The mechanism of caseinolytic protease (ClpP) inhibition

Article abstract of DOI:10.1002/anie.201204690

Catch me if you can: The ClpP protease mediates protein homeostasis and can be efficiently inhibited by β-lactones. A combination of molecular docking, mutagenesis, activity-based protein profiling, and kinetics studies now reveals the mechanism of ClpP inhibition. A hydrophobic pocket next to the active site allows binding of long aliphatic and aromatic residues. The preferred stereoisomer binds into the oxyanion hole. Copyright

Full text of DOI:10.1002/anie.201204690

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DOI: 10.1002/anie.201204690
Enzyme Inhibitors
The Mechanism of Caseinolytic Protease (ClpP) Inhibition**
Malte Gersch, Felix Gut, Vadim S. Korotkov, Johannes Lehmann, Thomas Bçttcher,
Marion Rusch, Christian Hedberg, Herbert Waldmann, Gerhard Klebe, and Stephan A. Sieber*
Maintaining homeostasis at the protein level is an important
prerequisite for cellular viability for which prokaryotes
exhibit several proteolytic machineries, including ClpXP.[1]
In 2008, we reported the first small-molecule inhibitor for the
proteolytic subunit ClpP and demonstrated that the inhibition
of the enzyme in living bacteria significantly attenuates their
capability to produce virulence factors, such as life-threat-
ening toxins.[2] Although ClpP has been extensively studied by
biochemical and structural methods,[3] the mechanism of
small-molecule inhibition of this enzyme is currently poorly
understood. Because chemical inhibition may lead to a novel
antibacterial therapy, it is important to systematically analyze
the binding site, the mechanism of inhibition, the stereogenic
preference of the enzyme for inhibitors, the chemical space of
putative inhibitors, and how other members of the ClpP
family can be inhibited. One major step towards these aims
was accomplished by the recently solved crystal structure of
homotetradecameric ClpP from Staphylococcus aureus
(SaClpP) in its active conformation.[4] With the structural
data at hand, we herein report an in-depth mechanistic
analysis of S. aureus ClpP inhibition by b-lactones. A screen of
a focused library of enantiopure b-lactones revealed the S,S-
stereopreference of the protease, which was rationalized by
molecular docking. Docking experiments also gave insight
into a hitherto unnoted deep hydrophobic pocket next to the
active site that accommodates b-lactone substituents in the a-
position to the carbonyl group. The binding hypothesis was
verified by binding studies with model compounds, detailed
kinetic analysis, and protein mutagenesis studies. Further-
more, the replacement of the b-lactone core by other scaffolds
resulted in the loss of inhibitory potency, thereby highlighting
the importance of a b-lactone moiety for mechanism-based
ClpP inhibition. Taken together, these results open intriguing
perspectives in the mechanistic understanding of ClpP
inhibition and provide direction for the design of potent and
pharmacologically optimized inhibitors.
We started by testing 22 enantiopure trans-substituted b-
lactones 1–22 for ClpP inhibition (Supporting Information,
Figure S1A).[5] These molecules share a high structural
similarity with our previous b-lactone candidates. They
feature a decyl chain as R1 substituent and structural
variations in chain lengths as well as in functional groups at
the R2 position (Figure 1A). For all of the compounds, both
trans-configured enantiomers (that is, R,R and S,S) were
tested for inhibition of recombinantly expressed SaClpP in an
assay monitoring the cleavage of a fluorogenic substrate.[4]
Almost all of the compounds inhibited SaClpP at 100 mm
concentration (100-fold excess over enzyme) after 15 min
incubation at 328C (Supporting Information, Figure S1A).
By lowering the inhibitor concentration to 10 mm, we were
able to differentiate the compounds tested. While most S,S-
configured lactones lead to inhibition below 10% residual
activity, R,R-configured lactones showed essentially no inhib-
ition (Figure 1B). Incubation of SaClpP with 1.3-fold molar
excess of the most potent compound, 2, led to modification of
all 14 subunits as revealed by intact-protein mass spectrom-
etry (Figure 1C).
To investigate if the potent in vitro inhibition correlates
with ClpP binding in living cells we applied the structurally
related alkynylated probe 23 with S,S-configuration for an
[*] M. Gersch, Dr. V. S. Korotkov,[+] J. Lehmann, Prof. Dr. S. A. Sieber
Center for Integrated Protein Science Munich (CIPSM)
Technische Universitꢀt Mꢁnchen, Department of Chemistry
Institute of Advanced Studies (IAS)
Dr. T. Bçttcher
Harvard Medical School, Department of Biological Chemistry and
Molecular Pharmacology
240 Longwood Ave., Boston, MA 02115 (USA)
[+] These authors contributed equally to this work.
Lichtenbergstrasse 4, 85747 Garching (Germany)
E-mail: stephan.sieber@tum.de
[**] We acknowledge funding from the Deutsche Forschungsgemein-
schaft, FOR1409, SFB749, SFB1035, CIPSM, and the European
Research Council (ERC Grant no. 268309 and ERC Starting Grant
no. 259024). We thank Martina Mꢁller, Matthias Stahl, Mona Wolff,
Jenny Sachweh, Daniela Bauer, and Jan Vomacka for help with
experiments, Arne Schrçder and Marco Balabajew for assistance
with docking experiments, and Matthew Nodwell for critical
evaluation of the manuscript. M.G. and F.G. wish to express their
thanks to the Heidelberg Life-Science Lab for providing a scientifi-
cally inspiring environment.
F. Gut,[+] Prof. Dr. G. Klebe
Institute of Pharmaceutical Chemistry, Philipps-University Marburg
Marbacher Weg 6, 35032 Marburg (Germany)
Dr. M. Rusch, Dr. C. Hedberg, Prof. Dr. H. Waldmann
Max Planck-Institut fꢁr Molekulare Physiologie
Abteilung Chemische Biologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
and
Technische Universitꢀt Dortmund, Fakultꢀt Chemie
Lehrbereich Chemische Biologie
Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
Supporting information for this article (detailed procedures for
chemical syntheses, biochemical methods, and docking experi-
ments) is available on the WWW under http://dx.doi.org/10.1002/
anie.201204690.
Dr. V. S. Korotkov,[+] Dr. T. Bçttcher, Prof. Dr. S. A. Sieber
Aviru Exist
Lichtenbergstrasse 4, 85747 Garching (Germany)
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formed with all known inhibitors
into the crystal structure of active
SaClpP (Figure 2A,B).[4] Remark-
ably, all well-ranked docking solu-
tions shared the placement of the
hydrophobic R1 chain in a deep
pocket of the protein, adjacent to
the active site (Figure 2B; Support-
ing Information, Figure S3A).
While the opening of the pocket is
narrow and corresponds to the S1
pocket of the protease,[9] the chan-
nel widens into an enclosed cavity,
the physiological role of which is
unclear. The occupation of this
channel along with the enclosed
cavity by
a ligand most likely
results in the replacement of water
molecules and reduces the solvent
accessible surface by about 165 ꢀ2,
of which 65% is apolar (Supporting
Information,
Table S1,
Fig-
ure S3B).[10] The binding of the
hydrophobic R1 ligand portion in
the pocket possibly provides the
driving force for the initial protein–
inhibitor association, prior to a fast
nucleophilic attack of Ser98 that is
due to spatial proximity to the b-
lactone core.[11]
To map the space available to
accommodate ligand side chains,
we performed covalent docking
using a virtual b-lactone library
Figure 1. A) General formula of the investigated b-lactones with S,S or R,R stereochemistry (1–11 and
12–22, respectively). R1 is n-C10H21; R2 is varied. (See the Supporting Information, Figure S1 for
a complete list of structures.) B) Residual activity of SaClpP (1 mm) after incubation with compounds
1–22 (10 mm) for 15 min at 328C. C) Intact-protein mass spectrometry, which reveals inhibition of all
14 SaClpP subunits by compound 2 (403.3 Da, 1.3 molar excess). D) Competitive labeling experiment.
S. aureus NCTC 8325 cells were preincubated with indicated compounds at 100 mm for 1 h at room
temperature. Labeling was performed with 50 mm of ClpP-specific probe 24 for 1 h. Following lysis,
rhodamine azide was appended to 24 by bioorthogonal click chemistry and the proteome was
separated by SDS-PAGE. See the Supporting Information, Figure S2 for a full image of the gel, and
the Supporting Information, Figure S4A for loading controls. E) Structures of compounds 2 and 3 as
well as 13 and 14 used in the competitive labeling experiment.
in situ labeling of S. aureus NCTC 8325 (Supporting Infor-
mation, Figure 2A).[5b,6] After incubation and cell lysis,
a fluorescent tag was attached by click chemistry[7] and the
proteome was analyzed by SDS-polyacrylamide gel electro-
phoresis. A fluorescent band was observed at the same height
as for the ClpP-specific probe 24, indicating that the
physiological target of the inhibitors tested is ClpP. A much
weaker band was observed for the cis-configured analogue of
probe 24,[2a] which is indicative of a strong preference for
trans-configured b-lactones (Supporting Information, Fig-
ure S2B). Pretreatment of living S. aureus cells with S,S-
configured lactones 2 and 3 followed by labeling with probe
24 lead to a decrease in fluorescence intensity of the ClpP
band, while the respective R,R-configured enantiomers 13
and 14 showed no effect (Figure 1D,E). This competitive
labeling experiment confirms the physiological relevance of
the in vitro data. Next, we utilized molecular docking studies
to unravel the binding mode and rationalize the observed
preferences for long chain aliphatic substituents and S,S-
configured lactones.
consisting of compounds with aliphatic chains as R1 substitu-
ents covering a chain length from one to twelve carbon atoms
(Figure 2C). Scoring values increased with the length of the
substituent with a maximum at eight atoms. Only side chains
with up to ten atoms could be accommodated in the pocket
(Figure 2C). Next, we sought to validate these results
experimentally by an inhibition assay. IC50 values of covalent
inhibitors highly depend on the incubation time and the
enzyme concentration used.[12] We therefore used kobs/[I]
values as a quantitative measure of potency.[13] We synthe-
sized compounds 25, 27–29 and developed an additional
fluorogenic substrate assay in which the increase in fluores-
cence during inhibition was directly monitored and from
which rate constants (kobs) were obtained. In agreement with
the scoring values, an increasing length of the substituent
from 3 (25) to 4 (26), 6 (27), 8 (28), and 9 (24) carbon atoms
led to an increase of kobs/[I] values (Figure 2C,D; Supporting
Information, Figure S5). In contrast, 29 with a dodecyl
substituent did not bind ClpP, as indicated by the inhibition
assay and intact-protein mass spectrometry.
b-Lactones[8] are known to react covalently with the
catalytic Ser98,[2b] which leads to ring opening and blockage of
the active site and furnishes a catalytically inactive b-
hydroxyacyl–enzyme complex. To determine the geometry
of the acyl–enzyme complex, a covalent docking was per-
To further validate the suggested binding mode, we
mutated several residues of the putative binding pocket and
looked for changes in inhibitor binding (for characterization
data on the mutants tested, see the Supporting Information,
Table S3). First, we enlarged the binding pocket to allow
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tyrosine did not bind compound 24 while
binding of smaller compound 26 was still
possible (Figure 2G). These data confirm
the excellent correlation between calculated
modeling predictions and experimental
results.
According to the suggested binding
mode the R1 side chain of b-lactones is
oriented into the hydrophobic pocket and
thus contributes significantly to the observed
binding affinity. To further validate the
importance of this substituent, we synthe-
sized analogous b-lactones with only one
substituent at either R1 or R2 position. In line
with the prediction, racemic lactone 30 with
a decyl group at the R1 position inhibited
SaClpP, while no inhibition could be
observed for racemic lactone 31 carrying
a decyl group at the R2 position (Figure 3A).
Longer incubation times up to 24 h and
higher excess of inhibitor (up to 1000-fold)
did not lead to binding of 31 as investigated
by mass spectrometry (see the Supporting
Information, Figure S6A for further details).
To translate our findings into the design
of ligands that fill up the hydrophobic
pocket, we applied a computational rescaf-
folding for R1 to identify putative substitu-
ents. We repeatedly found that aromatic
rings were placed into the pocket; we there-
fore synthesized 32 with a phenylethyl group
at the R1 position. In line with the rescaf-
folding, 32 turned out to be a potent SaClpP
inhibitor (Figure 3B). As expected, 33 with
the phenylethyl group at the R2 position did
not show inhibition.
In search of the structural basis of the
observed stereochemical preference of the
ClpP protease, we performed a non-covalent
docking mimicking the binding situation
before formation of the acyl–enzyme com-
plex (Supporting Information, Figure S6B).
All of the compounds could be docked with
the lactone moiety being placed near the
catalytic Ser98. We then compared the bind-
ing modes of representative enantiomeric
pairs of b-lactones (Figure 3F; Supporting
Information, Figure S7). We noted that the
S,S-isomers show better scoring values com-
pared to the respective R,R-isomers in most
cases (Supporting Information, Table S2).
Moreover, the carbonyl groups of S,S-con-
figured lactones were positioned towards the
oxyanion hole, while carbonyl groups of
Figure 2.
A) Molecular docking of 2 (light blue) covalently bound to the catalytic serine 98 (red) in
tetradecameric SaClpP (left image). Close-up view on one monomer (right image).
B) Stereoview of the docking pose that reveals the presence of a hydrophobic pocket (black
mesh) next to the active site (red) that accommodates the R1 side chain (n-C10H21).
C) Docking scores for virtual lactones substituted with aliphatic R1 side chains with
different lengths. kobs/[I] values for racemic compounds 24–28 as measured with recombi-
nantly expressed SaClpP (Supporting Information, Figure S5). ? denotes that compound 29
does not inhibit SaClpP. D) Structures of compounds 24–29 exhibiting aliphatic R1 side
chains with different lengths. E) The L154Y mutant is labeled by compound 26, but not
compound 24, which is consistent with a smaller binding pocket. F,G) Wild-type enzyme
reacts with probe 24, but not with probe 29. Mutant proteins N151A and L154M with an
artificially enlarged binding pocket are labeled by both probes 24 and 29. (See the
Supporting Information, Figure S4B–D, for loading controls.)
accommodation of 29. Replacement of Asn154 at the rear end
of the binding pocket by alanine as well as substitution of
Leu154 by a linear and more flexible methionine enabled
binding of 29 (Figure 2E,F). Conversely, a ClpP enzyme in
which Leu154 is replaced by a sterically more demanding
R,R-configured lactones were pointing into the opposite
direction in order to enable placement of the R1 substituent
into the hydrophobic pocket (Figure 3F). This steric consid-
eration presumably leads to a less favored positioning of R,R-
configured lactones for nucleophilic attack by Ser98, in
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Figure 3. A–C) Compound inhibition data for b-lactones 30–35 emphasizing the preference of SaClpP for R1-substituted b-lactones with S
configuration. D) Synthesis of enantiopure b-lactones 34 and 35. a) (CH2O)x, TiCl4, iPr2EtN, CH2Cl2, 50%; b) LiOH, H2O2, THF, H2O, 86%;
c) HBTU (O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate), iPr2EtN, CH2Cl2, 9%. E) kobs/[I] values for enantiomeric
compounds 2 and 13 (for structures see Figure 1E). F) Non-covalent docking of S,S-configured b-lactone 2, revealing binding into the active site
(red) with optimal geometry for attack of the active site serine (2.7 ꢂ). The carbonyl group of 2 (blue) is accommodated in the oxyanion hole built
up by amide backbone groups of Glu69 and Met99. Enantiomeric lactone 13 (green) points into opposite direction, thus likely explaining its lower
binding kinetics (see Figure 3E).
agreement with the predicted docking poses. The higher
reactivity of S,S-lactones as represented by their approxi-
mately 25-fold higher kobs/[I] value (Figure 3E) is most likely
a consequence of the better suited stereochemistry to address
the oxyanion hole. To test if this also applies to monosub-
stituted lactones, we synthesized enantiopure versions of 32
by a chiral-auxiliary-supported aldol reaction (Figure 3D).
As predicted by modeling, R-configured 34 did not inhibit
ClpP, while S-configured 35 led to concentration dependent
inhibition (Figure 3C) and covalent modification of the active
sites in all 14 subunits. Consistently, enantiopure S-configured
35 was approximately twice as active as racemic 32.
To date, b-lactones are the only non-peptidic inhibitor
scaffold that exhibits specificity for ClpP, raising the question
if other core structures could substitute and retain activity. We
therefore utilized our knowledge on ligands 24 and 36 that
satisfy the binding into the hydrophobic pocket and appended
these substituents on other core scaffolds. We synthesized
derivatives 37–40 in which the electrophilic b-lactone was
replaced by an unreactive but geometrically closely related
oxetane moiety (Figure 4A,B). However, no blockage of
SaClpP function was observed up to 1 mm for racemic
mixtures of both trans- and cis-configured oxetanes. As b-
lactones are cyclic esters, we also synthesized acyclic esters 41
and 42. However, evaluation by peptidase assay did not reveal
them as ClpP inhibitors. Although several carbamates are
reported as potent inhibitors of serine hydrolases, carbamates
45–47 were not successful as inhibitors. Interestingly, even a b-
lactam unit in 43 and 44, the closest mimetic possible, did not
result in any inhibitory effect. These results emphasize the
unique reactivity and geometry of b-lactones for mechanism-
based inhibition of ClpP.
We were also curious as to whether other members of the
ClpP family of proteins can be inhibited. We cloned,
expressed, and purified the ClpP proteins from Escherichia
coli (EcClpP) and Bacillus subtilis (BsClpP), which share
64% and 79% sequence identity and 76% and 85% sequence
similarity to SaClpP. In both enzymes, the binding pockets
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Figure 4. b-Lactones are privileged structures for ClpP inhibition. A) Core scaffolds investigated in this study. B) Residual activity of SaClpP (1 mm)
after incubation with compounds 37–47 (100 mm) for 15 min at 328C. C) Recombinant ClpP enzymes from different organisms were incubated
with indicated lactones (50 mm) for 1 h followed by addition of a rhodamine dye by click chemistry. (See the Supporting Information, Figure S4E
for loading controls.) D) Structures of lactones 48–52. E) Compound 24 inhibits SaClpP, EcClpP, and BsClpP peptidase activity while 29 does not,
thereby indicating similarly shaped binding pockets for all three organisms.
have a similar geometry to SaClpP and are predominantly
hydrophobic.[3b,g] Upon labeling with a diverse set of b-
lactones[2] (24–29, 48–52), we received similar patterns with
ClpP enzymes from all three organisms (Figure 4C,D) with
BsClpP tolerating also more bulky R1 substituents. Conse-
quently, treatment of EcClpP and BsClpP with 24 but not with
the dodecyl compound 29 corresponded to reduction of
enzyme activity, as revealed by fluorogenic substrate assays.
The existence of the hydrophobic binding pocket may not
only have significant impact on the design of potent inhibitors
and corresponding anti-virulence pharmaceuticals but also
contribute to our understanding of the natural cleavage site
specificity of ClpP.
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Keywords: ClpP · enzyme inhibitors · lactones ·
molecular docking · structure–activity relationship
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