Coelomycin, a highly substituted 2,6-dioxo-pyrazine fungal metabolite antibacterial agent discovered by Staphylococcus aureus fitness test profiling

Article abstract of DOI:10.1038/ja.2010.86

Bacterial resistance to antibiotics, particularly to multiple antibiotics, is becoming a cause for significant concern. The only really viable course of action to counter this is to discover new antibiotics with novel modes of action. We have recently implemented a new antisense-based chemical genetic screening technology to accomplish this goal. The discovery and antibacterial activity of coelomycin, a fully substituted 2,6-dioxo pyrazine, illustrates the application of the Staphylococcus aureus fitness test strategy to natural products discovery.

Full text of DOI:10.1038/ja.2010.86

The Journal of Antibiotics (2010) 63, 512–518
&
2010 Japan Antibiotics Research Association All rights reserved 0021-8820/10 $32.00
www.nature.com/ja
ORIGINAL ARTICLE
Coelomycin, a highly substituted 2,6-dioxo-pyrazine
fungal metabolite antibacterial agent discovered
by Staphylococcus aureus fitness test profiling
Michael A Goetz1, Chaowei Zhang1, Deborah L Zink1, Marta Arocho1, Francisca Vicente2,3, Gerald F Bills2,3,
Jon Polishook1, Karen Dorso1, Russell Onishi1, Charles Gill1, Emily Hickey1, Suzy Lee1, Richard Ball1,
Stephen Skwish1, Robert GK Donald1, John W Phillips1 and Sheo B Singh1
Bacterial resistance to antibiotics, particularly to multiple antibiotics, is becoming a cause for significant concern. The only
really viable course of action to counter this is to discover new antibiotics with novel modes of action. We have recently
implemented a new antisense-based chemical genetic screening technology to accomplish this goal. The discovery and
antibacterial activity of coelomycin, a fully substituted 2,6-dioxo pyrazine, illustrates the application of the Staphylococcus
aureus fitness test strategy to natural products discovery.
The Journal of Antibiotics (2010) 63, 512–518; doi:10.1038/ja.2010.86; published online 28 July 2010
Keywords: antibacterial; coelomycin; fitness test; fungal metabolite; pyrazine; staphylococcus aureus; target array
INTRODUCTION
extract stage.3 Second, we have expanded our efforts to isolate
Antibiotic-resistant bacterial strains continue to increase in frequency microorganisms from different geographical regions and habitats
and pose a serious threat to human lives.1 Infections by methicillin- (including marine microbes) in combination with improved
resistant Staphylococcus aureus alone are responsible for approximately high-throughput fermentation methods.4 The combination of these
18000 deaths per year in the United States.1 Most of the treatment two approaches resulted in the discovery of a large number of natural
options available today to treat bacterial infections are the result of products using either an rpsD-sensitized antisense strain5–10 or
incremental improvements to antibiotic lead structures discovered fabH/fabF antisense-sensitized strains, including discovery of the
during the ‘Golden Age’ of antibiotic discovery.2 Unfortunately, FabF inhibitors platensimycin and platencin.11–17
further improvements to those structural leads are proving to be
Recently, we have extended the antisense-based screening approach
more and more difficult, leading to diminishing return on investment. by constructing a S. aureus genome-wide fitness test assay and
Obviously, this problem could be alleviated by discovery of new lead validated its use by mechanistically profiling a diverse set of 59
classes of antibacterial agents with either known or novel modes of antibacterial compounds18 and by the discovery of a new class of
action that could be developed as effective treatment options against cell wall inhibitors.19 Included in the S. aureus fitness test assay are 245
drug-resistant bacteria. Most of the antibacterial leads are of natural inducible antisense RNA strains engineered for reduced expression of
product origin and natural products remain prolific sources of new genes essential for cell growth. Reduced expression of target genes
antibacterial leads. One of the biggest challenges in natural products leads to differential growth sensitivity of cells to compounds
discovery remains the rediscovery of known compounds. To improve that inhibit either the targeted gene product or related functions.
the probability of success for the discovery of novel natural products, When combined into pools and grown together for approximately
new screening technologies and new sources of natural products are 20 population doublings in the presence of test compounds, these
needed. We have tried to address both of these problems. First, we differences in growth lead to specific antisense strains, either being
have introduced a target-based whole-cell screening approach that depleted or enriched in the treated population. Using multiplex PCR,
uses inducible antisense to downregulate the expression of individual capillary electrophoresis and gene fragment analysis to compare
essential gene targets in S. aureus, thereby sensitizing the cells to the abundances of the strains at the end of the experiment with
inhibitors of those targets and enriching the pool of screening hits mock-treated controls generates characteristic antisense-induced
for inhibitors specific to those targets or pathways at the crude strain sensitivity (AISS) profiles that are indicative of the whole-cell
2
1Merck Research Laboratories, Rahway, NJ, USA and CIBE, Merck Sharp & Dohme de Espana, S. A. Josefa Valcarcel, Madrid, Spain
´
3
´
´
Current address: Fundacion Medina, Parque Tecnologico de Ciencias de la Salud, Armilla, Granada, Spain.
Correspondence: Dr SB Singh, RY33-612, Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, NJ 07065, USA.
E-mail: sheo_singh@merck.com
Dedicated to Professor Arnold Demain, Drew University, for his seminal and pioneering contributions to Fermentation Technology.
Received 22 March 2010; revised 29 April 2010; accepted 16 June 2010; published online 28 July 2010

Coelomycin discovered by S. aureus fitness test profiling
MA Goetz et al
513
(data not shown). As this was a unique profile and standard HRLCMS
analysis also did not reveal the presence of any obvious antibiotics
(data not shown) that could account for any or all of the aspects of the
AISS profile, this extract was selected for further studies.
The culture was grown on a glycerol-based liquid medium for
21 days and extracted with equal volume of acetone. Removal of
acetone followed by extraction with methyl ethyl ketone, concentra-
tion and trituration with methanol provided a semicrystalline solid
that was purified by reversed-phase HPLC to afford 47mg l–1 of
compound 1 as a yellow amorphous powder.
12
20
9
16
H
N
14
7
N
N
O
3
5
1
O
O
N
O
N
OR
O
N
O
OH
OR
1: R= H
2: R= Me
R1
4: R1= OH
5: R1= H
3
High-resolution electrospray ionization Fourier transformation
mass spectrometry analysis of compound 1 provided a molecular
formula of C18H14N2O4 (m/z 322.0956) indicating the presence of 131
Figure 1 Chemical structures of coelomycin (1) and its derivatives.
mechanism of action of the compounds. Compound mechanism of of unsaturation. The 13C NMR spectrum in CD2Cl2 (Table 1) showed
action can either be inferred directly from the AISS profile or by only 14 carbon signals with four overlapping aromatic carbon signals.
1
comparison with a database of AISS profiles of known inhibitors.18 The H NMR spectrum showed the presence of 10 aromatic protons
Both these phenomena are very useful for the discovery of new from dH 7.30 to dH 8.13, COSY correlations among these protons and
compounds by providing not only preliminary mechanistic informa- the presence of the four overlapping carbon signals, suggesting two
tion but also comparative information that is of great use in monosubstituted phenyl rings. The presence of a downfield shifted
dereplicating natural products. Similar to the recent discoveries of methylene singlet (dH 4.12, dC 39.3) and its heteronuclear multibond
parnafungin20,21 and other novel natural products22,23 using a Can- connectivity (HMBC) correlation with aromatic carbons at dC 130.1
dida albicans fitness test assay designed for antifungal discovery,24 and 136.9 showed that one of the phenyl groups was present as a
AISS profiling can be used in antibacterial discovery to prioritize benzyl unit. One of the protons resonating at dH 8.13 showed HMBC
extracts for natural products chemistry based on whether their correlation with a carbonyl resonating at dC 180.4, suggesting that the
activities are likely working through either known or novel mechan- second phenyl group was a benzoyl unit. Together, the benzyl and
isms of action. Those compounds showing a novel AISS profile and benzoyl units account for 14 of 18 carbons. The four unassigned
likely containing chemical inhibitors not observed before in the assay carbons resonated at dC 161.8, 115.6, 147.4 and 152.8. Taking into
can be assigned to isolation chemistry. Those showing a known AISS consideration the presence of two nitrogens and three additional
profile can be further analyzed with HRLCMS and other analytical oxygens indicated that this molecule is a diketopiperazine with
techniques to either confirm or rule out the presence of known extended conjugation similar to flutimide.25–27 This assumption was
inhibitors before any isolation chemistry steps are undertaken.
supported by the IR and UV spectra. The HMBC correlations of the
Through systematic screening a large number of crude natural methylene protons with dC 147.4 established the substitution of benzyl
product extracts in the S. aureus fitness test and comparison of their group at C-5. The NMR data did not allow to differentiate between a
AISS profiles to those of known inhibitors, we identified an extract 2,6-diketopiperazine and a 2,5-diketopiperazine and thus could not
from an unidentified Coelomycete fungal strain that produced a define the position of the benzoyl group (C-3 or C-2). It was clear
unique AISS profile. Bioassay-guided isolation of the extract using a from the presence of a OH group at dH 15.8 that the keto group was
S. aureus EP167 strain growth assay led to the isolation of pyrazine (1), enolized and formed a strong six-centered H-bond with the benzoyl
named herein coelomycin (Figure 1), which accounted for the AISS ketone. Methylation of compound 1 with diazomethane produced
profile. The isolation, structure elucidation and details of biological dimethyl ether 2. The methoxy group (dH 4.10, dC 64.7) showed
activity including in vivo activity of this compound are described.
HMBC correlation with dC 154.0 and the benzoyl keto group showed
HMBC correlation with H-9/13 dH 7.83, suggesting that the benzoyl
keto was not enolized. However, methylation still did not allow for
RESULTS AND DISCUSSION
As the bacterial target array is not a high-throughput assay, extracts distinction of 2,6- versus 2,5-diketo substitution.
were submitted for primary screening using standard wild-type LiBH4 reduction of compound 1 produced deoxy compound 3
1
bacterial strains such as S. aureus. Extracts that passed requisite (m/z 306.1007; C18H14N2O3) that showed H and 13C NMR spectra
antibacterial activity were selected for AISS profiling analysis. After identical to sclerominol.28 Hydrogenation of compound 1 with Pd/C
this triaging process, one of the extracts from an unidentified afforded tetrahydro deoxy compound 4 (C18H18N2O3) and tetrahydro
coelomycete belonging to the Dothideales, isolated from leaf litter of dideoxy compound 5 (C18H18N2O2). 13C NMR spectrum (Table 2) of
Juniperus phoenicea in salt marshes from Roquetas de Mar, Almeria, compound 4 showed only seven carbon resonances indicating
Spain, produced a distinct AISS profile (Figure 2) with significant C2 symmetry. The methine protons dH 3.70 (dC 60.2) of compound
depletions of antisense strains for genes involved in cell wall (mraY, 4 showed HMBC correlations with the methylene carbon (dC 36.8),
murG, uppS and pbpA) and fatty acid biosynthesis (fabI and hmrB). In aromatic carbon (dC 137.7) and carbonyl (dC 169.5) and most
addition, strong depletions were also observed for the map-antisense importantly with its carbon confirming the 2,6-diketopiperazine
strain (methionine aminopeptidase–protein synthesis) and to a lesser structure 4. The 13C NMR spectrum of compound 5 was similar to
extent the thyA-antisense strain (thymidylate synthase–DNA synth- the spectrum of 4 with the exception of 4 p.p.m. downfield shifted
esis). Although this profile contains some strain sensitivities shared carbonyl resonance (Table 2) due to loss of the N-oxy group. On the
with known cell wall or fatty acid synthase inhibitors, the combination basis of the structure of 4 and 5, benzoyl group was placed at C-3 and
of strain depletions is unique to this extract and the AISS profile did structure 1 was assigned to compound 1. This could be confirmed in
not correlate significantly with any AISS profiles for either known the following way: compound 1 was crystallized from aqueous
inhibitors or undescribed compounds or extracts in our profile methanol for X-ray crystallographic studies that showed enolization
database when analyzed using two-dimensional cluster analysis of benzoyl ketone (Figure 3), whereas in aprotic solvent the C-2 keto
The Journal of Antibiotics

Coelomycin discovered by S. aureus fitness test profiling
MA Goetz et al
514
Figure 2 S. aureus fitness test analysis of crude extract and coelomycin (1) samples. This two-dimensional cluster of antisense-induced strain sensitivity
(AISS) profiles shows the similarity between the profiles of the crude extract and pure compound. Strain depletions are represented in magenta and strain
resistances in cyan. The extract or compound name is followed by the treatment concentration. The 12 antisense strains shown in the cluster are those with
significant hypersensitivity to the treatments. The thresholds for including strains in the analysis are 45-fold depletion, P-value p0.01 in X3 experiments.
The data from a dose titration at sub-MIC concentrations are shown.
Table 1 1H (500 MHz) and 13C (125 MHz) NMR assignment of compounds 1–3 in CD2Cl2
1
2
3
No.
dH (mult, J in Hz)
dC
HMBC (H-C)
dH (mult, J in Hz)
dC
HMBC (H-C)
dH (mult, J in Hz)
dC
HMBC (H-C)
2
161.8
115.6
147.4
152.8
180.4
131.9
132.1
128.5
133.7
39.3
154.0
120.4
151.9
152.7
190.0
137.8
130.7
128.3
132.8
39.6
159.4
131.9
152.2
157.0
144.6
133.6
134.9
129.2
132.8
40.0
3
5
6
7
7.84 (s)
C-2, 9/13
8
9, 13
10, 12
11
8.13 (d, 7.6 Hz)
7.40 (m)
C-7, 11, 13/9
C-8, 11, 10/12
C-9/13, 10/12
C-5, 15, 16/20
7.83 (brd, 8.2 Hz)
7.44 (t, 7.7Hz)
7.57 (tt, 7.5 Hz)
4.06 (s)
C-7, 11, 13/9
C-10/12, 8
C-9/13
7.97 (brd, 7.8 Hz)
7.34 (m)
C-7, 11, 13/9
C-8
7.57 (t, 7.4Hz)
4.12 (s)
7.46 (t, 7.2Hz)
4.22 (s)
C-9/13
14
C-16/20, 15, 5
C-5, 6, 15, 16/20
15
136.9
130.1
128.8
127.1
137.6
129.8
128.7
126.9
135.9
130.4
129.0
127.3
16, 20
17, 19
18
7.31 (m)
7.34 (m)
C-14, 18
C-15, 19/17
C-16/20
7.32 (m)
C-18, 14, 20/16
C-15, 19/17
C-16/20
7.35 (m)
7.38 (m)
7.34 (m)
C-14, 18
C-15
7.28 (m)
7.30 (m)
7.22 (tt, 7.2 Hz)
OH
15.80 (brs)
N-OMe
OMe
4.08 (s)
4.10 (s)
65.5
64.7
C-2
Abbreviation: HMBC, heteronuclear multibond connectivity.
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Table 2 1H (500MHz) and 13C (125 MHz) NMR assignment of compounds 4 and 5 in CD2Cl2
4
5
No.
dH (mult, J in Hz)
dC
HMBC (H-C)
dH (mult, J in Hz)
dC
HMBC (H-C)
2/6
3/5
7
169.5
60.2
36.8
173.2
60.0
36.3
3.70 (dd, 8.5, 3.9 Hz)
2.93 (dd, 13.8, 8.5Hz)
3.36 (dd, 13.8, 3.9Hz)
C-2/6, 8, 5/3, 7
C-2/6, 8, 9, 3/5
3.55 (dd, 8.6, 3.9 Hz)
2.85 (dd, 13.9, 8.6 Hz)
3.33 (dd, 13.9, 3.9 Hz)
C-2/6, 8, 5/3, 7
C-2/6, 8, 9, 3/5
8
136.7
129.7
129.0
127.3
136.9
129.8
129.2
127.4
9
7.14 (m)
7.23 (m)
7.20 (m)
C-9, 11, 7
C-8, 10
C-9
7.12 (m)
7.20 (m)
7.17 (m)
C-9, 11, 7
C-8, 10
C-9
10
11
Abbreviation: HMBC, heteronuclear multibond connectivity.
antimycobacterial (MIC 310.5 mg ml–1) activities. These researchers
did not name the compound, and hence we named it coelomycin.
Biological activity
Coelomycin (1) was first evaluated in the S. aureus genome-wide
fitness test to establish the AISS profile for the purified compound 1.
Comparison of the AISS profile for coelomycin (1) with that of the
acetone extract revealed a similar profile with shared depletions in
antisense strains for cell wall, fatty acid, protein and DNA synthesis
(Figure 2). This step of the analysis confirms the isolation of the
sought-after compound that was responsible for the profile in
the extract and further establishes that this multipathway profile is
the result of the biological actions of a single antibacterial agent.
Coelomycin was further evaluated for its antibacterial activities
against a panel of clinically relevant Gram-positive and Gram-negative
bacterial strains and the data are summarized in Table 3. This
compound was essentially inactive when tested in standard CLSI
(Clinical and Laboratory Standards Institute) conditions in CAMHB
(cation-adjusted Mueller Hinton broth) medium. However, it showed
good activity when bacteria were grown in other media. For example,
it inhibited growth of S. aureus Smith with an MIC value(s) of
4 mg ml–1 in iso-sensitest medium and 0.25–0.5 mg ml–1 in nutrient
broth medium supplemented with 10% NaCl. It showed similar
activity against Streptococcus pneumoniae (MIC 4 mg ml–1) in iso-
sensitest medium. It was also active against Haemophilus influenzae
(MIC 8 mg ml–1) in HTM medium. It did not show any activity against
wild-type Escherichia coli but was effective against E. coli strain (MIC
0.5–2 mg ml–1) harboring mutations in envelope proteins and tolC
pump, suggesting that the compound either does not enter the cells or
is pumped out in the wild-type bacteria. To probe the effect of cations,
this compound was further tested in a few growth media in the
presence of Ca+2 or Mg+2 and the data are summarized in Table 4.
The presence of excess bivalent cations has a detrimental effect on the
activity of this compound in LB (Lysogeny broth) medium but not in
nutrient broth medium. In a macromolecular synthesis inhibition
assay, compound 1 inhibited all five (DNA, RNA, protein, peptido-
glycan and phospholipid) with a modest four-fold selectivity for cell
wall synthesis (Figure 5). These data are consistent with the AISS
Figure 3 X-ray crystal structure of coelomycin (1).
N
N
O
O
H
H
O
O
N
O
O
N
OH
OH
Aprotic
Protic
Figure 4 Tautomeric forms of compound 1 in protic and aprotic solvents.
group is enolized. Both enolizations lead to six-centered hydrogen profile data that showed depletion of a number of cell wall-associated
bond (Figure 4). The protic tautomeric form is likely the form that antisense strains along with depletions in antisense strains associated
predominates in aqueous systems and is perhaps the biologically active with several other biological processes (Figure 2). Compounds 2–5
form.
were significantly less active and did not show any antibacterial
While our studies were in progress, Chinworrungsee et al.29 activity at 64mg ml–1 against S. aureus EP167 strain.
reported the isolation of compound 1 from Menisporopsis theobromae
Coelomycin did not show efficacy in a murine disseminated target
BCC3975. They reported antimalarial (IC50 28.8mM) and organ assay against S. aureus. The compound was dosed twice daily by
The Journal of Antibiotics

Coelomycin discovered by S. aureus fitness test profiling
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516
Table 3 In vitro antibacterial activity of coelomycin (1) (MIC in mg ml–1
)
Organism
Strain no.
CAMHB
NB+10% NaCl
Iso-sensitest
HTM
S. aureus (MSSA)
MB2865
EP167
416
416
416a
416
416
416
416b
0.5
0.25
NT
4
4
NT
NT
NT
8
S. aureus
Streptococcus pneumoniae (penicillinS)
Haemophilus influenzae
Escherichia coli
CL2883
MB4572
MB2884
MB5746
MY1055
4
NT
NT
48
2
48
0.5
NT
NT
NT
NT
E. coli envA, tolC
Candida albicans
NT
Abbreviations: CAMHB, cation-adjusted Mueller Hinton broth; MSSA, methicillin-sensitive Staphylococcus aureus; NB, nutrient broth.
aMedium: CAMHB +2.5% lysed horse blood.
bIn YPD (richest) and Sab-Dex media.
Table 4 Effect of media on MIC (lg ml–1) of coelomycin (1)
linear and the compound produced better exposures at 25 (AUC
1.80mM h) and 12.5 (AUC 1.84mM h) mg kg–1. The compound was
not tested at higher doses because of poor solubility and lack
of PK linearity. Coelomycin did not show toxicity to a number of
mammalian cells (human embryo kidney, Chinese hamster ovary and
Hela cells) at 50mg ml–1.
In summary, in this study we report the application of an antisense-
based S. aureus fitness test platform to the discovery of new natural
product antibiotics. This led to the discovery of coelomycin, (at the
time it was a novel compound), a broad-spectrum Gram-positive
antibacterial agent. Both AISS profiling and a macromolecular synth-
esis inhibition assay suggested that coelomycin inhibits bacterial
growth by multiple modes of action with a slight preference for
peptidoglycan (cell wall) synthesis inhibition. Although the mechan-
ism of action of coelomycin remains to be resolved, this report shows
that a unique AISS profile in an acetone extract can lead to the
isolation of novel chemical entities. This compound did not show any
in vivo activity due to poor exposure.
Media
S. aureus EP167
NB
0.2–0.4
0.8
NB+0.2% YE
NB+0.2% YE+1% NaCl
LB
0.8–1.6
0.8–3.2
X25
6.25
CAMHB
BHI
+2a
NB+Mg+2 /Ca
0.4
a
+2a
LB+Mg+2 /Ca
25
a
Abbreviations: BHI, brain heart infusion broth; CAMHB, cation-adjusted Mueller Hinton broth;
LB, Lysogeny broth; NB, nutrient broth; YE, yeast extract.
aBoth Ca+2 (25mgl–1) and Mg+2 (12.5mgl–1) were used as chlorides.
Phospholipid
Peptidoglycan
RNA
DNA
Protein
120
100
80
EXPERIMENTAL PROCEDURE
General experimental procedures
coelomycin
HP1100 (Agilent, Santa Clara, CA, USA) was used for analytical HPLC. The
UV spectra were recorded in MeOH/THF 1:1 on a Perkin-Elmer Lambda 35
Spectrometer (Perkin Elmer, Waltham, MA, USA). IR spectra were recorded on
a Perkin-Elmer Spectrum One spectrometer. Electrospray ionization MS data
were recorded on an Agilent 1100 mass selective detector with electrospray
ionization. High-resolution electrospray ionization Fourier transformation mass
spectrometry was acquired on a Thermo Finnigan LTQ-FT (Thermo Fisher
Scientific, Waltham, MA, USA) with the standard Ion Max API source (without
the sweep cone) and electrospray ionization probe. Three scan events were used.
The ion trap was scanned from 150 to 2000, first in negative ion mode and then
in positive ion mode. The FT was scanned from 200 to 2000 in the positive ion
mode only. In all cases, the SID was set to 18 V to try to reduce multiple ion
clusters. Instrument resolution was set to 100 000 at m/z 400. No internal
calibration was required; the instrument was calibrated once a week and checked
daily to assure accuracy. All NMR spectra were recorded using a Varian Unity 500
(1H, 500 MHz, 13C, 125 MHz) spectrometer (Varian, Palo Alto, CA, USA). The
residual proton of CD2Cl2 was used as an internal reference (dH 5.32, dC 54.0).
The COSY, DEPT, heteronuclear multiple quantum coherence (HMQC) and
HMBC spectra were measured using standard Varian pulse sequences.
60
40
20
-20
0.1
1
10
µg/ml
Producing organism and fermentation
Figure 5 Inhibition of macromolecular synthesis by coelomycin.
The producing microfungus, an unidentified coelomycete, was isolated from
leaf litter of Juniperus phoenicea in the salt marshes of Roquetas de Mar,
Almeria, Spain. Sequencing of the ITS1/5.8S/ITS4 region of the ribosomal DNA
followed by sequence similarity searches in GenBank indicated that this strain
was remotely related to a number of fungi in the Dothideales. Among the most
similar internal transcribed spacer sequences retrieved were: Kabatina juniperi
subcutaneous route at 50mg kg–1 in cremaphor formulation. This lack
of efficacy may perhaps be due to poor exposure. At 50mg kg–1,
it showed an area under the curve (AUC) of 1.07mM h mg kg–1 with
normalized AUC of 0.28mM h mg kg–1. Unfortunately, the PK was not AF182376.2 (92%), Kabatina thujae AF013226 (91%), Dothidea berberidis CBS
The Journal of Antibiotics

Coelomycin discovered by S. aureus fitness test profiling
MA Goetz et al
517
186.58 (92%) and Dothidea muelleri CBS 191.58 (91%). Fermentation of this containing 0.1% TFA at a flow rate of 10mlmin–1. Lyophilization of fractions
culture was accomplished by inoculating several agar plugs with mycelia into a eluting at 60min afforded 4.2 mg of compound 2 as a light-yellow amorphous
seed broth flask (50 ml medium in 250 ml unbaffled flask). The formulation for powder. (2): UV (CH3OH/THF 1:1) lmax 209 (e 13210) 251 (e 6436) 306 (e
the seed broth was as follows (g per liter, unless specified): corn steep liquor, 8728) nm; IR (ZnSe) nmax 2943, 1682, 1651, 1598, 1450, 1380, 1324, 1181, 982,
5.0; tomato paste, 40.0; oat flour, 10.0; glucose, 10.0; trace element mix, 947 cmꢂ1; 1H and 13C NMR data, see Table 1; and HRESI-FTMS m/z 350.1263
10.0 mll–1; and agar, 4.0. The pH was adjusted to 6.8 with NaOH before (calcd for C20H18N2O4: 350.1267).
sterilization. The formulation for the trace element mix consisted of (g per
liter): FeSO4ꢀ7H20, 1.0; MnSO4ꢀH20, 1.0; CuCl2ꢀ2H2O, 0.025; CaCl2, 0.1;
Synthesis of 1-hydroxy-3-benzylene-5-benzyl-pyrazine-2,6-dione (3)
A 5 mg (0.015mmol) aliquot of 1 was added to 0.5 ml lithium borohydride
solution in THF and stirred for 2 h. Reaction was quenched with 2.5 ml 5%
acetic acid in MeOH and the product was chromatographed by gel filtration
H3BO3, 0.056; (NH4)6Mo6O24ꢀ4H2O, 0.019; and ZnSO4ꢀ7H20, 0.2, prepared
in 0.6N HCl. The flasks were incubated at 221C with 80% relative humidity
and shaken on a rotary shaker at 220 r.p.m. After the seed stage flasks have
grown for 4–7 days, a 1 ml aliquot was used to inoculate each flask of the
on Sephadex LH-20 (GE Healthcare, Uppsala, Sweden; 10ml column, gravity
production medium (50 ml medium in a 250 ml unbaffled flask). The for-
elution, eluted with 100% MeOH, 2 ml per fraction). Fractions 4 and 5 were
mulation of production medium consisted of (g per liter): glycerol, 125.0;
combined and concentrated to dryness. The residue was redissolved in 1.5 ml
glucose, 25.0; pectin, 20.0; ardamine pH, 5.0; (NH4)2ꢀSO4, 4.0; KH2PO4, 4.0;
MeOH, diluted with 8 ml H2O and chromatographed on Amberchrome resin
(Rohm and Haas, Philadelphia, PA, USA; 5 ml column, washed with 10%
and CoCl2ꢀ6H2O, 0.1. The pH was adjusted to 7.0 with NaOH before
sterilization. The flasks were incubated at 221C with 80% relative humidity
on a rotary shaker at 220 r.p.m. for 21 days.
MeOH and eluted with 100% MeOH). The fractions eluting with 100% MeOH
were concentrated to dryness and were further fractionated by semi-prep HPLC
using Zorbax C8 (9.4ꢁ250 mm) column eluting with a 45-min gradient of
25–35%, and a 15-min gradient of 35–95% aqueous CH3CN containing 0.1%
TFA at a flow rate of 5 mlmin–1. Lyophilization of fractions eluting at 51 min
afforded 1.2mg of compound 3 as light-yellow amorphous powder. (3): UV
(CH3OH/THF 1:1) lmax 242 (e 6104) 351 (e 6890) nm; IR (ZnSe) nmax 3248,
2926, 1674, 1615, 1580, 1563, 1495, 1454, 1365, 1252, 1179, 1072, 1031,
S. aureus fitness test
The S. aureus fitness test assay was performed as previously described.18 In brief,
245 strains each containing a different inducible antisense RNA interference
plasmid targeting an essential gene in S. aureus RN4220 were pooled into 24
different bins of 6–12 strains each. The bins were then grown each at a different
concentration of xylose inducer (ranging from 1.8 to 55 mM) for B20 popula-
tion doublings either in the presence of acetone extract or compound being
tested or as a 2% DMSO mock treatment control. Strain bin growth was
performed in 384-well plates (Costar 3680; Corning Life Sciences, Acton, MA,
USA) on a fully automated system using LB media containing chloramphenicol
(34 mgml–1) over three cycles of growth ranging from 5 to 7h each for a total
time of B18 h in a total volume of 50ml. At the end of the growth period, cells
from all 24 bins were pooled for each test treatment or DMSO mock treatment
control. Pooled cells were then lysed and subjected to multiplex PCR to amplify
specific antisense markers for each strain. Strain-specific markers were subse-
quently identified and peak areas quantified by DNA fragment analysis using an
ABI 3730 genetic analyzer (Applied Biosystems, Carlsbad, CA, USA). Peak areas
were then normalized and strain depletion ratios were calculated. Statistical
significance was determined as a P-value using an error model generated for
each individual strain across a discrete set of known standards and unknown test
samples selected to represent total coverage of all strains within the array set.
967 cmꢂ1 1H and 13C NMR data, see Table 1; and HRESI-FTMS m/z
;
306.1007 (calcd for C18H14N2O3: 306.0999).
Hydrogenation of coelomycin
A solution of 5mg (0.015 mmol) of 1 in 0.5ml THF was treated with 5% Pd/C
and was saturated with hydrogen filled in a balloon. Reaction was stirred for
days and catalyst was removed by filtration through a Whatman 0.2mm filter
(GE Healthcare, Piscataway, NJ, USA). The solvent was removed under a stream
of nitrogen and the residue was dissolved in MeOH and fractionated by semi-
prep HPLC using Zorbax C8 (9.4ꢁ250mm) column eluting with a 45-min
gradient of 25–40% aqueous CH3CN at a flow rate of 5 ml min–1. Lyophilization
of fractions eluting at 31min and 39min afforded 1.4mg of compound 4 and
0.4 mg of compound 5 as colorless amorphous powders, respectively. (4): UV
(CH3OH/THF 1:1) lmax 205 (e 55800) nm; IR (ZnSe) nmax 3274, 1699, 1684,
1559, 1496, 1436, 1256, 1203, 1135, 1073, 973 cmꢂ1; 1H and 13C NMR data, see
Table 2; and HRESI-FTMS m/z 310.1317 (calcd for C18H18N2O3: 310.1312). (5):
UV (CH3OH/THF 1:1) lmax 201 (e 18 404) nm; IR (ZnSe) nmax 2929, 1699,
1651, 1559, 1497, 1455, 1436, 1206, 973 cmꢂ1 1H and 13C NMR data, see
;
Extraction and isolation
Table 2; and HRESI-FTMS m/z 294.1367 (calcd for C18H14N2O2: 294.1363).
Acetone (5 l) was added to 5 l fermentation broth and shaken for 1 h and filtered
through celite to remove cells. The extract was concentrated under reduced
pressure to remove most of the acetone, and then partitioned into methyl ethyl
ketone (2ꢁ3 l). The organic extract was concentrated under reduced pressure to
dryness to yield a solid residue that was triturated with aqueous MeOH to give
semicrystalline solid. This was dissolved in DMSO and directly injected on a
preparative reversed-phase HPLC column (Zorbax RX C8, 21.2ꢁ250 mm;
Agilent, Santa Clara, CA, USA) eluting with a 90-min gradient of 10–100%
aqueous CH3CN +0.1% TFA at a flow rate of 10mlmin–1. Lyophilization of the
X-ray of coelomycin
Compound (1) C18H14N2O4, Mr¼322.310, orthorhombic, P212121,
3
˚
˚
a¼4.8388(2), b¼16.9567(7), c¼17.7886(8) A, V¼1459.56(11) A , Z¼4,
Dx¼1.467 gcmꢂ3, monochromatized radiation l(Mo)¼0.71073 A, m¼0.11 mm
,
ꢂ1
˚
F(000)¼672, T¼100K. Data were collected on a Bruker CCD diffractometer
(Bruker, Madison, WI, USA) to a y limit of 32.071 that yielded 17 858 reflections.
There are 5096 unique reflections with 4003 observed at the 2s level. The structure
was solved by direct methods (SHELXS-97; Sheldrick30) and refined using
full-matrix least-squares on F2 (SHELXL-97; Sheldrick31). The final model was
refined using 219 parameters and all 5096 data. All non-hydrogen atoms were
refined with anisotropic thermal displacements. The final agreement statistics
were: R¼0.046 (based on 4003 reflections with I42s(I)), wR¼0.087, S¼1.02
with (D/s)max¼0.01. The maximum peak height in a final difference Fourier
major peak from a number of runs yielded 377 mg of compound 1 (47.1 mg l–1
)
as an amorphous yellow powder. Coelomycin (1): UV (CHCl3) lmax 265 (e
13 298) 375 (e 21091) nm; IR (ZnSe) nmax 3437, 2926, 1639, 1674, 1585, 1519,
;
1489, 1436, 1356, 1220, 1073, 1035, 960 cmꢂ1 1H and 13C NMR data, see
Table 1; and HRESI-FTMS m/z 322.0956 (calcd for C18H14N2O4: 322.0954).
ꢂ3
˚
map was 0.308 eA and this peak was without chemical significance. CCDC
(Cambridge Crystallographic Data Centre) nnnnnn contains the supplemen-
tary crystallographic data for this paper. These data can be obtained free of
charge from the CCDC through www.ccdc.cam.ac.uk/data_request/cif.
Methylation of coelomycin (1)
An aliquot of 20mg (0.062mmol) was added to a mixture of 4 ml anhydrous
CH2Cl2 and 1 ml anhydrous ether. To this stirred solution was added 2.5 ml
(0.4M) ethereal solution of freshly prepared diazomethane. After 90 min,
reaction was stopped by addition of AcOH. Solution was concentrated under
a stream of nitrogen and the residue was redissolved in 500ml MeOH and
MIC
chromatographed by preparative reversed-phase HPLC using a Zorbax RX C8 The MIC against each of the strains was determined as previously described.32
(21.2ꢁ250 mm) column eluting with a 45-min gradient of 20–40%, followed The medium with a two-fold serial dilution of compounds in BHI (brain heart
by a 15-min gradient of 40–95%, and then holding at 95% aqueous CH3CN infusion) broth was inoculated with 105 CFU per ml, and was incubated at
The Journal of Antibiotics

Coelomycin discovered by S. aureus fitness test profiling
MA Goetz et al
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The Journal of Antibiotics