Thailandepsins: Bacterial products with potent histone deacetylase inhibitory activities and broad-spectrum antiproliferative activities

Article abstract of DOI:10.1021/np200324x

Histone deacetylase (HDAC) inhibitors have emerged as a new class of anticancer drugs, with one synthetic compound, SAHA (vorinostat, Zolinza; 1), and one natural product, FK228 (depsipeptide, romidepsin, Istodax; 2), approved by FDA for clinical use. Our studies of FK228 biosynthesis in Chromobacterium violaceum no. 968 led to the identification of a cryptic biosynthetic gene cluster in the genome of Burkholderia thailandensis E264. Genome mining and genetic manipulation of this gene cluster further led to the discovery of two new products, thailandepsin A (6) and thailandepsin B (7). HDAC inhibition assays showed that thailandepsins have selective inhibition profiles different from that of FK228, with comparable inhibitory activities to those of FK228 toward human HDAC1, HDAC2, HDAC3, HDAC6, HDAC7, and HDAC9 but weaker inhibitory activities than FK228 toward HDAC4 and HDAC8, the latter of which could be beneficial. NCI-60 anticancer screening assays showed that thailandepsins possess broad-spectrum antiproliferative activities with GI₅₀ for over 90% of the tested cell lines at low nanomolar concentrations and potent cytotoxic activities toward certain types of cell lines, particularly for those derived from colon, melanoma, ovarian, and renal cancers. Thailandepsins thus represent new naturally produced HDAC inhibitors that are promising for anticancer drug development.

Full text of DOI:10.1021/np200324x

ARTICLE
pubs.acs.org/jnp
Thailandepsins: Bacterial Products with Potent Histone Deacetylase
Inhibitory Activities and Broad-Spectrum Antiproliferative Activities
Cheng Wang,† Leonhard M. Henkes,‡ Leah B. Doughty,† Min He,§ Difei Wang,^ Franz-Josef Meyer-Almes,‡
||
and Yi-Qiang Cheng*,†,
†Department of Biological Sciences, and Department of Chemistry and Biochemistry, University of WisconsinÀMilwaukee, P.O. Box
413, Milwaukee, Wisconsin 53201, United States
‡Department of Chemical Engineering and Biotechnology, University of Applied Sciences Darmstadt, 64287 Darmstadt, Germany
§Developmental Therapeutics Program, the US National Cancer Institute, Frederick, Maryland 21701, United States
^Laboratory of Cell Biology, the US National Cancer Institute, Bethesda, Maryland 20892, United States
MoE Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University School of Pharmaceutical Sciences,
Wuhan 430071, People's Republic of China
S Supporting Information
b
ABSTRACT: Histone deacetylase (HDAC) inhibitors have
emerged as a new class of anticancer drugs, with one synthetic
compound, SAHA (vorinostat, Zolinza; 1), and one natural
product, FK228 (depsipeptide, romidepsin, Istodax; 2), ap-
proved by FDA for clinical use. Our studies of FK228 bio-
synthesis in Chromobacterium violaceum no. 968 led to the
identification of a cryptic biosynthetic gene cluster in the
genome of Burkholderia thailandensis E264. Genome mining
and genetic manipulation of this gene cluster further led to the
discovery of two new products, thailandepsin A (6) and
thailandepsin B (7). HDAC inhibition assays showed that
thailandepsins have selective inhibition profiles different from
that of FK228, with comparable inhibitory activities to those of FK228 toward human HDAC1, HDAC2, HDAC3, HDAC6,
HDAC7, and HDAC9 but weaker inhibitory activities than FK228 toward HDAC4 and HDAC8, the latter of which could be
beneficial. NCI-60 anticancer screening assays showed that thailandepsins possess broad-spectrum antiproliferative activities with
GI50 for over 90% of the tested cell lines at low nanomolar concentrations and potent cytotoxic activities toward certain types of cell
lines, particularly for those derived from colon, melanoma, ovarian, and renal cancers. Thailandepsins thus represent new naturally
produced HDAC inhibitors that are promising for anticancer drug development.
pigenetic abnormalities participate with genetic mutations to
cause cancer,1 and consequently epigenetic intervention of cancer
FR901,375 (3),13 spiruchostatins A (4) and B (5),14 thailandep-
sins A (6) and B (7) discovered by us15 (and this article.15 is a US
patent application filed on August 19, 2010 and published online
on March 10, 2011, with a priority date of August 19, 2009), and
burkholdacs A (8) and B (identical to 6) reported recently by
Biggins et al. (appeared online on February 26, 2011).16 All members
of this family of natural products are produced by rare Gram-
negative bacteria, and each of them contains a signature disulfide
bond that is known or presumed to mediate its anticancer activity
via reduction of the disulfide bond to generate a free thiol group
(“warhead”) that chelates a Zn2+ in the catalytic center of class
I and class II HDACs, thereby inhibiting the enzyme activities.17
The biosynthesis of FK228 is proposed to follow a widely
accepted “assembly-line” mechanism18À20 in which simple building
E
has emerged as a promising avenue toward cancer therapy.
Selective inhibition of histone deacetylases (HDACs) by small
molecules often leads to a cascade of chromatin remodeling,
tumor suppressor gene reactivation, apoptosis, and regression of
cancer.2 HDAC inhibitors have thus gained much attention in recent
years as a new class of anticancer agents.2À6 One synthetic HDAC
inhibitor, SAHA (vorinostat, Zolinza; 1), and one natural product
HDAC inhibitor, FK228 (depsipepide, romidepsin, Istodax; 2), have
already been approved by the US Food and Drug Administration
(FDA) for the treatment of cutaneous T-cell lymphoma,7,8 and many
more HDAC inhibitors (mostly synthetic molecules) are in various
stages of preclinical or clinical trials as single agents or in combination
with other chemotherapy drugs for diverse cancer types.5,9,10
FK228 isproducedbyChromobacterium violaceum no. 968.11,12 It
belongs to a small family of natural products that also includes
Received: April 15, 2011
Published: July 27, 2011
r
Copyright
2011 American Chemical Society and
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dx.doi.org/10.1021/np200324x J. Nat. Prod. 2011, 74, 2031–2038
American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
blocks (amino acids, amino acid derivatives, and short carboxylic
acids from primary metabolism) are assembled stepwise by seven
modules of a hybrid nonribosomal peptide synthetase (NRPS)-
polyketide synthase (PKS) multifunctional pathway to afford a
linear intermediate, which is subsequently cyclized by a terminal
thioesterase (TE) domain to form an immediate FK228 precursor.21
An FAD-dependent oxidoreductase is responsible for a disulfide
bond formation as the final step of FK228 biosynthesis.22
’ RESULTS AND DISCUSSION
Identification of a Cryptic Biosynthetic Gene Cluster. The
goal of our research is to explore naturally produced and
biologically or chemically engineered members of the FK228-
family of HDAC inhibitors as new anticancer agents. To this
end, we have cloned and characterized a biosynthetic gene
cluster (designated dep for depsipeptide FK228) and three
discrete genes collectively responsible for FK228 biosynthesis in
C. violaceum.21,30,31 Those studies provided us the first-hand
opportunity to identify a cryptic biosynthetic gene cluster
(designated tdp for thailandepsins) in the published genome of
Burkholderia thailandensis E264 (GenBank accession no. CP000085
and CP000086)32 (Figure 1). The genes and their deduced pro-
teins of this tdp gene cluster exhibit a significant overall similarity
to those of the dep gene cluster (Table 1). In particular, the
deduced products of eight genes (tdpA, tdpB, tdpC1, tdpF, tdpG,
tdpH, tdpI, and tdpJ) share 67%/80% or higher sequence
identity/similarity with their respective counterparts from the
FK228 biosynthetic pathway. Like the dep gene cluster, this tdp
gene cluster does not contain any gene that encodes a phospho-
pantetheinyltransferase (PPTase) necessary for posttranslational
modification of carrier proteins33 or an acyltransferase (AT)
necessary for in trans complementing the three “AT-less” PKS
modules34 on TdpB, TdpC1, and TdpC2 proteins. A thorough
search of the B. thailandensis genome found multiple candidate
genes that may encode the missing PPTase or AT activity;
experimental verification of the responsible gene (tentatively
named tdp_sfp or tdp_AT) is in progress. A few differences
between the two parallel gene clusters are identified as follows:
(i) Unlike depR which is located downstream of the dep gene
cluster and encodes an OxyR-type transcriptional activator, tdpR
is located upstream of the tdp gene cluster and encodes a putative
AraC-type transcriptional regulator. These two deduced regula-
tory proteins do not have notable sequence homology. (ii) There
is no depM-equivalent in the tdp gene cluster. (iii) There are two
copies of a depC-like gene in the tdp gene cluster, the second copy
is fused to DNA encoding a likely inactive epimerase (E) domain
and is located after tdpDE1. (iv) A depE-like gene in the tdp gene
cluster is split into two parts, the first part is fused to the end of
tdpD, and the second part is transposed to a downstream location
between tdpG and tdpH. (v) Unlike pseudogene “depN”, the
deduced protein of tdpN appears to be a functional peptidyl
carrier protein (PCP) with a critical serine residue for phospho-
pantetheinylation.
Historically natural products have made tremendous contri-
butions to humanmedicines.23,24 Despite the controversial downsiz-
ing of natural product-based drug discovery efforts by the pharma-
ceutical industry in the past two decades, there has been a sign of
renaissance of natural product discovery activities in recent years,
largely due to new technology advances and unmet need of new
drugs for chronic and emerging diseases.20,25 Genome mining,
which takes advantage of the rapid growth of microbial genome
sequence information and in-depth understanding of natural
product biosynthetic logics, has emerged as an effective new
approach for the discovery of molecules encoded in cryptic
biosynthetic gene clusters.26,27
In this article, we report the discovery of two FK228-analogues, 6
and 7, from the fermentation broth of a genome-sequenced
bacterium, by a combination of several enabling technologies includ-
ing genome mining and metabolic profiling. Enzyme inhibition
assays showed that 6 and 7 are potent inhibitors of human HDACs.
NCI-60 anticancer screening28,29 demonstrated broad-spectrum
antiproliferative activities of 6 and 7 against an array of human
cancer cell lines. Therefore, 6 and 7 represent new naturally
produced HDAC inhibitors that are promising for anticancer
drug development.
Using the FK228 biosynthetic pathway as a reference,21 we
dissected the domain and module organization of six deduced
NRPS- and PKS-type enzymes (TdpA, TdpB, TdpC1, TdpDE1,
TdpC2, and TdpE2) encoded by the tdp gene cluster and
proposed a hybrid NRPS-PKS biosynthetic pathway model
which also includes three discrete enzymes (AT, TdpF, and
TdpH) (Supporting Information Figure S1). This proposed
pathway contains eight NRPS/PKS modules responsible for
seven consecutive steps of building block polymerization that
results in a full-length linear intermediate installed on a PCP
domain in the last module. A terminal TE domain is predicted to
cleave off the intermediate and subsequently cyclize it into a
macrolactam intermediate. Finally an FAD-dependent oxidore-
ductase (TdpH) is predicted to catalyze a disulfide bond forma-
tion as the final step of the biosynthesis of 6 and 7.
We initially predicted the putative chemical structures of com-
pounds that may be produced by the proposed biosynthetic pathway.
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Figure 1. Comparison of two homologous biosynthetic gene clusters and discrete genes necessary for the biosynthesis of FK228 (2) or thailandepsins A
(6) and B (7). Each gene cluster is depicted in a row, under which is the deduced respective modular biosynthetic pathway. A, ACP, AL, AT, C, DH, E,
KR, KS, PCP, and TE are standard abbreviations of the nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) domain names whose
full name and respective function can be found in Fischbach et al.18 Sfp and AT are the generic protein names of their respective genes defined in the
main text.
Table 1. Comparison of Two Homologous Biosynthetic Gene Clusters and Their Associated Discrete Genes Necessary for
Product Biosynthesis
thailandepsin biosynthetic (tdp) gene cluster
FK228 biosynthetic (dep) gene cluster21,30,31
percentage ident./
simil. btw prot. seq.
confirmed or deduced
protein functionc
genea
proteinb
Tdp_Sfp
geneb
proteinb
tdp_sfp (multiple candidates)
(discrete)d
dep_sfp (discrete)d
Dep_Sfp
--
--
--
phosphopantetheinyl
transferase (PPTase)
acyltransferase, malonyl
CoA-specific (AT)
AraC-type transcriptional
regulator
tdp_AT (multiple candidates)
(discrete)d
Tdp_AT
TdpR
dep_fabD1, dep_fabD2
(discrete)d
Dep_FabD1,
Dep_FabD2
--
BTH_I2369
--
--
--
depM
DepM
--
--
--
aminotransferase
BTH_I2368
TdpN
--
type II peptidyl carrier
protein (PCP)
BTH_I2367
BTH_I2366
BTH_I2365
BTH_I2364
TdpA
depA
depB
depC
DepA
DepB
DepC
74/83
78/86
76/84
NRPS (1 module)
PKS (1 module)
TdpB
TdpC1
PKS (1 module)
TdpDE1
Àfirst module
Àsecond module
TdpC2
depD
depE
depC
depF
DepD
DepE
DepC
DepF
56/67
39/52
39/50
88/93
NRPS (1 module)
NRPS (1 module)
PKS (1 module)
BTH_I2363
BTH_I2362
TdpF
FadE2-like acyl-CoA
dehydrogenase
BTH_I2361
BTH_I2360
BTH_I2359
TdpG
TdpE2
TdpH
depG
depE
depH
DepG
DepE
DepH
75/84
31/49
72/84
phosphotransferase
NRPS (partial module)
FAD-dependent disulfide
oxidoreductase
BTH_I2358
BTH_I2357
--
TdpI
TdpJ
--
depI
depJ
depR
DepI
DepJ
DepR
74/84
67/80
--
esterase/lipase
type II thioesterase
OxyR-type transcriptional
regulator
a Gene annotations from the GenBank. b Gene/protein names designated by the authors. c Standard abbreviations: NRPS, nonribosomal peptide
synthetase; PKS, polyketide synthase. d Detached from the perspective gene cluster; --: not available.
The predicted structures (Supporting Information Figure S1)
were slightly different from the experimentally determined
ones (6 and 7), reflecting the power and yet limitation of in silico
analysis.
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Figure 2. Metabolic profiling of the wild type strain (BthWT) and BthΔtdpAB mutant strain of B. thailandensis E264. (aÀc) HPLC traces of extract of
the medium control, BthWT strain or BthΔtdpAB strain, respectively. (d,e) ESI-LC-MS spectra of the HPLC peak 1 and peak 2 of the BthWT sample
(b). (f,g) ESI-LC-MS spectra of the reduced HPLC peak 1 and peak 2 of the BthWT sample (b), showing a +2 m/z shift of every ion signal.
Discovery of Thailandepsins A (6) and B (7). We executed a
series of experiments to purify and identify two new bacterial
products, named thailandepsin A (6) and thailandepsin B (7),
from the fermentation culture of B. thailandensis E264.
cryptic tdp gene cluster. Those peaks were collected and exam-
ined by electrospray ionization (ESI)-LC-MS.
Importantly, we observed critical chemical properties of the
putative target compounds. The material of peak 1 from HPLC
yielded a pair of ion signals of 547.9/530.0 m/z (Figure 2d),
and the material of peak 2 sample yielded 530.0/511.9 m/z
(Figure 2e). It is believed that the higher m/z signal from each
pair is the protonated adduct of a target molecule [M + H]+, and
the lower m/z signal is the protonated adduct of a respective
target molecule with a H2O molecule removed (dehydrated;
[M À H2O + H]+) by heat/electrovoltage (eV) during ESI-LC-
MS. Interestingly, when the materials of HPLC peaks were first
reduced with DTT and then subjected to ESI-LC-MS analysis,
both samples generated ion signals with a +2 m/z mass shift, a
polarity shift (more hydrophilic, as judged by an earlier elution
time), and a change of the relative abundance of parental
molecule/dehydrated derivative (Figure 2f,g). Those observa-
tions strongly suggested that we had identified two target
compounds that are most likely produced by the cryptic tdp
gene cluster in B. thailandensis under the fermentation conditions
tested, and those compounds probably contain a disulfide bond
(thus existing as a prodrug) which can be reduced/activated by
DTT in the same way as FK228 by DTT.
Finally, we fermented the BthWT strain in large volume in M9,
and subsequently purified and identified the target compounds
6 and 7 by natural product chemistry methods including organic
extraction, gravity chromatography, preparative HPLC, ami-
no acid analysis, MS, NMR, degradation analysis, and chemi-
cal derivatization (see Supporting Information).
Thailandepsins A (6) and B (7) are New FK228-Class
Natural Products. Compounds 6 and 7 are new chemical
entities with distinctive features that belong to the FK228-class
of natural products. Both 6 and 7 are bicyclic depsipeptides that
contain a 15-membered macrolactam ring and a second 15-
membered ring with a signature disulfide bond, whereas FK228
contains a 16-membered macrolactam ring and a 15-membered
side ring with a signature disulfide bond. Like FK228,17,21 the
disulfide bond in 6 and 7 is predicted to be reduced inside
mammalian cells to generate free thiol groups, of which one thiol
group interacts with the Zn2+ inside the catalytic pocket of human
First we identified by semiquantitative reverse transcription
(RT)-PCR method several cultivation conditions in which the
cryptic tdp gene cluster is highly expressed (Supporting Informa-
tion Figure S2a). We found that two representative structural
genes, tdpA and tdpJ, are expressed at significant levels at 30 ꢀC in
five (M1, M2, M3, M8, and M9) of the nine production media
tested (Supporting Information Table S1) at the 24 h time point,
with the highest level in M9, a modified minimal medium.
Furthermore, we found that tdpA is expressed at significant levels
in the five identified media at all four time points examined, with
the highest level in M9 at about 24 h (Supporting Information
Figure S2b); the level of tdpA expression begins to decrease after
24 h but persists beyond 72 h. Medium M9 was thus selected as
the optimal medium for subsequent bacterial fermentation.
We then created a gene-deletion mutant (BthΔtdpAB) and
detected metabolic profiling differences between the wild type
strain (BthWT) and the mutant strain of B. thailandensis E264.
We employed a well-established multiplex PCR method22,35 to
create the mutant strain in which a critical segment of the tdpAB
adjoining region was permanently deleted (Supporting Informa-
tion Figure S2c). This deletion truncated a part of the adenyla-
tion (A) domain and the entire PCP domain of the NRPS module
on TdpA and a part of the ketoacyl synthase (KS) domain of the
PKS module on TdpB of the thailandepsin biosynthetic pathway
(Supporting Information Figure S1). As a result, not only three
critical catalytic domains of the pathway have been impaired or
deleted by the gene deletion event but also the proteinÀprotein
communication between TdpA and TdpB was disrupted. It is
thus certain that the entire biosynthetic pathway must have been
disabled. Subsequently both BthWT and BthΔtdpAB strains
were cultivated in M9. Organic extracts of both bacterial cultures
and a medium control were analyzed by high performance liquid
chromatography (HPLC) and detected the disappearance of
several HPLC peaks in the BthΔtdpAB sample (part c vs b of
Figure 2). We anticipated that the corresponding peaks in the
BthWT sample could be the target compounds produced by the
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Table 2. The Calculated IC50 Value of Each Compound vs Each HDAC in μM Concentration
enzyme
inhibitor
HDAC1
HDAC2
HDAC3
HDAC4
HDAC6
HDAC7
HDAC8
HDAC9
2
2*a
6.7
1.5
0.018
>50
0.47
>50
42
12
>50
3.2
>50
11
>50
0.026
>50
1.2
>50
12
0.0053
7.5
0.0039
39
0.0053
0.087
0.33
9.9
6
>50
12
6*a
7
7*a
0.014
7.7
0.0035
4.5
0.0048
0.023
0.38
11
22
27
30
>50
30
0.0065
0.0067
0.0094
18
0.61
24
1.0
a Compounds were reduced/activated prior to being assayed.
Table 3. The Influence of Different H2O2 Concentrations on
the Potency of FK228 (2) against HDAC3/N-CoR2
reduced with DTT and assayed against HDAC1, HDAC2,
HDAC4, and HDAC6.17
Reduced thailandepsins 6* and 7* exhibited a similar ranking
of inhibitory potency against human HDACs: HDAC1, HDAC2,
and HDAC3 are strongly inhibited at low nanomolar concentra-
tions; inhibition of the distantly related HDAC8 is about 80À350
times weaker than the inhibition of HDAC1À3; HDAC6 is still
strongly inhibited but the potency of 2*, 6*, or 7* toward HDAC6
is about 30À100 times less than to HDAC1À3; HDAC4, HDAC7,
and HDAC9 are the least inhibited with IC50 values 3À4 orders
of magnitude higher than those for HDAC1À3. It is apparent
that all three activated compounds are more potent toward class I
HDACs than toward class II HDACs. Thus class I HDACs,
particularly HDAC1À3, are the primary targets of inhibition by
those natural products.
Interestingly there are significant differential inhibitory effects
among 2*, 6*, and 7* on HDAC4 and HDAC8. Both 6* and 7*
are 40À90-fold less potent against HDAC4 or HDAC8 than 2*.
This difference could be beneficial for drug development. Studies
have shown the involvement of HDAC4 in multiple vital cellular
regulation mechanisms.38À41 Although HDAC8 is a member of
class I HDACs but by sequence comparison it is far away from
the other members (Supporting Information Figure S7), there-
fore HDAC8 may presumably play a very different biological role
than the other members of class I HDACs. Because the toxicity of
HDAC inhibitors is often a bigger concern than potency in drug
development, and 6* and 7* are much less inhibitory than 2*
against HDAC4 and HDAC8, 6* and 7* may exhibit favorable
anticancer or cytotoxicity profiles.
Surprisingly FK228 and thailandepsins in their natural/oxi-
dized prodrug form (2, 6, and 7) appeared unexpectedly to be
strong inhibitors of the HDAC3/N-CoR2 complex with IC50
values ranging from 18 to 87 nM, whereas they are 80À450 times
less active against the closely related HDAC1 and HDAC2. To
exclude the potential influence of traces of thiol 2* on enzyme
inhibition, a series of doseÀresponse curves of 2 in the presence
of different concentrations of hydrogen peroxide (H2O2) was
carried out against HDAC3/N-CoR2 (Supporting Information
Figure S3c). Increasing concentrations of the oxidizing agent
up to 1.0 mM resulted in decreasing levels of enzyme activity of
the HDAC3/N-CoR2 complex in the absence of 2. However
when assayed with 2, the IC50 values of 2 remained similarly low
(Table 3), indicating that the oxidized form of 2 indeed has
pronounced inhibitory activity against the HDAC3/N-CoR2
complex, perhaps by a different mode of action. This seemingly
increased susceptibility of HDAC3 might be influenced by its
complex with corepressor N-CoR2. Guenther et al.42 have shown
compd
H2O2 [mM]
enzyme activity [%]
IC50 [μM]
2
2
2
2
0.0
0.2
0.4
1.0
100.0
70.8
68.9
57.3
0.018
0.020
0.039
0.041
HDACs, thus inhibiting the enzyme’s activity, as illustrated by
molecular modeling (Supporting Information Figure S8).
From a biosynthetic point of view, it is apparent that both 6
and 7 are composed of building blocks of short carboxylic acids,
amino acids, or amino acid derivatives, consistent with the
proposed model of a hybrid PKS-NRPS biosynthetic pathway
(Supporting Information Figure S1). Compound 6 differs from 7
by having a methionine (Met) moiety, where 7 has a norleucine
(NLeu) moiety. According to a comprehensive database of
nonribosomal peptides,36 the simultaneous presence of cysteine
(Cys) and Met, two proteogenic amino acids that contain a thiol
function, in nonribosomally synthesized 6, is unprecedented, and
7 appears to be the first natural product reported to contain
a nonproteogenic NLeu building block. Overall, 6 and 7 are
structurally more similar to 4 and 5 than to 2. In particular, 6 and
7 only differ from 5 at the Met/NLeu position, where 5 has an
alanine (Ala) moiety.
HDAC Inhibitory Activities of Thailandepsins A (6) and B
(7) Compared to FK228 (2). Because of a striking structural
similarity between 6/7 and 2, we predicted that both 6 and 7
would likely also possess HDAC inhibitory and antiproliferative
activities. To test this hypothesis, the inhibitory potency of both
the oxidized form (natural, prodrug form with a disulfide bond)
and the reduced form (activated form with two free thiol groups,
indicated by *) of 2 (as a reference compound), 6, and 7 against
recombinant human class I HDACs [HDAC1, HDAC2, HDAC3
(in complex with N-CoR2), and HDAC8], class IIa HDACs
(HDAC4, HDAC7, and HDAC9), and class IIb HDAC6 was
determined by a two-step fluorogenic assay.37 All HDAC doseÀ
response curves were bundled for each compound individually
(Supporting Information Figure S3a,b), and the IC50 values in
μM are summarized in Table 2.
Each of the three compounds in its reduced/activated form
(2*, 6*, 7*) is a much more potent HDAC inhibitor than its
natural/oxidized prodrug form (2, 6, 7). These observations are
in agreement with a previous report that an enhanced inhibitory
effect of the reference compound 2 was detected after being
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Figure 3. In vitro antiproliferative/cytotoxic activities of thailandepsins A (6) and B (7) in comparison with FK228 (2). (a) Heat map representation of
antiproliferative/cytotoxic activities50 of 6 and 7 across NCI-60 cell lines, with higher activity (lower index value) in warmer colors. GI50, growth-
inhibition indicator; TGI, cytostatic effect indicator; LC50, cytotoxic effect indicator. Compound concentration in M was converted to log10 value for
continuous color scale transformation. Mean panel response values were expressed in μM. (b) Statistics comparison of antiproliferation/cytotoxicity
profile between 2, 6, and 7 across NCI-60 cell lines.
that HDAC3 is completely inactive on its own but is activated by
forming a stable complex with the nuclear hormone receptor
corepressor N-CoR2. Therefore, N-CoR2 is required both for
activation of HDAC3 and for its recruitment of other nuclear
repressors. It is speculated that the FK228-family of HDAC
inhibitors may display their effects either through binding outside
of the active site of HDAC3 to its complex with N-CoR2 or by
interfering directly with the interaction between HDAC3 and
N-CoR2, yielding inactive uncomplexed HDAC3. The concept
of disturbing the formation of the HDAC3/N-CoR2 complex
could open new routes to selective inhibitors against HDAC3
because the ability of N-CoR2 to activate HDAC is specific to
HDAC3.42
In Vitro Antiproliferative/Cytotoxic Activities of Thailan-
depsin A (6) and Thailandepsin B (7). Compounds 6 and 7
were submitted to the US National Cancer Institute (NCI)’s
Developmental Therapeutics Program (DTP) on August 12, 2009
for screening against the NCI-60 human cancer cell lines28,29 and
were accepted by assigning entry numbers NSC D751510 and
NSC D751511, respectively. Both compounds exhibited broad-
spectrum antiproliferative activities similar to 2, with GI50 for
over 90% of the tested cell lines at low nanomolar concentrations
(Figure 3a). Differential activities toward specific cell lines were
observed at the TGI and LC50 level, particularly for those derived
from colon, melanoma, and renal cancers, while leukemia cell
lines were generally less sensitive. Matrix Compare analysis
showed close correlation in antiproliferation profile between 6,
7, and 2, with Pearson correlation coefficient above 0.7 across the
GI50 and LC50 levels (Figure 3b). Overall, 2 is slightly more
potent at the TGI and LC50 levels in most cell lines, consistent
with its slightly more potent in vitro HDAC inhibitory activity
compared with 6 and 7 (Table 2). However, 6 is more potent
than 2 or 7 in most cell lines at the GI50 level, and in certain
cell lines at the TGI and LC50 levels (colon cancer HCC-2998,
melanoma LOXIMVI and UACC-62, and renal cancers A498
and RXF393).
Further in vivo toxicities and efficacies of 6 and 7 are being
evaluated in the mouse xenograft models. It remains interesting
to see whether 6 and/or 7 could advance to clinical trials or how
far they could proceed in the drug development pipeline. For the
immediate future, 6 and 7 could be used as valuable epigenetic-
intervening reagents for research on cancer or other human
diseases.
During the preparation and journal review process43 of this
article, Biggins et al. published a rapid communication of two
compounds (named burkholdac A and burkholdac B) discovered
from the same bacterial strain produced by the same gene cluster but
using a different technical approach.16 Burkholdac B is identical to 6,
while burkholdac A (8) and7are different minor products related to 6.
Compound 7 is newly reported in this article.
’ EXPERIMENTAL SECTION
Bacterial Strains, Plasmids, and Reagents. Burkholderia thai-
landensis E264 (ATCC 700388), a Gram-negative β-proteobacterium
strain originally isolated from a rice paddy in central Thailand,44 was
purchased from the American Type Culture Collection (ATCC, Man-
assas, VA). This bacterial strain was cultured in LuriaÀBertani (LB)
medium or LB agar supplemented with 50 μg/mL apramycin (Am; this
bacterial strain is naturally resistant to up to 200 μg/mL of Am) at
30À37 ꢀC overnight for seed culture and for genetic and molecular
manipulations. Other general molecular biological procedures were as
described in refs 21 and 45 or in reagent supplier’s instruction.
RT-PCR Detection of Gene Expression Conditions. RT-PCR
for the detection of gene expression conditions of tdpA and tdpJ was
performed as described in the Supporting Information.
Construction of a Targeted Gene-Deletion Mutant of B.
thailandensis E264. A detailed description of the creation of a
BthΔtdpAB mutant is described in the Supporting Information.
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Journal of Natural Products
ARTICLE
Metabolic Profiling by LC-MS. B. thailandensis E264 wild type
(BthWT) strain and the BthΔtdpAB mutant strain were cultivated side
by side in 50 mL of Medium 9 (M9 in Supporting Information Table S1)
supplemented with 1% (w/v) of XAD16 resin and 1% (w/v) of Diaion
HP-20 resin (Sigma-Aldrich) at 30 ꢀC for 3 days. A blank M9 was set up
as medium reference. At the end of fermentation, resin and cell debris
were collected and lyophilized to dryness, and the dry mass was extracted
three times with 5 mL of ethyl acetate and pooled. A 100 μL aliquot of
the ethyl acetate extract was analyzed by HPLC, using an Eclipse XBD
compare/COMPARE_methodology.html) and were used for a matrix
COMPARE analysis.49
’ ASSOCIATED CONTENT
S
Supporting Information. Methods, results, NMR spectra,
b
structures, assays, and references. This material is available free of
charge via the Internet at http://pubs.acs.org.
C
18 column (5 μm particle size, 4.6 mm  250 mm; from Agilent) and
’ AUTHOR INFORMATION
a gradient elution from 20% to 100% of acetonitrile/water (v/v) in 30 min.
Flow rate was set at 1 mL/min and UV signal was monitored at 200 nm.
Peaks of interest from the BthWT sample were manually collected,
dried, and redissolved in acetonitrile. A 20 μL aliquot of such sample was
reanalyzed by LC-MS (1100 series LC/MSD Trap mass spectrometer
from Agilent). In addition, an equal volume of the sample was reduced
with 50 mM DTT at room temperature and reanalyzed by LC-MS.
Purification and Identification of Thailandepsins A (6) and
B (7). A detailed description of natural product purification and
identification is described in Supporting Information.
Fluorogenic Assays of HDAC Inhibition Activities. Trypsin
(from bovine pancreas), DMSO, and Pluronic were purchased from
Sigma-Aldrich (Germany). All reactions were performed in FB-188
buffer (15 mM Tris, 50 mM KH2PO4/K2HPO4, 250 mM NaCl, 250 μM
EDTA, pH 8.0; from Roth, Germany) supplemented with 0.001% (v/v)
Pluronic. The recombinant human HDACs were purchased from BPS
Bioscience Inc. (San Diego, CA) and diluted in corresponding buffers.
Compounds (2, 6, and 7) were dissolved in DMSO; half of each was
then reduced by tris(2-carboxyethyl)phosphine hydrocloride (TCEP)
(CalBioChem, Germany) in a molar ratio of 1:1.5 for 20 min at ambient
temperature prior to being assayed. The two-step fluorogenic assay was
performed in 96-well half area microplates (Greiner Bio-One, Germany)
in a total volume of 100 μL according to Wegener et al.37 In principle, an
ε-acetylated lysine substrate is first deacetylated by an HDAC in a
reaction which is subsequently quenched by SAHA (kindly provided by
Dr. A. Schwienhorst). Trypsin then is added to the reaction to cleave the
detectable 7-amino-4-methylcoumarin (AMC; excitation at 390 nm,
emission at 460 nm) off the deacetylated lysine. Fluorogenic signals were
detected with a Polarstar fluorescence plate reader (BMG). Blank
reactions showed that both DMSO and TCEP inhibit the investigated
HDAC activities less than 3% at the highest compound concentration
used. Therefore, the influence of solvent or reducing agent on the enzyme
activity could be neglected.
Corresponding Author
*Phone: (414) 229-4739. Fax: (414) 229-3926. E-mail: ycheng@
uwm.edu.
’ ACKNOWLEDGMENT
We thank D. DeShazer (Walter Reed Army Medical Center)
and H. Schweizer (Colorado State University) for providing
vector tools, K. Nithipatikom (Medical College of Wisconsin)
for assistance with HR-MS analysis, H. Foersterling (University
of Wisconsin-Milwaukee) for assistance with NMR analysis, the
NCI Developmental Therapeutics Program for conducting NCI-
60 anticancer screening, M. Kunkel (NCI-DTP) for assistance
with the COMPARE analysis of NCI-60 data, and D. Newman
(NCI-DTP) for critical reading of this manuscript. This work was
supported by a Research Growth Initiative Award from the
University of WisconsinÀMilwaukee and an NIH/NCI grant
R01 CA152212 (both to Y.-Q.C). D.W. also acknowledges the
NIH/NCI Intramural Research Program of the Center for
Cancer Research for a fellowship support.
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