Structure-activity studies of echinomycin antibiotics against drug-resistant and biofilm-forming Staphylococcus aureus and Enterococcus faecalis

Article abstract of DOI:10.1016/j.bmcl.2009.01.010

Four echinomycin antibiotics were isolated from the culture broth of a marine streptomycete, and their structures were determined by a combination of chemical and spectroscopic analyses. Antibiotic activities were measured against drug-resistant and biofi

Full text of DOI:10.1016/j.bmcl.2009.01.010

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1504–1507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity studies of echinomycin antibiotics against drug-resistant
and biofilm-forming Staphylococcus aureus and Enterococcus faecalis
Aaron M. Socha a, Kerry L. LaPlante b,c, David J. Russell d, David C. Rowley a,
*
a Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA
b Department of Pharmacy Practice, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA
c Veterans Affairs Medical Center, Providence, RI 02908, USA
d Applications Laboratory, Varian NMR Systems, Palo Alto, CA 94304, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Four echinomycin antibiotics were isolated from the culture broth of a marine streptomycete, and their
structures were determined by a combination of chemical and spectroscopic analyses. Antibiotic activi-
ties were measured against drug-resistant and biofilm-forming strains of Staphylococcus aureus and
Received 13 November 2008
Revised 26 December 2008
Accepted 6 January 2009
Available online 10 January 2009
Enterococcus faecalis. Minimum inhibitory concentrations ranging from 0.01
lM to greater than 14 lM
clearly defined structure–activity relationships for antibiotic potency. Echinomycin was the most active
compound with a MIC of 0.03
forming E. faecalis.
l
M against methicillin-resistant S. aureus and 0.01
lM against biofilm-
Keywords:
Antibiotic
Natural product
Streptomycete
Echinomycin
Ó 2009 Elsevier Ltd. All rights reserved.
Staphylococcus aureus
Enterococcus faecalis
Nosocomial infections caused by antibiotic-resistant staphylo-
cocci and enterococci present increasing treatment challenges,
often resulting in prolonged hospitalization.1 Biofilm growth of
methicillin-resistant Staphylococcus aureus (MRSA) is common dur-
ing hemodialysis2 and among patients bearing indwelling medical
devices.3,4 In the United States, 12–25% of MRSA catheter-related
blood stream infections result in mortality, with annual patient
care costs projected to be as high as $460 million.5 When embed-
ded within a biofilm, MRSA demonstrates enhanced resistance to
multiple classes of antibiotics.6 Clinical isolates of enterococci are
also well documented to form biofilms;7 however, no currently
available chemotherapeutic agent can completely eradicate bio-
films formed by vancomycin-resistant Enterococcus (VRE).8 Clearly
there is a critical need for new antibiotics to combat these evolving
pathogens.
Echinomycin is an antibiotic produced by Streptomyces bacte-
ria,9 and the first known bifunctional DNA bisintercalator.10,11
Resurgent interest in its antibacterial properties derives from pro-
ven efficacy against antibiotic-resistant pathogens.12,13 For exam-
ple, echinomycin exhibited seven times the potency of
vancomycin when treating systemic MRSA infections in mice.13
In this study, an echinomycin-producing bacterium was iso-
lated from a marine sediment sample collected from a depth of
13.5 m in Fisher’s Island Sound, New York (41°1505600N,
72°203000W). Strain URI-F39 was cultivated on marine agar and
identified as a Streptomyces sp. using 16S rRNA gene sequence
comparison (Genbank Accession No. EU998645).14 The organic
extracts of 1 L fermentations showed antimicrobial activity against
S. aureus (ATCC 43300). The broths from scale-up fermentations
(16ꢀ 1200 mL) were filtered to remove cells, extracted with HP-
20 resin (DiaionÒ, 5 ꢀ 36 cm resin bed) and eluted with 1:1,
MeOH:acetone. The cells were lyopholized and extracted with
1:1, MeOH:DCM. The combined extracts were concentrated to dry-
ness, and then partitioned between 15% aqueous MeOH and isooc-
tane. MeOH was removed from the polar fraction in vacuo, and the
remaining water was extracted sequentially with EtOAc and 9:1
DCM:isopropanol. The organic fractions were concentrated to dry-
ness (2.75 g) and separated by LH-20 (FlukaÒ) column chromatog-
raphy (100% MeOH, 2.6 ꢀ 67.5 cm resin bed). Early eluting
fractions were combined by LC–MS analysis and subsequently
purified by reversed-phase HPLC (Phenomenex Luna Phenyl Hexyl
column, 5 mm, 250 ꢀ 10 mm). A linear gradient of 50–100% MeCN
in H2O + 0.1% TFA (45 min, 5 mL/min) yielded pure compounds 1
(22.6 mg), 2 (5.5 mg), 3 (16.4 mg), and 4 (7.3 mg).
The molecular formula of 1 (C51H65N12O12S2+) was established
by HR-ESI-MS, and 1H and 13C NMR spectra resembled those
reported for echinomycin.15,16 Since chemical shift assignments
that distinguish the upper and lower hemispheres were not previ-
ously reported, the compound was completely characterized using
* Corresponding author. Tel.: +1 401 874 9228.
E-mail address: drowley@uri.edu (D.C. Rowley).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.010

A. M. Socha et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1504–1507
1505
O
S
O
N
N
S
O
N
N
N
S
O
HN
N
O
HN
O
N
O
H
H
N
N
O
O
O
O
N
O
O
N
O
O
O
S
N
O
N
N
O
O
N
N
O
N
H
O
N
H
N
NH
NH
O
N
O
O
O
2
1
O
O
S
N
N
N
S
N
N
N
S
HN
O
N
O
COOH
HO
HN
N
O
COOH
HO
H
N
H
N
O
N
O
N
O
O
O
O
OH
HOOC
S
N
O
N
N
O
O
N
N
O
N
N
H
H
NH
NH
O
N
O
O
4
3
1 Echinomycin
2 FD-991
3 Depsiechinoserine
4 Echinoserine
NMR spectral data. By referencing the cysteine b-protons of the
thioacetal bridge, 2D NMR experiments allowed assignment of all
resonances (Tables 1 and 2 in Supplemental data).
echinomycin peptides. A DEPT-135 experiment distinguished the
quinoxaline chromophores by their characteristic 13C resonances.
COSY, HSQC, and HMBC allowed for the identification of two
N-Me-Val, two Ala, and two modified Cys residues. The HSQC spec-
trum of 3 also identified a pair of upfield serine b-protons (dH,H
3.99, 4.09) that were distinguishable from its ester-linked serine
b-protons (dH,H 4.89, 4.44). Semi-synthetic preparation of both iso-
mers was achieved by mild hydrolysis of 1. A solution of 1
HR-ESI-MS measurements of 2 indicated a molecular formula
(C51H65N12O13S2+), which required the addition of an oxygen atom
to 1. A new IR band was observed at 1014 cmꢁ1, consistent with
the presence of a sulfoxide. Cys and Cys0 sulfoxide analogs of 1
have been reported in the semi-synthetic and natural product liter-
ature, respectively.17,18 To determine the site of oxidation in 2, ESI-
MS–MS experiments were conducted on both 1 and 2 under
identical fragmentation parameters. Product ions showing a loss
of SCH3 were observed for both parent compounds (m/z for
2 = 1068.4 [MꢁH]ꢁ), thus indicating that oxidation resided at the
Cys0 sulfur atom. The structure of 2 was confirmed by 2D NMR
(Fig. 1) and shown to match that of FD-991, a natural product.17
The new compound depsiechinoserine (3) displayed a HR-ESI-
MS of m/z 1119.4384, consistent with the addition of H2O to the
molecular formula of 1. The UV spectrum was nearly identical to
the aforementioned peptides, and selective ion monitoring LC–
MS showed a very slight separation of 3 into a pair of isomers in
an approximate 1:1 ratio. However, no conditions were found that
allowed sufficient resolution for individual analysis. Therefore, all
NMR spectra were recorded on the mixture of positional isomers,
which precluded some NMR chemical shift assignments. Despite
signal overlap, the spectra of 3 were highly similar to the other
(0.114
LiOH (65 mM) for 10 min at ambient temperature. The reaction
was quenched with 25 L of 0.1 N HCl, and analyzed by LC–MS–
MS (5-90% MeCN in H2O + 0.2% acetic acid, 500 L/min over
20 min, Waters X Terra C18 MS column, 5
m, 3 ꢀ 100 mm). Both
lmol in 50 lL THF) was treated with 125 lL of aqueous
l
l
l
the semi-synthetic derivatives and the natural products displayed
matching retention times (18.6 min/18.6 min), and produced sev-
eral identical mass fragments. Product ions in each MS2 spectra
showed loss of SCH3 (1071.4), N-Me-Val (988.3), Ala0 (805.3), and
Cys0 (444.1) from the parent ions (Fig. 2). Combined, these results
demonstrate that 3 is the N-Me-Val carboxylic acid analog of
echinomycin.
Compound
4 provided HR-ESI-MS data that matched the
metabolite echinoserine,19 the bis-N-Me-Val carboxylic acid analog
of 1. Tandem MS experiments displayed the same fragment ions as
its reported structure. Since complete chemical shift assignments
for this compound were missing from the literature, a full charac-
988.3
Cys O
O
Quinoxaline
Val
Ala
N
N
N
N
N
S
O
4
HN
O
N
COOH
NH
3
2
HN
N
H
N
H
N
O
N
OH
O
O
O
7
1
O
O
S
O
N
O
Ser
Ser'
S
S
N
1071.4
O
N
N
O
O
N
O
N
H
O
444.1
N
H
O
O
2'
3'
N
Ala' NH
O
N
N
O
Val'
Quinoxaline'
O
O
805.3
Cys'
398.2
Figure 1. Selected TOCSY and COSY (bold) and HMBC correlations (arrows)
observed for 2.
Figure 2. MS2 fragments of depsiechinoserine (3) observed for both the isolated
metabolite and the identical compound produced by semi-synthesis.

1506
A. M. Socha et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1504–1507
Table 1
Pharmacological activities for echinomycin analogs
Analog
MRSA-L32a
M) MBC (
MRSA-L44b
M) MBC (
MSSAc
EFd
HCT-116e
IC50 M)
MIC (l
l
M)
MIC (
l
l
M)
MIC (
lM)
MBC (
l
M)
MIC (
lM)
MBC (
l
M)
(l
1
2
3
4
0.03
1.79
>14
>14
0.91
7.17
>14
>14
0.03
1.79
>14
>14
0.45
7.17
>14
>14
0.01
0.90
>14
>14
0.45
3.58
>14
>14
0.01
0.22
14
0.11
>14
>14
>14
<0.003
0.4
4.6
>14
13.4
a
MRSA-L32 = clinical isolate.
MRSA-L44, clinical isolate.
MSSA, ATCC strain 35556.
EF, ATCC strain 29212.
b
c
d
e
HCT-116, human colon tumor cell line.26
terization was accomplished by examination of 2D NMR data
including COSY, TOCSY, HSQC and HMBC (Tables 1 and 2 in Supple-
mental data). As seen with 3, the serine b-protons of 4 were shifted
upfield (dH,H 4.00, 4.04), and integration of four protons indicated
that both esters were hydrolyzed in the latter compound. Addition-
ally, the 13C chemical shifts of the N-Me-Val and N-Me-Val0 carbox-
ylic acids were shifted approximately 4 ppm downfield (d 174.2,
174.7) as compared with 1 and 2.
and 280 nm and negative effects at approximately 230 and
315 nm, respectively. Therefore, the absolute configurations of 2–
4 were determined to be the same as echinomycin.22
Minimum inhibitory concentrations (MIC) and minimal bacteri-
cidal concentrations (MBC) were measured against biofilm-form-
ing (BF) strains of methicillin-susceptible S. aureus (MSSA, ATCC
35556) and E. faecalis (EF, ATCC 29212).23,24 Additionally, two clin-
ical BF strains of MRSA (L32 and L44) were tested.25 The clinical
strains were obtained from blood samples of patients at the Prov-
idence Veterans Affairs Medical Center. Assays conducted using
clinical isolates may provide a better indication of the antibiotic
potential of a new agent. Each compound was also tested for cyto-
toxicity against a human colon tumor (HCT-116) cell line (Table 1).
Based on these results, time-kill assays were conducted on 1
and 2 allowing measurement of their cell killing properties over
24 h. The advantage of this technique over traditional MIC and
MBC assays is that one can quantify how rapidly killing occurs
and then determine if a compound has bactericidal or bacterio-
static activity. The time-kill studies were conducted at two and
four times the respective MIC (Table 2).27 FD-991 (2) demonstrated
bactericidal activity against the biofilm-producing MSSA (ATCC
35556) with a decrease of 3.39 and 3.24 log10 CFU/mL at two and
four times the MIC, respectively. Both echinomycin (1) and FD-
991 (2) demonstrated bacteriostatic activity against the clinical
MRSA strains and Enterococcus faecium at four times the MIC.
The biofilm inhibitory effects of 1 and 2 were measured using a
colorimetric microtiter plate assay.25 Biofilm formation was quan-
tified by staining with crystal violet and measuring UV absorbance
at 570 nm. Compounds 1 and 2 were evaluated over a concentra-
Application of the advanced Marfey’s analysis established the
presence of L-Ala, D-Ser, and L-N-Me-Val in 1–4 by comparison with
the appropriate amino acid standards.20 Approximately 0.5 mg of
each compound was hydrolyzed with 0.8 mL of 6 N HCl in sealed
glass ampoules (22 h, 95 °C). The 2,4-dinitrophenyl-5-L-leucina-
mide derivatives were prepared according to previously reported
methods.21 Chromatography was performed on a Waters X-Terra
C18 MS column (5
l
m, 3 ꢀ 100 mm) using a linear gradient (10–
60% MeCN in H2O + 0.2% acetic acid, 500 mL/min over 18 min,
see Supplemental data for RT comparison). The optical rotation of
1 matched the reported value10 and the CD spectra of all com-
pounds displayed positive Cotton effects at approximately 205
Table 2
Time-kill assay results for 1 and 2 against S. aureus and E. faecalis strains
Test
Mean change in bacterial densitya (log10 CFU/mL)
MRSA-L32
3.44 0.22
MRSA-L44
3.21 0.05
MSSA
2.84 0.03
EF
2.87 0.09
Growth control
2ꢀ MIC
1
2
0.61 0.23
ꢁ1.35 0.20
0.423 0.09
ꢁ1.09 0.19
ꢁ1.54 0.06
ꢁ3.39 0.06
0.04 0.09
0.69 0.12
tion range of 0–14
film in the positive controls, and all test isolates provided an
acceptable optical density (OD570 between 0.5 and 0.8 AU.
lM. Each bacterial strain produced a robust bio-
4ꢀ MIC
)
1
2
ꢁ2.39 0.09
ꢁ2.15 0.17
ꢁ1.23 0.03
ꢁ2.24 0.07
ꢁ1.79 0.04
ꢁ3.24 0.17
ꢁ1.42 0.11
ꢁ1.04 0.09
Negative controls wells contained media only. Echinomycin (1)
and FD-991 (2) inhibited all S. aureus and E. faecium isolates at con-
centrations near their MIC (Fig. 3).
a
Inoculum change from starting inoculum of 5 ꢀ 105 CFU/mL (observed at 24 h).
CFU, colony forming units.
Echinomycin
1
FD-991
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Pos
Control
0.45
0.9
1.8
7.2
Neg
Control
Pos control
0.03125
0.0625
0.5
Neg
control
Concentration (µM)
Concentration (µM)
MRSA-L32
MRSA-L44
MSSA
EF
MRSA-3 2
MRSA-4 4
MSSA
EF
Figure 3. Effects of echinomycin (1) and FD-991 (2) on biofilm formation by S. aureus and E. faecalis strains (n = 2).

A. M. Socha et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1504–1507
1507
Table 3
Modified Calgary pin-lid bioassay data
Analog
MRSA-32
MRSA-44
MSSA
EF
MBIC (
lM)
MBEC (
lM)
MBIC (lM)
MBEC (lM)
MBIC (lM)
MBEC (lM)
MBIC (
l
M)
MBEC (lM)
1
2
>4
>4
>4
>4
>4
>4
>4
>4
0.03
3.6
0.22
>4
0.22
>4
0.22
>4
Antimicrobial susceptibilities of established biofilms were also
evaluated using a modified version of the Calgary Biofilm (Pin-
lid) Device.28 This assay determines the antibiotic effects on sessile
bacteria seeded from an established biofilm mass. The minimum
biofilm inhibitory concentration (MBIC) is defined as no visible
growth after incubation for 24 h in the presence of a preformed
biofilm and the antibiotic. The minimal biofilm eradication concen-
tration (MBEC) is defined as the minimal concentration of antibi-
otic that is required to eradicate the CFU/mL growth from the
biofilm. The MBIC and MBEC for 1 against MSSA and EF were sim-
ilar to the respective values for these bacteria when grown plank-
tonically (Table 3). Compound 2 did not demonstrate any notable
activity against the panel of isolates in this assay (highest concen-
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This research was supported by NOAA Grant NA04OAR4600193
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antibacterial bioassays and Roberta Sheffer for assistance with the
HCT-116 assay and phylogenetic identification of strain URI-F39.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.010.