The structural requirements of histone deacetylase inhibitors: SAHA analogs modified at the C5 position display dual HDAC6/8 selectivity

Article abstract of DOI:10.1016/j.bmcl.2017.06.033

Histone deacetylase (HDAC) proteins have emerged as important targets for anti-cancer drugs, with four small molecules approved for use in the clinic. Suberoylanilide hydroxamic acid (Vorinostat, SAHA) was the first FDA-approved HDAC inhibitor for cancer

Full text of DOI:10.1016/j.bmcl.2017.06.033

Accepted Manuscript
The structural requirements of histone deacetylase inhibitors: SAHA analogs
modified at the C5 position display dual HDAC6/8 selectivity
Ahmed T. Negmeldin, Mary Kay H. Pflum
PII:
S0960-894X(17)30637-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.033
BMCL 25067
Reference:
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Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 April 2017
8 June 2017
12 June 2017
Please cite this article as: Negmeldin, A.T., Pflum, M.K.H., The structural requirements of histone deacetylase
inhibitors: SAHA analogs modified at the C5 position display dual HDAC6/8 selectivity, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.033
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The structural requirements of histone
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Ahmed T. Negmeldin and Mary Kay H. Pflum

Bioorganic & Medicinal Chemistry Letters
The structural requirements of histone deacetylase inhibitors: SAHA analogs
modified at the C5 position display dual HDAC6/8 selectivity
a
a,
Ahmed T. Negmeldin and Mary Kay H. Pflum 
a
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Histone deacetylase (HDAC) proteins have emerged as important targets for anti-cancer
drugs, with four small molecules approved for use in the clinic. Suberoylanilide hydroxamic
acid (Vorinostat, SAHA) was the first FDA-approved HDAC inhibitor for cancer treatment.
However, SAHA inhibits most of the eleven HDAC isoforms. To understand the structural
requirements of HDAC inhibitor selectivity and develop isoform selective HDAC inhibitors,
SAHA analogs modified in the linker at the C5 position were synthesized and tested for
potency and selectivity. C5-modified SAHA analogs displayed dual selectivity to HDAC6
and HDAC8 over HDAC 1, 2, and 3, with only a modest reduction in potency. These findings
are consistent with prior work showing that modification of the linker region of SAHA can
alter isoform selectivity. The observed HDAC6/8 selectivity of C5-modified SAHA analogs
provide guidance toward development of isoform selective HDAC inhibitors and more
effective anti-cancer drugs.
Keywords:
Histone Deacetylase
HDAC inhibitors
Vorinostat
Isoform selectivity
HDAC6/8 selective inhibitor
2009 Elsevier Ltd. All rights reserved.
1
4-
Histone deacetylase (HDAC) proteins have a prominent
regulatory role in gene transcription and cell function. HDAC
proteins catalyze the removal of the acetyl group from ε-
acetyllysine residues on nucleosomal histones. Upon
deacetylation of histone proteins, the interaction between histone
proteins and DNA increases, which reduces DNA expression and
in leukemia, ovarian cancer, and oral squamous cell carcinoma.
16
Moreover, HDAC6 plays an important role in cancer cell
1
7
growth and survival through several non-epigenetic pathways.
Importantly, HDAC6 and HDAC8 were expressed at abnormally
high levels in various human breast cancer cell lines and were
associated with breast cancer invasiveness and metastasis,
1,2
18
gene transcription.
In addition, HDAC proteins affect
making them both interesting for anti-cancer drug design.
intracellular interactions, protein localization, and protein
stability through deacetylation of non-histone substrates.
3-6
Due to their fundamental role in cancer, inhibitors targeting
HDAC proteins have been developed. HDAC inhibitors showed
Beyond basic cell biology, HDAC proteins are overexpressed in
7
the ability to reduce proliferation and metastasis, and promote
several cancers, as well as other diseases.
14, 18-22
apoptosis in several cancers.
Four HDAC inhibitors have
+
20, 23-26
HDAC proteins require either metal ions or NAD as
been approved by the FDA for cancer treatment.
For
8
cofactors for catalysis. The eleven metal-dependent HDAC
example, SAHA (suberoylamide hydroxamic acid, Vorinostat,
isoforms are grouped as classes I, II, or IV, depending on their
size, cellular localization, and homology to other HDAC
proteins. The seven NAD dependent HDAC proteins are
grouped as class III. The metal-dependent HDAC proteins
comprise eleven isoforms (HDAC 1-11) and are the focus of this
work.
Zolinza™) and Belinostat (PXD101, Belodaq™)(Figure 1) were
2
3-25
approved for treatment of T-cell lymphoma,
while
9
+
Panobinostat (LBH-589, Farydak™)(Figure 1) was approved for
26
treatment of multiple myloma. SAHA inhibits most of the
eleven metal-dependent HDAC proteins, with only a modest
27, 28
selectivity against HDAC8.
The non-selectivity of SAHA
might explain the side effects observed in clinic, but certainly
Each metal-dependent HDAC isoform has been associated
with various cancers. For example, HDAC1 was overexpressed
limits the use of SAHA to study individual HDAC isoforms in
29
1
0, 11
cancer biology.
In the last four years, several dual
in lung and colon cancers,
expression in colorectal and gastric cancer. Abnormal activity
of HDAC8 was observed in acute myeloid leukemia, T-cell
while HDAC2 displayed aberrant
30-33
12
HDAC6/HDAC8 selective inhibitors have been developed.
Dual inhibition of HDAC6 and HDAC8 can possibly have
synergistic therapeutic applications in treatment of various
13
lymphoma, and neuroblastoma. HDAC6 was highly expressed
—
——

Corresponding author. Tel.: +1-313-577-1515; fax: +1-313-577-8822; e-mail: pflum@wayne.edu

with minimal effect on potency.
Synthesis of C5-modified SAHA analogs: C5-modified
SAHA analogs 1a-e were synthesized as shown in Scheme 1.
The synthesis started from a coupling reaction of 4-pentenoic
acid 2 with aniline using TBTU to obtain amide 3, which was
then reacted with crotonaldehyde 4 via a cross metathesis
reaction using second generation Grubbs' catalyst to afford the
α,β-unsaturated aldehyde 5. Aldehyde 5 was substituted with
different groups through
a 1,4-conjugate addition using
organolithium or organomagnesium cuprates to yield
intermediates 6a-e. Horner–Wadsworth–Emmons olefination of
6a-e with trimethyl phosphonoacetate followed by reduction
gave amide esters 8a-e with a saturated linker. Finally, amide
esters 8a-e were reacted with hydroxylamine to afford the C5-
substituted SAHA derivatives 1a-e as racemic mixtures.
Figure 1. Chemical structures of the FDA-approved drugs SAHA,
Belinostat, and Panobinistat, along with the C5-modified SAHA
analogs reported here.
cancers, which can improve anti-cancer efficiency compared to
30-34
current non-selective HDAC inhibitors.
In vitro screening of C5-modified SAHA analogs. As a
preliminary screen, the new analogs were tested for their global
HDAC inhibition with HeLa cell lysates as the source of all
HDAC proteins (Table 1). SAHA was also tested as the parent
unsubstituted control molecule. The inhibitory activities of the
analogs were measured with the HDAC-Glo™ I/II substrate
SAHA and most of the HDAC inhibitors have a similar
pharmacophore that consists of three structural elements (Figure
). The capping group interacts with solvent-exposed residues of
1
the HDAC active site, while the metal binding group forms key
interactions with the catalytic metal deeply buried in the active
site (Figure 1). The linker region that connects the capping and
the metal binding groups is positioned in the narrow hydrophobic
active site channel. Both the capping and the metal binding
groups have been modified extensively in HDAC inhibitor
(Promega). C5-methyl SAHA analog 1a showed greater potency
compared to SAHA (100 nM vs 200 nM IC50 values, Table 1).
However, all other analogs showed weaker potency than SAHA
(
11- to 33-fold reduction in potency), with IC50 values from 2.2
to 6.5 µM (Table 1). The observed lower potencies of compounds
b-1e may be due to selectivity for specific HDAC isoform(s),
35-40
design.
linker region.
region, SAHA analogs substituted at carbon 2 (C2), 3 (C3), or 6
In contrast, few studies report modification of the
41-44
1
To study the effect of substitution on the linker
which lowered the potency against lysates that contains all
HDAC isoforms. The lower potency observed here was similar to
(
1
C6) of the linker region were synthesized and screened (Figure
44
44
what was observed with the C2-modified SAHA analogs.
). C2-hexyl SAHA displayed dual HDAC6/8 selectivity, C3-
42
ethyl SAHA showed HDAC6 selectivity, and C6-butyl SAHA
43
Table 1
inhibited HDAC1 and 6 over HDAC3. The conclusion of this
prior work is that modification of the SAHA linker can alter
inhibitor specificity.
IC50 values for SAHA and C5-modified SAHA analogs (1a-
a
1
e) with HeLa cell lysates.
Compounds
SAHA
R
H
IC50 (µM)
0.20 ± 0.02
0.10 ± 0.01
5.0 ± 0.4
6.5 ± 0.1
2.2 ± 0.1
Guided by this prior work, here we explored the effect of
substituents on the C5 position of SAHA (Figure 1). SAHA
analogs substituted at the C5 were synthesized and tested for
potency and selectivity both in vitro and in cellulo. Several
analogs showed dual HDAC6/8 selectivity over HDAC1, 2, and
, with a modest reduction in HDAC6 inhibition but enhanced
HDAC8 inhibition compared to SAHA. This study documents
that modifying the linker region of SAHA can alter its selectivity
1
1
a
b
methyl
n-butyl
n-hexyl
phenyl
benzyl
1
c
1
d
3
1
e
6.2 ± 0.2
a
Mean IC50 value and standard error of at least three independent trials are
shown (Figure S52 and Table S1).
Scheme 1. Synthesis of C5-modified SAHA analogs (1a-e)
To test isoform selectivity, the parent molecule, SAHA, and
all the C5-modified SAHA analogs were tested at a single
concentration using the recently developed ELISA-based HDAC
2
8
activity assay (Figure 2). SAHA, as expected, showed no
28
selectivity among HDAC1, 2, 3, and 6. Interestingly, several
C5-SAHA analogs displayed more potent inhibition against
HDAC6 compared to HDAC1, HDAC2, and HDAC3 (Figure 2).
The analogs that showed the greater difference in potency with
HDAC6 versus the other isoforms were C5-n-butyl (1b), C5-n-
hexyl (1c), and C5-benzyl (1e). The C5-methyl SAHA (1a) and
C5-phenyl SAHA (1d) showed only a small difference in
potency comparing HDAC6 to the others (Figure 2).
IC50 values for the most selective derivatives 1b, 1c, and 1e were
determined with HDAC1, HDAC2, HDAC3, HDAC6, and
HDAC8 isoforms to quantitatively assess the selectivity (Table
2). HDAC8 was also tested due to its similar active site compared
to HDAC6. The IC50 values of SAHA as the parent compound

Table 2
a
IC50 values for SAHA and C5-modified SAHA analogs 1b, 1c, and 1e against HDAC1, 2, 3, 6 and 8.
IC50 values (nM)
Compound
HDAC1
33 ± 1
HDAC2
96 ± 10
HDAC3
20 ± 1
HDAC6
33 ± 3
320 ± 30
410 ± 60
270 ± 20
HDAC8
540 ± 10
430 ± 10
420 ± 20
380 ± 20
b
SAHA
1
1
1
b (n-butyl)
c (n-hexyl)
e (benzyl)
1,100 ± 100
2,100 ± 100
2,900 ± 300
1,300 ± 100
2,500 ± 100
3,500 ± 100
1,600 ± 100
2,900 ± 300
5,800 ± 800
a
Mean IC50 value and standard error of at least three independent trials are shown (Figures S53-S55 and Tables S3-S5).
Previously reported IC50 values using the same assay procedure.
b
28
HDAC8 compared to HDAC1, 2 and 3. Steric clash with the
active sites of HDAC1, 2, and 3 might explain the reduction in
inhibition of HDAC1, 2, and 3 (36- to 290-fold reduction in IC50
HDAC1
HDAC2
HDAC3
HDAC6
100
4
4, 46
values, see Table 2).
Consistent with this analysis, docking
studies of the structurally similar C2-hexyl SAHA analog showed
that both enantiomers had a similar selectivity and IC50 values,
which suggests that the size of the substituent is the origin of the
observed selectivity rather than the chirality of the substituted
8
6
4
2
0
0
0
0
0
4
4
carbon.
In cellulo selectivity testing. To test the analogs in a more
biological context, the C5-benzyl (1e) SAHA analog was tested
for selectivity in cells. The inhibition of HDAC6 was monitored
by detecting the levels of its known substrate acetyl-α-tubulin
(
AcTub), whereas Class I HDAC (HDAC1, 2, and 3) inhibition
was monitored by observing the known substrate, acetyl-histone
(AcH3). SAHA or C5-benzyl SAHA 1e were incubated with
3
U937 leukemia cells before lysis and western blot analysis of
protein acetylation (Figure 3). As expected, SAHA increased the
levels of both acetyl-α-tubulin and acetyl-histone H3 to a similar
extent (Figure 3, lane 1), which is consistent with its non-
selective inhibition of HDAC1, 2, and 3 isoforms. On the other
hand, C5-benzyl SAHA analog 1e showed a dose dependent
selective increase in levels of acetyl-α-tubulin, which was greater
than the increased levels of acetyl histone H3 (Figure 3, lanes 3-
5) compared to the DMSO control (Figure 3, lane 2). The
observed HDAC6 selectivity of the C5-benzyl SAHA 1e in cells
is consistent with the selectivity observed in the in vitro
screening (Table 2 and Figure 3).
SAHA
1a
1b
1c
1d
1e
(Bn)
(
Me) (Bu) (Hex) (Ph)
Figure 2. In vitro isoform selectivity screening of C5-modified SAHA
analogs (1a-e) against HDAC1, HDAC2, HDAC3, and HDAC6 using the
ELISA-based HDAC activity assay. Analogs 1a-e were tested at 0.025,
.25, 1.25, 0.125, and 1.25 µM final concentrations, respectively. SAHA
was tested at 1 µM concentration in a previous report using the same
0
2
8
assay procedure. Mean percent deacetylase activities from a minimum
of two independent trials with standard errors were plotted (Table S2).
28
were included as well (Table 2). SAHA displayed similar IC50
values against HDAC1, 2, 3, and 6, with 6- to 27-fold selectivity
28, 44
against HDAC8.
Both C5-n-butyl (1b), and C5-n-hexyl (1c)
SAHA analogs displayed modest selectivity, with 3- to 5-fold
and 5- to 7-fold selectivities for HDAC6 and HDAC8 over
HDAC1, 2, and 3 (Tables 2 and S6). In addition, 1b and 1c
showed modest reductions in HDAC6 potency (IC50 values of
3
4
20 and 410 nM), but similar potency against HDAC8 (430 and
20 nM) compared to SAHA (Table 2). The most potent and
selective analog was C5-benzyl SAHA (1e), which displayed 8-
to 21-fold selectivity for HDAC6 and HDAC8 over HDAC1, 2,
and 3 and IC50 values of 270 and 380 nM with HDAC6 and
HDAC8, respectively (Table 2 and S6). The selectivity was due
to a dramatic reduction in potency with HDAC1, 2, and 3 (14- to
Figure 3. Western blots analysis of acetyl-lysine 9 of histone H3,
(AcH3) and acetyl-lysine 40 of α-tubulin (AcTub) after treatment with
SAHA or C5-benzyl SAHA 1e. U937 cells were treated with DMSO
(1%), SAHA (5 µM), or increasing concentrations of C5-benzyl SAHA
80-fold), but only a modest potency reduction with HDAC6 (8.5-
(
1e) analog (10-40 µM), before lysis, SDS-PAGE separation, transfer to
to 12-fold) and similar potency with HDAC8 (380 – 540 nM),
compared to SAHA. Modification of SAHA at the C5 position of
the linker led to selectivity for HDAC6 and 8 over HDAC1, 2,
and 3.
a PVDF membrane, and western analysis with AcH3 or AcTub
antibodies. GAPDH levels in the samples were also probed as a gel load
control. A DMSO control sample was included for comparison to
inhibitor-treated samples. Repetitive trials are shown in Figures S56.
The size of the substituent had a substantial effect on selectivity
(
Figure 2 and Table 2). More bulky or longer alkyl chain
In vitro cancer cell growth inhibition.
enhanced the selectivity to HDAC6 and 8 over HDAC1, 2, and 3.
4
5
As suggested by previous reports, larger substituents in the
linker region might have led to enhanced HDAC6/8 selectivity
due to the wider active site entrance of both HDAC6 and
To evaluate the ability of the C5-modified SAHA analogs to
influence cell growth, the most selective analogs were tested. C5-
n-butyl (1b), C5-n-hexyl (1c), and C5-benzyl (1e) SAHA analogs

were tested at 1 and 10 µM concentrations using MTT assay.
Jurkat cells, a T-cell lymphoma derived cancer cell line, were
selected due to the role of both HDAC6 and HDAC8 in
screening, and IC50 curves and tables) associated with this article
can be found with the online version.
22, 47
lymphoma.
The tested analogs displayed cytotoxicity against
Funding Sources
the cell line, with 39%, 24%, and 50% cell viability with C5-n-
butyl SAHA (1b), C5-n-hexyl SAHA (1c), and C5-benzyl SAHA
We thank the National Institutes of Health (GM121061) and
Wayne State University for funding. The content is solely the
responsibility of the authors and does not necessarily represent
the official views of the National Institutes of Health.
(1e) at 10 µM concentrations, respectively (Figure 4). On the
other hand, SAHA demonstrated higher cytotoxicity than the C5-
SAHA analogs, with 49% cell viability at 1 µM. The reduced
cytotoxicity of the analogs compared to SAHA can be due to
their lower potency (8- to 12-fold reduction in IC50 values with
HDAC6 compared to SAHA, Table 2). In addition, the
nonselective inhibition of most HDAC isoforms by SAHA might
contribute to its higher cytotoxicity (Table 2 and Figure 4).
Acknowledgments
We thank Lumigen Instrument Center at Wayne State
University for NMR and MS instrumentation, Dr. K. Honn and
Dr. Y. Ahn for use of the Alpha Innotech FluorChem imaging
systems, and I. Gomes, J. Knoff, D. Nalawansha, and Y. Zhang
for comments on the manuscript.
1
µM
10 µM
100
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