Evaluation of 5-(Trifluoromethyl)-1,2,4-oxadiazole-Based Class IIa HDAC Inhibitors for Huntington's Disease

Article abstract of DOI:10.1021/acsmedchemlett.0c00532

Using an iterative structure-activity relationship driven approach, we identified a CNS-penetrant 5-(trifluoromethyl)-1,2,4-oxadiazole (TFMO, 12) with a pharmacokinetic profile suitable for probing class IIa histone deacetylase (HDAC) inhibition in vivo.

Full text of DOI:10.1021/acsmedchemlett.0c00532

pubs.acs.org/acsmedchemlett
Letter
Evaluation of 5‑(Trifluoromethyl)-1,2,4-oxadiazole-Based Class IIa
HDAC Inhibitors for Huntington’s Disease
Andrew J. Stott, Michel C. Maillard,* Vahri Beaumont, David Allcock, Omar Aziz,
Alexander H. Borchers, Wesley Blackaby, Perla Breccia, Gillian Creighton-Gutteridge, Alan F. Haughan,
Rebecca E. Jarvis, Christopher A. Luckhurst, Kim L. Matthews, George McAllister, Scott Pollack,
̈
Elizabeth Saville-Stones, Amanda J. Van de Poel, Huw D. Vater, Julie Vann, Rachel Williams,
́
̃
Dawn Yates, Ignacio Munoz-Sanjuan, and Celia Dominguez
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ABSTRACT: Using an iterative structure−activity relationship driven approach, we identified a CNS-penetrant 5-(trifluoromethyl)-
1,2,4-oxadiazole (TFMO, 12) with a pharmacokinetic profile suitable for probing class IIa histone deacetylase (HDAC) inhibition in
vivo. Given the lack of understanding of endogenous class IIa HDAC substrates, we developed a surrogate readout to measure
compound effects in vivo, by exploiting the >100-fold selectivity compound 12 exhibits over class I/IIb HDACs. We achieved
adequate brain exposure with compound 12 in mice to estimate a class I/IIb deacetylation EC50, using class I substrate H4K12
acetylation and global acetylation levels as a pharmacodynamic readout. We observed excellent correlation between the compound
12 in vivo pharmacodynamic response and in vitro class I/IIb cellular activity. Applying the same relationship to class IIa HDAC
inhibition, we estimated the compound 12 dose required to inhibit class IIa HDAC activity, for use in preclinical models of
Huntington’s disease.
KEYWORDS: Class IIa HDAC inhibitors, trifluoromethyloxadiazole, CNS exposure, Huntington’s disease, CHDI-00484077
he histone deacetylase (HDAC) protein family (HDACs
1−11 and class III sirtuins 1−7) is involved in
deacetylation activity of class IIa enzymes is intrinsically
much weaker (∼1000-fold lower).2 No natural substrate of
class IIa enzymes has been conclusively identified that can be
ascribed purely to their catalytic activity rather than to their
class I/III HDAC binding partners.2−4 This questions the class
IIa HDACs’ bona fide deacetylase activity; the “catalytic
domain” may serve instead as a recognition domain for binding
to N-ε-acetyl lysine residues of proteins to orchestrate
T
transcriptional repression and chromatin condensation mech-
anisms, arising from the removal of acetyl groups from
acetylated ε-amino groups of lysine side chains present in
histones and other nonhistone proteins.1 The HDAC family is
comprised of 11 zinc-dependent enzymes grouped into three
classes (classes I, II, and IV). Class I enzymes (HDACs 1−3,
8) are structurally differentiated from the class II subfamily and
are considered “prototypical”, in that they exhibit efficient
deacetylase activity. HDAC8 has atypical catalytic activity,
resembling class IIa enzymes in substrate recognition.1 Class II
HDACs can be further subdivided into class IIa (HDACs 4, 5,
7, and 9) and class IIb (HDACs 6 and 10), with the latter
exhibiting catalytic deacetylation comparable to that of class I
enzymes. Compared to class I HDACs, the catalytic
Received: October 12, 2020
Accepted: February 3, 2021
Published: February 11, 2021
https://dx.doi.org/10.1021/acsmedchemlett.0c00532
© 2021 American Chemical Society
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protein−protein interactions and macromolecular protein
signaling.5,6 Class IIa HDACs have been implicated in
numerous diseases, but of interest to our group is the role
they play in Huntington’s disease (HD). Broad-spectrum/
Class I/IIb HDAC inhibitors are reported to partially improve
HD model phenotypes,7−10 but in some instances beneficial
effects are offset by toxicity following chronic dosing
paradigms.9 Our interest in selectively targeting Class IIa
HDACs arose from a report that HDAC4 genetic suppression
in the R6/2 mouse model of HD leads to amelioration of
neurological phenotypes and extended lifespan,11 although the
underlying mechanism for this improvement was unclear, given
the presumed independence on deacetylase activity and the
predominant cytoplasmic HDAC4 localization in the brain.12
To date, this beneficial effect appears selective to HDAC4
knockdown, as the use of genetic suppression to query
involvement of some other HDAC enzymes (HDAC3,
HDAC6, or the Class IIa HDAC7) has shown neither
beneficial nor detrimental effects in HD mouse models.13−15
Replication of beneficial effects in HD via occupancy of the
class IIa HDAC4 catalytic domain would provide confidence
for small molecule intervention in treating the human disease.
Here, we have focused on the identification of brain-penetrant
chemotype (Vorinostat, Belinostat, and Panobinostat) and
the fourth (Romidepsin) being a natural product.
Previously, we reported our research efforts toward
hydroxamic acid-based HDAC4 inhibitors including benzhydr-
yl analogues and novel cyclopropanes.17−19 The bidentate
interaction formed between the hydroxamic acid and the active
site zinc is critical from a potency standpoint; however, rapid
clearance arising from glucuronidation and hydrolysis, coupled
with low oral bioavailability, is an issue that needed to be
addressed in order to achieve adequate exposure in vivo.20
Toward this goal, we were intrigued by reports of novel 5-
(trifluoromethyl)-1,2,4-oxadiazole (TFMO) moieties for tar-
geting class IIa HDACs.21,22 The TFMO ring system in
compound 1 (Figure 1) was postulated to adopt a non-
chelating interaction with the active-site zinc, providing distinct
structural differentiation from hydroxamic acid-based HDAC
inhibitors.21 For targeting the central nervous system (CNS),
we anticipated that removal of two H-bond donors present in
the hydroxamic acid moiety would be beneficial for
physicochemical properties required for brain penetration
and provide compounds with superior pharmacokinetics.23
Herein, we describe our efforts to develop a series of TFMO-
based class IIa HDAC inhibitors and identify a molecule with a
suitable PK profile for evaluation in efficacy and proof-of-
concept studies in disease models where deregulation of class
IIa HDAC biology has been implicated.
TFMO-containing benzamides such as 1 and 2 have been
reported (Figure 1).24 We profiled compound 2 and confirmed
it was a potent HDAC4 inhibitor with good selectivity over
class I/IIb HDAC isoforms, exhibiting 150-fold selectivity for
class IIa over class I/IIb HDACs in cells (IC50 value of 0.02
μM vs 3.0 μM) (Table 1). The HDAC biochemical and Jurkat
E6.1 cell assays used to profile compound 2 have been
described previously,25 employing artificial substrates Boc-Lys-
(TFA)-AMC (class IIa/HDAC8-specific) and Boc-Lys-(Ac)-
AMC (class I/IIb-specific); also see Supporting Information
(SI). 2 also demonstrates high kinetic solubility, negligible P-
gp efflux, high permeability, but poor metabolic stability in
mouse liver microsomes (Table 1).
Figure 1. Examples of HDAC inhibitors containing the TFMO group.
small molecules targeting HDAC4 as a representative isoform
of the class IIa subfamily.
Class I and II HDAC inhibitors can be grouped into distinct
structural classes, including hydroxamic acids, carboxylic acids,
benzamides, electrophilic ketones, and cyclic peptides.16
Hydroxamic acids arguably represent the most studied
structural class of HDAC inhibitors, with three of four FDA-
approved (anticancer) HDAC inhibitors containing this
Surface plasmon resonance (SPR) experiments confirmed
binding of compound 2 to immobilized HDAC4 catalytic
domain with a KD(ss) of 0.044 μM and a short residence time
(tR) of 2.5 min (SI Table 1). To assess the importance of the
TFMO moiety for HDAC4 inhibition, close analogues (3−6)
a
Table 1. Compounds 2 and 7 HDAC Isoform Biochemical and Cellular Activity Data
Cellular
HDAC
activity Lys
d
Biochemical class IIa (catalytic Biochemical class I HDAC Class IIb HDAC
(substrate)
IC50 (μM)
MDCK-
f
domain) HDAC IC50 (μM)
IC50 (μM)
IC50 (μM)
MDR1
Kinetic
solubility
(μM)
MLM Clint
e
Papp
EER (nm/s)
b
c
Cpd
4
5
7
9
1
2
3
8
6
TFA
Ac
(mL/min/kg)
2
7
0.003
0.03
0.01
0.09
0.01
0.14
0.01
0.07
6.0
13
9.3
26
2.5
5.1
2.9
2.8
6.4
9.8
0.02
0.25
3.0
3.7
834
76
1.3
1.2
541
604
140
169
a
b
c
Geometric mean of three experiments as described previously;25 standard deviations are <25% of the mean. HDAC3-NCoR1. Determined
utilizing HDAC6 overexpression in HEK cell lysate (Supporting Information). Assay−substrate combinations are described in SI Table 4,
d
Supporting Information. TFA: the Boc-Lys-(TFA)-AMC substrate is specific to class IIa HDACs and HDAC8, with the majority of class IIa
e
HDAC activity in the Jurkat E6.1 cell line derived from HDAC4.19 Ac: the Boc-Lys-(Ac)-AMC substrate is specific to class I/IIb HDACs. Intrinsic
clearance (Clint) values of >257 mL/min/kg in mouse liver microsomes (MLM) indicate a rapid rate of oxidative metabolism by CYP450 enzymes
f
under the assay conditions. A Clint value of <65 mL/min/kg indicates a low rate of metabolism. EER is defined as the efflux ratio in MDCK-MDR1
cells/efflux ratio in MDCK wild-type cells with the efflux ratio being Papp(B to A)/Papp(A to B).
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The highly electron-deficient TFMO ring has been reported
to behave as a 1,3-dielectrophilic substrate susceptible to
nucleophilic attack.26 In order to assess the chemical stability
of this zinc-binding moiety in solution, compound 2 was
Table 2. TFMO Ring Modifications and the Effect on Class
IIa Biochemical and Cellular Activity
1
dissolved in DMSO-d6 and monitored by H NMR spectros-
copy over 9 days at room temperature, with no apparent
degradation observed after this time.
To address the poor metabolic stability observed for
compound 2, we focused our efforts on modifications to the
lipophilic basic amine region; a potential site for oxidative
metabolism. Introduction of a terminal pyrrolidine group24 (7)
reduced overall lipophilicity (AlogP = 4.0 for 2 vs AlogP = 2.4
for 7) and resulted in improved mouse metabolic stability
(Clint = 76 mL/min/kg body weight) over the acyclic amine 2
(Table 1). Although reduced class IIa cell activity (IC50 0.25
μM) and diminished (15-fold) cellular selectivity over HDAC
class I/IIb were observed for 7, its increased metabolic stability
became a key driver for future inhibitor optimization.
To further investigate the SAR in 7, several substituted
amino linker analogues were synthesized (Table 3). Class IIa
HDAC potency and selectivity over class I/IIb HDACs was
improved by incorporation of an (R)-methyl substituent in the
aliphatic linker; 8 exhibited a 3-fold improvement in HDAC4
biochemical and cellular potency over 7, with improved
cellular selectivity for class IIa over class I/IIb HDACs (cell
ratio = 143-fold); the (S)-enantiomer 9 was 10-fold less active
than 8 in the class IIa HDAC cell assay. Substitution of the
linker with a gem-dimethyl (10) or cyclopropyl (11) also
reduced HDAC class IIa cellular selectivity compared to 8.
Toward exploiting 8’s superior class IIa HDAC cellular
potency and selectivity, we focused efforts on modifications to
the terminal pyrrolidine ring (Table 4). We previously
disclosed X-ray structures of hydroxamic acid-based ligands
in complex with HDAC4, showing an aspartate residue
(Asp759) in close proximity to the ligand.18,19 The terminal
basic amines exemplified in Table 4 are likely to occupy this
region of the binding cavity and form a salt bridge interaction
were synthesized (Table 2). Replacement of the CF3 group in
2 with CHF2 (3) lead to a 300-fold decrease in HDAC4
potency. Introduction of a methyl group or hydrogen atom
onto the oxadiazole ring (4 and 5) led to complete loss of
HDAC4 activity. Furthermore, the regioisomeric oxadiazole 6
exhibited 4 orders of magnitude loss of HDAC4 potency.
These data demonstrate not only that the CF3 group is
required for class IIa HDAC cellular potency but also that the
position of the oxygen atom in the 1,2,4-oxadiazole
regioisomer is crucial for achieving adequate biochemical and
cellular activity.
Table 3. Linker Modifications and the Effect on Potency and Selectivity
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Table 4. Terminal Amine Modifications and the Effect on Potency and ADME Properties
a
pKa values calculated using ACD Laboratories Percepta Portal, Client Version 1.7.2.
with Asp759. Pyrrolidine, piperidine, and azepane derivatives
(8, 15, and 21) all demonstrate potent HDAC4 inhibition and
improved metabolic stability when compared to acyclic
analogue 2, and with respect to basicity, cyclic amines covering
a range of pKa values (6.9−9.1) are able to achieve very potent
HDAC4 inhibition. Finally, as demonstrated by diastereomeric
pairs 12 vs 13 and 19 vs 20, the absolute stereochemistry of
the substituent on the pyrrolidine and piperidine rings
respectively has a significant effect on HDAC potency.
We sought to identify compounds with ≥100-fold selectivity
for class IIa versus class I/IIb HDACs. Based on its optimal
potency, selectivity, and encouraging ADME profile, 12 was
progressed to further biochemical and cellular selectivity
assessment and mouse PK studies.
Additional profiling in HEK293 cells (Supporting Informa-
tion) revealed potent class IIa inhibition (0.01 μM) and
excellent selectivity against class I and class IIb HDACs (990-
fold and >2900-fold, respectively). To assess broader
selectivity and gain insight into potential cardiac liability, 12
was profiled against a panel of binding, cellular and nuclear
receptor functional and enzymatic assays (Cerep, France). 12
exhibited >80% inhibition at 10 μM single concentration
against HDAC4 and muscarinic M1-M5 receptors and 50−
80% inhibition for σ1, L-type calcium (diltiazem site) and
sodium channels (SI Table 2). In separate assays, Kd values for
M1−M4 were determined to be >5 μM, and the Kd value for
hERG was 3.9 μM (IonWorks) (SI Table 3). No cytochrome
P450 inhibition was observed in human liver microsomes for
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Figure 2. Brain (A) and blood (B) exposure of 12 following oral gavage of compounds to C57/BL6 mice (10, 30, and 100 mg/kg). Left Y axis
indicates the measured exposure in the matrix (determined by LC-MS/MS). Right Y axis indicates estimated free exposure, scaled based on fu
values returned in each matrix in vitro. Class IIa cellular IC50 is shown by the red arrowhead; class I/IIb cellular IC50 is shown by the blue
arrowhead. Affinity measures for other binding targets identified by the Cerep Diversity panel (KD range for M1−M4 receptors) indicated by the
purple bar, and KD for HERG channels (IonWorks) indicated by the green arrowhead (SI Table 3).
a
Table 5. HDAC Biochemical and Cellular Data for Compound 12
Biochemical potency
Subfamily
Class IIa
Class IIa
Class I
Class IIb
HDACn
4
5
7
9
1
2
3
8
6
a
IC50 (μM)
0.01
0.02
0.02
0.03
Cellular potency
17
27
10
2.0
22
Subfamily
Class I
HEK293
9.9
Class IIb
HEK293
>29
Class I and IIb
Cell type
Jurkat
0.04
HEK293
0.01
Jurkat
5.0
a
IC50 (μM)
a
Geometric mean of three experiments; standard deviations are <25% of the mean. Assays as previously described25 and as described in the SI.
any of the tested isoforms (1A2, 2C8, 2C9, 2C19, 2D6, 3A4,
all IC50 values >50 μM) (Supporting Information).
explaining the supraproportional increase in brain concen-
trations. Indeed, brain to blood ratios increased with increasing
dose with mean values ranging from 1.3−2.7 (10 mg/kg), 1.4−
5.6 (30 mg/kg), and 1.2−8.7 (100 mg/kg) over the 0.25 to 12
h following administration. All doses achieved total brain
exposures above the on-target class IIa cellular IC50 for ∼4, 8,
and 14 h, respectively, for the 10, 30, and 100 mg/kg doses,
whereas only the 30 and 100 mg/kg doses achieved significant
brain exposure predicted to engage off-target class I/IIb
cellular IC50 for ∼2 and 6 h, respectively.
12 was stable in mouse and human plasma and blood and
simulated gastric fluid (SI Figure 1 and 2). It preferentially
distributed into mouse blood versus plasma (ratio 3:1) and
displayed an unbound fraction (Fu) of 0.73 in blood,
determined by equilibrium dialysis. Fu of 12 in mouse brain
homogenate was 0.17, higher than previous hydroxamic acid-
based compounds tested in our hands, which were typically in
the range 0.026−0.0041.18,19
In PK studies, 12 was dosed intravenously (3.3 mg/kg) and
via oral gavage (11.5 mg/kg) to fed male C57Bl/6 mice (PK
parameters shown in SI Table 5) and demonstrated 100% oral
bioavailability, high volume of distribution (3.8 L/kg), high
distribution to brain (brain to blood exposure ratio up to 3; SI
Table 6), and high plasma clearance (4.2 L/h/kg). In oral dose
escalation studies (Figure 2), 12 was absorbed rapidly with a
Tmax of 0.5 h in blood and brain. An approximate linear
increase in Cmax and AUC was seen in blood across the dose
range investigated; however, a supra-proportional increase in
Cmax and AUC was seen in brain and muscle (SI Table 7). As
12 has moderate to high % unbound in blood, we speculated
that blood binding may be saturated at higher doses resulting
in increased free compound available to cross into the brain,
Consistent with the lack of precedent for endogenous class
IIa HDAC substrates4 and diminished deacetylase activity
observed for this HDAC subfamily, we were unable to identify
a specific class IIa biomarker to directly study compound
effects for selective class IIa deacetylase activity in vivo.
However, as 12 displays off-target class I/IIb inhibition at high
concentration in vitro (Jurkat cell IC50 = 5 μM) (Table 5) and
exhibits brain exposure sufficient to inhibit class I/IIb enzymes
at higher doses administered (Figure 2), studies assessing class
I/IIb substrate acetylation were undertaken in the brain
samples from these mice. Specific acetylation of histone
H4K12 (deacetylated by class I HDACs) or an increase in
global acetylation, measured using a pan antiacetylation
antibody, was quantified by the dot blot technique (Supporting
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Figure 3. (A−B) Correlation of HDAC class I/IIb substrate acetylation (broken line) to 12 brain exposure (continuous line). Left axis:
concentration of 12 measured by LC-MS/MS in brain (solid line). Right axis: H4K12 normalized to H4 histone levels (broken line) (A) or Pan
AcK normalized to tubulin (B) measured via dot blot assay from brain samples. Increasing doses of 12 administered p.o. resulted in increasing “off-
target” class I H4K12 acetylation (A) and increasing global acetylation levels (B). (C−D) To confirm specific class I/IIb substrate acetylation,
samples from the 2 h time point were probed by Western blot to determine H3, H4, and tubulin acetylation and compared to the 5 min 10 mg/kg
samples (C). (D) Quantitation of the pan-AcK band corresponding to H3 (left) and H4 (right) normalized to H3 and H4 total protein band
intensity. (E). Quantitation of the pan-AcK band corresponding to tubulin molecular weight normalized to total tubulin protein band intensity
(left). Quantitation of tubulin acetylation was equivalent when an Ab specific for acetylated tubulin was used (right). One-way ANOVA with
Dunnett’s multiple comparison test: p < 0.05*, p < 0.01**, p < 0.001***, p < 0.0001****.
Information) (Figure 3). Acetylated H4K12 levels were
normalized to total H4 levels (Figure 3A), and pan-acetylation
levels were normalized to tubulin (Figure 3B). H4K12
acetylation, reflective of class I HDAC activity, increased
dose-dependently. Global acetylation increase (reflective of
both class I and IIb HDAC activity) showed somewhat greater
acetylation at lower exposures when compared to H4K12
acetylation (Figure 3B, 10 mg/kg dose). In general, the
maximum effect on either H4K12 or pan-acetylation lagged
behind that of maximal compound exposure; compound tmax
was observed at ∼0.5 h, while maximal pan-acetyl lysine
intensity was observed at 2 h, and maximal acetylated H4K12
intensity was observed between 2 and 4 h. Acetylation of
specific substrates in these samples was confirmed by Western
blot assessment from 2 h postdose samples (Figure 3C).
Acetylation of histones H3, H4, and tubulin was quantified by
normalizing to respective total protein intensity. Dose-
dependent acetylation of the class I target histones H3/H4
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with 12 for oral doses ≥10 mg/kg, while H4K12 acetylation
was increased for 30 and 100 mg/kg oral doses (data pooled
for wild-type and R6/2 mice) (SI Figure 3A). The estimated
acetylation EC50 in brain for global acetylation (likely reflective
of both class I and IIb protein substrates) was 20 μM total
exposure (or 3.5 μM estimated free exposure based on a Fu
value of 0.17) (SI Figure 3B−C). This EC50 was consistent
with the IC50 values previously determined in HDAC class I/
IIb cellular assays (IC50 5−10 μM; Table 5). Western blots
were run on these samples to assess acetylation of the specific
class I substrates H3 (SI Figure 3D) AND H4 (SI Figure 3E)
and class IIb substrate tubulin (SI Figure 3F). In these
instances, although trends for increased acetylation of H4 and
tubulin were seen in the 30 mg/kg group, only significant
increased acetylation was seen in the highest dose group (100
mg/kg). Overall, the similar PK:PD profile obtained here after
15 days of po bid dosing compared to acute administration
confirmed the impact of 12 was largely sustained following
subchronic administration.
Given the good correlation of the 12 PD response
empirically determined in vivo and the class I and IIb cellular
activity measured in vitro, it is reasonable to assume this
relationship will also hold for occupancy of class IIa HDAC
enzymes. In oral b.i.d. dosing simulations, we calculated brain
concentrations of compound 12 at 3, 10, and 30 mg/kg
(Figure 4). Three and 10 mg/kg b.i.d. were predicted to
achieve sufficient brain exposure to effectively inhibit class IIa
HDACs (above the cellular IC50 value for class IIa HDACs for
∼4−6 h post each dose respectively) with low to negligible off-
target liability (<EC10 for acetylation of class I/IIb substrates).
The synthesis of compound 12 is illustrated in Scheme 1.
In conclusion, we optimized the in vitro HDAC class IIa
selectivity and metabolic stability of a series of TFMO HDAC
class IIa inhibitors, leading to identification of compound 12
(CHDI-00484077), which displays PK properties and CNS
exposure suitable for progression to efficacy studies in mouse
models of HD. Determination of observable class I and IIb-
mediated acetylation of proteins in CNS dosing simulations
indicated that doses of 3 and 10 mg/kg of 12 would likely
provide selective class IIa HDAC inhibition. Compound 12
offers higher unbound exposure in mouse brain over existing
hydroxamic-based inhibitors previously described by this
laboratory17−19 and represents a valuable compound to further
probe potential class IIa HDAC-targeted therapies for HD.
Figure 4. Oral bid dosing simulation (modeling a dose interval of 10
h), showing simulated total brain concentration of 12. Estimates of
a
final parameters from 10 mg/kg po were used to predict
concentrations achieved following 3 mg/kg po. Both doses are
predicted to provide brain exposure exceeding class IIa HDAC IC50
for ∼4−6 h post each dose and to have minimal effect on engaging
class I/IIb HDACs, with brain exposure failing to reach concen-
trations > EC10 for class I/IIb substrate acetylation, as determined
empirically (SI Figure 3B−C). *The in vivo class IIa IC50 (235 nM)
was estimated from the measured cellular IC50 (40 nM) adjusted for
12 Fu of 0.17 in brain homogenate.
a
Scheme 1. Synthesis of 12
a
Reagents and conditions: (a) MsCl, Et3N, DCM, 0 °C to r.t., 18 h.
(b) (S)-2-methylpyrrolidine hydrochloride, Cs2CO3, DMF, 60 °C, 17
h (35% two steps). (c) HCl, dioxane, DCM, r.t., 2 h (81%). (d) 4-(5-
(Trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzoic acid, EDC, HOPO,
DIPEA, DCM, r.t., 17 h (97%).
and acetylation of the class IIb substrate tubulin was confirmed
in all samples, dose-dependently increasing with increasing
compound exposure (Figure 3D−E).
We next examined the tolerability of 12 in the R6/2 mouse
model of HD alongside wild-type litter-mate controls. 12 was
stable in vehicle (10% [w/v] hydroxypropyl-β-cyclodextrin in
50 mM citrate buffer) over a 30-day period (SI Table 8). Mice
were dosed orally b.i.d. with 12 at 10, 30, or 100 mg/kg or
vehicle for 14.5 days, with collection of blood and brain
samples 2 h post final dose. 12 was well-tolerated over this
period with no adverse effects observed on body weight, body
temperature, or scores to passive observations or active
manipulations of the mice (SI Table 9). Concentrations of
12 in blood and brain were similar in wild-type and R6/2 mice
(male and female) at each dose level. In addition to
bioanalytical determination of 12 in brain, half-brain samples
from dosed mice were also assessed for acetylation signatures
using the dot blot technique, and acetylation of the specific
class I substrates H3 and H4 and the class IIb substrate tubulin
in these samples was subsequently assessed by Western blot
(Supporting Information).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00532.
■
sı
Methods, compound synthesis, and supplementary
tables and figures (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Michel C. Maillard − CHDI Management/CHDI Foundation
Inc., Los Angeles, California 90045, United States;
Email: michel.maillard@chdifoundation.org
Authors
Andrew J. Stott − Charles River Discovery, Saffron Walden,
Essex CB10 1XL, United Kingdom; orcid.org/0000-
0002-0266-6784
In agreement with acute dosing, global acetylation signatures
using the dot blot technique increased in response to treatment
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elusive catalytic activity of vertebrate class IIa histone deacetylases.
Bioorg. Med. Chem. Lett. 2008, 18 (6), 1814−9.
Vahri Beaumont − CHDI Management/CHDI Foundation
Inc., Los Angeles, California 90045, United States
David Allcock − Charles River Discovery, Saffron Walden,
Essex CB10 1XL, United Kingdom
Omar Aziz − Charles River Discovery, Saffron Walden, Essex
CB10 1XL, United Kingdom
Alexander H. Borchers − CHDI Management/CHDI
Foundation Inc., Los Angeles, California 90045, United
States
Wesley Blackaby − Charles River Discovery, Saffron Walden,
Essex CB10 1XL, United Kingdom
(3) Lahm, A.; Paolini, C.; Pallaoro, M.; Nardi, M. C.; Jones, P.;
Neddermann, P.; Sambucini, S.; Bottomley, M. J.; Lo Surdo, P.; Carfi,
A.; Koch, U.; De Francesco, R.; Steinkuhler, C.; Gallinari, P.
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Perla Breccia − Charles River Discovery, Saffron Walden,
Essex CB10 1XL, United Kingdom; orcid.org/0000-
0002-8552-9012
Gillian Creighton-Gutteridge − Charles River Discovery,
Saffron Walden, Essex CB10 1XL, United Kingdom
Alan F. Haughan − Charles River Discovery, Saffron Walden,
Essex CB10 1XL, United Kingdom
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Rebecca E. Jarvis − Charles River Discovery, Saffron Walden,
Essex CB10 1XL, United Kingdom
Christopher A. Luckhurst − Charles River Discovery, Saffron
Walden, Essex CB10 1XL, United Kingdom
Kim L. Matthews − Charles River Discovery, Saffron Walden,
Essex CB10 1XL, United Kingdom
George McAllister − Charles River Discovery, Saffron
Walden, Essex CB10 1XL, United Kingdom
Scott Pollack − Charles River Discovery, Saffron Walden,
Essex CB10 1XL, United Kingdom; orcid.org/0000-
0002-8176-0997
Elizabeth Saville-Stones − Charles River Discovery, Saffron
Walden, Essex CB10 1XL, United Kingdom
̈
Amanda J. Van de Poel − Charles River Discovery, Saffron
Walden, Essex CB10 1XL, United Kingdom
Huw D. Vater − Charles River Discovery, Saffron Walden,
Essex CB10 1XL, United Kingdom; orcid.org/0000-
0002-2904-1848
Julie Vann − Charles River Discovery, Saffron Walden, Essex
CB10 1XL, United Kingdom
Rachel Williams − Charles River Discovery, Saffron Walden,
Essex CB10 1XL, United Kingdom
Dawn Yates − Charles River Discovery, Saffron Walden, Essex
CB10 1XL, United Kingdom
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́
Ignacio Munoz-Sanjuan − CHDI Management/CHDI
̃
Foundation Inc., Los Angeles, California 90045, United
States
Celia Dominguez − CHDI Management/CHDI Foundation
Inc., Los Angeles, California 90045, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00532
(13) Benn, C. L.; Butler, R.; Mariner, L.; Nixon, J.; Moffitt, H.;
Mielcarek, M.; Woodman, B.; Bates, G. P. Genetic knock-down of
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P. Genetic knock-down of HDAC3 does not modify disease-related
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Notes
The authors declare no competing financial interest.
DEDICATION
■
§The authors dedicate this manuscript to the memory of Dr.
Alexander H. Borchers, who passed away on August 3rd, 2019.
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