β-lactones as privileged structures for the active-site labeling of versatile bacterial enzyme classes

Article abstract of DOI:10.1002/anie.200705768

(Chemical Equation Presented) A chemical proteomic strategy has been applied directly to bacterial proteomes, and b-lactones have been identified as important natural product derivatives with a high affinity to various enzyme classes (see picture). This a

Full text of DOI:10.1002/anie.200705768

Communications
DOI: 10.1002/anie.200705768
Medicinal Chemistry
b-Lactones as Privileged Structures for the Active-Site Labeling of
Versatile Bacterial Enzyme Classes**
Thomas Böttcher and Stephan A. Sieber*
Evolution of multiresistant bacterial strains has meant that
infectious diseases once again pose a major threat to public
health. Since many antibiotics still target only a limited set of
cellular functions, it is a desirable goal to expand the number
and breadth of therapeutic targets as well as to gain a deeper
understanding of the molecular mechanisms responsible for
pathogenesis.[1] To approach this goal, a chemical proteomic
strategy (activity-based protein profiling, ABPP), developed
by Cravatt and co-workers,[2] that uses active-site-directed
probes was directly applied to bacterial proteomes. ABPP
probes consist of at least two general elements: 1) a reactive
group for binding and covalently modifying the active site of a
certain enzyme class, and 2) a reporter tag for the detection,
enrichment, and identification of probe-labeled proteins.[3]
Many ABPP probes have so far utilized electrophilic
reactive groups,[2] including fluorophosphonates,[4] sulfonate
esters,[5] and epoxides,[6] which exhibit preferences for nucle-
ophilic groups in the active site of several distinct enzyme
classes. For bacterial ABPP, we selected a new reactive group
based on the b-lactone (2-oxetanone) structure derived from
natural products. b-Lactones represent promising biologically
active privileged structures that can react covalently with the
active sites of certain enzymes.[7] Although b-lactones such as
obafluorin[8] and hymeglusin[9] have been proven to exhibit
antibiotic activity, their molecular targets remain largely
unknown (see Figure S1 in the Supporting Information).
Here, we apply ABPP with b-lactones to prokaryotes to
identify dedicated target enzymes, with special emphasis on
those which are crucial for bacterial viability and virulence.
To maximize the number of labeled enzymes we synthe-
sized a small library of probes with an alkyne tag at the C-4-
position and with diversity introduced at the C-3-position of
the 2-oxetanone ring (Figure 1A).[10] The modification of the
alkyne tag through a 1,3-dipolar Huisgen cycloaddition (click
chemistry) allows a fluorophor reporter group to be appended
for visualization of the target enzyme by SDS-gel electro-
phoresis after proteome labeling,[11] as reported by Cravatt
and co-workers (Figure 1B).
Most b-lactones derived from natural products are
biologically active in the trans configuration,[8,9,12] but do not
show a clear preference for the absolute configuration within
the trans stereochemistry.[13] Therefore, we focused our
attention on developing a synthetic strategy to yield trans-b-
lactones as racemic mixtures, which show similar labeling
profiles as the corresponding cis isomers (see Figure S2 in the
Supporting Information). Inspired by naturally occurring b-
lactones, the biomimetic library comprised 10 compounds
with aliphatic or aromatic substitutions of different length and
branching. To evaluate the selectivity for the target enzymes,
the library was screened against the proteomes of several
Gram-positive and Gram-negative bacteria—Pseudomonas
putida, Listeria welshimeri, Bacillus licheniformis, Bacillus
subtilis, and Escherichia coli—which are phylogenetically
related to pathogenic strains. Mouse liver cytosol was also
included in the study as an eukaryotic reference proteome.
Initial labeling experiments were carried out by adding
individual probes at a concentration of 50 mm to the pro-
teome; this concentration is sufficient to achieve full satu-
ration of most target enzymes. Interestingly, individual
members of the probe library showed highly distinct reactivity
profiles against the native proteomes investigated (see
Figure S3 in the Supporting Information), thus indicating
that the substitution at the C-3-position exerted a strong
influence over specific probe–protein interactions. As an
example, the labeling of L. welshimeri cytosolic and mem-
brane proteomes as well as of B. subtilis cytosolic proteome
with a subset of the most complementary probes is shown in
Figure 2A and B. Only one unspecific binding event was
observed in the heat-denatured control proteome of L. wel-
shimeri, which emphasizes the predominant preference of b-
lactones for native proteins (Figure 2C, and see Figure S4 in
the Supporting Information). Most of the targeted proteins
were of low abundance, as shown by direct comparison of the
relative intensities observed with coomassie staining and
fluorescence scanning (see Figure S5 in the Supporting
Information).
[*] T. Böttcher, Dr. S. A. Sieber
Center for integrated Protein Science Munich, CiPSM
Department of Chemistry and Biochemistry
Ludwig-Maximilians-Universität München
Butenandtstrasse 5–13, 81377 Munich (Germany)
Fax: (+49)89-2180-77756
E-mail: stephan.sieber@cup.uni-muenchen.de
[**] We thank Prof. Thomas Carell and his group for their generous
support and fruitful scientific discussions. We gratefully acknowl-
edge funding by the Emmy Noether Program of the DFG), the
SFB 749 (DFG grant), a stipend from the Römer Stiftung, the Fonds
der chemischen Industrie, and the Center for integrated Protein
Science Munich, CiPSM. T.B. acknowledges funding from the
Studienstiftung des deutschen Volkes. We thank Prof. Dr. Mohamed
Marahiel and Alan Tanovic for providing recombinant SrfAC
enzyme. We thank Kerstin Kurz for excellent technical assistance
and Maximilian Pitscheider for help with recombinant expression.
Supporting information for this article (details on the synthesis and
characterization of probes as well as proteome preparation and
labeling) is available on the WWW under http://www.angewand-
te.org or from the author.
Subsequent identification of the labeled target enzymes
by LC-MS analysis (see the Supporting Information) revealed
the labeling of about 20 different enzymes (Table 1). The MS
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Figure 2. Labeling profiles of the b-lactone library against bacterial
proteomes. A) Fluorescent gel of L. welshimeri cytosol and membrane
proteomes after treatment with selected library members. The enzyme
identities are assigned to the corresponding gel band (for a list of
abbreviations, see Table 1). B) Fluorescent gel of B. subtilis cytosol
proteome showing the identified enzyme targets. C) All reactions were
carried out with a heat control (DT) to identify unspecific labeling. An
example is shown for G2 in the membrane proteome of L. welshimeri
(boxed). D) Examples of recombinantly expressed enzymes (À: before
induction, +: after induction, P: native proteome, DT: after induction/
heat control, C: after induction/no probe).
ring. Indeed, preincubation of several proteins with phenyl-
methylsulfonyl fluoride (PMSF) and cerulenin, which are
known active-site inhibitors of serine proteases[14] and b-
ketoacyl acyl carrier protein synthases (KAS),[15] respectively,
prevented subsequent labeling with the probe (see Figure S7
in the Supporting Information). In addition, substrate inhib-
ition assays with S-formylglutathione hydrolase (SFGH)
demonstrated probe-mediated inhibition of enzyme activity
(IC50 = 5 mm). The inhibition of enzyme activity and the broad
coverage of mechanistically distinct enzyme classes empha-
sizes the unprecedented utility of b-lactones as new proteomic
tools for ABPP.
Interestingly, a large number of the identified enzymes are
involved in important cellular functions, such as primary
(KAS I and II) and secondary metabolism (surfactin A
synthetase C, SurfAC), nucleotide synthesis (CTP synthase),
detoxification (SFGH), antibiotic resistance (penicillin-bind-
ing protein (PBP) 4*) as well as in virulence (ATP-dependent
caseinolytic protease, ClpP). With the exception of SFGH, no
homologue of these enzymes was detected in the mouse liver
proteome.
Figure 1. b-Lactones as privileged structures for bacterial ABPP.
A) Synthetic route and biomimetic structures of the b-lactone library.
LDA=lithium diisopropylamide B) Proteomes are first treated with the
b-lactone library and subsequently appended with a fluorescent dye by
click chemistry. Labeled proteomes were separated on sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized
by fluorescence scanning.
results were confirmed by recombinant expression of all
major hits and subsequent labeling by the corresponding
probes (Figure 2D, and see Figure S6 in the Supporting
Information). The identified enzymes belong to four major
families comprising ligases, oxidoreductases, hydrolases, and
transferases (Table 2). All of these families require a nucle-
ophilic residue in their active site for catalysis (Cys or Ser),
with this residue likely attacking the electrophilic b-lactone
Several targets are of special medicinal interest: KAS II,
an essential component of the fatty acid biosynthetic path-
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Table 1: Enzymes identified (clustered by function).
ing S. aureus, L. monocytogenes,
and P. aeruginosa.[19–21]
Function Enzyme (abbreviation)
Proteomes identified
To estimate the strength of
primary metabolism
probe–protein interactions we com-
pared the reactivity profiles of
selected b-lactones across a broad
concentration range from 10 mm to
5 nm. Several target enzymes dis-
played a very robust labeling behav-
ior. For example, the aliphatic b-
lactone G2 is a very sensitive probe
for lipase (Lip, down to 20 nm probe
concentration), but does not exhibit
a strong interaction with lipase/
acylhydrolase (LipAc) in L. welshi-
meri (Figure 3A). In contrast, the
aromatic b-lactone P1 shows the
opposite selectivity (down to
160 nm for LipAc), thus illustrating
the sensitivity and target enzyme
selectivity of the b-lactones G2 and
P1. Interestingly, ADB shows a
strong preference only for aliphatic
b-lactones (D3, G2) at 100 nm con-
centration in E. coli cytosol (Fig-
ure 3B). Similarly, several other
enzymes could be clustered on the
basis of their preferred binding
partners (Figure 3C). This informa-
tion is useful to gain insight into the
native substrate preferences of all
labeled enzymes, especially for
those which have been less inves-
tigated and are uncharacterized. In
this context, it is interesting to note
that unsubstituted b-lactones (A1)
seem to be poor at labeling
enzymes. Steric constraints in
probes such as O1 and L1 also
decreased the number of specific
labeling events. Therefore, b-lac-
tones with aliphatic and aromatic
acetylCoA hydrolase (ACoAH)
aldehyde dehydrogenase B (ADB)
formate-C-acetyltransferase (FCA)
lipase (Lip)
lipase/acylhydrolase (LipAc)
lysophospholipase (LPL)
b-ketoacyl acyl carrier protein synthase I
(KAS I)
b-ketoacyl acyl carrier protein synthase II
(KAS II)
b-ketoacyl acyl carrier protein synthase III
(KAS III)
P. putida
E. coli
B. licheniformis
L. welshimeri
L. welshimeri
Mus musculus
E. coli, P. putida
B. subtilis, B. licheniformis,
E. coli, L. welshimeri
B. licheniformis
b-ketothiolase (BKT)
P. putida
B. subtilis
Secondary metabolism
surfactin A synthetase subunit C(SrfAC)
nucleotide synthesis
CTP synthase (CTPS)
B. subtilis, B. licheniformis,
E. coli, L. welshimeri
L. welshimeri
thymidylate synthase (ThyS)
resistance/cell-wall biosynthesis
penicillin-binding protein 4* (PBP4*)
B. subtilis
virulence associated
ATP-dependent Clp protease (ClpP)
proline iminopeptidase (PIP)
P. putida, L. welshimeri
P. putida
detoxification
S-formylglutathione hydrolase (SFGH)
dienelactone hydrolase (DLH)
B. subtilis, E. coli, L. welshimeri, Mus mus-
culus
P. putida
unknown function
AB hydrolase (ABH)
para-nitrobenzyl esterase (PNBE)
peptidase S66 (Pep66)
putative ATP dependent protease (PADP)
putative esterase (PutE)
Mus musculus
B. subtilis
B. subtilis
P. putida
B. licheniformis
way,[16] is highly conserved among key pathogens,[17] and was
labeled by the aliphatic b-lactone probes D3 and G2
(Figure 2A and B). In addition, PBP4*, an enzyme which is
reported to exhibit b-lactamase
moieties seem to be the most potent probes, which might
further help to guide the design of specific inhibitors in the
future.
activity,[18] was labeled in B. subtilis
(Figure 2B). Although we used
nonpathogenic bacterial proteomes
for our screens, two virulence-asso-
ciated enzymes, ClpP and proline
iminopeptidase (PIP), which play
crucial roles in many pathogenic
strains were detected in L. welshi-
meri and P. putida. In particular,
ClpP has attracted much attention
because of its fundamental role for
stress tolerance and virulence in
many pathogenic bacteria, includ-
Table 2: Enzyme classes labeled by b-lactone probes.
Enzyme class
ECActive site
Example
ligase
oxidoreductases
hydrolases
EC6.3.4.
EC1.2.1.
EC3.1.2.
EC3.1.1.
EC3.4.11.
EC3.4.21.
EC2.3.1.
EC 2.3.1.
EC2.3.1.
EC2.1.1.
Cys, His, Glu
Cys, Glu
CTP synthase
aldehyde deyhdrogenase B
S-formylglutathione hydrolase
para-nitrobenzyl esterase
proline iminopeptidase
ATP-dependent Clp protease
b-Ketoacyl-ACP synthase II
formate-C-acetyltransferase
b-Ketothiolase
Ser, Asp, His
Ser, Glu, His
Ser, Asp, His
Ser, His
Cys, His, Asn
Gly, Cys, Cys
Cys, His, Cys
Cys
transferases
thymidylate synthase
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molecules. In view of the labeling of several important
bacterial enzymes, this approach might represent a promising
starting point for the identification of novel antibacterial
targets together with their corresponding inhibitors.
Received: December 17, 2007
Published online: March 28, 2008
Keywords: biomimetic synthesis · enzymes · lactones ·
.
medicinal chemistry · proteomics
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The active-site labeling of essential enzymes such as
KAS II raised the question of a possible antibacterial effect.
Several probes were tested for growth inhibition, but no
antibiotic activity could be observed. One possible reason
could be a limited cellular uptake of probes by intact bacteria.
To clarify this, we incubated L. welshimeri with various
concentrations of probes in vivo. These experiments revealed
that KAS II, the most important target for viability, was only
weakly labeled at high concentrations (100 mm) and, there-
fore, probably did not reach saturation for full inhibition. In
contrast, strong labeling occurred for the virulence-associated
enzyme ClpP with probe concentrations as low as 5 mm
(Figure 3D). This specific and sensitive in vivo labeling of a
common virulence factor, which is not essential for viability
but indispensable for bacterial pathogenesis, shows that
probes are applicable to in vivo studies and represents an
attractive strategy for the inhibition of new target enzymes
from bacteria in the future. Inhibitors of virulence-associated
enzymes display many advantages over conventional anti-
biotics, such as preserving the host endogenous microbiome
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promising privileged structures for the design of novel ABPP
probes which can be utilized to screen and compare enzyme
activity of different bacterial proteomes. We were able to
identify a variety of mechanistically distinct target enzymes
that show sensitive labeling as well as inhibition by the probe
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