Discovery of potent and selective histone deacetylase inhibitors via focused combinatorial libraries of cyclic α3 β-tetrapeptides

Article abstract of DOI:10.1021/jm900850t

Histone deacetylase (HDAC) inhibitors are powerful tools in understanding epigenetic regulation and have proven especially promising for the treatment of various cancers, but the discovery of potent, isoform-selective HDAC inhibitors has been a major chal

Full text of DOI:10.1021/jm900850t

7836 J. Med. Chem. 2009, 52, 7836–7846
DOI: 10.1021/jm900850t
Discovery of Potent and Selective Histone Deacetylase Inhibitors via Focused Combinatorial Libraries
of Cyclic r3 β-Tetrapeptides
Christian A. Olsen and M. Reza Ghadiri*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
Received June 10, 2009
Histone deacetylase (HDAC) inhibitors are powerful tools in understanding epigenetic regulation and
have proven especially promising for the treatment of various cancers, but the discovery of potent,
isoform-selective HDAC inhibitors has been a major challenge. We recently developed a cyclic
R3β-tetrapeptide scaffold for the preparation of HDAC inhibitors with novel selectivity profiles
(J. Am. Chem. Soc. 2009, 131, 3033). In this study, we elaborate this scaffold with respect to side chain
diversity by synthesizing one-bead-one-compound combinatorial libraries of cyclic tetrapeptide analo-
gues and applying two generations of these focused libraries to the discovery of potent HDAC ligands
using a convenient screening platform. Our studies led to the first HDAC6-selective cyclic tetrapeptide
analogue, which extends the use of cyclic tetrapeptides to the class II HDAC isoforms. These findings
highlight the persistent potential of cyclic tetrapeptides as epigenetic modulators and possible anticancer
drug lead compounds.
Introduction
5, 7, and 9; class IIb: HDACs 6 and 10; class IV: HDAC11).
Class III HDACs comprise a series of NADþ-dependent
enzymes (sirtuins 1-7).
Histone deacetylases (HDACsa) are a family of enzymes
found in bacteria, fungi, plants, and animals that profoundly
affect cellular function. The natural substrates of most
HDACs are believed to be Nε-acetyl lysine residues in the
tails of histones H2A, H2B, H3, and H4 (Supporting Infor-
mation (SI) Figure S1). The degree of acetylation of these sites
affects the packing of nucleosomes in chromatin complexes as
well as the recruitment of transcription activator- and repres-
sor-proteins to histone tails, which in turn modulate the
extent of gene transcription.1-4 The HDAC enzymes are not
restricted to deacetylation of histones, however, and are
known to regulate the function of other proteins within cells.5
For example, HDAC6 has been associated with deacetylation
of R-tubulin6 as well as peroxiredoxin I and II7 and has been
shown to rescue neurodegeneration in Drosophila melano-
gaster.8 To date, 11 Zn2þ-dependent human HDAC enzymes
have been identified and classified according to their sequence
similarity (class I: HDACs 1, 2, 3, and 8; class IIa: HDACs 4,
Although the majority of HDAC inhibitors in clinical trials
are nonselective,9,10 small-molecule HDAC inhibitors have
proven to be promising candidates for therapeutic interven-
tion, especially as tumor suppressors for potential cancer
chemotherapies.3,11-15 Indeed, several candidates have ad-
vanced to clinical trials9 and vorinostat/SAHA was recently
approved by the FDA for the treatment of cutaneous T-cell
lymphoma.16 Nonselective HDAC inhibitors have also re-
cently been shown to elicit positive effects related to neuro-
logical disorders.17-20 Despite these successes with nonselec-
tive HDAC inhibitors, the ability to selectively perturb in-
dividual HDACs with small molecules would be exceedingly
useful in unravellingthe complex andhighlydynamicnetwork
of HDAC signaling and in the design of new and safer drug
candidates. Structure-based efforts to design class- or iso-
form-selective inhibitors have been hampered by the limited
structural information available. To date, there is only a
structural understanding of "HDAC-Like Protein"
(HDLP),21 HDAC8,22,23 and the catalytic domains of
HDAC724 and HDAC4.25 Although the class IIa HDACs
have considerably lower intrinsic deacetylase activity com-
pared to class I HDACs against standard substrates,4,26-28
classIIaHDACsplaypivotalroles in numerous pathwaysand
they are therefore equally important targets for future selec-
tive therapeutic intervention in various diseases.4,29-32
Nature provides a number of related cyclic scaffolds with
HDAC inhibitory activity, including nonribosomal depsipep-
tides,33 the marine natural product largazole,34-39 and
tetrapeptide natural products such as the trapoxins,40,41 HC
toxins,42,43 chlamydocin,43 apicidins (1),44-46 and the azuma-
mides (2)47-51 (Figure 1). Numerous analogues of these
*To whom correspondence should be addressed. Phone: (+1) 858-
784-2700. Fax: (þ1) 858-784-2798. E-mail: ghadiri@scripps.edu.
a Abbreviations: AMC, 7-amino-4-methylcoumarin; Aoda, (S)-2-
amino-8-oxo-decanoic acid; Asu, (S)-2-amino-suberic acid; DIC,
N,N0-diisopropylcarbodiimide; FBS, fetal bovine serum; Fmoc, fluor-
enylmethyloxycarbonyl; HATU, 2-(1H-azabenzotriazol-1-yl)-1,1,3,3,
-tetramethyluronium hexafluorophosphate; HDAC, histone deacety-
lase; HDLP, HDAC-like protein; HOBt, hydroxybenzotriazole; HPLC,
high
performance
liquid
chromatography;
MALDI-TOF,
matrix-assisted laser desorption/ionization time-of-flight; MS, mass
spectrometry; NAD, nicotinamide adenine dinucleotide; Nal, 1-naph-
thylalanine; NCoR2, nuclear coreceptor 2; NMR, nuclear magnetic
resonance; Pa, 3-propanoic acid; Pip, pipecolic acid; PMS, N-methyldi-
benzopyrazine methyl sulfate; SAR, structure-activity relationship;
SPPS, solid-phase peptide synthesis; SPS, solid-phase synthesis; TFA,
trifluoroacetic acid; TSA, trichostatin A; XTT, 30-[1-(phenylamino-
carbonyl)-3,4-tetrazolium]-bis-(4-methoxy-6-nitro)-sulfonic acid.
r
pubs.acs.org/jmc
Published on Web 08/25/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7837
finding was encouraging because the carboxylic acid Zn2þ
natural compounds have been investigated as well.52-63 For
the medicinal chemist, cyclic tetrapeptides built from all R-
amino acids often present significant challenges as drug
targets due to poor macrolactamization yields for closing
the 12-membered ring and multiple three-dimensional con-
formations on the NMR time scale.64-66 We were recently
able to minimize these shortcomings by developing synthetic
HDAC inhibitor scaffolds (3, Figure 1) that have an Rfβ-
amino acid substitution in the backbone to give a 13-mem-
bered ring.67 By systematically changing the chirality of the
amino acids and the position of the β-amino acid, we opti-
mized the scaffold for inhibition of HeLa extract HDAC
activity. Moreover, we correlated compound potencies with
their high-resolution NMR structures, which allowed us to
construct a three-dimensional pharmacophore model. In our
previous study, the identities of the side chains were fixed
so that we could focus on understanding the effects of
peptide backbone modifications on structure and potency.
In contrast, here we investigate a variety of side chain
functionalities by designing and carrying out total solid-phase
synthesis (SPS) of one-bead-one-compound libraries68 of
cyclic peptides for the purpose of inhibiting HDAC enzymes.
The present study focuses on HDACs with potent deacetylase
activity against histones and standard substrates, i.e., class
I HDACs 1, 3, and 8 as well as class IIb HDAC6. HDAC2
was omitted from this investigation due to its high degree
of sequence similarity to HDAC1.
-
coordinating group would provide a useful handle for resin
anchoring to synthesize focused libraries of inhibitors via
efficient and robust chemical SPS techniques.
To reduce the synthetic effort required for assessing the
effect of different lengths of the Zn2þ-coordinating side
chain, we designed a series of alkylated cysteine-derived
analogues (4a-d), which enabled evaluation of the distance
requirements for the different enzyme isoforms (Figure 1 and
SI Figure S3). The use of alkylated cysteine containing
compounds has precedence in the literature for probing
HDACs.70,71 It was previously shown that changing the
linker length by plus/minus one methylene unit significantly
decreased the potency of inhibition of HDAC activity in
HeLa cell extract;52 however, the effect on individual HDAC
isoforms was not investigated in that study. Furthermore,
largazole analogues with different linker lengths have re-
cently been investigated.61 With the sulfur atom in com-
˚
pound 4a extending the linker length by only ∼0.28 A
compared to the methylene in 3b, it was not surprising to
find that this compound behaved similarly to 3b. The
corresponding compound with a linker of one fewer methy-
lene unit, 4b, showed total loss of activity. Likewise, the
shortened linkers in the carboxylic acids 4c and 4d also
rendered these peptides less potent than their parent com-
pound (3a) across our selection of enzymes. It may be noted,
however, that a comparison of compounds 3a vs 4c reveals
a ∼40-fold decrease in potency against HDAC1 and only a
∼3-fold decrease against HDAC8. The length of the linker
may thus be a factor to consider when designing isoform
selective HDAC ligands. The design of so-called “linker-
less” hydroxamic acid HDAC inhibitors with selectivity for
HDAC8 was recently reported,72 supporting the suggestion
that shortened linkers might prove effective against this
isoform. Partial decomposition of these thioether com-
pounds was observed upon prolonged storage (presumably
due to oxidation of the thioether); nonetheless, they are
useful as a discovery tool giving easy access to a variety of
different linker lengths and Zn2þ-coordinating moieties.
First-Generation Library Design. Our first-generation
library comprised a focused 252-member one-bead-one-
compound library aimed at exploring the importance of the
side chain functionalities for potent HDAC inhibition.
On the basis of our preliminary studies and to facilitate solid
phase synthesis, we opted to employ the carboxylic acid
Zn2þ-coordinating residue for this library. Albeit not the
most potent Zn2þ-coordinating moiety, the carboxylic acid
was considered more appropriate than the hydroxamic acid
because the latter gave rise to poor isoform selectivity for the
cyclic tetrapeptides tested.52 The ethylketone containing
ligands seemed ideal for inhibition of class I HDACs 1-3
but showed no inhibitory activity against HDAC6, and the
total solid-phase synthetic route to give the ethylketone
containing ligands required postcleavage purification (SI
Figure S4), which was not ideal for a high-throughput library
screening scheme.
Results and Discussion
Preliminary SAR Information. To guide the design of the
first-generation library, we initially surveyed the effect of the
Zn2þ-coordinating group and its distance from the peptide
core on our selection of HDAC enzymes. Although we had
previously established that changing the Zn2þ-coordinating
functionality in peptides 3a-c accommodated leaps in po-
tency using HeLa cell nuclear extract,67 we wished to deter-
mine more specifically how the inhibitors acted against the
individual HDAC isoforms. We therefore tested 3a-c
against our panel of recombinant human HDACs as well
as against a HeLa cell cytosolic extract (Table 1). The
inhibition trend described previously for 3a-c against HeLa
nuclear extract (hydroxamic acid > ethylketone > acid
Zn2þ-coordinating functionality) was also observed for the
cytosolic extract as well as for HDACs 1 and 3. The IC50
values indicated that the predominant source of HDAC
activity in HeLa nuclear extract is HDAC1, while HDAC3
seems to be more dominant in HeLa cytosolic extract, which
corresponds well with the fact that HDAC3 is known to
shuttle into the cytoplasm.69 Furthermore, the observed IC50
values indicated that the requirements for inhibition of
HDAC8 are different from the other HDACs in class I,
in agreement with previous findings.10,22,23 Interestingly,
ketone 3b, like apicidin, did not inhibit HDAC6 in the
concentration range tested, while acid 3a proved to be a
micromolar inhibitor of HDAC6. This might be explained
by an interaction between the carboxylate of 3a and a
free binding site His in HDAC6 (SI Figure S2). For compar-
ison, HDAC8 contains an active site His residue (His143)
that forms a contact with an adjacent Asp (Asp183).
Although HDAC6 does contain a corresponding active
site His residue, the corresponding Asp is absent, so it
is possible that the active site His in HDAC6 is free
to interact with the carboxylate in 3a (SI Figure S2). This
Previous SAR studies involving cyclic tetrapeptides as
HDAC inhibitors have indicated that the aromatic residue
vicinal to the Zn2þ-coordinating amino acid (position aa2 in
Figure 2) is important for potent inhibition.58,59 We there-
fore decided to probe this position extensively in our initial
library. Aromatic residues of varied bulkiness and polarity as
well as nonaromatic hydrophobic residues were included.
For position aa3, four different β3-amino acids were chosen,

7838 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Olsen and Ghadiri
Figure 1. HDAC inhibitors, natural products, and scaffolds. Arrows show the amide chain directionality. Scaffold 3 shows the structure of our
previously developed R3β ring type. Compound 4 is based on the optimized synthetic R3β ring type but contains a cysteine residue that readily
allows for introduction of different Zn2þ-coordinating moieties.
Table 1. Potencies of 3a-3c, 4a-d, Apicidin, and TSA against HeLa
Extracts and Recombinant Enzymes (IC50 [nM])a
HeLa extracts
class I
class IIb
compd nuclear cytosolic HDAC1 HDAC3b HDAC8 HDAC6
3a
3b
3c
4a
4b
4c
4d
324 ( 4 630 ( 30 116 ( 4 608 ( 3 2300c
2310 ( 20
>10000
31 ( 2
19c
85 ( 6 26 ( 3
3 ( 1.5 2 ( 1.4 18 ( 3 133c
54 ( 5 (20) 62 ( 7 (4920)
143 ( 10 2200c
<1c
54c
NDe
ND
ND
>10000
ND
ND
ND
>10000 >10000 >10000 >10000
>10000
>10000
(4840)
>10000 >10000 (9700)
(7080)
(6050)
1a apicidin 22 ( 6 43 ( 8 22 ( 4
TSAd
29 ( 4 760 ( 20 >10000
3 ( 1.3 3 ( 1.0 3.3 ( 1.5 11 ( 1.4 25 ( 3 6 ( 1.9
a IC50 values are means of at least two experiments performed in
duplicate unless otherwise noted. Values in parentheses are from a single
assay performed in duplicate. b In complex with NCoR2. c From our
previous report.67 d TSA (trichostatin A) was included as a positive control
inhibitor in the assays with HDAC6 and HDAC8 because apicidin is not a
potent inhibitor of these enzymes. e ND = not determined.
including a β3-Phe residue to mimic the Phe side chain found
in trapoxin and chlamydocin. Residues commonly found in
natural product HDAC inhibitors (e.g., Aib, D-Pip, Pro, and
Val) were included along with Phe at position aa4. To
synthesize the library, the first residue (N-Fmoc-(S)-Asu-
O-Allyl) was anchored to a macrobead 2-chlorotrityl solid
support via its carboxylic acid side chain using protocols
previously developed for regular SPS resins.67,73 The choice
of macrobeads as solid support afforded facile handling
during the split-pool synthesis procedure and enabled an
economical small-scale preparation of each compound while
still affording enough material to test each product in several
assays in 96-well microtiter plates (Figure 2). After the total
synthesis of the 252-member library of ligands including on-
resin cyclization, individual beads were arrayed in 96-well
plates, cleaved by treatment with a cocktail containing
trifluoroacetic acid (TFA), and dissolved in DMSO to give
stock solutions. After screening the library for HDAC
inhibitory activity using HeLa nuclear extract, the obtained
hits were analyzed and sequenced using an LC-MS/MS
procedure previously developed in our lab.74
Figure 2. Synthetic scheme and amino acids used in the first-
generation library.
we obtained eight confirmed hits (six different structures) as
shown in Figure 3A. Semilogarithmic plots were obtained
from the inhibition data at two different dilutions of the
From the initial screen and retesting of the first-generation
library for inhibition of HDAC activity in HeLa cell extract,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7839
Figure 3. First-generation library screening results. The library screening in 96-well plates initially provided 15 “hits” that satisfied a criteria of
>75% inhibition at 10 μM and >25% inhibition at 1 μM against HeLa cell extract. Identified hits were then retested in duplicate and
sequenced using LC-MS/MS, which confirmed six different hit structures. (A) Structures of hits and approximate IC50 values were obtained
from plots of the inhibition data; the dose-response curve for apicidin (blue) is shown in each plot for comparison. (B) Table showing the
abundance of amino acids by position in the hit series.
initial stock solutions, from which approximate IC50 values
for these peptides were calculated using GraphPad Prism.
Interestingly, the two most potent compounds (8a and 13)
closely resembled our parent compound (3a), having only a
single substitution in the aromatic residue at the aa2 site
(Trpfnaphthylalanine in 8a or TrpfPhe in 13). Further-
more, one of the hits with intermediate potency (compound
12) contained both the aa2-Trp and the aa4-D-Pip residues
as found in the natural product apicidin A (1b). The abun-
dance of the various side chains by position on the scaffold
(Figure 3B) revealed that the aa2 position required an aromatic
side chain as anticipated; the naphthylalanine residue, which is
similar to Trp in size, was the most abundant in the hit series.
At position aa3, the β3Leu residue was more abundant than
the aromatic residues included in the library, and at position
aa4, no consensus could be derived, indicating that the identity
of the side chain at this position is less significant for inhibition
of HeLa cell HDAC activity.
To confirm the results from the library screening, the
four identified naphthylalanine containing compounds (8a,
9-11) were resynthesized on ∼50 μmol scale, purified, and
retested to obtain IC50 values against HeLa nuclear extract
(Table 2). Their IC50 values confirmed the approximated
values from the initial screening data, supporting that the
library screening format is appropriate for the discovery of
novel HDAC inhibitors. To derive more detailed SAR
information from this initial hit series, compound 14 was
also prepared along with the ethylketone analogue (8b) of
compound 8a (SI Figure S5 and Table 2). The unnatural
naphthylalanine residue was confirmed to be a good alter-
native to Trp at position aa2. Furthermore, the relative IC50
values for carboxylic acid (8a) vs ethylketone (8b) showed the
expected trend, as ethylketone 8b was 10-30-fold more
potent against HeLa extract and HDACs 1 and 3. A decrease
in potency was observed for HDAC6, providing further
evidence that ketones are not favorable for inhibition of this
enzyme isoform. The SAR also suggested that aromatic side
chains at position aa3 are not favorable for inhibition of class
I HDACs (9 vs 10 and 11 vs 14). Interestingly, compound 10
was 2-fold more potent against HDAC1 compared to our
parent carboxylic acid (3a) and was somewhat less potent
against HDAC3, thus providing a ∼10-fold selectivity index
for HDAC1 vs HDAC3 as well as potent inhibition of
HDAC1 (68 nM). The substitution of the AlafPhe residue
in position aa4 may therefore give rise to an increased
selectivity for HDAC1.
Compounds 15 and 16, in which the least structurally
restrictive residue (aa4) was substituted with Nε-Ac-lysine or
Aoda, respectively, were also prepared and tested (Table 2)
so that we could ascertain how such substitutions would
influence binding to the HDACs before designing a second-
generation library. The aa4-Lys(Ac) residue in compound 15
was inspired by the two consecutive lysine residues K381 and
K382 known to be involved in acetylation/deacetyla-
tion events in the transcription factor p53. Moreover, the
Lys(Ac)-Lys(Ac) motif has been used in a commercially
available fluorescent HDAC8 substrate (His-Arg-Lys(Ac)-
Lys(Ac)-AMC, www.biomol.com), which led us to hypothe-
size that such a motif might provide an improved degree of
recognition by HDAC8. The Aoda-Aoda sequence in 16
provides a double isosteric version of this feature. Indeed,
improved activity against HDAC8 was observed for
compound 15 (150 nM) and to a much lesser extent for 16
(1400 nM); however, the IC50 values for inhibition of
HDACs 1 and 3 were still in the same range as that observed
for parent compounds 3b and apicidin (Table 2).
Second-Generation Library Design, Synthesis, and Screen-
ing. On the basis of the SAR information gathered from the
first-generation library, we decided to focus on the diversi-
fication of the aa3 and aa4 positions in the second-generation
library in the hopes of reducing bias for class I HDAC
inhibitory activity. In addition to amino acids found in the

7840 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Olsen and Ghadiri
Table 2. IC50 Values [nM] of Purified Compounds against HeLa Nuclear Extract and Recombinant Enzymesa
class I
class IIb
HDAC6
compd
HeLa nuclear extract
HDAC1
HDAC3b
HDAC8
8a c[Asu-Nal-β3Leu-Ala]
8b c[Aoda-Nal-β3Leu-Ala]
9 c[Asu-Nal-β3Trp-Phe]
10 c[Asu-Nal-β3Leu-Phe]
11 c[Asu-Nal-β3Trp-Aib]
14 c[Asu-Nal-β3Leu-Aib]
15 c[Aoda-Trp-β3Leu-Lys(Ac)]
16 c[Aoda-Trp-β3Leu-Aoda]
17 c[Asu-Nal-β3Leu-Arg]
18 c[Asu-Nal-β3Ser-Arg]
19 c[Asu-Nal-β3Tyr-Arg]
20 c[Asu-Nal-β3Tyr-Lys(Ac)]
21 c[Asu-Nal-β3Tyr-Phe(F5)]
22 c[Asu-Asp-β3Tyr-Arg]
23 c[Asu(NHOH)-Asp-β3Tyr-Arg]
24 c[Aoda-Trp-β3Leu-Phe]c
25 c[Asu(NHOH)-Trp-β3Leu-Phe]
26 c[Asu-Nal-β3Leu-Lys]
27 c[Asu-Nal-β3Leu-Lys(Ac)]
28 c[Cys(Pa)-Trp-β3Leu-Lys(Ac)]
1a apicidin
205 ( 5
17 ( 2
1040 ( 10
253 ( 4
1010 ( 20
500 ( 20
ND
200 ( 10
8 ( 1
460 ( 20
68 ( 3
(751)
990 ( 20
32 ( 1
1670 ( 30
690 ( 20
(2990)
ND
ND
4900 ( 70
>10000
(8000)
ND
530 ( 10
(2670)
(870)
5800 ( 40
(3700)
(7000)
136 ( 1
42 ( 5
150 ( 20
(178)
970 ( 10
54 ( 5
150 ( 10
1400 ( 40
(607)
>10000
>10000
1400 ( 20
2320 ( 20
1080 ( 20
4400 ( 30
>10000
>10000
39 ( 3
ND
ND
260 ( 10
262 ( 3
1090 ( 10
694 ( 4
1040 ( 20
3630 ( 20
>10000
164 ( 4
ND
ND
ND
ND
ND
ND
(1400)
(1380)
(6700)
ND
ND
ND
288 ( 4
(∼1000)
>10000
120 ( 20
ND
ND
ND
240 ( 10
ND
ND
ND
ND
ND
70 ( 6
ND
ND
31 ( 5
ND
ND
242 ( 5
190 ( 10
>10000
29 ( 4
ND
1120 ( 10
1670 ( 20
>10000
>10000
6 ( 1.9
56 ( 4
ND
1000 ( 20
7040 ( 50
760 ( 20
25 ( 3
ND
22 ( 6
3 ( 1.3
22 ( 4
3.3 ( 1.5
TSA
11 ( 1.4
a IC50 values are means of at least two experiments performed in duplicate. Values in parentheses are from a single assay performed at least in duplicate.
ND = not determined. Pa = 3-propanoic acid (see Figure 1 for structure). b In complex with NCoR2. c This compound was too hydrophobic for assaying.
various nonribosomal cyclic tetrapeptide inhibitors and their
analogues (mostly hydrophobic residues), a number of hy-
drophilic residues (i.e., β3Ser, Thr, Arg, and Lys) were
included due to the presence of these side chains in the
histone tails (SI Figure S1). Aspartic acid was also included
due to its presence vicinal to an Nε-acetylated lysine in
R-tubulin, which is known to be a substrate for HDAC6.
A 525-member second-generation library (Figure 4) was
next synthesized on macrobeads as described for the initial
library. The compounds were initially screened for HDAC
inhibition activity in HeLa cell extract, and then both
libraries were counter-screened against HDAC6. As before,
the hits were confirmed by retesting the stock solutions in
duplicate as well as performing LC-MS/MS sequencing and
MALDI-TOF MS, which furnished another nine hit struc-
tures (for full hit series, see SI Figures S6 and S7). All the hits
contained either Trp or naphthylalanine at the aa2 position.
In addition to the results already discussed for the first
generation library above, Tyr at the aa3 position was ob-
served in a number of hits, while Phe(F5) or Arg appeared at
the aa4 position. Unfortunately, no hits against HDAC6
were observed that were not also inhibitors of HeLa HDAC
activity. Three peptides that were hits against both HeLa
extract and HDAC6, all of which had an Arg residue at the
aa4 position, were resynthesized (compounds 17-19) along
with two analogues having either a Lys(Ac) or a Phe(F5)
residue at the aa4 position, respectively (compounds 20, 21)
(Figure 5 and Table 2). The obtained IC50 values revealed
that the unnatural Phe(F5) residue, which was present in a
number of hits from the HeLa HDAC activity screening,
resulted in low potency of compound 21 over the whole
selection of enzyme isoforms tested, so other hit structures
containing this residue were not pursued further.
Structure-Activity Relationships and HDAC Isoform
Selectivity. The compounds differing only in the identity of
the residue at position aa3 (17-19) revealed interesting SAR
Figure 4. Amino acids included in the second-generation tetrapep-
tide library (525 members).
information. Notably, the compound having the β3Tyr-Arg

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7841
Lys-aa4 and the Lys(Ac)-aa4 residues, respectively. The
potencies of these compounds confirm that an aromatic
residue at position aa3 results in decreased potency against
the class I HDACs, as previously observed for 9-14.
Furthermore, the results show that substitution of the Lys-
(Ac)-aa4 residue (27) for an Arg-aa4 (17) or a Lys-
aa4 residue (26) had little effect on the inhibitory activi-
ties measured against the entire selection of HDACs. It
was recently reported that an Arg residue vicinal to the
Lys(Ac) promoted binding of synthetic histone substrates
to HDAC8,83 which also corresponds well with the observa-
tion that most of the lysine residues known to participate in
acetylation/deacetylation events in the tails of histones H3,
H4, and H2A have Arg residues in their vicinity (SI Figure
S1). Taking into account that compounds 17, 26, and 27 are
also 4-5-fold more potent against HDAC3 and 3-4-fold
more potent against HDAC6 when compared to their
Ala-aa4 analogue (8b), our results suggest that the presence
of an Arg, Lys, or Lys(Ac) residue vicinal to the Zn2þ
-
coordinating amino acid may also promote binding to other
HDAC isoforms (i.e., HDACs 3 and 6 in this study). To
check if the Lys(Ac) residue in 27 was deacetylated under the
conditions of our assay, we carried out the standard HeLa
extract assay with this compound (at concentrations of 10
μM or 1 μM), but instead of the trypsin development step, we
quenched the reaction with MeOH and analyzed the reaction
mixtures by LC-MS. These experiments showed the peak
expected for compound 27 but no trace of the corresponding
deacetylated analogue (26).
Finally, the composition of our most potent inhibitor of
HDAC8 (compound 15) was combined with the cysteine
derived Zn2þ-coordinating amino acid containing one
methylene unit fewer than Asu (as in 4c) to give peptide 28.
Although the HDAC3/HDAC8 selectivity was reversed, the
potency against HDAC8 was not considered satisfactory.
Efforts to design cyclic tetrapeptide inhibitors with improved
potency as well as selectivity for HDAC8 are now in progress
in our laboratory. The SAR information obtained is sum-
marized in Figure 6.
Western Blot Analysis and Cell Growth Inhibition. To
assess the effectiveness of our novel inhibitors in cells, we
treated Jurkat cells (T-cell leukemia) with a selection of
compounds (apicidin 1a, 10, 15, and 23) for 24 h and Western
blotted for histone H3, acetylated histone H3 (K9 þ K14),
and acetylated R-tubulin as previously described.84 The
presence of acetylated tubulin is an indicator of HDAC6
inhibition, whereas the presence of acetylated histone H3
indicates inhibition of the class I HDACs. Similarly,
as expected based on the IC50 values obtained with the
recombinant enzyme isoforms, the two ketone containing
compounds (apicidin and 15) exhibited potent histone dea-
cetylase activity and only minor tubulin deacetylase activity.
We were also pleased to find that library hit 10, containing
the carboxylic acid Zn2þ-coordinating group, affected the
acetylated histone levels in the cells compared to the DMSO
control, whereas the effect on acetylated R-tubulin levels was
considerably less pronounced in relation to the correspond-
ing DMSO control. As expected, considering the in vitro
enzyme inhibition data, 10 was less potent than both apicidin
and 15 (Figure 7). Finally, the HDAC6 selectivity of com-
pound 23 was confirmed by the presence of relatively high
levels of acetylated R-tubulin, with comparably low levels of
histone H3 acetylation.
Figure 5. Chemical structures of peptide ligands.
motif at positions aa3-aa4 (19) was the most potent car-
boxylic acid-containing inhibitor of HDAC6 identified so far
and had also the best selectivity index (HDAC3/HDAC6 =
0.65) in the series, albeit still in favor of the class I HDACs.
Because HDAC6 is known to deacetylate R-tubulin, we
decided to combine the β3Tyr-Arg motif with an Asp
residue at the aa2 position to give compounds 22 and 23.
Interestingly, a successful reversal of the isoform selectivity
pattern was achieved with compound 23, as the selectivity
indices for HDAC1/HDAC6, HDAC3/HDAC6, and
HDAC8/HDAC6 were approximately 3, 4, and 6, respec-
tively, with potent inhibition of HDAC6 (IC50 = 39 nM).
Compound 23 is, to the best of our knowledge, the first cyclic
tetrapeptide analogue exhibiting isoform selectivity toward a
class II HDAC. Moreover, its selectivity index is comparable
to that observed for tubacin (HDAC1/HDAC6 selectivity
index of 7),75-77 and its potency is in the nanomolar range as
recently reported for a series of thiolate containing inhibi-
tors78,79 inspired by an HDAC6 selective substrate.80,81
More potent (picomolar) HDAC6 selective inhibitors contain-
ing a phenylisoxazole surface recognition group have also been
reported recently.82 The ethylketone analogue 24 containing
the aa4-Phe residue as observed in compound 10 was pre-
pared in the hopes that higher potency against HDAC1 could
be obtained while still retaining the selectivity profile discussed
for 10, but peptide 24 was unfortunately too hydrophobic and
hence insoluble under the assay conditions. As expected based
on previously observed trends for hydroxamic acid compound
3c, the hydroxamic acid containing homologue 25 was a potent
inhibitor of both classes of HDACs.
To derive more detailed SAR related to compounds 17, 19,
and 20, we also prepared peptides 26 and 27 containing the

7842 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Olsen and Ghadiri
potent against HDAC1, was less cytotoxic toward HeLa cells
than 3b, indicating that HDAC6 is less important than
HDAC1 for cell survival. Clearly, these findings require
further support, and differences in cell permeability may
also play a major role in the different cytoselectivity profiles
observed. In Jurkat cells, for example, compound 8b proved
slightly more potent than the hydroxamic acid inhibitor 3c,
even though 3c is a more potent inhibitor of all the HDACs
tested in vitro. This observation may be explained by an
expected more favorable logP value for compound 8b. The
trend observed for these compounds against HeLa, K-562,
and KYO-1 cells seem to better reflect the HDAC inhibitory
activities, confirming that several factors play important
roles in the growth inhibition process. Taken together with
the Western blots, these results show that our compounds
may find use as specific small-molecule probes to regulate
histone and/or tubulin acetylation at subcytotoxic concen-
trations in cells. Finally, we were pleased to find that by
reintroducing the strong Zn2þ-coordinating hydroxamic
acid moiety in one of the hit compounds to give 25, we were
able to obtain potent growth inhibition in a number of cell
lines. This provides further evidence that the effects of the
cyclic peptide core and the Zn2þ-coordinating moiety on
potency may be considered somewhat additive in this class of
compounds.
Figure 6. Summary of the obtained SAR information. The shown
high-resolution NMR structure of lead compound 3b in DMSO-d6
was determined previously by our group.67 The hydrogen atoms are
omitted for clarity.
Figure 7. Western blot analysis of lysates from Jurkat cells treated
with selected compounds and controls. Concentrations of com-
pounds 10 and 23 are shown in μM. The presence of acetylated
tubulin is an indicator of HDAC6 inhibition, whereas the presence
of acetylated histone H3 indicates inhibition of the class I HDACs.
Histone H3 (bottom) serves as a control for protein loading.
Conclusions
A synthetic tetrapeptide scaffold having an Rfβ-amino
acid substitution in the backbone has been employed for the
identification of moderately selective inhibitors for the
HDAC isoforms 1 and 6. Using an all (S)-configured R3β
ring type as the lead compound, a semihigh-throughput
screening scheme was developed for assaying ligands from
focused one-bead-one-compound libraries. The present study
shows that this screening platform in combination with
sequence information from natural as well as synthetic
HDAC substrates and inhibitors is useful for the discovery
of new HDAC inhibitors. The obtained SAR information
revealed a range of potent compounds displaying novel side
chain patterns and containing unnatural amino acid residues.
Depending on the HDAC isoform in question, different
amino acid positions on the scaffold proved more or less
important for potency, which will aid the future design of
selective ligands, help identify optimal sites for labeling, and
enable the design of efficient probes for activity-based pro-
teomics or affinity matrix pull-down studies.41,86,87
Table 3. GI50 Values [μM] for Selected Compounds against Various
Cancer Cellsa
compd
HeLa
MCF-7
Jurkat
K-562
KYO-1
vinblastine
1a apicidin
0.06
3
3.5b
5.6b
18
∼10
7
<0.01
4
0.3
0.9
<0.1
2.5
10.5b
0.8b
43
3b
3c
8b
10
15
23
25
>20b
0.6b
ND
>20
>20
>20
ND
NDc
1.7
2b
1.6b
30
0.3
>20
>20
9
>20
>20
>20
1.3
>20
ND
ND
1.9
>20
ND
ND
1.9
ND
a HeLa (cervical cancer), MCF-7 (breast cancer), Jurkat (T-cell
leukemia), K-562 and KYO-1 (chronic myeloid leukemia). b From our
previous report.67 c ND = not determined.
Growth inhibition of various cancer cell lines by selected
compounds was next determined using the XTT assay
(Table 3).67,85 Except for the highly potent hydroxamic-
acid-containing broad spectrum HDAC inhibitors (3c and
25), which were also broadly cytotoxic, the general trend
seemed to be that cytotoxicity correlated largely with potent
HDAC1 inhibition. Our most potent carboxylic acid HDAC
inhibitor (10) did not potently inhibit the growth in any of
our series of cancer cells, which is in accord with the relatively
weak inhibition of HDAC activity in Jurkat cells compared
to compound 15 and apicidin indicated by the Western blot.
Interestingly, compound 15, which is an analogue of 3b with
slightly lower potency against HDAC1 (∼2-fold) and sig-
nificantly higher potency against HDAC8 (∼15-fold), was
less cytotoxic than 3b toward HeLa cells, indicating that
HDAC8 may not be important for cell survival in this cell
line. Moreover, compound 23, which is a >250-fold more
potent inhibitor of HDAC6 than 3b and only ∼5-fold less
Our data also indicate that the cyclic tetrapeptide motif not
only provides a highly modular scaffold but also that this class
of compounds can be further modulated by changing the
Zn2þ-coordinating moiety in order to fine-tune potency,
HDAC isoform selectivity, or cytoselectivity. Importantly,
compounds exhibiting potent HDAC inhibitory activity and
isoform selectivity were identified and shown to work in cell
culture. It is remarkable that from this relatively small selec-
tion of compounds, it was possible to design ligands with
completely reversed selectivity profiles as compared to the
known natural product tetrapeptide inhibitors. We therefore
envision that this discovery platform may find additional use
for the design ofclass- and isoform-selective HDACinhibitors
targeting other enzyme isoforms. It will be interesting to
explore trends in cytoselectivity between different cell lines
in greater detail with these ligands because such studies
will help delineate the significance of individual HDACs in

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7843
Table 4. Data for Resynthesized Peptides
various cancers. This knowledge may aid the design of
targeted drug candidates with lower toxicity and fewer side
effects.
compd
HPLC230 nm, %
ESI-TOF HRMS calcd/found (m/z)
4a
4b
4c
99
95
96
90
99
99
90
99
95
95
99
99
99
96
99
99
95
95
90
95
95
95
92
95
608.2883/608.2893a
594.2721/594.2721a
582.2357/582.2348a
568.2200/568.2198a
589.2997/589.2996a
579.3541/579.3537b
738.3268/738.3280a
665.3310/665.3298a
654.3286/654.3285b
603.3153/603.3197a
689.3997/689.3992a
702.4201/702.4197a
652.3812/652.3814b
626.3297/626.3300b
702.3610/702.3617b
716.3654/716.3658b
783.2812/783.2821b
620.3039/620.3027b
635.3148/635.3147b
644.3806/644.3794b
647.3557/647.3552b
624.3756/624.3759b
666.3861/666.3867b
681.3041/681.3039a
Experimental Section
4d
8a
8b
9
Enzymes and Chemicals. HDAC1, HDAC3-NCoR2,
HDAC6, and HDAC8 were purchased from BPS Biosciences
(San Diego, CA). Trypsin (TPCK treated, from bovine pan-
creas; 12500 units/mg) was from Sigma-Aldrich (Milwaukee,
WI). 2-Cl-Trityl chloride resin macrobeads were from Peptides
International (Louisville, KY), Weinreb amide resin was from
Novabiochem (San Diego, CA), and 2-chlorotrityl N-Fmoc-
hydroxylamine resin was from Sigma-Aldrich (Milwaukee, WI).
β-Amino acids were purchased from Peptech Corporation
(Burlington, MA). All R-amino acids, chemicals, and solvents
were used as received from suppliers. HDAC assay buffer was
prepared as described in the Biomol International protocol
AK-503 (Tris/Cl (50 mM), NaCl (137 mM), KCl (2.7 mM),
MgCl2 (1 mM), pH 8.0). Histone H3 antibody was from Abcam
(Cambridge, MA), acetylated K9 þ K14 histone antibody was
from Upstate (Temecula, CA), and acetylated R-tubulin anti-
body was from Sigma (St. Louis, MO).
Library Synthesis. N-Fmoc-(S)-2-Asu(t-Bu)-OAllyl (3 equiv
with respect to the resin) was stirred in TFA-CH2Cl2 for 1 h,73
the solvents were evaporated in vacuo, and the residue was
coevaporated several times with CH2Cl2-hexane and dried for
2 h under high vacuum. The TFA-free residue was taken up in
CH2Cl2 (2 mL/100 mg) and i-Pr2EtN (20 equiv) and was shaken
with 2-Cl-trityl chloride macrobeads in a fritted syringe for 16 h.
The resin was washed with DMF, MeOH, and CH2Cl2 (3ꢀ
each), and the loading was determined by UV measurement
(290 nm) of the chromophore in an Fmoc deprotected sample
(Novabiochem procedure). The bulk of the resin was then Fmoc
deprotected with DMF-piperidine (3:1, 2 ꢀ 20 min), washed
with DMF, MeOH, and CH2Cl2 (3ꢀ each), and dried in vacuo.
Three cycles of combinatorial split-pool Fmoc peptide synth-
esis were then performed in 3 mL fritted syringes, applying the
Fmoc protected amino acids (5 equiv), DIC (5 equiv), HOBt
(5 equiv), and bromophenol blue (2 equiv) as internal indicator
in each coupling step.88 Standard Fmoc deprotection and
washing protocols were applied. After coupling of the fourth
amino acid, the resins were washed with DMF, MeOH, CH2Cl2,
and CHCl3-N-methylmorpholine degassed with argon (3ꢀ
each). The allyl group was then cleaved with Pd(Ph3P)4 (0.2
equiv) in degassed CHCl3-N-methylmorpholine (4 mL) by
shaking for 16 h in the dark, after which time the resin was
washed with DMF, 1% sodium dimethylthiocarbamate in DMF,
and then neat DMF again (2ꢀ each), followed by Fmoc depro-
tection and standard washing. Finally, the resin-bound peptides
were cyclized (2 ꢀ 12 h) with HATU (5 equiv) and
i-PrEt2N (20 equiv) in DMF (3 mL), washed extensively, and
dried under high vacuum. The beads were then divided into
individual wells in round-bottomed 96-well polypropylene
microtiter plates (Fisherbrand), leaving a column free for the
standards and controls during screening. To each well was added
∼200 μL of a cleavage cocktail (TFA-CH2Cl2-i-Pr3SiH-H2O
(48:48:2:2)) and the plates were left for 16 h. Residual solvents
were removed by placing the plates under vacuum in a desiccator
for 24 h, before DMSO was added to give stock solutions (1 mM
theoretical concentration based on the initial loading and beads/
mg) of each peptide for screening and sequencing.
10
11
14
15
16
17
18
19
20
21
22c
23c
24c
25
26c
27c
28
a [M þ Na]þ. b [M þ H]þ. c 1H NMR spectra are included in the
Supporting Information
preparative reversed-phase HPLC (C-18 column), and the ob-
tained fractions were lyophilized to give the compounds as fluffy
solids that were characterized by LC-MS and high resolution
ESI-TOF MS (Table 4) as well as 1H NMR for selected
compounds. The purities of the compounds were determined
by analytical HPLC with UV detection at 230 nm, using a
Phenomenex Luna 150 mm ꢀ 4.6 mm C18(2) column (5 μ)
eluted at a rate of 1.5 mL/min. Injection volumes were 20 μL of
approximately 1 mg/mL solution. The gradient elution system
consisted of eluent A (H2O-MeCN-TFA 99:1:0.1) and eluent
B (MeCN-H2O-TFA 90:10:0.07) rising linearly from 0% to
100% of B during 30 min. All cyclic peptides were g95% pure,
with the exceptions of compounds 4d, 9, 23, and 27, which
were g90% pure. The HPLC purity of each compound is given
in Table 4. DMSO stock solutions were prepared using UV
(ε(Trp) = 5690 M-1 ꢀ cm-1 (280 nm) and ε(Tyr) = 1280 M-1
ꢀ
cm-1 (280 nm)) to adjust the concentrations. Stock solutions of
compounds lacking these aromatic chromophores were pre-
pared by weight.
IC50 Determination. The IC50 values against HeLa extracts
were determined using the standard Biomol HDAC fluorimetric
assay protocol (AK-503).85 For inhibition of recombinant hu-
man HDACs, the dose-response experiments with internal
controls were performed in black or white low binding Nunc
96-well microtiter plates. The dilution series were prepared in
Milli-Q water from 1 mM DMSO stock solutions. The appro-
priate dilution of the inhibitor (10 μL of a 5ꢀ concentra-
tion solution) was added to each well followed by HDAC
assay buffer (37.5 μL) containing Ac-Lys(Ac)-AMC substrate
(67 μM) and bovine serum albumin (0.75 mg/mL). For HDAC8,
the Arg-His-Lys(Ac)-Lys(Ac)-AMC substrate (27 μM) was
used. Finally, a solution of the appropriate HDAC (2.5 μL)
was added and the plate was incubated at 37 °C for 30 min in the
dark (HDAC1, 0.09 μg/μL; HDAC3, 0.03 μg/μL; HDAC6,
0.011 μg/μL; HDAC8, 0.4 μg/μL). Then trypsin (50 μL,
0.4 mg/mL) was added and the assay development was allowed
to proceed for 15 min at RT before the plate was read using a
Tecan GENios plate reader with excitation at 360 nm and
detecting emission at 460 nm.
Peptide Synthesis. Compounds were prepared on solid phase
with cyclization on the resin (for hydroxamic acids) or in
solution (for ethylketones) as previously described.67 Linear
precursors for carboxylic-acid-containing pseudopeptides were
prepared by standard Fmoc SPPS and cleaved using 2,2,2-
trifluoroethanol-HOAc-CH2Cl2 (2:2:6) in order to keep the
side chain protecting groups in place until after cyclization in
solution. All compounds were purified to homogeneity by

7844 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Olsen and Ghadiri
Library Screening and Hit Identification. Dilutions of 10 and
1 μM were prepared from each of the 1 mM DMSO stock
solutions, and 10 μL of each were added to white Nunc assay
plates. In each plate, standards and controls consisted of dupli-
cate wells of: (1) no enzyme (blank), (2) 1 μM of inhibitor 3a, (3)
1 μM of apicidin, or (4) no inhibitor (max read-out) were installed
in each plate. For screening of HDAC6, we used TSA (0.1 μM)
instead of apicidin as one of the internal standards. Each plate
was assayed as described above for the purified peptides, and
compounds that wereconsideredhits, i.e., satisfied our criteria for
inhibition at both concentrations, were rediluted and retested in
duplicate to weed out false positives. Hits that also satisfied this
round of screening were then sequenced using automated
LC-MS/MS as previously described,74,89-91 and parent ion
masses were confirmed by either negative mode sonic spray
ionization (SSI) MS or MALDI-TOF MS.
(2) Allis, C. D.; Berger, S. L.; Cote, J.; Dent, S.; Jenuwien, T.;
Kouzarides, T.; Pillus, L.; Reinberg, D.; Shi, Y.; Shiekhattar, R.;
Shilatifard, A.; Workman, J.; Zhang, Y. New nomenclature for
chromatin-modifying enzymes. Cell 2007, 131, 633–636.
(3) Biel, M.; Wascholowski, V.; Giannis, A. Epigenetics;an epicenter
of gene regulation: histones and histone-modifying enzymes.
Angew. Chem., Int. Ed. 2005, 44, 3186–3216.
(4) Verdin, E.; Dequiedt, F.; Kasler, H. G. Class II histone deacety-
lases: versatile regulators. Trends Genet. 2003, 19, 286–293.
(5) Yuan, Z. L.; Guan, Y. J.; Chatterjee, D.; Chin, Y. E. Stat3
dimerization regulated by reversible acetylation of a single lysine
residue. Science 2005, 307, 269–273.
(6) Hubbert, C.; Guardiola, A.; Shao, R.; Kawaguchi, Y.; Ito, A.;
Nixon, A.; Yoshida, M.; Wang, X. F.; Yao, T. P. HDAC6 is a
microtubule-associated deacetylase. Nature 2002, 417, 455–458.
(7) Parmigiani, R. B.; Xu, W. S.; Venta-Perez, G.; Erdjument-Bromage,
H.; Yaneva, M.; Tempst, P.; Marks, P. A. HDAC6 is a specific
deacetylase of peroxiredoxins and is involved in redox regulation.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 9633–9638.
Western Blot Analysis. Jurkat cells were cultivated in RPMI
1640 medium supplemented with 10% fetal bovine serum and
antibiotics. At confluency, cells were seeded in six-well plates
and incubated with various concentrations of the inhibitor
compounds at 37 °C for 24 h. The cells were then washed,
harvested, and lysed. The lysates were run on 4-12% poly-
acrylamide SDS PAGE gels (Invitrogen), transferred to nitro-
cellulose membranes, and visualized as described previously.84
TSA and the vehicle (DMSO) were used as positive and negative
controls, respectively.
Cancer Cell Cytotoxicity Assays. Cells were cultivated in flat
96-well tissue culture plates in 90 μL of medium supplemented
with 10% FBS and antibiotics. HeLa cells were seeded at a
density of 5000 cells/well, Jurkat and MCF-7 at a density of
10000 cells/well, and K-562 and KYO-1 at a density of 15000
cells/well. Twenty-four hours later, 10 μL of medium containing
various concentrations of the desired compounds were added in
triplicate, and the cells were incubated at 37 °C for 72 h in a 5%
CO atmosphere. Cell survival was determined using the XTT
(Sigma-Aldrich, St. Louis, MO) colorimetric assay. Imme-
diately prior to use, a mixture of XTT (1 mg/mL) and PMS
(N-methyl dibenzopyrazine methyl sulfate, 0.383 mg/mL) in
phenol-red-free RPMI was prepared (0.1 mL of PMS per 5 mL
of XTT solution). After adding 50 μL of this mixture to each
well, plates were incubated at 37 °C for 2 h in the case of HeLa
and MCF-7 cells, and 4 h for Jurkat, K-562, and KYO-1 cells.
Plates were shaken to evenly distribute the dye in the wells, and
the absorbance was measured using a Tecan GENios plate
reader spectrometer at a wavelength of 485 nm.
(8) Pandey, U. B.; Nie, Z.; Batlevi, Y.; McCray, B. A.; Ritson, G. P.;
Nedelsky, N. B.; Schwartz, S. L.; DiProspero, N. A.; Knight, M.
A.; Schuldiner, O.; Padmanabhan, R.; Hild, M.; Berry, D. L.;
Garza, D.; Hubbert, C. C.; Yao, T. P.; Baehrecke, E. H.; Taylor, J.
P. HDAC6 rescues neurodegeneration and provides an essential
link between autophagy and the UPS. Nature 2007, 447, 859–863.
(9) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone
deacetylase inhibitors: from bench to clinic. J. Med. Chem. 2008,
51, 1505–1529.
(10) Bieliauskas, A. V.; Pflum, M. K. Isoform-selective histone deace-
tylase inhibitors. Chem. Soc. Rev. 2008, 37, 1402–1413.
(11) Cole, P. A. Chemical probes for histone-modifying enzymes. Nat.
Chem. Biol. 2008, 4, 590–597.
(12) Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Anticancer activities
of histone deacetylase inhibitors. Nat. Rev. Drug Discovery 2006, 5,
769–784.
(13) Minucci, S.; Pelicci, P. G. Histone deacetylase inhibitors and the
promise of epigenetic (and more) treatments for cancer. Nat. Rev.
Cancer 2006, 6, 38–51.
(14) Marks, P.; Rifkind, R. A.; Richon, V. M.; Breslow, R.; Miller, T.;
Kelly, W. K. Histone deacetylases and cancer: causes and thera-
pies. Nat. Rev. Cancer 2001, 1, 194–202.
(15) Marks, P. A.; Richon, V. M.; Breslow, R.; Rifkind, R. A. Histone
deacetylase inhibitors as new cancer drugs. Curr. Opin. Oncol.
2001, 13, 477–483.
(16) Marks, P. A.; Breslow, R. Dimethyl sulfoxide to vorinostat:
development of this histone deacetylase inhibitor as an anticancer
drug. Nat. Biotechnol. 2007, 25, 84–90.
(17) Kazantsev, A. G.; Thompson, L. M. Therapeutic application of
histone deacetylase inhibitors for central nervous system disorders.
Nat. Rev. Drug. Discovery 2008, 7, 854–868.
(18) Fischer, A.; Sananbenesi, F.; Wang, X.; Dobbin, M.; Tsai, L. H.
Recovery of learning and memory is associated with chromatin
remodelling. Nature 2007, 447, 178–182.
(19) Herman, D.; Jenssen, K.; Burnett, R.; Soragni, E.; Perlman,
S. L.; Gottesfeld, J. M. Histone deacetylase inhibitors reverse
gene silencing in Friedreich’s ataxia. Nat. Chem. Biol. 2006, 2,
551–558.
(20) Tsankova, N. M.; Berton, O.; Renthal, W.; Kumar, A.; Neve, R.
L.; Nestler, E. J. Sustained hippocampal chromatin regulation in a
mouse model of depression and antidepressant action. Nat. Neu-
rosci. 2006, 9, 519–525.
(21) Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.; Rifkind,
R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P. Structures of a
histone deacetylase homologue bound to the TSA and SAHA
inhibitors. Nature 1999, 401, 188–193.
(22) Somoza, J. R.; Skene, R. J.; Katz, B. A.; Mol, C.; Ho, J. D.;
Jennings, A. J.; Luong, C.; Arvai, A.; Buggy, J. J.; Chi, E.; Tang, J.;
Sang, B. C.; Verner, E.; Wynands, R.; Leahy, E. M.; Dougan, D.
R.; Snell, G.; Navre, M.; Knuth, M. W.; Swanson, R. V.; McRee,
D. E.; Tari, L. W. Structural snapshots of human HDAC8 provide
insights into the class I histone deacetylases. Structure 2004, 12,
1325–1334.
Acknowledgment. We thank the TSRI Mass Spectrometry
Facility for high resolution mass spectral analyses, Dr. Ana
Montero for cell lines and HeLa cell extracts, Andrei Loutchni-
kov and Devon Cayer for MCF-7 and Jurkat cell lines, respec-
tively, Prof. Joel M. Gottesfeld, Dr. James C. Chou, and Dr.
David Herman for helpful discussions and assistance with
western blot analyses, as well as Dr. Luke J. Leman for fruitful
discussions and invaluable comments on this manuscript. C.A.
O. thanks the Benzon Foundation and the Lundbeck Founda-
tion for postdoctoral fellowships as well as the Danish Indepen-
dent Research Council for a Young Researcher0s Award (274-
06-0317). This work was supported in part by a grant from the
National Institute of General Medical Sciences (GM52190) and
the Skaggs Institute for Chemical Biology.
(23) Vannini, A.; Volpari, C.; Filocamo, G.; Casavola, E. C.; Brunetti,
M.; Renzoni, D.; Chakravarty, P.; Paolini, C.; De Francesco, R.;
Gallinari, P.; Steinkuhler, C.; Di Marco, S. Crystal structure of a
eukaryotic zinc-dependent histone deacetylase, human HDAC8,
complexed with a hydroxamic acid inhibitor. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 15064–15069.
Supporting Information Available: Figures S1-S7 and 1H
NMR spectra for selected compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
(24) Schuetz, A.; Min, J.; Allali-Hassani, A.; Schapira, M.; Shuen, M.;
Loppnau, P.; Mazitschek, R.; Kwiatkowski, N. P.; Lewis, T. A.;
Maglathin, R. L.; McLean, T. H.; Bochkarev, A.; Plotnikov, A. N.;
Vedadi, M.; Arrowsmith, C. H. Human HDAC7 harbors a class IIa
References
(1) Kouzarides, T. Chromatin modifications and their function. Cell
2007, 128, 693–705.

Article
histone deacetylase-specific zinc binding motif and cryptic deace-
tylase activity. J. Biol. Chem. 2008, 283, 11355–11363.
(25) Bottomley, M. J.; Lo Surdo, P.; Di Giovine, P.; Cirillo, A.; Scarpelli,
R.; Ferrigno, F.; Jones, P.; Neddermann, P.; De Francesco, R.;
Steinkuhler, C.; Gallinari, P.; Carfi, A. Structural and functional
analysis of the human HDAC4 catalytic domain reveals a regulatory
structural zinc-binding domain. J. Biol. Chem. 2008, 283, 26694–
26704.
(26) Fischle, W.; Dequiedt, F.; Hendzel, M. J.; Guenther, M. G.; Lazar,
M. A.; Voelter, W.; Verdin, E. Enzymatic activity associated with
class II HDACs is dependent on a multiprotein complex containing
HDAC3 and SMRT/N-CoR. Mol. Cell 2002, 9, 45–57.
(27) Fischle, W.; Dequiedt, F.; Fillion, M.; Hendzel, M. J.; Voelter, W.;
Verdin, E. Human HDAC7 histone deacetylase activity is asso-
ciated with HDAC3 in vivo. J. Biol. Chem. 2001, 276, 35826–35835.
(28) Lahm, A.; Paolini, C.; Pallaoro, M.; Nardi, M. C.; Jones, P.;
Neddermann, P.; Sambucini, S.; Bottomley, M. J.; Lo Surdo, P.;
Carfi, A.; Koch, U.; De Francesco, R.; Steinkuhler, C.; Gallinari, P.
Unraveling the hidden catalytic activity of vertebrate class IIa histone
deacetylases. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17335–17340.
(29) Vega, R. B.; Matsuda, K.; Oh, J.; Barbosa, A. C.; Yang, X.;
Meadows, E.; McAnally, J.; Pomajzl, C.; Shelton, J. M.; Richardson,
J. A.; Karsenty, G.; Olson, E. N. Histone deacetylase 4 controls
chondrocyte hypertrophy during skeletogenesis. Cell 2004, 119, 555–
566.
(30) Chang, S.; Young, B. D.; Li, S.; Qi, X.; Richardson, J. A.; Olson, E.
N. Histone deacetylase 7 maintains vascular integrity by repressing
matrix metalloproteinase 10. Cell 2006, 126, 321–334.
(31) Tao, R.; de Zoeten, E. F.; Ozkaynak, E.; Chen, C.; Wang, L.;
Porrett, P. M.; Li, B.; Turka, L. A.; Olson, E. N.; Greene, M. I.;
Wells, A. D.; Hancock, W. W. Deacetylase inhibition promotes the
generation and function of regulatory T cells. Nat. Med. 2007, 13,
1299–1307.
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7845
(45) Singh, S. B.; Zink, D. L.; Polishook, J. D.; Dombrowski, A. W.;
Darkin-Rattray, S. J.; Schmatz, D. M.; Goetz, M. A. Apicidins:
Novel cyclic tetrapeptides as coccidiostats and antimalarial agents
from Fusarium pallidoroseum. Tetrahedron Lett. 1996, 37, 8077–
8080.
(46) Kranz, M.; Murray, P. J.; Taylor, S.; Upton, R. J.; Clegg, W.;
Elsegood, M. R. Solution, solid phase and computational struc-
tures of apicidin and its backbone-reduced analogs. J. Pept. Sci.
2006, 12, 383–388.
(47) Nakao, Y.; Yoshida, S.; Matsunaga, S.; Shindoh, N.; Terada, Y.;
Nagai, K.; Yamashita, J. K.; Ganesan, A.; van Soest, R. W.;
Fusetani, N.; Azumamides, A-E histone deacetylase inhibitory
cyclic tetrapeptides from the marine sponge Mycale izuensis.
Angew. Chem., Int. Ed. 2006, 45, 7553–7557.
(48) Izzo, I.; Maulucci, N.; Bifulco, G.; De Riccardis, F. Total synthe-
sis of azumamides A and E. Angew. Chem., Int. Ed. 2006, 45,
7557–7560.
(49) Maulucci, N.; Chini, M. G.; Micco, S. D.; Izzo, I.; Cafaro, E.;
Russo, A.; Gallinari, P.; Paolini, C.; Nardi, M. C.; Casapullo, A.;
Riccio, R.; Bifulco, G.; Riccardis, F. D. Molecular insights into
azumamide E histone deacetylases inhibitory activity. J. Am.
Chem. Soc. 2007, 129, 3007–3012.
(50) Wen, S.; Carey, K. L.; Nakao, Y.; Fusetani, N.; Packham, G.;
Ganesan, A. Total synthesis of azumamide A and azumamide E,
evaluation as histone deacetylase inhibitors, and design of a more
potent analogue. Org. Lett. 2007, 9, 1105–1108.
(51) Nakao, Y.; Narazaki, G.; Hoshino, T.; Maeda, S.; Yoshida, M.;
Maejima, H.; Yamashita, J. K. Evaluation of antiangiogenic
activity of azumamides by the in vitro vascular organization model
using mouse induced pluripotent stem (iPS) cells. Bioorg. Med.
Chem. Lett. 2008, 18, 2982–2984.
(52) Furumai, R.; Komatsu, Y.; Nishino, N.; Khochbin, S.; Yoshida,
M.; Horinouchi, S. Potent histone deacetylase inhibitors built from
trichostatin A and cyclic tetrapeptide antibiotics including trapo-
xin. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 87–92.
(32) Wang, S.; Li, X.; Parra, M.; Verdin, E.; Bassel-Duby, R.; Olson, E.
N. Control of endothelial cell proliferation and migration by
VEGF signaling to histone deacetylase 7. Proc. Natl. Acad. Sci.
U.S.A. 2008, 105, 7738–7743.
(33) Schreiber, S. L., Kapoor, T.; Wess, G., Eds. Chemical Biology:
From Small Molecules to Systems Biology and Drug Design; Wiley-
VCH: Weinheim, 2007; Vol. 2, 303 pp.
(34) Taori, K.; Paul, V. J.; Luesch, H. Structure and activity of
largazole, a potent antiproliferative agent from the Floridian
marine cyanobacterium Symploca sp. J. Am. Chem. Soc. 2008,
130, 1806–1807.
(35) Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. Total synthesis
and molecular target of largazole, a histone deacetylase inhibitor.
J. Am. Chem. Soc. 2008, 130, 8455–8459.
(53) Meinke, P. T.; Colletti, S. L.; Doss, G.; Myers, R. W.; Gurnett, A.
M.; Dulski, P. M.; Darkin-Rattray, S. J.; Allocco, J. J.; Galuska, S.;
Schmatz, D. M.; Wyvratt, M. J.; Fisher, M. H. Synthesis of
apicidin-derived quinolone derivatives: parasite-selective histone
deacetylase inhibitors and antiproliferative agents. J. Med. Chem.
2000, 43, 4919–4922.
(54) Colletti, S. L.; Myers, R. W.; Darkin-Rattray, S. J.; Gurnett, A. M.;
Dulski, P. M.; Galuska, S.; Allocco, J. J.; Ayer, M. B.; Li, C.; Lim,
J.; Crumley, T. M.; Cannova, C.; Schmatz, D. M.; Wyvratt, M. J.;
Fisher, M. H.; Meinke, P. T. Broad spectrum antiprotozoal agents
that inhibit histone deacetylase: structure-activity relationships of
apicidin. Bioorg. Med. Chem. Lett. 2001, 11, 107–111.
(36) Seiser, T.; Kamena, F.; Cramer, N. Synthesis and biological
activity of largazole and derivatives. Angew. Chem., Int. Ed. 2008,
47, 6483–6485.
(37) Bowers, A.; West, N.; Taunton, J.; Schreiber, S. L.; Bradner, J. E.;
Williams, R. M. Total synthesis and biological mode of action of
largazole: a potent class I histone deacetylase inhibitor. J. Am.
Chem. Soc. 2008, 130, 11219–11222.
(38) Nasveschuk, C. G.; Ungermannova, D.; Liu, X.; Phillips, A. J. A
concise total synthesis of largazole, solution structure, and some
preliminary structure-activity relationships. Org. Lett. 2008, 10,
3595–3598.
(39) Ghosh, A. K.; Kulkarni, S. Enantioselective total synthesis of (þ)-
largazole, a potent inhibitor of histone deacetylase. Org. Lett. 2008,
10, 3907–3909.
(40) Kijima, M.; Yoshida, M.; Sugita, K.; Horinouchi, S.; Beppu, T.
Trapoxin, an antitumor cyclic tetrapeptide, is an irreversible
inhibitor of mammalian histone deacetylase. J. Biol. Chem. 1993,
268, 22429–22435.
(41) Taunton, J.; Hassig, C. A.; Schreiber, S. L. A mammalian histone
deacetylase related to the yeast transcriptional regulator Rpd3p.
Science 1996, 272, 408–411.
(42) Kawai, M.; Rich, D. H.; Walton, J. D. The structure and con-
formation of HC-toxin. Biochem. Biophys. Res. Commun. 1983,
111, 398–403.
(43) Shute, R. E.; Dunlap, B.; Rich, D. H. Analogues of the cytostatic
and antimitogenic agents chlamydocin and HC-toxin: synthesis
and biological activity of chloromethyl ketone and diazomethyl
ketone functionalized cyclic tetrapeptides. J. Med. Chem. 1987, 30,
71–78.
(55) Colletti, S. L.; Myers, R. W.; Darkin-Rattray, S. J.; Gurnett, A. M.;
Dulski, P. M.; Galuska, S.; Allocco, J. J.; Ayer, M. B.; Li, C.; Lim,
J.; Crumley, T. M.; Cannova, C.; Schmatz, D. M.; Wyvratt, M. J.;
Fisher, M. H.; Meinke, P. T. Broad spectrum antiprotozoal agents
that inhibit histone deacetylase: structure-activity relationships of
apicidin. Bioorg. Med. Chem. Lett. 2001, 11, 113–117.
(56) Taunton, J.; Collins, J. L.; Schreiber, S. L. Synthesis of natural and
modified trapoxins, useful reagents for exploring histone deacety-
lase function. J. Am. Chem. Soc. 1996, 118, 10412–10422.
(57) Berst, F.; Ladlow, M.; Holmes, A. B. Solid-phase synthesis of
apicidin A and a cyclic tetrapeptoid analogue. Chem. Commun.
2002, 508–509.
(58) Shivashimpi, G. M.; Amagai, S.; Kato, T.; Nishino, N.; Maeda, S.;
Nishino, T. G.; Yoshida, M. Molecular design of histone deacety-
lase inhibitors by aromatic ring shifting in chlamydocin frame-
work. Bioorg. Med. Chem. 2007, 15, 7830–7839.
(59) Gomez-Paloma, L.; Bruno, I.; Cini, E.; Khochbin, S.; Rodriquez,
M.; Taddei, M.; Terracciano, S.; Sadoul, K. Design and synthesis
of cyclopeptide analogues of the potent histone deacetylase inhi-
bitor FR235222. ChemMedChem 2007, 2, 1511–1519.
(60) Horne, W. S.; Olsen, C. A.; Beierle, J. M.; Montero, A.; Ghadiri,
M. R. Probing the bioactive conformation of an archetypal natural
product HDAC inhibitor with conformationally homogeneous
triazole-modified cyclic tetrapeptides. Angew. Chem., Int. Ed.
2009, 48, 4718–4724.
(61) Ying, Y.; Liu, Y.; Byeon, S. R.; Kim, H.; Luesch, H.; Hong, J.
Synthesis and activity of largazole analogues with linker and
macrocycle modification. Org. Lett. 2008, 10, 4021–4024.
(62) Bowers, A. A.; Greshock, T. J.; West, N.; Estiu, G.; Schreiber,
S. L.; Wiest, O.; Williams, R. M.; Bradner, J. E. Synthesis and
conformation-activity relationships of the peptide isosteres of
FK228 and largazole. J. Am. Chem. Soc. 2009, 131, 2900–2905.
(63) Bowers, A. A.; West, N.; Newkirk, T. L.; Troutman-Youngman,
A. E.; Schreiber, S. L.; Wiest, O.; Bradner, J. E.; Williams, R. M.
Synthesis and histone deacetylase inhibitory activity of largazole
(44) Darkin-Rattray, S. J.; Gurnett, A. M.; Myers, R. W.; Dulski, P. M.;
Crumley, T. M.; Allocco, J. J.; Cannova, C.; Meinke, P. T.; Colletti,
S. L.; Bednarek, M. A.; Singh, S. B.; Goetz, M. A.; Dombrowski,
A. W.; Polishook, J. D.; Schmatz, D. M. Apicidin: a novel
antiprotozoal agent that inhibits parasite histone deacetylase. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 13143–13147.

7846 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Olsen and Ghadiri
analogs: alteration of the zinc-binding domain and macrocyclic
scaffold. Org. Lett. 2009, 11, 1301–1304.
(64) Schumann, F.; Muller, A.; Koksch, M.; Muller, G.; Sewald, N. Are
beta-amino acids gamma-turn mimetics? Exploring a new design
principle for bioactive cyclopeptides. J. Am. Chem. Soc. 2000, 122,
12009–12010.
selective histone deacetylase 6 inhibitors designed based on a
small-molecular substrate. J. Med. Chem. 2006, 49, 4809–4812.
(79) Itoh, Y.; Suzuki, T.; Kouketsu, A.; Suzuki, N.; Maeda, S.; Yoshida,
M.; Nakagawa, H.; Miyata, N. Design, synthesis, structure-
selectivity relationship, and effect on human cancer cells of a novel
series of histone deacetylase 6-selective inhibitors. J. Med. Chem.
2007, 50, 5425–5438.
(80) Heltweg, B.; Dequiedt, F.; Marshall, B. L.; Brauch, C.; Yoshida,
M.; Nishino, N.; Verdin, E.; Jung, M. Subtype selective substrates
for histone deacetylases. J. Med. Chem. 2004, 47, 5235–5243.
(81) Because the IC50 values for HDAC8 were obtained using a different
substrate that gave a better signal-background ratio for this
enzyme, we also measured the inhibition constant for 23 against
HDAC8 by applying the same substrate as used for HDACs 1, 3,
and 6. This IC50 value was in the same range as observed with the
HDAC8 specific substrate Ac-Lys(Ac)-AMC (IC50 = 343 nM),
which further confirmed the HDAC8/HDAC6 selectivity for com-
pound 23.
(82) Kozikowski, A. P.; Tapadar, S.; Luchini, D. N.; Kim, K. H.;
Billadeau, D. D. Use of the nitrile oxide cycloaddition (NOC)
reaction for molecular probe generation: a new class of enzyme
selective histone deacetylase inhibitors (HDACIs) showing pico-
molar activity at HDAC6. J. Med. Chem. 2008, 51, 4370–4373.
(83) Gurard-Levin, Z. A.; Mrksich, M. The activity of HDAC8 depends
on local and distal sequences of its peptide substrates. Biochemistry
2008, 47, 6242–6250.
(65) Glenn, M. P.; Kelso, M. J.; Tyndall, J. D.; Fairlie, D. P. Con-
formationally homogeneous cyclic tetrapeptides: useful new three-
dimensional scaffolds. J. Am. Chem. Soc. 2003, 125, 640–641.
(66) Norgren, A. S.; Buttner, F.; Prabpai, S.; Kongsaeree, P.;
Arvidsson, P. I. Beta2-amino acids in the design of conformation-
ally homogeneous cyclo-peptide scaffolds. J. Org. Chem. 2006, 71,
6814–6821.
(67) Montero, A.; Beierle, J. M.; Olsen, C. A.; Ghadiri, M. R. Design,
synthesis, biological evaluation, and structural characterization of
potent histone deacetylase inhibitors based on cyclic alpha/beta-
tetrapeptidearchitectures. J. Am. Chem. Soc. 2009, 131, 3033–3041.
(68) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski,
W. M.; Knapp, R. J. A new type of synthetic peptide library for
identifying ligand-binding activity. Nature 1991, 354, 82–84.
(69) Yang, W. M.; Tsai, S. C.; Wen, Y. D.; Fejer, G.; Seto, E. Functional
domains of histone deacetylase-3. J. Biol. Chem. 2002, 277, 9447–9454.
(70) Glenn, M. P.; Kahnberg, P.; Boyle, G. M.; Hansford, K. A.; Hans,
D.; Martyn, A. C.; Parsons, P. G.; Fairlie, D. P. Antiproliferative
and phenotype-transforming antitumor agents derived from cy-
steine. J. Med. Chem. 2004, 47, 2984–2994.
(71) Simon, M. D.; Chu, F.; Racki, L. R.; de la Cruz, C. C.; Burlingame,
A. L.; Panning, B.; Narlikar, G. J.; Shokat, K. M. The site-specific
installation of methyl-lysine analogs into recombinant histones.
Cell 2007, 128, 1003–1012.
(72) Krennhrubec, K.; Marshall, B. L.; Hedglin, M.; Verdin, E.; Ulrich,
S. M. Design and evaluation of “Linkerless” hydroxamic acids as
selective HDAC8 inhibitors. Bioorg. Med. Chem. Lett. 2007, 17,
2874–2878.
(73) Kahnberg, P.; Lucke, A. J.; Glenn, M. P.; Boyle, G. M.; Tyndall, J.
D.; Parsons, P. G.; Fairlie, D. P. Design, synthesis, potency, and
cytoselectivity of anticancer agents derived by parallel synthe-
sis from alpha-aminosuberic acid. J. Med. Chem. 2006, 49,
7611–7622.
(74) Redman, J. E.; Wilcoxen, K. M.; Ghadiri, M. R. Automated mass
spectrometric sequence determination of cyclic peptide library
members. J. Comb. Chem. 2003, 5, 33–40.
(84) Chou, C. J.; Herman, D.; Gottesfeld, J. M. Pimelic diphenylamide
106 is a slow, tight-binding inhibitor of class I histone deacetylases.
J. Biol. Chem. 2008, 283, 35402–35409.
(85) Wegener, D.; Hildmann, C.; Riester, D.; Schwienhorst, A. Im-
proved fluorogenic histone deacetylase assay for high-throughput
screening applications. Anal. Biochem. 2003, 321, 202–208.
(86) Salisbury, C. M.; Cravatt, B. F. Activity-based probes for proteo-
mic profiling of histone deacetylase complexes. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 1171–1176.
(87) Salisbury, C. M.; Cravatt, B. F. Optimization of activity-based
probes for proteomic profiling of histone deacetylase complexes.
J. Am. Chem. Soc. 2008, 130, 2184–2194.
(88) Lam, K. S.; Lebl, M.; Krchnak, V. The “One-bead-one-
compound” combinatorial library method. Chem. Rev. 1997, 97,
411–448.
(89) Dartois, V.; Sanchez-Quesada, J.; Cabezas, E.; Chi, E.; Dubbelde,
C.; Dunn, C.; Granja, J.; Gritzen, C.; Weinberger, D.; Ghadiri, M.
R.; Parr, T. R., Jr. Systemic antibacterial activity of novel synthetic
cyclic peptides. Antimicrob. Agents Chemother. 2005, 49, 3302–
3310.
(75) Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Butcher, R. A.;
Schreiber, S. L. Multidimensional chemical genetic analysis of
diversity-oriented synthesis-derived deacetylase inhibitors using
cell-based assays. Chem. Biol. 2003, 10, 383–396.
(76) Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Grozinger, C. M.;
Schreiber, S. L. Domain-selective small-molecule inhibitor of
histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4389–4394.
(77) Estiu, G.; Greenberg, E.; Harrison, C. B.; Kwiatkowski, N. P.;
Mazitschek, R.; Bradner, J. E.; Wiest, O. Structural origin of
selectivity in class II-selective histone deacetylase inhibitors.
J. Med. Chem. 2008, 51, 2898–2906.
(90) Horne, W. S.; Wiethoff, C. M.; Cui, C.; Wilcoxen, K. M.; Amorin,
M.; Ghadiri, M. R.; Nemerow, G. R. Antiviral cyclic D,L-alpha-
peptides: targeting a general biochemical pathway in virus infec-
tions. Bioorg. Med. Chem. 2005, 13, 5145–5153.
(91) Fletcher, J. T.; Finlay, J. A.; Callow, M. E.; Callow, J. A.; Ghadiri,
M. R. A combinatorial approach to the discovery of biocidal six-
residue cyclic D,L-alpha-peptides against the bacteria methicillin-
resistant Staphylococcus aureus (MRSA) and E. coli and the
biofouling algae Ulva linza and Navicula perminuta. Chem.;Eur.
J. 2007, 13, 4008–4013.
(78) Suzuki, T.; Kouketsu, A.; Itoh, Y.; Hisakawa, S.; Maeda, S.;
Yoshida, M.; Nakagawa, H.; Miyata, N. Highly potent and