Short-chain HDAC inhibitors differentially affect vertebrate development and neuronal chromatin

Article abstract of DOI:10.1021/ml1001954

Carboxylic acids with known central nervous system and histone deacetylase (HDAC) inhibitory activities were converted to hydroxamic acids and tested using a suite of in vitro biochemical assays with recombinant HDAC isoforms, cell based assays in human cervical carcinoma HeLa cells and primary cultures from mouse forebrain, and a whole animal (Xenopus laevis) developmental assay. Relative to the parent carboxylic acids, two of these analogues exhibited enhanced potency, and one analogue showed altered HDAC isoform selectivity and in vivo activity in the Xenopus assay. We discuss potential uses of these novel hydroxamic acids in studies aimed at determining the utility of HDAC inhibitors as memory enhancers and mood stabilizers.

Full text of DOI:10.1021/ml1001954

pubs.acs.org/acsmedchemlett
Short-Chain HDAC Inhibitors Differentially Affect
Vertebrate Development and Neuronal Chromatin
Daniel M. Fass,†, Rishita Shah,‡ Balaram Ghosh,†, Krista Hennig,†, Stephanie Norton,†,
Wen-Ning Zhao,†, Surya A. Reis,† Peter S. Klein,‡ Ralph Mazitschek, Rebecca L. Maglathlin,§
Timothy A. Lewis,§ and Stephen J. Haggarty*,†,
†Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge,
Massachusetts 02142, United States, ‡Department of Medicine, Division of Hematology-Oncology, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania 19104, United States, §Chemical Biology Program, Broad Institute of Harvard
and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142, United States, and Center for Human Genetic Research,
Massachusetts General Hospital, Department of Neurology, Harvard Medical School, Boston,
Massachusetts 02114, United States
ABSTRACT Carboxylic acids with known central nervous system and histone
deacetylase (HDAC) inhibitory activities were converted to hydroxamic acids and
tested using a suite of in vitro biochemical assays with recombinant HDAC
isoforms, cell based assays in human cervical carcinoma HeLa cells and primary
cultures from mouse forebrain, and a whole animal (Xenopus laevis) develop-
mental assay. Relative to the parent carboxylic acids, two of these analogues
exhibited enhanced potency, and one analogue showed altered HDAC isoform
selectivity and in vivo activity in the Xenopus assay. We discuss potential uses of
these novel hydroxamic acids in studies aimed at determining the utility of HDAC
inhibitors as memory enhancers and mood stabilizers.
KEYWORDS HDAC inhibitors, hydroxamic acid, valproate, butyrate, phenylbuty-
rate, histone acetylation
t present there is great optimism for the development
of inhibitors of histone deacetylases (HDACs) as novel
Atherapeutics for memory and mood disorders. Much
of this interest derives from studies reporting the enhance-
ment of memory in rodent behavioral models by the short-
chain carboxylic acid HDAC inhibitorsodium butyrate.1-5 The
butyrate analogue phenylbutyrate, an FDA-approved drug
known to inhibit HDACs,6 enhances cognition in an animal
model of Alzheimer's disease.7 Finally, another short-chain
carboxylic acid, valproate (VPA), originally developed as an
antiepileptic,8 has been shown to inhibit HDAC activity9,10
and is currently also in clinical use as a mood stabilizer for
the treatmentof bipolardisorder.11 However, these three com-
pounds suffer from low potency and, in the case of VPA, a
narrow therapeutic index.12
In an attempt to improve potency, these short-chain car-
boxylic acid HDAC inhibitors were converted to hydroxamic
acid analogues. We reasoned that the carboxylic acid moiety
itself is critical for inhibition of HDACs because the corre-
sponding amide analogue of VPA is inactive.13 Hydroxamic
acid is the zinc-chelating moiety from suberoyl hydroxamic
acid, SAHA (Vorinostat; Merck), a highly potent HDAC in-
hibitor that has been approved by the FDA for the treatment
of cutaneous T-cell lymphoma.14 Other hydroxamic acid HDAC
inhibitors are currently in clinical development, including
Panobinostat (LBH-589; Novartis AG), Belinostat (PDX-10;
TopoTarget), and IF2357 (Italfarmaco SpA). Three hydroxamic
acids (2,15 4,16 617) were made from the corresponding car-
boxylic acids butyrate, phenylbutyrate, and valproate (1, 3, 5)
by esterification with methanol followed by treatment of the
resulting esters with hydroxylamine in methanol under basic
conditions (Scheme 1).
Based on structural homology, zinc-dependent HDAC iso-
forms have been divided into three classes: I (HDACs 1, 2, 3,
and 8); IIa (4, 5, 7, and 9); IIb (6 and 10); and IV (11). To
determine the HDAC inhibitory activity of compounds 1-6,
we used a panel of in vitro enzymatic trypsin-coupled assays
with recombinant human HDACs 1-9. In these assays, HDAC
isoforms were incubated with an acetyl-lysine tripeptide sub-
strate linked to a coumarin fluorophore that is designed to
mimic acetyl-lysine 12 of histone H4. Deacetylation of the
substrate renders it sensitive to trypsin cleavage; this releases
the aminomethylcoumarin (AMC) moiety that has greatly
increased fluorescence in the free form. Critical to these studies
was the use of a newly developed trifluoro-acetyl-lysine-AMC
substrate for class IIa HDACs, which allows the measurement of
Received Date: August 18, 2010
Accepted Date: October 15, 2010
Published on Web Date: November 08, 2010
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the enzymatic activity of these HDACs free from contaminating
activities of class I HDACs that have previously confounded
many studies.18 With the concentrations of these substrates set
to the Km for each enzyme, IC50 values were determined for
each compound (Ta bl e 1).
were similar (Figure 1; data for AcH3 not shown), and reflec-
tive of the in vitro potencies of the compounds toward class
I HDACs. For a representative non-histone substrate, we used
tubulin acetylation as a measure of inhibition of class IIb
HDAC isoforms, as tubulin is deacetylated specifically by
HDAC6.20 In these assays, the hydroxamic acids 2 and 4 were
much more potent at inducing histone acetylation (Figure 1a)
than their parent carboxylic acids. In contrast, VAHA (6) had no
effect on histone acetylation, consistent with its low potency
toward class I HDAC isoforms in vitro. However, 2, 4, and, to a
lesser extent, 6 induced tubulin acetylation, consistent with
their abilities to inhibit HDAC6 in vitro, whereas the carboxylic
acids 1, 3, and 5 had no effect (Figure 1b). Thus, these data
indicate that the hydroxamic acids 2, 4, and 6 are cell active
HDAC inhibitors with altered potency or HDAC target selectivity
that correlates with their in vitro activity.
The data in Table I indicate that carboxylic acids (1, 3, 5)
are selective inhibitors of class I HDACs. Notably, in these new
and improved assays, we found the potency of the inhibitors
to be significantly greater than indicated in previous reports.13
In particular, butyrate(1) was the most potent of the carboxylic
acids. While a previous report indicated that conversion of
1to hydroxamic acid (2) increased its potency toward a mixture
of HDACs isolated from rat liver,23 our data indicate increased
potency toward class II enzymes, but no effect on inhibition of
class I enzymes. The hydroxamic acid analogue of phenyl-
butyrate (4) had greatly enhanced potency toward all enzymes.
In contrast, the hydroxamic acid of VPA (6; which we call
VAHA) had reduced potency toward class I HDACs, and instead
gained potency toward class II enzymes. This discovery is con-
sistent with another recent report describing class IIa selective
hydroxamates.19
We next tested the ability of compounds 1-6 to inhibit
HDAC activity in cells (Figure 1). We used immunofluores-
cence assays to study the effects of these compounds on
histone and non-histone substrates in human HeLa cells.
Specifically, we measured acetylation of the N-terminal tails
of histones H3 and H2A: in both cases, compound effects
Scheme 1a
Figure 1. Induction of histone acetylation (a) or tubulin acetyla-
tion (b) in HeLa cells by carboxylic acid- and hydroxamic acid-
based HDAC inhibitors. Histone H2A and tubulin acetylation were
measured by immunofluorescence assays after 24 h compound
treatments at the indicated doses.
a Reagents and conditions: (a) H2SO4, MeOH, reflux. (b) NH2OH HCl,
NaOMe, MeOH.
3
Table 1. In Vitro Potency of Carboxylic and Hydroxamic Acid HDAC Inhibitorsa
class I
class IIa
HDAC7
class IIb
HDAC6
compd
HDAC1
HDAC2
12 ( 6
HDAC3
HDAC8
HDAC4
HDAC5
HDAC9
1
2
3
4
5
6
16 ( 11
7 ( 9
9 ( 6
15 ( 6
26 ( 20
93 ( 110
4 ( 1
>2000
>2000
>2000
140 ( 70
>2000
170 ( 95
>2000
360 ( 240
>2000
25 ( 13
>2000
37 ( 4
>2000
>1000
>2000
150 ( 27
>2000
99 ( 4
>2000
>2000
>2000
430 ( 304
>2000
91 ( 30
>2000
2 ( 2
9 ( 10
10 ( 11
64 ( 18
0.6 ( 0.4
39 ( 5
65 ( 9
260 ( 160
2 ( 2
240 ( 95
0.5 ( 0.1
>2000
16 ( 1
0.6 ( 0.8
62 ( 4
161 ( 81
340 ( 170
103 ( 8
39 ( 9
560 ( 90
680 ( 280
a IC50 (μM) values for inhibition of enzymatic activityof HDACs 1-9. Data from two experiments (each performed in duplicate) are shown as mean ( SD.
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Figure 2. Induction of developmental defects in frog embryos by VPA and VAHA, and effect of VAHA on tubulin acetylation. (a) Xenopus
developmental assay: left, untreated; center, VAHA (2 mM); right, 2 VPA (2 mM). (b) Induction of tubulin or histone acetylation in frog
embryos by VAHA (6) or VPA (5). Western blot analysis of tubulin or histone acetylation in frog embryos treated at the indicated
concentrations from the completion of neurulation (stage 18) through the tailbud stages (32) for 18 h (tubulin acetylation; as described in
ref 21) or 2 h (histone acetylation).
Clinical use of VPA (5) is tempered by concern over its tera-
togenic effects,21 and the relationship of the isoform specificity
of HDAC inhibitory effects of VPA to teratogenicity remains
to be clarified. Thus, we tested the effects of VPA (5) and VAHA
(6) in a frog embryo-based whole organism development
assay (Figure 2). VPA (5), at 2 mM, induced dramatic devel-
opmental defects in frog embryos, including loss of anterior
structures, shortening of the anterior-posterior axis, and
heart looping and pigment defects, as reported previously.21,22
In contrast, at the same dose, VAHA (6) did not induce these
developmental defects. VAHA (6) did induce tubulin acetyla-
tion, but not histone acetylation (Figure 2b), showing that it
was active and selective in the embryos. These data further
support the conclusion that conversion of VPA (5) to VAHA (6)
results in a novel, class II selective HDAC inhibitor that is active
in cells. Moreover, these data suggest that inhibition of class I
HDACs by VPA (5) underlies its teratogenicity.
Finally, we tested the effects of compounds 1-6 on histone
andtubulinacetylation inprimaryculturesofmouse forebrain
neurons (Figure 3). The forebrain contains several regions
(cortex, hippocampus, striatum) known to play key roles in
mood and memory.24,25 At a concentration of 92.5 μM, the
hydroxamic acid derivatives 2 and 4 induced robust histone
acetylation, whereas the carboxylic acids (1, 3, 5), as well
as VAHA (6), were inactive at this concentration. In contrast,
Figure 3. Induction of histone acetylation (a) and tubulin acetyla-
VAHA (6) was able to induce tubulin acetylation in forebrain
neurons, as did carboxylic acid 3.
tion (b) in mouse primary forebrain cultures. Acetylation of histone
H3, lysine 9 or tubulin was measured by immunofluorescence after
24 h compound treatments (compounds 1-6, 92.5 mM). * signifies
statistical significance (p < 0.05).
In conclusion, here we report the synthesis and activ-
ity testing of novel hydroxamic analogues of short-chain
carboxylic acid HDAC inhibitors. To date, the carboxylic acids
butyrate (1) and phenylbutyrate (3) have been shown to
enhance memory in rodent behavioral models.1-5 We pro-
pose that the hydroxamic acid analogues of butyrate and
phenylbutyrate (2 and 4) might show even greater activity in
these memory models, given their enhanced in vitro potency
and cellular activity (Table 1 and Figure 1 ). The hydroxamic acid
analogue of valproate, VAHA (6), had an in vitro and cellular
activity profile consistent with it being a class II selective inhib-
itor. Interestingly, similar hydroxamic acid analogues of VPA
have been shown to readily cross the blood-brain barrier,22
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and VAHA has been shown to have anticonvulsant activity
in vivo.17 Comparison of the activities of VPA (5) and VAHA (6)
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SUPPORTING INFORMATION AVAILABLE Experimental
procedures for the synthesis of hydroxamic acids 2, 4, and 6, and
procedures for the in vitro HDAC inhibition assays, cellular histone
and tubulin acetylation assays, and Xenopus embryonic develop-
ment assays. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *Tel: 617-643-3201. Fax: 617-643-
3202. E-mail: haggarty@chgr.mgh.harvard.edu.
Funding Sources: This project has been funded in whole or in part
with Federal funds from the National Cancer Institute's Initiative for
Chemical Genetics, NIH, under Contract No. N01-CO-12400. P.S.K. is
supported by a grant from the NIH (1RO1MH58324). S.J.H is sup-
ported by a grant from the NIDA/NIH (5R01DA028301-02) and the
Stanley Medical Research Institute.
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