Design and evaluation of 'Linkerless' hydroxamic acids as selective HDAC8 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.02.064

In this report, we describe new HDAC inhibitors designed to exploit a unique sub-pocket in the HDAC8 active site. These compounds were based on inspection of the available HDAC8 crystal structures bound to various inhibitors, which collectively show that the HDAC8 active site is unusually malleable and can accommodate inhibitor structures that are distinct from the canonical 'zinc binding group-linker-cap group' structures of SAHA, TSA, and similar HDAC inhibitors. Some inhibitors based on this new scaffold are >100-fold selective for HDAC8 over other class I and class II HDACs with IC₅₀ values <1 μM against HDAC8. Furthermore, treatment of human cells with the inhibitors described here shows a unique pattern of hyperacetylated proteins compared with the broad-spectrum HDAC inhibitor TSA.

Full text of DOI:10.1016/j.bmcl.2007.02.064

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2874–2878
Design and evaluation of ‘Linkerless’ hydroxamic acids as
selective HDAC8 inhibitors
Keris KrennHrubec,a Brett L. Marshall,b Mark Hedglin,a
Eric Verdinb and Scott M. Ulricha,*
aDepartment of Chemistry, Ithaca College, Ithaca, NY 14850, USA
bGladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94158, USA
Received 19 December 2006; revised 16 February 2007; accepted 21 February 2007
Available online 25 February 2007
Abstract—In this report, we describe new HDAC inhibitors designed to exploit a unique sub-pocket in the HDAC8 active site.
These compounds were based on inspection of the available HDAC8 crystal structures bound to various inhibitors, which collec-
tively show that the HDAC8 active site is unusually malleable and can accommodate inhibitor structures that are distinct from the
canonical ‘zinc binding group-linker-cap group’ structures of SAHA, TSA, and similar HDAC inhibitors. Some inhibitors based on
this new scaffold are >100-fold selective for HDAC8 over other class I and class II HDACs with IC50 values <1 lM against HDAC8.
Furthermore, treatment of human cells with the inhibitors described here shows a unique pattern of hyperacetylated proteins com-
pared with the broad-spectrum HDAC inhibitor TSA.
Ó 2007 Elsevier Ltd. All rights reserved.
Post-translational e-acetylation of lysine residues was
first identified as a post-translational modification of
histones and has since emerged as a central mechanism
of transcriptional control in eukaryotes; hypoacetyla-
tion is correlated with transcriptional repression and
hyperacetylation with transcriptional activation.1–4 Pat-
terns of lysine acetylation along regions of nucleosomes
have distinct biological meanings, through recruitment
of specific proteins as well as structural changes to the
chromatin fiber.5,6 Histone lysine acetylation patterns
affect diverse cellular processes including differentiation,
cellular response to stimuli, and tumorigenesis. More
recently, it has been shown that non-histone proteins
such as tubulin and p53 are also targets of reversible
lysine acetylation, suggesting that acetylation may play
a much broader role in controlling cellular events akin
to protein phosphorylation.7,8
classes of HDACs. Class I (HDACs 1, 2, 3, and 8) and
class II (HDACs 4, 5, 6, 7, 9, and 10) HDACs are zinc-
dependent amidohydrolases with a conserved catalytic
core but differing in size, domain structure, and tissue
expression pattern. Class III HDACs are NAD+ depen-
dent, unrelated in sequence and mechanism to classes I
and II.9 Zinc-dependent HDACs have received much
attention as anticancer drug targets. Inhibitors of these
enzymes have demonstrated a remarkable ability to
induce terminal differentiation of transformed cells,
presumably by altering patterns of gene expression
through influencing the acetylation state of select histone
lysine residues.10 HDAC inhibitors are also exceedingly
useful tools to study the biology of histone deacetylases.
Indeed, determining whether a cellular process involves
HDACs is readily ascertained by using the many potent,
cell-permeable molecules available.
The lysine acetylation state of cellular proteins is deter-
mined by the action of histone acetyl transferases (HATs)
and histone deacetylases (HDACs). There are three
Most HDAC inhibitors such as TSA and SAHA closely
resemble the aliphatic acetyl-lysine substrate and deliver
a hydroxamic acid or other zinc binding group to the
catalytic zinc ion at the bottom of a narrow active site
pocket as seen in co-crystal structures of inhibited
HDLP (HDAC-like protein),11 HDAH (HDAC-like
amidohydrolase),12 and human HDAC8.13 These inhib-
itors all have a zinc binding group and a polar ‘cap
Keywords: Histone deacetylases; Hydroxamic acids; HDAC8.
*
Corresponding author. Tel.: +1 607 274 7977; fax: +1 607 274
8135; e-mail: sulrich@ithaca.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.064

K. KrennHrubec et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2874–2878
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Figure 1. Common structural features of the broad-spectrum HDAC
inhibitors SAHA and TSA.
Figure 2. The HDAC8:TSA co-crystal structure, showing the con-
served hydrophobic residues (F152, F208, and M274) that form the
narrow active site channel, at the bottom of which is the catalytic zinc
ion.
group’ connected by a straight chain alkyl, vinyl or aryl
linker (Fig. 1). Attempts to generate isozyme-specific
HDAC inhibitors generally focus on varying the cap
group to exploit variability in the HDAC surface sur-
rounding the active site.14 Despite much effort, truly
selective compounds remain difficult to find. Screens of
large compound libraries have yielded selective inhibi-
tors of HDACs 1, 6, and 8.15,16 However, structural
determinants of selective HDAC inhibition remain un-
known. In this report, we describe the rational design
of HDAC inhibitors which show selectivity toward hu-
man HDAC8 by targeting an active site pocket which
may be unique to this family member.
The HDAC8 structure was solved with four different
hydroxamate inhibitors bound.13 The active site topo-
logy of HDAC8 showed large structural differences
depending on which inhibitor is bound. In the SAHA:
HDAC8 co-crystal structure, the active site is deep
and narrow similar to the HDLP and HDAH structures.
However, when a hydroxamate inhibitor with an aryl
linker (CRA-A) is bound to HDAC8, a large sub-pocket
forms in the side of the active site, adjacent to M274.
This pocket is created by movement of F152 away from
its normal position packed against M274 to form the lip
of the active site tunnel (Fig. 3). This shift may be a con-
sequence of the more sterically demanding aryl hydroxa-
mate CRA-A versus the aliphatic hydroxamate SAHA
binding the active site.
HDAC8 is an unusual HDAC family member. Recent
data suggest that HDAC8 is constitutively localized to
the cytoplasm and its expression in primary cells is
restricted to smooth muscle.17 Interestingly, RNAi
ablation of HDAC8 in these cells results in a contrac-
tion-deficient phenotype.18 Thus, identifying the sub-
strates of HDAC8 may expand the range of targets
and functions of the HDAC family. Furthermore, a
common form of acute myeloid leukemia (AML) results
from a chromosomal translocation creating an abnor-
mal fusion protein, Inv1. Inv1 binds HDAC8 and is
associated with aberrant, constitutive genetic repres-
sion.19 As such, specific HDAC8 inhibitors may assist
a medicinal chemistry effort against AML.
We reasoned that this sub-pocket may be targeted by a
new inhibitor scaffold. If this pocket is unique to
HDAC8, inhibitors that bind this pocket should be
selective for HDAC8. To test this idea, we synthesized
a panel of six bulky aryl hydroxamic acids that are
unlike the canonical ‘zinc binding group-linker-cap
group’ structure of most HDAC inhibitors (Fig. 4).
These inhibitors are aryl hydroxamates like CRA-A
and the new scaffold also displays aryl groups to bind
this pocket a short distance from the hydroxamic acid.
Such molecules should be excluded from HDACs that
lack the sub-pocket seen in HDAC8.
Our approach to generate specific HDAC8 inhibitors is
founded upon analysis of the HDAC8, HDAH, and
HDLP structures with bound hydroxamate inhibitors.
The amino acid sequences and overall active site
topology of the enzymes are similar. In HDAC8, the
zinc ion that facilitates amide hydrolysis is found at
the bottom of a narrow pocket; just above which are
conserved and catalytically important Y306 and H180
residues. The rim of the pocket is formed by three
conserved hydrophobic residues, F152, F208, and
M274 (Fig. 2). These form the tunnel that the acetyl-
lysine substrate and straight-chain hydroxamate
inhibitors penetrate to access the catalytic machinery.
Similar architecture is found in HDLP and HDAH.
Compounds 1–6 were synthesized in two steps from the
corresponding carboxylic acids by conversion to the acid
chlorides followed by treatment with hydroxylamine in
water/THF under basic conditions and purified by
recrystallization from ethanol/water.20 To access struc-
ture 6, we first performed a Suzuki coupling between
1-naphthyl boronic acid and p-bromobenzoic acid to
make p-(1-naphthyl)benzoic acid which was converted
to its corresponding hydroxamate in the same manner
1
(Scheme 1). All hydroxamates gave H and 13C NMR
spectra consistent with the structures shown.21 The

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K. KrennHrubec et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2874–2878
Figure 4. Structures of linkerless hydroxamates designed to bind the
sub-pocket of the HDAC8 active site.
Table 1. IC50 values of compounds 1–6 against HDACs 1, 6, and 8
Compound
IC50 values
HDAC8(lM)
HDAC1(lM)
HDAC6(lM)
1
2
3
4
5
6
20.0
14.0
6.6
>100
>100
—
>100
>100
—
66.0
0.7
—
>100
>100
—
82
0.3
55
Values are the average of three experiments.
Figure 3. (a) Structure of HDAC8 bound to SAHA, an alkyl-linker
HDAC inhibitor. M274 (red) and F152 (cyan) pack against each other
to form the wall of the active site pocket. (b) Structure of HDAC8
bound to CRA-A, an aryl-linker HDAC inhibitor. In this case F152
rotates away from M274, exposing a large sub-pocket.
HDACs 1 and 6 to determine their selectivity toward
HDAC8. The data show that all hydroxamates based
on the linkerless scaffold are selective for HDAC8 over
HDACs 1 and 6. The most potent compounds 5 and 6
are >100-fold selective against HDAC8 versus HDACs
1 and 6. The less potent compounds may be even more
so, since they present steric bulk in closer proximity to
the zinc binding group, potentially clashing with the
narrow active site seen in HDAH and HDLP.
compounds were then tested as HDAC inhibitors using
a tritiated acetyl histone peptide assay22 against recom-
binant human HDAC8. The selectivity of the com-
pounds was determined by similar inhibition assays
against immunoprecipitated human HDACs 1 (class I)
and 6 (class II) as representatives of the larger family.
We designed compounds 1–6 based on a simple blocking
effect, where the malleability of the HDAC8 active site
with the inducible sub-pocket would allow the bulky
hydroxamates to access the catalytic zinc while HDACs
with more rigid, narrow active sites prohibit zinc chela-
tion. Inspection of the CRA-A:HDAC8 co-crystal struc-
ture reveals that the primary pocket between F152 and
F208 where the aryl group bearing the hydroxamate
binds is at a right angle to the induced sub-pocket
The inhibition data show that the linkerless, sterically
demanding aryl hydroxamates are indeed HDAC8
inhibitors (Table 1 and Fig. 5). Some of the compounds
are also moderately potent; compounds 5 and 6 have
submicromolar IC50 values against HDAC8. We also
tested compounds 1, 2, 5, and 6 as inhibitors against
Scheme 1. Synthesis of hydroxamates shown in Figure 4. Crude acid chlorides were not isolated, and yields from the starting carboxylic acids were
>80%.

K. KrennHrubec et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2874–2878
2877
Figure 5. Inhibition plots of compounds 2, 5, and 6 against HDACs 1, 6, and 8.
(Fig. 6). Interestingly, the more potent molecules against
HDAC8 (5 and 6) have such conformations accessible to
them, which may explain why the conformationally rigid
compounds 1–4 are poorer inhibitors of HDAC8 than
compounds 5 and 6.
In order to determine the effects of these compounds on
the levels of lysine acetylated proteins in cells, we treated
HeLa and HEK293 cells with compounds 2, 5 and the
broad-spectrum HDAC inhibitor TSA. The cell lysate
was then subjected to western blotting with anti-acetyl-
lysine antibodies (Fig. 7). The data show that treating
either cell type with TSA increased the lysine acetylation
level of three proteins. The same three proteins became
hyperacetylated upon treatment with compound 5.
While compound 5 does show selectivity among purified
HDACs 1, 6, and 8, it has nearly identical effects on pro-
tein acetylation levels in cells as TSA. Thus, it is likely
that compound 5 has targets among the HDAC family
that we have not included in our selectivity assay. How-
Figure 7. Anti-acetyl lysine Western blot of HeLa and HEK293 cell
lysates pretreated with TSA (relatively nonspecific HDAC inhibitor),
no inhibitor, and increasing concentrations of compounds 2 and 5.
ever, when either cell type was treated with compound 2,
only the high molecular weight protein became hyper-
acetylated. The high molecular weight protein was deter-
mined to be tubulin when the experiment was repeated
with antibodies specific toward acetyl tubulin and acetyl
histone (Fig. 8). HDAC6 has previously been shown to
deacetylate tubulin specifically.8 Our data show that
compound 2 weakly inhibits HDAC6, which would
account for this result, but tubulin hyperacetylation is
seen at concentrations of 2 (0.8 lM) that showed
minimal inhibition of purified HDAC6. These data do,
however, clearly indicate that compound 2 inhibits a
restricted subset of HDACs (possibly only HDAC8) in
cells when compared with TSA, and the target(s) of
compound 2 do not deacetylate histones. Rather, the
target(s) of compound 2 deacetylate tubulin and
possibly other non-histone proteins.
Figure 6. The HDAC8 CRA-A co-crystal structure showing the right-
angle orientation of the aryl linker relative to the induced sub-pocket.
Note that the aryl group of CRA-A attached to the aryl linker does not
bind the sub-pocket but rather is positioned well above it, as can be
seen in Figure 3b.
These compounds represent rationally designed inhibi-
tors specific for an individual HDAC family member

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Figure 8. Western blotting of the same lysates with antibodies specific
for lysine acetylated histone H4 and lysine acetylated tubulin.
based on available structural data. We hypothesize that
the relatively simple structures described here exploit the
malleability of the HDAC8 active site and its unique
sub-pocket, resulting in selective inhibition. As more
HDAC family members are structurally characterized,
it will be seen if HDACs that are insensitive to these
compounds also lack the sub-pocket seen in HDAC8.
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Acknowledgments
We thank Chris Roessler for early work on this project.
Figures 2, 3, and 6 were generated with PyMol (Delano
Scientific). This work was supported by the Research
Corporation’s Cottrell College Science Award
#CC5955 (S.M.U.) and The Gladstone Institutes (E.V.).
21. Compound 2: 1H NMR (DMSO-d6, 400 MHz) d = 7.51
(m, 4H), 7.94 (m, 2H), 8.15 (m, 1H), 9.24 (s, 1H), 11.09 (s,
1H). 13C NMR (DMSO-d6, 100 MHz) d = 166.18, 133.65,
132.77, 130.56, 128.79, 127.33, 126.87, 126.02, 125.71,
125.54 ppm.
Compound 5: 1H NMR (400 MHz, DMSO-dÀ6) d = 10.86
(s, 1H), 9.11 (s, 1H), 8.20 (d, J = 16 Hz, 1H), 7.95 (m, 2H),
7.77 (m, 1H), 7.60 (m, 4H), 6.55 (d, J = 16 Hz, 1H). 13C
NMR (100 MHz, DMSO-dÀ6) d = 163.11, 135.31, 133.85,
132.45, 131.26, 130.12, 129.21, 127.47, 126.79, 126.30,
124.94, 123.69, 122.76 ppm.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.02.064.
Compound 6: 1H NMR (400 MHz, DMSO-dÀ6) d = 11.32
(s, 1H), 9.11 (s, 1H), 7.97 (m, 4H), 7.75 (d, J = 7.4 Hz,
1H), 7.52 (m, 6H). 13C NMR (100 MHz, DMSO-d
)
À6
d = 164.54, 143.30, 139.16, 133.94, 132.33, 131.11, 130.34,
128.98, 128.60, 127.61, 127.52, 127.12, 126.60, 126.12,
125.55 ppm.
References and notes
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