Rugulactone and its Analogues Exert Antibacterial Effects through Multiple Mechanisms Including Inhibition of Thiamine Biosynthesis

Article abstract of DOI:10.1002/cbic.201200265

Rugulactone is a dihydro-α-pyrone isolated from the plant Cryptocarya rugulosa in 2009. It has been reported to display IkB kinase (IKK) inhibitory activity, as well as antibiotic activity in several strains of pathogenic bacteria. However, its biological

Full text of DOI:10.1002/cbic.201200265

DOI: 10.1002/cbic.201200265
Rugulactone and its Analogues Exert Antibacterial Effects
through Multiple Mechanisms Including Inhibition of
Thiamine Biosynthesis
Matthew B. Nodwell,[a] Helge Menz,[b, c] Stefan F. Kirsch,[b, d] and Stephan A. Sieber*[a]
Rugulactone is a dihydro-a-pyrone isolated from the plant
Cryptocarya rugulosa in 2009. It has been reported to display
IkB kinase (IKK) inhibitory activity, as well as antibiotic activity
in several strains of pathogenic bacteria. However, its biological
targets and mode of action in bacteria have not yet been ex-
plored. Here we present enantioselective syntheses of rugulac-
tone and of some corresponding activity-based protein profil-
ing (ABPP) probes. We found that the ABPP probes in this
study are more potent than rugulactone against Staphylocco-
cus aureus NCTC 8325, S. aureus Mu50, Listeria welshimeri
SLCC 5334 and Listeria monocytogenes EGD-e, and that mole-
cules of this class probably exert their antibacterial effect
through a combination of targets. These targets include cova-
lent inhibition of 4-amino-5-hydroxymethyl-2-methylpyrimidine
phosphate (HMPP) kinase (ThiD), which is an essential compo-
nent of the thiamine biosynthesis pathway in bacteria. This
represents the first example of a small-molecule inhibitor of
ThiD.
Introduction
Natural products possess unique molecular properties that dis-
tinguish them from libraries of synthetic compounds, and by
virtue of their coevolution with biological systems, the struc-
tures can be considered “pre-validated”. Heroic efforts have
been made in the field of the isolation and synthesis of natural
products, but in many cases their application as drugs is hin-
dered because the precise mechanisms of action and suites of
targets are unknown. Rugulactone (Scheme 1) was isolated in
2009 from the plant Cryptocarya rugulosa[1] and reported to ex-
hibit antibacterial activities.[2] The scaffold includes two poten-
tial Michael acceptors: an a,b-unsaturated g-lactone, which is
a system known to modify proteins, as in the cases of leptomy-
cin B and ratjadone,[3] together with an a,b-unsaturated
ketone. The presence of two electrophilic groups suggests that
rugulactone exerts its biological effects through inhibition of
its target proteins based on a covalent mechanism. There are
several published syntheses of rugulactone, but its biological
targets or mechanism of action in bacteria have not to date
been explored. In this account we report new asymmetric syn-
theses of rugulactone and of related probes and subsequently
the application of these tool compounds to unravelling of
their corresponding cellular targets by activity-based protein
profiling.[4]
step is advantageous because of the commercial availability of
the catalyst, as well as the ease of experimental setup.
One key consideration was the introduction of an alkyne
handle for subsequent target analysis. In the interests of keep-
ing the ABPP probes as structurally similar to rugulactone as
possible, we attached the alkyne directly to the C-16 position
of the appended aryl ring—distant from the Michael acceptors
in the molecule. A retrosynthetic analysis of rugulactone and
ABPP analogues is shown in Scheme 2A.
Results and Discussion
Our synthesis commenced with trichloroacetimidate 1[10]
(Scheme 2B). Attempts to form the desired product 4 through
asymmetric Overman esterification of 1 with vinylacetic acid
failed. Treatment of 1 with benzoic acid (3 equiv) in the pres-
ence of (S)-(+)-COP-OAc (1.5 mol%) thus provided ester 2 in
excellent yield and enantioselectivity as determined by chiral
[a] M. B. Nodwell, S. A. Sieber
Department Chemie, Centre for Integrated Protein Science CIPSM
Institute of Advanced Studies, Technische Universitꢀt Mꢁnchen
Lichtenbergstrasse 4, 85747, Garching (Germany)
E-mail: stephan.sieber@tum.de
Several syntheses of rugulactone have already appeared in
the scientific literature. The key step—the introduction of the
single chiral centre at C-6—has been achieved variously by use
of a chiral pool approach,[5] proline-catalysed a-aminooxylation
of alcohols,[2] Jacobsen’s hydrolytic kinetic resolution of epox-
ides,[6] Keck asymmetric allylation[7] or allylation of carbonyl
compounds with chiral boronic esters.[8] In our syntheses of ru-
gulactone and of probes, we utilized asymmetric catalytic
Overman esterification[9] as the crucial step (Scheme S1 in the
Supporting Information). The use of this reaction as the key
[b] H. Menz, S. F. Kirsch
Department Chemie und Catalysis Research Center Munich
Lichtenbergstrasse 4, 85747, Garching (Germany)
[c] H. Menz
Current address: BASF SE
Carl-Bosch-Strasse 38, GMT/S-B001 67056, Ludwigshafen (Germany)
[d] S. F. Kirsch
Current address: Bergische Universitꢀt Wuppertal, Organic Chemistry
Gausstrasse 20, 42119, Wuppertal (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200265.
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S. A. Sieber et al.
Scheme 1. A) Rugulactone (Ru0) structure. B) Thiol groups in the target enzyme’s binding site can participate in Michael reactions either with the a,b-unsatu-
rated g-lactone in rugulactone (pathway a) or with the a,b-unsaturated ketone (pathway b). C) An alkyne function in an appropriately designed rugulactone
probe allows the attachment of a tag to labelled enzymes. The conjugation of a rhodamine tag by click chemistry after cell lysis, allowing for fluorescent pro-
teome visualization, is shown.
HPLC (see the Supporting Information). Cleavage of the ester
followed by coupling of the resultant alcohol, 3, with vinylace-
tic acid yielded 4 in three steps. Standard ring-closing meta-
thesis of 4 with the second-generation Grubbs catalyst yielded
lactone 5, which was isomerised to the desired a,b-unsaturat-
ed lactone 6 and deprotected to yield primary alcohol 7. To
complete the synthesis, treatment of phosphonate 9 with alde-
hyde 8 (generated in situ) gave (À)-rugulactone (Ru0) in mod-
erate yield (Scheme 3A).
For the synthesis of the alkyne-containing rugulactone
probes, phosphonate 11 was assembled by means of a reac-
tion[11] between dimethyl methylphosphonate and ester 10.
With 11 to hand, the oxidation of 7 and a subsequent Horner–
Wadsworth–Emmons (HWE) reaction, followed by deprotection
of the alkyne group in 12, yielded probe Ru1.
We also synthesized reduced analogues Ru2, Ru3, and Ru4
in order to explore the contributions of the two Michael ac-
ceptors to the labelling and biological activity of rugulactone.
The synthesis of these probes is shown in Schemes S2–S4. The
structures of all the rugulactone probes used in this study are
shown in Scheme 3B.
With rugulactone (Ru0) and probes Ru1–4 to hand, we as-
sessed their biological properties in Staphylococcus aureus
NCTC 8325, multidrug-resistant S. aureus Mu50,[12] Listeria mon-
ocytogenes EGD-e, Listeria welshimeri SLCC 5334, Pseudomonas
aeruginosa PAO1 and Pseudomonas putida KT2440. An exami-
nation of the MIC values for Ru0–Ru4 (Table 1) reveals that ad-
dition of an alkyne tag (Ru0 to Ru1) results in a twofold in-
crease in potency for the Staphylococcus and Listeria strains
tested (800 mm for Ru0 vs. 400 mm for Ru1 in both strains). Re-
duction of the lactone Michael acceptor yields a molecule
(Ru2) that is still more active than the natural product
Scheme 2. A) Retrosynthetic analysis of rugulactone. B) Synthesis of primary
alcohol 7. a) (S)-(+)-COP-OAc, BzOH, CH2Cl2; b) K2CO3, MeOH; c) DIPC, DMAP,
CH2Cl2; d) Grubbs II, D, CH2Cl2; e) DBU, CH2Cl2; f) HF/CH3CN.
(600 mm), indicating that the ketone Michael acceptor is pri-
marily responsible for antibacterial activity. Probes Ru3 and
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Antibacterial Effects of Rugulactone and its Analogues
In order to unravel the molec-
ular mechanisms responsible for
the antibacterial activity, we ex-
amined whole-cell labelling with
our rugulactone probes. The
probes were incubated with
living bacteria and after one
hour, excess probe was removed
and the cells were lysed by soni-
cation. A fluorescent azide-con-
taining tag (RhN3, Figure S1) was
then appended to the alkyne-la-
belled proteome through a Huis-
gen–Sharpless–Meldal cycloaddi-
tion (click chemistry),[13] and the
labelling was analysed by in-gel
fluorescence scanning (Figure 1).
An in vivo dose-down experi-
ment determined that 200 mm
was a sufficient concentration
for the full saturation of most of
the observed protein targets
(Figure S2). Additionally, thermal
denaturation of the bacterial
proteomes resulted in a loss of
the visible protein bands, dem-
onstrating that an active, folded
protein is necessary for probe la-
belling (Figure S3). In order to
unravel the identities of all
target proteins, we applied
a quantitative enrichment proce-
dure by which living cells were
incubated with Ru1, lysed and
clicked to trifunctional rhod-
amine-biotin-azide linker (Rh-
biotin-N3, Figure S1); this allows
the selective enhancement of
probe-labelled proteins through
the use of avidin beads. Subse-
Scheme 3. A) Synthesis of rugulactone (Ru0) and ABPP probe Ru1. B) Structures of the rugulactone ABPP probes
used in this study. a) Pyr-SO3, DMSO, DIEA, CH2Cl2, À208C; b) NaH, THF, À788C; c) LDA, THF, 08C; d) Pyr-SO3,
DMSO, DIEA, CH2Cl2, À208C; e) 11, NaH, THF, À788C; f) TBAF, THF, HOAc.
Ru4 were found to be inactive against all tested bacteria. Ru-
gulactone and its ABPP probes were inactive against both
P. aeruginosa and P. putida, which came as a surprise because
rugulactone had previously been reported to show an MIC of
12.5 mgmLÀ1 (46 mm) against P. aeruginosa.[2] The lack of anti-
bacterial activity could be due either to a lack of appropriate
targets in these organisms or to limited cell permeability.
quent SDS-PAGE revealed protein bands, and these were isolat-
ed, digested with trypsin and subjected to mass spectrometry
(LC-MS/MS analysis). The obtained peptide fragments were an-
alysed with the aid of the SEQUEST search algorithm and iden-
tified enzymes are summarized in Figure 1 and Table S2.
A strong band observed only in S. aureus was identified as
formate acetyltransferase (FAT), which contains eight cysteine
Table 1. MICs of Ru0–4 versus selected organisms in mm (mgmLÀ1).
S. aureus NCTC 8325
S. aureus Mu50
L. monocytogenes EGD-e
L. welshimeri SLCC 5334
P. aeruginosa PAO1
P. putida KT2440
Ru0
Ru1
Ru2
Ru3
Ru4
800 (0.22)
400 (0.12)
600 (0.18)
>1000 (0.3)
>1000 (0.3)
800 (0.22)
400 (0.12)
600 (0.18)
>1000 (0.3)
>1000 (0.3)
800 (0.22)
400 (0.12)
600 (0.18)
>1000 (0.3)
>1000 (0.3)
800 (0.22)
400 (0.12)
600 (0.12)
>1000 (0.3)
>1000 (0.3)
>1000 (0.3)
>1000 (0.3)
>1000 (0.3)
>1000 (0.3)
>1000 (0.3)
>1000 (0.3)
>1000 (0.3)
>1000 (0.3)
>1000 (0.3)
>1000 (0.3)
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S. A. Sieber et al.
showdomycin; this probably
contributes heavily to the anti-
bacterial activity of this com-
pound.[14a] However, upon mea-
surement of MurA1 inhibition
with Ru0–Ru4, only very weak
inhibition was observed: Ru1, for
example, shows ꢀ50% inhibi-
tion at 300 mm (Figure S5). To
evaluate the effect on MurA2,
we recombinantly expressed the
enzyme (solubilised in the mem-
brane fraction, Figure S6) and
measured its inhibition with
probes Ru0–4. The IC50 values
and dose response curves are
presented in Figure 2 and
Table 2.
Neither Ru3 nor Ru4 displayed
any activity versus MurA2 at any
of the concentrations tested.
Ru0–Ru2 inhibited MurA2 to
much greater extents than
MurA1, and the IC50 values from
this assay correlate with MIC
values, indicating that inhibition
of MurA2, in concert with weak
Figure 1. Labelling of bacterial proteomes with rugulactone probes as visualized by in-gel fluorescence scanning
after click chemistry reaction with azide RhN3. Cytosolic fractions shown. Membrane fractions showed no bands.
A) In vivo labelling of S. aureus NCTC 8325 and Mu50 with Ru1 (200 mm). B) In vivo labelling of L. welshimeri
SLCC 5334 and L. monocytogenes EGD-e with Ru1 (200 mm). C) In vivo labelling of S. aureus NCTC 8325 with Ru1–
Ru4 (200 mm). For abbreviations please refer to Table S2.
residues and is known to react strongly with Michael-acceptor-
based probes.[14] Furthermore, heat denaturation of this protein
does not negate its reactivity towards Michael acceptors, indi-
cating a predominantly unspecific labelling event. Further as-
sessment of Figure 1A and B reveals several interesting bacte-
rial targets for probe labelling. MsrA (methionine sulfoxide re-
ductase), for example, is absent from S. aureus NCTC 8325 and
L. welshimeri SLCC 5334, yet is strongly labelled in these organ-
isms’ more virulent counterparts S. aureus Mu50 and L. mono-
cytogenes EGD-e (Figure 1A and B). Interestingly, MsrA has re-
peatedly been identified as a virulence factor in pathogenic or-
ganisms,[15] whereas pathogens deficient in MsrA have been re-
ported to have reduced ability to adhere with eukaryotic
cells[16] and to survive inside hosts.[17] The key global regulator
MgrA has been reported to control the expression of
ꢀ350 genes in S. aureus[18] and is labelled by Ru1in both strains
of S. aureus (Figure 1A). As with MsrA, MgrA is also implicated
in virulence regulation in pathogenic bacteria.[19][20] The MsrA
and MgrA hits were verified by recombinant expression, and
subsequent labelling of these proteins with Ru1 indicated
a specific binding event (Figure S4). In addition to these viru-
lence regulators, several essential bacterial enzymes were also
identified. MurA (UDP-N-acetylglucosamine-1-carboxyvinyl-
transferase) catalyses the first committed step in cell wall bio-
synthesis. Low C+G Gram-positive bacteria possess two
copies of MurA: MurA1 and MurA2.[21] Gene deletion experi-
ments demonstrate that removal of either gene results in
a viable organism, but a double murA1/murA2 knockout is
lethal.[21–22] We had previously demonstrated potent inhibition
of MurA1 by the Michael-acceptor-containing compound
MurA1 activity, is a possible contributor to the antibacterial ac-
tivities of the rugulactone compounds.
Finally, we turned our attention to the strongly labelled
band identified as ThiD. ThiD (HMPP kinase) is an essential
dual-function enzyme in the thiamine biosynthesis pathway
that catalyses the phosphorylation reactions of HMP and
HMPP (Scheme 4) to form HMPPP, which is then used in a cou-
pling reaction to form thiamine phosphate.[23]
Thiamine (vitamin B1) is present in all living organisms as an
essential cofactor of several key enzymes. Humans and other
mammals depend solely on uptake of thiamine from their diet,
whereas plants, bacteria and the protozoan parasite Plasmodi-
um obtain thiamine by a combination of de novo biosynthesis
and various salvage pathways.[23] As shown in Figure 1A and B,
ThiD is labelled strongly by Ru1 in L. monocytogenes and
S. aureus. Furthermore, an examination of Figure 1C reveals
that in susceptible bacteria (S. aureus NCTC 8325 shown), only
the biologically active probes Ru1 and Ru2 label ThiD. These
data indicate that the inhibition of ThiD might play a role in
the antibacterial activity of rugulactone and its ABPP probes.
To date, there are no reported small-molecule inhibitors of
ThiD, but inhibition of thiamine biosynthesis is a potential
source of antibacterial targets,[24] particularly in organisms such
as Mycobacterium tuberculosis, which lack salvage pathways as
well as transporters of thiamine, eliminating the possibility of
environmental uptake.[25] The significance of ThiD in L. monocy-
togenes metabolism has been established: deletion mutants of
thiD in this organism displayed a 3.3-fold reduction in epithe-
lial intracellular growth—presumably a consequence of a thia-
mine-depleted environment.[26] The inhibition of recombinant
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Antibacterial Effects of Rugulactone and its Analogues
Table 2. IC50 values [mm] for inhibitors Ru0–Ru4.
IC50 [mm]
S. aureus Mu50 Mur A2
Compound
S. aureus Mu50 ThiD
Ru0
Ru1
Ru2
Ru3
Ru4
~400
90
150
25
14
32
n.a.
n.a.
n.a.
n.a.
n.a.: not applicable.
purified ThiD from S. aureus Mu50 by rugulactone and its ABPP
probes was assayed with the aid of a pyruvate kinase/lactate
dehydrogenase coupled enzyme system.[27] The dose–response
curves and IC50 values for this assay are shown in Figure 2 and
Table 2.
Neither Ru3 nor Ru4 showed any HMPP kinase inhibition at
any of the concentrations tested. Interestingly, though, potent
inhibition, ranging from 14 to 32 mm, was observed for Ru0–
Ru2; this correlates well with the observed labelling pattern.
These compounds therefore represent the first known small-
molecule inhibitors of ThiD. In order to assess the contribution
of ThiD inhibition by Ru1 to antibacterial activity, we compared
MIC values of L. monocytogenes EGD-e in chemically defined
media (CDM) with and without thiamine. L. monocytogenes is
known to be capable of thiamine uptake, so a drop in MIC for
Ru1 should be observed if no environmental thiamine can be
salvaged. To our satisfaction, the MIC of Ru1 for L. monocyto-
genes EGD-e grown in CDM without thiamine was approxi-
mately four times lower than that of L. monocytogenes grown
in thiamine-containing CDM (25 and 100 mm, respectively),
demonstrating the significance of ThiD inhibition for antibacte-
rial activity (Figure S7). In biological systems, Michael acceptors
Figure 2. Dose–response curves for rugulactone probes against MurA2 and
~
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*
ThiD, together with IC50 values for each inhibitor. : Ru1, : Ru0, : Ru2, N/
A: not applicable.
Scheme 4. Prokaryotic thiamine biosynthetic pathway. Inhibition of the essential enzyme ThiD by Ru1 would block de novo thiamine biosynthesis.
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S. A. Sieber et al.
Figure 3. Identification of the binding site or Ru1 by mass spectrometry. A) MS fragmentation pattern for ThiD chymotryptic peptide VVDPVMVC*KGE-
DEVLNPGNTEAMI. B) Sequence alignment of ThiD from various organisms.
Conclusions
overwhelmingly react with the thiol moieties in cysteine resi-
dues.[28] It is therefore very likely that rugulactone and its
probes modify ThiD at a cysteine residue. A crystal structure of
ThiD from Salmonella typhimurium reveals an active site cys-
teine (Cys213) that is highly conserved amongst HMPP kinas-
es.[29] However, labelling of recombinant ThiD with Ru1 fol-
lowed by digestion and examination of the (chymo)tryptic
peptides by LC-MS/MS reveals that Cys110 is the site of modifi-
cation of ThiD (Figure 3A). These data suggest allosteric inhibi-
tion of ThiD by Ru1. Interestingly, BLAST comparisons of
S. aureus (Mu50 and NCTC 8325), L. monocytogenes EGD-e,
P. aeruginosa PAO-1, P. putida KT2440 and Escherichia coli K12
ThiD show that in the susceptible bacteria, Cys110 is con-
served, whereas in P. aeruginosa and other non-susceptible or-
ganisms it is not (Figure 3B). Unfortunately, Cys110 is not con-
served in M. tuberculosis, but further studies are needed to
confirm a lack of ThiD inhibition in this organism.
In conclusion, we report syntheses of the natural product ru-
gulactone and of its related ABPP probes. Probes Ru1 and Ru2
display substantially more antibacterial activity than the natural
product, despite relatively small perturbations in structure. The
observed bioactivity and labelling patterns of Ru1–Ru4 suggest
that the chemical reactivity and inhibitory power of rugulac-
tone is largely contributed by the a,b-unsaturated ketone Mi-
chael acceptor systems present in Ru1 and Ru2. We have de-
scribed probes Ru0–Ru2 as MurA2 inhibitors, as well as the
first known examples of small-molecule inhibitors of ThiD. Fur-
thermore, we have demonstrated that the inhibition of this
enzyme contributes to its antibacterial activity against L. mono-
cytogenes in a chemically defined medium; this is consistent
with the growth behaviour of DthiD L. monocytogenes in epi-
thelial cells.[26] Rugulactone therefore represents an important
core scaffold that exerts its antibacterial activity through multi-
ple target interactions. With regard to antibacterial therapy,
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Antibacterial Effects of Rugulactone and its Analogues
ed, and click chemistry was carried out on the cytosolic fraction as
described above with the rhodamine-biotin-azide Rh-biotin-N3
[amounts used: Rh-biotin-N3 (10 mm, 4 mL), TCEP (60 mm, 4 mL),
TBTA (1.6 mm, 12 mL), CuSO4 (50 mm, 4 mL)]. Reactions for enrich-
ment were carried out together with a control lacking the probe
to compare the results for the biotin-avidin-enriched samples with
the background of unspecific protein binding on avidin-agarose
beads. After click chemistry, proteins were precipitated by use of
an equal volume of precooled acetone. Samples were stored on
ice for 60 min and centrifuged at 16200ꢁg for 30 min at 48C. The
supernatant was discarded, and the pellet was washed two times
with cold methanol (08C, 500 mL), with resuspension by sonication
each time (2ꢁ10 s, 40% power) and collection of the pellet each
time by centrifugation (16200ꢁg, 30 min, 48C). Subsequently, the
pellet was dissolved in SDS in PBS (0.2%, 1 mL) by sonication (3ꢁ
10 s, 40% power) and incubated with gentle mixing with avidin-
agarose beads (Sigma–Aldrich, 50 mL) for 1 h at room temperature.
The beads were washed three times with SDS in PBS (0.2%, 1 mL),
twice with urea (6m, 1 mL), and three times with PBS (1 mL). Beads
were collected after each wash by centrifugation at 2500 rpm for
1 min. Aliquots of 2ꢁ BME buffer (50 mL) were added and the pro-
teins were released for preparative SDS-PAGE by 6 min incubation
at 958C. The whole aliquot was then applied to a preparative SDS-
PAGE gel. Gel bands were isolated, washed and tryptically digested
as described previously.[30]
this mode of action is preferred because resistance develop-
ment against several targets is more difficult to achieve.
Experimental Section
Bacterial strains: Staphylococcus aureus strains NCTC 8325 (Insti-
tute Pasteur, France), Mu50/ATCC 700699 (Institute Pasteur,
France), were maintained in brain–heart broth (BHB) medium at
378C. Listeria monocytogenes strains EGD-e (Institute Pasteur,
France) and the nonpathogenic strain Listeria welshimeri SLCC 5334
serovar 6b (DSMZ, Germany) were maintained in BHB medium,
whereas Pseudomonas aeruginosa PAO1 (Institute Pasteur, France)
and Pseudomonas putida KT2440 (ATCC, USA) were maintained in
lysogeny broth (LB) medium. All strains were grown at 378C.
MIC measurements: Overnight cultures of bacteria were diluted in
fresh BHB or LB medium to give OD600 =0.01, and aliquots (99 mL)
were incubated in Nunclon round-bottomed 96-well plates with
the corresponding DMSO stocks of rugulactone and rugulactone
probes (1 mL) in varying concentrations. The samples were incubat-
ed overnight at 378C and the optical densities were obtained by
visual inspection for MIC calculation. All experiments were con-
ducted at least in triplicate, and DMSO served as control.
In vivo experiments
Analytical labelling: S. aureus NCTC 8325, S. aureus Mu50, L. mono-
cytogenes EGD-e and L. welshimeri SLCC 5334 were grown in BHB
(5 mL). All bacteria were harvested by centrifugation 1 h after
reaching stationary phase. After washing with 1ꢁPBS (pH 7.5), the
cells were resuspended in PBS (100 mL). The bacteria were then in-
cubated at room temperature for 60 min with varying concentra-
tions of probe in DMSO, with the final DMSO concentration not ex-
ceeding 2%. Subsequently, the cells were washed twice with PBS
to remove excess probe, resuspended in PBS (100 mL) and lysed by
sonication with a Bandelin Sonopuls instrument (10ꢁ30 s, 80%
power) and ice cooling. The proteomes were then separated into
cytosolic and membrane fractions by centrifugation (16200ꢁg,
30 min, 48C). The membrane fraction was washed with PBS (2ꢁ
500 mL), with collection by centrifugation (16200ꢁg, 30 min, 48C)
after each wash, then resuspended in PBS (100 mL). Click chemistry
was then carried out on both fractions with rhodamine azide RhN3.
RhN3 (10 mm, 2 mL), tris(2-carboxyethyl)phosphine (TCEP, 60 mm,
2 mL) and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA,
1.6 mm, 6 mL) were added in succession to the lysate solution. The
samples were gently vortexed, and the cycloaddition was initiated
by the addition of CuSO4 (50 mm, 2 mL). The cycloaddition reaction
was allowed to proceed for 1 h at room temperature, followed by
the addition of 100 mL 2ꢁBME buffer [TRIS·HCl (496 mg), glycerine
(5 mL), Bromophenol blue (1.25 mg), SDS (1 g), b-mercaptoethanol
(2.5 mL) in H2O (50 mL), pH 8.3]. This final solution (50 mL) was ap-
plied to an SDS-PAGE gel, and the developed gel was visualized by
in-gel fluorescence scanning with use of a Fujifilm Las-4000 lumi-
nescent image analyzer, a Fujinon VRF43LMD3 lens and a 575DF20
filter. Coomassie staining was then preformed on the gels to
gauge levels of protein expression.
Acknowledgements
We thank Mona Wolf for excellent scientific support and Max
Pitcheider for the E. coli clones. We also thank Tadhg Begley for
the kind gift of HMP. S.A.S. was supported by the Deutsche For-
schungsgemeinschaft, SFB749, FOR1406, an ERC starting grant
and the Center for Integrated Protein Science, Munich (CIPSM).
M.B.N. thanks the Alexander von Humboldt foundation for finan-
cial support.
Keywords: antibiotics · chemical biology · natural products ·
proteomics · rugulactone
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Received: April 20, 2012
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FULL PAPERS
Target acquisition: Rugulactone,
M. B. Nodwell, H. Menz, S. F. Kirsch,
S. A. Sieber*
a plant natural product isolated in 2009,
has been reported to display interesting
biological properties, but its protein tar-
gets in biological systems have not
been examined. We have applied activi-
ty-based protein profiling to examine
the targets of rugulactone in bacteria
and have found that inhibition of thia-
mine biosynthesis contributes to its an-
tibacterial activity.
&& – &&
Rugulactone and its Analogues Exert
Antibacterial Effects through Multiple
Mechanisms Including Inhibition of
Thiamine Biosynthesis
ChemBioChem 0000, 00, 1 – 9
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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These are not the final page numbers!