Tropolones as lead-like natural products: The development of potent and selective histone deacetylase inhibitors

Article abstract of DOI:10.1021/ml400158k

Natural products have long been recognized as a rich source of potent therapeutics but further development is often limited by high structural complexity and high molecular weight. In contrast, at the core of the thujaplicins is a lead-like tropolone scaf

Full text of DOI:10.1021/ml400158k

Letter
pubs.acs.org/acsmedchemlett
Tropolones As Lead-Like Natural Products: The Development of
Potent and Selective Histone Deacetylase Inhibitors
Sophia N. Ononye,†,‡ Michael D. VanHeyst,† E. Zachary Oblak,‡ Wangda Zhou, Mohamed Ammar,
Amy C. Anderson,* and Dennis L. Wright*
Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, Storrs, Connecticut 06269, United
States
S
* Supporting Information
ABSTRACT: Natural products have long been recognized as
a rich source of potent therapeutics but further development is
often limited by high structural complexity and high molecular
weight. In contrast, at the core of the thujaplicins is a lead-like
tropolone scaffold characterized by relatively low molecular
weight, ample sites for diversification, and metal-binding
functionality poised for targeting a range of metalloenzyme
drug targets. Here, we describe the development of this
underutilized scaffold for the discovery of tropolone derivatives that function as isozyme-selective inhibitors of the validated
anticancer drug target, histone deacetylase (HDAC). Several monosubstituted tropolones display remarkable levels of selectivity
for HDAC2 and potently inhibit the growth of T-cell lymphocyte cell lines. The tropolones represent a new chemotype of
isozyme-selective HDAC inhibitors.
KEYWORDS: Tropolone, HDAC, isozyme-selectivity, thujaplicin, metalloenzyme, T-lymphocyte cancer cell lines
atural products have long served as a rich source of drugs
there are a variety of natural products that inhibit HDACs such
Nfor a variety of indications ranging from anticancer to as trichostatin A (TSA; Figure 1), romidepsin, and trapoxin.7
antimicrobial to neurological disorders. Typically these natural
products are characterized by high molecular weight and
potency as well as high levels of structural complexity with
limited sites for diversification. In contrast, thujaplicins,
members of the tropolone family of natural products, can be
regarded as lead-like natural products. β-Thujaplicin (also
known as hinokitiol) is characterized by low molecular weight
(MW = 164) and a relatively lower level of complexity that
allows more extensive structural modification. Thujaplicins are
Figure 1. Vorinostat and the natural product, TSA, inhibit HDACs; β-
thujaplicin is a lead-like natural product.
monoterpene natural products isolated from the heartwood of
trees in the Cupressaceae family1 that are associated with
antiproliferative activity.2−4 There have been few attempts to
utilize these lead-like compounds in drug discovery, perhaps
exacerbated by limited synthetic accessibility to these non-
benzenoid aromatics.
Both romidepsin and vorinostat were approved by the FDA for
the treatment of cutaneous T-cell lymphoma; the latter
possesses a zinc-targeting hydroxamate, similar to TSA.
Developing an alternative class of HDAC inhibitors based on
tropolones may impart greater metabolic stability and isozyme
selectivity relative to the hydroxamates. It is well-known that
hydroxamates such as vorinostat suffer from relatively short
metabolic half-life owing to an easily reduced N−O bond, a
hydrolytically labile amide linkage and the formation of
glucoronides.8,9 In contrast, the metal-targeting moiety of the
tropolone can be viewed as an extended vinylogous carboxylic
acid8 and would not be subject to reductive or hydrolytic
The tropolone functionality is uniquely disposed to strongly
chelate metal ions, which may be a hallmark of the biological
activity of these compounds.3 Substituted tropolones are a
compelling and distinct chemotype for the development of
inhibitors of metalloenzyme drug targets. Herein, we describe
our efforts to use β-thujaplicin as a lead-like natural product to
develop a novel class of inhibitors of histone deacetylase, a
validated target in the treatment of cancer.5,6
Of the 18 HDAC isoforms, 11 are metalloenzymes that use
zinc to remove a terminal acetyl group from lysine residues
present in histones and other client proteins. The reversible
acetylation and hydrolysis of the ε-acetamide in histones is
associated with regulation of gene expression. Interestingly,
Received: April 29, 2013
Accepted: June 9, 2013
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
transformations. Additionally, the tropolone scaffold offers
several points of modification to access pockets near the metal-
binding site that may impart isozyme selectivity, a feature
believed to be associated with increased efficacy and lower
toxicity.10 Although some hydroxamate-bearing compounds
show some selectivity,11 most of the compounds in clinical
trials are considered to be pan-HDAC inhibitors and exhibit
some associated toxicity that may be related to the inhibition of
the many functions of the HDAC isozymes.10 Herein, we
describe first-generation tropolones that function as potent
inhibitors of the HDAC enzyme and display significant
cytotoxicity against T-lymphocyte cancer cell lines.
followed by basic workup. This route was utilized to generate
six analogues (7a−f). β-Thujaplicin and the corresponding
methylated analogue (8, a mixture of positional isomers) were
also included for evaluation.
In order to accurately compare inhibition values of different
HDAC isozymes, we first performed a thorough Michaelis−
Menten analysis of six different HDAC isozymes: 1, 2, 4, 5, 6,
and 8 (Supporting Information Table S1). We then evaluated
12 substituted tropolones and show that these compounds
function as inhibitors of HDACs and, excitingly, seven (4b−e,
7c,7d, and 7f) show high levels of selectivity (>100-fold) for
the inhibition of HDAC2 relative to HDACs 1, 4, 5, 6, and 8
(Table 1). In fact, all of the free tropolones, with the exception
Structural analysis of HDACs12,13 revealed hydrophobic
pockets near the bound Zn2+ ion that would be accessible to
lipophilic substituents placed in proximity to the putative metal-
binding headgroup. In order to evaluate this design for HDAC
inhibitors, an initial series of monosubstituted tropolones was
prepared, focusing on placing small lipophilic substituents at
the α- or β-positions (Figure 1).
a
Table 1. Inhibition of HDAC Isozymes
HDAC isozyme (Ki values in nM)
compd
1
2
8
4
5
6
TSA
4a
4b
4c
4d
4e
7a
7b
7c
7d
7e
7f
0.87
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1.06
0.26
0.25
0.81
0.42
0.23
0.06
0.12
0.51
0.13
0.22
0.04
15.44
NA
69.65
1.09
14547
NA
4120
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
3.02
527
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
A concise route was developed for α-substituted tropolones
from commercially available 2-chlorotropone (1). The α-chloro
substituent proved very amenable to palladium-catalyzed cross-
coupling conditions such that a series of 2-aryl tropones (2a−e)
were easily prepared by Suzuki reactions. On the basis of a
protocol initially reported for the synthesis of α-thujaplicin,14
the addition of hydrazine led directly to the corresponding 7-
aryl 2-amino tropones (3a−e). Final hydrolysis of the amino-
tropones under basic conditions yielded the α-substituted
tropolones (4a−e) in good overall yield (Scheme 1).
186.30
83.80
811.50
123.65
1.47
NA
NA
NA
NA
10860
8361
11204
806.13
6115
990.23
NA
2.38
266.30
12.81
2.27
122.70
177.95
7.87
β-tj
8
Scheme 1. Synthesis of α-Tropolones
11,641
a
For all HDAC isozymes except HDAC4, not active (NA) refers to Ki
values >2500 nM, the highest concentration of inhibitor; for HDAC4,
NA refers to Ki values >20 000 nM, the highest concentration of
inhibitor. β-tj refers to β-thujaplicin.
of β-thujaplicin, were more potent inhibitors of HDAC2 than
TSA, which inhibited HDACs 1, 2, and 6 with similar levels of
potency. As methylated tropolone 8 shows significantly lower
activity than the free tropolones, it is likely that the ionizable α-
hydroxy ketone may facilitate strong metal (Zn2+) chelation in
the HDAC active site. Overall, both α- and β-substituted
tropolones function as very potent inhibitors of HDAC2 with
groups larger than the isopropyl group found in β-thujaplicin
preferred in the β-position. The selectivity demonstrated by the
tropolones is in sharp contrast to the pan-HDAC inhibition
exhibited by the hydroxamates TSA and vorinostat.16
The selective and potent inhibition of HDAC2 is particularly
exciting as the overexpression of this isozyme has been shown
to be significant in aggressive forms of some cancers.17,18
Additionally, specific knockdown of HDAC2 in estrogen
receptor-positive cells potentiated tamoxifen-induced apopto-
sis,19 while an HDAC2 knockout mouse showed increased
synaptic plasticity and memory facilitation.20 Moreover, it is
observed that the tropolones specifically do not inhibit
HDAC5; this lack of inhibition is potentially beneficial as
deletion of HDAC5 may impair cardiac function.21
We have also recently reported a direct and flexible route to
β-substituted tropolones from the oxabicyclic dibromoenone
515 (Scheme 2). Substituents can be selectively introduced
through cross-coupling or cuprate chemistry to give the
bromoenone 6 that is converted in a single step to the β-
tropolone by reductive ring-opening with samarium diiodide
Scheme 2. Synthesis of β-Tropolones
The addition of HDAC8 inhibition by some of the
tropolones (4a, 7a, 7b, and 7e) is also noteworthy as TSA
and vorinostat have much higher Ki values at 69.65 and 480
nM,16 respectively. Inhibition of HDAC8 was more sensitive to
patterns of substitution with only one α-substituted compound
B
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ACS Medicinal Chemistry Letters
Letter
(4a) showing good inhibition (Ki = 1.09 nM); increasing steric
bulk at this position correlated with loss of potency, while β-
substitution showed variable effects against HDAC8.
substituted derivatives show high potency for HDAC2 than for
HDAC8; increasing the size of this group relative to the
naturally occurring isopropyl substituent is associated with
increased potency. The α-substituted derivatives also per-
formed well as inhibitors of HDAC2 and were selective over
HDAC8. As the pocket comprising Tyr 308, Leu 276, and Phe
155 may be hindered for these compounds, it is possible that
the alpha-tropolones bind in the pocket comprising Cys 156
and Leu 144 (Supporting Information Figure S2). Others have
also exploited this foot pocket of HDAC2.12
The β-phenyl derivative, 7a, was docked to the structure of
HDAC822 (PDB ID 1W22; Figure 2a) using Surflex-Dock. The
In order to understand the origins of the observed selectivity,
structural analysis was extended to HDAC423 (Figure 2b), an
enzyme that was not inhibited by any of the tropolones.
HDAC4 possesses His 332 at the analogous position as Tyr
308 in HDAC2 and appears to be poorly positioned to form a
hydrogen bond to the tropolone oxygens. Additionally, major
differences appear in two key active site loops. One loop,
Pro296−Leu 299 (HDAC4) projects away from the active site
relative to the same loop, Asp 274−Leu 276 (HDAC2; only
L276 shown for clarity) that projects toward the inhibitor. The
second loop in HDAC2, Tyr 27−Pro 37 (containing Pro 34
that interacts with the tropolone), appears to be missing in
HDAC4.
The highly potent derivative, compound 4a, was chosen to
validate the competitive mode of inhibition of HDAC8 by the
tropolones (Figure S3, Supporting Information). These experi-
ments indicate that compound 4a affects the binding of
substrate and is a competitive inhibitor. Additionally, the
measured Ki value for compound 4a is 0.53 nM, which
correlates well with a calculated Ki derived from the measured
KM and measured IC50 value shown in Table 1 (1.09 nM). The
competitive nature of the inhibition and control experiments
with the methylated tropolone (8) provide strong evidence that
the tropolones inhibit HDAC activity by targeting the bound
metal at the active site.
While it was exciting that several of the substituted
tropolones were excellent HDAC inhibitors, it was critical to
evaluate whether these compounds are indeed drug-like and
could translate enzyme inhibition to cellular effects. As it is
well-known that HDAC inhibitors have pronounced anti-
proliferative effects against hematological cancer cell lines,24
two T-cell lymphocyte lines (HuT-78 and Jurkat) were chosen
for analysis. Additionally, a colon cancer cell line (HCT116)
and a pancreatic cancer line (BxPC-3) were also selected as
representative solid tumor lines. The tropolone derivatives
showed significant half maximal growth inhibition (GI50) values
in both T-lymphocyte cell lines (Table 2) with analogues 4c,
4d, 7a, and 7e showing increased activity relative to vorinostat
with GI50 values below 1 μM against the Jurkat cell line.
Although results against HCT-116 showed that the tropolones
were less active, two derivatives (4a and β-thujaplicin) did show
GI50 values less than 20 μM. Likewise, four derivatives (4b, 4d,
7a, and β-thujaplicin) have GI50 values less than 20 μM against
the BxPC-3 cells. Evaluation of the compounds against a
nonmalignant human dermal fibroblast line indicated a lack of
general cytotoxicity with most of the derivatives showing no
activity at 100 μM. This selectivity index is significantly greater
than that exhibited by vorinostat in the same line.
Figure 2. Docked complexes of (a) compound 7a bound to HDAC2
(blue) and HDAC8 (orange) and (b) compound 7a bound to
HDAC2 (blue) and HDAC4 (green).
docked complex suggests that the bulky, hydrophobic β-
substituent preferentially occupies the pocket formed by
residues Phe 152, Tyr 306, Met 274, and Lys 33. In fact, a
potential hydrogen bond may be formed between the
tropolone carbonyl oxygen and the hydroxyl group of Tyr
306 (2.51 Å). It is noteworthy that this pocket is the same one
that houses the alkyl chain of vorinostat. As this pocket is
relatively large, there may be fewer interactions between other
β-substituted tropolones and residues in the pocket, partially
explaining the lower potency of β-thujaplicin and 7f. Branching
at this position (compounds 7d and 7e) or larger aryl
substituents (7a and 7b) is associated with improved
HDAC8 inhibition. While the α-phenyl substituent of
compound 4a appears to be accommodated in the pocket
comprising Tyr 306, Met 274, and Phe 152, substituted (4c−e)
or bulker (4b) phenyl groups would create destabilizing
interactions (Supporting Information Figure S1).
For HDAC2, the pocket defined by Tyr 308, Leu 276, Pro
34, and Phe 155 is analogous to that described above for
HDAC8, including the same potential hydrogen bond formed
with Tyr 304 (Figure 2a). A key substitution of Leu 276 for
Met 274 and the addition of Pro 34 produce additional
potential for hydrophobic interactions. As such, more β-
In order to further characterize the drug-likeness of the leads,
we measured the metabolic stability of a representative active
tropolone toward phase I and II transformations. Incubation of
compound 4a with mouse liver microsomes showed good
metabolic stability with a half-life of 93 min, which compares
C
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ACS Medicinal Chemistry Letters
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Table 2. Inhibition of Cell Growth
AUTHOR INFORMATION
Corresponding Author
*(A.C.A.) E-mail: amy.anderson@uconn.edu. Phone: (860)
486-6145. (D.L.W.) E-mail: dennis.wright@uconn.edu. Phone:
(860) 486-9451.
■
cell line (GI50 values in μM)
compd
Jurkat
HuT-78
HCT-116
BxPC-3
hDF
4a
3.33
1.15
0.62
0.76
1.86
0.67
4.62
5.89
4.45
0.59
6.30
1.10
>100
0.90
7.83
4.11
2.87
3.05
4.74
4.14
8.95
17.09
13.11
3.25
11.36
4.99
>100
2.10
15.24
32.06
56.99
46.65
34.98
53.44
43.67
>100
62.61
26.86
>100
6.92
29.39
17.1
35.93
14.1
21
96.46
93.07
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
18.95
4b
4c
Present Address
‡(S.N.O.) Yale School of Medicine, 333 Cedar Street, New
Haven, Connecticut 06520, United States. (E.Z.O.) Princeton
University, Washington Road, Princeton, New Jersey 08544,
United States.
4d
4e
7a
18.5
91.6
34.79
43.02
104
7b
7c
Author Contributions
†These authors contributed equally to this work.
7d
7e
Funding
Funding was provided by NIH CA162470 to D.L.W.
Notes
The authors declare no competing financial interest.
7f
180
β-tj
8
19
>100
2.50
>100
5.56
Vorin.
ACKNOWLEDGMENTS
■
The authors thank Dr. Kyle Hadden and Dr. Theodore
Rasmussen for providing the HCT116 and BxPC-3 and hDF
cell lines as well as Dr. Janet Paulsen for docking compound 7a
to HDAC8 and Dr. Charles Giardina for advice.
favorably with the 28 min half-life found for vorinostat in a
similar system.9 We also studied the potential for the
tropolones to be metabolized through phase II conjugation
reactions by the addition of UDPGA to the microsomal
incubation and determined a half-life of 60 min and observed
the formation of the gluconoride by mass spectrum analysis.
To validate that the tropolones modulate the acetylation
state of histones in cells, treated Jurkat cells were probed using
antibodies to acetylated histone H4K12. Using flow cytometric
analysis to quantify histone modulation,25 two tropolones (β-
thujaplicin and aryl tropolone 4d) were compared to vorinostat
and an untreated control. The geometric mean of the
fluorescence intensities (GMFI) show that both vorinostat
(GMFI = 100) and the tropolone derivatives (GMFI = 45.40
and 42.20) produce increased levels of histone acetylation for
H4K12 as compared to control (GMFI = 7.13). Although these
experiments do not exclude the possibility that there are
additional targets, the data validate the modulation of HDAC
enzymes in cells.
In conclusion, we investigated whether thujaplicins could
serve as powerful lead-like natural products targeting HDACs
as they possess relatively low molecular weight, ample sites of
diversification, and a key metal-directing functional group.
Structural analysis suggests that the tropolones form a strong
complex with the bound zinc ion and project pendant
functionality into different hydrophobic pockets that confer
isozyme specificity at the active site. In addition to activity at
the enzyme level, further evaluation shows that the compounds
exhibit significant cytotoxicity in cancer cells and good
metabolic stability and modulate histone acetylation levels.
These initial investigations into tropolone-based HDAC
inhibitors suggest that this new chemotype could give rise to
potent and selective inhibitors that may find application in the
study of HDAC function or even as versatile leads for new
therapeutic development.
ABBREVIATIONS
■
HDAC, histone deacetylase; TSA, trichostatin A; hDF, human
adult fibroblasts; UPDGA, uridine diphosphosphate glucoronic
acid
REFERENCES
■
(1) Erdtman, H.; Gripenberg, J. Antibiotic substances from the
heartwood of Thuja plicata. Nature 1948, 161, 719.
(2) Liu, S.; Yamauchi, H. p27-Associated G1 arrest induced by
hinokitiol in human malignant melanoma cells is mediated via down-
regulation of pRb, Skp2 ubiquitin ligase, and impairment of Cdk2
function. Cancer Lett. 2009, 286 (2), 240−249.
(3) Liu, S.; Yamauchi, H. Hinokitiol, a metal chelator derived from
natural plants, suppresses cell growth and disrupts androgen receptor
signaling in prostate carcinoma cell lines. Biochem. Biophys. Res.
Commun. 2006, 351 (1), 26−32.
(4) Morita, Y.; Matsumura, E.; Okabe, T.; Fukui, T.; Shibata, M.;
Sugiura, M.; Ohe, T.; Tsujibo, H.; Ishida, N.; Inamori, Y. Biological
activity of α-thujaplicin, the isomer of hinokitiol. Biol. Pharm. Bull.
2004, 27, 899−902.
(5) Lee, M.-J.; Kim, Y.; Kummar, S.; Giaccone, G.; Trepel, J. Histone
deaceylase inhibitors in cancer therapy. Curr. Opin. Oncol. 2008, 20,
639−649.
(6) Minucci, S.; Pelicci, P. Histone deacetylase inhibitors and the
promise of epigenetic (and more) treatments for cancer. Nat. Rev.
Cancer 2006, 6, 38−51.
(7) Seidel, C.; Schmnekenburger, M.; Dicato, M.; Diederich, M.
Histone deacetylase modulators provided by Mother Nature. Genes
Nutr. 2012, 7, 357−367.
(8) Flipo, M.; Charton, J.; Hocine, A.; Dassonneville, S.; Deprez, B.;
Deprez-Poulain, R. Hydroxamates: Relationships between structure
and plasma stability. J. Med. Chem. 2009, 52, 6790−6802.
(9) Venkatesh, P.; Goh, E.; Zeng, P.; New, L.; Xin, L.; Pasha, M.;
Sangthongpitag, K.; Yeo, P.; Kantharaj, E. In vitro phase I cytochrome
P450 metabolism, permeability and pharmacokinetics of SB639, a
novel histone deacetylase inhibitor in preclinical species. Biol. Pharm.
Bull. 2007, 30, 1021−1024.
(10) Balasubramanian, S.; Verner, E.; Buggy, J. J. Isoform-specific
histone deacetylase inhibitors: The next step? Cancer Lett. 2009, 280
(2), 211−221.
(11) Binaschi, M.; Boldetti, A.; Gianni, M.; Maggi, C.; Gensini, M.;
Bigioni, M.; Parlani, M.; Giolitti, A.; Fratelli, M.; Valli, C.; Tereo, M.;
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, data for enzyme kinetic parameters,
additional figures describing protein:ligand interactions, and
compound characterization. This material is available free of
charge via the Internet at http://pubs.acs.org.
D
dx.doi.org/10.1021/ml400158k | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Garattini, E. Antiproliferative and differentiating activities of a novel
series of histone deacetylase inhibitors. ACS Med. Chem. Lett. 2010, 1,
411−415.
(12) Bressi, J.; Jennings, A. J.; Skene, R. J.; Wu, Y.; Melkus, R.; De
Jong, R.; O’Connell, S.; Grimshaw, C.; Navre, M.; Gangloff, A.
Exploration of the HDAC2 foot pocket: synthesis and SAR of
substituted N-(2-aminophenyl)benzamides. Bioorg. Med. Chem. Lett.
2010, 20, 3142−3145.
(13) 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.
Structural snapshots of human HDAC8 provide insights into the class
I histone deacetylases. Structure 2004, 12 (7), 1325−1334.
(14) Noyori, R.; Makino, S.; Okita, T.; Hakyakawa, Y. A method for
the generation of a synthetic equivalent of unsubstituted oxyallyl via
the bromo ketone-iron carbonyl reaction. A new route to thujaplicins.
J. Org. Chem. 1975, 40, 806−807.
(15) Oblak, E. Z.; Bolstad, E.; Priestley, N.; Hadden, M. K.; Wright,
D. The furan route to tropolones: Probing the antiproliferative effects
of β-thujaplicin analogs. Org. Biomol. Chem. 2012, 10, 8597−8604.
(16) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone
deacetylase inhibitors: From bench to clinic. J. Med. Chem. 2008, 51
(6), 1505−1529.
(17) Marquard, L.; Gjerdrum, L.; Christensen, I.; Jensen, P.;
Sehested, M.; Ralfkiaer, E. Prognostic significance of the therapeutics
targets histone deacetylase 1, 2 and 6 and acetylated histone H4 in
cutaneous T-cell lymphoma. Histopathology 2008, 53, 267−277.
(18) Song, J.; Noh, J.; Lee, J. W.; Eun, J.; Ahn, Y.; Kim, S.; Lee, S.;
Park, W.; Yoo, N.; Lee, J.; Nam, S. Increased expression of histone
deacetylase 2 is found in human gastric cancer. APMIS 2005, 113,
264−268.
(19) Bicaku, E.; Marchion, D.; Schmitt, M.; Munster, P. Selective
inhibition of histone deacetylase 2 silences progesterone receptor-
mediated signaling. Cancer Res. 2008, 68, 1513−1519.
(20) Guan, J.-S.; Haggarty, S.; Giacometti, E.; Dannenberg, J.-H.;
Joseph, N.; Gao, J.; Nieland, T.; Zhou, Y.; Wang, X.; Mazitschek, R.;
Bradner, J.; DePinho, R.; Jaenisch, R.; Tsai, L.-H. HDAC2 negatively
regulates memory formation and synaptic plasticity. Nature 2009, 459,
55−60.
(21) Chang, S.; McKinsey, T.; Zhang, C.; Richardson, J.; Hill, J.;
Olson, E. Histone deacetylases 5 and 9 govern responsiveness of the
heart to a subset of stress siganls and play redundant roles in heart
development. Mol. Cell. Biol. 2004, 24, 8467−8476.
(22) Vannini, A.; Volpari, C.; Filocamo, G.; Casavola, E.; Brunetti,
M.; Renzoni, D.; Chakravarty, P.; Paolini, C.; DeFrancesco, R.;
Gallinari, P.; Steinkuhler, C.; Di Marco, S. Crystal structure of a
eukaryotic zinc-dependent histone deacetylase, human HDAC8,
complexed with a hydroxamic acid inhibitor. Proc. Natl. Acad. Sci.
2004, 101, 15064−15069.
(23) Bottomley, M. J.; Lo Surdo, P.; Di Giovine, P.; Cirillo, A.;
Scarpelli, R.; Ferrigno, F.; Jones, P.; Neddermann, P.; De Francesco,
R.; Steinkuhler, C.; Gallinari, P.; Carfí, A. Structural and functional
̈
analysis of the human HDAC4 catalytic domain reveals a regulatory
structural zinc-binding domain. J. Biol. Chem. 2008, 283 (39), 26694−
26704.
(24) Hagelkruys, A.; Sawicka, A.; Rennmayr, M.; Seiser, C. The
biology of HDAC in cancer: The nuclear and epigenetic components.
Handb. Exp. Pharmacol. 2011, 206, 13−37.
(25) Obier, N.; Muller, A. Chromatin flow cytometry identifies
changes in epigenetic cell states. Cells Tissues Organs 2010, 191, 167−
174.
E
dx.doi.org/10.1021/ml400158k | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX