Virtual screening of peptide and peptidomimetic fragments targeted to inhibit bacterial dithiol oxidase DsbA

Article abstract of DOI:10.1371/journal.pone.0133805

Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB - or peptides - complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

Full text of DOI:10.1371/journal.pone.0133805

RESEARCH ARTICLE
Virtual Screening of Peptide and
Peptidomimetic Fragments Targeted to
Inhibit Bacterial Dithiol Oxidase DsbA
Wilko Duprez☯, Prabhakar Bachu☯, Martin J. Stoermer, Stephanie Tay,
Róisín M. McMahon, David P. Fairlie, Jennifer L. Martin*
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland,
Brisbane, Queensland, Australia
☯ These authors contributed equally to this work.
* j.martin@imb.uq.edu.au
a11111
Abstract
Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately
needed to address the growing problem of antibiotic resistance. The periplasmic oxidative
folding system in Gram-negative bacteria represents a possible target for anti-virulence
antibacterials. By targeting virulence rather than viability, development of resistance and
side effects (through killing host native microbiota) might be minimized. Here, we undertook
the design of peptidomimetic inhibitors targeting the interaction between the two key
enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence
factor assembly. Structures of DsbB - or peptides - complexed with DsbA revealed key inter-
actions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the
active site. The present work aimed to discover peptidomimetics that target the hydrophobic
groove to generate non-covalent DsbA inhibitors. The previously reported structure of a
Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the hep-
tapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomi-
metic fragment library. The highest scoring fragment compound and nine derivatives were
synthesized and evaluated for DsbA binding and inhibition. These experiments discovered
peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only
weakly potent relative to larger covalent peptide inhibitors that interact through the active
site cysteine, these fragments offer new opportunities as templates to build non-covalent
inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with
sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.
OPEN ACCESS
Citation: Duprez W, Bachu P, Stoermer MJ, Tay S,
McMahon RM, Fairlie DP, et al. (2015) Virtual
Screening of Peptide and Peptidomimetic Fragments
Targeted to Inhibit Bacterial Dithiol Oxidase DsbA.
PLoS ONE 10(7): e0133805. doi:10.1371/journal.
pone.0133805
Editor: L. Michel Espinoza-Fonseca, University of
Minnesota, UNITED STATES
Received: May 12, 2015
Accepted: June 13, 2015
Published: July 30, 2015
Copyright: © 2015 Duprez et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: Australian Research Council Laureate
Fellowship FL0992138 to JLM. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Introduction
The recent emergence of ‘extremely drug resistant’ bacterial pathogen strains is a major public
health concern [1] exacerbated by the low number of newly approved drugs to treat bacterial
infections [2–4]. The World Health Organization has warned that we are entering a “post-
Competing Interests: The authors have declared
that no competing interests exist.
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Virtual Screening for DsbA Inhibition
antibiotic era” where minor infections will be deadly [5], while US President Obama issued an
Executive Order to combat antimicrobial resistance [6]. Since the early 1980s newly approved
antibiotics, with the exception of six classes, have been analogues of previously released scaf-
folds [7]. Moreover the six new classes [8–13] target Gram-positive pathogenic bacteria, con-
tributing to an urgent need to develop new treatments aimed at infectious Gram-negative
bacteria, particularly those among the “ESKAPE” pathogens [1]. Due to a shortage of novel
naturally-occurring antibiotics, efforts have been made to design new antimicrobial scaffolds
with different modes of action [14–18]. One approach is to target bacterial virulence pathways,
which are thought to be less likely to induce resistance mechanisms [19]; neutralizing pathoge-
nicity without impeding bacterial viability might limit adaptive resistance to the drug [20] and
also reduce the impact on host native microbiota [21].
The periplasmic oxidative folding machinery that catalyses protein folding through disulfide
bond formation is a potential target for antivirulence therapeutics and is widespread in Gram-
negative bacteria [22]. Substrates of the pathway include components essential for pili forma-
tion and motility, host cell adhesion, toxin production, and secretion [23]. The archetypal
machinery characterized in Escherichia coli K12 (Fig 1A) involves Dsb (disulfide bond form-
ing) proteins [24, 25]. DsbA is a dithiol oxidase comprising a thioredoxin (TRX) domain and
an inserted α-helical domain [26, 27]; a CXXC motif (30CPHC33 in E. coli) forms the catalytic
site [28] together with a cisPro loop and a groove formed from hydrophobic residues including
Phe36, Phe174 and Tyr178. In the active oxidized state, E. coli DsbA (EcDsbA) has a disulfide
bond between Cys30 and Cys33 which is transferred to a substrate through bimolecular nucle-
ophilic transfer (SN2) (Fig 1A) [29–31]. Oxidative folding of the substrate converts EcDsbA to
Fig 1. The DsbA-DsbB interaction. A. Schematic showing the proposed mechanism of oxidative folding in the periplasm of Gram-negative bacteria. DsbA
catalyses the formation of a disulfide bond in a protein substrate, then interacts with DsbB to which it transfers electrons so that DsbA is regenerated into its
active oxidized state. The electrons are subsequently transferred from DsbB to ubiquinone (UQ) and ultimately to the respiratory complex. B. The binding
interface between EcDsbA (black and red) and EcDsbB loop P2 (blue) derived from the crystal structure of the EcDsbAC33A:EcDsbBC130S complex [37].
The EcDsbA hydrophobic groove residues are highlighted in orange shading, the intermolecular disulfide bond is shown as a solid red line and the hydrogen
bond with the cisPro loop is shown as a dashed red line. C. The binding interface between PmDsbA (black and red) and the peptide PWATCDS (blue) from
the crystal structure of the complex [38]. In this complex there is no disulfide bond as the active site cysteine of PmDsbA was mutated to Ser (S30). The
peptide Cys5 residue points away from the binding interface. Residues W2 and P1 of the peptide both interact with the hydrophobic groove (in orange) and
these interactions were used as the target for this peptidomimetic design.
doi:10.1371/journal.pone.0133805.g001
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Virtual Screening for DsbA Inhibition
the inactive reduced form, which can then interact with the periplasmic loop P2 of transmem-
brane partner EcDsbB (Fig 1A) [32]. The EcDsbA-EcDsbB interaction regenerates the oxidized
state of EcDsbA through SN2 transfer of electrons to EcDsbB [33, 34]. Inhibition of the EcDs-
bA-EcDsbB interaction would block oxidation of EcDsbA and thereby block oxidative folding
of virulence factors. Accordingly, the phenotype of dsbA/dsbB null uropathogenic E. coli
(UPEC) cells is severe attenuation of virulence in a mouse infection model, though bacteria
remain viable [35]. Similarly, mice infected with a dsbA mutant of B. pseudomallei all survived
whereas mice infected with wildtype all died [36].
The crystal structure of the EcDsbA-EcDsbB complex has been determined, through use of
a covalent complex that trapped the otherwise transient interaction between the two proteins
[39–41]. The structure revealed details of the intermolecular disulfide bond between EcDsbA
Cys30 and EcDsbB Cys104, as well as a hydrogen bond between main chain atoms of EcDsbA
Arg148 (on the cisPro loop) and EcDsbB Phe106, and hydrophobic contacts between EcDsbB
Pro100 and Phe101 and the EcDsbA hydrophobic groove (Fig 1B). We have identified peptides
that bind to oxidized EcDsbA with low micromolar affinity (Kd values 2–20 μM), but they all
required a cysteine for inhibition of EcDsbA, suggesting that they targeted an active site cyste-
ine in order to inhibit the enzyme [42]. We have also co-crystallized the peptide PWATCDS
with a Cys30Ser mutant of Proteus mirabilis DsbA to generate a non-covalent complex (no
disulfide bond is formed) (Fig 1C) [38]. In this non-covalent complex, the Pro1 and Trp2 resi-
dues of PWATCDS interact with the hydrophobic groove in a manner similar to that described
for the EcDsbB periplasmic loop P2 interaction with EcDsbA (Fig 1C).
In the present work, we explored the importance of the DsbA hydrophobic groove for inhi-
bition by designing and developing small peptide-derived molecules predicted by computer
modeling to bind to this region. EcDsbA and PmDsbA are very similar proteins [38] so we
used the high-resolution PmDsbAC30S-PWATCDS non-covalent protein-heptapeptide com-
plex structure as the starting point for an in silico virtual screen of a peptidomimetic library.
Our goal was to move from peptides to more ‘drug-like’ compounds, by designing and screen-
ing peptidomimetics. The resulting in silico hit and nine derivatives were synthesized and their
affinities and inhibitor potencies for native EcDsbA were measured using a combination of dif-
ferential scanning fluorimetry, isothermal titration calorimetry (ITC) and an enzyme assay.
The compounds were weak inhibitors of EcDsbA, suggesting that additional binding interac-
tions will be required to generate significant inhibitor potency.
Materials and Methods
Virtual Docking
The 1.6 Å resolution crystal structure of the PmDsbAC30S-PWATCDS protein-heptapeptide
complex (PDB code 4OD7 [38]) was used to dock putative ligands in the substrate-binding
active site of DsbA. The bound peptide was removed from the structure and hydrogen atoms
were added using the Hermes interface in GoldSuite 5.1 [43]. Additionally the indole ring sys-
tem of Trp2 of the bound peptide was separately stored in the same XYZ coordinate space to
serve as a template file for the template docking mode within GOLD. The PWA tripeptide was
replaced with a small targeted library of 10 compounds based on both D- and L-tryptophan
cores, and both hydrophobic and hydrophilic C- and N-terminal capping groups. These candi-
date virtual ligands were prepared from 2D ChemDraw (CS ChemBioDraw Ultra 12.0. Cam-
bridge Scientific Computing) representations via SMILES strings, and minimum energy
conformers were prepared using OMEGA2 v2.4.6 [44–46] and the mmff94s forcefield. Dock-
ing with GOLD 5.1 was performed using the standard precision settings and the default
CHEMPLP scoring function. Docking results were visualized in Pymol [47–49]. The initial
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Virtual Screening for DsbA Inhibition
template docking with the indole fragment in its previously determined location [38] was car-
ried out with the template constraint set to the default values and progressively reduced to
allow for better fit docking and to not overly bias in favour of the observed crystal structure
bound conformation of the Trp. The backbone amine and carbonyl of Trp2 in the bound full
length peptide do not form hydrogen bonds with PmDsbAC30S [38], allowing replacement
and removal to explore whether simpler small molecules could bind with conformations differ-
ing from the full length peptide, with only the indole ring of Trp2 used as a hydrophobic
anchor. The steady reduction in the template constraint also allowed subtle changes to arise
that may provide additional interactions in the case of smaller molecules, which would reason-
ably be assumed to adopt multiple possible binding conformations. Indeed if the constraint
was completely reduced, other conformations were observed (up to 4 out of 10 docking poses),
some of which included extra H-bonds with the protein, particularly with the charged mole-
cules. Nevertheless, the dominant interactions in this portion (PWA) of the heptapeptide
(PWATCDS) were the hydrophobic interactions of the Pro and Trp side chain fragments.
Peptidomimetic synthesis
All purchased chemicals were used without further purification. All solvents were HPLC grade.
Boc-Trp-OH (1.00 mmol) was dissolved in 10 mL of dioxane under nitrogen. Carbonyldiimi-
dazole (1.02 mmol) was then added in a stepwise manner and the resulting mixture stirred for
3 h at room temperature and then heated to 50°C for 30 min. The desired amine (1.03 mmol)
was added to the reaction mixture at room temperature and stirring was continued for 48 h
(Fig 2). The solvent was removed under reduced pressure and crude product was extracted
with ethyl acetate (3 x 20ml). The organic extracts were washed with 1M hydrochloric acid
(15 mL), saturated sodium bicarbonate solution (15 mL) and water (15 mL), then dried over
anhydrous MgSO4 and the solvent was removed under reduced pressure. To this crude com-
pound in water (1 mL) was added anisole (2.00 mmol). The solution was then cooled to 0°C
prior to addition of trifluoroacetic acid (30.00 mmol) in 1 mL of water. After stirring for 1 h at
0°C, the reaction mixture was allowed to warm to room temperature and stirring was contin-
ued for 12 h. The reaction mixture was then diluted with ethyl acetate (15 mL) and washed
with saturated sodium bicarbonate solution (15 mL), water (15 mL) and brine solution
(15 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure
to afford the crude free amine which was directly used in the next step without further purifica-
tion. To a solution of the above crude amine in dichloromethane (5 mL) was added carbonyl-
diimidazole (1.2 mmol) under nitrogen at room temperature. After 3 h stirring, morpholine or
1-Boc-piperazine (1.55 mmol) was added to the reaction mixture and stirred for another 12 h
at room temperature. The solvent was removed under reduced pressure, diluted with ethyl
Fig 2. Synthetic route for the tripeptide peptidomimetics.
doi:10.1371/journal.pone.0133805.g002
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Virtual Screening for DsbA Inhibition
acetate (15 mL) and washed with 1M hydrochloric acid (15 mL), saturated sodium bicarbonate
solution (15 mL), brine solution (15 mL) and dried over anhydrous MgSO4. The crude product
was then purified by preparative HPLC (Gradient 0 to 100% of 95/5 acetonitrile/water solution
over 25 min) and freeze-dried. NMR spectral data of all synthesized compounds 1–10 are
included in the supporting information (S1–S10 Figs). NMR spectra were recorded on Bruker
Avance DRX-600 and Varian 400 MHz spectrometers at 298 K with TMS as internal standard.
High-resolution mass spectrometry (HRMS) was performed on a Bruker micro-TOF by direct
infusion in acetonitrile/H2O 70:30 at 3 μL/min using sodium formate clusters as an internal
calibrant. Semi-preparative RP-HPLC purification of the compounds was performed using a
Waters Delta 600 chromatography system fitted with a Waters 486 tuneable absorbance detec-
tor with detection at 214 nm. Purification was performed by eluting with solvents A (0.1% TFA
in water) and B (9:1 CH3CN:H2O, 0.1% TFA) on a Vydac C18 250 x 22 mm (300 Å) steel jac-
keted column at 20 mL/min. NH peak of Indole is not observed in some of the compounds in
CDCl3 due to peak broadness.
Protein expression and purification
Wild type E. coli DsbA was expressed and purified in our laboratory as previously described
[42, 50].
E. coli membrane preparations containing over-expressed EcDsbB (GenBank accession
number AAC74269) were produced as previously reported [51], and resuspended in PBS buffer
containing 10% glycerol.
Differential Scanning Fluorimetry
All differential scanning fluorimetry experiments were performed on a ViiA 7 Real-Time PCR
instrument (Life Technologies, Applied Biosystems Division) using 384 well MicroAmp Clear
Optical Plates (Life Technologies, Invitrogen Division). Oxidised EcDsbA (25 mM HEPES pH
7.4, 50 mM NaCl) was prepared at a final concentration of 2.5 μM, in the presence of either 5%
(v/v) DMSO (reference) or 2, 1, 0.5, 0.25, 0.125 or 0.0625 mM of the test compound (also in a
final concentration of 5% DMSO (v/v.)) A 5000x stock of SYPRO Orange Protein Gel Stain
(Life Technologies, Invitrogen Division) was diluted to a final concentration of 5x. The final
reaction volume was 20 μL. For each condition, five technical replicates were included. Non-
protein control reactions (DMSO or compound, and dye in the absence of EcDsbA) were also
included to monitor compound-dye interactions. Plates were sealed with Axygen Ultra Clear
Pressure Sensitive Sealing Film (Fisher Biotec) and were centrifuged immediately prior to anal-
ysis (1000 x rpm, 2 min) to draw down all liquid and break any bubbles. A standard melt curve
analysis was conducted using a temperature ramp from 25°C to 99°C at a rate of 0.05°C/s. Fluo-
rescence was detected using the in built ‘m4’ filter set (excitation λ 470 15 nm, emission λ 586
10 nm) and data were analyzed using Prism Software (v6.0a, GraphPad). Tm was determined
by fitting a Boltzmann equation to the data.
Isothermal titration calorimetry
Oxidized EcDsbA was diluted to 50 μM in 25 mM HEPES pH 7.4, 50 mM NaCl and 5%
DMSO. Compounds were dissolved in 100% DMSO, and then diluted to a final concentration
of 2 mM into 25 mM HEPES pH 7.4 50 mM NaCl with a final DMSO concentration of 5%. All
experiments were performed using the MicroCal Auto-ITC200 instrument (GE Healthcare,
USA), loading 200 μL of EcDsbA in the sample cell and 40 μL of compound in the syringe.
Titrations were set up at 25°C with 19 injections of 2 μL separated by 180 seconds and a con-
stant stirring speed of 1000 rpm. A preliminary injection of 0.5 μL was added to avoid slow
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Virtual Screening for DsbA Inhibition
leakage of titrant into the sample cell before the first 2 μL injection and the corresponding data
point was excluded from analysis. Every compound was tested with three technical replicates.
An additional titration of compound into buffer only (25 mM HEPES pH 7.4, 50 mM NaCl
and 5% DMSO) was performed to measure the background heat of dilution. Analysis was per-
formed with the MicroCal Origin software (version 7.0552).
Enzyme assay
Lyophilized EcDsbA synthetic substrate (peptide CQQGFDGTQNSCK) which contains a C-
terminal methylcoumarin and an N-terminal 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid (DOTA) group (Anaspec, USA) was dissolved in 100 mM imidazole pH 6.0.
100 mM of europium trifluoromethanesulfonate (Sigma Aldrich, Australia) was added to the
synthetic substrate peptide at a molar ratio of 2:1 and incubated for 5 min at room temperature
to allow europium chelation. Experiments were performed in white 384-well plates (Perkin
Elmer OptiPlate-384, Part #: 6007290) in 50 mM MES, 50 mM NaCl and 2 mM EDTA pH 5.5
buffer. Each well contained 80 nM EcDsbA, 1.6 μM EcDsbB, one of the tested compounds at
concentrations ranging from 16 μM to 2 mM and 10 μM synthetic substrate peptide (added
last to initiate the reaction) for a final volume of 50 μL per well. Positive controls were set up in
which compound was replaced with buffer, and negative controls lacked EcDsbA or EcDsbB.
Fluorescence emitted by substrate folding was measured with a Synergy H1 multimode plate
reader (excitation λ = 340 nm and emission λ = 615 nm). Analysis was performed using the
Prism software (v6.0a, GraphPad). A very low compound concentration value was used (1 nM)
to allow plotting the activity of the positive control (the native EcDsbA activity without com-
pound) on a logarithmic scale of compound concentration.
Results
Virtual screening and compound synthesis
The 1.6 Å resolution PmDsbAC30S-PWATCDS crystal structure PDB code 4OD7 [38], rather
than the 3.7 Å resolution EcDsbB-EcDsbA crystal structure (PDB 2ZUP [40]), was used to ana-
lyse the hydrophobic groove interactions (Fig 3A). Virtual screening of a peptidomimetic
library was performed by focusing on the interaction of Trp2 in heptapeptide PWATCDS with
Tyr173 in PmDsbAC30S within the hydrophobic groove (Fig 3B). The compound with the
best fit, hereafter named compound 1, was a tryptophan residue flanked by a C-terminal mor-
pholine functional group and an N-terminal benzyl moiety (Fig 3C). Apart from the pi-stack-
ing interaction between the Trp indole and Tyr173 of PmDsbAC30S, the ligand docking model
showed the oxygen atom of the morpholine group at similar distances from the PmDsbAC30S
Pro150 backbone amide, His32 imidazole ring amines, and the Asn162 side chain amide
(ꢀ 4.5 Å). Compound 1 scored well in docking experiments performed using three different
programs Goldscore, Chemscore and ChemPLP. Secondly, it had a good balance between
aqueous solubility and "drug-like" properties (CLogP = 2.6, 6 rotatable bonds, 3 hydrogen
bond acceptors, 3 hydrogen bond donors, MW = 406 Da, and is neutral at physiological pH).
Compound 1 was an attractive first target for synthesis because it was predicted to extend its
indole ring into the Trp-binding pocket, form hydrophobic interactions from its benzylamide
group with the same region as the proline of the original peptide, and insert its morpholine
ring into the groove.
Nine derivatives of 1 were also synthesized (Fig 4) to explore the binding groove and exploit
potential optimization possibilities. N-Boc-tryptophan was coupled with a variety of amines
using carbonyliimidazole and then deprotected using trifluoroacetic acid and anisole to give
free amines at room temperature. This was subjected to treatment with carbonyldiimidazole to
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Virtual Screening for DsbA Inhibition
Fig 3. Comparison of the docked designed peptidomimetic with the EcDsbA-EcDsbB and PmDsbA-PWATCDS crystal structures. Calculated
electrostatic surfaces of the enzymes are shown, with acidic regions in red, basic regions in blue and non-polar (hydrophobic) regions in white. Electrostatics
cut-offs used are +/- 7.5 keV. A. Detail of the EcDsbA complex with EcDsbB from the crystal structure (PDB code 2ZUP [37]) centred on the
97YPSPFATCDFMVR109 sequence of EcDsbB (in light blue) showing Phe101 (F101) binding in the EcDsbA hydrophobic groove (circled). B. Detail of the
PmDsbAC30S:PWATCDS crystal structure (PDB code 4OD7) with PWATCDS in magenta. Residue Trp2 (W2) of the peptide binds in the PmDsbA
hydrophobic groove (circled). C. Virtual screening identified compound 1 as a potential hit. Three optimal conformations of 1 are shown (in differing shades of
green), in their predicted binding mode to the PmDsbAC30S hydrophobic groove. Potential hydrogen bonds between the morpholine moiety and DsbA
Pro150, His32 and Asn162 are shown as yellow dashed lines.
doi:10.1371/journal.pone.0133805.g003
afford the activated carbonyl imidazole urea intermediate which reacted smoothly with mor-
pholine or 1-Boc-piperazine at room temperature to afford compounds 1, and 3–10. The
N-Boc of compound 3 was removed with trifluoroacetic acid to afford compound 2.
Differential scanning fluorimetry
The peptidomimetic compounds were first evaluated using differential scanning fluorimetry
(DSF) to detect binding to EcDsbA through a concentration-dependent increase (ΔTm) of
EcDsbA melting temperature Tm. The peptidomimetics were tested over a concentration
range from 64 μM to 2 mM and checked for potential compound-dye interferences that could
lead to unwanted background fluorescence. When mixed with SYPRO orange on a tempera-
ture gradient from 25°C to 99°C, compounds 7 and 8 indicated strong background fluores-
cence at 0.5 mM, 1 mM and 2 mM concentrations. Compounds 1, 3, 4 and 5 showed some
interaction with the dye at the highest concentration (2 mM) but the resulting fluorescence
decreased to background level well before the DsbA unfolding point (<50°C), and thus would
not be expected to interfere with the analysis. Finally, compounds 2, 6, 9, and 10 did not dem-
onstrate substantial fluorescence from interaction with the dye. For each compound, concen-
trations that demonstrated interference with the dye were excluded from the following binding
experiments with EcDsbA.
To be considered significant, a binding-induced thermal shift value ΔTm should be at least
twice that of the estimated standard deviation of the native protein Tm [52], in this case 0.1°C.
We found that the measured ΔTm values were less than 0.2°C, and the compounds also failed
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Virtual Screening for DsbA Inhibition
Fig 4. Chemical structures of the 10 peptidomimetic compounds synthesized and tested in this work.
Compound 1 is the hit from the virtual screening from which derivatives 2–10 were designed.
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Virtual Screening for DsbA Inhibition
Fig 5. Compound 10 demonstrated weak inhibitory activity. A. Differential scanning fluorimetry profile with increasing concentrations of compound 10.
Similar to all other compounds tested, there was no significant shift in the unfolding temperature of EcDsbA up to 2 mM of compound 10. B. ITC profile of
EcDsbA titration by compound 10, which shows no detectable binding under the conditions used (see methods for details). A similar outcome was found for
the other 9 compounds. C. Compound 10 was the only one of the ten tested peptidomimetics that exhibited detectable activity in the DsbA assay, inducing a
reduction in DsbA folding activity. D. Plotting the log of the peptidomimetic concentration against the rate of fluorescence increase measured in the enzyme
assay allowed fitting of a sigmoidal curve and an estimated IC50 value of ~1 mM for compound 10. The positive control with no compound is shown as a white
circle.
doi:10.1371/journal.pone.0133805.g005
to demonstrate a concentration-dependent effect on Tm (Fig 5A). These data suggested that
the peptidomimetic compounds did not bind tightly to EcDsbA. However, DSF results can be
biased by the intrinsic affinity of the compounds for the dye, by virtue of their hydrophobicity.
To further investigate the potential of this series of peptidomimetic, their affinity was also eval-
uated using a label-free approach, namely isothermal titration calorimetry.
Isothermal titration calorimetry
The affinity and thermodynamics of the 10 compounds were measured using ITC allowing
comparison with the original peptide sequence PWATCDS (Kd 3.0 0.1 μM, ΔH -14.3 0.5
kcal.mol-1, ΔH -22.6 2.8 cal.mol-1.deg-1 for EcDbsA [42] and Kd 8.3 0.4 μM, ΔH -13.7 0.3
kcal.mol-1, ΔS -22.3 1.1 cal.mol-1.deg-1 for PmDsbA [38]). Titration of EcDsbA with com-
pound 1 revealed little evidence of binding (low release of energy -0.60 μcal.sec-1 for the highest
peak) or EcDsbA saturation. Similarly for derivatives 2–10 no substantial binding was detected
under the conditions used (Fig 5B). The ITC profiles were similar to the negative controls, sug-
gesting that the compounds did not bind to or had at best weak (millimolar range) affinity for
EcDsbA. The low ITC signals could also result from a low enthalpic contribution to binding
which might be a consequence of ligand interaction with a hydrophobic binding site [53]. We
therefore assessed the ability of the compounds to inhibit the enzymatic activity of EcDsbA.
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Virtual Screening for DsbA Inhibition
Enzyme assay
EcDsbA activity can be assessed using a synthetic substrate and a fluorescent assay [50, 54]
Inhibition of EcDsbA activity is demonstrated by a reduction in the rate of fluorescence signal
emission. Inhibition by the peptidomimetics could be due to competition with the EcDsbA-
substrate interaction, the EcDsbA-EcDsbB interaction or the EcDsbB-ubiquinone electron
exchange. None of the compounds 1–9 showed any effect on EcDsbA activity at the concentra-
tions used. However, compound 10 exhibited a reduction in fluorescence signal at high con-
centrations (1 mM and 2 mM, Fig 5C). Plotting the concentration versus the rate of
fluorescence increase enabled an IC50 of 1.1 mM to be estimated for compound 10 (Fig 5D).
This value is ~200-fold lower than the value for the 9-residue peptide from native EcDsbB
(PSPFATCDF, 6.7 1.1 μM, [42]) or the optimized 7-residue peptide PWATCDS
(5.7 0.4 μM [42]) for EcDsbA. Taken together, the data we present here indicate that these
much smaller peptidomimetic compounds were poor inhibitors of EcDsbA activity with IC50
values estimated in the millimolar range.
Discussion
The antibiotic pipeline is in urgent need of new antibacterials especially for Gram-negative
pathogens. EcDsbA is a potential antibacterial target because it catalyzes the assembly of viru-
lence factors. The present work is part of an effort towards the development of EcDsbA inhibi-
tors. It follows up on the observation that a peptide PWATCDS binds non-covalently to a
cysteine mutant of P. mirabilis DsbA (PmDsbAC30S), making interactions with the hydropho-
bic groove of the enzyme [38]. The series of peptidomimetic fragments described here was
designed to target the hydrophobic peptide- and DsbB-binding groove of the EcDsbA protein.
Peptidomimetics have been highlighted as an important source of novel antimicrobial scaffolds
[55–62]. They offer the advantage of being able to mimic protein-protein interactions, but hav-
ing better properties as drug candidates than peptides (improved stability and pharmacokinet-
ics, lower immunogenicity, lower molecular mass [63–67]).
To start this line of fragment-focused research, we employed virtual screening of the
PmDsbAC30S-PWATCDS crystal structure, focusing on the hydrophobic groove fitted by the
7-residue peptide, and identified a virtual hit fragment from a peptidomimetic library (com-
pound 1), around which nine derivatives were also synthesized (compounds 2–10). No evi-
dence of binding was detected by differential scanning fluorimetry or by ITC, but a functional
enzyme assay indicated weak inhibition of EcDsbA by compound 10 (IC50 1.1 mM). Although
these small fragment compounds had > 200-fold reduction in affinity and inhibition of
EcDsbA compared with the much larger 7-residue covalent peptide PWATCDS, they might
represent useful templates for rationally deriving more drug-like small organic and peptidomi-
metic compounds to bind non-covalently to the enzyme.
Three possibilities could account for these findings. First, these small peptidomimetics built
around tryptophan may simply not bind well to the DsbA hydrophobic groove, perhaps due to
being so small or conformationally restricted. Second, the small peptidomimetics bind weakly
and reversibly to the hydrophobic groove, and are easily displaced by substrate or DsbB. This
possibility would imply that the hydrophobic contacts formed between the peptidomimetic
and the EcDsbA groove are insufficient in number or too weak to compete with the longer pep-
tide substrate or the protein substrate EcDsbB, both of which also make an additional covalent
interaction with the EcDsbA active site cysteine. Third, the peptidomimetic binds tightly to the
groove (but is not detected by DSF because of interference, or by ITC because of a major entro-
pic component to binding), and disulfide bond transfer occurs between EcDsbA/substrate and
EcDsbA/EcDsbB despite the hydrophobic groove interaction. The role of the hydrophobic
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Virtual Screening for DsbA Inhibition
groove as the sole binding interaction for peptidomimetic drugs may therefore be limited in
this system. However, inhibitors of DsbA-mediated cellular motility in E. coli have recently
been identified using a fragment-screening approach that bind to the hydrophobic groove with
a Kd of 200 μM [68]. We therefore consider that the second of the three explanations is proba-
bly true, and that additional interactions are needed for inhibitor potency. In the future, it may
be possible to combine such inhibitors with other peptidomimetic fragments that interact with
enzyme active site regions, the CXXC motif or the cisPro loop to realize the necessary drug-like
potencies being sought.
Supporting Information
S1 Fig. NMR spectral data of compound 1 (S)-2-[N-(Morpholine-1-carbonyl)amino]-3-
(1H-indol-3-yl)-N-(benzyl)propanamide. 1H NMR (400 MHz, CDCl3): δ 7.96 (br, 1H), 7.72
(d, J = 7.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.23–7.19 (m, 5H), 7.13(t, J = 6.6 Hz, 1H), 6.99
(d, J = 7.8 Hz, 1H), 6.93 (br, 1H), 5.99 (s, 1H), 5.28 (d, J = 7.8 Hz, 1H), 4.68–4.64 (m, 1H), 4.32
(dd, J = 13.2, 4.8 Hz, 1H), 4.24 (dd, J = 13.2, 4.8 Hz, 1H), 3.64–3.59 (m, 4H), 3.37 (dd, J = 13.2,
6.6 Hz, 1H), 3.33–3.30 (m, 2H), 3.26–3.22 (m, 2H), 3.13(d, J = 12.6 Hz, 1H). 13C NMR (101
MHz, CDCl3): δ 172.03, 157.09, 137.71, 136.16, 128.55, 127.63, 127.41, 123.12, 122.49, 119.88,
118.82, 111.28, 110.93, 66.46, 55.31, 43.95, 43.58, 28.89. HRMS (ESI+): C23H27N4O3+ [MH]+
calcd: 407.2078, found: 407.2079.
(PNG)
S2 Fig. NMR spectral data of compound 2 (S)-2-[N-(Piperazine-1-carbonyl)amino]-3-(1H-
indol-3-yl)-N-(benzyl)propanamide. 1H NMR (600 MHz, DMSO-d6): δ 10.82 (br, 1H), 8.79
(br, 1H), 8.47 (t, J = 6.6 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.3 (t, J = 7.8
Hz, 2H), 7.21 (t, J = 7.8 Hz, 1H), 7.16 (t, J = 7.2 Hz, 3H), 7.06 (t, J = 7.2 Hz, 1H), 6.97 (t, J = 7.8
Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 4.44–4.40 (m, 1H), 4.32–4.25 (m, 2H), 3.51–3.42 (m, 4H),
3.13 (dd, J = 14.4, 4.8 Hz, 1H), 3.02–2.98 (m, 5H). 13C NMR (151 MHz, DMSO-d6): δ 172.57,
156.70, 139.39, 136.06, 128.16, 127.37, 126.95, 126.58, 123.75, 120.79, 118.58, 118.12, 111.27,
110.59, 55.56, 42.57, 41.98, 40.66, 27.91. HRMS (ESI+): C23H28N5O2+ [MH]+ calcd: 406.2238,
found: 406.2233.
(PNG)
S3 Fig. NMR spectral data of compound 3 (S)-2-[N-(tert-Butyl-4-piperazine-1-carbonyl)
amino]-3-(1H-indol-3-yl)-N-(benzyl)propanamide. 1H NMR (600 MHz, CDCl3): δ 8.07
(br, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.24–7.21(m, 3H), 7.20 (t, J = 7.8 Hz,
1H), 7.10 (t, J = 7.8 Hz, 1H), 6.97 (dd, J = 7.0, 3.0 Hz, 2H), 6.93 (d, J = 1.8 Hz, 1H), 6.42
(br, 1H), 5.45 (d, J = 6.6 Hz, 1H), 4.69 (q, J = 7.8 Hz, 1H), 4.34 (dd, J = 15.6, 6.6 Hz, 1H), 4.23
(dd, J = 15.6, 6.6 Hz, 1H), 3.31–3.14 (m, 10H), 1.47 (m, 9H). 13C NMR (151 MHz, CDCl3):
δ 172.48, 156.95, 154.59, 137.45, 136.15, 128.55, 127.62, 127.44, 127.35, 123.22, 122.43, 119.90,
118.70, 111.31, 110.68, 80.36, 55.46, 43.61, 43.40, 28.70, 28.35. HRMS (ESI+): C28H36N5O4
[MH]+ calcd: 506.2762, found: 506.2761.
(PNG)
+
S4 Fig. NMR spectral data of compound 4 (S)-2-[N-(Morpholine-1-carbonyl)amino]-3-
(1H-indol-3-yl)-N-(phenyl)propanamide. 1H NMR (400 MHz, CDCl3): δ 8.09 (br, 1H), 7.88
(br, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.30–7.21 (m, 3H), 7.16–7.12
(m, 2H), 7.06 (t, J = 7.2 Hz, 1H), 5.27 (d, J = 7.8 Hz, 1H), 4.78 (q, J = 7.8 Hz, 1H), 3.64–3.57
(m, 4H), 3.47 (dd, J = 14.4, 5.4 Hz, 1H), 3.33–3.20 (m, 5H). 13C NMR (151 MHz, CDCl3):
δ 170.34, 157.36, 137.40, 136.22, 128.93, 127.42, 124.39, 123.40, 122.62, 120.10, 119.94, 118.82,
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Virtual Screening for DsbA Inhibition
111.42, 110.93, 66.40, 55.83, 44.02, 28.04. HRMS (ESI+): C22H25N4O3+ [MH]+ calcd: 393.1921,
found: 393.1921.
(PNG)
S5 Fig. NMR spectral data of compound 5 (S)-2-[N-(Morpholine-1-carbonyl)amino]-3-
(1H-indol-3-yl)-N-(phenethyl)propanamide. 1H NMR (600 MHz, CDCl3): δ 8.07 (br, 1H),
7.67 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.21(t, J = 7.8 Hz, 1H), 7.16–7.12 (m, 4H), 6.96
(d, J = 2.4 Hz, 1H), 6.89–6.87 (m, 2H), 5.79 (br, 1H), 5.29 (d, J = 7.8 Hz, 1H), 4.60–4.56 (m, 1H),
3.59 (t, J = 5.4 Hz, 4H), 3.42–3.20 (m, 7H), 3.09 (dd, J = 15.6, 9.0 Hz, 1H), 2.60–2.55 (m, 1H),
2.51–2.46 (m, 1H). 13C NMR (151 MHz, CDCl3): δ 172.18, 157.01, 138.49, 136.18, 128.60,
128.54, 127.46, 126.47, 123.18, 122.49, 119.92, 118.84, 111.38, 110.97, 66.38, 55.33, 43.87, 40.53,
35.28, 28.68. HRMS (ESI+): C24H29N4O3+ [MH]+ calcd: 421.2234, found: 421.2232.
(PNG)
S6 Fig. NMR spectral data of compound 6 (S)-2-[N-(Morpholine-1-carbonyl)amino]-3-
(1H-indol-3-yl)-N-(1-phenylethyl)propanamide. 1H NMR (600 MHz, CDCl3): δ 7.77 (br,
1H), 7.71 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.24–7.23 (m, 2H), 7.18 (t, J = 7.8 Hz, 1H),
7.12 (t, J = 7.8 Hz, 1H), 7.24–7.23 (m, 2H), 6.67 (d, J = 2.4 Hz, 1H), 5.92 (d, J = 7.8 Hz, 1H), 5.40
(d, J = 7.8 Hz, 1H), 4.97 (q, J = 6.0 Hz, 1H), 4.66–4.62 (m, 1H), 3.63–3.59 (m, 4H), 3.34–3.25
(m, 5H), 3.04 (dd, J = 15.0, 9.0 Hz, 1H), 1.33 (d, J = 7.2 Hz, 3H). 13C NMR (151 MHz, CDCl3):
δ 171.23, 157.10, 142.72, 136.12, 128.54, 127.32, 127.20, 126.11, 123.27, 122.38, 119.90, 118.99,
111.24, 110.76, 66.37, 54.94, 49.02, 43.94, 29.13, 21.58. HRMS (ESI+): C24H29N4O3+ [MH]+
calcd: 421.2234, found: 421.2231.
(PNG)
S7 Fig. NMR spectral data of compound 7 (S)-2-[N-(Morpholine-1-carbonyl)amino]-3-
(1H-indol-3-yl)-N-(2-(trifluoromethyl)benzyl)propanamide. 1H NMR (600 MHz, CDCl3):
δ 8.01 (br, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.35–
7.31 (m, 2H), 7.19 (t, J = 7.2 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.09 (t, J = 7.2 Hz, 1H), 6.90
(d, J = 1.8 Hz, 1H), 6.45 (br, 1H), 5.44 (br, 1H), 4.73 (q, J = 6.6 Hz, 1H), 4.51 (dd, J = 15.6, 6.0
Hz, 1H), 4.44 (dd, J = 15.6, 6.0 Hz, 1H), 3.60–3.56 (m, 4H), 3.32–3.13 (m, 6H). 13C NMR (151
MHz, CDCl3): δ 172.70, 157.22, 136.20, 135.88, 132.15, 129.96, 127.98 (q, J = 30.2 Hz), 127.51,
127.26, 125.89 (q, J = 5.9 Hz), 124.29 (q, J = 272 Hz), 123.11, 122.48, 119.94, 118.61, 111.30,
110.50, 66.28, 55.30, 43.89, 40.10, 28.53. HRMS (ESI+): C24H26F3N4O3+ [MH]+ calcd:
475.1952, found: 475.1951.
(PNG)
S8 Fig. NMR spectral data of compound 8 (S)-2-[N-(Morpholine-1-carbonyl)amino]-3-
(1H-indol-3-yl)-N-(3-(trifluoromethoxy)benzyl)propanamide. 1H NMR (600 MHz, CDCl3):
δ 8.02 (br, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.24 (t, J = 6.6 Hz, 1H), 7.20 (t,
J = 6.6 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.07 (d, J = 7.8 Hz, 1H), 6.97 (s, 1H), 6.91 (d, J = 7.8 Hz,
1H), 6.84 (s, 1H), 6.45 (br, 1H), 5.44 (br, 1H), 4.71–4.68 (m, 1H), 4.32 (dd, J = 15.6, 6.6 Hz,
1H), 4.25 (dd, J = 15.6, 6.6 Hz, 1H), 3.63–3.57 (m, 4H), 3.34–3.14 (m, 6H). 13C NMR (151
MHz, CDCl3): δ 172.79, 157.27, 149.38, 139.97, 136.21, 129.98, 127.27, 125.92, 123.15 (q, 270.1
Hz), 122.57, 121.26, 120.01, 119.96, 119.76, 118.62, 111.40, 110.58, 66.31, 55.44, 43.90, 42.99,
28.56. HRMS (ESI+): C24H26F3N4O4+ [MH]+ calcd: 491.1901, found: 491.1902.
(PNG)
S9 Fig. NMR spectral data of compound 9 (S)-2-[N-(Morpholine-1-carbonyl)amino]-3-
(1H-indol-3-yl)-N-(2,4-(dimethoxybenzyl)propanamide. 1H NMR (400 MHz, CDCl3): δ
7.91 (br, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 10.4 Hz, 1H), 7.14(t, J = 10.4 Hz, 1H), 7.08(t,
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Virtual Screening for DsbA Inhibition
J = 10.4 Hz, 1H), 6.92 (d, J = 10.4 Hz, 1H), 6.77 (s, 1H), 6.53 (s, 1H), 6.35 (d, J = 10.4 Hz, 1H),
6.32 (s, 1H), 5.75 (br, 1H), 4.67 (br, 1H), 4.27 (dd, J = 13.8, 6.8 Hz, 1H), 4.14 (dd, J = 13.8, 6.8
Hz, 1H), 3.79 (s, 3H), 3.59 (t, J = 4.7 Hz, 4H), 3.55 (s, 3H), 3.29–3.19 (m, 5H), 3.11–3.04
(m, 1H). 13C NMR (101 MHz, CDCl3): δ 172.49, 160.56, 158.39, 157.21, 136.11, 130.22, 127.12,
127.11, 123.30, 122.21, 119.75, 118.62, 117.82, 111.16, 110.13, 103.73, 98.39, 98.35, 66.29, 55.44,
54.97, 44.90, 39.31, 28.90. HRMS (ESI+): C25H31N4O5+ [MH]+ calcd: 467.2289, found:
467.2287.
(PNG)
S10 Fig. NMR spectral data of compound 10 S)-2-[N-(Morpholine-1-carbonyl)amino]-3-
(1H-indol-3-yl)-N-(2,4-(dimethoxybenzyl)propanamide. 1H NMR (400 MHz, CDCl3):
δ 8.04 (br, 1H), 7.62 (d, J = 6.4 Hz, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.20 (t, J = 6.6 Hz, 1H), 7.12
(d, J = 7.6 Hz, 1H), 6.92 (s, 1H), 6.72 (d, J = 8.5 Hz, 1H), 6.59 (s, 1H), 6.55 (d, J = 9.48 Hz, 1H),
6.40 (br, 1H), 5.60 (br, 1H), 4.67 (s, 1H), 4.29–4.16 (m, 3H), 3.86 (s, 3H), 3.79 (s, 1H), 3.61
(t, J = 4.2 Hz, 4H), 3.32–3.13 (m, 6H). 13C NMR (101 MHz, CDCl3): δ 172.72, 157.32, 149.06,
148.51, 136.24, 132.67, 129.80, 127.33, 123.26, 122.54, 120.06, 119.90, 118.63, 111.29, 111.22,
111.18, 111.10, 110.49, 66.25, 55.98, 55.57, 43.91, 43.60, 28.64. HRMS (ESI+): C25H31N4O5
[MH]+ calcd: 467.2289, found: 467.2287.
(PNG)
+
Acknowledgments
Open Eye Scientific Software kindly made available the OMEGA software for no cost. Fig 1B
and 1C were generated using LigPlot+.
Author Contributions
Conceived and designed the experiments: WD PB MJS DPF JLM. Performed the experiments:
WD PB MJS ST RMM. Analyzed the data: WD PB MJS ST RMM DPF JLM. Wrote the paper:
WD PB MJS RMM DPF JLM.
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