α-Amino Diphenyl Phosphonates as Novel Inhibitors of Escherichia coli ClpP Protease

Article abstract of DOI:10.1021/acs.jmedchem.8b01466

Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem, and new classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target. ClpP function is involved in protein turnover and homeostasis, stress response, and virulence among other processes. The focus of this study was to identify new inhibitors of Escherichia coli ClpP and to understand their mode of action. A focused library of serine protease inhibitors based on diaryl phosphonate warheads was tested for ClpP inhibition, and a chemical exploration around the hit compounds was conducted. Altogether, 14 new potent inhibitors of E. coli ClpP were identified. Compounds 85 and 92 emerged as most interesting compounds from this study due to their potency and, respectively, to its moderate but consistent antibacterial properties as well as the favorable cytotoxicity profile.

Full text of DOI:10.1021/acs.jmedchem.8b01466

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Article
#-Amino diphenyl phosphonates as novel
inhibitors of Escherichia coli ClpP protease
Carlos Moreno-Cinos, Elisa Sassetti, Irene G. Salado, Gesa Witt, Siham Benramdane, Laura
Reinhardt, Cristina Durante Cruz, Jurgen Joossens, Pieter Van der Veken, Heike Brotz-Oesterhelt,
Päivi Tammela, Mathias Winterhalter, Philip Gribbon, Björn Windshügel, and Koen Augustyns
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01466 • Publication Date (Web): 20 Dec 2018
Downloaded from http://pubs.acs.org on December 21, 2018
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-Amino diphenyl phosphonates as novel inhibitors of Escherichia coli ClpP protease
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Carlos Moreno-Cinos,1,§ Elisa Sassetti,2,5,§ Irene G. Salado, Gesa Witt, Siham Benramdane, Laura
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Reinhardt, Cristina Durante Cruz, Jurgen Joossens, Pieter Van der Veken, Heike Brötz-Oesterhelt,
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Päivi Tammela, Mathias Winterhalter, Philip Gribbon, Björn Windshügel, * and Koen Augustyns *.
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Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610
Antwerp, Belgium.
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Fraunhofer Institute for Molecular Biology and Applied Ecology, ScreeningPort,
Schnackenburgallee 114, 22525, Hamburg, Germany.
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Interfaculty Institute for Microbiology and Infection Medicine, University of Tübingen, Auf der
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Morgenstelle 28, 72076, Tübingen, Germany.
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Drug Research Program, Division of Pharmaceutical Biosciences, University of Helsinki,
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Viikinkaari 5E, FI-00014 Helsinki, Finland.
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Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus
Ring 1, 28759 Bremen, Germany.
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Shared first author.
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* Shared corresponding author.
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ABSTRACT
Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem and new
classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit
P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target.
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ClpP function is involved in protein turnover and homeostasis, stress-response and virulence among
other processes. The focus of this study was to identify new inhibitors of Escherichia coli ClpP and to
understand their mode of action. A focused library of serine protease inhibitors based on diaryl
phosphonate warheads was tested for ClpP inhibition and a chemical exploration around the hit
compounds was conducted. Altogether 14 new potent inhibitors of E. coli ClpP were identified.
Compounds 85 and 92 emerged as most interesting compounds from this study due to their potency
and, respectively, to its moderate but consistent antibacterial properties as well as the favorable
cytotoxicity profile.
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INTRODUCTION
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Antibiotic resistance is a major global problem in both developed and developing countries. The
selection pressures on microorganisms when in contact with antibacterial agents underlies the
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emergence of resistance, and the efficacy of first and second line antibiotics is decreasing at an
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alarming rate. The importance of antimicrobial drug discovery was underlined by the World Health
Organization’s (WHO) first global report on antibiotic resistance which attributed 25,000 deaths in
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Europe and 2 million worldwide per year to bacterial infections. Of particular concern are Gram-
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negative multidrug-resistant bacteria (MDR) which are becoming more prevalent. Among the
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antibiotic drugs launched since the year 2000, only five new classes were introduced and only one was
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directed against Gram-negative bacteria in combination with -lactams. To avoid key resistance
mechanisms to pre-existing antibiotics, drug discovery research has focussed on addressing alternative
targets with novel mechanisms of antibacterial action.7
The antibacterial drug target caseinolytic protease proteolytic subunit (ClpP) is a widely conserved
protein which is present in bacteria, in many eukaryotes (including humans, localised in mitochondria),
but is absent in archaea and mollicutes.8-9 ClpP, a chymotrypsin-like serine protease, is thought to play
an important role in determining virulence and stress response by modulating virulence factor
expression in several bacteria including Staphylococcus aureus, Listeria monocytogenes and
Streptococcus pneumoniae.11-14 ClpP degrades mistranslated, misfolded or aggregated proteins, arising
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as a result of stress factors (e.g. heat stress and antibiotics). In Listeria monocytogenes ClpP was found
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to be essential for bacterial survival in macrophages. In Streptococcus pneumoniae the levels of ClpP
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were demonstrated to be correlated with nitric oxide stress.14-15
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ClpP proteases in Gram-positive bacteria have been more thoroughly studied as drug targets, but also
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advances in Gram-negative ClpPs were recently reported. Robinson et al. identified ClpP as potential
target for antivirulence therapies by showing differences between growth curves of wild-type and clpP-
defective E. coli under nitric oxide stress conditions. It has also been demonstrated that in E. coli is
responsible for the cleavage of proteins involved in metabolism, transcription factors, as well as in
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oxidative stress response and starvation. Furthermore, clpP-deficient Legionella pneumophila showed
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impaired virulence and reduced translocation of effector proteins in the studies from Zhao et al. and
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ClpX and ClpP2 were identified by Qiu et al.19 as part of the proteolytic network of the
exopolysaccharide alginate biosynthesis in Pseudomonas aeruginosa, a marker for the onset of chronic
lung infection in cystic fibrosis.
ClpP is a tetradecamer with a cylindrical shape. The 14 subunits are arranged in two heptameric rings
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and a central chamber which contains the active sites of each subunit. Each active site comprises the
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canonical Ser-His-Asp catalytic triad (for most bacteria). The peptidase activity, a characteristic of a
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chymotrypsin-like serine protease,21 typically results in peptides of 7-8 residues length,22 with cuts
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occurring after non-polar residues. ClpP proteolytic activity requires the presence of specific ATPases
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(ClpX and ClpA in the case of E. coli), of the AAA+ enzyme superfamily, whose function is to
recognize, unfold and then transfer the substrates into the chamber, thus forming the Clp complex
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together with ClpP. The interface between ClpP and the AAA+ partners has been investigated as a
drug targeting site and several antibacterial peptides were identified, which activate and deregulate
ClpP.24-25 These acyldepsipeptides (ADEPs) prevent ATPases binding to the heptameric rings of ClpP,
resulting in uncontrolled proteolysis of essential bacterial proteins and eventually in bacterial cell
death.8
, 26
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A promising approach to target the virulence-related functions of ClpP is to develop enzyme inhibitors
used in combination with existing antibiotics. The pioneering efforts of Böttcher and Sieber to target
ClpP led to the development of a series of -lactone inhibitors (among them D3, Figure 1) for S. aureus
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ClpP. These inhibitors bind covalently to the catalytic serine, leading to irreversible inhibition of
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proteolytic activity. Further characterization proved their ability to reduce bacterial virulence expression
not only in S. aureus but also in L. monocytogenes.28-29 The potency of this inhibitor was improved 3-
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to 5-fold with the optimized -lactone U1 (Figure 1). However, the reduced plasma stability of these
compounds, due to the fast hydrolysis of the cyclic ester, impeded further clinical development.31
Thereafter, a new class of potent ClpP inhibitors with better plasma stability was discovered by the
Sieber group.31 The phenyl esters (AV170, Figure 1) irreversibly inhibited S. aureus ClpP, and
triggered deoligomerization of the ClpP tetradecamer into inactive heptamers. Their higher potency,
inhibition kinetics and plasma lifetime, compared to the -lactone series, were countered by their lower
anti-virulence activity. Furthermore, attempts to further improve their acyl-enzyme complex stability
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unfortunately let to a loss of ClpP reactivity. A non-covalent inhibitor against S. aureus ClpP has been
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also identified in a high-throughput screening (HTS) campaign. The inhibitor (AV145, Figure 1)
bound to the handle region near the active site, locking S. aureus ClpP in a novel and inactive
conformation. However, binding of ClpX to ClpP revoked the inhibitory effect of AV145 and its
analogues in bacteria.32
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Boron derived compounds have also shown evidence of successfully inhibiting ClpP in Mycobacterium
tuberculosis as demonstrated for bortezomib by Moreira et al. or the substrate-based peptide boronate
inhibitors by Akopian et al. (Figure 1).33,34 Nevertheless, proteasome inhibition, short half-life, poor
pharmacokinetics and its high cost limited the direct use of bortezomib, the most potent in cellulo of
previously described compounds at M. tuberculosis treatment.33
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Recently, also pyrimidines have been shown to inhibit ClpP. Compounds (P33, Figure 1) targeting
Plasmodium falciparum ClpP achieved inhibition of growth and segregation of the apicoplast during
the cell cycle, leading to parasite death.
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Although ClpPs have been investigated in several organisms, inhibition of Gram-negative bacteria ClpP
remains untapped and the chloromethyl ketone (Z-LY-CMK, Figure 1) co-crystallized by Szyk and
Maurizi was the only reported inhibitor for E. coli ClpP reported so far.36
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O
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-lactone D3
-lactone U1
Phenyl ester AV170
AV145
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S
NH
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S
NH2
N-(Picolinoyl)-Trp-Lys-boroMet
Bortezomib
Pyrimidine 33
Z-LY-CMK
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Figure 1. Examples of ClpP reported inhibitors.
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Despite the demonstration of the potential of irreversible inhibitors on different ClpPs, inhibitors with
a classical -amino diaryl phosphonate warhead remained unexplored.37 Thus far, several diaryl
phosphonate compounds have been identified as potent, irreversible serine protease inhibitors. Some
illustrative examples are a urokinase plasminogen activator (uPA) inhibitor reported by Joossens et
al.,38-39 a dipeptidyl peptidase 8 (DPP8) inhibitor by Van der Veken et al., an elastase inhibitor by
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Winiarski et al., a subtilisin inhibitor by Pietrusewicz et al., and the GluC and SplA inhibitors by
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Burchacka et al.43-44
The mode of action for this class of inhibitors (Figure 2) involves a nucleophilic attack by the hydroxyl
of the active site serine on the electrophilic phosphorus atom, leading to the formation of a phosphonate
ester. The initial enzyme-inhibitor complex is unstable. Therefore, hydrolysis of the aryl ester (with a
half-life ranging from few hours to few days) leads to the formation of the “aged complex”.
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S1 pocket
a)
S1 pocket
b)
OPh
OPh
R2
HN
R2
HN
R1
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HO
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Unreacted Inhibitor
Ph Ph
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PENTACOORDINATE
TRANSITION STATE
S1 pocket
S1 pocket
Aging
S1 pocket
c)
d)
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R1
R2
R1
R2
R1
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HN
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P
O
O
P
O
O
Ser
O
Ser
OH Ser
Ph Ph
OPh
Ph
AGED COMPLEX
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Figure 2. Binding mechanism of diphenyl phosphonates with serine proteases. a) The unreacted
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inhibitor enters the active site, with the R moiety filling the S1 pocket while the phosphonate sits at a
reachable distance from the oxyanion hole and the catalytic serine. b) Nucleophilic attack of the serine
to the phosphonate facilitated by the hydrogen bonds of this group with the oxyanion hole residues to
form the pentacoordinate transition state. c) Formalised bonds between the serine oxygen and
phosphorus of the phosphonate lead to a negative charge on the oxygen that, when recovering the
tetrahedral geometry, leads to the release of the phenol group. d) Stabilised configuration after covalent
bonding between ligand and serine protease. e) Slow hydrolysis of the remaining phenolate leads to
formation of the aged complex.
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The aim of this work was to identify new classes of compounds as inhibitors of ClpP activity and
investigate their mechanisms of action. We describe a series of -amino diphenyl phosphonate esters
as the first potent inhibitors of E. coli ClpP, using this species as a model organism for Gram-negative
bacteria, encouraged by the availability of a crystal structure and by the previous studies where a clpP-
defective strain showed a decreased growth under nitric oxide stress conditions.16, 36
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RESULTS
Chemical explorations and enzymatic activity screening. The existing diarylphosphonate library of
the Medicinal Chemistry group of the University of Antwerp (UAMC) was highly enriched in
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hydrophilic and polar residues in R position, since it was mainly focused on targeting trypsin–like
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serine proteases. Based on the specificity of chymotrypsin-like serine proteases for lipophilic residues
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in the S1 pocket, a library of hydrophobic moieties in R was designed. Some analogues of the
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previously described inhibitors (Z-LY-CMK and Lys-boroMet in Figure 1) were also included,
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together with variations on the warhead (diversity of arylphosphonates and nitriles).3
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Synthesis of the analogues with -Cbz in R position and a diphenyl phosphonate as warhead (10-23)
was carried out following the general synthesis described in Scheme 1, where protection of the hydroxyl
groups on some of the aromatic rings was carried out in order to improve the yield of the following
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steps: Dess-Martin oxidation and a modified alternative of the Birum-Oleksyszyn reaction previously
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reported by Van der Veken et al. Those protected compounds, were finally debenzylated following
the conditions of Okano et al.47
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n = 0-2
OH
n
OH
n
O
n
a
b
c
d
PO(OPh)2
Cbz
PO(OPh)2
Cbz
R
R
(Bn)
R
R
R
N
n
N
n
(Bn)
(Bn)
H
H
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-2
3-9
10-21
22-23
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Scheme 1. Reagents and conditions. a) K CO , BnBr, DMF, rt, 4 h. b) Dess-Martin periodinane, DCM,
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0-25 °C, 2 h. c) CbzNH , P(OPh) , Cu(OTf) , DCM, rt, 16 h. d) Pentamethylbenzene, BCl , DCM, -
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3
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3
78 °C, 15 min.
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Synthesis of the compounds with modifications on the phosphonate warhead or its substitution by a
nitrile (23-30) were undertaken as described in Scheme 2, while dipeptidic diphenyl phosphonates (32-
41) were obtained by prior cleavage of -Cbz and subsequent peptidic coupling. These protocols can be
found in the experimental section.
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O
OPh
O
HN
O P OH
R
Cbz
HN
O
N
H
c
a
b
OPh
OPh
Cbz
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O
O P
O
O
R
R
d
OPh
OMe
P
R
N
O
P
Cbz
H
R
N
H
Cbz
N
H
23
21
25
HO
O
e
HO
O
H2N
O
f
g
CN
R
R
R
R
Cbz
Cbz
Cbz
NH2
N
N
H
N
H
H
26
27-28
29-30
1
2
2
9
0
1
Scheme 2. Reagents and conditions. a) CbzNH , tris(4-acetamidophenyl) phosphite, Cu(OTf) , DCM,
2
2
rt, 16 h. b) CbzNH , P(OPh) , Cu(OTf) , DCM, rt, 16 h. c) KOH, H O:dioxane (1:1), rt, 16 h. d) NH ,
2
3
2
2
3
7
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
NH Cl, MeOH, rt, 72 h. e) CbzCl, NaOH, H O, 0-25 °C, 2 h. f) Isobutylchloroformate, N-
4
2
methylmorpholine, NH , DCM, 0-25 °C, 16 h. g) Burgess reagent, DCM, rt, 16 h.
3
3
4
5
6
7
8
9
The compounds 10-41 were evaluated for ClpP inhibition together with a subset of diaryl
phosphonates from the UAMC library (42-74), selected in order to expand the variety of R1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
and R residues (Table 1). ClpP inhibition was assessed by a high-throughput screen in 384-
well format using a fluorescence assay with Suc-LY-AMC as fluorogenic substrate. The
compounds were screened at 200 μM concentration. Compounds were considered as active if
the percentage of inhibition (compared to a control without compounds) was higher or equal
to 75 % (or ≤ 25% remaining activity).
1
0
1
1
1
1
2
3
Figure 3. Generic compound structure.
Table 1. Enzymatic inhibition of the apolar exploration and first library screening.
E. coli ClpP inhibition
Compd.
R1
R2
Warhead
%I (200 M) IC (M)
5
0
O
HO
Cl
O
Z-LY-CMK
100
14.4
HN
Cbz
1
1
0
1
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
7
7
ND
ND
Me
MeO
1
1
2
3
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
6
6
ND
ND
O
8
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8
9
1
1
4
5
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
<1
13
ND
ND
F
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F3C
1
1
6
7
-Cbz
-Cbz
-PO(OPh)2
PO(OPh)2
100
14.2 ± 1.3
S
9
ND
1
8
-Cbz
-PO(OPh)2
<1
ND
HO
HO
2
2
1
2
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
<1
3
ND
ND
HO
O
O
HN
HN
2
2
3
4
-Cbz
9
ND
O
P
O
O
HO
HO
HO
OPh
OH
-Cbz
-Cbz
O
O
P
P
11
14
ND
ND
OPh
25
OMe
2
3
9
0
-Cbz
-Cbz
-CN
-CN
<1
<1
ND
ND
HO
O
3
3
4
4
2
6
1
2
-PO(OPh)2
-PO(OPh)2
-CN
<1
4
ND
ND
ND
ND
HN
Cbz
O
NH2
S
HN
Cbz
HO
O
13
13
HN
Cbz
OMe
O
H2N
N
-PO(OPh)2
NH
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
4
4
4
3
4
5
H2N
N
N
-PO(OPh)2
-PO(OPh)2
13
24
ND
ND
NH
O
O
H2N
N
NH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
H2N
H2N
H2N
H2N
N
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
18
27
6
ND
ND
ND
ND
NH
O
O
S
46
47
N
O
NH
O
N
NH
NH
O
O
O
4
4
5
8
9
0
N
4
NH
O
H2N
N
O
O
-PO(OPh)2
-PO(OPh)2
24
12
ND
ND
NH
N
H2N
H2N
N
N
O
NH
5
5
5
5
5
1
2
3
4
5
N
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
11
27
90
23
68
ND
ND
NH
H2N
HN
NH
NH
NH
NH
O
O
H2N
HN
HO
O
O
H
N
S
49.5 ± 0.5
ND
N
H
O
H2N
HN
O
O
H2N
HN
O
O
ND
O
N3
O
F
H2N
HN
NH
F
F
O
5
6
-PO(OPh)2
25
ND
F
F
1
0
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H2N
HN
NH
O
CF3
CF
F2
C
F2
C
5
5
7
8
O
C
C
CF3
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
55
96
1
ND
39.8 ± 2. 9
ND
F2
F2
-Cbz
H2N
H2N
H2N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
59
O
O
6
6
0
1
<1
ND
O
O
CF3
O
F
F
F
F
-PO(OPh)2
28
ND
H2N
F
CF3
NH2
O
S
O
6
6
2
3
-PO(OPh)2
-PO(OPh)2
27
29
ND
ND
HN
HN
N
H
NH2
O
S
O
N
H
O
F
F
F
F
NH2
O
6
6
4
5
-PO(OPh)2
48
ND
HN
N
H
F
O
CF3
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
64
93
73
ND
8.2 ± 0.8
ND
O
O
N
H
O
O
CF3
O
O
66
O
N
H
O
CF3
6
7
NH2
O
O
N
H
H
N
Me2N
68
-Cbz
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
9
ND
ND
O
6
7
9
0
15
H2N
O
100
13.1 ± 1.2
O2N
O
O
HN
7
7
1
2
-PO(OPh)2
-PO(OPh)2
93
48.1 ± 1.7
NH2
O
O
O2N
17
ND
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
HN
N
7
7
7
3
4
5
-PO(OPh)2
12
23
25
ND
ND
ND
N
NH2
NH
N
HO
N
-PO(OPh)2
H
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
-PO(OPh)2
N
O
O
HN
1
2
3
4
5
6
7
8
9
0
Six compounds emerged as active in the primary screen (16, 53, 58, 66, 70 and 71). Dose-response
experiments confirmed all initial hits and the IC values ranged between 8.2 and 49.5 M (Table 1).
5
0
The biochemical tests revealed that the S1 pocket showed a preference for hydrophilic moieties, while
1
2
16 represents the only active compound with a lipophilic R moiety. Regarding the R substitution, a
variety of simple carbamates were tolerated, -Cbz being the most common and chemically accessible.
However, methyl carbamates and benzodioxol carbamates were also taken into account for future
investigations.
After learning that the S1 pocket accepted a wider range of side chains, every remaining compound
2
1
from the library with -Cbz in R position (76-95) was submitted to a second round of experimental
11
12
testing (Table 2).
1
3
Table 2. Enzymatic inhibition of second library screening.
E. coli ClpP inhibition
Compd.
R1
R2
Warhead
%
I (200 M)
IC (M)
5
0
H
HN
N
76
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
15
ND
ND
NH2
O
7
7
7
8
6
O
-Cbz
-PO(OPh)2
100
27
0.6 ± 0.1
Me2N
79
-Cbz
-PO(OPh)2
ND
N
1
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O2N
80
-Cbz
-PO(OPh)2
6
ND
8
8
1
2
Me
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
3
4
ND
ND
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
83
-Cbz
PO(OPh)2
29
ND
HN
N
H
8
8
8
4
5
6
O
-Cbz
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
4
ND
H2N
MeO
O2N
100
100
0.5 ± 0.0
38.0 ± 2.4
N
8
8
7
8
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
6
ND
ND
Cl
HN
30
NH2
Cl
8
9
-Cbz
-PO(OPh)2
88
100.5 ± 8.0
N
HN
9
9
9
0
1
2
-Cbz
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
81
71
79.7 ± 7.2
ND
NH2
O2N
NH2
HN
100
0.5 ± 0.0
9
9
3
4
-Cbz
PO(OPh)2
<1
ND
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
6
ND
ND
95
51
1
1
3
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2
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2
2
2
2
2
2
2
3
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3
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4
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9
The biochemical tests resulted in the identification of six additional ClpP inhibitors (78, 85, 86, 89, 90
and 92), of which three inhibited the enzyme with a sub-micromolar IC value. From this and the
5
0
previous screening, we concluded that hydrophilicity in the S1 pocket is preferred, and we therefore
1
continued with a chemical exploration of polar groups in R together with further modifications around
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
three selected R residues.
First, based on the polarity of the most active compounds identified so far, the scope of hydrophilic
1
moieties for R was enlarged, leaving the rest of the structure unchanged (96-127). Some of these
compounds (96-98) were directly obtained from the commercial aldehydes after a Birum-Oleksyszyn
reaction as stated in Scheme 3.
PO(OPh)2
O
a
Cbz
N
R
H
R
96-98
1
1
0
1
Scheme 3. Reagents and conditions. a) CbzNH , P(OPh) , Cu(OTf) , DCM, rt, 16 h.
2
3
2
1
1
1
1
1
2
3
4
5
6
Still, most of them required a higher synthetic effort. The remaining compounds can be summarized in
two synthetic schemes. For the first group (107-116), the Birum-Oleksyszyn reaction was conducted on
the selected commercial aldehydes with Boc-protected amine, with the subsequent deprotection and
guanylation for 115 and 116. This group comprises a variety of aniline and piperidine related moieties
1
in the R position (Scheme 4).
O
a
PO(OPh)2
b
PO(OPh)2
PO(OPh)2
c, b
Boc
Boc
Cbz
H
Cbz
R2
Cbz
R1
R1
N
H
R1
N
H
R1
N
H
9
9-106
107-114
115-116
NC
R1 =
H
N
N
N
N
N
N
N
N
H
H
NH
O
R2 =
H2N
Me
1
1
1
7
8
9
Scheme 4. Reagents and conditions. a) CbzNH , P(OPh) , Cu(OTf) , DCM, rt, 16 h. b) TFA, DCM, rt,
2
3
2
1 h. c) N,N'-bis-Boc-1-guanylpyrazole, Et N, DCM, rt, 48 h.
3
2
2
0
1
For a second group of aniline-related compounds and aromatic guanidines (117-127), the starting
materials were a variety of commercial nitroaryl aldehydes that, after a Birum-Oleksyszyn reaction,
1
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1
were reduced and subsequently substituted in some cases to generate methyl sulphoxyamines,
guanydines, dimethylarylureas and methyl amides (Scheme 5). The biochemical tests for ClpP
inhibition of these compounds revealed two inhibitors with IC values of 0.6 and 71.3, respectively
2
7
3
8
5
0
9
4
(Table 3).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
a
PO(OPh)2
b
PO(OPh)2
PO(OPh)2
c
H
O2N
O2N
Cbz
H2N
Cbz
N
Cbz
Ar
Ar
N
H
Ar
N
H
R
Ar
N
H
7
0, 80, 91, 117-118
58, 85, 119-121
122-127
Ar =
R =
Me2N
H2N
Me
Me2N
Me
S
O O
NH
O
O
5
6
7
8
Scheme 5. Reagents and conditions. a) CbzNH , P(OPh) , Cu(OTf) , DCM, rt, 16 h. b) Zn, THF:NH Cl
2
3
2
4
(sat. sol.) (1:1), 0 ˚C, 1 h. c) 124-125: N,N'-bis-Boc-1-guanylpyrazole, Et N, DCM, rt, 48 h, then TFA,
3
DCM, rt, 1 h; 122-123, 126-127: RCl, DIPEA, DCM, rt, 2 h.
1
9
0
Finally, further investigation around the two most potent R moieties (aniline 85 and amidine 92) and
1
the only lipophilic structure with activity (4-(trifluoromethyl)benzyl 16) was undertaken, with
substitution of the -Cbz by other active substituents from the first screening, together with some
warhead alternatives (paracetamol-like phosphonates and nitriles). The chemistry regarding this
exploration can be found in Scheme 6 and Scheme 7. None of the 10 tested compounds revealed any
pronounced ClpP inhibition (Table 4). A summary of all the compounds initial screening is reported in
Figure S1 in the supporting information.
11
12
1
1
1
1
3
4
5
6
1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Scheme 6. Reagents and conditions. a) Dess-Martin periodinane, DCM, 0-25 °C, 2 h. b) CbzNH (129,
2
133, 140)/methyl carbamate (128, 130-132, 134, 139, 141)/benzo[d][1,3]dioxol-5-ylmethyl carbamate
(132), P(OPh) (128, 131-132, 139)/ tris(4-acetamidophenyl) phosphite (129-130, 133-134, 140-141),
3
Cu(OTf) , DCM, rt, 16 h. c) Zn, THF:NH Cl (aq. sat. sol.) (2:1), 0-25 °C, 16 h. d) NH OH⸱ HCl,
2
2
4
6
7
DIPEA, EtOH, 95 °C, 30-72 h, then acetic anhydride, MeCN, rt, 1 h; e) Pd(II)/C 10%, H gas, AcOH,
2
rt, 30 h.
8
9
0
1
Scheme 7. Reagents and conditions. a) HNO , H SO , rt, 2 h. b) CbzCl, NaOH, H O, 0-25 °C, 2 h. c)
3
2
4
2
1
1
Isobutylchloroformate, N-methylmorpholine, NH , DCM, 0-25 °C, 16 h. d) Burgess reagent, DCM, rt,
3
16 h. e) Zn, THF:NH Cl (aq. sat. sol.) (2:1), 0-25 °C, 16 h.
4
12
Table 3. Enzymatic inhibition of the hydrophilic exploration.
E. coli ClpP inhibition
Compd.
R1
R2
Warhead
-PO(OPh)2
-PO(OPh)2
%I (200 M)
IC (M)
5
0
NC
9
9
6
7
-Cbz
-Cbz
15
ND
ND
<1
Me2N
1
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CF3
9
8
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
15
ND
O
N
H
1
1
07
08
N
100
0.6 ± 0.0
HN
HN
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NC
N
-Cbz
-PO(OPh)2
10
ND
1
1
09
10
HN
-Cbz
-Cbz
-Cbz
-PO(OPh)2
PO(OPh)2
-PO(OPh)2
30
16
15
ND
ND
ND
HN
111
H N
2
1
1
14
15
-Cbz
-PO(OPh)2
<1
ND
H2N
H2N
N
-Cbz
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
-PO(OPh)2
100
18
71.3 ± 2.4
ND
NH
H
Me
N
116
O
1
17
O2N
49
ND
O2N
1
1
1
18
20
21
-Cbz
-Cbz
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
PO(OPh)2
-PO(OPh)2
39
13
22
8
ND
ND
ND
ND
Me2N
H2N
H2N
Me2N
Me
122
O
O
N
H
NMe2
1
23
-Cbz
-PO(OPh)2
22
ND
N
H
1
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H
N
HN
1
1
24
25
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
15
38
ND
ND
NH2
NH2
HN
N
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Me
1
1
26
27
O
S
-Cbz
-Cbz
-PO(OPh)2
-PO(OPh)2
10
46
ND
ND
N
O
H
O
S
Me
O
HN
Me2N
1
2
3
Table 4. Enzymatic inhibition of the exploration around 16, 85 and 92.
E. coli ClpP inhibition
Compd.
R1
R2
Warhead
%
I (200 M)
IC (M)
5
0
F3C
O
O
1
1
28
29
-PO(OPh)2
4
ND
ND
HN
O
F3C
O
-Cbz
1
NH
O
O
P
O
HN
O
O
F3C
O
O
O
1
1
30
35
20
ND
NH
O
P
O
H2N
O
O
-PO(OPh)2
-PO(OPh)2
7
ND
ND
O
H2N
O
O
136
137
O
20
HN
O
H2N
O
-Cbz
21
ND
NH
O
O
P
O
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HN
O
O
H2N
O
O
1
1
1
38
45
46
12
30
29
ND
ND
ND
O
NH
O
P
O
NH2
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HN
-PO(OPh)2
O
HN
O
NH2
O
HN
Cbz
NH
O
O
P
O
HN
O
O
NH2
O
O
O
HN
1
1
47
51
12
8
ND
ND
NH
O
P
O
H2N
-Cbz
-CN
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
The hydrophilic exploration resulted in two additional (107 and 115), with 107 having an IC in the
5
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sub-micromolar range. Unfortunately, every alteration on the structure of our reference compounds (16,
85 and 92) led to loss of activity.
Biological evaluation of hits. The 14 hits identified after the different stages were submitted to further
in vitro profiling. The selectivity properties versus chymotrypsin-like serine proteases were evaluated
by screening the 14 compounds against -chymotrypsin (bovine) at 200 M concentration. Most
compounds showed no significant inhibition of chymotrypsin with the exception of 85, which resulted
in a residual enzyme activity of 18 % (Table 5).
1
1
1
1
1
1
Cytotoxicity was tested against the human cell lines A549 (lung), HepG2 (liver) and HeLa (cervical
cancer) in dose response (Table 5). Compounds 89, 90 and 107 were toxic for all cell lines while
compounds 66 and 58 exerted high cytotoxicity (<10-fold of compound IC ) against the lung cell line
5
0
A549. Compounds 78 and 85 showed moderate toxicity against A549 and HepG2 cell lines compared
with their in vitro potency against the target (IC ). The cytotoxicity effects reported here against human
5
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
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4
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cell lines could in principle be caused by the interaction with ClpP present in the human mitochondria
as well by ClpP unrelated mechanisms.
Table 5. Enzymatic activity, cytotoxicity and activity against chymotrypsin of the selected hits.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
E. coli ClpP
IC (M)
Cytotoxicity EC (µM)
Chymotrypsin
5
0
%
of remaining
activity (200 M)
93.9 ± 0.8
Compd.
HeLa
HepG2
A549
5
0
1
6
14.2 ± 1.3
49.5 ± 0.5
39.8 ± 2. 9
8.2 ± 0.8
≥100
≥100
≥100
≥100
≥100
≥100
≥100
≥100
≥100
≥100
53
58
66
≥ 100
≥ 100
≥100
57.8 ± 6.7
41.5 ± 3.8
≥100
≥100
46.0 ± 2.9
≥ 100
7
7
7
8
8
8
9
0
1
8
5
6
9
0
13.1 ± 1.2
48.1 ± 1.7
0.6 ± 0.1
≥100
≥100
≥100
≥ 100
≥100
28.4 ± 3.5
23.8 ± 2.8
≥100
65.6 ± 8.3
27.5 ± 2.3
≥100
≥ 100
0.5 ± 0.0
≥100
17.9 ± 1.6
≥ 100
38.0 ± 2.4
100.5 ± 8.0
79.7 ± 7.2
0.5 ± 0.0
≥100
19.9 ± 1.8
19.9 ± 2.3
≥100
10.5 ± 1.4
28.3 ± 2.5
≥100
5.9 ± 8.5
25.6 ± 2.7
≥100
≥ 100
≥ 100
92
≥ 100
1
07
15
0.6 ± 0.0
8.6 ± 1.2
≥100
1.1 ± 0.1
≥100
0.4 ± 0.0
≥100
≥ 100
1
71.3 ± 2.4
≥ 100
5
6
In order to investigate the mode of interaction between ClpP and selected compounds, surface plasmon
resonance measurements were conducted. The known covalently binding compound chloromethyl
7
3
6
8
ketone (Z-LY-CMK) was used as positive control for irreversible binding.
9
Compounds with IC values <10 µM were tested in a range of concentrations. In addition, the known
5
0
10
covalent inhibitor Z-LY-CMK was tested as positive control. Z-LY-CMK clearly showed irreversible
binding to ClpP, since the compound signal in the sensogram did not return to the baseline (0 RU), even
1
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after stop of the compound injection (at ~350 seconds in all experiments) (Figure 4A). In contrast, all
compounds from this study (Figure 4B-F) bound reversibly to the protein, as shown by the signal drop
to the baseline after stopping injection. Moreover, the sensorgrams revealed rapid on- and off-rates for
all newly identified ClpP inhibitors.
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1
1
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1
1
1
1
2
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2
2
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2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
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4
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5
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5
5
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5
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0
1
2
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4
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9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
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8
9
0
1
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9
0
1
2
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9
0
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9
1
1
1
1
1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
4
4
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1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
4
5
6
7
Figure 4. Surface plasmon resonance sensorgrams. A) Known covalent inhibitor Z-LY-CMK as control
3
6
for irreversible binding. B-F) Selected screening hits, B) 78, C) 107, D) 85, E) 66, F) 92.
Antibacterial assays. Since ClpP is not an essential protease in E. coli, an assay was required to
investigate the influence of the ClpP inhibitors on bacterial growth rates. We utilized a method reported
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1
by Robinson et al., who observed that a ClpP deletion mutant recovered more slowly from nitric oxide
stress than the corresponding wild type, and adapted this assay to a HTS format. Nitric oxide stress was
induced by addition of DPTA NONOate (2 mM) to the E. coli WT and the isogenic E. coli ClpP deletion
strain (ΔclpP). Although ΔclpP strain grew less well under our assay conditions (M9 minimal medium,
96-well format) compared to the wild type, we observed a small but significant difference in time to
growth recovery after nitric oxide stress for the ΔclpP strain compared to the WT strain (Figure 5).
Statistical analysis indicated that the ClpP deletion strain required approximately one hour longer than
the wild type to for growth recovery (see Figure S2 in the supporting information).
2
7
3
8
9
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
7
8
9
0
1
2
3
4
1
1
1
1
1
Figure 5. Comparison of the the growth curves, depicted by OD , of E. coli WT and the isogenic
6
00
mutant E. coli JW0427-1 (clpP-defective mutant), both in absence and presence of the •NO chemical
donor DPTA NONOate (2 mM) in minimal M9 medium supplemented with 10 mM of glucose. Effect
of compound 85 on bacterial growth is quantified by OD600 of E. coli BW25-113 (WT) in presence and
in absence of DPTA NONOate (2 mM). Each value represents the mean of three independent
experiment ± standard deviation.
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1
1
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2
2
2
2
2
2
2
2
3
3
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3
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3
3
3
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2
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5
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7
8
9
0
1
2
3
4
The fourteen hits identified in this study were tested in WT strain in presence of nitric oxide stress. Only
compound 85 showed a remarkable effect in the WT growth (Figure 5), opposite to all the other
compounds which were also tested but without showing any effect. Therefore, while not every hit
achieved the desired inhibition, the effects of compound 85 on the stressed WT strain showed a
comparable growth delay observed to the ΔclpP strain exposed to nitric oxide stress conditions,
consistent with a potential ClpP protease inhibition mediated effect of compound 85.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5 shows that in the presence of nitric oxide stress the growth rate of the WT strain is reduced,
and requires additional 4 h to reach maximum absorbance (orange circle versus red triangle). The
growth rate of the ΔclpP strain compared to the WT strain is reduced, and is further reduced in the
presence of nitric oxide stress, taking an additional 6 h to reach maximum absorbance (light blue square
versus dark blue inverted triangle). The addition of compound 85 at 100 M does not affect the growth
rate of the WT strain (green ring versus red triangle). However, under nitric oxide stress conditions, the
WT strain growth is reduced in presence of compound 85 compared to nitric oxide stress only (green
diamond versus orange circle) and, interestingly, the growth of ΔclpP is similar to that of the WT strain
in the presence of compound 85 (blue square versus green diamond). Moreover, the effect of compound
85 in ΔclpP growth was tested in presence and absence of nitric oxide stress to ensure that the
compound-mediated effect on the WT bacteria growth was due to ClpP inhibition and not due to off-
target effect. As shown in Figure S3 (in the supporting information), the compound did not significantly
affect the growth of the ΔclpP bacteria either in absence or presence of nitric oxide stress conditions.
This confirms that the effect of 85 on WT bacteria under nitric oxide stress conditions is most likely
mediated through its inhibition of ClpP.
1
1
1
1
1
15
16
1
1
1
2
2
2
2
7
8
9
0
1
2
3
Selected compounds were also screened against the wild-type strains of S. aureus, Acinetobacter
baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and E. coli in a standard bacterial growth
assay. Two E. coli mutants with either lpxC defect (impaired in lipidA synthesis) or tolC defect (efflux
pump defect) were also included. At 100 M compound concentration, only 115 inhibited the growth
of S. aureus WT (% inhibition of growth 99.9 ± 0.04), while 90 (98.9 ± 0.05), 107 (69.9 ± 5.6) and 115
(99.0 ± 0.5) inhibited the efflux pump deficient E. coli strain. The mode of action underlying this growth
24
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inhibition remains elusive and could be caused by mechanisms unrelated to E. coli ClpP. In order to
investigate whether the compounds are efflux pump substrates, the growth of E. coli wild type was
examined in presence of test compounds (concentration 50 µM) and 25 µg/ml of phenylalanine-arginine
beta-naphthylamide (PAβN), a known efflux pump substrate. At 24 h compound 90 inhibited bacterial
growth (98.5 ± 1.2). In order to verify whether the observed effect of compound 90 was due to ClpP
inhibition, the assay was repeated using the E.coli ΔclpP strain. The same output of the assay with the
wild type strain was obtained (growth inhibition 99.4 ± 0.7), we can therefore assest that compound 90
addresses a different target that is influencing bacterial growth in presence of the efflux pump substrate.
A summary of the compounds active in bacteria can be found in the supporting information (Table S1).
2
7
3
8
9
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
7
8
9
0
1
2
3
4
5
1
1
1
1
1
1
Molecular docking. Potential binding modes of the most potent inhibitors (92 and 85) within the active
site of ClpP were investigated by molecular docking of the compounds into the X-ray crystal structure
of E. coli ClpP (PDB ID 2FZS) using GOLD.
Clustering of the docking poses of 92 revealed two preferred binding modes (Figure 6A-D). The top-
ranked pose of the first cluster (Figure 6A&B) shows the benzamidine group to be positioned deeply
within the S1 pocket, while a hydrogen bond network between the phosphonate and residues Gly68
(constituent of the oxanion hole) and Leu125 is well established. However, the distance between the
side chain oxygen atom of Ser97 and the phosphorus atom of the ligand is larger than required for the
expected nucleophilic attack (3.35 Å). The second predominant binding mode revealed the benzamidine
group to be solvent-exposed and the docked ligand shares several hydrogen bonds with the protein
(Figure 6C&D). The interaction energy between docked ligand and ClpP was calculated using the
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Amber10:EHT force field. The top-ranked docking pose of cluster 1 (R moiety placed in the S1 pocket)
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revealed a more favorable interaction energy (-62.5 kcal mol ) compared to the top-ranked pose of
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cluster 2 (-54.6 kcal mol ).
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The predicted binding mode of 85 is shown in Figure 6E&F. Only one cluster was identified and the
binding mode revealed the phenyl groups of the diarylphosphonate to be solvent-exposed, whilethe
aniline moiety is positioned inside the S1 pocket. Again, several hydrogen bonds are formed between
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the phosphonate group and the protein, but Ser97 did not display a favorable position for the nuclephilic
attack.
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Figure 6. Computational prediction of potential binding modes for 92 and 85 within the E. coli ClpP
crystal structure (PDB ID: 2FZS). A and C) Docking poses of the two main docking clusters of
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compound 92. E) Pose of compound 85. Black dotted lines indicate hydrogen bonds between the ligands
and the protein. B, D and F) 2D interaction plots between protein and 92 of cluster 1 (B), cluster 2 (D)
and 85 (F) where the polar residues are indicated in purple, the non-polar or charged residues in green,
and the solvent exposure by blue shadow. The black dotted line designates the proximity contour. The
green dotted arrows indicate hydrogen bonds involving amino acid side chain atoms (donors and
acceptors) while blue dotted arrows indicate hydrogen bonds accepted or donated by protein backbone
atoms. Moreover, arene-H interactions are shown as green dotted line.
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DISCUSSION AND CONCLUSIONS
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An extensive chemical exploration and enzymatic screening identified 14 compounds inhibiting E. coli
ClpP in vitro with sub-micromolar IC50 values for 78, 85, 92 and 107. Despite the expected
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hydrophobicity of the protease recognition pocket, compounds containing polar residues in R position
displayed the highest inhibitory activity. Molecular docking analysis of compound 85 and 92 revealed
that the most favorable poses had the aniline or benzamidine group deeply positioned within the
recognition pocket. The diphenyl phosphonate warhead was crucial, with none of the replacements or
small modifications attempted maintaining the inhibitory activity.
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Surface plasmon resonance demonstrated a reversible binding for all tested compounds. With help of
the docking studies of 85 and 92, it can be hypothesized that the inhibitory poses do not allocate the
phosphonate esters of the ligands in a favorable position to form the pentacoordinate transition state
(Figure 2) after the attack by Ser97. Albeit unexpected, this reversible binding of the chemical family
has a precedent in the KLK4 inhibitors reported by Van Soom et al.48
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Benzamidine 92 emerges as the safest option for further optimization due to its potent enzymatic
inhibition, absence of activity against chymotrypsin and lack of toxicity against the tested eukaryotic
cell lines. Even though the latter could be related to a compound’s incapability to enter eukaryotic cells
or to the possibility of being substrate of an efflux pump and would also explain the lack of activity in
the nitric oxide stress assay. At the same time, aniline 85 showed reduction of growth in E. coli WT
under nitric oxide stress conditions, consistent with a ClpP-mediated effect. However, it requires further
improvement in terms of limiting its toxicity against human cell lines and decreasing activity against
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chymotrypsin. Both compounds, as well as 16, 66, 70, 78 and 107 are significantly more potent ClpP
inhibitors compared to the so far only known inhibitor Z-LY-CMK. In order to enlarge the scope and
to understand the lack of effect of some of our inhibitors in the nitric oxide stress assay, further
exploration of chemical alternatives is needed. Given the already known potential of covalent binding
compounds as antimicrobial agents, for example the huge success of -lactam antibiotics (e.g. penems,
cephalosporins, carbapenems, monobactams), the development of a covalent binder for E. coli ClpP
should focus on the replacement of the diaryl phosphonate by a different covalent warhead for serine
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proteases. The comprehensive R moiety library developed in this study may guide future work in the
field combining them with warheads such as boronates, based on the success of bortezomib with M.
tuberculosis ClpP and its inhibition of human 26S proteasome.33
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EXPERIMENTAL SECTION
CHEMISTRY. Reagents were obtained from commercial sources and were used without further
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purification. Characterization of all compounds was done with H and C NMR and mass spectrometry.
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H and C NMR spectra were recorded on a 400 MHz Bruker Avance III Nanobay spectrometer with
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Ultrashield working at 400 MHz and 100 MHz respectively; and analyzed by use of MestReNova
analytical chemistry software. Chemical shifts are in ppm, and coupling constants are in hertz (Hz). The
UPLC (Ultra Performance liquid chromatography), used to quantify the purity of the products was
an ACQUITY UPLC H-Class System with a TUV detector Waters coupled to a MS detector Waters
QDa. An Acquity UPLC BEH C18 1.7 µm (2.1 x 50 mm) column was used and as eluent a mixture of
0.1% FA in H O, 0.1% FA in MeCN, H O and MeCN. The wavelengths for UV detection were 254 nm
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and 214 nm. Key target compounds for the activity were analysed by High Resolution Mass: 10 μL of
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each sample (conc. = 10 M) was injected using the CapLC system (Waters, Manchester, UK) and
electrosprayed using a standard electrospray source. Samples were injected with an interval of 5 min.
Positive ion mode accurate mass spectra were acquired using a Q-TOF II instrument (Waters,
Manchester, UK). The MS was calibrated prior to use with a 0.2% H PO solution. The spectra were
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lock mass corrected using the known mass of the nearest H PO cluster. Where necessary, flash column
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chromatography was performed on a Biotage ISOLERA One flash system equipped with an internal
variable dual wavelength diode array detector (200−400 nm). For normal phase purifications SNAP
cartridges (4 − 100 g, flow rate of 10 − 100 mL/min) were used, and reverse phase purifications were
done making use of KP-C18 cartridges (4 - 30 g, flow rate of 10 − 50 mL/min). Dry sample loading
was done by self-packing samplet cartridges using Celite 545. Gradients used varied for each
purification.
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The following sections comprise the synthetic procedures and analytical data for all compounds
reported in this manuscript. Every reaction was performed under N atmosphere if not stated otherwise.
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Several synthetic procedures that were used in the preparation of intermediates and final products are
summarized here as “General Procedures”. Target compounds were obtained with a purity >95% and
as amorphous solids, unless stated otherwise.
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