Dual-Mode HDAC Prodrug for Covalent Modification and Subsequent Inhibitor Release

Article abstract of DOI:10.1021/acs.jmedchem.5b00539

Histone deacetylase inhibitors (HDACi) target abnormal epigenetic states associated with a variety of pathologies, including cancer. Here, the development of a prodrug of the canonical broad-spectrum HDACi suberoylanilide hydroxamic acid (SAHA) is describ

Full text of DOI:10.1021/acs.jmedchem.5b00539

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Article
A Dual-Mode HDAC Prodrug for Covalent
Modification and Subsequent Inhibitor Release
Kevin B Daniel, Eric D Sullivan, Yao Chen, Joshua C. Chan,
Patricia A. Jennings, Carol A. Fierke, and Seth M. Cohen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00539 • Publication Date (Web): 14 May 2015
Downloaded from http://pubs.acs.org on May 18, 2015
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A Dual-Mode HDAC Prodrug for Covalent
Modification and Subsequent Inhibitor Release
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Kevin B. Daniel,1 Eric D. Sullivan,2 Yao Chen,1 Joshua C. Chan,1 Patricia A. Jennings,1 Carol A.
Fierke,2,3,4* and Seth M. Cohen1*
1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA
92093, United States
2Interdepartmental Program in Chemical Biology, 3Department of Biological Chemistry, and
4Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, United States
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ABSTRACT. Histone deacetylase inhibitors (HDACi) target abnormal epigenetic states
associated with a variety of pathologies including cancer. Here, the development of a prodrug of
the canonical broad-spectrum HDACi suberoylanilide hydroxamic acid (SAHA) is described.
Although hydroxamic acids are utilized universally in the development of metalloenzyme
inhibitors, they are considered poor pharmacophores with reduced activity in vivo. We
developed a prodrug of SAHA by appending a promoiety, sensitive to thiols, to the hydroxamic
acid warhead (termed SAHA-TAP). After incubation of SAHA-TAP with an HDAC, the thiol of
a conserved HDAC cysteine residue becomes covalently tagged with the promoiety, initiating a
cascade reaction that leads to the release of SAHA. Mass spectrometry and enzyme kinetics
experiments validate that the cysteine residue is covalently appended with the TAP promoiety.
SAHA-TAP demonstrates cytotoxicity activity against various cancer cell lines. This strategy
represents an original prodrug design with a dual-mode of action for HDAC inhibition.
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Transcription is a tightly regulated biological process that is the first step in gene expression.1-3
In eukaryotic cells, sequence-specific DNA binding factors control the flow of genetic
information from DNA to RNA, thereby regulating transcription. In cells, DNA is tightly
compacted into chromatin, a highly organized and dynamic complex between DNA and proteins.
When gene transcription is activated, the DNA is made accessible to transcription factors via
nucleosome modification.1, 2 The local architecture of chromatin, which is influenced by post-
translational modifications of histones, can regulate gene expression. These modifications
include methylation, phosphorylation, and acetylation of core histones. Histone acetylation
occurs at the ε-amino groups of conserved lysine residues near the N-termini. Acetylation levels
of core histones are a result of the balance between histone acetyltransferases (HATs) and
histone deacetylases (HDACs).1-4 Increased levels of histone acetylation are generally associated
with transcriptional activity, whereas decreased levels of histone acetylation are associated with
repression of transcription. Additionally, acetylation of specific lysines on histone tails
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facilitates the recruitment of bromodomain-containing chromatin remodeling factors.5,
Furthermore, acetylated lysines have been observed in many cellular proteins indicating that
HATs and HDACs do not function solely to modify histones.7
Histone deacetylase inhibitors (HDACi) have been developed as a class of therapeutic agents
intended to target aberrant epigenetic states associated with a variety of pathologies, most
notably cancer.8 Recent findings have shown that the relief of oncogenic transcriptional
repressors by HDACi can lead to cell cycle arrest and apoptosis.1-4 This is because many cancers
have evolved such that pro-apoptotic pathways are transcriptionally repressed via histone
deacetylation. HDACi prevent deacetylation of the lysine residues of the histone tails, which in
turn leads to transcriptional activation, gene expression, and cell death.1, 8
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The development of HDACi has been ongoing, and >10 candidates have progressed to clinical
trials.3 HDACi can be subdivided into structural classes including hydroxamic acids, cyclic
peptides, aliphatic acids, and benzamides.9 The HDACi Vorinostat (suberoylanilide hydroxamic
acid, SAHA) received approval by the United States Food and Drug Administration (FDA) in
2006 for the treatment of cutaneous T-cell lymphoma (CTCL).10 Crystallization of SAHA with
HDAC8 supported a model involving the linkage of a metal-binding pharmacophore (MBP) to a
capping group designed to form favorable interactions with amino acid residues at the entrance
to the active site tunnel (Figure 1a).11 Three other HDACi have been approved by the FDA
including Panobinostat and Belinostat, both broad-spectrum, hydroxamate-based HDACi for the
treatment of multiple myeloma or relapsed/refractory peripheral T-cell lymphoma, respectively
(Figure 1a).12, 13 Romidepsin (FK228), a cyclic peptide HDACi that uses a thiol group to
coordinate the active site metal ion, is approved for CTCL treatment (Figure 1a).10
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SAHA, Romidepsin, and Panobinostat act to inhibit most isoforms of the metal-dependent
HDAC family and are regarded as broad-spectrum HDAC inhibitors. Despite promising clinical
results for HDACi, these drugs have not been effective in clinical trials involving solid tumors.
In fact, these FDA-approved drugs have been associated with the onset of serious side effects
including: fatigue, gastrointestinal issues (diarrhea, nausea, vomiting), and hematologic
complications (thrombocytopenia, anemia, neutropenia).8, 10 Both SAHA and Romidepsin have
also been associated with cardiotoxicity.8 Clinical studies in humans determined the major
metabolic pathways of SAHA degradation involve glucoronidation by UDP-
glucoronosyltransferases (UGTs) to generate inactive 1 (Figure 1b). Alternatively, hydrolysis of
SAHA to the carboxylic acid analog (2), followed by β-oxidation generates the inactive
metabolite 4-anilino-4-oxoxbutanoic acid (3, Figure 1b).10, 14 Clinical studies determined that the
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mean steady state serum exposures of 1 and 2 were 4-fold and 13-fold higher than SAHA,
respectively. Additionally, the apparent t1/2 of SAHA in human serum was ~1.5 h for patients
receiving single doses of 400 mg SAHA.8, 10, 14 The poor pharmacokinetic (PK) properties of
SAHA are similar for other hydroxamic acid-based compounds and involve chemical instability
and rapid elimination.8, 15 In fact, the FDA has approved SAHA for CTCL only in patients with
persistent or recurrent disease who have already followed two systemic therapies.8 Similarly,
the FDA has only approved Romidepsin for CTCL treatment in patients who have received at
least one prior systemic therapy, and Panobinostat is only administered after two prior standard
therapies have failed.16 The onset of these deleterious side effects is proposed to originate, in
part, from the lack of selectivity of these drugs for a specific HDAC isozyme.8
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Figure 1. FDA-approved HDAC inhibitors. (a) The hydroxamic acid and sulfhydryl MBP
donor atoms of SAHA, Panobinostat, Belinostat, and Romidepsin are shown in red. (b)
Metabolism of SAHA. Upon systemic circulation, UGT enzymes localized in the liver can
convert SAHA to a SAHA β-D-glucuronide (1) rendering the drug inactive. A different pathway
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involves initial hydrolysis of SAHA to the corresponding carboxylic acid (2), followed by
oxidation to 3.
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Chemically modified versions of active drugs have been developed in an effort to overcome
barriers to drug formulation and delivery. The modified, latent version of the drug, termed the
prodrug, undergoes a transformation in the presence of a desired chemical or enzymatic stimulus
in vivo to generate the active agent.17, 18 The chemical group appended to the active drug
rendering it inactive is termed the promoiety. Only a handful of reports have investigated HDAC
prodrugs, with most studies focused on developing acyl derivatives of SAHA or similar
hydroxamic acid-based HDACi to enhance cell permeability and hydrolytic stability.19 As
expected, these prodrugs showed little activity as HDAC inhibitors and biochemical assays
suggest that the acylated prodrugs are more cell-permeable than the hydroxamic acid parent
drugs. A similar report investigated a carbamate prodrug concept for hydroxamate HDACi
(including SAHA) to improve drug-like properties, including cellular permeability.20 However,
both of these strategies rely on hydrolysis in vivo to release the active drug and do not improve
drug-target specificity for selected disease states or sites of disease.
Initially, we sought to develop new HDAC inhibitor prodrugs (proinhibitors) that become
activated in the presence of thiols such as glutathione in its reduced form (GSH), which is
frequently more abundant at the site of disease (e.g. cancer).21 Previously, Huang and coworkers
reported the development of a long-wavelength fluorescent probe involving a quinone-methide
reaction that can detect physiologically relevant thiols including GSH.22 Although the quinone
promoiety functions as an electrophilic Michael acceptor, it was determined that other
biologically relevant nucleophiles, including serine and lysine, were unreactive with this
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functionality. Our prodrug approach considered the covalent appendage of this quinone
promoiety to the hydroxamate of an HDACi, since the alkylation of hydroxamates has shown to
be effective in improving PK properties including hydrolytic stability, cellular permeability, and
glucoronidation.19, 23, 24
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As described below, we found that even in the absence of nucleophilic thiols, we observed
activation of our prodrug (SAHA-TAP); sequence homology analysis revealed a single cysteine
(Cys) residue is conserved in all metal-dependent HDAC isoforms, which we found was reactive
with our prodrug (Supporting Information, Figure S1).25 The crystal structure of HDAC8
complexed with SAHA reveals that the conserved Cys (Cys153 for HDAC8) is located in the
catalytic active site pocket ~5.6 Å away from the hydroxamic acid moiety of SAHA (Figure 2).
Thus, we have concluded that our prodrug is cleaved by the sulfhydryl moiety of the conserved
Cys of HDAC, leading to drug activation and a dual mode of inhibition: covalent modification of
the conserved Cys leading to the formation of an inactive, covalently modified enzyme and
release of the competitive inhibitor SAHA.
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Figure 2. Protein crystal structure of HDAC8 complexed with SAHA. The distance between
the sulfhydryl moiety of Cys153 and the nitrogen atom of the MBP of SAHA was determined to
be 5.6 Å. SAHA and Cys153 are shown as sticks in color code (carbon = grey, nitrogen = blue,
oxygen = red, sulfur = yellow) and the Zn2+ ion is shown as an orange sphere (PDB: 1T69).
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Results and Discussion
Development of a Unique HDACi Prodrug.
SAHA was chosen as the drug of interest because it is FDA-approved for the treatment of
CTCL and has been well studied. A prodrug of SAHA containing a quinone-based, thiol-
sensitive promoiety was designed, termed SAHA-TAP (TAP = thiol activated prodrug) (Figure
3). To develop SAHA-TAP, the promoiety was first synthesized using a modified literature
procedure.22 The promoiety was appended to SAHA under basic conditions to generate SAHA-
TAP. A control compound, SAHA-OBn, was synthesized in a similar manner. This compound
contains a benzyl moiety appended to SAHA via the N-hydroxyl functionality. SAHA-OBn was
designed to have a similar structure to SAHA-TAP, but to be unreactive toward nucleophilic
thiols.
Assessment of SAHA-TAP Reactivity with GSH.
To evaluate the reactivity of SAHA-TAP and SAHA-OBn with nucelophilic thiols, analytical
HPLC was utilized under simulated physiological conditions (50 mM HEPES, pH 7.4). SAHA-
TAP was treated with GSH (2 mM, 2 eq) to confirm that the prodrug is indeed reactive with
thiols. After incubation of SAHA-TAP with GSH, three distinct peaks were apparent in the
HPLC chromatogram (Figure 3a). Liquid chromatography-mass spectrometry (LCMS) confirms
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that the identity of the first peak (Rt = 10.5 min) is the quinone-methide side product, the second
peak is SAHA (Rt = 10.7 min) and the third peak is unreacted SAHA-TAP (Rt = 14.7 min).
Treatment of SAHA-OBn with GSH under the same conditions resulted in no change in the
HPLC chromatogram, indicative of the expected lack of reactivity (Figure 3b). Having shown
that SAHA-TAP reacts rapidly with GSH, the aqueous stability of SAHA-TAP was evaluated
under simulated physiological conditions (50 mM HEPES, pH 7.4). An HPLC chromatogram
was obtained immediately after preparation in aqueous buffer and a second trace was collected
after incubation at 37 °C for 24 h. SAHA-TAP was determined to be >97% stable to hydrolysis
in the absence of thiols (Figure S2). The activation of SAHA-TAP by reaction with GSH is
summarized in Figure 3c.
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Figure 3. (a) Activation of SAHA-TAP by GSH. In the presence of GSH, the sulfhydryl moiety
can attack the electrophilic quinone moiety. Subsequent rearrangement releases SAHA along
with a quinone-methide adduct. (b) HPLC trace of SAHA (black), SAHA-TAP (blue), and
SAHA-TAP after treatment with GSH (2 mM, 2 eq) for 2 h at 37 °C (red). Retention times are
10.7 min for SAHA, 14.7 min for SAHA-TAP, and 10.5 min for the quinone-methide GSH
adduct generated from the reaction. (c) HPLC trace of SAHA (black), SAHA-OBn (blue), and
SAHA-OBn after incubation in HEPES (50 mM, pH 7.4) for 24 h at 37 °C (red). Retention
times are 10.7 min for SAHA and 14.9 min for SAHA-OBn.
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HDAC Inhibition by SAHA-TAP.
To determine the efficacy of the SAHA-TAP prodrug strategy, the ability of SAHA-TAP and
SAHA-OBn to inhibit HDAC-1, -2, -3, -6, and -8 was evaluated using an optimized homogenous
fluorescence-based assay (BPS Bioscience). Surprisingly, even in the absence of exogenous
thiols, SAHA-TAP was an effective inhibitor of all HDACs tested with an apparent IC50 value
that is slightly less potent (2- to 50-fold) than the parent inhibitor SAHA (Table S1). This result
was unexpected because the metal-binding ability of the hydroxamic acid MBP of SAHA-TAP is
blocked by the promoiety, which should render the drug nearly inactive. Recent studies indicate
that the metal-free form of HDAC8 has a low affinity for SAHA analogs, further demonstrating
the importance of metal binding for HDAC inhibition.26 To determine if a component of the
biochemical assay resulted in SAHA-TAP activation, analytical HPLC was utilized. SAHA-
TAP was incubated with either BSA (5 mg/mL) or trypsin (5 mg/mL) at 37 °C for 2 h; however,
SAHA-TAP was found to be >95% stable in the presence of either of these assay components
(data not shown).
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Because SAHA-TAP is a larger molecule than SAHA and the metal-binding hydroxamate
group is blocked, it is feasible that the promoiety may be positioned very close to Cys153 when
bound to HDAC8. This positioning could be ideal for nucleophilic attack by the sulfhydryl
moiety, leading to covalent modification and SAHA release. We hypothesized that the Cys153
residue of HDAC8 reacts with bound SAHA-TAP, resulting in a covalent modification of the
protein and subsequent release of SAHA, a competitive inhibitor (Figure S3). It is important to
note that there are many other Cys residues in the metal-dependent HDAC isoforms (e.g. 10 Cys
residues in HDAC8), and activation of SAHA-TAP by these residues may also be responsible in
part for the release of SAHA we observe (vide supra).
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Mass Spectrometry Analysis
To investigate whether the active site Cys153 is covalently modified by the SAHA-TAP
promoiety, mass spectrometry (MS) techniques were utilized. Digestion of wild type (WT)
HDAC8 with trypsin yields an 18 amino acid peptide containing Cys153, which can be used to
monitor the modification via mass spectrometry (Figure 4a). If a covalent modification occurs at
this position after treatment of WT HDAC8 with SAHA-TAP, the expected MS ion for this
peptide fragment will be different from the unmodified parent ion.
HDAC8 (WT, with or without incubation with a 12-fold excess of SAHA-TAP for 60 min at
37 °C) was digested with trypsin, and the resulting peptides were analyzed by LCMS. The
expected parent ion for the WT HDAC8 Cys153 peptide was consistent with a peak at Rt = 84.5
min (Figure 4b). Similarly, a peak at Rt = 88.5 min corresponds to the expected parent ion for
the covalently modified Cys153 HDAC8 peptide after SAHA-TAP treatment (Figure 4c). To
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eliminate other digestion products that could account for this ion, further MS techniques were
applied to verify that these ion peaks correspond to the peptide sequence of interest.
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Figure 4. HDAC8 tryptic fragment MS data. (a) Expected HDAC8 Cys153 peptide upon
digestion with trypsin (Cys153 in bold). The expected MS ion for the WT protein is shown
along with the expected ion for the peptide including the covalent addition of the TAP promoiety
on Cys153. (b) The tryptic fragment of WT HDAC8 (Rt = 84.5 min) is consistent with the
expected [M+2H]2+ ion. (c) After treatment of HDAC8 with SAHA-TAP (12 eq) and digestion
with trypsin, a peak aligning with the expected [M+2H]2+ ion is observed (Rt = 88.5 min).
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Tandem mass spectrometry (MS2 or MS/MS) is routinely used in proteomics to characterize
amino acid sequences of proteins, where peptides undergo further fragmentation to amino acid
aggregates.27 The fragmentation patterns observed in the MS/MS spectra of tryptic peptides for
WT HDAC8 and SAHA-TAP treated HDAC8 were investigated to obtain additional insight into
the possibility of covalent modification of Cys153. The expected monoisotopic masses for the y
ion series in both WT HDAC8 and the SAHA-TAP treated sample are summarized in Table S2.
The expected y fragment ions for both peptides align until Cys153 (y12), where this ion and each
subsequent ion have different masses. Indeed, the MS/MS fragmentation spectrum for the WT
HDAC8 tryptic peptide (parent ion m/z=1006.98, [M+2H]2+) shows many of the expected y ions
(Figure S4). Similarly, the MS/MS fragmentation spectrum for the SAHA-TAP treated HDAC8
tryptic peptide (parent ion m/z=1059.53 [M+2H]2+) shows many y ions including the
characteristic peak of m/z=1511.82 (Figure S5). This peak is indicative of a covalent
modification of m/z=162.1 for the HDAC8 tryptic fragment at Cys153. These data prove that the
covalent modification of Cys153 is occurring to form an adduct containing the SAHA-TAP
promoiety, as shown in Fig. 2c.
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MS analysis also indicated that Cys102, Cys244, and Cys314 could be modified by the TAP
moiety from SAHA-TAP. Importantly, the peptides containing surface cysteines (Cys275 and
Cys352) were not modified with TAP, suggesting that activation is not non-specific.
Nonetheless, the hypothesized mechanism of activation involving a covalent modification of Cys
residues in HDAC8, including Cys153, was observed, which can aid in explaining the inhibition
of HDACs by SAHA-TAP even in the absence of exogenous nucleophilic thiols, as observed in
the in vitro assays.
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In vitro Time Dependence of SAHA-TAP HDAC Inhibition.
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With the MS data in hand confirming covalent modification of Cys residues in HDAC8, we
sought to investigate the kinetics of inhibition of HDAC8 by SAHA-TAP using the Fluor-de-Lys
activity assay (Enzo Life Sciences). A previous study determined that the catalytic activity and
thus inhibition of HDAC8 is dependent on the identity of the active site metal ion (e.g. Co2+,
Fe2+, Zn2+, and Ni2+).28 To obtain the most accurate results, apo-HDAC8 (human, recombinant)
was initially prepared before the addition of Zn2+ in a 1:1 stoichiometry. An initial test of
HDAC8 inhibition by preincubation of the enzyme with SAHA-TAP demonstrated that activity
loss occurred within the first 0.5 h (data not shown). To determine the kinetics for the time-
dependent inhibition by SAHA-TAP, HDAC8 progress curves were measured through a range of
inhibitor concentrations (Figure 5a). For these reactions, the Fluor-de-Lys HDAC8 substrate
(150 ꢀM) and SAHA-TAP (0-20 ꢀM) were added to each assay prior to initiating the reactions
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with WT HDAC8 (0.5 ꢀM). Over a time course, aliquots of the reactions were stopped by
dilution into a solution of Trichostatin A and trypsin, and product formation was analyzed from
the resulting change in fluorescence. Analysis of the HDAC8 progress curves in the presence of
increasing concentrations of SAHA-TAP demonstrates a non-linear formation of product with
respect to time. Equation 1, which describes the time-dependent decrease in initial velocity
under steady state turnover conditions,29, 30 was fit to the progress curves:
(Equation 1)
where P represents production formation, vs and v0 represent final and initial velocities,
respectively, t is time, C is the initial fluorescent ratio, and kobs is the rate constant describing the
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transition from the initial velocity to the final steady state velocity, reflecting the time-dependent
enzyme inactivation.
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This fit reveals that both the initial velocity and the rate constant for inactivation, kobs, have a
hyperbolic dependence on the concentration of SAHA-TAP (Figures 5b-c). The initial velocity
decreases with an apparent Ki = 7 ± 4 ꢀM and the rate constant for inactivation increases with a
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K1/2 = 8 ± 2 ꢀM to a maximal rate constant of 0.0013 s-1 at saturating SAHA-TAP. This type of
inhibition is characteristic of a two-step mechanism (Scheme 1) in which a rapid reversible step,
such as binding of SAHA-TAP to HDAC8, is followed by a time dependent step, consistent with
irreversible inactivation.29, 30 These data demonstrate that the prodrug, SAHA-TAP, is capable
of binding to and inhibiting HDAC8. The time-dependent decrease in activity is consistent with
the MS data demonstrating the formation of a covalent enzyme adduct.
kobs
KI
E + I
E I
E I
(Scheme 1)
For comparison, progress curves for inhibition of HDAC8 with the competitive inhibitor
SAHA and a negative control, SAHA-OBn, were evaluated. These assays were performed in the
same manner as the SAHA-TAP progress curves, where substrate and inhibitor were added to
the assay prior to the addition of HDAC8 to initiate the reaction. In contrast to the data with
SAHA-TAP, these progress curves are linear with no observable curvature for all concentrations
of SAHA (Figure S6a) and SAHA-OBn (Figure S6b). As expected, HDAC8 (0.5 ꢀM) is
inhibited >90% by SAHA in the concentration range tested (2-8 ꢀM), which is in agreement with
the 250 nM Ki reported for Zn2+-HDAC8.28 SAHA-OBn (2-10 ꢀM) does not inhibit the activity
of HDAC8. The lack of inhibition by SAHA-OBn is consistent with previous IC50 data (Table
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S1). Taken together, these data show that SAHA-TAP has a unique mode of inhibition for
HDAC8 when compared to SAHA. This inhibitor functions both as a competitive inhibitor, and
as a time-dependent inactivator, in contrast to the linear, time-independent inhibition observed
for SAHA.
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To determine the role of Cys153 on the time dependent inhibition of HDAC8, a Cys153Ala
(C153A) HDAC8 mutant was prepared and purified. In vitro assays for the mutant were
conducted under the same conditions used for WT HDAC8. Progress curves for these assays
reveal dose-responsive inhibition with Ki = 8±4 ꢀM for SAHA-TAP (Figure 5d and Figure S7).
Furthermore, these progress curves are linear for all concentrations of inhibitor, showing a loss
of the time-dependent inhibition observed with WT HDAC8. This data suggests that SAHA-
TAP, containing the same linker spacer and capping group as SAHA, can bind HDAC8 in a non-
covalent manner, inhibiting the enzyme (Scheme 1). The linear progress curves also
demonstrate that Cys153 is important for the time-dependent inactivation, eliminating an
alternate explanation that the time-dependent inhibition is due to the slow formation of SAHA
from SAHA-TAP. Progress curves with C153A HDAC8 measured with SAHA and SAHA-OBn
data reveal that the HDAC8 mutant remains susceptible to inhibition by SAHA and not SAHA-
OBn (Figure S8).
Collectively, these data indicate both that SAHA-TAP binds non-covalently to HDAC8 to
inhibit the activity and that the time-dependence mainly reflects the reaction of SAHA-TAP with
Cys153. Although the MS data suggests that SAHA-TAP can react with other Cys residues in
HDAC8 leading to SAHA release, C153A HDAC8 is not inactivated in a time-dependent
manner, demonstrating the importance of this particular Cys in the mechanism of inhibition for
the WT enzyme. The combination of the enzyme kinetics and MS data provides evidence that
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the inactivation of HDAC8 by SAHA-TAP involves two steps: non-covalent binding of SAHA-
TAP to HDAC8 followed by covalent modification of Cys153.
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a"
c"
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1.5
1.0
0.5
0.0
0.0012
0.0008
0.0004
0.0000
0"µM"
2"µM"
4"µM"
6"µM"
8"µM"
12"µM"
20"µM"
0
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SAHAꢀTAP (ꢁM)
Time (s)
b"
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0.0003
0.0002
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4"µM"
6"µM"
8"µM"
12"µM"
16"µM"
0
5
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25
2,000
4,000
6,000
SAHAꢀTAP (ꢁM)
Time (s)
Figure 5. Time dependence of HDAC8 inhibition. (a) WT HDAC8 (0.5 ꢀM) progress curves at
varying concentrations (0-20 ꢀM) of SAHA-TAP. Dependence of both the (b) initial rate, v0,
and (c) kobs on the concentration of SAHA-TAP. (d) C153A HDAC8 (2 ꢀM) progress curves at
varying concentrations (0-20 ꢀM) of SAHA-TAP.
Plasma Stability
As mentioned earlier, SAHA suffers from poor PK properties, including hydrolytic instability
with t1/2 ~1.5 h. To determine the stability of SAHA-TAP in a biologically relevant model,
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human plasma stability studies were conducted as previously reported.31 After incubating SAHA
or SAHA-TAP in human plasma, aliquots were withdrawn at various time points (0, 15, 30, 60,
and 120 min), quenched with acetonitrile, filtered, and evaluated via analytical HPLC. The
percent parent compound remaining was determined by integrating the area under the curve and
comparing this number with the initial sample of parent compound at an incubation time of 0
min. Approximately 72% of SAHA-TAP remained after 1 h incubation at 37 °C while only
~60% of SAHA remained under identical conditions. Only ~50% of either parent compound
remained after a 2 h incubation at 37 °C (Figure 6a). After 2 h incubation at 37 °C the HPLC
trace of SAHA-TAP showed that the major degradation peak (~23%) corresponds to SAHA with
~52% SAHA-TAP remaining and ~25% other products. This suggests that hydrolysis of SAHA-
TAP to SAHA is a major component of the degradation process in human plasma (Figure S9).
For comparison, after a 2 h incubation in human plasma under identical conditions, the HPLC
chromatogram for SAHA shows the emergence of a series of new unidentifiable peaks (~45%)
with ~55% SAHA remaining (Figure S9). Even though SAHA-TAP gradually degrades over
this 2 h period, it is relatively slow and results in the release of the active drug SAHA. Efforts to
identify other product peaks via LCMS were inconclusive. Overall, this study indicates that
SAHA-TAP has a moderately improved stability profile than SAHA in human plasma.
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Cell Proliferation Studies
With the kinetics of activation and plasma stability of SAHA-TAP elucidated, we then studied
the effect of SAHA-TAP on the proliferation of a variety of cell lines. Because SAHA is FDA
approved for CTCL, we selected HH (CTCL) and Jurkat (T-cell leukemia) cell lines for analysis.
The viability of NIH/3T3 (mouse embryo fibroblast) was also tested to determine the toxicity of
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each compound for a non-cancer cell line. The EC50 values of SAHA, SAHA-TAP, and SAHA-
OBn are shown in Figure 6b for each cell line. The observed EC50 values of SAHA for HH and
for Jurkat cell lines were 1.03±0.21 ꢀM and 1.66±0.14 ꢀM, respectively, consistent with
previously reported data.32, 33 SAHA-TAP is ~3-4-fold less potent than SAHA, but is still an
active compound, with calculated EC50 values of 3.38±0.30 ꢀM and 6.05±0.14 ꢀM for HH and
Jurkat cells, respectively. This difference in potency may be attributed to the alkylated Cys
affecting the binding of SAHA to HDAC8; further structural studies are needed to confirm this
hypothesis. Interestingly, SAHA also is toxic for NIH/3T3 cell lines, with a calculated EC50 of
4.80±0.99 ꢀM. Other studies also report cytotoxicity of healthy kidney cells (Vero) after
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treatment with SAHA (EC50 = 5.20±0.96 ꢀM).28 Unfortunately, cell proliferation studies
indicate that SAHA-TAP is also slightly toxic for NIH/3T3 cells with an apparent EC50 value of
9.37±1.21 ꢀM. As expected, cell proliferation is unaffected by the addition of SAHA-OBn for
all cells studied.
Intracellular Target Validation
With the cell proliferation data in hand for both Jurkat and HH cells, we sought to validate the
intracellular target of SAHA-TAP using western blotting techniques. Broad-spectrum HDACi
are known to increase the steady-state accumulation of tubulin, an endogenous HDAC substrate
and a common marker for intracellular HDAC6 activity.34, 35 For these experiments, SAHA was
used as a positive control, since it has been shown to dramatically increase tubulin acetylation in
a variety of cells.33, 36 Indeed, we observed that SAHA-TAP increased tubulin acetylation in
both Jurkat and HH cells without disturbing actin levels (Figure 6c), suggesting that the
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antiproliferative mechanism of action for SAHA-TAP, like SAHA, involves nonspecific HDAC
inhibition.
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Figure 6. (a) Plasma stability for SAHA (red circles) and SAHA-TAP (black squares) over time
(mean ± sd). (b) Cellular EC50 values (ꢀM) obtained from the MTS cell proliferation assay. NI
= no inhibition at 50 ꢀM. (c) Western blot analysis of tubulin acetylation for Jurkat (lanes 1-5)
and HH (lanes 6-10) cells. Lane 1, control (no treatment); lane 2, SAHA (1.5 ꢀM); lane 3,
SAHA (20 ꢀM); lane 4, SAHA-TAP (6 ꢀM); lane 5, SAHA-TAP (20 ꢀM); lane 6, control (no
treatment); lane 7, SAHA (1.5 ꢀM); lane 8, SAHA (20 ꢀM); lane 9, SAHA-TAP (6 ꢀM); lane
10, SAHA-TAP (20 ꢀM).
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Conclusions
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A thiol-sensitive prodrug of the FDA-approved HDACi SAHA has been developed that
displays a time-dependent inhibition of HDAC8. SAHA-TAP functions as a dual-mode HDAC
inhibitor with both a covalent modification and a non-covalent, conventional mode of action.
SAHA-TAP is susceptible to nucleophilic attack by Cys residues on the target HDAC,
particularly the conserved Cys153 residue in the catalytic domain. These Cys residues are
covalently modified with the promoiety, inactivating the enzyme, followed by the release of the
competitive inhibitor SAHA. Proteomic MS confirms that this modification occurs at Cys153,
and the kinetics of inhibition show unambiguous time-dependent inhibition of HDAC8 by
SAHA-TAP, indicative of a covalent modification with release of SAHA. The HDAC8 C153A
mutant retains the non-covalent mode of inhibition by SAHA-TAP (Scheme 1), while the time-
dependent mode of inhibition disappears. This result demonstrates the importance of the active
site Cys in the inactivation of HDAC8 by SAHA-TAP. The stability of SAHA-TAP in human
plasma is slightly improved with slow conversion to SAHA observed. In contrast, SAHA is
rapidly degraded to several products under identical conditions, consistent with previous
literature studies. Finally, cellular proliferation studies show a clear dose-response relationship
with SAHA-TAP for two distinct cancer cell lines, with only moderately inferior EC50 values
compared to SAHA; immunoblotting confirms that the antiproliferative mechanism of action of
SAHA-TAP involves HDAC inhibition. To the best of our knowledge, SAHA-TAP is the first
dual-mode HDAC proinhibitor that exploits the modification of an endogenous, conserved Cys
residues, namely the catalytic site Cys153 residue in HDAC8, to generate a covalent adduct in
addition to releasing a competitive inhibitor.
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Experimental Section
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Enzyme Inhibition Assays. HDAC-1, -2, -3, -6 and -8 activity was determined in vitro with
an optimized homogenous assay performed in a 384-well plate. Recombinant, full-length HDAC
protein (BPS Biosciences) was incubated with fluorophore conjugated substrate, MAZ1600 and
MAZ1675, at [Substrate] = Km (MAZ1600; 11 ꢀM for HDAC1, 18 ꢀM for HDAC2, 9 ꢀM for
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HDAC3, 4 ꢀM for HDAC6; MAZ1675; 263 ꢀM for HDAC8). Reactions were performed in
assay buffer (50 mM HEPES, 100 mM KCl, 0.001% Tween-20, 0.05% BSA and pH 7.4.
Additional 200 ꢀM TCEP was added for HDAC6) and followed by fluorogenic release of 7-
amino-4-methylcoumarin from substrate upon deacetylase and trypsin enzymatic activity.
Flourescence measurements were obtained every five minutes using a multilabel plate reader and
plate-stacker (Envision; Perkin-Elmer). Each plate was analyzed by plate repeat, and the first
derivative within the linear range was imported into analytical software (Spotfire DecisionSite).
Replicate experimental data from incubations with inhibitor were normalized to DMSO controls
([DMSO] < 0.5%). IC50 values are determined by logistic regression with unconstrained
maximum and minimum values.
Mass Spectrometry Experiments. Preparation. An aliquot of HDAC8 (1.7 ꢀM) was
incubated with SAHA-TAP (12 eq, 20 ꢀM) at 37 ºC for 1 h. The protein was purified by SDS-
PAGE followed by Commassie staining prior to analysis.
In Gel Digest. The gel slices of interest were cut to 1 mm cubes and destained 3 times by first
washing with 100 mM ammonium bicarbonate (100 ꢀL) for 15 minutes, followed by the addition
of ACN (100 ꢀL) for 15 minutes. The supernatant was collected and samples were dried in a
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speedvac. The samples were then reduced by the addition of 100 ꢀM ammonium bicarbonate-10
mM DTT (200 ꢀL) and incubated at 56 °C for 30 min. The liquid was removed and 100 mM
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ammonium bicarbonate-55 mM iodoacetamide (200 ꢀL) was added to gel pieces and incubated
at RT in the dark for 20 min. After the removal of the supernatant and one wash with 100 mM
ammonium bicarbonate for 15 min, the same volume of ACN was added to dehydrate the gel
pieces. The solution was then removed and the samples were dried in a speedvac. For digestion,
enough solution of ice-cold trypsin (0.01 mg/mL) in 50 mM ammonium bicarbonate was added
to cover the gel pieces, and then incubated on ice for 30 min. After complete rehydration, the
excess trypsin solution was removed, replaced with fresh 50 mM ammonium bicarbonate, and
incubated overnight at 37 °C. The peptides were extracted twice by the addition of 0.2% formic
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acid and 5% ACN (50 ꢀL) and vortex mixing at RT for 30 min. The supernatant was removed
and saved. A total of 50 ꢀL of 50% ACN-0.2% formic acid was added to the sample, which was
vortexed again at RT for 30 min. The supernatant was removed and combined with the
supernatant from the first extraction. The combined extractions were analyzed directly by LCMS.
LC-MS/MS. Trypsin-digested peptides were analyzed by HPLC coupled with tandem mass
spectrometry (LC-MS/MS) using nano-spray ionization. The nanospray ionization experiments
were performed using a TripleTOF 5600 hybrid mass spectrometer (SCIEX) interfaced with
nano-scale reversed-phase HPLC (Tempo) using a 10 cm-100 micron ID glass capillary packed
with 5-mm C18 ZorbaxTM beads (Agilent Technologies). Peptides were eluted from the C18
column into the mass spectrometer using a linear gradient (5–60%) of ACN at a flow rate of 250
mL/min for 1 h. The buffers used to create the ACN gradient are: Buffer A (98% H2O, 2%
ACN, 0.2% formic acid, and 0.005% TFA) and Buffer B (0.2% formic acid and 0.005% TFA in
ACN). MS/MS data were acquired in a data-dependent manner in which the MS1 data was
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acquired for 250 ms at m/z of 400 to1250 Da and the MS/MS data was acquired from m/z of 50
to 2,000 Da. For Independent data acquisition (IDA) parameters MS1-TOF 250 milliseconds,
followed by 50 MS2 events of 25 milliseconds each. The IDA criteria, over 200 counts
threshold, charge state +2-4 with 4 seconds exclusion. Finally, the collected data were analyzed
using MASCOT® (Matrix Sciences).
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Protein Expression and Purification. Recombinant human HDAC8 in a pET-20b-derived
plasmid with an added c-terminal TEV protease cleavage site and His6 tag (termed pHD)28 was
expressed and purified in E.coli BL21(DE3) according to Gantt and coworkers,28 with the
following modification: elution from the nickel columns was performed using a linear gradient
(10-250 mM imidazole). In the preparation of apo-enzyme, HDAC8 was dialyzed twice at 4 °C
against 4 L of 25 mM MOPS, 1 mM EDTA, 5 mM KCl, 1 mM TCEP, pH 7.5 followed by four
times against 2 L of 25 mM MOPS, 5 mM KCl, 1 mM TCEP, pH 7.5. All components were free
of transition metals and dialysis occurred in plasticware that had been washed with EDTA and
rinsed with Milli-Q ddH2O. Apo-enzyme was stored at -80 °C in the same metal-free buffer.
The Cys153Ala HDAC8 mutant was constructed in a pHD4 TEV-His plasmid, using the
QuikChange site-directed mutagenesis protocol and kit, and the mutation was confirmed by the
UM DNA sequencing facility. This construct was expressed and purified in the same manner as
wild-type enzyme.
HDAC8 Time-course Inhibition Experiments. Recombinant WT or C153A mutant human
HDAC8 was reconstituted for 1 h on ice at a 1:1 stoichiometry (at 10 ꢀM) with Zn2+ in 1×
HDAC assay buffer (25 mM HEPES, 3 mM KCl, 137 mM NaCl, pH 8.0). The 5 and 50 mM
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SAHA-TAP, SAHA-OBn, and SAHA stocks were serially diluted into decreasing concentrations
of DMSO to maintain solubility of the compounds. Reaction mixtures of 1× HDAC assay
buffer, 150 ꢀM Fluor-de-Lys peptide substrate (R – H – K(Ac) - K(Ac) - fluorophore) (Enzo
Life Sciences), and various concentrations of inhibitors (SAHA-TAP, SAHA-OBn, or SAHA)
were prepared and allowed to equilibrate at 30 °C. The final DMSO content was <1%. Assays
were initiated by addition of wild-type (0.5 ꢀM) or Cys153Ala mutant (2 ꢀM) HDAC8. At
various time points a reaction aliquot (5 ꢀL) was diluted into a Fluor-de-Lys quench solution (45
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ꢀL) containing trypsin and trichostatin A (TSA). Assays were read in 96-well plates (Corning
3686) using a PolarStar Fluorescent plate reader. The fluorescence corresponding to product
formation (λex = 340 nm, λem = 450 nm) and remaining substrate (λex = 340 nm, λem = 380 nm)
was measured and the ratio of product formed/remaining substrate is reported. Standard curves
demonstrate that this fluorescent ratio linearly reflects product under these conditions.
Cell Proliferation Studies. HH and Jurkat cell lines were obtained from ATCC (Manassas,
VA, USA), and grown on RPMI 1640 medium supplemented with 10% fetal bovine serum
(Gibco, Grand Island, NY, USA). The NIH/3T3 cell line was kindly donated by Dr. Richard
Klemke and grown on DMEM medium supplemented with 10% fetal bovine serum (Gibco,
Grand Island, NY, USA) at 37 °C in an incubator with 5% CO2. The CellTiter 96 AQueous One
Solution Cell Proliferation assay (MTS) kit was purchased from Promega (Madison, WI, USA).
Cell viability was measured using the MTS assay according to the manufacturer protocol. To
start the assay, cells were counted with a hemocytometer, diluted with fresh medium to the
proper concentration and seeded in 96 well plates (5000 cells/well for NIH/3T3 and 20000
cells/well for HH and Jurkat). Jurkat and HH cells were then directly incubated in media
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containing the various concentrations of drugs for 70 h (ranging from 0.5 ꢀM to 128 ꢀM).
NIH/3T3 cells were first incubated at 37 °C with 5% CO2 for 16 h prior to the drug treatment for
cell attachment. The cells were then treated with various concentrations of drugs for 70 h. The
CellTiter 96 AQueous One Solution was added (20 ꢀL per well) and the plate was incubated at
37 °C for 2 h (NIH 3T3) or 4 h (HH and Jurkat). The absorbance was recorded at 490 nm using
the BioTek Synergy HT microplate reader. Each concentration of drug treatment was conducted
in triplicate for each trail with 2-3 trials conducted.
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Plasma Stability. The plasma stability of SAHA and SAHA-TAP was investigated with
pooled normal human plasma (Innovative Research, Novi, MI). In duplicate, plasma (1.0 mL)
was pre-incubated for 2 min at 37 °C followed by the addition of 20 ꢀL of a 5.0 mM stock
solution (DMSO). Aliquots (100 ꢀL) were withdrawn at 0, 15, 30, 60, and 120 min and
immediately quenched with 100 ꢀL ACN to precipitate the proteins. The samples were vortexed
thoroughly and centrifuged for 2 min at 13,000 RPM. The supernatant was collected and
centrifuged through 0.2 ꢀm spin filters (Corning) for 5 min at 8,000 RPM. Samples were then
frozen until analyzed by HPLC with the following method: Analytical HPLC was performed on
a HP Series 1050 System equipped with a Poroshell 120 reverse-phase column (EC-C18,
4.6×100 mm, 2.7 ꢀm). Separation was achieved with a flow rate of 1 mL min-1 and the
following mobile phase: 2.5% ACN + 0.1% formic acid in H2O (A) and 0.1% formic acid in
ACN (B). Starting with 95% A and 5% B, a linear gradient was run for 15 min to a final solvent
mixture of 5% A and 95% B, which was held for 5 min before ramping back down to 95% A and
5% B over the course of 2 min, with constant holding at this level for 4 additional min.
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Western Blot Analysis. Log phase growing HH and Jurkat cell lines were cultured until 70%
percent confluent and treated with specified concentrations of compounds for 4 hours prior to
harvesting. Cells were spun at 250 x g and washed with DPBS buffer (Life Technologies) before
lysis using RIPA Buffer (50 mM Tris-HCl (pH 7.5), 150 mM Na2EDTA, 1% Nonidet P-40, 1%
sodium deoxycholate, 0.1% sodium dodecyl sulphate) supplanted with cOmplete protease
inhibitors cocktail (Roche) for 30 min on ice. Samples were then spun at 12,000 x g before
quantification of total protein concentration using a BCA assay (Thermo). Dilutions were made
to normalize total protein concentration for each gel sample. Diluted samples were then run on a
10% SDS-PAGE gel at 100 V for 2 h before transfer onto Immobilon-P PVDF Membrane (EMD
Millipore) at 100 V for 45 min before blocking in 5% (v/v) Casein-TBST (Tris-buffered saline
Tween-20; 0.05% Tween-20 v/v) at 4 °C. Blots were then incubated with monoclonal mouse
anti-acetylated tubulin (Life Technologies), polyclonal rabbit anti-tubulin (Sigma), or anti-actin-
HRP (Santa Cruz) in 5% (v/v) BSA-TBST at dilutions according to manufacturer instructions
overnight at 4 °C. Blots were then washed by three 5 min washes in TBST (0.05% v/v) anti-
acetylated tubulin and anti-tubulin antibodies were then incubated with HRP-conjugated anti-
mouse (Santa Cruz) or anti-rabbit (Pierce). Detection was performed using SuperSignal West
Pico Substrate (Pierce) with 10% (v/v) SuperSignal West Femto Substrate (Pierce) and imaged
on ChemiDoc XRS+ System with Image Lab Software (Bio-rad).
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ASSOCIATED CONTENT
Supporting Information. Synthetic procedures, NMR and MS characterization, analytical
HPLC chromatograms, enzyme inhibition assay results, progress curves, and proteomic MS data.
This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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* To whom correspondence should be addressed.
E-mail: scohen@ucsd.edu. Telephone: (858) 822-5596
E-mail: fierke@umich.edu. Telephone: (734) 936-2678
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Author Contributions
K.B.D. conceived the project, synthesized all compounds, and designed and performed
experiments. E.D.S. designed experiments, expressed/purified WT HDAC8 and C153A mutant
HDAC8, and preformed all time-dependent progress curve experiments. Y.C. performed all cell
proliferation experiments. J.C.C. performed all western blot experiments. K.B.D., E.D.S., Y.C.,
J.C.C., P.A.J., C.A.F., and S.M.C. analyzed the data and wrote the manuscript.
ACKNOWLEDGMENT
The authors would like to acknowledge the James E. Bradner laboratory for performing
preliminary HDAC inhibition studies, and Dr. Majid Ghassemian (UCSD Biomolecular and
Proteomics MS Facility) for completing proteomic MS experiments. The authors would also like
to thank Dr. David P. Martin and Dr. David T. Puerta for helpful discussions. This work was
supported by grants from the US National Institutes of Health (NIH) through the National
Institute of General Medical Sciences (NIGMS) (R01 GM101467; P.A.J), (R01 GM40602;
C.A.F.), and (R01 GM098435; S.M.C.).
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ABBREVIATIONS
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HDAC, Histone Deacetylase; HDACi, Histone Deacetylase Inhibitor; PK, pharmacokinetics;
CTCL, Cutaneous T-Cell Lymphoma; GSH, Glutathione in its reduced form; Cys, Cysteine.
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REFERENCES
1.
Haberland, M.; Montgomery, R. L.; Olson, E. N. The many roles of histone deacetylases
in development and physiology: implications for disease and therapy. Nat. Rev. Genet. 2009,
10, 32ꢀ42.
2.
De Ruijter, A. J. M.; Van Gennip, A. H.; Caron, H. N.; Kemp, S.; Van Kuilenburg, A. B. P.
Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem. J.
2003, 370, 737ꢀ749.
3.
Marks, P. A.; Rifkind, R. A.; Richon, V. M.; Breslow, R.; Miller, T.; Kelly, W. K. Histone
deacetylases and cancer: Causes and therapies. Nat. Rev. Cancer 2001, 1, 194ꢀ202.
4.
Johnstone, R. W. Histoneꢀdeacetylase inhibitors: Novel drugs for the treatment of
cancer. Nat. Rev. Drug Discov. 2002, 1, 287ꢀ299.
5.
Agalioti, T.; Chen, G.; Thanos, D. Deciphering the Transcriptional Histone Acetylation
Code for a Human Gene. Cell 2002, 111, 381ꢀ392.
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