Structural Requirements of HDAC Inhibitors: SAHA Analogues Modified at the C2 Position Display HDAC6/8 Selectivity

Article abstract of DOI:10.1021/acsmedchemlett.6b00124

Histone deacetylase (HDAC) proteins are epigenetic regulators that deacetylate protein substrates, leading to subsequent changes in cell function. HDAC proteins are implicated in cancers, and several HDAC inhibitors have been approved by the FDA as antica

Full text of DOI:10.1021/acsmedchemlett.6b00124

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Letter
The structural requirements of HDAC inhibitors: SAHA analogs
modified at the C2 position display HDAC6/8 selectivity
Ahmed T Negmeldin, Geetha Padige, Anton V Bieliauskas, and Mary Kay H. Pflum
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00124 • Publication Date (Web): 07 Feb 2017
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The structural requirements of HDAC inhibitors: SAHA analogs
modified at the C2 position display HDAC6/8 selectivity
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Ahmed T. Negmeldin, Geetha Padige, Anton V. Bieliauskas,[a] Mary Kay H. Pflum*
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, United States
KEYWORDS: histone deacetylase, HDAC inhibitor, HDAC6 HDAC8 selective inhibitor, HDAC isoform selectivity, Vori-
nostat
ABSTRACT: Histone deacetylase (HDAC) proteins are epigenetic regulators that deacetylate protein substrates, leading to subse-
quent changes in cell function. HDAC proteins are implicated in cancers, and several HDAC inhibitors have been approved by the
FDA as anti-cancer drugs, including SAHA (suberoylanilide hydroxamic acid, Vorinostat, Zolinza™). Unfortunately, SAHA inhib-
its most HDAC isoforms, which limits its use as a pharmacological tool and may lead to side effects in the clinic. In this work
SAHA analogs substituted at the C2 position were synthesized and screened for HDAC isoform selectivity in vitro and in cells. The
most potent and selective compound, C2-n-hexyl SAHA, displayed sub-micromolar potency with 49- to 300-fold selectivity for
HDAC6 and HDAC8 compared to HDAC1, 2, and 3. Docking studies provided a structural rationale for selectivity. Modification
of the non-selective inhibitor SAHA generated HDAC6/HDAC8 dual selective inhibitors, which can be useful lead compounds
towards developing pharmacological tools and more effective anti-cancer drugs.
Histone deacetylase (HDAC) proteins play an important
role in the epigenetic regulation of transcription. HDAC pro-
teins deacetylate acetyllysine residues on nucleosomal histone
proteins, which influences gene expression.1 The HDAC fam-
ily contains eighteen proteins, which are grouped into four
classes according to their size, cellular localization, and
phylogenetic analysis.2 Classes I, II, and IV are metal depend-
ent, while class III are NAD+ dependent.2 The metal dependent
HDACs are the focus in this work.
cell signaling dysregulation, transcription and expression
changes, and protein degradation. Through these effects on
tumor cells, HDAC inhibitors can reduce proliferation, migra-
tion, and angiogenesis, enhance differentiation and immuno-
genicity, and promote apoptosis.16 In fact, several HDAC in-
hibitors are used for treatment of T-cell lymphoma, including
SAHA (suberoylanilide hydroxamic acid, Vorinostat, Figure
1) and Belinostat (Figure S41).12-14 Panobinostat (Figure S41)
was recently approved for treatment of multiple myloma.15
Unfortunately, these FDA approved drugs are relatively non-
selective and inhibit most of the eleven metal-dependent
HDAC isoforms.17 Nonspecific inhibition may account for
several mild to severe side effects associated with treatment,
including dehydration, thrombocytopenia, anorexia, and car-
diac arrhythmia.17-18 In addition, the non-selectivity of these
drugs limits their use as biological tools to probe HDAC func-
tion in cancer biology.
Through the deacetylation of nucleosomal histones, HDAC
proteins regulate both the expression and activity of cancer-
related proteins that are involved in transcription, tumor sup-
pression, and cell signaling.3-4 Through the deacetylation of
non-histone substrates, HDAC proteins affect protein stability,
localization, and intracellular interactions, including protein-
protein interactions and protein-DNA interactions.4-5 Due to
their fundamental role in regulating gene expression and pro-
tein activity, overexpression of HDAC proteins is linked to
cancer formation.5 For example, HDAC1 was overexpressed
in prostate6 and breast,7 while HDAC8 has been implicated in
neuroblastoma and T-cell lymphoma and acute myeloid leu-
kemia.8 Among the class II proteins, HDAC6 was overex-
pressed in oral squamous cell carcinoma.9 In addition, HDAC6
is implicated in several non-epigenetic cancer-related intracel-
lular functions.10 Both HDAC6 and HDAC8 were found to be
highly expressed and implicated in the invasion and progres-
sion of breast cancer cells.11
Figure 1. Chemical structures of the FDA approved drug
SAHA (suberoylanilide hydroxamic acid, Vorinostat,
Zolinza™) and the C2-modified SAHA analogs reported here.
Several isoform selective inhibitors have been developed.
MS-275 (Figure S41) is in clinical trials and is class I selec-
tive, with 4- to 400- fold selectivity for HDAC1, 2, and 3 over
the other isoforms.17 RGFP966 (Figure S41) showed more
than 188-fold selectivity for HDAC3 over the other isoforms.19
Tubastatin (Figure S41) is HDAC6 selective with 87-fold or
1000-fold selectivity for HDAC6 over HDAC1, 2, and 3 ac-
With a role in cancer, HDAC proteins have emerged as im-
portant targets for cancer treatment, with a wide variety of
HDAC inhibitor drugs available.12-15 The effect of HDAC in-
hibitors on both histone and non-histone proteins can lead to
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cording to different reports.20-21 Selective inhibitors can be derived HDAC1, HDAC2, HDAC3, and HDAC6.21 As an
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used as biological tools to elucidate the function of each iso-
form in the development of cancer. In addition, modification
of non-selective inhibitors currently used in clinic can possibly
improve their selectivity and reduce their clinical side effects.
initial test of selectivity, the potency of each C2-modified
SAHA derivative was tested with HDAC1, 2, 3, and 6 at sin-
gle concentrations of either 5 or 10 µM. All analogs (1a-i)
displayed some selectivity for HDAC6 compared to HDAC1,
HDAC2, and HDAC3 (Figure 2). Among them, the C2-benzyl
(1g), C2-n-pentyl (1h), and C2-n-hexyl (1i) analogs showed
the greatest difference in inhibitory activity comparing
HDAC6 to HDAC1, HDAC2, and HDAC3. In contrast, the
C2-methyl SAHA analog was least selective, with similar
activity against all four isoforms (Figure 2).
Towards development of selective HDAC inhibitors, we
previously created SAHA analogs containing substituents in
the linker region between the hydroxamic acid and the anilide
ends (Figure 1). A C3-modified SAHA analog showed modest
preference for HDAC3, while C6-modified SAHA analogs
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displayed selectivity for HDAC1 and
6 compared to
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HDAC3.22-24 In addition, modifying the amine of the hydrox-
amic acid reduced potency, but enhanced preference for
HDAC1.25 C2-modified SAHA analogs (Figure 1) were also
generated and showed µM potency with HeLa cell lysates
(Table S1), but no selectivity assessment was performed.22 We
report here a selectivity assessment of C2-modified SAHA
analogs both in vitro and in cells. Modification at the C2 posi-
tion led to reduced potency but enhanced selectivity compared
to SAHA, with preference for HDAC6 and 8 over HDAC1, 2,
and 3. HDAC6/8 dual inhibitors can be used as biological
tools to study breast cancer metastasis.11, 26 In addition, the
SAHA analogs reported here are useful lead compounds for
further development of pharmacological agents and anti-
cancer drug targeting HDAC6 and 8. More generally, these
studies confirm that modification of the SAHA linker region
can enhance isoform selectivity.
Figure 2. Isoform selectivity screening of C2-modified SAHA
analogs (1a-i) against HDAC1, 2, 3, and 6 using the ELISA-
based HDAC activity assay.21 All analogs were tested at 5 µM
concentration, except for 1d, which was tested at 10 µM.
SAHA was tested at 1 µM.21 Mean percent deacetylase
activities from a minimum of three independent trials with
standard errors were plotted (Table S2). The substituent
below each compound number corresponds to the R group in
C2-modified SAHA (Figure 1).
Synthesis of C2-modified SAHA analogs. The syntheses
of seven C2-modified SAHA analogs (1a-g) were previously
described.22 Two new derivatives, C2-n-pentyl SAHA (1h)
and C2-n-hexyl SAHA (1i) are reported here. Synthesis began
with ring opening of ε-caprolactone 2 with aniline to give
anilide alcohol 3. Activation of the hydroxyl as a mesylate
with subsequent substitution with dimethyl malonate gave
intermediate diester 5. Substitution with 1-bromopentane or 1-
bromohexane generated the substituted diesters 6h-i. Decar-
boxylation and subsequent saponification, followed by cou-
pling with N-benzyl protected hydroxylamine afforded pro-
tected hydroxamic acids 7h-i. Finally, hydrogenolysis gave
the desired C2-n-pentyl (1h) and C2-n-hexyl SAHA (1i) ana-
logs (Scheme 1).
To further assess selectivity, IC50 values for the most selec-
tive compounds in the initial screen, compounds 1g-i, were
determined with HDAC1, 2, 3, 6, and 8 (Table 1). As controls,
the IC50 values of both SAHA and tubastatin were included.
As expected, SAHA displayed similar IC50 values with
HDAC1-6, but 5-fold reduced activity with HDAC8, which is
consistent with prior reports.17, 27 Tubastatin showed at least
87-fold selectivity for HDAC6 over class I HDAC1, 2, and 3,
but only 10-fold selectivity versus HDAC8.21 Interestingly, the
C2-modified SAHA analogs showed selectivity for HDAC6
and HDAC8, with IC50 values in the sub-micromolar to mi-
cromolar range (0.6-2.0 µM, Table 1). The C2-benzyl 1g and
C2-n-pentyl 1h analogs displayed 33 to 92-fold selectivity for
HDAC6 and HDAC8 over the Class I isoforms (Tables 1, S5,
and S6, and Figures S44 and S45). The most selective com-
pound, C2-n-hexyl SAHA 1i displayed 49- to 300-fold selec-
tivity for HDAC6 and HDAC8 compared to the class I iso-
forms (Tables 1 and S7 and Figure S46).
Scheme 1. Synthesis of C2-n-pentyl and C2-n-hexyl SAHA
analogs (1h-i).
It is notable that the selectivity of C2-n-hexyl SAHA 1i for
HDAC6 (˃163-fold) is elevated compared to tubastatin (˃87-
fold), while it showed 20-fold less potency than tubastatin
(0.60 vs. 0.031 µM IC50 values). The conclusion is that C2-
substituents impart selectivity by discriminating against
HDAC1, HDAC2, and HDAC3.
In-vitro screening of C2-modified SAHA analogs. Prior
work showed that C2-modified SAHA analogs displayed weak
µM potency with the HDAC activity from HeLa cell lysates.22
However, no selectivity assessment was performed. In this
work we used the recently developed ELISA-based HDAC
activity assay to screen the analogs against mammalian-
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Table 1. IC50 values for SAHA, Tubastatin, SAHA analogs 1g-1i against HDAC1, 2, 3, 6, and 8. a
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IC50 values (µM)
Compound
HDAC1
0.033 ± 0.001
2.7 ± 0.2
84 ± 6
HDAC2
0.096 ± 0.01
3.9 ± 0.4
110 ± 10
58 ± 2
HDAC3
0.020 ± 0.001
2.9 ± 0.5
91 ± 4
HDAC6
0.033 ± 0.003
0.031 ± 0.004
1.5 ± 0.2
0.85 ± 0.05
0.60 ± 0.05
ND
HDAC8
0.54 ± 0.01
0.33 ± 0.01
1.2 ± 0.1
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SAHA21
Tubastatin21
1g (benzyl)
1h (pentyl)
1i (hexyl)
48 ± 2
43 ± 2
1.3 ± 0.1
180 ± 20
330 ± 30
ND
180 ± 30
580 ± 30
ND
98 ± 10
2.0 ± 0.1
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(S)-1i (hexyl)
(R)-1i (hexyl)
530 ± 50
ND
3.1 ± 0.1
ND
0.71 ± 0.01
a
Mean IC50 value and standard error of at least two independent trials are shown (Figures S42-S48 and Tables S3-S9). ND = not deter-
mined
In-cell selectivity testing. To assess the HDAC6 selectiv-
ities of the analogs in a biologically relevant context, C2-
benzyl (1g), C2-n-pentyl (1h), and C2-n-hexyl (1i) SAHA
were tested for their abilities to increase the acetylation levels
of HDAC substrates. Acetylated-α-tubulin (AcTub) was moni-
tored as a known substrate of HDAC6, whereas acetylated-
histone H3 (AcH3) was observed as a substrate for HDAC1, 2,
and 3. U937 myeloid leukemia cells were used in these cellu-
lar HDAC6 selectivity study. HDAC6 is overexpressed in
several acute myeloid leukemia (AML) cell lines, suggesting
that HDAC6 is a promising target for development of anti-
leukemic drugs.28 SAHA or the analogs were incubated with
U937 cells before lysis and western blot analysis of protein
acetylation. As expected, SAHA increased the acetylation
levels of both α-tubulin and histone H3 (Figure 3a, lane 2),
consistent with its broad inhibition. In contrast, the HDAC6
selective inhibitor tubastatin affected only α-tubulin acetyla-
tion (Figure 3a, lane 3). Similar to tubastatin, the three analogs
1h-i increased acetylation levels of α-tubulin to a greater level
than histone H3 (Figure 3a, lanes 4-6). Quantification con-
firmed that 1h-i significantly increased acetylation of α-
tubulin compared to DMSO, but not acetyl histone H3 levels
(Figure 3b and Table S10). In addition, the C2-n-hexyl analog
1i promoted a dose-dependent increase in acetylation of α-
tubulin, but not histone H3 (Figure 3c, lanes 2-7), compared to
the DMSO control (Figure 3c, lane 1). The HDAC6-dependent
acetylation of tubulin observed in cells is consistent with the
HDAC6 selectivity observed in-vitro (Table 1 and Figure 2).
Figure 3. Cell-based selectivity testing of the SAHA analogs.
U937 cells were treated with (a) DMSO (1%), SAHA (2 µM),
tubastatin (2 µM), C2-benzyl SAHA (1g, 30 µM), C2-n-pentyl
SAHA (1h, 30 µM), C2-n-hexyl SAHA (1i, 30 µM), or (c)
increasing concentrations of C2-n-hexyl SAHA analog (1i, 10-
60 µM) before lysis, SDS-PAGE separation, and western blot
analysis of acetyl-histone H3 (AcH3) and acetyl-α-tubulin
(AcTub). GAPDH was a load control. Repetitive trials are
shown in Figures S49 and S50. (b) Fold increase in AcH3 or
AcTub after quantification of bands intensity from part a,
with mean fold increase from four independent trials and
standard error (Table S10).
Inhibitor cytotoxicity. To test the anti-cytotoxic properties
of the HDAC6-selective inhibitors, analogs 1g-i were tested in
cell-based cytotoxicity assays using leukemia cell lines.28
First, the analogs were tested with the Jurkat cell line at 1 and
10 µM concentrations using an MTT assay (Figure 4, Table
S11). SAHA was also tested as a control. All compounds
showed reduced cytotoxicity compared to the SAHA (Figure
4). Of the analogs, C2-n-hexyl SAHA (1i) showed the great-
est cytotoxic effect, with only 47% viability at 10 µM concen-
tration.
To further assess cytotoxicity, both SAHA and the most po-
tent analog 1i were tested to determine EC50 values against
three leukemia cancer cell lines: Jurkat, AML MOLM-13, and
U937 cells. SAHA showed potent cytotoxicity, with EC50 val-
ues of 0.72, 1.2, and 0.88 µM with Jurkat, AML MOLM-13,
and U937 cell lines, respectively (Table 2). The observed EC50
values are consistent with previous reports.29-31
Figure 4. Cytotoxicity screening of 1g, 1h, 1i, and SAHA at 1
and 10 µM concentrations using an MTT assay with Jurkat
cells. Mean percent viability from at least three independent
trials with standard error were plotted (Table S11).
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The high potency of SAHA may be due to its non- Docking studies. To rationalize the HDAC6 selectivity of
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selectivity, as well as the high inhibitory activity against class
1 HDAC1, 2, and 3. The C2-n-hexyl SAHA analog 1i showed
roughly 10-fold reduced cytotoxicity compared to SAHA, with
EC50 values of 11.8, 10.5, and 13.8 µM with Jurkat, AML
MOLM-13, and U937 cell lines, respectively (Table 2). The
reduced cytotoxicity is consistent with the 18-fold reduction in
potency against HDAC6 compared to SAHA (Table 1). In
addition, the selectivity for HDAC6 and 8 over HDAC1, 2,
and 3, might also contribute to the lower cytotoxicity.
the C2-n-hexyl SAHA (1i) analog, we performed docking
analysis using the AutoDock 4.2 program.33 Both enantiomers
of the analog were docked into the recently published HDAC6
crystal structure (pdb: 5EEM)34 and both displayed similar
binding interactions (Figure 5 and Figure S53), consistent with
the similar IC50 values observed experimentally. For exam-
ple, the hydroxamic acid was positioned in bonding distance
(1.9-2.9 Å) of three active site residues (H573, H574, and
Y745) and the catalytic zinc atom in HDAC6 active site (Fig-
ures 5a and S53A). For comparison, docking of the parent
SAHA compound with HDAC6 showed similar distances be-
tween the hydroxamic acid and the active site (1.6-2.4 Å, Fig-
ure S54A). To explore the HDAC6 selectivity, compound 1i
was also docked into the HDAC2 crystal structure (pdb ID:
3MAX).35 In contrast to the bonding distances observed with
HDAC6, elongated distances between the hydroxamic acid
group and H145 (5.7-5.9 Å), H146 (3.8 Å), and Y308 (3.0-5.5
Å) were observed (Figures 5b and S53B). Metal binding was
also weakened with longer bond distances (3.5-4.7 Å, Figures
5b and S53B). One possibility accounting for the weak bind-
ing with HDAC2 is that the bulky C2-n-hexyl substituent can-
not favorably fit into the relatively narrow catalytic active
channel of HDAC2.20 Consistent with this possibility, super-
imposition of the docked poses of compound 1i and SAHA
with HDAC2 showed that the C2-n-hexyl substituent is posi-
tioned towards the solvent exposed surface of the active site,
which consequently places the hydroxamic acid distant from
the metal (Figure S55C and D). In contrast, the relatively wide
catalytic pocket in HDAC6 allowed compound 1i and SAHA
to similarly position the hydroxamic acid within bonding dis-
tances of the catalytic metal and nearby residues (Figure S55A
and B).
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Table 2: EC50 values for SAHA and C2-n-hexyl (1i) SAHA
analog against Jurkat, AML MOLM-13, and U937 cells
using MTT assay. a
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Cellular EC50 values (µM)
Compound
AML MOLM-
Jurkat
U937
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SAHA
0.72 ± 0.13
11.8 ± 2.2
1.2 ± 0.06
10.5 ± 3.1
0.88 ± 0.13
13.8 ± 1.7
1i (hexyl)
a Mean EC50 value and standard error of at least three independent
trials are shown (Figures S51-S52 and Tables S12-S13).
Synthesis and Screening of (R)- and (S)-C2-n-hexyl
SAHA (1i). C2-n-hexyl SAHA (1i) contains a stereocenter at
the 2 position and the compounds tested to this point were
racemic mixtures. To test the selectivity of each enantiomer,
an enantioselective synthesis of C2-n-hexyl SAHA (1i) was
employed using Evans chiral auxiliary 8 and octanoyl chloride
(Schemes 2 and S1). Allyl bromide was added to the resulting
amide 9 to generate chiral compound (R)-10 from auxiliary
(R)-8 (Scheme 2) or (S)-10 from auxiliary (S)-8 (Scheme S1).
After olefin metathesis with Grubbs' second generation cata-
lyst32 and removal of the auxiliary, the olefin was reduced to
generate (S)-11 and (R)-11. Finally, coupling with hydroxy-
lamine generated the two enantiomers of C2-n-hexyl SAHA,
(S)-1i or (R)-1i in 95 and 92% ee, respectively.
Scheme 2. Synthesis of (S)-C2-n-hexyl SAHA, (S)-1i.
With the two C2-n-hexyl SAHA enantiomers in hand, IC50
values were determined (Table 1). As expected, both enanti-
omers displayed low micromolar to submicromolar potency
with HDAC8 (3.1±0.1 or 0.71±0.01 µM), similar to racemic 1i
(2.0±0.1). The data suggested that (R)-1i is more potent than
(S)-1i, although only by 4-fold. The (S)-1i enantiomer was
further tested for selectivity against HDAC 1, 2, and 3. (S)-1i
displayed 106- to 187-fold selectivity for HDAC8, which is
greater than that observed with racemic 1i (49- to 300-fold). In
total, studies with the enantiomers of C2-n-hexyl SAHA indi-
cated that both are low micromolar to submicromolar potency
HDAC8 inhibitors, with the expected HDAC8 selectivity
compared to HDAC1, 2, and 3.
Figure 5. Docked pose of (S)-C2-hexyl SAHA ((S)-1i)) in the
(a) HDAC6 (pdb:5EEM)34 or (b) HDAC2 (pdb:3MAX)35
crystal structures (b) using Autodock 4.2.33 Binding distances
between the hydroxamic acid atoms and active site residues
(numbered in figure) or the metal are displayed in Angstroms.
The (R) enantiomer is shown in Figure S53. The atomic
radius of the metal (Zn2+) was set at 0.5 Å for clarity. Atom
color-coding: (S)-C2-n-hexyl SAHA (C=purple/white; O=red;
N=blue; H=white); amino acids (C=deep teal; O=red,
N=blue); Zn2+ metal ion (grey sphere).
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synthesized (S)-1i and (R)-1i. M.K.H.P. conceived of the project
and assisted in experimental design and interpretation. All
Because all HDAC isoforms show high conservation among
their active site residues,36-37 previous studies suggested that
the shape of the active sites might explain the HDAC6 selec-
tivity of reported compounds.38 In particular, HDAC6 main-
tains a wider active site entrance compared to the class I iso-
forms.20 In previous work, HDAC6-selective inhibitors were
generated by replacing the solvent-exposed anilide group of
SAHA with bulky aryl groups.20, 39-40 In addition, compounds
with an aryl, cyclic, or unsaturated group attached in the linker
region directly adjacent to the hydroxamic acid demonstrated
HDAC6 selectivity.40 For example, tubastatin is a highly
HDAC6-selective inhibitor that displays a series of bulky aryl
groups near the hydroxamic acid (Figure S41).20 More closely
related to this work, valpropylhydroxamic acid (Figure S41)
positions an alkyl group adjacent to a hydroxamic acid and
showed HDAC6/8 dual selectivity, with 16 and 39 µM IC50
against HDAC6 and 8, respectively, but only 9-17-fold re-
duced potency with HDAC1, 2, and 3.41 The data with valpro-
pylhydroxamic acid are consistent with our data showing that
the linker can influence potency and selectivity. Our docking
analysis and these prior studies suggest that the selectivity of
the C2-modified SAHA analogs is due to the bulky substituent
adjacent to the hydroxamic acid takes advantage of the wider
active site entrance of HDAC6 and HDAC8.20, 42
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authors contributed to the writing of the manuscript.
Funding Sources
We thank National Institutes of Health (GM067657) and Wayne
State University for funding. The content is solely the responsibil-
ity of the authors and does not necessarily represent the official
views of the National Institutes of Health.
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank the Lumigen Instrument Center at Wayne State Univer-
sity for NMR and MS instrumentation, Dr. Y. Ge for the AML
MOLM-13, and U937 cell lines, Dr. K. Honn for use of the Alpha
Innotech FluorChem imaging system, Dr. M. Allen for use of the
chiral HPLC column, Dr. Y. Danylyuk for HRMS analysis, and
Dr. D. Nalawansha for helpful comments.
ABBREVIATIONS
HDAC, histone deacetylase; SAHA, suberoylanilide hydroxamic
acid; NAD, Nicotinamide adenine dinucleotide; DNA, deoxyribo-
nucleic acid; FDA, Food and Drug Administration; ELISA, en-
zyme-linked immunosorbent assay; SDS-PAGE, sodium dodecyl
sulfate polyacrylamide gel electrophoresis; GAPDH, Glyceralde-
hyde 3-phosphate dehydrogenase; AML, Acute Myloid Leuke-
mia; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide; CDI, carbonyldiimidazole; TEA, triethylamine; EDCI,
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;
NaHMDS, Sodium bis(trimethylsilyl)amide; DCM, Dichloro-
methane.
In conclusion, we report the synthesis and screening of sev-
eral SAHA analogs substituted at the C2 position. C2-
modified SAHA analogs displayed selectivity for HDAC6 and
HDAC8 over HDAC1, 2, and 3. The highest selectivity ob-
served was with C2-n-hexyl SAHA analog 1i, which displayed
49- to 300-fold selectivity for HDAC6 and 8 over HDAC1, 2,
and 3. Importantly, the selectivity of C2-n-hexyl SAHA is
elevated compared to the widely used HDAC6-selective in-
hibitor, tubastatin. Cell-based selectivity testing of analogs
1g-i reproduced the selectivity observed in vitro. The dual
HDAC6/8 selective C2-modified SAHA analogs reported in
this work can be useful as lead compounds to develop phar-
macological tools and anti-cancer drugs targeting HDAC6 and
HDAC8. More generally, these studies with SAHA analogs
suggest that modifying known drugs can significantly improve
their properties.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website. Experimental procedure, procedure for
biological screening, docking procedure, compound characteriza-
tion, IC50 curves and tables, and docking figures. (PDF)
AUTHOR INFORMATION
Corresponding Author
* Phone: 313-577-1515; Fax: States Fax: 313-577-8822;
E-mail: pflum@wayne.edu
Present Addresses
[a] Dr. A.V. Bieliauskas, Ash Stevens, Inc., 18655 Krause Street,
Riverview, MI 48193
Author Contributions
A.T.N purified and tested compounds 1g-i and performed all in
cells selectivity, cytotoxicity, and docking studies. G.P. tested
compounds 1a-f. A.V.B. synthesized analogs (1a-i), while A.T.N
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Chemical structures of the FDA approved drug SAHA (suberoylanilide hydroxamic acid, Vorinostat, Zolinza™)
and the C2-modified SAHA analogs reported here
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Isoform selectivity screening of C2ꢀmodified SAHA analogs (1aꢀi) against HDAC1, 2, 3, and 6 using the
ELISAꢀbased HDAC activity assay.21 All analogs were tested at 5 µM concentration, except for 1d, which
was tested at 10 µM. SAHA was tested at 1 µM.21 Mean percent deacetylase activities from a minimum of
three independent trials with standard errors were plotted (Table S2). The substituent below each compound
number corresponds to the R group in C2ꢀmodified SAHA (Figure 1).
Figure 2
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Cellꢀbased selectivity testing of the SAHA analogs. U937 cells were treated with (a) DMSO (1%), SAHA (2
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separation, and western blot analysis of acetylꢀhistone H3 (AcH3) and acetylꢀαꢀtubulin (AcTub). GAPDH was
a load control. Repetitive trials are shown in Figures S49 and S50. (b) Fold increase in AcH3 or AcTub after
quantification of bands intensity from part a, with mean fold increase from four independent trials and
standard error (Table S10).
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Cytotoxicity screening of 1g, 1h, 1i, and SAHA at 1 and 10 µM concentrations using an MTT assay with
Jurkat cells. Mean percent viability from at least three independent trials with standard error were plotted
(Table S11).
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Docked pose of (S)-C2-hexyl SAHA ((S)-1i)) in the (a) HDAC6 (pdb:5EEM)34 or (b) HDAC2 (pdb:3MAX)35
crystal structures (b) using Autodock 4.2.33 Binding distances between the hydroxamic acid atoms and
active site residues (numbered in figure) or the metal are displayed in Angstroms. The (R) enantiomer is
shown in Figure S53. The atomic radius of the metal (Zn2+) was set at 0.5 Å for clarity. Atom color-coding:
(S)-C2-n-hexyl SAHA (C=purple/white; O=red; N=blue; H=white); amino acids (C=deep teal; O=red,
N=blue); Zn2+ metal ion (grey sphere).
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Synthesis of C2-n-pentyl and C2-n-hexyl SAHA analogs (1h-i).
Scheme 1
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Synthesis of (S)-C2-n-hexyl SAHA, (S)-1i.
Scheme 2
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