetate product release
channel. More specifically, the phenyl rings of these molecules
p-stack with the indole ring of Trp-141. These inhibitors differ
from previously determined structures of HDAC8 (PDB codes
1T64, 3F0R) bound to hydroxamate inhibitors, such as TSA (1),
in which interactions only take place at the active site zinc
and acetyl-lysine substrate tunnel.16 To accommodate binding
of 3 and 4 to the acetate release channel, the side chains of
Trp-141, Phe-152 and Tyr-306 have an alternative geometry rel-
ative to the TSA(1)-HDAC8 protein–ligand complex. This side-
chain movement is demonstrated in Figure 5. Additionally,
second shell movements are also seen for Leu-31, Tyr-111, and
Tyr-154.
Figure 3. Novelty of the
b = 2.9 Å, c = 2.0 Å, d = 2.0 Å, e = 2.3 Å, f = 2.95 Å.
a-amino-ketone binding motif in HDAC8. a = 2.2 Å,
Two monovalent potassium cations are also solved in this
HDAC8 structural disclosure. Activation and inhibition of HDAC8
function with these cations has been experimentally validated.34
A site 1 potassium ion resides approximately 7 Å from the catalytic
zinc, coordinated by Asp-176, Asp-178, His-180, Ser-199 and Leu-
200. Site 2 potassium resides ꢀ15 Å from site 1, coordinated by
Phe-189, Thr-192, Val-195, Tyr-225 and Ser-226.
motif is essential. This binding feature has been experimentally ob-
served in several complexes via inspection of small molecule X-ray
crystal structure solutions within the Cambridge structural data-
base.28 In this database, reference code numbers ISODOJ and
ISODUP,29 MAFGUW,30 MIVDEB,31 TEXTOF32 and TUFHEI33 demon-
strate the ability of the carbonyl oxygen and amine functionalities
to coordinate with zinc, however in all but one (ISODOJ) of these
examples the hydrogen bond donating capability of the amine is
not observed, because it cannot be satisfied in these chemical com-
plexes due to the absence of a suitable acceptor.
2.3. Describing the binding data
To explain the inhibitory data of the molecules disclosed in
Table 1, a series of homology models of HDAC1 were generated,
multiple sequence alignments performed and X-ray crystallo-
This zinc binding motif is accommodated in the presence of an
uncharged amine. We suspect that during diffusion of 3 and 4 in
their protonated state from the aqueous bulk solvent towards the
HDAC enzymatic surface and the hydrophobic walls of the acet-
yl-lysine tunnel, that is, moving from a high to low dielectric phys-
icochemical environment, de-protonation of the amine allows the
graphic resources leveraged. Within HDAC8, molecule
4
(Mw = 335.24 g/mol) has a ligand binding efficiency of 0.32 for this
isozyme compared to 0.24 for compound 3 (Mw = 441.41 g/mol),
and an order of magnitude improved binding affinity over 1
(Mw = 302.38 g/mol) with ligand efficiency of 0.27. In the acetate
release channel the chlorophenyl moieties of 3 and 4 reside as
shown in Figure 6, buried in a hydrophobic pocket flanked by res-
idues described previously. It appears that 3 exploits more of a
a-amino-ketone moiety to perform this zinc binding pose. An
alternative argument could be the basic His-142 extracts a proton
which may be shuttled away from the binding area via the aspartic
acid residues that are coordinating a site 1 mono-valent potassium
cation.16
face-to-face phenyl moiety
p-stacking interaction with Trp-141
than 4, but the ortho substituted chloro of 4 exploits favorable
hydrophobic pocket volume at the same time as influencing the
shape of the benzylic functionality to place the para-chloro atom
within 3.3 Å of Ile-34 and 3.2 Å of Arg-37. The meta-chloro on 3
reaches to within 3.8 Å of Ile-34 and 4.1 Å of the Arg-37 side-chain.
This difference in proximity to the side-chain residues of Ile-34 and
Arg-37 and the small difference in attractive van der Waals and
coulombic forces that represents, is one rationalization for HDAC8
affinity of 3 and 4, with compound 1 unable to exploit these favor-
able acetate release tunnel interactions in any way.
In the acetyl-lysine binding tunnel the piperazine and isoindole
moieties overlay as shown in Figures 4 and 6b. The difference in
the hydrophobic nature of these compounds’ interactions with
Phe-152 and Phe-208 may simply be due to the presence of unfavor-
able polar piperazine nitrogen and carbonyl oxygen atoms for mol-
ecule 3 in comparison to more favorable apolar aromatic sp2
carbons of molecule 4 exploiting this region of favorable hydropho-
2.2. X-ray crystallography
X-ray crystal structures of HDAC8 in complex with compounds
3 and 4 were determined at 2.0 Å and 2.7 Å resolution, respec-
tively. Figures 3 and 4 display the structures revealing a novel-
binding mode in which the inhibitors make interactions with
HDAC8 on both sides of the catalytic zinc cation, occupying volume
in both the acetyl-lysine substrate tunnel and the acetate product
release channel.
At the core of both molecules is a primary amine moiety that
completes a pseudo-tetrahedral coordination with the active site
zinc cation, which is also coordinated by protein residues Asp-
178, His-180, and Asp-267. In addition, the central amino group
donates a proton to the N(2) atom of His-142 forming a hydro-
gen bond. The adjacent carbonyl oxygen in 3 and 4 participate in
a weakened electrostatic interaction with zinc at a distance of
2.8 Å and 2.95 Å, respectively in the structural complexes com-
pared to 2.4 Å for TSA. In addition, both species form a water-
mediated hydrogen bond with the backbone amide of Gly-305.
bic binding volume. Also, a 29.4° difference in the
of Phe-152 between both structures provides 4 with a favorable T-
shaped -stacking interaction that 3 cannot form. In Figure 7 the
v2 dihedral angle
p
overlay of the HDAC8-3 complex with recently solved structures
of HDAC8-compound 1 (PDB code 3F0R) and HDAC8-substrate

L. Whitehead et al. / Bioorg. Med. Chem. 19 (2011) 4626–4634
4629
Figure 4. (A) Position of 3 in the active site of HDAC8. (B) Position of 4 in the active site of HDAC8.
clashing, suggesting that loop 2 movement is altered in such a way
that 3 may have modified Kon/off binding rate behavior in relation
to 4 because the acetyl-lysine tunnel will be more open and thus
exposed to the solvent accessible surface. Note the positioning of
Phe-152 between HDAC8 structures, if Asp-101 is un-impeded, the
benzylic amino-acid side chain of Phe-152 sits deeper within the
acetyl-lysine binding tunnel. As 4 does not exploit the solvent ex-
posed rim of HDAC8, it clearly avoids steric clashing with mobile
side-chains like Asp-101 at the surface of the enzyme, stabilizing
the tunnel wall resulting in stronger HDAC8 binding affinity.
2.4. HDAC1 and 2
The observed motion of the loop 2 amino-acid side-chain resi-
dues at the HDAC solvent accessible surface, combined with side-
chain differences in the acetate release channel, can explain the
Figure 5. Overlay of 1 and 3 illustrating movement of residues Tyr-306, Trp-141
and Phe-152 to accommodate 3 penetrating into the acetate release tunnel.
binding behavior of 3 and 4 with HDAC1. A 1.7 lM binding affinity
for 4 in HDAC1 is evidence for the formation of the acetate release
channel we have described in HDAC8. The most prominent amino-
acid differences between HDAC8 and HDAC1 in this channel are
Trp-141 to leucine and Ile-34 to lysine shown in Figure 8, creating
a smaller channel area available for binding. The loss of favorable
(PDB code 3EWF)35 shows the proximity of the positioning of the
difluorophenyl fragment to loop 2 residues, specifically Asp-101
for which we were unable to determine the atomic coordinates. In
this alignment Asp-101 and the difluorophenyl moiety are steric
p-stacking interactions between phenyl rings due to the absence
Figure 6. Alignment of 3 and 4 with prominent amino-acid interactions. (A) Acetate release channel close contacts, complex 3 shown in magenta, 4 is in light blue.
(B) Movement of the benzylic rotamer of the Phe-152 side-chain between crystallographic coordinates.

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L. Whitehead et al. / Bioorg. Med. Chem. 19 (2011) 4626–4634
residue differences between isozymes are distant from the active
site.
2.5. The class IIb HDAC isoform HDAC6
HDAC6 is a class IIb histone deacetylase with 19.7% sequence
identity with HDAC1.5 Trichostatin A (1) IC50 binding data to
HDAC6 implies that the acetyl-lysine binding tunnel and the cata-
lytic zinc areas are structurally similar to the class I HDAC1 and 2
isozymes because the data is consistently below the 100 nM mark.
The acetate release channel in HDAC8 is formed with amino-acid
side chains from loops 1, 2, 3 and 7. On inspecting the molecular
models of the HDAC1/8 channels and the published sequence iden-
tity to HDAC65 a number of interesting observations are evident.
For instance comparing HDAC6 to HDAC1 primary amino-acid se-
quences, loop 1 is one amino-acid shorter, whereas loops 2 and 3
are four and two residues shorter, respectively. The shorter length
of the HDAC6 loops will effect the cavity shape and physicochem-
ical nature of the acetate release channel, and consequently the
selectivity data we observe. Also, residues Leu-137 in HDAC1 and
Trp-141 in HDAC8 have no equivalent residue in the HDAC6 se-
quence as this position is within a two residue gap in the align-
ment shown by Finnin et al. Indeed in order for His-210 in
HDAC6 to retain a zinc binding contact, Gly-209 would occupy
the position of Leu-137 in HDAC1 with very little offered in terms
of favorable intramolecular interactions for the substituted ben-
zylic functionalities of molecules 3 and 4. In addition, Arg-98 in
the HDAC6 loop 1 would be the equivalent residue to Lys-24 in
HDAC1, and Ile-34 in HDAC8. It has already been noted for HDAC1
that the inclusion of a basic amino-acid in the acetate release chan-
nel is likely to be detrimental to the binding data shown for mole-
cule 4. HDAC6 selectivity is clearly demonstrated in the SAR,
however we are unable to provide evidence at this time that syn-
thetic modifications to this novel family of HDAC binders are capa-
ble of activity on HDAC6, but suspect that the acetate release
channel has a unique three dimensional geometry yet to be deter-
mined and chemically exploited.
Figure 7. Alignment of the HDAC8-ligand 3 complex with published HDAC8 PDB
structures. Magenta and dark blue side chains of Asp-101, Phe-152, His-180, Phe-
208 and Tyr-306 correspond to HDAC8 complexes with PDB codes 3EWF and 3F0R,
respectively. Note the proximity of the fluorophenyl moiety with the acidic Asp-101
side-chains identified by the asterisks.
of the indole side-chain of Trp-141, along with the longer lysine
side-chain probing deeper into the channel towards the catalytic
zinc will be detrimental to ligand binding and cause for loss in
binding affinity for 4 in relation to its HDAC8 activity of 90 nM.
HDAC1 data for 3 suggests that interactions within the acetyl-
lysine binding tunnel and the solvent accessible surface are
responsible for the complete lack of activity. Asp-101 in HDAC8
is Asp-99 in HDAC1, and as our previous discussion details, the
difluorophenyl group of 3 is unlikely to fit comfortably in HDAC1
or the motion of loop 2 halts this molecule’s progression into the
HDAC1 active site completely as the IC50 data reports.
Data for the HDAC2 enzyme suggests molecule 4 is capable of
accessing the acetate release channel for this isozyme, binding
with an IC50 of 3.9 lM. As the sequence homology between HDAC1
and 2 is 83%, there is little evidence in the surrounding atomic
binding neighborhood of compound 4 to explain the minor dispar-
ity in this structure activity relationship between HDAC isozymes 1
and 2, IC50 values that are relatively similar for binding to both en-
zymes. The IC50 data for compound 1 suggests that the atomic
environment it experiences with HDAC1 is essentially equivalent
in HDAC2, which is supported by the knowledge that amino-acid
2.6. Mechanism of acetate release
A comparison of the size and shape of the internal acetate re-
lease channel observed in HDAC8 with the HDLP structural homo-
log may have revealed more insights into the mechanism of acetate
Figure 8. Prominent amino-acid side-chain differences between HDAC8 (magenta) and HDAC1 in the acetate-release channel. Crystal structure coordinates of the HDAC8-
ligand 4 complex aligned with an HDAC1 homology model.

L. Whitehead et al. / Bioorg. Med. Chem. 19 (2011) 4626–4634
4631
Figure 9. (A) A comparison between the published HDLP acetate release channel
(red mesh with foreground clipped for clarity), with this reported HDAC8 structure
(green solid surface). Measurement criteria discussed in Supplementary data.
Gating amino-acid residues are defined as Leu-31, Ile-34, Arg-37, Ser- 138 and Trp-
141. (B) Schematic of 4 within HDAC8 showing key binding interactions and the
amino-acid side-chain environment in HDAC1/8.
release from histone deacetylases. These authors note that the ori-
gin of the measurement criterion of the internal 14 Å channel has
not been reported. For instance the distance between the amino-
acids that define the start and end of the channel (terminal hydro-
xyl functionality on Ser-29 and His-131 imidazole side-chain) is
ꢀ20 Å for the first HDLP reported structure.5 In Figure 9 we com-
pare the shape of the ‘14 Å’ acetate release channel between HDLP
and HDAC8. Notice that the volume available in the HDAC8 acetate
release channel is considerably smaller than HDLP. There are
clearly two volume components in HDAC8, and this internal ace-
tate release channel cavity appears to be divided at a junction be-
tween amino-acid residues Leu-31, Ile-34, Arg-37, Ser-138 and
Trp-141 into an ‘12 Å+14 Å’ channel pair.
The existence of the 12 Å+14 Å channel arrangement leads us to
speculate about the mechanism of acetate release within HDACs. In
Figure 1b the re-population of the acetyl-lysine tunnel with water
molecules from the solvent accessible surface is an assumption. On
approach of another acetylated lysine histone residue one could
speculate that the active diffusional forces at play at the molecular
level shunt the acetic acid product from the previous enzymatic
event from the 12 Å ‘release’ channel into the 14 Å ‘disposal’ chan-
nel. In this process Arg-37 would play a role in charge stabilization
of the acetic acid and in partnership with Trp-141 relocating the
side-chain indole moiety to close the 12 Å channel, both residues
Figure 11. A proposed docking mode of acetic acid released from the previous
catalytic cycle transferred into the 14 Å disposal channel. H-Bonding and hydro-
phobic matching distances are noted, with a transparent contour of the channel
shown in green. Compound 3 and zinc are also displayed.
are responsible for actively transporting the acid into the second
channel. Figure 10 is a schematic of the process we describe with
acetic acid switching between channels in order for HDAC to pre-
pare for another catalytic cycle. As a further curiosity, a molecular
docking experiment with acetic acid was completed to assess the
likelihood of the 14 Å disposal channel accommodating this chem-
ical species, the result of which is shown in Figure 11.
The acetic acid settles well at the base of the 14 Å disposal chan-
nel making hydrogen bonding contact with Ile-19 and Trp-137. The
acidic species also makes favorable hydrophobic contacts via its
methyl group with the side-chain residues of Ile-45, Ala-38 and
Val-41. This may indicate the final resting place of the acetic acid
group within the histone deacetylase prior to its release via a
mechanism mediated between residues Tyr-20, Ala-38, Val-41
and His-42 whose side-chain residues separate the acetic acid moi-
ety in this disposal cavity from the aqueous bulk solvent as shown
in Figure 12.
Figure 10. A proposed mechanism of HDAC8 catalytic behavior. (A) Acetylated lysine re-occupies the tunnel along with water (B) acetic acid released from the previous
catalytic cycle is transferred into the 14 Å ‘disposal’ channel.

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L. Whitehead et al. / Bioorg. Med. Chem. 19 (2011) 4626–4634
50% power at 50% duty cycle, and clarified by centrifugation at
200,000g at 4 °C for 1 h. The supernatant was batch bound onto
3 ml of Ni Sepharose High Performance resin (GE) that was pre-
equilibrated in lysis buffer for 1 h at 4 °C. Resin was washed with
lysis buffer plus 5 mM imidazole, and protein eluted with
250 mM imidazole. Following elution the sample was cleaved
with AcTev protease (Invitrogen) yielding full length HDAC8 with
an extra 5 N terminal amino acids of GAMGS. The cleaved sample
was further purified on a Mono Q 10/100 GL column (GE) pre-
equilibrated in buffer A (20 mM TRIS pH 8, 100 mM KCl, 1 mM
TCEP), protein was eluted using a 20 column volume gradient to
1 M KCl. The fractions containing HDAC8 were pooled and assayed
for zinc content using the colorimetric chelator 4-(2-pyridy-
lazo)resorcinol, PAR.36 The zinc content was adjusted by adding
zinc acetate to 1.5-fold the HDAC8 concentration and incubating
for 15 min at room temperature followed by a cleanup of excess
zinc with EDTA. The zinc loaded HDAC8 was purified by size exclu-
sion on a Superdex 75 26/60 prep grade column (GE) which was
equilibrated in SEC buffer (20 mM TRIS pH 8, 50 mM NaCl, 50 mM
KCl, 50 mM ammonium acetate, 1 mM TCEP). The final yield of
HDAC8 was 66 mg and the purity was estimated to be greater than
98%. The zinc content of the purified HDAC8 was measured to be 1
zinc per protein molecule.
Figure 12. The proposed docking mode of acetic acid in the 14 Å disposal channel.
Transparent green contour of the disposal channel has been clipped in the
foreground to aid visualization. Tyr-20, Ala-38, Val-41 and His-42 form the barrier
between the interior of the enzyme and the exterior solvent.
4.3. Chemistry. Methods and materials
3. Conclusions
1-Hydroxybenzotriazole (HOBT), N-ethyl-N’-(3-dimethylami-
nopropyl)carbodiimide hydrochloride (EDCÁHCl), diisopropylethyl-
amine (DIEA) and isoindoline were purchased from Sigma–Aldrich
The hereby-presented novel binding mode of an isoform selec-
tive, non-hydroxamic acid containing inhibitor of class I human
histone deacetylase isoforms is presented. This chemical function-
ality may also provide useful as an alternative metal-binding war-
head motif for other targets of interest to practitioners of small
molecule drug-discovery. Implications and full exploration of our
recent findings for the design of isoform selective histone deacetyl-
ase inhibitors for the treatment of anti-cancer and anti-parasitic
disease indications shall be disclosed and discussed in full account
in a future communication.
and used as such. BOC-3-chloro-
D-phenyl alanine and BOC-2,4-
dichloro- -phenylalanine were purchased from Chem-Impex. Tri-
D
chostatin A (1) was purchased from Sigma–Aldrich. 1H and 13C
NMR spectra were recorded on Bruker Avance 400, equipped with
BBO 5 mm NMR probe, operating at frequency of 400 MHz for 1H
and 100 MHz for 13C. All chemical shifts were referenced to stan-
dard frequency of SiMe4 set at 0 ppm. High resolution mass spectra
were recorded using Agilent 1100 HPLC system (5–95% acetonitrile
in water, 0.1% formic acid gradient, using Intersil ODS3 C18
column).
4. Methods
4.1. HDAC fluorescence assay for IC50 determination
4.3.1. (R)-2-Amino-1-[4-(2,5-difluoro-benzoyl)-piperazin-1-yl]-
3-(3-chloro-phenyl)-propan-1-one (3)
All reaction components are prepared in fluorescent assay buf-
fer (25 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM
MgCl2). Add HDAC enzyme (fivefold activity) and diluted inhibitors
To the solution of BOC-3-chloro-D-phenylalanine (0.10 g,
0.340 mmol) in dichloromethane (1.2 mL) were added EDC.HCl
(0.074 g, 0.387 mmol), HOBT (0.059 g, 0.387 mmol) and DIEA
(0.162 ml, 0.928 mmol) at 0 °C under nitrogen atmosphere. The
solution was stirred for 15 min and (2,5-difluoro-phenyl)-pipera-
zin-1-yl-methanone37 (0.070 g, 0.309 mmol) was added. After
16 h, reaction mixture was diluted with 25 mL of dichloromethane
and washed with 25 mL of satd NaCl solution. Organic layer was
separated, dried using MgSO4 and solvent was removed under re-
duced pressure. Resulting crude was purified using flash column
chromatography (10–90% ethyl acetate/hexanes) to afford the de-
sired coupled product (0.144 g, 92% yield). Purified product
(0.140 g, 0.276 mmol) was dissolved in dichloromethane (1 mL)
and to clear solution was added 4 M HCl in dioxane (1.723 ml,
6.89 mmol) via a syringe. Solution was stirred for 16 h. Upon com-
pletion of reaction, solvent was removed under reduced pressure,
and resulting crude was crystallized using ethyl acetate/hexanes
mixture to afford the title compound 3 (0.095 g, 85% yield). 1H
NMR (400 MHz, CD3OD): d 2.98–3.25 (m, 5H), 3.30–3.41 (m, 1H),
3.43–3.51 (m, 1H), 3.52–3.74 (m, 3H), 4.54–4.77 (m, 1H),
7.12–7.41 (m, 7H) 13C NMR (100.6 MHz, CD3OD): d 38.1, 42.6,
42.7, 42.8, 43.5, 46.1, 46.6, 47.5, 47.6, 51.8, 116.5, 116.7, 118.6,
118.7, 118.8, 118.9, 119.6, 119.7, 119.8, 119.9, 129.4, 130.9,
131.8, 135.9, 137.7, 154.3, 156.7, 158.9, 161.5, 165.9, 168.3 HRMS
(total volume is 25
reactions by adding 25
HDAC1 and 2 assay, 8 M for HDAC6 and 8 assay). The background
well contains panobinostat at 30 M to completely inhibit HDAC
ll) to clear bottom 96-well isoplate. Initiate
l
l Rhodamine substrate (15 M for the
l
l
l
enzyme activity. Enzyme reactions with DMSO are used as positive
control. The reaction is run for 1 h at 37 °C and stopped with 50
ll/well of developer (1.35 mg/ml Trypsin) with TSA(1). Develop
at room temperature for 10 min and read plate with Cytofluor
Fluorescence Reader at excitation 485 nm and emission 530 nm.
4.2. Expression and protein purification
The gene for human HDAC8 was cloned into the pFastbacHTb
vector (Invitrogen) and a high titer baculo virus was obtained using
the Bac-to-Bac system (Invitrogen). Spodopterafrugiperda, Sf9, cells
were infected with the baculo virus containing the full length
HDAC8 with an N terminal hexahistidine tag followed by a TEV
cleavage site for 65 h. Forty grams of frozen sf9 cell pellet was re-
suspended in 200 ml of room temperature lysis buffer (100 mM
TRIS pH 8, 250 mM KCl, 10 mM zinc acetate, 1 mM TCEP) by stirring
for 10 min at 4 °C. The cells were lysed by sonication for 5 min at

L. Whitehead et al. / Bioorg. Med. Chem. 19 (2011) 4626–4634
4633
(TOF-MS) Exact mass calcd for C20H20ClF2N3O2 [M+H]+; 408.1290,
Table 2
Crystallographic data, phasing and refinement information
found:408.1292.
Molecule
3
4
4.3.2. (R)-2-Amino-3-(2,4-dichloro-phenyl)-1-(1,3-dihydro-
isoindol-2-yl)-propan-1-one (4)
Space group
Unit cell
P21
P21
a = 57.81 Å
b = 53.67 Å
c = 61.34 Å
a = 57.64 Å
b = 53.37 Å
c = 60.89 Å
To
a stirred solution of Boc-2,4-dichloro-D-phenylalanine
(0.280 g, 0.84 mmol), EDCÁHCl (0.161 g, 0.84 mmol), HOBt (0.170 g,
1.26 mmol) and DIPEA (1.12 mL, 6.29 mmol) in DMF (2.0 mL), iso-
indoline (0. 10 g, 0.84 mmol) was added. The reaction was stirred
at room temperature for 24 h. Upon completion, solvent was re-
moved under reduced pressure and the crude was purified using col-
umn chromatography to provide desired coupled product in 78%
yield. To the purified compound (0.21 g, 0.48 mmol) was added
1 M HCl (9.5 mL, 1 M in Et2O). After 4 h of stirring, the solvent was
removed under reduced pressure to afford target compound 4 as a
white solid. 1H NMR (400 MHz, CD3OD): d 3.42 (d, 2H, 8.0 Hz),
4.24 (d, 1H, 12 Hz), 4.63 (t, 1H, 8.0 Hz), 4.72 (d, 1H, 16 Hz), 4.89 (d,
1H, 16 Hz), 5.00 (d, 1H, 12 Hz), 7.26 (m, 1H), 7.35 (m, 4H), 7.44 (d,
1H, 8.0 Hz), 7.54 (d, 1H, 4.0 Hz) 13C NMR (100.6 MHz, CD3OD): d
35.3, 52.1, 53.2, 53.6, 123.7, 123.9, 128.9, 129.1, 129.2, 130.7,
132.1, 132.4, 136.0, 136.3, 136.4, 136.6, 168.1 HRMS (TOF-MS) Exact
mass calcd for C17H16Cl2N2O [M+H]+: 335.0718, found: 335.0712.
a
=
c
= 90°
a = c = 90°
b = 108.99°
29.06–2.00
86,153
b = 109.47°
29.82–2.70
35,430
Resolution range (Å)
Total observations
Unique reflections
Completeness (%)a
I/ra
23253
8238
99.6 (97.9)
11.3 (1.8)
0.071 (0.293)
0.177/0.221
2663
31
234
27.5
100.0 (100.0)
12.3 (2.8)
0.159 (0.469)
0.179/0.244
2673
25
183
24.4
a,b
Rsym
Rcryst/Rfree
c
Protein atoms
Heterogen atoms
Solvent molecules
Average B-factor (Å2)
Rms deviations from ideal values
Bond lengths (Å)
Bond angle (°)
0.010
1.03
0.004
0.7
a
b
c
Numbers in parenthesis are for the highest resolution shell.
Rsym over all h, where Ih is the intensity of reflection h.
|Ih À <Ih>|/R h
Rcryst and Rfree |Fo|, where Fo and Fc are observed and calculated
=
R
I
=
R
||Fo| À |Fc||/
R
4.4. Crystallization and structure determination
amplitudes, respectively. Rfree was calculated using 5% of data excluded from the
refinement.
HDAC8–inhibitor complexes were formed by incubating HDAC8
at 25 mg/mL with either 1 mM compound 1 or 2 for 1 h at 4 °C.
Crystals of HDAC8–inhibitor complexes grew within one week by
external research groups.35,44 Sequence alignments were per-
formed with ClustalW45 and used alongside published HDAC align-
ments to verify PRIME’s assessment of amino-acid sequence
matching prior to model building. Structural alignments were per-
formed with the protein alignment feature in Maestro and inter-
nally and externally available protein–ligand X-ray crystal
structures were accessed with Proasis software.46
hanging drop vapor diffusion using 2 ll complex + 2 ll well solu-
tion over 1 ml of well solution (100 mM TRIS pH 8, 12–15% PEG
3350, 500 mM NDSB-201, 18% isopropanol). The crystals were
cryo-protected with the addition of 15% glycerol and 1 mM com-
pound for 5–10 min at 4 °C prior to flash freezing in liquid
nitrogen.
Diffraction data for the HDAC8-inhibitor 3 crystals were col-
Acknowledgments
lected on
a Saturn92 CCD detector using Cu-Ka radiation
(l = 1.5418) from a Rigaku FR-E microfocus rotating anode X-ray
generator operating at 45 kV and 45 mA, with Varimax optics. Dif-
fraction data for the HDAC8-inhibitor 4 crystals were collected on
the IMCA 17BM line at the Advanced Proton Source (APS, Argonne
National Laboratory). Each data set was measured from a single
crystal and the reflections were indexed, integrated, and scaled
using the HKL2000 package.38 The space group for each
HDAC8–inhibitor complex was P21 with 1 HDAC8–inhibitor com-
plex in the asymmetric unit. Molecular replacement was performed
using PHASER39 with the initial model of HDAC8-TSA complex (PDB
entry 1T64) with the water, ion, and inhibitor molecules removed.
Structure determination was achieved through iterative cycles of
both positional and simulated annealing refinement using CNX40
or BUSTER41 and model building using Coot42 Individual B-factors
were refined using an overall anisotropic B-factor refinement along
with bulk solvent correction. The inhibitors along with solvent and
ion molecules were built into the model in later rounds of
refinement. The final coordinates for HDAC8 contain residues Leu-
14-Ser-83 (or Glu-85 in cpd 3 structure) and Gly-107-Val-377, the
N-terminal 13 residues and the loop containing Gln-84-Glu-106
were not modeled due to lack of electron density. Data collection
and refinement statistics are shown in Table 2.
Use of the IMCA-CAT beam-line 17-BM at the Advanced Photon
Source was supported by the companies of the Industrial Macro-
molecular Crystallography Association through a contract with
the Center for Advanced Radiation Sources at the University of
Chicago. Use of the Advanced Photon Source was supported by
the US Department of Energy, Office of Science, Office of Basic
Energy Sciences, under Contract No. W-31-109-Eng-38.
T.C. is a B3 baccalaureate student shared between the Massa-
chusetts Institute of Technology and N.I.B.R., funded through the
N.I.B.R. Diversity and Inclusion Office. L.W. and T.S. would like to
thank Chris Harwell, Steven Litster, Michael Derby and Michael
Steeves of NIBR IT for Linux hardware maintenance and software
installation support. L.W. thanks 2010 Seeding Labs visiting fellow
Dr. Evans Ogwagwa of Kenyatta University in Nairobi for enlight-
ening HDAC discussions.
Supplementary data
Supplementary data (atomic coordinates for HDAC8 in complex
with inhibitors 3 and 4 have been deposited in the Protein Data
Bank with accession codes 3SFF and 3SFH, respectively. Additional
images are contained) associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.030.
4.5. Computational methods
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