Dissecting structure-activity-relationships of crebinostat: Brain penetrant HDAC inhibitors for neuroepigenetic regulation

Article abstract of DOI:10.1016/j.bmcl.2016.01.022

Targeting chromatin-mediated epigenetic regulation has emerged as a potential avenue for developing novel therapeutics for a wide range of central nervous system disorders, including cognitive disorders and depression. Histone deacetylase (HDAC) inhibitor

Full text of DOI:10.1016/j.bmcl.2016.01.022

Accepted Manuscript
Dissecting Structure-Activity-Relationships of Crebinostat: Brain Penetrant
HDAC Inhibitors for Neuroepigenetic Regulation
Balaram Ghosh, Wen-Ning Zhao, Surya A. Reis, Debasis Patnaik, Daniel M.
Fass, Li-Huei Tsai, Ralph Mazitschek, Stephen J. Haggarty
PII:
S0960-894X(16)30022-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.01.022
BMCL 23487
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
23 November 2015
7 January 2016
8 January 2016
Please cite this article as: Ghosh, B., Zhao, W-N., Reis, S.A., Patnaik, D., Fass, D.M., Tsai, L-H., Mazitschek, R.,
Haggarty, S.J., Dissecting Structure-Activity-Relationships of Crebinostat: Brain Penetrant HDAC Inhibitors for
Neuroepigenetic Regulation, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/
j.bmcl.2016.01.022
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Title: Dissecting Structure-Activity-Relationships of Crebinostat: Brain Penetrant HDAC
Inhibitors for Neuroepigenetic Regulation
Authors:
Balaram Ghosh*,a,b,^, Wen-Ning Zhao*,a,b, Surya A. Reisa,b, Debasis
Patnaika,b, Daniel M. Fassa,b, Li-Huei Tsaic, Ralph Mazitschekd, Stephen J. Haggartya,b,#
*These authors contributed equally
#Correspondence: Stephen J. Haggarty (shaggarty@mgh.harvard.edu)
Addresses:
a. Chemical Neurobiology Laboratory, Center for Human Genetic Research,
Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114,
USA
b. Departments of Psychiatry & Neurology, Massachusetts General Hospital &
Harvard Medical School, Boston, MA 02114, USA
c. Picower Institute for Learning and Memory, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, USA
d. Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge
Street, Boston, Massachusetts 02114, USA
^ Present Address: Department of Pharmacy, Birla Institute of Technology and
Sciences at Hyderabad Campus, India 500 078
ABSTRACT: Targeting chromatin-mediated epigenetic regulation has emerged as a
potential avenue for developing novel therapeutics for a wide range of central nervous
system disorders, including cognitive disorders and depression. Histone deacetylase
(HDAC) inhibitors have been pursued as cognitive enhancers that impact the regulation
of gene expression and other mechanisms integral to neuroplasticity. Through
systematic modification of the structure of crebinostat, a previously discovered cognitive
enhancer that affects genes critical to memory and enhances synaptogenesis,
combined with biochemical and neuronal cell-based screening, we identified a novel
hydroxamate-based HDAC inhibitor, here named neurinostat, with increased potency
compared to crebinostat in inducing neuronal histone acetylation. In addition,
neurinostat was found to have a pharmacokinetic profile in mouse brain modestly
improved over that of crebinostat. This discovery of neurinostat and demonstration of its
effects on neuronal HDACs adds to the available pharmacological toolkit for dissecting
the molecular and cellular mechanisms of neuroepigenetic regulation in health and
disease.
KEYWORDS Cognitive enhancer, nootropic, histone deacetylases, epigenetic,
chromatin, acetylation, CREB, neuroepigenetic
1

Chromatin-mediated epigenetic regulation of gene expression is an integral part
of neuroplasticity [1], dysregulation of which is associated with aging and a broad range
of central nervous system (CNS) disorders, including Alzheimer’s disease (AD) [2], and
mood disorders [3, 4]. Inhibition of histone deacetylases (HDACs) has emerged as a
promising new therapeutic avenue for CNS disorders [5, 6]. As summarized in Figure 1,
there are three main chemical structure classes of HDAC inhibitors that have been
developed as pharmacological probes with more recent use in the context of CNS
disorders: 1) carboxylic acids (e.g., butyric acid, valproic acid); 2) hydroxamic acids
(e.g., suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA)); and 3) ortho-amino
anilides (e.g., RGFP136, CI-994). All three classes of HDAC inhibitors have been
shown to enhance learning and memory in normal, aged, or age-associated
neurodegenerative animal models, and also to increase histone acetylation and change
gene expression related to neuroplasticity in relevant brain regions [7-10]. An anti-
depressant-like effect has also been shown in preclinical settings in rodent models for
several HDAC inhibitors including sodium butyrate [11, 12], SAHA and MS-275 [13], a
HDAC1/2-selective inhibitor Cpd-60 [14], and HDAC6-selective inhibitors ACY-738 and
ACY-775 [15].
Hydroxamic Acids
Carboxylic Acids
Ortho-amino Anilides
O
H
O
O
O
N
OH
NH
F
OH
N
O
H
NH2
N
trichostatin A
butyric acid
RGFP136
O
O
O
H
N
OH
N
O
NH
OH
H
O
NH2
N
H
valproic acid
SAHA
CI-994
Figure 1. Three main chemical structure classes of HDAC inhibitors under
investigation in the context of central nervous system disorders.
While accumulating evidence indicates certain HDACs facilitate, while others
play a suppressive role, in cognitive processes [2, 4, 5], the understanding of the
precise relationship between HDAC subtype activity, critical epigenetic substrates, the
activity of specific HDAC complexes, and therapeutically beneficial effects relevant to
neurodegenerative and neuropsychiatric disorders remains incomplete. Thus, the
development of potent and subtype-selective HDAC inhibitors that are functionally
2

active toward relevant HDAC complexes and that can achieve sufficient brain exposure
to be biochemically and behaviorally active is required [2, 16-18]. Toward this goal, by
screening for enhancers of CREB-mediated transcription and modulators of chromatin-
mediated neuroplasticity, our previous studies identified a potent hydroxamate HDAC
inhibitor, crebinostat, which produced robust enhancement of cognition in mice [19].
Here, through systematic dissection of the structure-activity-relationship (SAR) of
crebinostat, we identified an improved analog of crebinostat with enhanced cellular
activity in inducing histone acetylation in mouse neuronal cultures and a modestly
improved in vivo pharmacokinetic (PK) profile in mice.
Structural studies of hydroxamate HDAC inhibitors such as SAHA and TSA have
revealed three key features: a surface-recognition cap region that interacts with the
HDAC enzyme surface adjacent to the catalytic site; a linker region that extends into the
catalytic site; and a terminal metal-chelating hydroxamic acid moiety that interacts with
the catalytic site zinc ion [20, 21] (Figure 2A). The cognitive enhancer crebinostat,
discovered by a high-throughput, cell-based screen of small molecule libraries, shares
this “cap-linker-zinc chelator” pharmacophore model [16] (Figure 2A). Structurally,
although both are hydroxamic acids, crebinostat differs from SAHA in the cap moiety
and length of the aliphatic linker. Functionally, crebinostat demonstrated improved
activities over SAHA in in vitro assays and in neuronal cellular assays of histone
acetylation [19].
Given interest in identifying novel HDAC inhibitors with improved potency, HDAC
subtype selectivity and suitable brain PK properties, we synthesized a series of
crebinostat derivatives. Crebinostat contains a 5-methylene chain linker while SAHA
has a 6-methylene linker. The surface-recognition cap moieties are also different for
crebinostat and SAHA. To dissect the structural composition that contributes to the
advantageous properties in crebinostat, we first assessed the effect of linker length by
synthesizing crebinostat analogs with 4-6 carbon aliphatic chain linkers (Figure 2B).
This range of linker lengths was considered appropriate to explore considering the 5-
and 6-methylene linker distance in the prototype hydroxamic inhibitors TSA and SAHA
(Figure 1), the distance between cap and catalytic site observed in co-crystal structures
of HDACs and inhibitors, and based on previous reports in the literature [22, 23]
Synthesis of Compounds 9a, 9b and 9c
Chemical Schemes 1-3 describe the procedures used to synthesize three
compounds (9a, 9b, 9c) with 4, 5, or 6-methylene linker length respectively, but with the
same biphenyl cap and hydroxamate zinc-binding motifs. In order to obtain 9a, 9b, 9c,
we synthesized intermediate linker compounds (3a, 3b, 7) terminated by a hydrazide on
one side and a hydroxamic acid on the other side (Schemes 1, 2). Acyl hydrazones 9a,
9b and 9c were synthesized according to Scheme 3 by difluoroacetic acid catalyzed
3

condensation of the biphenyl aldehyde (8) with hydrazides 3a, 3b and 7, respectively.
Compound 9b corresponds to the previously characterized crebinostat. The
physicochemical properties of synthesized compounds are shown in Figure 2B and
Supplemental Table S1. Analytical LC-MS spectra for these compounds are included
in Supplemental Figure S1.
A
B
Figure 2. Novel HDAC inhibitors with 4-, 5-, and 6-methylene linkers. (A) Potent
HDAC inhibitors SAHA and crebinostat possess three structural components: surface
recognition cap, linker, and a divalent metal (Zn2+) binding element. (B) Novel HDAC
inhibitors with 4-, 5-, and 6-methylene linkers and their physicochemical properties.
tPSA: topological polar surface area. cLogP: calculated LogP.
4

Optimal Linker Length for HDAC Inhibitory Activities in Biochemical Assays
After having synthesized compounds 9a, 9b (crebinostat), and 9c, which share
the same cap and zinc-binding elements, and differ in the linker length, we first
determined the ability of these compounds to inhibit the enzymatic activity of HDAC1, 2
or 3. The inhibitory activities of compounds 9a, 9b, and 9c were measured in dose
responses with the IC50 values reported in Figure 3A. Consistent with our previous
report [19], crebinostat (9b) was a more potent inhibitor than SAHA of HDAC1, 2 or 3,
with more than a 5-fold reduction in its IC50. 9c exhibited further reduced IC50’s; its IC50
values (0.7, 1.0 and 2.0 nM for HDAC1-3 respectively) are half of those measured for
crebinostat (9b). Apparently, the 4-methylene linker conferred a disadvantage to the
inhibitory activity; 9a exhibited an approximately 20-fold increase in IC50’s over those of
crebinostat (9b). Presumably, the cap to chelator distance produced by the 4-methylene
linker precluded correct positioning of the surface-recognition and zinc-interacting
elements. These data, though no magnitude difference of IC50 values between 9c and
crebinostat (9b), indicate that the 6-methylene linker length is optimal among the three
compounds, with ~2 fold improved potency for inhibiting HDAC1-3 activities in the
biochemical reaction. Previous work examining linker length of benzothiazole-containing
analogs of SAHA reported that a 6-carbon linker showed better inhibition of HDAC3 and
HDAC4 than a 4-carbon linker [22]. Studies involving thirty-six acylurea connected
aliphatic linker hydroxamates identified close analogs of SAHA with 5-methylene linkers
to be more potent than SAHA against HDAC1 or HDAC6 [23].
5

Figure 3. In vitro and ex vivo HDAC inhibitory activities of novel HDAC inhibitors
with 4-, 5-, and 6-methylene linkers. (A) In vitro IC50 (nM) with recombinant human
HDAC1-3. (B) Increased acetylation of histone H3K9 and H4K12 in mouse forebrain
primary neuronal culture (DIV14) detected at both 10 M and 1 M (24 hrs). Cellular
activity of SAHA at 10 M was set to 100% as a reference. N=3. Error bars are SEM.
For all three compounds, we did not observe significant isoform specific activities
between HDAC1-3; inhibitory activities for HDAC1-3 were in the same magnitude for
each compound albeit inhibitory activities for HDAC1 were about half of inhibitory
activities for HDAC2, which were about half of inhibitory activities for HDAC3. Therefore,
changing the linker length did not lead to HDAC1, 2 or 3 isoform selective inhibitory
activities in this structural series.
Optimal Linker Length for HDAC Inhibitory Activities in Neuronal Assays
Given interest in developing novel HDAC inhibitors as therapeutics of CNS
disorders, we examined cellular activities of our novel hydroxamate HDAC inhibitors in
induction of neuronal histone acetylation [19, 24, 25]. After 24 hour compound
treatments, neuronal cultures were fixed and immunostained for histone acetylation
marks H3K9ac and H4K12ac. The acetylation levels of H3K9 and H4K12 have been
implicated in cognition enhancement in normal and aged mice [8, 26, 27]. Inhibition of
HDAC activities by inhibitors leads to elevated H3K9ac and H4K12ac fluorescence
intensities in neuronal nuclei. To assess the HDAC inhibitory activities in neurons, we
tested 9a, 9b (crebinostat) and 9c, as well as SAHA, at two concentrations: 1 and 10
6

M, and their cellular activities are reported relative to those of SAHA (Figure 3B).
Crebinostat (9b) and 9c treatment resulted in greater increases in both H3K9ac and
H4K12ac than SAHA at both 1 M and 10 M. Despite the higher potencies measured
for 9c containing the 6-methylene linker in the in vitro HDAC enzymatic assays
described in the previous section, the cellular activities of 9b (crebinostat) were
modestly greater than those of 9c, suggesting that the 5-methylene linker may make
hydroxamate HDAC inhibitors with biphenyl moieties more accessible to endogenous
HDAC complexes.
In vitro enzymatic assays assessed HDAC inhibitory activities of the reported
compounds by direct enzyme engagement whereas the ex vivo, neuron-based
functional assays enabled us to evaluate these compounds for additional properties
such as cell membrane permeability and target accessibility. The degree of
hydrophilicity/hydrophobicity and the size of small molecules can affect their diffusion
into intact cells through membrane penetrance as well as their ability to reach target
enzymes existing in multi-protein complexes. Crebinostat (9b) and 9c have the same
topological polar surface area (tPSA), and crebinostat (9b) has slightly lower calculated
LogP than 9c (Figure 2B), the latter of which may give crebinostat (9b) better cell
membrane permeability, and therefore better cellular activity. Our results presented a
case where 9c, containing a 6-methylene linker, was modestly more potent than
crebinostat (9b), containing a 5-methylene linker, in the biochemical assay, but less
potent in the cell based assay. Given the necessity for HDAC inhibitors to have strong
intracellular activity in order to be effective when used in vivo, and that crebinostat (9b)
is well characterized, the 5-methylene linker was chosen for further study of the
structure-activity relationship of the crebinostat series.
All of our novel compounds are hydrazone derivatives, which are known to be
labile in the cellular environment at lower pH [28]. To ensure that the cellular activities of
induction of neuronal histone acetylation that we observed were from the intact
molecule, but not from the fragmented linker with zinc-chelating moiety, we also tested
the intermediate linker compounds 3a, 3b, and 7 in the neuronal histone acetylation
assay (Supplemental Figure S2). No induction of histone acetylation of H3K9 or
H4K12 was detected for 3a, 3b, and 7, indicating that the observed functional activities
for 9a-c were from the intact synthesized compounds. As these linker-only compounds
also contain hydroxamates that may chelate divalent metal ions, this also indicates that
the presence of a chelating moiety alone on a linker is insufficient to cause a
measurable change in histone acetylation.
Synthesis of Compounds 9d-9k
7

After evaluation of the effect of aliphatic linker length on HDAC inhibitory
activities, and having identified the 5-methylene linker as most promising for further
SAR studies, we proceeded to varying the surface-recognition caps for potentially
improved potency or HDAC subtype selectivity. The biphenyl cap moiety in crebinostat
(9b) was replaced with eight different substituted and hetero aromatic moieties (Table
1) that cover a wide range of calculated LogP from 1.26 to 4.06, as LogP is one of the
key factors that determine the ability of small molecules to penetrate the cell membrane.
Scheme 4 was followed to synthesize eight compounds (9d-9k) using the same
strategy as in Scheme 3, in which various aldehydes were coupled with hydrazide 3b.
Physicochemical properties of synthesized compounds are reported in Supplemental
Tables S1 and S2, and analytical LC-MS spectra for key compounds are shown in
Supplemental Figure S1.
Compound 9f Surpasses 9b (crebinostat) in Induction of Neuronal Histone Acetylation
All of the synthesized final analog compounds were tested in the in vitro HDAC1-
3 inhibition assays and the results showed that most were potent with low nanomolar
IC50 values (Table 1). All analogs except 9k maintained single-digit IC50’s against
HDAC1 and 3, around the same magnitude as crebinostat (9b), while four out of seven
of these analogs had decreased potency against HDAC2 to double-digit IC50’s.
Compounds 9f and 9j surpassed crebinostat (9b) in inhibiting HDAC1 and 3, but both
had decreased potency against HDAC2. Changing the terminal phenyl group of the
biphenyl moiety of crebinostat structure to a pyridinyl group fine tuned HDAC inhibitory
activities (9e, 9f, 9g and 9h). The pyridinyl nitrogen at meta position (9f) improved
potency for HDAC1 and 3 inhibition. The result of 9j having improved potency against
HDAC1 and 3, and comparable potency against HDAC2 is intriguing in that 9j contains
2-bromo-5-methoxy benzylidene group as a cap element, one of three cap moieties in
the series least similar to the biphenyl group of crebinostat. Also interesting is that 9i,
which has a thiophenyl phenyl group in the cap region, scored as a potent HDAC
inhibitor as well. The least favorable substitution occurred in 9k, with a dimethoxyphenol
moiety replacing the biphenyl group, resulting in dropped potency to double-digit IC50’s
for HDACs 1 and 3, and a three-digit IC50 for HDAC2.
8

Table 1. In vitro HDAC inhibitory activities of crebinostat analogs.
IC50 values (nM) ± SD
n
tPSA cLogP
Compound
SAHA
Ar
HDAC1
HDAC2
HDAC3
7.0 ± 1.10
10.2 ± 1.10 21.0 ± 2.00
9b
5
5
90.79
90.79
3.58
4.06
1.4 ± 0.05
2.1 ± 0.08
3.4 ± 0.07
5.4 ± 0.07
(crebinostat)
9d
9e
9f
8.0 ± 1.01
2.3 ± 0.05
1.1 ± 0.01
18.8 ± 2.06
5
5
5
5
103.15
103.15
103.15
115.51
2.66
2.24
2.24
1.64
6.9 ± 1.03
10.8 ± 2.03
14.2 ± 1.12
21.0 ± 3.2
1.4 ± 0.03
0.8 ± 0.01
1.8 ± 0.02
2.4 ± 0.04
9g
9h
3.1 ± 0.02
3.2 ± 0.06
9i
9j
5
5
90.79
3.56
2.06
2.1 ± 0.03
0.6 ± 0.01
6.3 ± 0.08
5.0 ± 0.61
1.6 ± 0.02
1.2 ± 0.03
100.02
9k
5
129.48
1.26
31.4 ± 3.21 108.0 ± 8.07 25.6 ± 2.33
Figure 4. Induction of histone H3K9 and H4K12 acetylation in mouse forebrain
primary neuronal cultures by crebinostat analogs. Neurons (DIV13) were treated at
9

the indicated concentration (n=3) for 24 hrs and then imaged after immunofluorescence-
based detection. Error bars are SEM.
After evaluating HDAC inhibitory activities of cap-modified compounds in
enzymatic assays, we again assessed their cellular activities of induction of neuronal
histone acetylation using the ex vivo primary neuronal culture assay. As shown in
Figure 4, all compounds except 9k showed cellular activities comparable to SAHA, with
several exceeding SAHA’s activities, at both concentrations (with the exception of 9d
which had low H3K9ac activity). 9k was the least potent in the neuronal cell assay,
consistent with its relative low biochemical activities for HDAC1-3. While all three
compounds (crebinostat (9b), 9f, 9j) were more effective in increasing both H3K9ac and
H4K12ac than SAHA, 9f was found to be the most potent. The activity differences
between 9f and crebinostat (9b) are statistically significant for H3K9ac at 1 M and for
H4K12ac at both 1 and 10 M (n= 3, unpaired t-test, p<0.05). Based on these neuronal
cell-based assay data, demonstrating 9f as the most improved analog of crebinostat
(9b) identified in our series, 9f became the focus of additional studies, and we named it
neurinostat.
To better evaluate the cellular potency of neurinostat (9f), we determined its EC50
by treating neuronal cultures in a 10-point dose response (1 nM - 5 M) and measuring
the acetylation level of H3K9. Figure 5 shows that neurinostat (9f) was modestly more
potent than SAHA and crebinostat (9b) in induction of neuronal histone acetylation; it
had an EC50 of 140 nM, whereas crebinostat (9b) and SAHA had EC50s of 210 nM and
950 nM, respectively. The difference in the bioactivities of neurinostat (9f) versus
crebinostat (9b) is statistically significant (comparison of 10-point dose curves, exact
sum-of-squares F test, p=0.0006). These data indicate that the pyridinyl nitrogen at
meta position in the terminal aromatic ring enhances HDAC inhibitory activities in
neurons.
Figure 5. Dose response profiles of histone H3K9 acetylation in mouse forebrain
primary neuronal cultures. Neurons (DIV13) were treated at the indicated
concentrations (n=2) for 24 hrs and then imaged after immunofluorescence-based
detection. Error bars are SEM.
10

To comprehensively assess inhibitory activities of neurinostat (9f) toward
different class categories of HDACs, we tested neurinostat (9f) in HDAC5 (class IIa) and
HDAC6 (class IIb) in vitro assays. For both neurinostat (9f) and crebinostat (9b), no
activity was observed toward HDAC5, whereas IC50 values were obtained as 0.5 nM
and 0.6 nM, respectively, toward HDAC6 (Supplemental Figure S3). Collectively, our
in vitro enzymatic assay data suggest that neurinostat (9f) and crebinostat (9b) have
similar HDAC subtype selectivity, and that further SAR studies will be needed to yield
any subtype selectivity within this chemical series.
Neurinostat (9f) Possesses Modestly Improved Pharmacokinetic Profiles and Brain
Penetration
Given our intention to develop neurinostat (9f) as a cognitive enhancer, we
proceeded to evaluate its brain PK properties. Neurinostat (9f) was administered
intraperitoneally at 25 mg/kg to mice, and its concentration in the brain was monitored
over a 6-point time course by collecting brains from three mice at each time point and
measuring neurinostat (9f) concentrations in the processed brain samples. As shown in
Figure 6 and Supplemental Table S3, neurinostat (9f) reached peak concentration
(Cmax) of 79 nM at one hour after systemic administration of the compound. This Cmax
exceeds the IC50 concentrations measured for HDAC1-3 in biochemical assays and at
the comparable level of the EC50 concentration measured for H3K9ac in cultured mouse
neurons. These data indicate that potentially functionally relevant levels of neurinostat
(9f) were achieved in the brain after its systemic administration at 25 mg/kg. Though
neurinostat’s Cmax was higher than that of crebinostat (9b) administered at the same
dose, previously reported to be 60 nM [19], their PK profiles are similar.
Figure 6. Mouse brain pharmacokinetics of compound neurinostat (9f). Neurinostat
(9f) and Crebinostat (9b) (as published previously [19]) were injected intraperitoneally at
25 mg/kg. N=3 for each time point. Error bars are SEM.
11

In addition to brain tissue, peripheral blood samples were also collected at each
time point and analyzed for neurinostat (9f) concentration (Supplemental Tables S4
and S5). Around one hour after injection, neurinostat (9f) reached a maximum
concentration of 1.9 M with a half life of 1.28 hours. Individual brain-to-plasma
concentration ratios were between 0.03-0.07 from 0.5 to 2 hours (Supplemental Table
S6). At time points 0.5, 1 and 2 hours, the brain-to-plasma ratios of neurinostat (9f)
were about twice of those measured for crebinostat (9b), indicating that neurinostat (9f)
was distributed to the brain more effectively than crebinostat (9b). The overall low ratios
of brain-to-plasma concentrations suggest brain-penetrant properties of neurinostat (9f)
and crebinostat (9b) require further improvement.
In pursuit of a better understanding of the role of HDACs in chromatin-mediated
neuroplasticity and improved CNS disease therapeutics, we systematically dissected
the SAR of the hydroxamate-based cognitive enhancer HDAC inhibitor crebinostat (9b),
and optimized its linker length and surface recognition cap moieties. These SAR studies
identified novel HDAC inhibitors that possess improved potency in HDAC inhibition,
especially in functional neurons. The discovery of the neurinostat (9f), which exhibited
nanomolar IC50 in HDAC1-3 inhibition, a lower EC50 for induction of neuronal histone
acetylation than those of crebinostat (9b) and SAHA, and modestly improved brain PK
properties compared to crebinostat (9b) provides a new chemical tool to dissect the role
of neuroepigenetic mechanisms in regulating neuroplasticity in health and in disease.
Acknowledgements
We would like to thank Dr. Florence F. Wagner for helpful discussions and critical
reading of the manuscript. S.J.H. was supported through funding from the NIH
(R01DA028301, R01DA030321), and the Tau Consortium. R.M. was supported through
funding from the NIH (P50CA086355).
Disclosure Statement
R.M. has financial interests in SHAPE Pharmaceuticals and Acetylon Pharmaceuticals.
He is also the inventor on IP licensed to these two entities that is unrelated to this study.
S.J.H. has financial interest in Rodin Therapeutics and is an inventor on IP licensed to
this entity that is unrelated to this study.
12

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