Computer-aided identification of new histone deacetylase 6 selective inhibitor with anti-sepsis activity

Article abstract of DOI:10.1016/j.ejmech.2016.03.046

Histone deacetylase (HDAC) inhibitors have been recognized as promising approaches to the treatment of various human diseases including cancer, inflammation, neurodegenerative diseases, and metabolic disorders. Several pan-HDAC inhibitors are currently approved only as anticancer drugs. Interestingly, SAHA (vorinostat), one of clinically available pan-HDAC inhibitors, shows an anti-inflammatory effect at concentrations lower than those required for inhibition of tumor cell growth. It was also reported that HDAC6 selective inhibitor tubastatin A has anti-inflammatory and anti-rheumatic effect. In our efforts to develop novel HDAC inhibitors, we rationally designed various HDAC inhibitors based on the structures of two hit compounds identified by virtual screening of chemical database. Among them, 9a ((E)-N-hydroxy-4-(2-styrylthiazol-4-yl)butanamide) was identified as a HDAC6 selective inhibitor (IC₅₀ values of 0.199 μM for HDAC6 versus 13.8 μM for HDAC1), and it did not show significant cytotoxicity against HeLa cells. In vivo biological evaluation of 9a was conducted on a lipopolysaccharide (LPS)-induced mouse model of sepsis. The compound 9a significantly improved 40% survival rate (P = 0.0483), and suppressed the LPS-induced increase of TNF-α and IL-6 mRNA expression in the liver of mice. Our study identified novel HDAC6 selective inhibitor 9a, which may serve as a potential lead for the development of anti-inflammatory or anti-sepsis agents.

Full text of DOI:10.1016/j.ejmech.2016.03.046

Accepted Manuscript
Computer-Aided Identification of New Histone Deacetylase 6 Selective Inhibitor with
Anti-sepsis Activity
Jakyung Yoo, So-Jin Kim, Dohyun Son, Heewon Seo, Seung Yeop Baek, Cheol-
Young Maeng, Changsik Lee, Insu Kim, Young Hoon Jung, Sun-Mee Lee, Hyun-Ju
Park
PII:
S0223-5234(16)30228-8
10.1016/j.ejmech.2016.03.046
EJMECH 8471
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 November 2015
Revised Date: 16 March 2016
Accepted Date: 17 March 2016
Please cite this article as: J. Yoo, S.-J. Kim, D. Son, H. Seo, S.Y. Baek, C.-Y. Maeng, C. Lee, I. Kim,
Y.H. Jung, S.-M. Lee, H.-J. Park, Computer-Aided Identification of New Histone Deacetylase 6 Selective
Inhibitor with Anti-sepsis Activity, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.03.046.
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Computer-Aided Identification of New Histone
Deacetylase 6 Selective Inhibitor with Anti-sepsis
Activity
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Jakyung Yoo , So-Jin Kim , Dohyun Son , Heewon Seo , Seung Yeop Baek , Cheol-Young
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Maeng , Changsik Lee , Insu Kim , Young Hoon Jung , Sun-Mee Lee , and Hyun-Ju Park *
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School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do 16419, Republic of
Korea
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SK Biopharmaceuticals Co. Ltd., 325 Exporo Yuseong-gu, Daejeon 305-712, Republic of
Korea
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KEYWORDS.HDAC6 selective inhibitor,computer-aided drug design, anti-sepsis
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ABSTRACT. Histone deacetylase (HDAC) inhibitors have been recognized as promising
approaches to the treatment of various human diseases including cancer, inflammation,
neurodegenerative diseases, and metabolic disorders. Several pan-HDAC inhibitors are currently
approved only as anticancer drugs. Interestingly, SAHA (vorinostat), one of clinically available
pan-HDAC inhibitors, shows an anti-inflammatory effect at concentrations lower than those
required for inhibition of tumor cell growth. It was also reported that HDAC6 selective inhibitor
tubastatin A has anti-inflammatory and anti-rheumatic effect. In our efforts to develop novel
HDAC inhibitors, we rationally designed various HDAC inhibitors based on the structures of
two hit compounds identified by virtual screening of chemical database. Among them, 9a ((E)-
N-hydroxy-4-(2-styrylthiazol-4-yl)butanamide) was identified as a HDAC6 selective inhibitor
(IC50 values of 0.199 µM for HDAC6 versus 13.8 µM for HDAC1), and it did not show
significant cytotoxicity against HeLa cells. In vivo biological evaluation of 9a was conducted on
a lipopolysaccharide (LPS)-induced mouse model of sepsis. The compound 9a significantly
improved 40% survival rate (P=0.0483), and suppressed the LPS-induced increase of TNF-α and
IL-6 mRNA expression in the liver of mice. Our study identified novel HDAC6 selective
inhibitor 9a, which may serve as a potential lead for the development of anti-inflammatory or
anti-sepsis agents.
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Introduction
Histone deacetylases (HDACs) are epigenetic enzymes that regulate several proteins such as
transcription factors, chaperones and signaling molecules as well as histone.[1-3] HDACs are
generally associated with condensation of chromatin[4] and transcriptional silencing of genes
implicated in the pathogenesis of many diseases.[5, 6] Therefore, development of HDAC
inhibitors (HDACi) is an attractive strategy for the treatment of cancer,[7] inflammation,[8]
neurodegenerative diseases,[9] and metabolic disorders [10], but main effort has been made to
develop HDACi only as anticancer therapeutics. To date, synthetic three hydroxamate HDAC
inhibitors (SAHA, belinostat, and panobinostat),[11-13] one benzamide HDAC inhibitor
chidamide,[14] and the natural product cyclic depsipeptide romidepsin,[15] have been approved
for the treatment of cancer. Some studies show that HDACi do not simply increase histone
acetylation across the genome thereby generally upregulating gene expression. Many genes are
suppressed by HDACi, because non-histone proteins, especially transcription factors, are also
reversely acetylated and their functions are noticeably affected. For example, the pan-HDAC
inhibitor trichostatin A (TSA) suppresses IL (interleukin)-1β/LPS (lipopolysaccharide)/IFNγ
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(interferon γ)-induced nitric oxide synthase 2 (NOS2) expression in murine macrophage-like
cells.[16] Another pan-HDAC inhibitor SAHA also decreased the LPS-induced tumor necrosis
factor-α (TNF-α) and IFNγ mRNA expression. SAHA is particularly effective for reducing
inflammatory cytokines at nanomolar concentration, relatively lower doses compared to the
doses required for inhibition of tumors. [17]
Among 4 classes (class I through IV) of classified HDACs,[10] class II HDACs are divided
into 2 groups, i.e., class IIa and IIb. Class IIb includes 2 isoforms, HDAC6 and HDAC10.[18]
Unlike HDAC isozymes in other classes, HDAC6 contains 2 homologous catalytic domains and
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the ubiquitin binding zinc finger domain at the C-terminal region.[18, 19] HDAC6 is primarily
localized in the cytoplasm[20] and affect the function of cytoplasmic nonhistone substrates,
including tubulin,[21, 22] cortactin,[23] and Hsp90.[24] Therefore, HDAC6 selective inhibitors
may show limited toxicity by avoiding histone acetylation-induced global gene expression
change.[25] As a deacetylase or ubiquitinase, HDAC6 causes many diseases such as
neurodegenerative diseases[18, 26], cancer[27] and the inflammatory diseases.[28] As shown in
Figure 1, known HDAC6 inhibitors generally contain a zinc-binding group (ZBG), a linker, and
a cap group. Tubacin was first reported as a HDAC6 selective inhibitor in 2003 by Haggerty et
al.[29] ZBG and linker of tubacin are structurally similar to the pan-HDAC inhibitor SAHA.[30]
The bulky cap group is composed of 6 lipophilic rings and the stereochemistry of the dioxane
ring. It enables to affect α-tubulin acetylation and interaction with HDAC6.[31] Tubastatin A
was developed through computational approach using HDAC6 homology model by Kozikowski
and co-workers.[32] It demonstrates about 1,000-fold selectivity for HDAC6 over other HDAC
isoforms.[33] In recent studies, tubastatin A showed anti-inflammatory and anti-rheumatic
activities in mouse models through inhibiting the release of inflammatory cytokines like TNF-α,
IL-6, and chemokines such as NO.[34, 35] These results led to an increased interest in the
therapeutic use of HDAC6 selective inhibitors for autoimmune diseases and inflammation.
Existing HDAC inhibitors primarily exhibit inhibition against pan- or class I HDACs.[7]
ACY-1215 (rocilinostat) is the first HDAC6-selective inhibitor, which entered clinical trial, and
it is under phase I/II clinical evaluation in combination with bortezomib or lenalidomide for
relapsed and refractory multiple myeloma.[36] The development of selective HDAC6 inhibitors
may be useful for better understanding and their therapeutic applications to target diseases other
than cancer, with minimizing adverse effects.[37] Herein we report the development of HDAC6-
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selective inhibitor compound via computational approaches and compound synthesis, and
evaluation of their disease-modifying activity in mouse sepsis models.
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Results and Discussion
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Identification of Novel HDAC Inhibitor by Structure-based Virtual Screening and Design.
The procedure for structure-based virtual screening (VS) in this study is shown in Scheme 1.
Preliminary VS of a commercially available chemical database including drug-like compounds
identified 2 hit compounds (9D and 3C in Scheme 2) with mild HDAC inhibitory activities.[38]
The key interactions of TSA with the active site of HDLP served as the starting point to
construct structure-based pharmacophore constraints (Supplementary Figure S1).[39] First,
UNITY 3D search using pharmacophore query reduced initial set (80,600 entries) to 2503 entries.
In the next step, rigid and flexible docking methods were applied to detect regions favorable for
protein-ligand interactions. The 2503 compounds were docked into the binding pocket using
DOCK 4.0 and FlexX.[40] The final binding modes at the active site of HDLP were ranked for
their expected binding affinity by the scoring function implemented in FlexX. Of the 2503
compounds selected in the first step, only 315 entries were selected by visual inspection of the
final binding modes together with the scoring values of FlexX. Finally, 164 compounds were
selected for biological assay.
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We performed a preliminary screening of the pan-HDAC inhibitory activities of the 164
entries obtained from the virtual screening, using MS-275 as a reference compound. Among the
164 candidates, 2 compounds were found to have HDAC inhibitory activity comparable to MS-
275 (Supplementary Figures, S2 and S3, and Table 1). The structures of two hits, referred to as
3C and 9D are shown in Scheme 2. Hit compounds share 3-(styrylthio) propanamide group that
corresponds to the capping and spacer group of known hydroxamte HDAC inhibitors, such as
SAHA and TSA. In follow-up work to confirm the role of this moiety as a capping and spacer
group, we synthesized the analogue 9D-HA by introducing a hydroxamate functional group as a
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Zn-binding group. The compound 9D-HA exhibited improved HDAC inhibitory potency with an
IC value of 3.7 ꢀM (Supplementary Figure S4), in comparison with 9D and 3C. To optimize
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the HDAC inhibitory activity, we needed a more stable scaffold than thio-linker. Therefore, we
replaced thioether linker, which can be metabolized into sulfoxide in the body,[41] with rigid
thiazole moiety as a bioisostere of sulfide and extended carbon linker unit considering
appropriate spacer length as shown in Scheme 2. This hydroxamate analog 9a was designed to
examine whether thiazole linker deteriorates the HDAC inhibitory activity of 9D-HA containing
hydroxamate group. As expected, the thiazole linker maintained the HDAC inhibitory activity,
showing IC50 value of 1.48 ꢀM and 0.37 ꢀM in enzymatic (Table 1) and cell-based assay,
respectively (Supplementary Figure S5). Our efforts to discover novel HDAC inhibitors, gave a
new hydroxamate HDAC inhibitor 9a, and provided a novel scaffold involving styryl linked
thiazole moiety as a capping and spacer unit of HDAC inhibitors (Scheme 2).
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As summarized in Scheme 3, the synthesis of desired hydroxamate derivative 9a was initiated
from cinnamamide 5. The amide group was converted into thioamide 6 using Lawesson’s
reagent.[42, 43] The thioazole derivative 7 was formed by cyclization of 6 with bromoketone
4.[44] To prepare bromoketone 4, glutaric anhydride 1 was hydrolyzed with NaOMe, followed
by chlorination to give acid chloride 3, which converted into bromokeone 4 via Nierenstein
reaction with hydrobromic acid.[45-47] The hydrolysis of ester 7 with 1N-NaOH gave
carboxylic acid 8. Finally, carboxylic acid was reacted with hydroxyl amine to generate
hydroxamate derivative 9a.
We first examined the cytotoxicity of 9a in HeLa cells by MTS assay and the IC50 value was
52 ꢀM, implying that the drug may not be highly cytotoxic against cancer cells. However,
significant anti-inflammatory activity of 9a was observed in LPS-stimulated RAW 264.7 cells.
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Treatment of 9a (0.5-2.5 ꢀM) showed about 50% inhibition of LPS-induced increase of TNF-α,
IL-6, and NO production, without affecting cell viability.[48] Since severe sepsis is also related
with overproduction of TNF-α and NO, we tested the effect of 9a in mice with LPS-induced
endotoxemia. As shown in Figure 2, in the LPS-treated control group, the survival rate on the
first day of observation was 70% and reached a stable 20% survival on the fifth day after LPS
injection. Log-rank analysis of 10-days survival demonstrated that 9a (20 mg/kg i.p. 2 h before
and immediately after LPS injection) significantly improved survival rate of sepsis-induced mice,
as compared to the control group (P = 0.0483). The reduction of LPS-induced mortality by 9a
may be associated with the suppression of LPS-induced pro-inflammatory cytokine release. Our
result is comparable to the recent study reported by Alam et al., demonstrating that Tubastatin A
improves the survival of cecal ligation and puncture (CLP)-induced sepsis mice models and
attenuates the CLP-induced increase of local and systemic pro-inflammatory cytokine level
(TNF-α and IL-6). [49]
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Next, we examined whether 9a inhibits the production of TNF-α and IL-6 at the transcription
level. Production of TNF-α and IL-6 mRNA induced by LPS injection in mouse liver, was
measured using real time RT-PCR. Six-hour after LPS injection, mRNA expression of TNF-α
and IL-6 were significantly elevated by 2.3- and 1.8-fold compared with those of the control
group, respectively. Treatment of 9a (10mg/kg) attenuated the LPS-induced over-expression of
TNF-α and IL-6 mRNA (Figure 3). Taken together, we conclude that 9a may inhibit the HDAC6
selectively, repress the LPS-induced over-expression of pro-inflammatory cytokines, and
eventually prolong the survival of sepsis-induced mice.
HDAC enzymatic assays and western blot analysis were performed to confirm that in vivo anti-
sepsis activity of 9a is correlated with selective inhibition of HDAC6. HDAC inhibitory activity
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of 9a was examined with HDAC6 and HDAC1, in comparison with tubastatin A. Compound 9a
showed inhibitory potency against HDAC6 with IC50 values of 199.3 nM and about 70-fold
selectivity over HDAC1 (Figure 4). One of specific substrates for HDAC6 is known as α-tubulin,
whereas HDAC1 is mostly involved in histone deacetylation. To corroborate selective inhibition
of HDAC6 by 9a at a cellular level, hyperacetylation of histone H3 and α-tubulin were examined
by western blot analysis. As shown in Figure 5, the treatment of 2 µM tubastatin A and 10 µM
9a did not lead to hyperacetylation of histone-H3 significantly, whereas 2 µM TSA, a pan-
HDAC inhibitor, produced hyper-acetylated histone H3. Tubastatin A and 9a produced dose-
dependent increase of hyperacetylated tubulin. The results supported that 9a selectively inhibits
cellular HDAC6 over HDAC1.
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Docking study was performed to examine the binding mode of HDAC6 selective inhibitor 9a.
Homology model of HDAC6 was established using HDAC4 (PDB id: 4CBY) as a template. The
active site of HDAC6 consists of small zinc binding pocket, hydrophobic linker site (Phe620 and
Phe680),[50] and large cap group binding site that is able to accommodate structurally diverse
moieties.[51] This cap group binding site, which consists of residues such as His499, His500,
and Phe566, is the entrance of HDAC6 with wider rim than HDAC1 (Supplementary Figure
S6).[52] Our HDAC6 homology model well defines the binding grooves on the protein surface
to accommodate hydrophobic and bulky cap groups of selective inhibitors. As shown in Figure
6a, the hydroxamate group of 9a forms hydrogen bonds with His610, His611, and Tyr782 and
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coordinates with catalytic zinc ion in the active site (Zn -O of OH: 1.47Å and Zn -O of
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carbonyl: 1.96Å). The thiazole group of linker is positioned in the tubular access channel and
sandwiched between Phe620 and Phe680. In particular, the distance between thiazole ring and
Phe620 is 3.8Å, which is able to form close van der Waals interaction. The cinnamyl cap group
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interacts with an open cavity at the entrance of the binding pocket surrounded by several
aromatic and hydrophobic residues. The hydroxamate group of reference compound tubastatin A
also forms hydrogen bonds with His611 and Tyr782, and the cap group and phenyl linker occupy
a region similar to the cinnamyl cap group of 9a (Figure 6b). Docking results well demonstrates
that 9a can bind to the active site of HDAC6 with intermolecular interactions similar to those
formed by highly selective HDAC6 inhibitor tubastatin A.
Conclusions
In the present study, we successfully demonstrated the use of computational drug design
approaches combined with synthesis and in vitro/in vivo assays to identify selective HDAC6
inhibitor 9a with anti-sepsis activity. The structure of 9a was designed based on mild HDAC
inhibitor hit compounds identified from virtual screening of chemical database. A selective
HDAC6 inhibitory activity of 9a was confirmed by enzymatic HDAC assays and western blot
analysis showing dose-dependent increase of tubulin acetylation without noticeable increase in
acetylated histone H3. In addition, recombinant human HDAC isoform profiling was carried out
by the Reaction Biology Corp. (www.reactionbiology.com), and the results confirmed the
HDAC6-selectivity of 9a (Supplementary Figure S7). 9a attenuates the expression of LPS-
induced pro-inflammatory cytokines such as TNF-α and IL-6, and thereby improves the survival
of sepsis mice.
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Compound 9a has not only smaller capping group but also more flexible linker unit than
tubastatin A, which may give lower selectivity of 9a toward HDAC6 compared to tubastatin A.
Considering that Wagner et al’s study suggesting that a bulky capping group is not required for
selective inhibition of HDAC6, [55] modification of thiazole-linked alkyl group in 9a may
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improve the selectivity. Conclusively, 9a can be used as a lead compound for the design of
selective and potent HDAC6 inhibitor. Further structural optimization of 9a to improve the
HDAC6 selectivity and in vivo activity, in parallel with biological mechanistic study, is in
progress to develop antiseptic drug candidates.
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Materials and Methods
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Structure-based Virtual Screening
The virtual screening strategy was shown in Scheme 1. To conduct the virtual screening, the
LeadQuest database (80,600 entries) obtained from Tripos, was prepared using SYBYL 8.1.[38]
The crystal structure of HDLP bound to TSA was retrieved from the Protein Data Bank (PDB
entry 1C3R).[39] 3D database screening using UNITY module implemented in SYBYL
packages performed a pre-filtering of database compounds discarding all molecules that do not
match pharmacophore queries based on X-ray crystal structure of HDLP. As shown in
Supplementary Figure S1, the following queries were set up: (1) OH of Tyr297 as a receptor
donor and corresponding ligand acceptor site (2) the phenyl ring of TSA as hydrophobic feature
(3) R1, R2, and R3 can be substitute with any other group or atoms. (4) distance constraints, R2-
C1: 2.45 ± 0.10 Å, and hydrophobic feature-R1 and C1: 3.97 ± 0.10 Å and 9.05 ± 0.10 Å,
respectively. DOCK 4.0[53] and FlexX[54] implemented in SYBYL 6.81 were used as the
docking tool targeting at TSA binding site of HDLP. The candidate compounds with best docked
poses and scores were selected by visual inspection for experimental test.
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Procedures for Synthesis of compounds 2, 3, 4, 6, 7, 8 and 9a
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Proton and carbon nuclear magnetic resonance ( H and C NMR) spectra were recorded with
a Varian Unity Inova 300 and 500 MHz spectrometer for CDCl and DMSO-d solutions and
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chemical shifts (δ) are reported as part per million (ppm). Coupling constant (J) is reported in
hertz (Hz). Thin layer chromatography (TLC) was carried out using Kiesegel 60F254 (Merk Co.)
silica gel plates. Flash column chromatography was performed with E. Merck Kiesegel 60 (230-
400 mesh ASTM). All reactions were carried out under nitrogen or argon atmosphere.
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Glutarate (2)
To a solution of glutaric anhydride (5.0 g, 44 mmol) in methanol (15 mL), sodium methoxide
(50 mg, 1 mmol) was added, and the mixture was stirred for 6 h at room temperature. After
complete evaporation of the solvent under reduced pressure, the residue was used in the next step
directly without any further purification.
Methyl 5-chloro-5-oxopentanoate (3)
To a solution of crude monomethylglutarate in absolute benzene (20 mL) dimethylformamide
(2 drops) was added and thionyl chloride (6.9 mL, 94.3 mmol) was dropwise at room
temperature for 30 min. The reaction mixture was refluxed for 3 h. Solvent was removed in
vacuo, and the residue was used in the next step directly without any further purification.
Methyl 6-bromo-5-oxohexanoate (4)
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Methyl 5-chloro-5-oxopentanoate (4.0 g, 24.3 mmol) was added to a solution of diazomethane
(121.6 mmol) in diethylether (400 mL) at 0 °C under N2. After 2 h, N2 was bubbled through the
solution to remove excess CH2N2. The residue was treated with glacial acetic acid (10 mL) and
hydrobromic acid (33% in acetic acid, 5 mL) was dropwise. The mixture was allowed to warm to
room temperature for 24 h. The reaction mixture was washed with saturated sodium bicarbonate
solution and water. The organic layer was dried over MgSO4, filtered and evaporated. The
residue was purified by column chromatography (Hexane: EtOAc = 4:1) to give the 4. (2.4 g,
25% for 4 steps); 1H NMR (300 MHz, CDCl3): δ 3.88 (s, 2H), 3.68 (s, 3H), 2.75 (t, J = 4.0 Hz,
2H), 2.37 (t, J= 4.0 Hz, 2H), 1.95 (q, J = 4.0 Hz, 2H)
(E)-3-Phenylprop-2-enethioamide (6)
Phosphorus pentasulfide (1.11 g, 5 mmol) was added portionwise to a stirred solution of
cinnamamide (1.47 g, 10 mmol) in anhydrous pyridine (4.4 mL). The resulting yellow/orange
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solution was heated at reflux for 2 h, and then allowed to cool to room temperature. The mixture
was poured into water (10 mL) and then extracted with diethyl ether (3×10 mL). The combined
extracts were washed with 1N-HCl, then water, and dried over MgSO . After filtration, the
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solvent was removed by evaporation in vacuo to give a brown solid. Crystallisation from
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benzene gave yellow needles (1.07 g, 66%); H NMR (300 MHz, CDCl ): δ 7.78 (d, J = 15.0 Hz,
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1H), 7.58-7.55 (m, 2H), 7.45-7.37 (m, 5H), 6.88 (d, J = 15.0 Hz, 1H).
(E)-Methyl 4-(2-styrylthiazol-4-yl)butanoate (7)
The mixture of (E)-3-phenylprop-2-enethioamide (79 mg, 0.48 mmol) and methyl 6-bromo-5-
oxohexanoate (72 mg, 0.32 mmol) in MeOH (2 mL) was stirred overnight at room temperature.
The reaction mixture was concentrated by evaporation and the residue was purified by column
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chromatography (Hexane : EtOAc = 4:1) to give 7 (72 mg, 78%); H NMR (300 MHz, CDCl3):
δ 7.53 (s, 1H), 7.51 (s, 1H), 7.40-7.23 (m, 5H), 6.84 (s, 1H), 3.68 (s, 3H), 2.83 (t, J = 7.5 Hz,
2H), 2.40 (t, J = 7.5 Hz, 2H), 2.13-2.03 (m, 2H).
(E)-4-(2-Styrylthiazol-4-yl)butanoic acid (8)
To a solution of (E)-methyl 4-(2-styrylthiazol-4-yl)butanoate (1.0 g, 3.48mmol) in THF (6 mL)
and MeOH (6 mL) 1N-NaOH (6 mL) was added. The reaction mixture was stirred at room
temperature for 2 h, then the solvent was evaporated. The aqueous layer was acidified with 10%
aqueous citric acid solution (pH 3.0) and extracted with ethyl acetate. The organic layer was
dried (MgSO ), filtered and solvent was evaporated. The residue was used in the next step
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directly without any further purification.
(E)-N-Hydroxy-4-(2-styrylthiazol-4-yl)butanamide (9a)
To a stirred solution of crude (E)-4-(2-styrylthiazol-4-yl)butanoic acid in anhydrous DMF (22
mL) 1-hydroxybenzotriazole hydrate (0.66 g, 4.9 mmol) was added at room temperature
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followed by 1-(3-dimethylamino propyl)-3-ethyl-carbodiimide hydrochloride (EDC, 0.85 g, 5.7
mmol). After stirring for 1 h, hydroxylamine hydrochloride (0.29 g, 4.15 mmol) and Et N (574
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ꢀL, 4.15 mmol) were added, and stirring was continued at room temperature overnight. The
solvent was removed in vacuo, and the residue was diluted with ethyl acetate and then was
washed with saturated aqueous NaHCO3. The organic layer was dried (MgSO4), filtered and
concentrated in vacuo. The residue was purified by flash chromatography (10% methanol in
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chloroform) to give 9a (0.4 g, 40% for 2 steps). H NMR (500 MHz, DMSO-d
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): δ10.36 (s, 1H),
8.67 (s, 1H), 7.69 (s, 1H), 7.67 (s, 1H), 7.41-7.32 (m, 5H), 7.26 (s, 1H), 2.70 (t, J = 7.5 Hz, 2H),
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2.02 (t, J = 7.5 Hz, 2H), 1.89 (t, J = 7.5 Hz, 2H); C NMR (125 MHz, DMSO-d ): δ169.57,
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166.07, 136.32, 133.92, 129.55, 129.50, 127.88, 122.18, 116.54, 114.62, 32.49, 31.08, 25.52;
ESI-HRMS: calc. for C H N O S; [M+H]+ = m/z 288.0932 , found: [M+H] = m/z 288.0930
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HDAC Enzymatic Assay
Pan-HDAC fluorescent activity was measured in vitro Fluor de Lys substrate (Biomol,
Plymouth Meeting, PA), which contains an acetylated lysine side chain, according to
manufacturer's instructions and Glomax multi plus (Promega), microplatespectrofluorometer
was used with excitation at 355 nm and emission at 460 nm. The total pan-HDAC assay volume
was 25 ꢀl and all the assay components were diluted in HDAC assay buffer (50 mM Tris/Cl, pH
8.0, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl ). The reaction was carried out in half-volume
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white 96-well plates (Costar 3693). In brief, the pan-HDAC assay mixture contained HDAC
substrate (116 ꢀM, 12.5 ꢀl), HeLa nuclear extract (0.5ꢀl of extract diluted to 7.5ꢀl final volume),
and drug (diluted to 5ꢀl final volume). Positive controls contained all the above components
except the inhibitor. The negative controls contained neither enzyme nor inhibitor. In each case
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these were replaced with an equivalent volume of buffer. The assay components were incubated
at room temperature for 20 min. The reaction was quenched with 25 ꢀl HDAC-FDL Developer
(KI-105; Biomol) diluted to 20-fold in cold assay buffer containing 2 ꢀM TSA. The plates were
incubated for 20 min at room temperature (22–22 °C) to allow the fluorescence signal to
develop.
The HDAC subtype selectivity was tested to compare the inhibitory effect on the HDAC6
versus HDAC1, by using HDAC6 and 1 Fluorimetric Drug Discovery kit (Enzo life science,
BML-AK511-0001, BML-AK516-0001). HDAC6 selective inhibitor tubastatin A was used as a
reference. Assays were carried out two steps in accordance with protocol of the kit. Total HDAC
assay volume is 50 µl per well. Developer is diluted in HDAC assay buffer (50 mMTris / Cl, pH
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8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl ). The rest of assay components are prepared with
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HDAC assay bufferⅡ (50 mM Tris/Cl, pH8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2,
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2mg/ml BSA). All reactions were conducted in half-volume white 96-well plates (Costar 3693).
The first step can be done by reacting HDAC subtype enzyme with HDAC assay mixture.
HDAC reaction mixture includes substrate solution (1x, 12.5 µl per well) which comprises an
acetylated lysine side chain, diluted HDAC1 or 6 (7.5 µl contains 0.3 ug HDAC1 or 6 enzyme
per well) (except for no enzyme control). Compounds were diluted with DMSO (1 µl final
volume per well) and delivered to HDAC mixture with incubation of 15 min at room
temperature. After incubation, in the second step, we treated developer (1x, 25 ꢀl/well) which
produces a fluorophore by reacting with deacetylated substrate and incubated plate at 37 ˚C for
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45 min. The fluorescence are determined using Glomax multi plus (Promega) at a wavelength
with excitation in the range 350-380 nm and emission in the range 440-460 nm. The relative
enzymatic activity (in percent) was shown as the mean value of triplicate experiments. Dose-
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response curves and values were generated from resulting plots using GraphPad Prism 6
program.
Western blot analysis
HeLa cells were treated with compound at a dose range of 100 nM to 100 ꢀM for 24 hours.
Sample proteins were processed using Nuclear extract kit (Active motif) and quantified using
Bradford protein assay (Bio-Rad). Protein samples were electrophoresedon 15% SDS–
polyacrylamide gels and transferred to a membrane. Membranes were blocked with 8% skim
milk in TBS–Tween (10mM Tris–HCl (pH 7.5), 150 mMNaCl and 0.1% Tween 20) and
probed with anti-acetylated tubulin (6-11B-1; Sigma) or anti-α-tubulin (B-5-1-2; Sigma) at a
dilution of 1:2000 and anti-acetylated histone H3 (06-599; Millipore). Both tubulin and acetyl-
tubulin have a molecular mass of about 50 kDa. To re-probe the same membrane with acetyl-
tubulin, membrane was incubated for 20 min at room temperature in stripping buffer (ATTO).
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Results of blots were imaged using ChemiDoc (Davinch-chemi ).
Animals and Experimental Models of Sepsis
Male ICR mice (27-29 g) were supplied by Daehan-Biolink Co. (Eum-Sung, Korea). The
animals were housed in cages located in temperature-controlled rooms with a 12 h light-dark
cycle and recieved water and food ad libitum. All animal procedures were approved by the
Sungkyunkwan University Animal Care Committee and were performed in accordance with the
guidelines of the National Institutes of Health (NIH publication No. 86-23, revised 1985).
Endotoxemia was induced by intraperitoneal injection of lipopolysaccharide 10 mg/kg (LPS;
Escherichia coli serotype O111:B4; Sigma-Aldrich, St. Louis, MO, USA). The mice
intraperitoneally received vehicle (5% DMSO-contained saline), 9a, 5, 10, or 20 mg/kg 2 h
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before and immediately after LPS injection. The animals were randomly assigned to the
following 5 groups: (a) vehicle-treated control (control), (b) vehicle-treated LPS (LPS), (c-e) 9a,
5, 10, or 20 mg/kg-treated LPS (LPS + 9a, 5, 10, or 20 mg/kg). The animals were monitored for
survival for up to 10 days. In parallel experiments, mice were euthanized to collect blood
samples from the abdominal inferior vena cava at 6 h of LPS injection.
Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from liver tissue using RNAiso (Takara Bio Inc., Shiga, Japan)
according to the manufacturer's protocol. Reverse transcription of total RNA was performed for
synthesis of cDNA using EcoDry Synthesis Premix (Takara Bio Inc.). RT-PCR was performed
using the LightCyclerNano instrument (Roche Applied Science, Mannheim, Germany). The
gene-specific primers used are listed in Table 2. The PCR amplification cycling conditions were
as follows: holding 94 °C for 300 s; 94 °C for 30 s, 65 °C for 30 s, 72 °C for 30 s, total of 28
cycles for TNF-α; 94 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s, total of 25 cycles for IL-6 and β-
actin.
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Molecular Modeling
Homology modeling of HDAC6
Human HDAC6 sequence was obtained from UniProt database (www.uniprot.org). For
HDAC6, modeling focused on the catalytic domain II subunit (Gly481-Gly801), a functional
domain of HDAC6. Homology models were generated for HDAC6 by Sybyl-X 2.1.1 using
template of HDAC4 (PDB id: 2VQQ, 2VQV, 2VQW, 4CBT, and 4CBY). To arrange zinc at
catalytic site, zinc ion and chelating residues were extracted from the template HDAC4, and
merged into the constructed models. An energy minimization was performed by fixing the
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backbone and zinc ion. The homology models were validated by examining correlation between
biological activity for known HDAC6 inhibitors and Surflex-dock score. Among them, the
model using 4CBY (sequence identify 49.57%) as a template, provided the most reliable re-
docking results, and was used for docking analysis for 9a
Docking modeling
Docking study were carried out on HDAC6 model by Surflex-dock in Sybyl-X 2.1.1, operating
under Linux OS (CentOS release 5.9). The program generated the protomol in the active site of
HDAC6 including zinc ion and key residues within a 3 Å radius, such as His610, His611,
Tyr782, Phe620, and Phe680, with a threshold of 0.50 and bloat set to 0. Each atom on the
protein and ligand is labeled as nonpolar (e.g., the H of a C-H) or polar (e.g., the H of an N-H or
the O of a C=O), and generated protomol mimics the intermolecular interactions between drug
and HDAC6. The structures of known inhibitors and 9a were energy-minimized using the
conjugated gradient method with Gasteiger-Hückel charge until a convergence value of 0.001
kcal/(Å•mol), using the Tripos force field. The main setting was 50 solutions per each compound
and other parameters accepted the Surflex-Dock Geom settings. Surflex-Dock’s scoring function
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d d
was trained to estimate the dissociation constant (K ) expressed in –log(K ) unit. Then, the best
poses from each run were combined and re-ranked using a consensus scoring (CScore). We
selected good binding pose, with Cscore over 4 and Surflex-Dock total score higher than 5.0.
Docking of 9a and tubastatin A against HDAC1 (PDB id: 4BKX) were performed following the
same method described above.
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ASSOCIATED CONTENT
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Supplementary Materials. The information about virtual screening hit compound 9D and 3C,
and synthetic derivative 9D-HA, involving pharmacophore query used for virtual screening,
HDAC inhibition dose assay, and kinetics assay. The homology model of HDAC6. The
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recombinant human HDAC isoform profiling data for 9a. The H and C NMR spectra and HR
mass spectrum for 9a.
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AUTHOR INFORMATION
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Corresponding Author
*Phone: +82-31-290-7719. E-mail: hyunju85@skku.edu
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ACKNOWLEDGMENT
This research was supported by Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education (NRF-
2012R1A5A2A28671860).
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ABBREVIATIONS
HDAC, histone deacetylase; LPS, lipopolysaccharide; HDACi, , histone deacetylase inhibitors;
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IL, interleukin; IFNγ, interferon γ;TNF-α, tumor necrosis factor-α; ZBD, zinc-binding group.
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Figure captions:
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Figure 1. Chemical structures of HDAC6-selective inhibitors.
Figure 2. Effects of 9a on LPS-induced lethality.
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Figure 3. Effects of 9a (10mg/kg) on the LPS-induced mRNA expression of TNF-α and IL-6.
Figure 4. Selective inhibition of HDAC6 over HDAC1 by 9a.
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Figure 5. Western blot analysis of HeLa cell extracts after treated with TSA (Trichostatin A),
tubastatin A, and 9a for 24 hrs. Tubastatin A and 9a selectively increase the acetylated tublin.
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Figure 6. Docking poses (left) and top views (right) of (A) 9a (carbon atoms in cyan) and (B)
tubastatin A (carbon atoms in yellow). The hydrophobic channels for linker unit of 9a and
tubastatin A are shown as mesh contour surface. Zinc ion is represented by magenta ball.
Hydrogen bonds between the ligand and side chains are shown in dashed yellow lines.
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Scheme 1. Flow chart for identifying HDAC6 selective inhibitor 9a.
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Scheme 2. Structure-based design of styrylthiazolylhydroxamate 9a
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Scheme 3. Synthesis of 9a. Reagents and conditions: (a) NaOMe, MeOH, room temperature (rt),
o
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6h; (b) SOCl , benzene, reflux, 2h; (c) i) CH N , Et O, 0 C, 2h, ii) HBr, AcOH, rt, 12h; (d) P S ,
2
2
2
2
2 5
pyridine, 70 °C, 2h; (e) 4, MeOH, rt, 24h; (f) 1N-NaOH, THF, MeOH, rt, 2h; (g) NH OH·HCl,
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EDCl, HOBt, Et N, DMF, rt, 24h.
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Table 1. Pan-HDAC inhibition activities of the compounds in Scheme 1.
compound
IC (µM)
50
MS-275
8.02
19.18
29.80
3.70
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3
D
C
9
D-HA
9
a
1.48
2
3
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