Inhibitors selective for HDAC6 in enzymes and cells

Article abstract of DOI:10.1016/j.bmcl.2010.09.100

Histone deacetylase inhibitors with anticancer or anti-inflammatory activity bind to Class I or Class I and II HDAC enzymes. Here we compare selectivity of inhibitors of a Class II HDAC enzyme (HDAC6) and find one that retains high selectivity in macrophages.

Full text of DOI:10.1016/j.bmcl.2010.09.100

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7067–7070
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitors selective for HDAC6 in enzymes and cells
Praveer K. Gupta, Robert C. Reid, Ligong Liu, Andrew J. Lucke, Steve A. Broomfield, Melanie R. Andrews,
⇑
Matthew J. Sweet, David P. Fairlie
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone deacetylase inhibitors with anticancer or anti-inflammatory activity bind to Class I or Class I and
II HDAC enzymes. Here we compare selectivity of inhibitors of a Class II HDAC enzyme (HDAC6) and find
one that retains high selectivity in macrophages.
Received 10 August 2010
Revised 15 September 2010
Accepted 16 September 2010
Available online 12 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
HDAC6
Enzyme inhibitor
Anti-inflammatory
Inflammation
Acetylation of lysine residues in proteins is a reversible post-
translational modification that impacts on many cellular processes,
including signaling, RNA splicing, gene expression, protein stability
and transport.1 A recent proteomic analysis of three human cell
lines identified 1750 nuclear and non-nuclear proteins modified
by lysine acetylation at 3600 individual sites.2 Lysine acetylation
has been most extensively studied for histone proteins, where it
regulates chromatin architecture and gene transcription.1,3,4 An
Phenotypic analysis of HDAC6À/À mice suggests roles for this
protein in bone homeostasis, immune functions and protection
against neurodegenerative diseases.15,16 At the cellular level,
HDAC6 regulates autophagy,16 motility17 and adhesion.18 Many
synthetic HDAC inhibitors are known, but few are selective for
HDAC6. TSA (1) and SAHA (2) are pan-inhibitors of HDACs,
acetyl group is transferred to the e-amino group of lysine residues
in histones by enzymes called histone acetyl transferases (HATs)
and removed by histone deacetylases (HDACs).
Inhibitors of HDAC enzymes have diverse effects on cell func-
tion, causing differentiation, growth inhibition, apoptosis and
immunomodulation. Such pleiotropic effects may reflect distinct
functions of specific HDAC enzymes, which fall into four classes
based on sequence similarity and cofactor requirement. In humans,
there are 18 HDAC genes, and 11 of these encode the classical
(Classes I, II, IV) Zn2+-dependent HDAC enzymes.5 Class I enzymes
(HDACs 1, 2, 3, 8) reside mainly in the nucleus,6,7 Class IIa enzymes
(HDACs 4, 5, 7, 9) translocate between the nucleus and cytoplasm
in response to cellular signals,4,8 while Class IIb enzymes (HDACs 6,
10) have two independent deacetylase domains and HDAC6 at
least may be exclusively cytoplasmic.9,10 Class III HDACs called Sir-
tuins (SIRT1–7) require NAD+ for activity.11–13 All other HDACs,
including the only known human enzyme of Class IV, HDAC11,14
are Zn2+-dependent enzymes.5
⇑
Corresponding author. Tel.: +61 733461989; fax: +61 733461990.
E-mail address: d.fairlie@imb.uq.edu.au (D.P. Fairlie).
Figure 1. Relevant reported human HDAC inhibitors.22–25
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.100

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P. K. Gupta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7067–7070
Scheme 1. Synthesis of carboxylic acid, ester and hydroxamate inhibitors.
MS275 (3) is selective for HDAC1, while hydroxamate 4 and thiol 5
are reportedly HDAC6 inhibitors (Fig. 1). Tubacin is known as a
selective HDAC6 inhibitor but has poor drug like properties (mol
wt 721), is not readily available and is difficult to synthesize.19
Some HDAC inhibitors are anti-inflammatory,20 5 showing anti-
Table 1
Inhibitor potency for analogues of 5 at 1
l
M against rh-HDAC6
inflammatory properties in mouse macrophages21 but only at
lM
concentrations due to poor cell permeability and instability of
the thiol group. Here we compare 5 and analogues for inhibition
of recombinant human HDAC6 versus rh-HDAC1 enzymes and in
human cells.
Inhibitors
R1
R2
n
% Inhibitiona
5
6
7
8
9
10
11
12
13
14
15
16
Boc
Boc
Boc
Cbz
Boc
Cbz
Cbz
Boc
Boc
Boc
Boc
Boc
SH
OH
OH
5
5
4
4
4
5
5
5
5
6
6
5
98
80
50
20
40
65
40
60
45
60
3
The synthesis of inhibitors 8–13 and 16 is shown in Scheme 1.
Diethyl acetamidomalonate 19 was alkylated with methyl 5-
bromopentanoate (or 6-bromohexanoate) giving 20 then hydroly-
sis with HCl and decarboxylation gave the racemic amino acid 21.
After esterification and Cbz protection, enzymatic resolution of 22
with papain26 gave the (S)-amino acid 23. Cyclopentylamine was
coupled giving 10 then the Cbz group was cleaved by hydrogenol-
ysis and replaced with Boc giving 9 and 12. The side chain methyl
ester was either hydrolyzed to give the acids 8, 11 and 13 or con-
verted to the hydroxamate 16 with hydroxylamine. Inhibitors with
thiol (5, 14) or alcohol (6, 7, 15) zinc binding groups were prepared
by similar methods (Supplementary data) and the enantiopurity of
CO2H
CO2CH3
CO2CH3
CO2H
CO2CH3
CO2H
SH
OH
CONHOH
99
a
Assayed in triplicate in 25 mM Tris–HCl, pH 8, 37 °C.
Table 2
Comparative inhibition of rh-HDAC1 versus rh-HDAC6 enzymes
a
Inhibitor
IC50 SEM (nM)
rh-HDAC1
SIb
HDAC1/HDAC6
C log P
rh-HDAC6
1
2
3
4
5
2.06
0.98
À0.11
2.41
3.53
2.12
1.23
2.56
2.86
10
76
44
340
1731 18
>10,000
1420 10
48
21
4
3
4
56
85
>10,000
40
54
240 10
26
107
943
1
5
0.2
0.9
0.004
8.5
32
>40
55
0.4
2
3
2
6
16
17
18
2
1
6
2
2
0.02
Figure 2. Lineweaver–Burk plot for HDAC processing of substrates. For HDAC6,
a
Compounds assayed in triplicate in 25 mM Tris–HCl (pH 8, 37 °C).
SI = selectivity index.
Boc-Lys(Ac)-AMC (N, KM 20
Lys(TFAc)-AMC and Ac-RGK(Ac)-AMC which all gave KM P 150
Cbz-Lys(TFAc)-AMC (d, KM 15 M) was more effective than Boc-Lys(Ac)-AMC (KM
ꢀ100 M). Protein concentration was constant for all substrates (HDAC6, 0.35 ng/
L; HDAC1, 0.21 U/ L). Error bar = mean SEM (n = 3).
lM) was a better substrate than Z-Lys(TFAc)-AMC, Boc-
b
lM. For HDAC1,
l
the enzymatically resolved amino acids was confirmed by NMR
analysis of diastereomeric derivatives (Supplementary data).
l
l
l

P. K. Gupta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7067–7070
7069
A variety of different assays have been reported in the literature
to determine HDAC activity27 thus making a comparison of inhib-
itors difficult. No standard substrates have been adopted, different
concentrations of enzyme/substrate are often used and KM is usu-
ally not reported. Commercial assay kits often do not disclose the
substrate or reagents. We evaluated several HDAC1 and HDAC6
substrates (Supplementary data) before standardizing assays
(Fig. 2), to allow reliable potency/selectivity comparisons between
inhibitors to be made (Table 2).
Thiol 5 was reported as a selective HDAC6 inhibitor (IC50
29 nM),25 and we found comparable potency (IC50 54 nM) and
32-fold selectivity over rh-HDAC1 in our in vitro assay. HDAC6
inhibition translates to hyperacetylation of
was induced in mouse macrophages by 5 at >1
tions.21 To improve potency at the cellular level, we made deriva-
a-tubulin in cells, and
lM concentra-
tives of 5 in which the N-terminal capping group (R1), the Zn2+
-
binding group (R2) and/or linker length (n) were varied (6–16,
Scheme 1, Tables 1 and 2).
The optimal linker length (n) was consistently found to be
five methylenes regardless of the Zn2+-binding group (Table 1).
We had considered it possible that the six methylenes present
in the alcohol 15 may better mimic the linker length of the thiol
5 than the shorter chain alcohol 6 by compensating for the sig-
nificantly longer carbon–sulfur bond length but this was not so
(Table 1). Similarly we thought that the carboxylic acid/esters
8 and 9 with four methylenes plus the carboxyl carbon may con-
stitute a sufficiently long linker, however, this series also bene-
fited from the longer five methylene linker. With the optimized
linker of five methylenes, the thiol zinc binding group 5 con-
ferred greater potency than alcohol 6, the methyl esters (10
and 12) followed by the carboxylic acids (11 and 13) were less
active. Replacing Boc with Cbz at the N-terminus (R1) had little
effect on potency.
Figure 3. Concentration–response curves for inhibitors 1 (.), 2 (N), 4 ( ), 5 (j), 16
*
(d), 17 (ꢀ) against rh-HDAC6 and rh-HDAC1. Error bar = mean SEM for three
independent experiments.
Figure 4. (A) Comparison of compounds 2, 5 and 16 in PMA-differentiated THP-1 cells after 4 h treatment. Cell lysates were immunoblotted using antibodies against
acetylated alpha-tubulin, acetylated histone H3 and STAT-1 (n = 2 experiments). (B) Semi-quantitative analysis (densitometry) of data from A.

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P. K. Gupta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7067–7070
Finally the hydroxamic acid 16 was found to be the most potent
References and notes
and selective inhibitor of this series. We were initially concerned
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structurally related compound 17, a potent HDAC inhibitor with
anticancer activity28 does not discriminate between HDAC6 and
HDAC1 (IC50 107 nM, HDAC6; 48 nM, HDAC1).
Furthermore, hydroxamate 18 showed more potent (45Â) inhi-
bition of rh-HDAC1 than rh-HDAC6 (Table 2). Suzuki et al. previ-
ously noted that loss of selectivity for HDAC6 resulted when the
aliphatic cyclopentane ring of 5 was replaced by an aromatic
ring.25 Therefore, the combined effects of both Boc and cyclopentyl
groups for HDAC6 selectivity together with the potency gains
afforded by the hydroxamate group have synergised giving 16 with
optimal HDAC6 potency and selectivity; IC50 26 nM (rh-HDAC6)
versus 1420 nM (rh-HDAC1) corresponding to 55-fold selectivity
for rh-HDAC6 over rh-HDAC1 (Table 2, Fig. 3).
Since HDAC6 and HDAC1 deacetylate alpha-tubulin and histone
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tein substrates can be used to assess HDAC6 and HDAC1 inhibition
in cells. Using these readouts, we have previously found some inhib-
itor selectivity for 5 at HDAC6 over HDAC1 in primary mouse
macrophages.21 Here we compare compounds 4, 5 and 16 in PMA-
differentiated THP-1 human macrophages. Replacement of the thiol
in 5 with hydroxamate 16 greatly enhanced inhibition of HDAC6 in
cells (>10-fold, Fig. 4), and 16 selectively inhibited HDAC6 since
ꢀ10-fold lower concentrations were required for tubulin hyperacet-
ylation than for histone H3 hyperacetylation. Compound 5 did not
display any selectivity for HDAC6 over HDAC1 in human PMA-differ-
entiated THP-1 cells (Fig. 4), in contrast to our finding in mouse mac-
rophages.21 Compound4 displayedsimilar potencytocompound16,
but had only modest selectivity in cells (Supplementary data). Thus,
16 may be a valuable in vivo probe for HDAC6 function.
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Acknowledgments
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We thank the National Health Medical Research Council for
grant 569735 and the Australian Research Council for a Federation
Fellowship to D.F.
Supplementary data
Supplementary data associated (experimental methods, synthe-
sis/modeling, enzyme/cell assays) with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.100.
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Compound 4 was reported as HDAC6-selective with pM,23 then nM,30 inhibitory
potency. We re-examined an analogue (compound 1 in Ref. 30 Supplementary data
34) in our assay and find IC50 150 nM (HDAC6) versus 2980 nM (HDAC1) or 20-fold
selectivity (Supplementary data). Using the substrate Ac-RHKK(Ac)-ACC as used in
Ref. 30, we find their compound 1 to have IC50 41 nM (HDAC6) versus 3000 nM
(HDAC1) or 75-fold selective, whereas 16 gave IC50 27 nM (HDAC6) versus 3567 nM
(HDAC1) or 132-fold selectivity (Supplementary data). Thus, we are confident that 16
is more HDAC6-selective.