Delayed and prolonged histone hyperacetylation with a selective HDAC1/HDAC2 inhibitor

Article abstract of DOI:10.1021/ml4004233

The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone hyperacetylation and

Full text of DOI:10.1021/ml4004233

Letter
pubs.acs.org/acsmedchemlett
Delayed and Prolonged Histone Hyperacetylation with a Selective
HDAC1/HDAC2 Inhibitor
Joey L. Methot,* Dawn Mampreian Hoffman, David J. Witter, Matthew G. Stanton, Paul Harrington,
Christopher Hamblett, Phieng Siliphaivanh, Kevin Wilson, Jed Hubbs, Richard Heidebrecht,
Astrid M. Kral, Nicole Ozerova, Judith C. Fleming, Hongmei Wang, Alexander A. Szewczak,
Richard E. Middleton, Bethany Hughes, Jonathan C. Cruz, Brian B. Haines, Melissa Chenard,
Candia M. Kenific, Andreas Harsch, J. Paul Secrist, and Thomas A. Miller*
Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis
indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone
hyperacetylation and gene expression changes were also observed in cell culture, and histone acetylation was observed in vivo
beyond disappearance of drug from plasma. In vivo studies further demonstrated that continuous target inhibition was well
tolerated and efficacious in tumor-bearing mice, leading to tumor growth inhibition with either once-daily or intermittent
administration.
KEYWORDS: Histone acetylation, histone deacetylase inhibitor, isoform selectivity, kinetics
istone deacetylases (HDACs) catalyze the removal of
Hacetyl groups from lysine residues of proteins, most
notably nucleosomal histones as well as a variety of nonhistone
proteins (e.g., p53, E2Fs, nuclear receptors, NFκB, HSP90, α-
tubulin, KU70).1,2 Consequently, small molecule HDAC
inhibitors have been shown to regulate gene transcription,
cell cycle progression, and induce differentiation and/or
apoptosis in cancer cells.3−6 HDACs 1 and 2 in particular are
both overexpressed in human cancers and knockdown leads to
increased apoptosis in certain cellular contexts.7−10 They share
Figure 1. Early HDAC1/HDAC2-selective biaryl spirocyclic nicotina-
mide inhibitor.
92% sequence homology and 82% sequence identity. Moreover,
emerging preclinical studies suggest roles for HDACs in
inflammatory and neurodegenerative diseases.11
3-yl analogue 3 was potent and devoid of hERG binding, we
found that 4-methylthiophen-2-yl, phenyl, and 4-flourophenyl
analogues 4−6 did bind to hERG, indicating that the spirocyclic
carbamate moiety was not a general solution to the hERG
problem in this series. As seen in the SAR profile of this series,
polar biaryls containing pyrazole or imidazole moieties (7 and
8) had promising biochemical potency, but poor cell potency.
In our previous communication, we described the discovery
of spirocyclic nicotinamide 1, a potent and efficacious selective
SHI-1:2 (Figure 1).12 Unfortunately, the safety profile of 1 was
unacceptable due to hERG binding.
Additional studies demonstrated that the incorporation of a
cyclic carbamate functional group, to afford compound 2, was
well tolerated and that this modification attenuated hERG
activity (Table 1). Since 2 was inactive in an hERG binding
assay, we varied the biaryl substituent for further HDAC
inhibition potency optimization (Table 1). While the thiophen-
Received: October 26, 2013
Accepted: January 2, 2014
Published: January 2, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Table 1. SAR Profile of the Spirocyclic Carbamate
Nicotinamides
Figure 4. Treatment of HCT116 cells with 2 (500 nM) led to a
delayed increase in histone H2BK5 acetylation.
HDAC1 IC50 HCT−116 Gl50
heRG Ki
(μM)
compound: substituent
(nM)
(nM)
2: thiophen-2-yl
6
97
>30
>30
2.0
3: thiophen-3-yl
11
14
17
20
18
51
580
270
890
490
710
2120
4650
5080
4: 4-methylyhiophen-2-yl
5: phenyl
3.4
6: 4-fluorophenyl
7: 1-methylpyrazol-4-yl
8: 1-methylpyrazol-4-yl
9: hydrogen
0.62
>10
>10
>10
Figure 5. Effects of 2 on gene expression of DHRS2 and FAM46c in
HCT116 colon carcinoma cells. TaqMan real-time PCR was used to
evaluate time and dose-dependent regulation of DHRS2 and FAM46c
in vitro. The concentrations of 2 used represent GI25 (50 nM), GI50
(97 nM), GI75 (188 nM), and GI90 (367 nM) values for inhibition of
HCT116 cell proliferation in a 72 h proliferation assay.
Figure 2. In vitro profile of key HDAC1/HDAC2 inhibitor 2.
HCT116, human colon carcinoma; HEL, human erythroleukemia;
Jurkat, T cell lymphoma.
which we believe represent the biaryl benzamides as a whole,
and warrants this separate communication.
In vitro HDAC potency and selectivity were measured by
monitoring the deacetylation of a synthetic acetyl-lysine-
containing peptide by subtype-specific HDAC complexes
purified from mammalian cells overexpressing recombinant
human enzyme. The in vitro profile of 2 is summarized in
Figure 2.13 Compound 2 was a potent inhibitor of both
HDAC1 (IC50 6 nM) and HDAC2 (IC50 45 nM). Character-
istic to all SHI-1:2 biaryl benzamides,14−16 it was also highly
selective versus HDAC3 (IC50 7.0 μM), which itself may be
important for cardiac function.17 Inhibition of HDAC3 also
produces mitotic defects.18 It was a weak inhibitor of HDACs 8
and 11 (IC50 20−25 μM), and inactive in HDACs 4−7 assays.
In vitro kinetic analysis indicated that 2 was a time-
dependent, substrate-competitive, and reversible inhibitor of
HDAC1 and HDAC2 with relatively slow binding kinetics.19
While the IC50 values reported in Figure 2 were obtained after
10 min of preincubation time, compound 2 required ≥4 h to
maximally inhibit HDAC1 and HDAC2 enzymatic activities
(Figure 3). Additional kinetic experiments indicated that 2
Figure 3. Time-dependent inhibition of HDACs 1 and 2 with 2.
Preincubation time (0−240 min) of enzyme and 2 prior to initiating
deacetylase reactions with peptide substrate.
Removal of the internal cavity binding motif, such as simple
benzamide 9, diminished potency considerably. Remarkable in
vitro and in vivo properties of key inhibitor 2 were identified,
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ACS Medicinal Chemistry Letters
Letter
Figure 6. Cancer cell proliferation assay panel in the presence of 2.
NSCLC, nonsmall cell lung cancer; SCLC, small cell lung cancer.
Table 2. Pharmacokinetic Profile of 2
mouse
rat
dog
monkey
iv dose(mg/kg)
Clp (mL/min/kg)
Vdss (L/kg)
5
2
0.5
22
0.5
27
Figure 7. Acute oral administration of 2 induced significantly elevated
and prolonged tumor histone H2BK5 acetylation in the HCT116
tumor model (above) and associated average plasma levels (below).
27
3.6
0.7
4.2
4
2.1
2.0
10
2.2
1.4
1
7.8
7.1
1
t1/2 (h)
po dose(mg/kg)
AUCN (μM·h·kg/mg)
F (%)
HCT116 cells with 3 μM of 2 (24 h/day) for four days gave
>90% reduction in cell viability, while treatment with 3 μM of 2
for 8 h/day (4 days) gave only 50% reduction in cell viability.
A radioligand-based HDAC binding assay indicated that the
binding affinity of 2 for HDAC binding sites in intact HCT116
cells was 16 nM; close to the biochemical activity assay IC50 of
6 nM. At 97 nM, the GI50 value with HCT116 cells,
approximately 80% occupancy of HDAC binding sites was
achieved. This was consistent with RNAi-based observations
that HDAC1 knockdown must approach 80−90% before
significant cell death is observed in tumor cell lines.
Following treatment with 500 nM of 2, 97% of HCT-116
colon carcinoma cells were TUNEL positive, indicating that 2
had induced significant apoptosis after 72 h treatment. After 48
h of continuous treatment with 500 nM of 2, HCT116 cells
undergo a significant decrease in the percentage of cells in S-
phase from 29% in vehicle-treated cells to 6% in the group
treated with 2, indicating that fewer cells are undergoing DNA
replication. Concomitant with the decrease in the S phase cell
population was a modest increase in both the G1 and G2 cell
populations. After 96 h of treatment a significant proportion of
cells (61%) accumulated in the subG1 fraction, consistent with
the induction of apoptosis.
1.1
82
3.0
28
0.3
17
1.8
100
98.4
plasma protein binding (%, 2 μM)
98.5
99.8
97.6
dissociated from HDAC1 with an apparent half-life exceeding
100 min; significantly slower than that observed with
unselective hydroxamic acid inhibitors that typically have a
dissociation half-life of ∼1 min.20 A similar observation was
reported by Gottesfeld using a simple nonbiaryl benzamide in a
HDAC3 assay.21 Compound 2 maintained excellent selectivity
versus HDAC3 even with longer preincubation times. We
hypothesize that protein movement required to accommodate
the biaryl into the internal cavity of HDAC1 and HDAC2 gives
rise to the observed slow binding kinetics. A model of the boot-
like active site and proposed binding into the internal cavity is
described in greater detail in ref 15.
Histones are classic substrates for several HDAC subtypes,
including HDAC1 and HDAC2, representing proximal
pharmacodynamic biomarkers for target engagement. Treat-
ment of HCT116 colon carcinoma cells with 500 nM of 2
followed by cell lysis indicated a time-dependent hyper-
acetylation of several histone residues, including lysine 5 of
histone H2B (H2BK5). H2BK5 hyperacetylation becomes
apparent within 4−8 h of treatment and plateaus at 96 h
(Figure 4). At 24 h, the half maximal response was observed at
a 400 nM concentration of 2. It also inhibited HCT116 cell
growth with a GI50 of 97 nM with concomitant apoptosis (72 h
assay).
Since histone acetylation alters gene expression, we evaluated
whether gene expression changes induced by 2 occur at
biologically relevant concentrations. Two genes that were
previously identified from siRNA gene profiling experiments as
biomarkers of HDAC1 inhibition are DHRS2 and FAM46c.
FAM46c encodes a gene of unknown function, while DHRS2 is
an NADPH-dependent dicarbonyl reductase that is reported to
be upregulated in response to cellular stress. Indeed, robust
dose- and time-dependent increases in the expression of both
In separate cell proliferation experiments, continuous
exposure to 2 was shown to be more efficacious than
intermittent exposure. For example, continuous treatment of
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ACS Medicinal Chemistry Letters
Letter
Figure 8. Top: 21-Day treatment of HCT116 xenograft mice with once daily (qd) or biweekly (biw) 2. Middle: Tumor histone H2BK5 acetylation
after a single dose of 2. Bottom: Xenograft model summary and plasma levels of 2. Dosing formulation was 40% v/v PEG-400 and 25% w/v
HPβCD, 20 μL/g.
DHRS2 and FAM46c were noted following treatment of
HCT116 cells with 2 (Figure 5). These results indicated that at
times and concentrations that inhibit cell proliferation and
induce apoptosis, changes were also occurring at the level of
gene expression.
Given that 2 potently inhibited the growth of other cell lines,
such as HEL human erythroleukemia and Jurkat T cell
lymphoma, we proceeded to screen it in a cancer cell
proliferation assay panel (Figure 6). Potent antiproliferative
activity was observed with numerous multiple myeloma, colon,
nonsmall cell lung, breast, and small cell lung cancer cell lines.
Compound 2 was inactive in the hERG binding assay (Ki
>30 μM) as well as the potassium channel voltage-clamp assay
h) with excellent bioavailability giving a moderate oral exposure
(AUCN 1.1 μM·h·kg/mg), which would facilitate character-
ization in our mouse models. In rat plasma, lower clearance was
masked by higher plasma protein binding. The PK in dog and
monkey was characterized by moderate plasma clearance with
particularly good bioavailability in monkey.
Clearance of 2 in rats was primarily through metabolism,
including hydrolysis of the anilide bond (major), oxidation of
the thienyl group to form a monooxidized metabolite and
glucuronidation of monooxidized metabolite; these were
consistent with observations in rat hepatocytes. In dog
hepatocytes, the major metabolic pathway for 2 was hydrolysis
of the oxazolidinone group. In human hepatocytes, the major
metabolite was a direct glucuronide conjugate. Additional
metabolites in human hepatocytes included a carboxylic acid
product formed by hydrolysis of anilide bond, a product
derived from oxidation of the piperidine ring and a glucose
conjugate. Compound 2 did not undergo extensive oxidative
metabolism in liver microsomes in the presence of NADPH,
in human embryonic kidney cells stably expressing Ikr (IC50
>
30 μM). Importantly, there was no effect on electrocardio-
graphic intervals in anesthetized dogs up to 150 μM.
Given the overall favorable profile of 2, we profiled the PK
properties of the compound in male CD-1 mouse, male
Sprague−Dawley rat, beagle dog, and rhesus monkey (Table
2). In mouse, we observed moderate clearance and half-life (2.0
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ACS Medicinal Chemistry Letters
Letter
a
or reflect a limited role of protein acetylation in the
proliferation of the HCT116 cell line.
Scheme 1. Synthesis of Compound 2
In order to better understand the impact of prolonged
histone hyperacetylation observed beyond 72 h (see Figure 7),
biweekly oral administration of 2 was explored to assess the
relationship between tumor growth inhibition and histone
acetylation induction. In this study, a dose-dependent tumor
growth inhibition was observed, with 525 mg/kg biweekly
providing 59% tumor growth inhibition, although increasing
the dose to 875 mg/kg biweekly did not lead to improved
growth inhibition. These efficacious doses of 2 were well-
tolerated, with minimal effects on body weight loss, platelet, or
lymphocyte count in mice. In separate studies, inhibitor 2 was
also efficacious and well tolerated in a 32-day transgenic NeuT
breast model (86% tumor growth inhibition with oral 150 mg/
kg qd). In addition, 2 exhibited tumor growth inhibition when
dosed orally in several xenograft models of small and nonsmall
cell lung cancer (e.g., A549, NCI-H69, NCI-H2122; data not
shown).
a
Reagents and conditions: (a) Boc2O, NEt3, DMAP, DCM, 62%; (b)
thiophen-2-yl boronic acid, Pd(PPh3)4, K2CO3, dioxane, water, 90 °C,
92%; (c) 1 atm H2, Pd/C, EtOAc, 76%; (d) 6-chloronicotinoyl
chloride, pyridine, 96%; (e) LiAlH4, THF; (f) CDI, DCM, 51% two
steps; (g) 1 atm H2, Pd(OH)2/C, EtOAc, MeOH, 100%; (h) 11, 13
(2.5 equiv), NEt3, DMA, 90 °C, 70%; (i) TFA, DCM, 87%.
In connection with earlier in vitro HCT116 proliferation
experiments, which suggest continuous exposure to 2 was more
efficacious than intermittent exposure, we sought to address
whether a Cmax threshold was required or a continuous Ctrough
was sufficient for tumor growth inhibition. Tumor bearing mice
were implanted subcutaneously (sc) with an osmotic minipump
that slowly released compound 2. A dose of 20 mg/mL
maintained a continuous plasma level of 2.6 μM, was well-
tolerated, and provided a 43% tumor growth inhibition over a
four-week treatment period. When this result is compared to
the 50 mg/kg qd dose group in Figure 8, which was not
efficacious reaching a comparable Cmax of 3.5 μM, it is clear that
continuous exposure of 2 is optimal in vivo, as predicted from
in vitro studies.
suggesting that 2 was not significantly metabolized by CYP
isoforms.
A mouse HCT116 xenograft tumor model was used to
corroborate the compelling in vitro data. HCT116 cells (5 ×
106 per mouse) were injected subcutaneously into the right
flank of CD1 nu/nu mice. When the tumors reached ∼325
mm3, the mice were randomized into groups of 12 animals and
treated with oral doses of 2 at 67 mg/kg tid, 100 mg/kg bid,
and 200 mg/kg qd. At 24, 48, and 72 h postdose, tumors were
harvested and histones isolated. Histone 2B lysine 5 (H2BK5)
acetylation was measured by Elisa (Figure 7). After 24 h, ≥6-
fold increase in histone acetylation was observed, which was
maintained by ∼4-fold after 72 h; indicating that 2 exhibited an
extended duration of target inhibition. While the three times
per day regimen gave the greatest histone hyperacetylation at
24 h, by 72 h all three regimens gave comparable response.
Hence, in subsequent efficacy experiments, we chose once daily
administration. Interestingly, histone hyperacetylation was
observed at plasma levels as low at 0.2 μM. This was in
sharp contrast to the nonselective hydroxamic acid-containing
inhibitor class that ellicit only a short duration histone
hyperacetylation (<12 h) that mirrors compound exposure.
This behavior was consistent with 2 exhibiting slow-off rate
binding kinetics. A similar observation was reported using
MGCD0103 in human whole blood.22−24
We next examined the effect of three-week oral admin-
istration of 2 on tumor size, as estimated using caliper
measurements (Figure 8). Tumor growth inhibition was
observed in a dose-dependent manner. At 150 mg/kg/day a
78% tumor growth inhibition was achieved without body
weight loss. Higher doses, however, did not provide greater
tumor growth inhibition. With 150 mg/kg/day, plasma levels of
2 reached 16 μM (Cmax) with a trough level of 3.2 μM (C24h),
and a 0−24 h exposure of 208 μM·h. On the basis of the
protein binding in mouse, the unbound Cmax and C24h
concentrations were 240 and 48 nM, respectively. This
correlates well with the in vitro hyperacetylation of H2BK5
in HCT116 cells, with an IC50 of 400 nM. Tumor histone
hyperacetylation increased in a dose-dependent fashion up to a
plateau of 4-fold, although maximal efficacy was achieved with a
2-fold increase in acetylation. The ∼78% tumor growth
inhibition plateau may be a result of limited tumor penetration
A synthesis of 2 is outlined in Scheme 1.25 4-Bromo-2-
nitroaniline was protected and coupled with thiophene-2-
boronic acid using Pd(PPh3)4 and aqueous K2CO3. The nitro
group was reduced and the resulting aniline coupled to give
chloronicotinamide 11. Separately, the spirocycle was formed
by reduction of nitrile 12 and the resulting amino alcohol
cyclized with carbonyl diimidazole. Precursors 11 and 13 were
coupled at elevated temperature and the resulting amide
deprotected giving 2.
In summary, characterization of the HDAC1/HDAC2
inhibitor 2 revealed that this selective biaryl SHI-1:2 offers
unique slow in vitro HDAC binding kinetics, as well as delayed
and prolonged cell hyperacetylation.26 These results were
consistent with the extended histone hyperacetylation for >72 h
found in vivo, which continues even after the disappearance of
drug from plasma. It is plausible that the prolonged PD is a
result of the intrinsic slow binding kinetics of 2 to HDAC1/
HDAC2 enzymes. These studies also demonstrated that
continuous target inhibition is well tolerated in mice and may
offer enhanced antitumor efficacy in the clinic versus the short-
acting hydroxamic acid-based inhibitors.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for assay protocols, in vivo studies,
and synthesis of compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Letter
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AUTHOR INFORMATION
Corresponding Authors
*(J.L.M.) E-mail: joey_methot@merck.com.
*(T.A.M.) E-mail: thomas.miller.tm@gmail.com.
Notes
■
The authors declare no competing financial interest.
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