Design and Synthesis of Dihydroxamic Acids as HDAC6/8/10 Inhibitors

Article abstract of DOI:10.1002/cmdc.202000149

We report the synthesis and evaluation of a class of selective multitarget agents for the inhibition of HDAC6, HDAC8, and HDAC10. The concept for this study grew out of a structural analysis of the two selective inhibitors Tubastatin A (HDAC6/10) and PCI-

Full text of DOI:10.1002/cmdc.202000149

Accepted Article
Title: Design and Synthesis of Dihydroxamic Acids as HDAC6/8/10
Inhibitors
Authors: Aubry Kern Miller, Michael Morgen, Raphael R. Steimbach,
Magalie Géraldy, Lars Hellweg, Peter Sehr, Johannes
Ridinger, Olaf Witt, Ina Oehme, Corey J. Herbst-Gervasoni,
Jeremy D. Osko, Nicholas J. Porter, David W. Christianson,
and Nikolas Gunkel
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000149
Link to VoR: https://doi.org/10.1002/cmdc.202000149
01/2020

10.1002/cmdc.202000149
ChemMedChem
FULL PAPER
Design and Synthesis of Dihydroxamic Acids as HDAC6/8/10
Inhibitors
Michael Morgen,[a] Raphael R. Steimbach,[a,b] Magalie Géraldy,[a] Lars Hellweg,[a] Peter Sehr,[c]
Johannes Ridinger,[d,e,f] Olaf Witt,[d,e,f,g] Ina Oehme,[d,e,f] Corey J. Herbst-Gervasoni,[h] Jeremy D. Osko,[h]
Nicholas J. Porter,[h] David, W. Christianson,[h] Nikolas Gunkel,[a,g] Aubry K. Miller*[a,g]
[a]
Dr. M. Morgen, R. R. Steimbach, Dr. M. Géraldy, L. Hellweg, Dr. N. Gunkel, Dr. A. K. Miller
Cancer Drug Development Group, German Cancer Research Center (DKFZ)
Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
E-mail: aubry.miller@dkfz-heidelberg.de
[b]
[c]
[d]
[e]
[f]
R. R. Steimbach
Faculty of Biosciences, University of Heidelberg
69120 Heidelberg, Germany
Dr. P. Sehr
Chemical Biology Core Facility, European Molecular Biology Laboratory (EMBL)
69117 Heidelberg, Germany
Dr. J. Ridinger, Dr. O. Witt, Dr. I. Oehme
Hopp Children’s Cancer Center Heidelberg (KiTZ)
69120 Heidelberg, Germany
Dr. J. Ridinger, Dr. O. Witt, Dr. I. Oehme
Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ)
69120 Heidelberg, Germany
Dr. J. Ridinger, Dr. O. Witt, Dr. I. Oehme
Department of Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg
69120 Heidelberg, Germany
[g]
[h]
Dr. O. Witt, Dr. N. Gunkel, Dr. A. K. Miller
German Cancer Consortium (DKTK)
69120 Heidelberg, Germany
Dr. C. J. Herbst-Gervasoni, Dr. J. D. Osko, Dr. N. J. Porter, Dr. D. W. Christianson
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania
Philadelphia, Pennsylvania, 19104-6323, USA
Supporting information for this article is given via a link at the end of the document.
Abstract: We report the synthesis and evaluation of a class of Introduction
selective multi-target agents for the inhibition of HDAC6, HDAC8 and
HDAC10. The concept for this study grew out of a structural analysis
of the two selective inhibitors Tubastatin A (HDAC6/10) and PCI-
34051 (HDAC8), which we recognized share the same N-benzylindole
core. Hybridization of the two inhibitor structures resulted in
dihydroxamic acids with benzyl-indole and -indazole core motifs.
These substances exhibit potent activity on HDAC6, HDAC8 and
HDAC10, while retaining selectivity over HDAC1, HDAC2 and HDAC3.
The best substance inhibited viability of the SK-N-BE(2)C
neuroblastoma cell line with an IC50 value similar to a combination
treatment with Tubastatin A and PCI-34051. This compound class
establishes proof of concept for such hybrid molecules and may serve
In the past decades, drug discovery efforts have focused intensely
on the development of inhibitors with high target selectivity. At the
same time it is well recognized that successful drugs typically
exhibit polypharmacology, and that the “one-target-one-disease”
approach often oversimplifies the complex biology underlying
most pathologies.[1] Combination therapy approaches against
multiple targets are clinically successful, but there are advantages
to developing a single drug that engages multiple targets,
particularly when mono-targeted drugs are not already clinically
available. Such advantages include guaranteed action against
both targets in drug-exposed tissues, as well as simplified
pharmacodynamics, manufacture, and regulatory approval.[2]
Histone deacetylases (HDACs) regulate the acetylation state of
lysine residues of histones as well as other protein substrates,
and therefore play a pivotal role in many cellular processes.
Modulation of HDAC activity with inhibitors is known to be
effective in treating different pathologies, and four HDAC inhibitor
as
a starting point for the further development of enhanced
HDAC6/8/10 inhibitors.
1
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10.1002/cmdc.202000149
ChemMedChem
FULL PAPER
Linker
Cap group
Me
N
Cap/Linker
H
N
A
A
A
H
N
HO
ZBG
H
OH
N
N
HO
O
H
OMe
N
N
O
ZBG
OH
O
Cap group
O
Linker
Hybrid
HDAC6/8/10 inhibitor
PCI-34051 (2a)
HDAC8 inhibitor
Tubastatin A (1c)
HDAC6/10 inhibitor
1; X = H, Y = C(O)NHOH
Me
2;
3
X = C(O)NHOH, Y = OMe
; X = Y = C(O)NHOH
N
HO
NH
OH
HN
R
O
N
X
A
O
A
A
HDAC8
inhibition
A
HDAC6/10
inhibition
A
Z
Y
A
N
X
1c 2c 3c
/
/
Y
R
N
NH
O
HN
O
O
O
1a 2a 3a
/
/
;
Z = CH, R = H
Z = CH, R =
Z = N, R = H
Zn
Zn
1b/2b/3b;
1f/2f/3f;
NMe2
NMe2
X
Y
1d 2d 3d
; R = H
/
/
1g 2g 3g Z = N, R =
/
/
;
NMe2
2e/3e; R =
Figure 1. Concept to merge the scaffolds of PCI-34051 and Tubastatin A. Top: PCI-34051 (left) and Tubastatin A (right) are depicted with their respective
zinc binding group (ZBG) in red, linker in blue, and cap group in orange. Recognition that both inhibitors share an N-benzylindole scaffold inspired the
design of hybrid inhibitors (center) with two ZBGs (red) and a central core that would function as cap group and linker (green). Lower left and right:
Inhibition of HDAC8 and HDAC6/10 would result from engagement of one of the two ZBGs, respectively. Lower middle: Synthesized mono- and hybrid
dihydroxamic acids used in this study.
(HDACi) drugs have been approved by the FDA with many more
being evaluated in clinical studies.[3]
IDO1,[9] –proteasome,[10] -PDE5,[11] –kinase,[12] –IMPDH,[13]
–
BET,[14] –SERM,[15] –topoisomerase,[16] and other inhibitors.[17]
HDACi have also been incorporated into PROTACs.[18] We
envisioned developing a new HDACi, with activity against HDAC8
and HDAC10 by combining two isozyme-specific and highly
potent HDAC inhibitors into a chimeric inhibitor.
Many HDACi, including the four approved drugs, inhibit most
HDAC isozymes and are known to have severe side effects,
particularly due to inhibition of HDACs 1, 2, and 3.[4] Isozyme-
specific HDACi are expected to alleviate these liabilities, and
numerous selective HDACi have been described.[5] Specifically
targeting two or more distinct HDACs can also be beneficial, but
this presents a particular challenge when those two HDACs
belong to different isozyme classes with different structural
requirements for efficient binding (the Zn2+-dependent HDACs are
grouped into Class I (HDACs 1,2,3,8), Class IIa (HDACs 4,5,7,9),
Class IIb (HDACs 6,10) and Class IV (HDAC11)). Such a situation
exists for late-stage neuroblastoma, where high HDAC8 (Class I)
and HDAC10 (Class IIb) expression levels strongly correlate with
poor outcomes, and the two enzymes are considered as targets
for treatment.[6] On the one hand, inhibition and knock-down of
HDAC8 favors cell-cycle arrest and differentiation, retards cell
growth, and induces cell death in vitro and in vivo.[7] On the other
hand, inhibition and knock-down of HDAC10 halts autophagic flux
and impairs DNA damage repair mechanisms, leading to an
We recently showed that Tubastatin A, which is annotated as a
selective HDAC6 inhibitor, is also a highly potent HDAC10
binder.[19] We additionally recognized that the selective HDAC8
inhibitor PCI-34051[20] bears a structural similarity to Tubastatin A:
both compounds share an N-benzylindole core. Whereas in PCI-
34051 (Figure 1, top left), the indole moiety (blue color) functions
as the linker with a ZBG at C6, it is part of the -carboline cap
group (orange) of Tubastatin A (Figure 1, top right). Similarly, the
N-benzyl moiety in PCI-34051 (orange) is the cap group, while
functioning as the linker (blue) for Tubastatin A. Interestingly, the
two compounds present their ZBGs at opposing positions of this
core, which is presumably responsible for their very different
selectivity profiles. We postulated that these two known inhibitors
could be merged, to make a hybrid HDACi (Figure 1, top middle).
Because Tubastatin A inhibits both HDAC6 and HDAC10, these
hybrids would likely be HDAC6/8/10 inhibitors.[6] While HDAC6
expression does not significantly correlate with prognosis in
neuroblastoma,[7a, 21] HDAC6 inhibitors have been found to be well
tolerated in clinical studies,[22] and selective HDAC6 inhibition has
been shown to be non-cytotoxic in cancer settings.[23] We
increased
sensitivity
to
chemotherapy.[8]
Furthermore,
simultaneous inhibition of HDAC8 and HDAC10 has been shown
to be effective in killing neuroblastoma cells alone and in
combination with retinoic acid treatment.[6]
HDACi are usually described as containing three structural
modules: a zinc-binding group (ZBG), a “linker” moiety, and a “cap
group”. The cap groups in most HDACi are solvent exposed and
often tolerate a variety of chemical modifications. Arming of
HDACi cap groups with other targeted scaffolds to make chimeras
has been particularly successful, producing combination HDAC-
therefore allowed HDAC6 inhibition as
compounds.
a feature of our
In our strategy, the hybrid inhibitors would contain two ZBGs,
each one responsible for selectively binding to different enzymes.
Thus, the hydroxamic acid on the heterocycle would serve as the
2
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10.1002/cmdc.202000149
ChemMedChem
FULL PAPER
Br
N
a.
c.
CH2O
CO2Me
b.
NH2OH
N
N
R
R
H
N
HNMe2
H
OMe
N
OH
N
NaH
R
N
H
OH
N
O
O
4; R = H
5; R = CO2Me
6
; R = H
7; R = CO2Me
1a
3a
; R = H
; R = C(O)NHOH
O
1b
X
N
d.
O
O
e.
HNMe2
H
b.
NH2OH
R'
MeO
N
N
N
HO
MeO
MeO
N
NaBH(OAc)3
K2CO3
N
O
O
H
O
R'
R'
O
R'
8
9; R' = OMe
10
11 ; R' = OMe
12
2b; R' = OMe
3b
; R' = CO2Me
; R' = CO2Me
; R' = C(O)NHOH
X
Me
N
Me
N
Me
N
Me
h.
n-BuLi
–100 °C
a.
b.
N
f. H2SO4
g. Boc2O
then
+
R'
NH2
H
N
Br
N
O
H
MeO
N
Br
N
N
H
HO
NaH
NH2OH
O
N
Boc
H
O
O
O
13
15
R'
14
16
i.
NaCN, MnO2
MeOH
2c
; R' = OMe
3c; R' = C(O)NHOH
j. TFA
Scheme 1. Synthesis of N-benzylated derivatives 1a,b, 2b,c and 3a,b,c. Reagents and conditions: (a) NaH, 4-carbomethoxybenzyl bromide or PMBCl, DMF, rt, 1–
2 h, 43–99% ; (b) NH2OH (50 wt% in H2O), KCN or KOH, 1,4-dioxane, rt, 24–48 h, 11–72%; (c) HNMe2, CH2O, HOAc, MeOH/H2O (5:1), 0 °C to 70° C, 48 h, 46%;
(d) K2CO3, 4-carbomethoxybenzyl bromide or PMBCl, DMF, rt, 12–16 h, 85% (9), 74% (10); (e) HNMe2, NaBH(OAc)3, MeOH/H2O, 0 °C to rt, 48 h, 50% (11), 39%
(12); (f) H2SO4, 1,4-dioxane, 60 °C, 20 h, 94%; (g) Boc2O, DMAP, CH2Cl2, rt, 15 min, 71%; (h) t-BuLi, N-formylmorpholine, THF, –100 °C, 10 min, 76%; (i) NaCN,
MnO2, HOAc, MeOH, rt, 2 h, 73%; (j) TFA, CH2Cl2, 0 °C to rt, 2 h, 96%; PMBCl , 4-methoxybenzyl chloride; Boc2O, di-t-butyl dicarbonate; DMAP, 4-
dimethylaminopyridine.
Br
ZBG with respect to HDAC8 inhibition (Figure 1, lower left), with
the phenyl hydroxamate functioning as part of the cap group.The
situation would be reversed in the case of HDAC6/10 inhibition
(Figure 1, lower right).
a.
H
N
H
N
CO2Me
H
H2O, 150 °C
-wave
N
OH
We have previously have found that a basic nitrogen in the cap
group of Tubastatin A analogs is necessary for potent HDAC10
binding.[19] Therefore, we expected that a hybrid inhibitor bearing
a PCI-34051-like indole, with no substitution at C2 or C3 of the
indole, would be unlikely to give potent HDAC10 activity. Little
information is available in the literature with respect to SAR
around the PCI-34051 linker indole, but we postulated that the
bulky -carboline cap group of Tubastatin A might be too large to
serve as linker (linkers are usually relatively slender) for a hybrid
dual-inhibitor. We therefore synthesized a variety of phenyl-
hydroxamic acids 1 as HDAC6/10 inhibitors and indolyl/indazolyl-
hydroxamic acids 2 as HDAC8 inhibitors to serve as benchmark
comparisons to the corresponding dihydroxamic acids 3, which
should inhibit HDAC6/8/10 (Figure 1, bottom middle). In this
numbering scheme, PCI-34051 is labelled as 2a and Tubastatin
A as 1c.
4
O
b.
NH2OH
1d
X
NO2
NO2
c.
O
O
R
O
N
OMe
OMe
MeCN
reflux
17
R
18
19
; R = OMe
; R = CO2Me
H
N
b.
NH2OH
d.
H
Fe(0)
AcOH
130 °C
N
HO
O
R
H
N
2d
; R = OMe
; R = C(O)NHOH
3d
MeO
O
R
N
N
e.
Cl
20
; R = OMe
21; R = CO2Me
NMe2
R'
NaH
DMF, 100 °C
R
Chemistry
22; R = OMe, R' = CO2Me
b.
NH2OH
NH2OH
2e
23
3e
; R = OMe, R' = C(O)NHOH
; R = R' = CO2Me
; R = R' = C(O)NHOH
b.
The synthesis of N-benzylated indole derivatives was performed
in two to three steps starting with nucleophilic substitution of
indole building blocks 4, 5, or 8 with 4-methoxybenzyl chloride
(PMBCl) or 4-carbomethoxybenzyl bromide to give 6, 7, 9, and 10
(Scheme 1, top and middle). Indoles 1a and 3a were obtained by
Scheme 2. Synthesis of C3-benzylated derivatives 1d, 2d,e and 3d,e.
Reagents and conditions: (a) 4-carbomethoxybenzyl bromide, H2O, 150 °C,
-wave, 5 min, 45%; (b) NH2OH (50 wt% in H2O), KCN or KOH, 1,4-dioxane, rt,
24 h, 15–51%; (c) 4-methoxybenzyl bromide or 4-carbomethoxybenzyl bromide,
MeCN, 90 °C, 5 h, 78% (18), 91% (19); (d) Fe powder, AcOH/PhMe (3:4),
130 °C, 4 h, 54% (20), 44% (21); (e) 2-chloroethyl-N,N-dimethylamine•HCl, NaH,
DMF, 100 °C, 65% (22), 17% (23).
treatment of methyl esters 6 and
7 with hydroxylamine,
respectively. Gramine derivative 1b[24] was made from 1a via
Mannich reaction using formaldehyde and dimethylamine.
Gramines 2b and 3b were obtained from formylindoles 9 and 10,
3
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10.1002/cmdc.202000149
ChemMedChem
FULL PAPER
X
Table 1. HDAC8 and HDAC10 inhibitory data.
a.
N
1
: X = H; Y = C(O)NHOH
N
R
R'
N
N
2: X = C(O)NHOH; Y = OMe
3: X = Y = C(O)NHOH
R
base
H
R'
24; R = H
27; R = H, R' = COOMe
1f; R = H, R' = C(O)NHOH
b.
NH2OH
HDAC8 HDAC10
Scaffold
Cmpd.
R = CHO, R' = OMe
25; R = CHO
28;
29; R = COOMe, R' = OMe
2f;
30;
3f;
a,b
a,c
c.
NaCN, MnO2, MeOH
pIC50
pIC50
b.
NH2OH
R = C(O)NHOH, R' = OMe
R = R' = COOMe
R = R' = C(O)NHOH
26
; R = CO2Me
1a
2a
3a
6.42
7.29
7.17
7.18
4.38
8.59
b.
NH2OH
N
X
X
X
Y
Y
N
N
a.
d.
O
NMe
2
R'
1b
2b
3b
6.02
7.31
6.78
8.28
5.79
8.55
N
N
R
N
base
R
H
N
R'
HNMe2
NaBH(OAc)3
31
32
33;
R = H, R' = CO2Me
; R = H
b.
b.
b.
NH2OH
NH2OH
NH2OH
1g;
34;
2g;
35;
3g;
R = H, R' = C(O)NHOH
R = CO2Me, R' = OMe
R = C(O)NHOH, R' = OMe
R = R' = CO2Me
Me
N
; R = CO2Me
1c
2c
3c
5.70
6.85
6.29
7.90
5.19
7.91
R = R' = C(O)NHOH
N
X
X
Scheme 3. Synthesis of indazole derivatives 1f,g, 2f,g and 3f,g. Reagents and
conditions: (a) KOtBu or K2CO3 or NaH, 4-carbomethoxybenzyl bromide or 4-
methoxybenzyl chloride, DMF, rt, 1–18 h, 25–79%; (b) NH2OH (50 wt% in H2O),
KCN or KOH, 1,4-dioxane, rt, 24–48 h, 14–93%; (c) NaCN, MnO2, AcOH, MeOH,
rt, 2 h, 95%; d) HNMe2, NaBH(OAc)3, MeOH/H2O, 0 °C to rt, 48 h, 25–73%.
Y
Y
H
N
1d
2d
3d
N.M.
7.16
7.01
7.56
5.50
8.49
NMe
2
respectively, via reductive amination with dimethylamine to give
11 and 12, followed by hydroxamic acid formation.[25] The -
carboline scaffold of Tubastatin A analogues 2c and 3c was
prepared by Fischer indole synthesis using hydrazine 13 and
1-methylpiperidin-4-one (14), with subsequent BOC protection to
give 15 (Scheme 1, bottom). Bromide 15 was converted to ester
16 via carbonylation, Corey–Gilman–Ganem oxidation, and then
BOC removal.[26] Lastly, alkylation of 16 with PMBCl or 4-
carbomethoxybenzyl bromide before hydroxamic acid formation
provided 2c and 3c, respectively.
We utilized different approaches to access the C3-benzylated
indole derivatives. Selective C3-alkylation of indole (4) under
microwave irradiation in H2O,[27] followed by hydroxamic acid
formation gave 1d (Scheme 2, top). Alternatively, the C3-benzyl
indole scaffold was constructed starting from enamine 17
(Scheme 2, bottom). Alkylation of 17 with 4-methoxybenzyl
N
2e
3e
7.36
7.18
6.18
8.52
X
X
Y
Y
1f
2f
3f
6.41
7.68
7.37
6.63
4.72
8.47
N
N
N
N
NMe
2
1g
2g
3g
6.07
7.53
7.05
7.71
5.16
8.40
X
Y
a See Table S1 for error values associated with these calculations. b pIC50 values
determined with an enzymatic HDAC-GloTM I/II assay. c pIC50 values
determined with a ligand-displacement FRET assay. N.M. = not measured.
bromide
or
4-carbomethoxybenzyl
bromide
afforded
nitroaldehydes 18 and 19, respectively, which, after Fe-mediated
reduction, directly cyclized to the C3-substituted indoles 20 and
21.[28] These substances were either directly converted to
Results and Discussion
As our primary interest was to target HDAC8 and HDAC10, the
substances were first tested against the two proteins in an
enzymatic (HDAC-GloTM I/II) and a ligand displacement FRET
assay,[19] respectively (Table 1). Starting with the set of
compounds based on the simple PCI-34051 scaffold (1a/2a/3a),
we were pleased to see confirmation of our design concept:
Monohydroxamic acid 1a showed good activity against HDAC10
(pIC50 = 7.18) and moderate activity against HDAC8 (pIC50 = 6.42).
As expected, PCI-34051 (2a) was found to be highly selective for
HDAC8 (pIC50 = 7.29) over HDAC10 (pIC50 = 4.38). Critically,
dihydroxamic acid 3a had good activity against HDAC8
(pIC50 = 7.17) and excellent HDAC10 (pIC50 = 8.59) activity. The
other two non-basic C-3 benzyl (1d/2d/3d) and indazole (1f/2f/3f)
scaffolds also showed internally consistent profiles.
hydroxamic acids 2d and 3d, or equipped with
a
dimethylaminoethyl group prior to installation of the hydroxamic
acid ZBG to yield 2e and 3e.[29][30]
We synthesized indazole derivatives 1f, 2f and 3f in a similar
fashion to the N-benzylated indole derivatives by nucleophilic
substitution of indazoles 24–26 with benzyl halides to give 27, 28,
and 30 (Scheme 3, top). Ester 29 was obtained from 28 via
Corey–Gilman–Ganem oxidation, and 27, 29, and 30 were
converted to hydroxamic acids 1f, 2f, 3f.
In analogy to the gramine derivatives 2b/3b, an additional step for
the synthesis of the hydroxamic acids 1g, 2g and 3g was
performed (Scheme 3, bottom). Starting from indazoles 31 and
32,[31] benzylation followed by reductive amination gave 33–35,
which were converted to the corresponding hydroxamic acids
1g/2g/3g as before.
4
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Table 2. Data for hybrid inhibitors against Class I and Class IIb HDACs.
a
pIC50
Cmpd
HDAC1b
4.33
5.91
6.58
6.21
6.30
6.28
6.09
7.06
6.18
HDAC2b
<4.0
4.89
5.65
5.27
5.38
5.33
5.15
6.14
5.02
HDAC3b
4.24
5.78
5.26
5.16
4.87
5.19
5.18
5.88
5.18
HDAC6b
5.15
7.70
7.74
7.12
7.32
7.65
7.35
7.82
7.52
HDAC8b,c HDAC10c,d
PCI-34051 (2a)
7.29
5.70
7.17
6.78
6.29
7.01
7.18
7.37
7.05
4.38
7.90
8.59
8.55
7.91
8.49
8.52
8.47
8.40
Tubastatin A (1c)
3a
3b
3c
3d
3e
3f
3g
a See Table S1 for error values associated with these calculations. b Enzymatic HDAC-GloTM I/II assay. c Values taken from Table 1. d Ligand displacement
FRET assay.
Within these three scaffolds, inhibition of HDAC8 was slightly
diminished by the introduction of a second hydroxamic acid
moiety when compared to its parent monohydroxamic acid
inhibitor (e.g. 3a compared to 2a). We were intrigued to find that
HDAC10 activity was strongly increased by the addition of a
second hydroxamic acid in all three cases, with 3f showing the
largest increase in potency by a factor of ~70 over 1f. The high
potency of these substances, despite their lack of a basic amine
group, was surprising in light of our previous findings with
Tubastatin A derivatives.[19]
We also examined the -carboline series 1c/2c/3c and found the
HDAC10 activity of 3c is similar to 1c (Tubastatin A). The bulky -
carboline as a linker group (2c) produced the weakest HDAC8
inhibitor from the six monohydroxamic acids 2a–2g. Furthermore,
the corresponding dihydroxamic acid derivative 3c showed even
further diminished inhibition values toward HDAC8 (pIC50 = 6.29).
This was consistent with our hypothesis that -carboline
derivatives would be too bulky.
We have previously shown that “ring-opened” Tubastatin A
derivative 1b is a potent HDAC10 binder, with >100-fold
selectivity over HDACs 1, 2, and 3.[19] Consequently, the gramine-
type derivatives 1b/2b/3b and 1g/2g/3g were investigated.
Furthermore, the conceptually similar 3e and its monohydroxamic
acid analog 2e were prepared.[29] All three hybrids (3b/3e/3g)
gave high HDAC10 pIC50 values of 8.55, 8.52, and 8.40
respectively. As with the other scaffolds, HDAC8 inhibition was
detrimentally affected by the second hydroxamic acid, but
compounds 3e and 3g showed acceptable profiles with HDAC8
pIC50 values of 7.18 and 7.05, respectively.
Having shown that our design plan succeeded with a variety of
scaffolds, we tested the dihydroxamic acids (3a–3g) against all
the remaining Class I and Class IIb HDACs to establish selectivity
profiles (Table 2). HDACs 1, 2, 3, and 6 were assayed with the
HDAC-GloTM I/II system. As expected, all of the dihydroxamic
acids are excellent HDAC6 inhibitors, with pIC50 values of 7.12–
7.82. They also show some increased activity against HDAC1–3,
when compared to PCI-34051 and Tubastatin A. Compounds 3b
and 3c have relatively weak HDAC8 and moderate HDAC1
activity, disqualifying them for further biological investigation
along with 3a and 3f, which have the highest HDAC1–3 activities
of all the inhibitors. Substances 3d, 3e, and 3g each have low
HDAC2 and HDAC3 and moderate HDAC1 activity, and have the
best selectivity profiles overall.
Parallel to our biochemical evaluation, we attempted to co-
crystallize the dual inhibitors with D. rerio (zebrafish) HDAC10,[32]
where we had introduced A24E and D94A substitutions to more
closely resemble the human HDAC10 active site. The crystal
structure of the “humanized” zebrafish HDAC10-3a complex was
determined at 2.05 Å resolution, whereas other inhibitors either
gave no crystals, or their resulting crystals diffracted poorly. The
overall protein structure is quite similar to that of wild-type
zebrafish HDAC10 in its complex with a slender trifluoroketone
inhibitor,[33] with a root-mean-square deviation (rmsd) of 0.24 Å for
514 C atoms (Table S2). However, due to the rigidity and bulk
of 3a there are significant local structural changes in the active
site. Specifically, the 310 helix containing the P23(E,A)CE motif that
protrudes into the active site shifts, on average, by 1.4 Å
(maximum shift = 1.9 Å). In other HDAC10 structures, the
P23(E,A)CE motif sterically constricts the active site, presumably
to favor the binding of long slender polyamine substrates.
However, the current structure reveals that this motif can shift to
accommodate the binding of certain bulky inhibitors.
Zinc coordination by the ionized hydroxamate group of 3a is
achieved by a mixture of two different monodentate binding
modes (Figure 2A). The hydroxamate of the major conformer
(67% occupancy) coordinates to zinc through the N–O– group (O-
--Zn2+ separation = 2.1 Å) (Figure 2B). The phenolic hydroxyl
group of Y307 is within hydrogen bonding distance to both the
hydroxamate NH and N–O– groups (O---N and O---O separations
= 2.6 and 2.7 Å, respectively). A Zn2+-bound water molecule is
also observed (O---Zn2+ separation = 2.2 Å), which donates a
hydrogen bond to the hydroxamate C=O group (O---O separation
= 3.1 Å) and forms hydrogen bonds with H136 and H137 (O---N
separations = 2.3 and 2.7 Å, respectively).
At first glance, the hydroxamate group of the minor conformer of
3a (33% occupancy) appears to coordinate to Zn2+ in a manner
similar to that observed for bidentate hydroxamate-zinc
interactions as observed in other HDAC10-inhibitor complexes
5
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10.1002/cmdc.202000149
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Figure 2. Stereoviews of Polder omit maps of 3a in complex with HDAC10 (contoured at 6.0 ). Atoms are color-coded as follows: C = light gray
(HDAC10), orange (major conformer of 3a), or maroon (minor conformer of 3a), N = blue, O = red, Zn2+ = gray sphere, and solvent = small red spheres.
Metal coordination interactions are shown as solid black lines and hydrogen bonds are shown as dashed black lines. (A) Both conformers in complex
with HDAC10. (B) Major conformer in complex with HDAC10. (C) Minor conformer in complex with HDAC10.
(Figure 2C).[33] However, the C=O---Zn2+ separation of 2.7 Å is not
consistent with inner sphere metal coordination. Thus, inner
sphere Zn2+ coordination is achieved solely by the hydroxamate
N–O– group (O---Zn2+ separation = 2.4 Å). The Zn2+-bound N–O–
group accepts a hydrogen bond from H136 (O---N separation =
2.6 Å) and the hydroxamate NH group donates a hydrogen bond
to H137 (N---N separation = 2.5 Å). Additionally, Y307 forms an
anomalously short hydrogen bond (2.2 Å) with the hydroxamate
C=O group. The phenyl ring of 3a makes favorable offset 
stacking interactions in an aromatic crevice defined by F146 and
W205, as well as the Zn2+ binding residue H176. Interestingly, the
side chain of E24 packs against the indole ring of the inhibitor.
The A24E substitution was made to “humanize” the active site of
zebrafish HDAC10, so the packing interaction between E24 and
the indole ring of 3a is presumably important for binding to human
HDAC10. The indole hydroxamate is not fully defined by electron
density, potentially due to disorder, but it clearly forms a hydrogen
bond with N93, which in turn forms a hydrogen bond with W205.
Of note, N93 of zebrafish HDAC10 aligns with D91 of human
HDAC10, so these hydrogen bond interactions may be somewhat
6
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Figure 3. (A) Stereoview of the Polder omit maps of the HDAC6-3a complex (monomer A, contoured at 3.5 σ). Atoms are color-coded as follows: C =
light blue (HDAC6), dark gray (symmetry mate), or wheat (inhibitor), N = blue, O = red, Zn2+ = gray sphere, and solvent = small red spheres. Metal
coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively. (B) Stereoview of a superposition of the 3a
complexes with HDAC6 and HDAC10. Atoms are color-coded as follows: C = light blue (HDAC6) or white (HDAC10), dark gray (symmetry mate of
HDAC6), wheat (3a bound to HDAC6), orange (3a bound to HDAC10), N = blue, O = red, and Zn2+ = gray sphere.
altered upon inhibitor binding to human HDAC10.
N645 as well as R788 of a symmetry mate in the crystal lattice.
Since the inhibitor capping group has a specific binding site in a
pocket in the active site of HDAC10 that is not conserved in the
active site of HDAC6, this feature must contribute to selectivity for
binding to HDAC10.
We next measured viability of the HDAC8/10 sensitive SK-N-
BE(2)C neuroblastoma cell line[6] after treatment with our hybrid
inhibitors. On the basis of our biochemical profiling and previous
experience with Tubastatin A derivatives,[19] we began with the
two amine-containing inhibitors 3e and 3g. We were surprised to
find that both hybrid molecules showed little effect up to 100 M,
While no dihydroxamic acid in this study provided crystals of
sufficient quality with HDAC8, the crystal structure of 3a
complexed with D. rerio HDAC6 was determined at 1.94 Å
resolution (Table S3). The structure of this complex reveals that
3a binds to HDAC6 as a single conformer (Figure 3A). The
catalytic Zn2+ ion is coordinated in monodentate fashion by the
hydroxamate N–O– group of 3a (average O---Zn2+ separation =
2.1 Å), and the hydroxamate C=O group accepts a hydrogen bond
from the Zn2+-bound water molecule (average O---O separation =
2.4 Å). The Zn2+ coordination geometry is similar to that observed
for the major conformer of 3a bound to HDAC10 (Figure 2B). Also
similar to the HDAC10-3a complex, the aromatic ring of the
phenylhydroxamate nestles in an aromatic crevice, here defined
by F583 and F643.
Interestingly, superposition of the two enzyme-inhibitor
complexes reveals that the capping group conformation of 3a
differs between the HDAC6 and HDAC10 complexes (Figure 3B).
As observed in the HDAC10-3a complex, the capping group is
clamped down by E24 of the PEACE motif; however, the capping
group of 3a in the HDAC6 complex is oriented toward solution;
the indole hydroxamate makes hydrogen bond interactions with
although
a
1:1 molar ratio of PCI-34051 (2a) and
Tubastatin A (1c) gave an IC50 value of 7.3 M (6.5–8.1 M 95%
C.I.) (Figure 4A, Figure S1). In order to explain this discrepancy,
we measured cellular markers/phenotypes which are indicative of
target engagement. Whereas PCI-34051 (2a) and HDAC8
inhibitor 2g increased acetylation of the HDAC8 substrate SMC3
in a dose-dependent manner,[6] hybrid inhibitor 3g showed no
effect relative to solvent control (Figure 4B). Furthermore,
dihydroxamates 3e and 3g failed to produce an HDAC10
knockdown phenotype, i.e. increased lysosomal staining with the
acidotropic LysoTracker DND-99 dye (Figure 4C).[8b] Tubastatin A
7
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10.1002/cmdc.202000149
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(1c) and monohydroxamate 1g were effective as positive controls.
This data pointed toward poor cell permeability of the highly polar
dihydroxamic acids 3e and 3g (Table S4). We examined this
further by testing target engagement of selected inhibitors in a
cellular BRET HDAC10 target engagement assay.[34] Substances
1d and 1g had very similar FRET and BRET pIC50 values,
indicating good cell permeability for monohydroxamic acids with
or without a basic amine side chain (Figure 4D). Dihydroxamic
acid 3g, on the other hand, had a pIC50 value in the BRET assay
which is more than 60 fold weaker than in the FRET assay,
consistent with poor cell permeability. Compound 3d, which lacks
a basic amino side chain and is slightly less polar, showed only a
moderate ~5-fold loss of potency between the FRET and BRET
assays, pointing toward improved cell permeability.
This was supported by the fact that 3d induces lysosomal
acidification similar to Tubastatin A (1c) (Figure 4C). In light of this
finding, we tested 3d in cells and found it increases acetylation of
SMC3 (HDAC8 substrate) and tubulin (HDAC6 substrate), while
having significantly weaker effects on histone H3 (HDAC1
substrate) (Figure 4E). Pleasingly, 3d alone, was as effective
(IC50 = 7.0 M; 6.5–7.5 M 95% C.I.) as combination treatment
with Tubastatin A (1c) and PCI-34051 (2a) in a SK-N-BE(2)C cell
viability assay (Figure 4F).
As discussed above, previous data from our group showed that
only those Tubastatin A derivatives which contain a basic nitrogen
in the cap group are potent (pIC50 ≥ ~8.0) HDAC10 binders.[19] At
the outset of this study, we assumed that our hybrid molecules
would also require a basic nitrogen in their cap/linker group. While
all the compounds in this study which contain such a nitrogen are
indeed potent HDAC10 binders (i.e. 3b, 3c, 3e, and 3g), we were
surprised to find that this functionality was not required (i.e. 3a,
3d, and 3f). In the latter case, the addition of a hydroxamic acid
at C6 of the indole ring was sufficient to produce highly potent
HDAC10 binders (e.g. 3f versus 1f). The hydrogen bond found
between the cap group hydroxamic acid in 3a with N93 in
zebrafish HDAC10 may also be formed with D91 in human
HDAC10, potentially explaining the tight HDAC10 binding of the
dihydroxamic acids.
Conclusion
In
summary,
the
similar
central
scaffolds
of
PCI-34051 (2a) and Tubastatin A (1c) were used as the basis to
develop proof of concept hybrid molecules which target HDAC8
and HDAC10 (and HDAC6). Highly potent inhibitors could be
synthesized, but not all showed cellular activity. We attribute this
discrepancy to poor cell permeability, potentially due to the high
polarity of the substances. While 3d is sufficiently cell permeable
to inhibit the growth of SK-N-BE(2)C cells, its efficiency could
potentially be improved by a pro-drug strategy that masked one
or both of the hydroxamic acid groups, or via the replacement of
at least one of the hydroxamic acids with a different ZBG.
Figure 4. (A) Dose-response data for hybrid inhibitors and a 1:1 molar
ratio of PCI-34051 (2a) and Tubastatin A (1c) in SK-N-BE(2)C cells. (B)
Western blots of Ac-SMC-3 with actin as a loading control (SK-N-BE(2)C).
(C) Fluorescence readout from SK-N-BE(2)C cells treated with 10 M of
each inhibitor, normalized to cells treated with DMSO. (D) Comparison of
FRET and BRET HDAC10 binding data. FRET values are taken from
Table 1. BRET values are determined from an experiment run in triplicate.
(E) Western blots of Ac-SMC-3, Ac-tubulin, and Ac-Histone 3. The pan-
HDACi panobinostat is used as a positive control for Histone 3 acetylation.
(F) Dose-response data for 3d and a 1:1 molar ratio of PCI-34051 (2a) and
Tubastatin A (1c). The cell proliferation assays were run in triplicate. Error
bars represent S.D. Data is normalized to a vehicle control, which was also
performed in triplicate.
Experimental Section
Expression and purification of TwinStrepII-GST-HDAC10: A
synthetic gene encoding TwinStrepII-GST-HDAC10 (human) was
ordered from GeneArt (Thermo Fischer Scientific) and sub-cloned
into the pFastBac1 vector. The resulting construct was used for
8
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10.1002/cmdc.202000149
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transposition in E. coli DH10EMBacY cells. The isolated bacmid
DNA was then utilized to generate the recombinant baculovirus.
For protein expression, 10 mL of baculovirus was added to 1 L of
Sf21 cells at a density of 1 x 106 cells/mL. The infected Sf21 cells
were grown for 72 h in Sf-900 III SFM medium (Thermo Fischer
Scientific) at 27 ºC. Cells were harvested by centrifugation and re-
suspended in running buffer (100 mM Tris pH 8.0, 150 mM NaCl,
1 mM EDTA and 1 mM DTT) supplemented with 10 mM MgCl2,
benzonase and cOmplete protease inhibitors (Merck). The cells
were lysed using a Dounce homogenizer and the resulting lysate
was centrifuged for 30 min at 4 ºC at 125000 x g in an
ultracentrifuge. The clarified lysate was then loaded onto a 5 mL
Strep-Tactin Superflow high capacity column (IBA) pre-
equilibrated in running buffer. After sample loading and washing,
the TwinStrepII-GST-HDAC10 protein was eluted in running
buffer supplemented with 5 mM desthiobiotin (IBA). The elution
fractions containing TwinStrepII-GST-HDAC10 were pooled and
concentrated before being injected onto a HiLoad 16/600
Superdex 200 pg size exclusion chromatography column (GE
Healthcare) pre-equilibrated with 25 mM HEPES/NaCl pH 7.5,
150 mM NaCl, 0.5 mM EDTA, 1 mM DTT and 10% glycerol.
Samples were eluted from the size exclusion chromatography
column in the same buffer, flash-frozen in liquid N2 and stored at
–80 ºC.
TR-FRET assay (used with dihydroxamic acids): TR-FRET
assays were performed in white 384-well plates (4512, Corning)
using 50 mM HEPES pH 8.0, 150 mM NaCl, 10 mM MgCl2, 1 mM
EGTA and 0.01% Brij-35 as buffer. The concentrations of reagent
in 15 L final assay volume were 5 nM TwinStrep-GST-HDAC10
(preparation described below), 25 nM “Tubastatin-AF647-
Tracer” (S11, synthesis in Supporting Information) and 0.1 nM
DTBTA-Eu3+-labelled Streptactin (synthesis in Supporting
Information). Inhibitors were tested at eight serial dilutions in
triplicates ranging from 50 M – 86.7 pM and dosed from 10 mM
and 0.1 mM DMSO stock solutions with a D300e Digital Dispenser
(Tecan). After drug dosing to the premixed assay reagents in
buffer, plates were shaken (800 rpm orbital shaker, 30 s),
centrifuged (300 g, 1 min) and incubated at room temperature in
the dark for 60 min. TR-FRET was measured with a CLARIOstar
(BMG Labtech) plate reader, equipped with TR-FRET filters.
Sample wells were excited with 100 flashes and fluorescence
emission detected at 665 nm and 620 nm. FRET ratios were
calculated from 665 nm/620 nm ratio and normalized for each
plate using 50 M SAHA treated negative controls and uninhibited
positive controls. pIC50-values were calculated using nonlinear
regression log(inhibitor) four parameters least squares fit in
GraphPad Prism version 7.04 for Windows, GraphPad Software,
La Jolla California USA, www.graphpad.com.
Note on the TR-FRET Assay: We have made slight
modifications to the TR-FRET assay since the original publication
where we described it.[19] Control experiments indicate that pIC50
values for a given inhibitor tested in both assay formats are not
statistically different. Therefore, data from the two assay formats
can be reliably compared. The TR-FRET measurements of the
monohydroxamic acids in this manuscript were measured in the
original assay format, which is described directly below this
paragraph. The TR-FRET measurements of the dihydroxamic
acids in this manuscript were measured in the modified format,
which is described two paragraphs below this one.
Production of mono-clones stably expressing HDAC-
nanoBRET proteins: Plasmids expressing a fusion of HDAC6
(containing only the 2nd catalytic domain) or HDAC10 with
nanoluciferase were obtained from Promega (N2170). HeLa cells
(0.75 x 106) were seeded in a 6 cm dish and after 24 h were
transfected with a mix of 10 g plasmid and 3 L Fugene in
200 L OptiMEM. In detail, cells were washed with pre-warmed
OptiMEM and subsequently overlaid with 2.3 mL of OptiMEM.
After addition of 200 L transfection mix, cells were incubated for
24 h at 37 °C. Cells were than trypsinized and 0.2 x 105 cells were
seeded into both 10 cm and 15 cm dishes. Transformants were
selected with 1mg/mL G-418 for 6 days with a media change after
3 days. Clones which formed colonies were picked by rinsing
plates with 3 mL Trypsin/EDTA (Sigma T3924) followed by a 2
min incubation with 300 L Trypsin/EDTA at 37 °C. Colonies were
than loosened and aspirated with a 10 L filter tip and transferred
to 24-well plates containing selection medium. Clones exhibiting
a range of nanoluciferase activities were expanded and selected
according to the highest BRET ratio.
TR-FRET Assay (used with the monohydroxamic acids): All
TR-FRET experiments were performed in white 384-well
ProxiPlates (PerkinElmer) using 50 mM HEPES pH 8.0, 150 mM
NaCl, 10 mM MgCl2, 1 mM EGTA and 0.01% Brij-35 (+2%
DMSO) as buffer. The concentrations of reagents in 10 L final
assay volume were 3 nM GST-HDAC10, 30 nM “Tubastatin-
Alexa647-Tracer” and 0.5 nM Eu-anti-GST. GST-HDAC10 and
LanthaScreen™ Eu-anti-GST Antibody were purchased from Life
Technologies, and the Tubastatin-Alexa647-Tracer was
synthesized in-house as previously described.[19] An eleven-fold
1:3-serial dilution of compounds starting at 2 mM was prepared in
384 well pp-plates (Greiner) from 10 mM stocks in DMSO and
1 L was transferred to assay plates. Nine L of the reagent-mix
was added and the plate was incubated for 1 h at rt before TR-
FRET was measured in an EnVision™ plate reader equipped with
a TR-FRET Laser module. Sample wells were exited with 3
flashes of the TRF-Europium Laser, and emission was measured
at 620 nm and 665 nm to get the 665 nm/620 nm ratio. Percent
inhibition was calculated for each well from negative control wells
containing 2% DMSO and positive control wells containing 20 M
SAHA. The resulting dose-response curves were fitted in
ActivityBase (IDBS) using a four-parameter logistic model and
IC50-values were calculated.
Culture of stable BRET cell lines: Stably transfected HeLa cells
were cultivated under sterile conditions in polystyrene cell culture
flasks (658170, Greiner) at 37 °C and 5% CO2 in a humidified
atmosphere. D-MEM growth medium (D6049, Sigma) was
supplemented with 10% FCS (FBS-12A, Capricorn Scientific), 1%
Penicillin-Streptomycin (P4333, Sigma) and 1 mg/mL Geneticin
(2039.3, Roth). At confluency, cells were passaged by removing
old medium, DPBS (14190-094, gibco) wash, trypsination (T4049,
Sigma) and seeding in fresh growth medium.
BRET Assay: The intracellular target engagement assay on
HDAC6 and HDAC10 was performed as described by the kit
manufacturer in a 96-well plate (3600, Corning) format with
1.9 x 104 cells per well and a tracer concentration of 0.3 M.
Inhibitors were tested at ten 1:4 serial dilutions in triplicates
ranging from 129 pM to 40 M. Drug dosing was performed from
9
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10.1002/cmdc.202000149
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10 mM and 1 mM DMSO stock solutions with a D300e Digital
Dispenser (Tecan), DMSO concentrations were normalized to
0.5% for all wells. Assay plates were incubated at 37 °C for 2 h
followed by measurement of 450 nm and 650 nm luminescence
(80 nm bandwidth) at room temperature with a CLARIOstar (BMG
Labtech) plate reader 2 min after NanoLuc substrate addition.
Cells were washed with ice-cold RPMI without phenol-red and
trypsinized for 3 min at 37 °C. Detached cells were centrifuged for
3 min at 8600 x g and re-suspended in ice-cold RPMI without
phenol red. Mean LysoTracker fluorescence was quantified on a
BD FACSCanto II platform using the PE filter setting. Data were
normalized to mean LysoTracker fluorescence of solvent (DMSO)
treated cells.
BRET ratios were calculated from 650 nm/450 nm luminescence
and normalized for each plate using 50 M SAHA treated
negative controls and uninhibited positive controls. pIC50-values
were calculated from normalized BRET ratios using nonlinear
regression log(inhibitor) four parameters least squares fit in
GraphPad Prism version 7.04 for Windows, GraphPad Software,
La Jolla California USA, www.graphpad.com.
Cell viability assay: SK-N-BE(2)C were cultured in Dulbecco’s
Modified Eagle Medium (DMEM, Lonza, Basel, Switzerland)
supplemented with 10% fetal calf serum (FCS, Sigma-Aldrich,
Munich, Germany) and 1% non-essential amino acids (NEAA,
Lonza). SK-N-BE(2)C cells were seeded from a confluent flask
into 96-well plates in 100 L full growth medium at a density of
5 x 103 cells per well the day before treatment. Cells were treated
in triplicates with drug concentrations as indicated in the
respective figure. Drugs were dosed from 50 mM and 200 mM
stock solutions with a D300e Digital Dispenser (Tecan) and
DMSO concentrations were normalized to 0.2% for all wells.
Plates were shaken (1000 rpm orbital shaker, 30 s) and incubated
under standard culture conditions for 72 h. To each well 20 L of
CellTiter-Blue® reagent (G8081, Promega) was added, plates
were shaken (600 rpm orbital shaker, 20 s) and incubated at
HDAC-Glo Assay for HDAC 1, 2, 3, 6 and 8: HDAC6 and class
I inhibition was tested using the HDAC-Glo™ I/II Assay and
Screening System (G6421, Promega) with recombinant human
HDACs (BPS Bioscience; HDAC1 cat. # 50051; HDAC2 cat. #
50002; HDAC3/NcoR2 complex cat. # 50003; HDAC6 cat. #
50006; HDAC8 cat. # 50008). The assay was carried out in a 384-
well plate (4512, Corning) format according to the manufacturer’s
description. Inhibitors were tested at eight serial dilutions in
triplicate ranging from 50 M – 86,7 pM (HDAC6) or 100 M –
8,67 nM (HDAC1,2,3,8). Drug dosing was performed from 10 mM
and 0.1 mM DMSO stock solutions with a D300e Digital Dispenser
(Tecan). HDACs (7 ng/mL for HDAC1, 10 ng/mL for HDAC2, 200
ng/mL for HDAC3/Ncor2 complex, 100 ng/mL for HDAC6, 200
ng/mL for HDAC8) and inhibitors were incubated together for 30
min at room temperature. After addition of the HDAC-Glo™ I/II
reagent, plates were shaken (800 rpm orbital shaker, 30 s),
centrifuged (300 g, 1 min) and incubated at room temperature for
30 min. Luminescence was detected with a CLARIOstar (BMG
Labtech) plate reader. Luminescence signal was normalized with
100 M SAHA treated negative controls and uninhibited positive
controls. pIC50-values were calculated as described in the BRET
assay.
37 °C
overnight.
Fluorescence
was
measured
(extinction/emission: 570 nm/590 nm) on a FluoStar Optima
(BMG Labtech) plate reader. Cell viability was normalized with
untreated positive controls and cell-free negative controls. IC50-
values were calculated as described in the BRET assay.
Expression, Purification, and Crystallization of HDAC10. A
“humanized” version of HDAC10 was designed by making the
A24E and D94A substitutions in Danio rerio (zebrafish) HDAC10
so as to more closely resemble the active site of human HDAC10.
The preparation of this HDAC10 construct using standard PCR
mutagenesis techniques will be described separately; purification
was achieved as described for the wild-type enzyme.[32-33] For
crystallization, the protein solution [10 mg/mL HDAC10, 50 mM
HEPES pH 7.5, 300 mM KCl, 5% glycerol (v/v), and 1 mM tris-(2-
carboxyethyl)phosphine (TCEP) was augmented with 2 mM 3a
and incubated for 1 h on ice. Trypsin was added (1:1000
trypsin:HDAC10) and the mixture was allowed to digest at
ambient temperature for 1 h and then filtered using a 0.22 m
centrifuge filter. Utilizing a Mosquito crystallization robot (TTP
Labtech), a 100 nL drop of protein solution was added to a 100
nL drop of precipitant solution [0.168 M KH2PO4, 0.032 M K2HPO4,
and 20% PEG 3350] and microseeded with crystals of the
HDAC10-Tubastatin A complex. The 200 nL sitting drop was
equilibrated against 80 L of precipitant buffer in the well reservoir
at 4 ºC. Crystals appeared within one day.
Western blot analysis of SMC3, tubulin and histone H3
acetylation via western blot: For the analysis of protein
acetylation, 1.5–2 x 106 SK-N-BE(2)-C cells were seeded per 10
cm dish and allowed to adhere overnight. Cells were treated for
18 h (Figure 4B) or 6 h (Figure 4E) with inhibitors at
concentrations indicated in the respective figure. Cells were lysed
in SDS lysis buffer (2% w/v SDS, 10% v/v glycerol, 1 mM DTT,
62.5 mM TRIS, pH 6.8) and proteins were denatured at 95 °C for
10 min. Cell debris was removed by centrifugation (15,000 x g for
10 min at 12 °C). Western blot analysis was performed as
described before.[7a] The following antibodies were used: anti-
acetylated tubulin (6-11B-1; Sigma) anti-acetylated SMC3 (kindly
provided by Katsuhiko Shirahige, Institute for Molecular and
Cellular Biosciences, University of Tokyo, Japan),[35] anti
acetylated histone H3 (#06-911, Millipore), anti histone H3 (#9715,
Cell Signaling Technology) and anti -actin (#5441, Sigma-
Aldrich).
Crystal Structure Determination of the HDAC10-3a Complex.
X-ray diffraction data for the HDAC10-3a complex were collected
on NE-CAT beamline 24-ID-C at the Advanced Photon Source
(APS), Argonne National Laboratory. Data were indexed using
iMosflm[36] and scaled with Aimless[37] in the CCP4 program
suite.[38] The initial electron density map was phased by molecular
replacement using Phaser;[39] the structure of Y307F HDAC10
(PDB 5TD7)[32] with solvent and ligand atoms removed was used
as a search model. An iterative process of model building using
Coot[40] and crystallographic refinement with Phenix[41] yielded the
final model of the HDAC10-3a complex. The inhibitor was built
LysoTracker assay: SK-N-BE(2)-C cells were seeded into 6-well
dishes at a density of 1.5 x 105 cells per well. Cells were treated
with inhibitor over night at concentrations indicated in the figure
and stained the following day for 1 h with 50 nM LysoTracker®
Red DND-99 in medium under standard cell culture conditions.
10
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10.1002/cmdc.202000149
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into the electron density map during the final stages of refinement.
MolProbity[42] was used to validate the final refined structure. All
data reduction and refinement statistics are recorded in Table S2.
Financial support from the Helmholtz Drug Initiative and the
German Cancer Consortium (DKTK) is gratefully acknowledged.
D.W.C. thanks the NIH for grant GM49758, and N.J.P. received
financial support from the NIH through Chemistry-Biology
Interface Training Grant T32 GM071339. We thank Dr. Karel
Klika and Gabriele Schwebel for NMR spectroscopy support. We
thank Kim Remans and Julia Flock of the EMBL protein
production facility for preparation of recombinant HDAC10 for the
FRET assay. We thank Asat Baischew for the synthesis of 2f.This
work is based upon research conducted at the Northeastern
Collaborative Access Team beamlines, which are funded by the
National Institute of General Medical Sciences from the National
Institutes of Health (P30 GM124165). The Pilatus 6M detector on
beamline 24-ID-C is funded by a NIH-ORIP HEI grant (S10
RR029205). This research used resources of the Advanced
Photon Source, a U.S. Department of Energy (DOE) Office of
Science User Facility operated for the DOE Office of Science by
Argonne National Laboratory under Contract No. DE-AC02-
06CH11357.
Expression, Purification, and Crystallization of HDAC6.
HDAC6 catalytic domain 2 (CD2) from Danio rerio (zebrafish) was
used for the X-ray crystal structure determination of the complex
with 3a. The active sites of human and zebrafish HDAC6 CD2 are
essentially identical, with the exception of N530 and N645 at the
mouth of the active site of zebrafish HDAC6 CD2, which appear
as aspartate and methionine, respectively, in the human
enzyme.[43] The expression and purification of zebrafish HDAC6
CD2 (henceforth simply “HDAC6”) was achieved by modification
of the originally reported preparation as recently described.[44] For
co-crystallization of the HDAC6-3a complex by the sitting drop
method at 4 °C using a Mosquito crystallization robot (TTP
Labtech), a 500-nL drop of protein solution [10 mg/mL HDAC6,
50 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol (v/v), 1 mM
TCEP, and 2 mM 3a] was added to a 500-nL drop of precipitant
solution [0.02 M citrate/0.08 M Bis-Tris propane (pH 8.8) and 24%
w/v PEG 3350] and equilibrated against 80 μL of precipitant
solution in the well reservoir. Crystals appeared within 2 days.
Keywords: HDAC inhibitor, HDAC8, HDAC10,
polypharmacology, targeted therapy
Crystal Structure Determination of the HDAC6-3a Complex.
X-ray diffraction data were collected on NE-CAT beamline 24-ID-
E at APS. Data indexing was achieved with iMosflm[36] and
Aimless[37] was utilized for data scaling, as implemented in the
program suite CCP4.[38] The initial electron density map of the
enzyme-inhibitor complex was phased by molecular replacement
using Phaser[39] with the atomic coordinates of unliganded
HDAC6 CD2 (PDB 5EEM)[43] used as a search model. The
interactive graphics program Coot[40] was used to build and
manipulate the atomic model, and Phenix[41] was used for
crystallographic refinement. The inhibitor was built into the
electron density map during the final stages of refinement.
MolProbity[42] was used to validate the final structure prior to
deposition in the Protein Data Bank. All data reduction and
refinement statistics are recorded in Table S3.
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and coordinated the project. All authors have given approval to
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Entry for the Table of Contents
Cap group
Linker
Me
N
A
H
H
A
N
N
N
A
ZBG
OH
HO
HO
H
N
N
H
O
O
OH
OMe
N
ZBG
ZBG
O
Cap/Linker
ZBG
O
Cap group
Linker
Tubastatin A
selective HDAC6/10inhibitor
PCI-34051
selective HDAC8inhibitor
Hybrid HDAC6/8/10
inhibitor
TOC Text: A new HDAC inhibitor class was developed by hybridizing two structurally related, but functionally distinct, inhibitors: PCI-
34051 (HDAC8 inhibitor) and Tubastatin A (HDAC6/10 inhibitor). The resulting dihydroxamic acids are capable of inhibiting HDACs 6,
8, and 10, and are characterized by a benzyl-indole/indazole scaffold, which functions as both cap and linker group.
Twitter handle: @DrugDiscov_DKFZ
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