Chemical Validation of DegS As a Target for the Development of Antibiotics with a Novel Mode of Action

Article abstract of DOI:10.1002/cmdc.201900193

Despite the availability of hundreds of antibiotic drugs, infectious diseases continue to remain one of the most notorious health issues. In addition, the disparity between the spread of multidrug-resistant pathogens and the development of novel classes of antibiotics exemplify an important unmet medical need that can only be addressed by identifying novel targets. Herein we demonstrate, by the development of the first in vivo active DegS inhibitors based on a pyrazolo[1,5-a]-1,3,5-triazine scaffold, that the serine protease DegS and the cell envelope stress-response pathway σE represent a target for generating antibiotics with a novel mode of action. Moreover, DegS inhibition is synergistic with well-established membrane-perturbing antibiotics, thereby opening promising avenues for rational antibiotic drug design.

Full text of DOI:10.1002/cmdc.201900193

Accepted Article
Title: Chemical validation of DegS as a target for the development of
antibiotics with a novel mode of action
Authors: Jens Bongard, Anna Laura Schmitz, Alex Wolf, Gunther
Zischinsky, Michel Pieren, Birgit Schellhorn, Kenny Bravo-
Rodriguez, Jasmin Schillinger, Uwe Koch, Peter Nussbaumer,
Bert Klebl, Jörg Steinmann, Jan Buer, Elsa Sanchez-Garcia,
Michael Ehrmann, and Markus Kaiser
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900193
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900193
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10.1002/cmdc.201900193
ChemMedChem
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Chemical validation of DegS as a target for the development of
antibiotics with a novel mode of action
Jens Bongard, Anna Laura Schmitz, Alex Wolf, Gunther Zischinsky, Michel Pieren, Birgit Schellhorn,
Kenny Bravo-Rodriguez, Jasmin Schillinger, Uwe Koch, Peter Nussbaumer, Bert Klebl, Jörg
Steinmann, Jan Buer, Elsa Sanchez-Garcia, Michael Ehrmann* and Markus Kaiser*
Abstract: Despite the availability of hundreds of antibiotic drugs,
infectious diseases continue to remain one of the most notorious
health issues. In addition, the disparity between the spreading of
multidrug resistant pathogens and the development of novel classes
of antibiotics exemplify an important unmet medical need that can
only be addressed by identifying novel targets. Here we demonstrate
by the development of the first in vivo active DegS inhibitors based
on a pyrazolo[1,5-a]-1,3,5-triazine scaffold that DegS and the cell
envelope stress response pathway E represent a target for
generating antibiotics with a novel mode-of-action. Moreover, DegS
inhibition is synergistic to well-established membrane perturbating
antibiotics thereby opening promising avenues for rational antibiotic
drug design.
chemotherapies explore only a very limited number of molecular
mechanisms,[2] resulting in the unmet medical need to identify
and validate mechanistically novel strategies and targets for
developing alternative antibiotics.
The bacterial cell envelope stress response pathway E is
essential for bacterial survival under optimal growth but also
under stress conditions such as infection of a human host.[3] Its
impairment may thus represent an alternative approach for
generating antibiotics with a novel mode-of-action. In this
pathway, DegS, a S1 serine protease of the High temperature
requirement A (HtrA) protease family characterized by the
presence of at least one PDZ domain located C-terminal to the
protease domain,[4] serves as the molecular stress sensor.[5] To
this end, DegS binds the hydrophobic C-termini of misfolded or
mislocalized outer membrane proteins via its PDZ domain.[5-6]
This binding event triggers a reversible and allosteric activation
of the DegS protease domain, allowing DegS to perform the
rate-limiting event of stress response, i.e. cleavage of the
transmembrane protein and anti-sigma factor "regulator of sigma
E", RseA. Further downstream processing then results in
transcriptional activation of the stress response.[5] Although the
The persistent rise in resistances to current antibiotic therapies
requires the development of novel antibiotics.[1] Despite the
availability of
hundreds
of
antibiotic
drugs,
these
underlying activation mechanism seems to be conserved in
7]
[*]
Dr. J. Bongard, Dr. K. Bravo-Rodriguez, Dr. J. Schillinger, Prof. Dr.
M. Ehrmann
Microbiology, Faculty of Biology, Center of Medical Biotechnology,
University Duisburg-Essen, Universitätsstr. 2, 45117 Essen
(Germany)
E-mail: michael.ehrmann@uni-due.de
Dr. A. L. Schmitz, Prof. Dr. M. Kaiser
Chemical Biology, Faculty of Biology, Center of Medical
Biotechnology, University Duisburg-Essen, Universitätsstr. 2, 45117
Essen (Germany)
E-mail: markus.kaiser@uni-due.de
Dr. A. Wolf, Dr. G. Zischinsky, Dr. U. Koch, Dr. P. Nussbaumer, Dr.
B. Klebl
Lead Discovery Center GmbH, Otto-Hahn-Str. 15, 44227 Dortmund
(Germany)
Dr. M. Pieren, Dr. B. Schellhorn
other HtrA proteases,[4,
its elaboration i.e. a pronounced
substrate selectivity for RseA turns DegS into a unique protease
that offers the opportunity to develop selective inhibitors.
Proteases are well-established drugable targets.[8] After
many years of limited research activity, the potential of
proteases as targets for developing antibiotics has recently been
rediscovered.[9] For example, inhibitors for bacterial proteases
such as ClpXP, the bacterial proteasome or intramembrane
proteases such as rhomboids were generated recently.[10]
Although small molecule modulators of DegS have already been
reported,[11] these cannot be used in vivo. Therefore, more
suitable DegS inhibitors are required to chemically validate
inhibition of the bacterial cell envelope stress pathway as a new
strategy for antibiotic development.
BioVersys AG, Hochbergerstrasse 60C, CH-4057 Basel
(Switzerland)
To identify suitable DegS inhibitors, we performed an
Alpha-screen-based high-throughput screen (HTS) comprising
185.000 chemical compounds, 75 nM DegS, 18 nM of the
periplasmic fragment of RseA (later denoted as RseA) as the
substrate, and 37.5 nM Boc-FFF-OH as an activating peptide
mimicking the C-termini of misfolded hydrophobic proteins
(Supporting Fig. 1). 26 compounds displayed 30% DegS
inhibition of DegS at 100 µM and were thus chosen as primary
hits. Two subsequent concentration-dependent counter screens,
i.e. a second RseA digestion assay (10 µM DegS, 30 µM RseA,
Dr. K. Bravo-Rodriguez, Prof. E. Sanchez-Garcia
Computational Biochemistry, Faculty of Biology & Faculty of
Chemistry, Center of Medical Biotechnology, University Duisburg-
Essen, Universitätsstr. 2, 45117 Essen (Germany)
Prof. Dr. J. Steinmann, Prof. Dr. J. Buer
University Hospital Essen, University of Duisburg-Essen, Institute of
Medical Microbiology, Hufelandstr. 55, 45122 Essen (Germany)
Prof. Dr. J. Steinmann
Institute of Clinical Hygiene, Medical Microbiology and Infectiology,
Paracelsus Medical University, Prof.-Ernst-Nathan-Straße 1, 90419
Nürnberg (Germany)
†
equal contribution
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cmdc.201900193
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Figure 1. Suitably modified pyrazolo[1,5-a]-1,3,5-triazines are DegS inhibitors. A) Chemical structure of the HTS hit compound 1. B)
Dose-inhibition curves and corresponding IC50 values for indicated pyrazolo[1,5-a]-1,3,5-triazines obtained from gel-based digestion
experiments. C) Chemical synthesis route employed for generating modified pyrazolo[1,5-a]-1,3,5-triazines for structure-activity
relationships. a) i) ethoxycarbonyl isothiocyanate (1 eq.), dichloromethane, 0 °C to rt, 2 h, ii) K2CO3 (1.2 eq.), 90 °C, 2 h, b) methyl
iodide (1-2 eq.), 2 N NaOH/EtOH (1:2), 0 °C to rt, o/n, c) N, N-diethyl aniline (3 eq.), POCl3, 90 °C, 2 – 18 h, d) amine ‘A’ (3 eq.), di-
isopropyl ethyl amine (5-20 eq.), acetonitrile, rt, 17 – 82 h, e) meta-chlorperbenzoic acid (2 – 6 eq.), dichloromethane, rt, 1 – 2.5 h, f)
amine ‘B’ (3 eq.), N-methyl-2-pyrrolidone, 120 °C, 18 – 20 h. D) The docking of compounds 1, 2 and 4 into the PDZ domain of DegS
leads to three main binding poses (C1 – C3) for 1 and 4 filling the binding pockets of the PDZ domain; for 2, only C2 and C3 can be
realized. These binding poses are here exemplarily shown for compound 4.
50 µM Boc-FFF-OH) and a colorimetric digestion assay (2.5 µM
DegS, 500 µM of the DegS substrate VFNTLPMMGKASPV-
pNA) then resulted in the identification of the pyrazolo[1,5-a]-
1,3,5-triazine analogue 1 as the most promising hit (Fig. 1A).
Subsequently, the gel-based RseA degradation assay
comprising 5 µM DegS, 10 µM RseA and 50 µM of the activating
peptide Boc-FFF-OH was used to determine inhibition of DegS
Moderate to no inhibition of these proteases indicated a
promising selectivity (Supporting Fig. 3). Furthermore, 1 was
inactive vs. a DegS mutant lacking the PDZ domain (DegSPDZ
)
suggesting a desirable allosteric mode of inhibition (Supporting
Fig. 4).[12] The observed selectivity allowed us to test whether 1
is able to inhibit DegS activity in living bacteria. To this end, we
used our previously established E. coli-based DegS activity
reporter strain.[11] In this engineered strain, DegS activity
correlates to beta-galactosidase expression which is measured
via hydrolysis of ortho-nitrophenyl-β-D-galactopyranoside in
whole cells. As 1 completely inhibited reporter and thus DegS
activity, we conclude that the inhibitor is reaching the periplasm
by selected compounds. In this assay,
1
displayed
concentration-dependent DegS inhibition with an IC50 value of
94 ± 10 µM (Fig. 1B and Supporting Fig. 2). To initially test
selectivity and potential off target activity, a panel of S1 serine
proteases, i.e. chymotrypsin, trypsin and elastase as well as
human HTRA1, HTRA2 and HTRA3 were investigated.
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by
a
nucleophilic substitution via an addition/elimination
mechanism to introduce the derivatives at the ‘B region’ then
yielded the final pyrazolo[1,5-a]-1,3,5-triazines. This synthesis
route was used to synthesize 31 different derivatives
(Supporting Information). Together with further 29 compounds
from a chemical library, these were subsequently tested for their
DegS inhibitory potential, thereby revealing structural
determinants for inhibition.
Figure 3. Pyrazolo[1,5-a]-1,3,5-triazine-based DegS inhibitors inhibit outer
membrane stress response and act synergistically to outer membrane stress
inducing antibiotics. A) An in vivo screen with a DegS activity reporter strain
demonstrates that active DegS inhibitors such as 3 impair outer membrane
stress response (that was triggered by addition of the outer membrane
perturbing agents Colistin (Col) and Polymyxin nanopeptide B (PmBN)) while
the inactive control compound 2 does not. B) Measurements of minimal
inhibitory concentrations for E. coli growth in absence or presence of the outer
Figure 2. Pyrazolo[1,5-a]-1,3,5-triazine-based DegS inhibitors show DegS-
dependent growth inhibitory properties. E. coli WT strain CAG16037 and the
degS knockout strain CAG33315[13] were grown in presence of the indicated
concentrations of 1 as well as the in presence of the inactive compound 2 and
the DegS inhibitor 3. Growth curves were normalized to the corresponding
DMSO control. DegS inhibitors show degS-dependent growth effects while the
inactive derivative 2 does has no effects.
membrane perturbing chemotherapeutic Colistin that was used at
a
concentration corresponding to 1/8 of its MIC demonstrates synergism
between compounds.
(Supporting Fig. 5). As DegS is widely conserved in bacteria, we
determined minimal inhibitory concentrations (MIC) for 1 by
measuring growth of various Gram positive and Gram negative
bacteria. For most bacterial strains, MICs ranged between 100-
600 µM (Supporting Table 1). These data suggest that DegS is
essential in these strains even under optimal growth conditions,
i.e. in rich media. Altogether, our results indicate that 1 may
indeed represent a valuable starting point for DegS inhibitor
development, although 1 displays overall only moderate DegS
inhibitory potency in vitro and in vivo.
To obtain more potent compounds, we started to
synthesize pyrazolo[1,5-a]-1,3,5-triazine derivatives. 1 features
three “main” substitution sites (labelled as A, B and C in Fig. 1A).
To allow structural variation at all three positions, our synthetic
route started from 2-amino pyrazole derivatives that were
reacted with ethoxycarbonyl isothiocyanate (Fig. 1C).
Methylation of the thiourea moiety with methyl iodide, followed
by phosphoroxy trichloride-mediated chlorination yielded the
intermediates for introduction of the different ‘A region’
substitutes. Oxidation via meta-chloroperbenzoic acid, followed
Most notably, all structural changes at the ‘B’ position of 1,
including minimal changes such as the introduction of additional
methyl groups at the aminopiperidine moiety as done in
derivative 2 led to inactive compounds with IC50 > 400 µM,
indicating that this region of the inhibitor is interacting with a
distinct binding pocket (Supporting Fig. 6 and Supporting Table
2). In contrast, modifications at the ‘C’ position were better
tolerated, although none of the synthesized compounds
displayed much improved inhibitory potential (Supporting Table
2). The introduction of meta-substituted benzyl amine derivatives
at the ‘A’ position led to significantly better inhibitors, while
substitutions at the ortho- or para-position or non-benzyl amine
derivatives did not increase or even result in inactive derivatives
(Supporting Table 2). Insertion of hydrophobic residues led to
the best inhibitory improvements. For example, a 3-bromo
benzyl amine in 3 resulted in an IC50 of 33 ± 3 µM. The best
inhibitory potential was observed for a 3-benzoxybenzyl amine in
4 that inhibited DegS with an IC50 of 1.1 ± 0.4 µM and thus was
almost 100-times more potent than the parent compound 1 (Fig.
1B). These studies therefore demonstrated that pyrazolo[1,5-a]-
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1,3,5-triazines represent a promising DegS inhibitor class with
defined structure-activity relationships and potent inhibitory
potential.
disrupting and bactericidal agent, PmBN is considerably less
antibacterial, lacks bactericidal activity and mainly acts to
disorganize and permeabilize the outer membrane. In
accordance with our findings with 1, these assays revealed that
also the more potent DegS inhibitor 3 completely impaired DegS
reporter activity at 30 µM while the inactive control compound 2
had no effects (Fig. 3A). Accordingly, these results not only
demonstrate that 3 targets DegS in vivo but also indicates that
DegS inhibitors may act synergistically with outer membrane
perturbing antibiotics. Moreover, our chemical approach
supports previous genetic evidence indicating that DegS is
essential even under optimal growth conditions.[13]
Given the well-established implications of DegS in cell
envelope protein stress response, we tested the synergy of
DegS inhibition by pyrazolo[1,5-a]-1,3,5-triazines with the outer
membrane perturbing and last resort antibiotic Col. To this end,
we first determined the minimal inhibitory concentration of Col
for E. coli ATCC25922 as 0.25 µg/mL (Supporting Table 3).
Subsequently, the corresponding MIC for various pyrazolo[1,5-
a]-1,3,5-triazines either in absence or in presence of 0.03 µg/mL
Col (corresponding to 1/8 MIC of Col) was determined (Fig. 3B).
From these values, we calculated the fractional inhibitory
concentration (FIC) index as a measure of synergy between two
compounds. For all active DegS inhibitors, a FIC index ≤ 0.5 was
determined, indicating synergy. In addition, we tested synergy
for further Gram negative bacteria. While the combination of 1
and colistin was synergistic for Acinetobacter baumanni,
Enterobacter cloacae and Klebsiella pneumoniae, no synergy
was detected for Pseudomonas aeruginosa (Supporting Table
4); as 1 alone had no effect on Pseudomonas aeruginosa but on
the three other strains (Supporting Table 1), this finding further
supports the suggested synergy mechanism. Moreover, the
synergy between the DegS inhibitor and membrane-perturbing
antibiotic is selective. We have tested the combination of 1 with
other antibiotic classes with different mechanisms of action and
could not see any increase in antibiotic susceptibility (Supporting
Table 5). Overall, these studies demonstrate that impairment of
the outer membrane stress response via DegS acts
synergistically in combination with established antibiotics
inducing outer membrane stress.
To rationalize the structure-activity relationship of the
pyrazolo[1,5-a]-1,3,5-triazines at the molecular level, we
explored the binding of 1 (parent compound with intermediate
activity) and 4 (best binder) to DegS. To validate our model, we
also used the inactive compound 2 as a negative control. The
first step was to perform docking calculations of these
compounds to the PDZ domain of DegS. Binding to the protease
domain was discarded since the parent compound 1 is not able
to inhibit DegSΔPDZ (Supporting Fig. 4). We thus identified three
main binding poses (C1, C2 and C3) of 1, 2 and 4 to the PDZ
domain of DegS (Fig 1D). The analysis of C1 and C2 allows
rationalizing why chemical modifications of positions ‘B’ and ‘C’
failed to improve the IC50 of the compounds since in these
binding poses both positions are placed in PDZ binding pockets
that are limited in space. Next, we performed molecular
dynamics (MD) simulations of the three docked compounds with
C1 and C2 as starting positions. The MD simulations then
showed that both conformations lead to protein-ligand
complexes that were conserved during the simulations (see
Supporting Information for further discussion). In C1 and C2, all
compounds also engage in interactions with loop L3 as indicated
by the dynamic network analysis (Supporting Fig. 7-9).[13] To
initially test this model, we reasoned that binding of 1 to the PDZ
domain of DegS could be contested by allosteric activators. We
therefore examined inhibition of DegS by 1 in the presence of
two peptides that are known allosteric activators i.e.
DNRLGLVYQF and DNRLGLVYWF, the affinity of which is 53
µM and 3.2 µM, respectively.[6] Both peptides prevent inhibition
of DegS, suggesting competitive binding to the PDZ domain and
thus demonstrating feasibility of the proposed binding mode
(Supporting Fig. 10).
We continued to evaluate this class of inhibitors by
measuring DegS target engagement in living E. coli cells using 1
as the starting compound as well as 3 as an inhibitor with better
inhibitor potential (note that the most active compound 4 did not
display sufficient water solubility at higher concentrations to
perform these assays) and the inactive compound 2 as a
negative control. To this end, we investigated the impact of the
three compounds on the growth of the DegS WT E. coli strain
CAG16037 as well as the degS knockout strain CAG33315
(note that strain CAG33315 must contain so far unknown
suppressor mutations allowing it to grow in the absence of degS)
(Fig. 2). In these assays, the DegS inhibitors 1 and, in
accordance with its higher inhibitory potential, to a larger extent
3 displayed dose-dependent growth defects; more importantly,
stronger effects were observed for the WT than for the degS
knockout strain. In contrast, the inactive control compound 2
was insensitive to the different genetic backgrounds. These
results demonstrate that the employed compounds target and
inhibit DegS in living bacteria, leading to differential DegS-
dependent growth defects.
In summary, we have developed the first non-covalent
small molecule inhibitors of DegS. These inhibitors are based on
a pyrazolo[1,5-a]-1,3,5-triazine scaffold and display distinct
structure-activity relationships. These compounds also inhibit
DegS in living bacteria, thus allowing for the first time to
demonstrate that in vivo target engagement induces growth
inhibitory effects, in particular under stress conditions. In fact,
chemotherapeutical induction of outer membrane stress is
synergistic to DegS inhibition. Together, these findings validate
DegS and the E stress pathway as new targets for the
development of antibiotics with a novel mode-of-action, thus
opening promising avenues for rational antibiotic drug design.
These results encouraged us to further test in vivo target
engagement. Accordingly, we modified the DegS activity
reporter strain by employing the outer membrane perturbating
antibiotics Colistin (Col) and Polymyxin B nonapeptide (PmBN)
for DegS activation. While Col is a highly potent membrane-
Acknowledgements
Financial support by CRC1093, EH 100/16-1 and KA 2894/4-1
by Deutsche Forschungsgemeinschaft to M.E., E.S.-G. and M.K.
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Science (MIWF) of the province North-Rhine Westphalia (NRW),
Germany to Lead Discovery Center GmbH. E.S-G.
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Keywords: antibiotics • drug discovery • small molecule •
synthesis • target
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J. Bongard, A. L. Schmitz, A. Wolf, G.
Zischinsky, M. Pieren, B. Schellhorn, K.
Bravo-Rodriguez, J. Schillinger, U.
Koch, P. Nussbaumer, B. Klebl, J.
Steinmann, J. Buer, E. Sanchez-Garcia,
M. Ehrmann*, M. Kaiser*
Page No. – Page No.
Text for Table of Contents
Chemical validation of DegS as a
target for the development of
antibiotics with a novel mode of
action
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