Phenyl Esters Are Potent Inhibitors of Caseinolytic Protease P and Reveal a Stereogenic Switch for Deoligomerization

Article abstract of DOI:10.1021/jacs.5b03084

Caseinolytic protease P (ClpP) represents a central bacterial degradation machinery that is involved in cell homeostasis and pathogenicity. The functional role of ClpP has been studied by genetic knockouts and through the use of beta-lactones, which remain the only specific inhibitors of ClpP discovered to date. Beta-lactones have served as chemical tools to manipulate ClpP in several organisms; however, their potency, selectivity and stability is limited. Despite detailed structural insights into the composition and conformational flexibility of the ClpP active site, no rational efforts to design specific non-beta-lactone inhibitors have been reported to date. In this work, an unbiased screen of more than 137000 compounds was used to identify five phenyl ester compounds as highly potent ClpP inhibitors that were selective for bacterial, but not human ClpP. The potency of phenyl esters largely exceeded that of beta-lactones in ClpP peptidase and protease inhibition assays and displayed unique target selectivity in living S. aureus cells. Analytical studies revealed that while phenyl esters are cleaved like native peptide substrates, they remain covalently trapped as acyl-enzyme intermediates in the active site. The synthesis of 36 derivatives and subsequent structure-activity relationship (SAR) studies provided insights into conserved structural elements that are important for inhibition potency and acylation reactivity. Moreover, the stereochemistry of a methyl-substituent at the alpha position to the ester, resembling amino acid side chains in peptide substrates, impacted ClpP complex stability, causing either dissociation into heptamers or retention of the tetradecameric state. Mechanistic insights into this intriguing stereo switch and the phenyl ester binding mode were obtained by molecular docking experiments.

Full text of DOI:10.1021/jacs.5b03084

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Article
Phenyl esters are potent inhibitors of caseinolytic protease
P and reveal a stereogenic switch for de-oligomerization
Mathias W. Hackl, Markus Lakemeyer, Maria Dahmen, Manuel Glaser, Axel Pahl, Katrin Lorenz-Baath,
Thomas Menzel, Sonja Sievers, Thomas Boettcher, Iris Antes, Herbert Waldmann, and Stephan Axel Sieber
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b03084 • Publication Date (Web): 17 Jun 2015
Downloaded from http://pubs.acs.org on June 22, 2015
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Phenyl esters are potent inhibitors of caseinolytic protease P and reveal a stereogenic switch
for de-oligomerization
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Mathias W. Hackl1,6, Markus Lakemeyer1,6, Maria Dahmen1, Manuel Glaser2, Axel Pahl1,
Katrin Lorenz-Baath1, Thomas Menzel1, Sonja Sievers3, Thomas Böttcher5, Iris Antes2,
Herbert Waldmann3,4, Stephan A. Sieber*1
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1
Center for Integrated Protein Science at the Department of Chemistry, Technische Universität München,
Lichtenbergstrasse 4, Garching, Dꢀ85747, Germany.
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Center for Integrated Protein Science at the Department of Life Sciences, Technische Universität
München, EmilꢀErlenmeyerꢀForum 8, Dꢀ85354 Freising, Germany
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MaxꢀPlanckꢀInstitut für Molekulare Physiologie, Abteilung Chemische Biologie, OttoꢀHahnꢀStrasse 11,
Dꢀ44227 Dortmund, Germany
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Technische Universität Dortmund, Fakultät für Chemie und Chemische Biologie, OttoꢀHahnꢀStrasse 6,
Dꢀ44221 Dortmund, Germany
5 Department of Chemistry, Universität Konstanz, Universitätsstrasse 10, Dꢀ78457 Konstanz, Germany
6 These authors contributed equally to this work.
Corresponding Author:
* stephan.sieber@tum.de
Abstract
Caseinolytic protease P (ClpP) represents a central bacterial degradation machinery that is
involved in cell homeostasis and pathogenicity. The functional role of ClpP has been studied by
genetic knockouts and through the use of betaꢀlactones, which remain the only specific inhibitors
of ClpP discovered to date. Betaꢀlactones have served as chemical tools to manipulate ClpP in
several organisms, however, their potency, selectivity and stability is limited. Despite detailed
structural insights into the composition and conformational flexibility of the ClpP active site, no
rational efforts to design specific nonꢀbetaꢀlactone inhibitors have been reported to date. In this
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work, an unbiased screen of more than 137,000 compounds was used to identify five phenyl ester
compounds as highly potent ClpP inhibitors that were selective for bacterial, but not human ClpP.
The potency of phenyl esters largely exceeded that of betaꢀlactones in ClpP peptidase and
protease inhibition assays and displayed unique target selectivity in living S. aureus cells.
Analytical studies revealed that while phenyl esters are cleaved like native peptide substrates,
they remain covalently trapped as acylꢀenzyme intermediates in the active site. The synthesis of
36 derivatives and subsequent structureꢀactivity relationship (SAR) studies provided insights into
conserved structural elements that are important for inhibition potency and acylation reactivity.
Moreover, the stereochemistry of a methylꢀsubstituent at the alpha position to the ester,
resembling amino acid side chains in peptide substrates, impacted ClpP complex stability,
causing either dissociation into heptamers or retention of the tetradecameric state. Mechanistic
insights into this intriguing stereo switch and the phenyl ester binding mode were obtained by
molecular docking experiments.
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Introduction
Proteolysis represents an essential physiological mechanism for diverse cellular functions
including postꢀtranslational processing, signaling and protein degradation.1,2 This important
process is catalyzed by a variety of proteases which together constitute one of the largest enzyme
classes in eukaryotic and prokaryotic cells. In prokaryotes, a variety of ATPꢀdependent proteases
are responsible for protein degradation, including FtsH3, Lon4 and HslUV.5ꢀ7 One additional
member of this group is the caseinolytic protease P (ClpP), a tetradecameric, barrelꢀshaped serine
protease that associates with AAA+ chaperones such as ClpA, ClpC and ClpX for protein
degradation.8ꢀ13 The chaperones recognize, unfold and direct SsrAꢀtagged protein substrates into
the proteolytic chamber of ClpP in an ATPꢀdependent manner.14 ClpP is essential for the
regulation of the cellular stress response, cell homeostasis and bacterial virulence.10,15,16
Virulence, i.e. the expression of bacterial toxins such as alphaꢀhemolysin, is regulated by the Agr
signaling network wherein ClpP is believed to be involved in the degradation of Rot, a repressor
of bacterial toxins.17 Genetic ClpP knockouts in Listeria monocytogenes and Staphylococcus
aureus revealed a reduction in virulence, resulting in attenuated infections in murine abscess
models.10,18 Similarly, the same phenotype was observed upon the chemical inhibition of ClpP
with longꢀchain aliphatic betaꢀlactones (e.g. D3, Figure 1B), the only specific inhibitors reported
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for ClpP to date.19ꢀ21 Crystal structures of S. aureus ClpP revealed a deep hydrophobic channel
next to the active site Ser98 which accommodates lactone side chains of maximum 8 atoms
length.22ꢀ24 Ser98 nucleophilically attacks the 4ꢀmembered lactone ring resulting in a covalent
acylꢀenzyme intermediate. Depending on the betaꢀlactone Cꢀ4 substitution, the occupancy of
ClpP varies from stoichiometric for aliphatic D3 to 50 % for aromatic E2.25 One reason for these
differing ratios is the transient stability of the ClpP tetradecamer, which dissociates into
heptamers via an unknown mechanism upon E2 binding.
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The interaction of subunits in the tetradecameric complex is facilitated by a dynamic Hꢀbond
network of adjacent helices (Eꢀhelix), betaꢀsheets (Glyꢀrich loop) and oligomerization sensors
(Asp170ꢀArg171).22 The Eꢀhelices exist in extended, compact and compressed conformations.
While an extended helix supports an aligned and active catalytic triad (Ser98, His123 and
Asp172), the compressed helix induces a rotation of the active site His123 and displaces the
catalytic residues.26 Moreover, in the compressed state all crucial interactions between the rings
are abrogated leading to the formation of inactive heptamers. From a pharmacological
perspective, inhibitorꢀinduced heptamerization is advantageous as the corresponding enzyme,
although not quantitatively acylated, is misaligned and inactive in all catalytic centers, effectively
maximizing the sustainability of deactivation. Thus, drug discovery efforts for ClpP and other
multimeric enzymes need to consider deꢀoligomerization as an attractive mode of action.
As betaꢀlactones are labile electrophiles which quickly hydrolyze in human plasma within
minutes,19 previous rational attempts focused on replacing this group with more stable moieties.
However, installation of lactam, carbamate, ester and oxetane moieties failed to inhibit the
enzyme suggesting a restricted active site tolerance.23 In order to expand the repertoire of ClpP
inhibitors as chemical biology tools and to identify novel pharmacological leads we performed an
unbiased highꢀthroughput screen (HTS) of more than 137,000 compounds and identified six
compounds as potent hits, which all contained activated ester or amide moieties. Inꢀdepth
biochemical characterization and structureꢀactivity relationship (SAR) studies revealed a new
class of deꢀoligomerizing ClpP inhibitors with superior potency, inhibition kinetics, plasma half
life, stability of the acylꢀenzyme intermediate and specificity. The (R)ꢀ or (S)ꢀconfiguration of a
methyl substituent in the alpha position to the ester served as a switch of the oligomeric state
which was further supported by molecular docking experiments.
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Results
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High throughput screen for ClpP inhibitors
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For the discovery of novel, nonꢀbetaꢀlactone
inhibitors of ClpP, we performed a HTS
with more than 137,000 compounds, which
were each tested for inhibition of S. aureus
ClpP
(SaClpP)
peptidase
activity.
Compounds that inhibited turnover of the
fluorogenic substrate NꢀsuccinylꢀLeuꢀTyrꢀ7ꢀ
amidoꢀ4ꢀmethylcoumarin (SLYꢀAMC) by
50% at 12.5 µM concentration were
regarded as a hit. Doseꢀresponse behavior of
the assay was validated using the ClpP
inhibitor Palmostatin M – a betaꢀlactone – as
a positive control (Figure S1). The overall Z’
factor of the primary screen was calculated
to be 0.69 ± 0.14 and the overall signalꢀtoꢀ
background ratio was 25 ± 9.
161 initial hits were identified (hit rate
0.017%) and their individual IC50 values
determined (Figure 1A, Table S2).
Compounds with an IC50 < 2 µM and
desirable pharmacological properties (see
Supporting Information for details on
filtering criteria) were selected for further
studies. Given the large number of
molecules screened, it is surprising that only
Figure 1. Identification of HTS primary hits. A. A screen of
more than 137,000 compounds identified 161 primary hits
which reduced turnover of the fluorogenic substrate Nꢀ
succinylꢀLeuꢀTyrꢀ7ꢀamidoꢀ4ꢀmethylcoumarin (SLYꢀAMC)
by 50% at 12.5 µM concentration. B. Structures of the six
final hits that had suitable pharmacological properties and
showed an IC50 < 2 µM, and structures of betaꢀlactone
compounds D3 and E2.
six hits met these criteria, demonstrating a restricted structural access to the ClpP active site
(Figure 1B, Table S1). Interestingly, five molecules contained a central phenyl ester (of
respective aromatic acids: AV126, AV168 or aliphatic acids: AV127, AV167, AV170) and one
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compound a triazole amide motif (AV166). All compounds exhibited potent IC50 values between
0.3 and 1.3 µM which are even at the lower limit of the assay (requires 1 µM ClpP).
Although previous attempts to replace the lactone scaffold with openꢀchain esters and carbamates
failed,23 phenyl esters and triazole amides exhibit a stabilized aromatic leaving group that
elevates electrophilicity, and thereby reactivity, towards Ser98. Remarkably, of the 1780 phenyl
esters present in the library only five (0.3%) were identified as hits, emphasizing that not only
reactivity, but also structural prerequisites, are important for binding and inhibition.
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Customized inhibitors of bacterial and human ClpP peptidase and protease activity
The panel of inhibitors was tested for potency and selectivity against S. aureus, E. coli,
L. monocytogenes and human ClpP. First, inhibition of ClpPꢀcatalyzed SLYꢀAMC peptide
hydrolysis was determined by the percent residual activity at 100, 10 and 1 µM compound
concentration across the panel of enzymes. In prokaryotic ClpP, AV170 exhibited the most
pronounced reduction in peptide hydrolysis followed by AV166 and AV126 (Figure 2A and
Figure S2). Importantly, at the lowest concentration tested (1 µM), AV170 was four times more
effective (for SaClpP) compared to the previously reported betaꢀlactone inhibitors D3 and E2,
which have served as a gold standard for ClpP inhibition so far.21 On the contrary, the best
inhibitors of bacterial ClpP did not significantly reduce human ClpP (hClpP) peptidase activity
even at low to moderate (1 to 10 µM) concentrations, pointing towards significant differences in
the binding sites (Figure 2B). Human and S. aureus ClpP share only a moderate sequence
similarity of 37%. Since the inactivation of human ClpP has been linked to cancer and the
Perrault syndrome27 the observed discrimination between human and bacterial enzymes is
beneficial for putative medicinal applications. In turn, AV167, with a large naphthoꢀfuran moiety,
was the only compound that significantly reduced human ClpP peptidase activity even at low
(1 µM) concentrations (Figure 2B).
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Figure 2. Inhibition of ClpP peptidase and ClpXP protease activity. Hit compounds and two lactone inhibitors (E2
and D3) were tested at three concentrations for their inhibition of peptidase and protease activity. Data are
normalized with respect to DMSO as a negative control (100% activity) A. S. aureus ClpP peptidase assay (1 µM
SaClpP in the presence of 200 µM SLYꢀAMC) B. H. sapiens ClpP peptidase assay (1 µM HsClpP in the presence of
200 µM SLYꢀAMC). C. S. aureus ClpP protease assay (0.2 µM SaClpP in the presence of 0.4 µM SaClpX and
0.4 µM GFPꢀSsrA) D. H. sapiens ClpP protease assay (0.2 µM HsClpP in the presence of 0.4 µM EcClpX# and
0.4 µM GFPꢀSsrA). Each data set represents six replicates obtained from two independent experiments (mean ±
standard deviation). **** represent pꢀvalue ≤ 0.0001 determined by Student’s tꢀtest.
# EcClpX was used in place of HsClpX as the AAA+ Chaperone to recognize, unfold and translocate the substrate as
described previously.28
§Internal fluorescence of compound AV166 precluded measurements at 100 µM.
To examine if the identified peptidase inhibitors also impair proteolysis, we monitored the digest
of green fluorescent protein (GFP) tagged with a SsrA peptide sequence. Bacterial ClpX
recognizes SsrA as a degradation signal, unfolds, and subsequently directs the protein into the
ClpP proteolytic chamber.14,29 Proteolytic activity of the SaClpXP complex was effectively
inhibited at 10 µM by phenyl esters AV126 and AV170 which is nearly 11ꢀfold more potent
compared to the lactones D3 and E2 (Figure 2C). In line with previous reports,29 the
proteolytically inactive ClpXP(S98A) complex reduced GFP fluorescence solely by ClpXꢀ
mediated unfolding (about 17 %). However, no reduction of fluorescence was observed in ClpXP
samples treated with phenyl esters, suggesting that compounds not only abolished GFP
degradation but also impaired its unfolding. None of the phenyl ester compounds inhibited
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human ClpXP proteolysis, demonstrating the desired selectivity for bacterial ClpP species
(Figure 2D).
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Figure 3. Phenyl esters covalently modify ClpP. A. Intact protein mass spectrometry of SaClpP after compound
treatment reveals incomplete (AV170) and full (AV167) modification whereas the respective active site mutant
SaClpP(S98A) lacks binding. B. Suggested mode of action, illustrated for AV170. C. Sizeꢀexclusion
chromatograms show SaClpP heptamer formation upon treatment with AV170. D. SaClpP inactivation rates
(kobs/[I] values) for the six hit compounds.
Phenyl esters covalently bind to the active site and induce ClpP de-oligomerization
Given the compelling activity of phenyl esters in peptidase and protease assays, we next focused
on elucidating the mechanism of inhibition of SaClpP as the biologically relevant target. Phenyl
esters and triazole amides represent electrophilic molecules that can inhibit ClpP by either
covalent or nonꢀcovalent binding. In order to investigate the binding mode and the degree of
modification, we utilized intact protein mass spectrometry and determined the exact mass of
SaClpP treated with inhibitor. All compounds active in S. aureus peptidase assays shifted the
protein mass to higher molecular weights, confirming a covalent binding mechanism (Figure 3A,
Figure S3). The extent of modification ranged from 25% for AV168 to 50% for AV170. Only the
human ClpP inhibitor AV167 achieved binding saturation (Figure 3A). None of the molecules
were able to bind the SaClpP(S98A) mutant, reaffirming the role of active site Ser98 as the
functional nucleophile (Figure 3A, Figure S3).
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Moreover, the adduct mass corresponded to the attachment of only the carboxy part of the esters.
These findings elucidate a mode of inhibition in which the inhibitor binds to the active site,
undergoes nucleophilic attack by Ser98 at the ester or amide and releases the phenol or triazole as
a stabilized leaving group (Figure 3B). Activated ester inhibitors thus act as substrate mimics
which are cleaved like native peptides but remain trapped in the acylꢀenzyme intermediate state.
Previous studies with betaꢀlactones D3 and E2 showed that binding to the active site of ClpP can
either result in acylation of all sites (D3) under retention of the oligomeric state, or in partial
modification and subsequent deꢀoligomerization (E2).25 Here, all phenyl esters and triazole
amides followed the deꢀoligomerization route leading to inactive heptamers (Figure 3C, Figure
S4). This conformational change disassembles the ClpP barrel, stalls proteins at ClpX, and thus
explains the surprising dual inhibition of unfolding and degradation activity with AV170 and
AV126.
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In order to determine the potency of the covalent phenyl ester and triazole amide inhibitors, we
measured the inactivation rates via kobs/[I] values. The highest kobs/[I] values, i.e. acylation
velocities, were obtained with AV170 (1200 ± 105 Mꢀ1 sꢀ1) and AV126 (781 ± 31 Mꢀ1 sꢀ1). This
accounts for approximately 20 times faster reaction kinetics in the case of AV170, compared to
the previous gold standard inhibitors D3 (78 ± 6 Mꢀ1 sꢀ1) and E2 (64 ± 3 Mꢀ1 sꢀ1) (Figure 3D,
Figure S5).25 Based on its superior activity, AV170 was selected as the most promising candidate
for in situ target validation.
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ClpP is the predominant intracellular target of phenyl esters in S. aureus
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Figure 4. Synthesis of ML16 and in situ target analysis. A. Scheme for the synthesis of activity-based probe
ML16. Reagents and conditions a) MgI2, neat, 80 °C, 1.5 h; b) propargyl bromide, K2CO3, KOtBu, DMF, rt, 72 h; c)
15 % aq. NaOH, MeOH, 50 °C, 2 h; d) methyl 4-hydroxybenzoate, EDC·HCl, DMAP, DCM, rt, 24 h. B + C. ABPP-
labeling of living S. aureus cells with ML16 and D3. B. Fluorescence SDS-PAGE of soluble fraction. C. Western
blot against SaClpP confirming the labeling of ClpP. See Supporting Figure S7 for full image and overlay of
western blot and fluorescence scan. Note: Probe binding slightly changes protein migration explaining the
double band in Western Blots of treated samples. D. Vulcano plot representation of gel-free quantitative ABPP
experiments with ML16. ClpP is identified as the only target of ML16 (13-fold enrichment over DMSO, p-value
of 0.0015). Data are derived from three independent experiments.
Prior to compound optimization, cell permeability and target selectivity of AV170 in living
S. aureus cells were analyzed. We utilized a chemical proteomic strategy termed activityꢀbased
protein profiling (ABPP)30,31 wherein we synthesized an alkyneꢀtagged AV170 derivative which
remains bound to the enzyme upon inhibition. The alkyne serves as a benign tag for subsequent
conjugation to a functionalized azide via the HuisgenꢀSharplessꢀMeldal cycloaddition (click
chemistry).32ꢀ34 In brief, the synthesis started with 3ꢀ(3,4,5ꢀtrimethoxyphenyl)propanoic acid
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which was selectively demethylated in the para position by means of magnesium iodide (Figure
4A).35 Global propargylation and ester hydrolysis, followed by esterification with methylꢀ4ꢀ
hydroxybenzoate yielded the final probe ML16. The compound showed similar potency in
SaClpP peptidase inhibition compared to AV170 (Figure 5B) and intact protein mass
spectrometry confirmed covalent modification of the enzyme with about 22% (Figure S6). The
probe was incubated with intact S. aureus cells for 1h, followed by cell lysis, proteome separation
into soluble and insoluble fractions, treatment with a rhodamine azide dye, separation on SDSꢀ
PAGE and visualization via fluorescent scanning. Importantly, only a single band appeared on
the fluorescent SDS gel, which could be identified as ClpP by Western blotting, (Figure 4B, C,
Figure S7 for entire blot). The lack of any offꢀtargets surpassed the selectivity of D3, which
weakly labeled additional bands, and thereby establishes phenyl esters as selective ClpP
inhibitors. Furthermore, the ClpP band in D3 treated samples could selectively be outcompeted
by AV170 while all offꢀtargets of D3 remained unchanged (Figure S8). Quantitative MSꢀbased
proteomic experiments further underlined the unprecedented selectivity of ML16 for ClpP as the
predominant target in living S. aureus cells (Figure 4D). ClpP was the only identified target and
was enriched about 13ꢀtimes over the DMSO control with a pꢀvalue of 0.0015 (see Supporting
Information for further experimental details).
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Structure-activity relationship studies
To explore the SAR of phenyl esters, we systematically varied the phenol group, the carboxylic
acid moiety and the ester bond of AV170 and AV127 (Figure 5A, Scheme S1ꢀS3). Substitution of
the ester bond by carbamates (ML20), amides (AV137) or sulfonamides (AV134) led to inactive
compounds (Figure 5B). Therefore, the ester was kept as an integral part of this inhibitor class.
The phenol of AV170 is substituted with an electronꢀwithdrawing group in the para position,
which increases its lability. Interestingly, all HTS hits contained electronꢀwithdrawing phenol
substituents such esters (AV126, AV170), lactams (AV127) or oxadiazoles (AV167, AV168),
suggesting that this electronic fine tuning is a substantial driving force for reactivity and ClpP
acylation. Accordingly, removal of the electronꢀwithdrawing group (ML06) or exchange to
electron donating groups such as methoxy (ML05) and N´, N´ꢀdimethylamine (ML28) resulted in
a significant drop in potency (Figure 5B). In addition, an ester derivative of cyclohexanol
(ML09) exhibited no activity. We thus increased the diversity of electronꢀwithdrawing
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substituents and altered their position on the phenol ring. Aldehydes (ML25), ketones (ML27)
and isopropylesters (ML03) in the para position were among the best inhibitors (Figure 5B).
While the ester group was tolerated in the meta postion (ML23), a ketoꢀgroup in the ortho
position (ML24) displayed almost no activity, suggesting that electronic and steric restrictions in
the active site alter inhibitor potency. This is also supported by the observation that electronꢀ
withdrawing pꢀnitro (ML12) and pꢀcarboxy (ML18) substituents were inactive, while the small,
nonꢀactivated aliphatic butanol ester (ML14) retained moderate inhibitory activity.
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Figure 5. Structureꢀactivity relationship studies. A. Chemical structures of AV170 and its synthesized derivatives
modified at the ester, alcohol or acid moiety. B. Inhibition of SaClpP peptidase activity by the compounds. Each data
set represents six replicates from two independent experiments (mean ± standard deviation).
Next, we explored the carboxylic acid moiety of AV170 and determined a very limited structural
flexibility in this part of the molecule. Variations of aromatic substituents (ML92, ML57) and
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replacement of the benzene ring by aliphatic residues (ML30 and ML31) were not tolerated for
ClpP inhibition. Interestingly, ML33, bearing an indole moiety, was among the best inhibitors
while ML68, with an aromatic thiophene, displayed no potency (Figure 5B).
In AV170, the trimethoxyphenyl moiety and the reactive ester are separated by two methylene
groups. We successively removed both methylene groups to generate compounds ML21 and
ML74. While ML21 was active, ML74 displayed only weak activity compared to AV170,
suggesting that chain length is a crucial parameter for activity.
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Chemical modifications increase stability and reveal a ClpP de-oligomerization switch
Betaꢀlactones exhibit limited stability in acylꢀenzyme complexes. Interestingly, the half life of the
AV170 acylꢀenzyme intermediate was higher compared to the respective D3 acylꢀenzyme
complex (t1/2 = 8.2 ± 0.8 h vs t1/2 = 5.0 ± 0.4 h) (Figure 6C, Figure S9). To improve compound
stability, we rationalized that substituents alpha to the carbonyl could sterically hinder hydrolysis.
We therefore synthesized derivatives of AV170 containing either methyl (ML38, ML43, ML89,
ML90), methylene (ML73) or ethyl substituents (ML95, ML96) at this position (Figure 6A).
Moreover, methyl groups were installed in the ortho position of the phenol ring (ML15, ML37)
to protect the ester from the opposite side (Figure 6A). While incorporation of methyl groups in
the phenyl ring abolished inhibitor activity, methylation at the alpha position (ML43, ML89,
ML90) enhanced acylꢀenzyme stability with a moderate reduction of potency compared to
AV170 (Figure 6B, C). ML73, containing a methylene substituent, was the most potent
compound in this series and also showed an improved half life. Extended conjugated systems
(ML38, ML91), leading to mesomeric stabilization, exhibited the most prominent prolongation
of acylꢀenzyme half lifes. Similar trends were observed for compound stability in human blood
plasma (Figure 6D, Figure S10). Mesomeric stabilization by the extended conjugated system
exhibited the best stabilizing effects (ML38, ML91) with half lifes of up to 65 ± 18 h. This
corresponds to a 20ꢀfold and 6ꢀfold improvement compared to AV170 (3.7 ± 0.1 h) and beta
lactone U1 (11 ± 2 h)19, respectively. Sterical shielding of the carbonyl via introduction of a
methyl (ML89, ML90) or methylene group (ML73) had a less pronounced influence on
stabilization.
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Figure 6. A. Chemical structures of stabilized AV170 derivatives. B. Inhibition of peptidase activity upon treatment
with respective compounds. Each data set represents six replicates from two independent experiments (mean ±
standard deviation). C. Stability of various acylꢀSaClpP complexes at 32 °C as determined by timeꢀdependent intact
protein mass spectrometry. Half life refers to the time needed for a 50% reduction in the extent of protein
modification (see Supporting Figure S9 for raw data). Each data set represents three biological replicates (mean ±
standard deviation). D. Compound stability in human blood plasma. Each data set represents three independent
experiments (mean ± standard deviation). E. Sizeꢀexclusion chromatograms illustrating heptamer formation upon
treatment with the (R)-enantiomer ML89. F. Intact protein mass spectrometry showing partial (ML89) and full
§
(ML90) modification of SaClpP after compound treatment. Internal fluorescence of compound ML91 precluded
measurements at 100 µM.
ML43 was initially tested as a racemic mixture. To discriminate between the properties of each
enantiomer, we stereoselectively synthesized both (S)-ML90 and (R)-ML89 using a chiral
auxiliary (Figure 6A, Scheme S4, S5). (R)ꢀML89 is moderately more potent than (S)-ML90
(Figure 6B) and exhibited 3ꢀfold enhanced acylꢀenzyme stability (Figure 6C). In agreement with
the mode of action of other phenyl esters, (R)ꢀML89 partially modified ClpP and induced
dissociation into heptamers (Figure 6E, F). Surprisingly, unlike all other phenyl esters studied so
far, the orientation of the methyl group in (S)ꢀML90 resulted in acylation of all 14 ClpP active
sites and retention of the tetradecameric assembly (Figure 6E, F). It is an intriguing feature of
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these inhibitors that the orientation of the methyl substituent acts as a switch that either stabilizes
or destabilizes the transient ClpP complex. Contrastingly, an ethyl group at this position ((R)ꢀ
ML95 and (S)ꢀML96) abolished activity, demonstrating that longer chains are not tolerated
(Figure 6B).
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As ClpP activity is important for bacterial virulence the inhibitors were tested in a hemolysis
assay. A reduction in hemolysis was observed with compounds AV170, ML21, ML89 and
ML90 through doseꢀresponse curves, with EC50 values ranging from 60 – 200 µM (Figure S11).
However, upon prolonged incubation of bacterial supernatants with red blood cells, residual
hemolytic activity could be detected, suggesting that alpha–hemolysin production was not
completely attenuated. No cytotoxic effects were observed at a concentration of 100 µM for
AV170, ML21, ML89 and ML90 (Figure S12) against human epithelial lung cancer cells A549.
This is in line with in situ labeling experiments which showed weak labeling of only two distinct
bands in the soluble protein fraction of A549 cells upon incubation with the phenyl ester probe
ML16 (Figure S13).
Molecular docking reveals the binding mode of phenyl esters
To elucidate the binding mode of phenyl esters to ClpP in more detail and to investigate the
underlying cause of the (R),(S)ꢀenantiomer switch, we completed molecular docking studies with
compounds AV170, ML21, ML89, ML90, and nonꢀbinding ML74. The docking solutions were
filtered using pharmacophoric constraints to ensure the correct placement of the carbonyl oxygen
in the oxyanion hole, which is a prerequisite for the nucleophilic attack to occur. Molecular
dynamics (MD) simulations were subsequently performed for the three best poses of each
compound. Figure 7 illustrates the most energetically favoured conformations upon MD
refinement. All inhibitors show the same general binding mode, as exemplified by AV170 in
Figure 7A. The trimethoxyphenyl moiety of AV170 binds to a distinct hydrophobic channel
extending from the catalytic center, while the reactive ester points towards the oxyanion hole (red
circle).
Previous mutation experiments have validated this pocket as an integral part of the binding mode
of betaꢀlactones.23 Here, mutation of Leu154 to Tyr restricted the pocket size and strongly
reduced binding of AV170, while the shorter ML21 derivative was unaffected (Figure S14). We
calculated the equilibrated bound structures of AV170 as well as ML21 and ML74 within this
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binding pocket. In order to fully accommodate the trimethoxyphenyl moiety, a flexible linker is
needed to span the constricted area between the catalytic center and the hydrophobic pocket. In
the case of AV170, the twoꢀcarbon linker appears to have an optimal length to accommodate the
trimethoxyphenyl ring. In ML21, the methylene linker still allows the ring to enter the pocket but
its conformation is more constricted, whereas ML74, with its shorter linker, places the bulky
trimethoxyphenyl ring directly at the pocket entrance and prohibits a stable conformation where
the reactive ester group is close enough to the oxyanion hole for the reaction to occur
(Figures 7F). Moreover, no hydrogen bond between the backbone amino group of the oxyanion
hole and the ester groups of ML74 is formed. This leads to the release of compound out of the
pocket during MD (final conformation given in green) for all three investigated poses, whereas
all three poses of AV170 and ML21 remained in their bound conformation (Figures 7BꢀE).
Next, we looked into the strikingly different binding and deꢀoligomerization behaviors of
compounds ML89 and ML90 (Figures 7DꢀE). A comparison of these compounds with AV170
(same linker length, but no methyl group) revealed two main differences. First, the methyl group
of ML89/ML90 strongly restricts possible binding conformations as it is located directly at the
constricted area between the catalytic center and the hydrophobic binding pocket. Second, the
relative orientation of the methyl group largely diverges in the two stereoisomers. In ML90, the
methyl group is located at the bottom of the constricted area. In the case of the ML89
stereoisomer, the methyl group points to the top of the binding site towards the histidine of the
catalytic triad. As there is more space in this direction, there seem to be less conformational
restrictions compared to ML90. Importantly, this histidine has previously been identified as part
of a hydrogen bonding network which plays a crucial role for the deꢀoligomerization process.26
Thus, the relative orientation of the inhibitor scaffold of ML89 compared to ML90 may trigger a
steric rearrangement which causes the observed deꢀoligomerization.
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Figure 7. A. Surface representation of the ClpP active site bound to AV170. B-F. Conformations of five selected
compounds after MD refinement. Important residues of the catalytic site and the oxyanion hole (M99, S98, H123), as
well as bound ligands are shown in stick representation. B. AV170. C. ML21. D. ML89. E. ML90. F. ML74. Final
conformations of ML74 are given in green.
Discussion
Functional and mechanistic analysis of the ClpXP proteolytic system represents an area of intense
research. Recent reports have focused on mechanistic aspects of the whole ClpXP unfolding
machinery.36ꢀ38 Betaꢀlactones, the only specific ClpP inhibitors reported to date, have been
applied as tools to probe the binding pocket and mechanism of inhibition, as well as the transient
stability of the ClpP tetradecamer and its conformational switching.23,25,39 One additional
consequence of betaꢀlactoneꢀbased ClpP inhibition is the reduction of virulence in pathogenic
bacteria such as MRSA and the eradication of Mycobacteria that express two essential ClpP
isoforms.19,20,40 Here, we expand the set of ClpP tools through a novel class of phenyl esters that
surpass betaꢀlactones in terms of potency in peptidase and protease inhibition, reaction kinetics,
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target selectivity in the soluble fraction of the proteome, plasma half life, acylꢀenzyme stability
and deꢀoligomerization as the preferred mechanism of inhibition. Contrary to betaꢀlactones,
phenyl esters closely mimic the natural peptide substrate in which the acylꢀenzyme intermediate
is irreversibly trapped and the phenol released. Our SAR studies showed that an electronꢀ
deficient phenol moiety is important in maintaining a level of reactivity that facilitates the attack
of the active site serine. The ester must be substituted with an aromatic system that is linked via
at least one methylene bridge in order to pass a constricted area in the binding pocket.
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Importantly, introduction of a methyl group in the alpha position not only enhances the acylꢀ
enzyme stability but also yields a switch that, depending on the absolute configuration, retains the
oligomeric state or induces dissociation into heptamers. Considering that the native peptide
backbone branches at the same position, it is intriguing to speculate that this inhibitorꢀtrapped
intermediate may reflect a transition of ClpPꢀmediated substrate turnover.
It is worth noting that the HTS only revealed a very limited set of potent inhibitors and that all of
these utilize a covalent mode of action. These potent inhibitors help to elucidate and dissect ClpP
action; however, their translation into pharmacologicallyꢀsuitable virulence blockers still
represents a major challenge. Inspired by the marketed phenyl ester protease inhibitor sivelestat,
protection of the ester group by adjacent methyl substituents increased the ester stability, at the
cost of ClpP reactivity. Thus, future studies have to consider the two opposing factors, stability
and reactivity, in order to identify an optimal pharmacological candidate.
Acknowledgements
The work was funded by the Deutsche Forschungsgemeinschaft, SFB749, SFB1035, FOR1406
and the ERC starting grant (250924ꢀantibacterials). We would like to acknowledge Eilika Weberꢀ
Ban (ETH Zürich) for generously providing the EcClpX plasmid. We thank Malte Gersch and
Matthias Stahl for providing purified EcClpX, EcClpP and hClpP proteins, as well as Ines
Hübner for support in the synthesis of compounds MLIH18 and MLIH19. Furthermore we would
like to acknowledge Ernst Bernges, Burghard Cordes and Mona Wolff for technical assistance,
Elena Kunold for help with analysis of quantitative mass spectrometry data and Vadim Korotkov
and Annabelle Hoegl for critical revision of the manuscript.
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Supporting Information. Additional text, figures, tables and schemes giving biochemical
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data, labeling results, synthetic strategies, compound characterization data and H and 13C
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NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
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(39) Gersch, M.; Famulla, K.; Dahmen, M.; Göbl, C.; Malik, I.; Richter, K.; Korotkov,
V. S.; Sass, P.; RubsamenꢀSchaeff, H.; Madl, T.; BrötzꢀOesterhelt, H.; Sieber, S. A.
Nat. Commun. 2015, 6, 6320.
(40) Compton, C. L.; Schmitz, K. R.; Sauer, R. T.; Sello, J. K. ACS Chem. Biol. 2013, 8,
2669.
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Figure 1. Identification of HTS primary hits. A. A screen of more than 137,000 compounds identified 161
primary hits which reduced turnover of the fluorogenic substrate NꢀsuccinylꢀLeuꢀTyrꢀ7ꢀamidoꢀ4ꢀ
methylcoumarin (SLYꢀAMC) by 50% at 12.5 µM concentration. B. Structures of the six final hits that had
suitable pharmacological properties and showed an IC50 < 2 µM, and structures of betaꢀlactone compounds
D3 and E2.
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Figure 2. Inhibition of ClpP peptidase and ClpXP protease activity. Hit compounds and two lactone inhibitors
(E2 and D3) were tested at three concentrations for their inhibition of peptidase and protease activity. Data
are normalized with respect to DMSO as a negative control (100% activity) A. S. aureus ClpP peptidase
assay (1 µM SaClpP in the presence of 200 µM SLYꢀAMC) B. H. sapiens ClpP peptidase assay (1 µM HsClpP
in the presence of 200 µM SLYꢀAMC). C. S. aureus ClpP protease assay (0.2 µM SaClpP in the presence of
0.4 µM SaClpX and 0.4 µM GFPꢀSsrA) D. H. sapiens ClpP protease assay (0.2 µM HsClpP in the presence of
0.4 µM EcClpX# and 0.4 µM GFPꢀSsrA). Each data set represents six replicates obtained from two
independent experiments (mean ± standard deviation). **** represent pꢀvalue ≤ 0.0001 determined by
Student’s tꢀtest.
# EcClpX was used in place of HsClpX as the AAA+ Chaperone to recognize, unfold and translocate the
substrate as described previously.
§Internal fluorescence of compound AV166 precluded measurements at 100 µM.
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Figure 3. Phenyl esters covalently modify ClpP. A. Intact protein mass spectrometry of SaClpP after
compound treatment reveals incomplete (AV170) and full (AV167) modification whereas the respective
active site mutant SaClpP(S98A) lacks binding. B. Suggested mode of action illustrated for AV170. C. Size-
exclusion chromatograms show SaClpP heptamer formation upon treatment with AV170. D. SaClpP
inactivation rates (kobs/[I] values) for the six hit compounds.
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Figure 4. Synthesis of ML16 and in situ target analysis. A. Scheme for the synthesis of activity-based probe
ML16. Reagents and conditions a) MgI2, neat, 80 °C, 1.5 h; b) propargyl bromide, K2CO3, KOtBu, DMF, rt,
72 h; c) 15 % aq. NaOH, MeOH, 50 °C, 2 h; d) methyl 4-hydroxybenzoate, EDC·HCl, DMAP, DCM, rt, 24 h.
B + C. ABPP-labeling of living S. aureus cells with ML16 and D3. B. Fluorescence SDS-PAGE of soluble
fraction. C. Western blot against SaClpP confirming the labeling of ClpP. See Supporting Figure S7 for full
image and overlay of western blot and fluorescence scan. Note: Probe binding slightly changes protein
migration explaining the double band in Western Blots of treated samples. D. Vulcano plot representation of
gel-free quantitative ABPP experiments with ML16. ClpP is identified as the only target of ML16 (13-fold
enrichment over DMSO, p-value of 0.0015). Data are derived from three independent experiments.
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Figure 5. Structure-activity relationship studies. A. Chemical structures of AV170 and its synthesized
derivatives modified at the ester, alcohol or acid moiety. B. Inhibition of SaClpP peptidase activity by the
compounds. Each data set represents six replicates from two independent experiments (mean ± standard
deviation).
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Figure 6. A. Chemical structures of stabilized AV170 derivatives. B. Inhibition of peptidase activity upon
treatment with respective compounds. Each data set represents six replicates from two independent
experiments (mean ± standard deviation). C. Stability of various acylꢀSaClpP complexes at 32 °C as
determined by timeꢀdependent intact protein mass spectrometry. Half life refers to the time needed for a
50% reduction in the extent of protein modification (see Supporting Figure S9 for raw data). Each data set
represents three biological replicates (mean ± standard deviation). D. Compound stability in human blood
plasma. Each data set represents three independent experiments (mean ± standard deviation). E. Sizeꢀ
exclusion chromatograms illustrating heptamer formation upon treatment with the (R)ꢀenantiomer ML89. F.
Intact protein mass spectrometry showing partial (ML89) and full (ML90) modification of SaClpP after
compound treatment. §Internal fluorescence of compound ML91 precluded measurements at 100 µM.
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Figure 7. A. Surface representation of the ClpP active site bound to AV170. B-F. Conformations of five
selected compounds after MD refinement. Important residues of the catalytic site and the oxyanion hole
(M99, S98, H123), as well as bound ligands are shown in stick representation. B. AV170. C. ML21. D. ML89.
E. ML90. F. ML74.
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