Structural basis of metallo-β-lactamase inhibition by captopril stereoisomers

Article abstract of DOI:10.1128/AAC.01335-15

β-Lactams are the most successful antibacterials, but their effectiveness is threatened by resistance, most importantly by production of serine- and metallo-β-lactamases (MBLs). MBLs are of increasing concern because they catalyze the hydrolysis of almost all β-lactam antibiotics, including recent-generation carbapenems. Clinically useful serine-β-lactamase inhibitors have been developed, but such inhibitors are not available for MBLs. L-Captopril, which is used to treat hypertension via angiotensin-converting enzyme inhibition, has been reported to inhibit MBLs by chelating the active site zinc ions via its thiol(ate). We report systematic studies on B1 MBL inhibition by all four captopril stereoisomers. High-resolution crystal structures of three MBLs (IMP-1, BcII, and VIM-2) in complex with either the L- or D-captopril stereoisomer reveal correlations between the binding mode and inhibition potency. The results will be useful in the design of MBL inhibitors with the breadth of selectivity required for clinical application against carbapenem-resistant Enterobacteriaceae and other organisms causing MBL-mediated resistant infections.

Full text of DOI:10.1128/AAC.01335-15

AAC Accepted Manuscript Posted Online 19 October 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.01335-15
Copyright © 2015 Brem et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
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Structural basis of metallo-β-lactamase inhibition by captopril
stereoisomers
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Running title: Captopril stereoisomers and antibiotic resistance
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Jürgen Brem1*, Sander S. van Berkel1*, David Zollman1*, Sook Y. Lee1, Opher Gileadi2, Peter J.
McHugh3, Timothy R. Walsh4, Michael A. McDonough1#, and Christopher J. Schofield1#
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Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, United
Kingdom.
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Structural Genomics Consortium, University of Oxford, Old Road Campus, Headington, Oxford
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OX3 7BN, United Kingdom
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Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John
Radcliffe Hospital, Oxford OX3 9DS, United Kingdom.
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Department of Microbiology and Infectious Diseases, Institute of Infection and Immunity, Heath
Hospital, Cardiff, CF14 4XN, United Kingdom.
* Co-first authors: Jürgen Brem, Sander S. van Berkel and David Zollman
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Corresponding Authors: Michael A. McDonough, Department of Chemistry, University of Oxford,
12 Mansfield Road, Oxford, OX1 3TA, United Kingdom, michael.mcdonough@chem.ox.ac.uk, and
Christopher J. Schofield, Department of Chemistry, University of Oxford, 12 Mansfield Road,
Oxford, OX1 3TA, United Kingdom, christopher.schofield@chem.ox.ac.uk
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22 Abstract
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β-Lactams are the most successful antibacterials, but their effectiveness is threatened by
resistance, most importantly by production of serine- and metallo-β-lactamases (MBLs).
MBLs are of increasing concern because they catalyse the hydrolysis of almost all β-lactam
antibiotics, including recent generation carbapenems. Clinically useful serine-β-lactamase
inhibitors have been developed, but such inhibitors are not available for MBLs. L-Captopril,
used to treat hypertension via angiotensin-converting enzyme inhibition, has been reported to
inhibit MBLs by chelating to the active site zinc ions via its thiol(ate). We report systematic
studies on B1 MBL inhibition by all four captopril stereoisomers. High resolution crystal
structures of three MBLs (IMP-1, BcII and VIM-2) in complex with either L-or D-captopril
stereoisomers reveal correlations between the binding modes and inhibition potency. The
results will be useful in the design of MBL inhibitors with the breadth of selectivity required
for clinical application against carbapenem-resistant Enterobacteriaceae and other MBL
mediated resistant infections.
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37 Introduction
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The increasing problem of antibiotic resistance is a global health concern (1), with the World
Health Organization (WHO) and the European Centre for Disease Prevention and Control
(ECDPC) reporting that several million people are infected with antibiotic resistant bacteria
annually. It is estimated that >50,000 patients die each year due to infections caused by
multidrug resistant bacterial pathogens in the United States of America alone (2).
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β-Lactam containing compounds remain the most important antibiotics in clinical use, but
their effectiveness is threatened by increasing resistance. β-Lactam resistance is most
importantly mediated by serine- and zinc dependent metallo-β-lactamases (SBLs and MBLs,
respectively), which catalyse β-lactam hydrolysis (3). In combination with an appropriate
penicillin antibiotic, Class A SBL (‘penicillinase’) inhibitors (i.e. clavulanic acid,
tazobactam, and sulbactam) have been widely used in the clinic, and recently, a Class C
(cephalosporinase) SBL inhibitor (4), Avibactam, in combination with a cephalosporin has
been approved for clinical use (5). In contrast, there are no reports of clinically useful MBL
inhibitors (6).
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A challenge with the development of useful MBL inhibitors is achieving the breadth of
inhibition against most MBL subtypes, whilst avoiding inhibition of structurally related
human MBL-fold enzymes (7). Crystal structures reveal that MBLs have a characteristic
αβ/βα sandwich fold, that they possess conserved zinc ion binding sites, and that loops
flanking the active site are involved in ligand binding (8). MBLs can be divided into three
subclasses (B1, B2 and B3) based on the number of zinc ions in their metal binding sites,
and/or sequence and structural similarities (6). B1 MBLs are the most clinically relevant
MBLs (e.g. IMP - Imipenemase, VIM - Verona integron–encoded MBL, and NDM - New
Delhi MBL types); B1 MBLs catalyse hydrolysis of almost all β-lactams (BLs), including the
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latest generations of cephalosporins and carbapenems (9). Several classes of known metallo-
enzyme inhibitors inhibit MBLs, including thiols, carboxylic acids, trifluoromethyl ketones,
hydroxamic acids and rhodanines (7, 10, 11) (for structures see Figure S1 in the supplemental
material).
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(2S)-1-[(2S)-2-Methyl-3-sulfanylpropanoyl] pyrrolidine-2-carboxylic acid (commonly
referred to as L-captopril) (Figure 1) is a thiol-containing small molecule which was
developed in the 1970s to target the zinc-ion utilizing human angiotensin-converting enzyme
(ACE) (12),(13). L-Captopril was successfully used for several decades to control high blood
pressure. The clinically used (2S,2S)-stereoisomer of captopril, i.e. L-captopril inhibits
several MBLs from all subclasses (14-18). However, the (2S,2R)-stereoisomer, (2S)-1-[(2R)-
2-methyl-3-sulfanylpropanoyl] pyrrolidine-2-carboxylic acid (Figure 1), commonly referred
to as D-captopril, has been reported to be more active than L-captopril against some MBLs
(e.g. NDM-1 (19), BcII (17), CcrA (20) and CphA (17)).
Crystal structures have been reported for some MBLs in complex with L- or D-captopril, i.e.
(i) in the case of the B1 subclass MBLs, for NDM-1 complexed with L-captopril (21) and
BlaB (Chryseobacterium meningosepticum) with D-captopril (22); (ii) in the case of the B2
MBLs, for CphA (Aeromonas hydrophilia) with D-captopril (18); and (iii) in the case of the
B3 MBLs, for FEZ-1 (Fluoribacter gormanii MBL) with D-captopril (23) and L1
(Stenotrophomonas maltophilia MBL) with D-captopril (15). Biophysical analyses employing
extended X-ray absorption fine structure (EXAFS), and perturbed angular correlation of X-
rays (PAC) spectroscopy have been reported for BcII and CphA complexed with D- and L-
captopril (17). Molecular dynamic calculations on D- and L-captopril complexed with BcII
and D-captopril with NDM-1 have also been reported (20, 24). These analyses imply that
both L- and D-captopril can bind with their thiol(ate) ligated to both active site Zn(II) ions
(Figure 2; see Figure S2-4 and 2D diagram in the supplemental material). Interestingly,
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despite BlaB and NDM-1 belonging to the same B1 MBL subclass, different binding modes
were observed for the L- and D-captopril stereoisomers (19). In the case of the mono-Zn(II)
ion binding B2 subclass a structure of the CphA:D-captopril complex (18) indicates that the
D-captopril carboxylate, rather than the thiol(ate), binds to the single Zn(II) ion, a binding
mode that possibly reflects the relatively weak inhibition of this enzyme by D-captopril (Ki =
72 µM). Finally, with the B3 MBL subclass, in a crystal structure of the FEZ-1:D-captopril
complex (23) the binding of captopril was modelled such that neither the D-captopril thiol nor
carboxylate interacts with the active site Zn(II) ions, a binding mode that was also proposed
to be consistent with the relatively weak inhibition observed in this case (Ki 400 µM) (see
Figure S2 in the supplemental material). To date there have been no reports on MBL
inhibition by (2R)-1-[(2S)-2-methyl-3-sulfanylpropanoyl] pyrrolidine-2-carboxylic acid,
subsequently referred to as epi-L-captopril (the 2R,2S-stereoisomer) and (2R)-1-[(2R)-2-
methyl-3-sulfanylpropanoyl] pyrrolidine-2-carboxylic acid, subsequently referred to as epi-D-
captopril (the 2R,2R-stereoisomer) (Figure 1).
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We report systematic studies on the inhibition of four clinically relevant MBLs (IMP-1, VIM-
2, SPM-1 and NDM-1) and the model MBL, BcII, by the four captopril stereoisomers and
both enantiomers of a captopril derivative 1-(2-mercaptobenzoyl)pyrrolidine-2-carboxylic
acid (D- and L-MBP) (Figures 1 and Table S1 in the supplemental material) (25, 26). The
combined kinetic and structural studies clearly reveal different binding modes for different
captopril stereoisomers, and will help to enable the future development of broad spectrum
MBL inhibitors.
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109 Materials and methods
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Synthesis
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The different captopril isomers and captopril derivatives were prepared according to literature
procedures (see Scheme S1 and S2 and the experimental section in the supplemental
material).
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Protein production and purification
Recombinant forms of NDM-1, VIM-2, VIM-4, SPM-1, IMP-1 and BcII MBLs were
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produced in Escherichia coli as described (27, 28). Purified proteins were dialysed into
freshly prepared crystallisation buffer (50 mM HEPES pH 7.5, 150 mM NaCl containing 1
µg ZnCl2) then concentrated [to 2 mM (BcII), 0.75 mM (IMP-1) and 0.36 mM (VIM-2)]
before use in crystallisation studies.
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Crystallography
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Crystals were grown using the conditions stated in Table S2 in the supplemental material, and
cryoprotected using well solution diluted with 25% glycerol before being flash cooled in
liquid nitrogen. All data sets were collected at 100K. All data were indexed, integrated and
scaled using HKL-3000 (29). The structures were solved by molecular replacement using
Phaser (30). The structures were then refined using PHENIX (31) and COOT (32) until Rwork
and Rfree no longer decreased. Data collection and refinement statistics are given in Table S3-
5 in the supplemental material.
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Coordinates and structure factors have been deposited with PDB accession codes: di-Zn(II)-
BcII apo (PDB ID: 4C09), BcII L-captopril (PDB ID: 4C1H), BcII D-captopril (PDB ID:
4C1C), IMP-1 L-captopril (PDB ID: 4C1F), IMP-1 D-captopril (PDB ID: 4C1G), di-Zn(II)-
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VIM-2 apo (PDB ID: 4BZ3), VIM-2 L-captopril (PDB ID: 4C1D), VIM-2 D-captopril (PDB
ID: 4C1E). The URL for coordinate deposition is http://rcsb-deposit.rutgers.edu/.
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Kinetic analyses
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Kinetic and inhibition assays with the bacterial MBLs, hACE-2 (Angiotensin-converting
enzyme 2) and hHAGH (Hydroxyacylglutathione hydrolase, human Glyoxylase II were
performed as described (7, 27).
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Nuclease assays
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Nuclease assays with DCLRE1A and DCLRE1B (DNA cross-link repair enzymes 1A and B)
(33) used the described method (33) employing a 21-nucleotide DNA oligonucleotide with a
fluorescein label at its 3’-end. In brief, exonuclease activity was measured using ΔN-
DCLRE1A (3.5 ng, 8 nM) or ΔC-DCLRE1B (1.5 ng, 4 nM) mixed with 1 pmol (1 μM) of 3′
fluorescein-labeled DNA substrate in 10 µL of 20 mM HEPES pH 7.9, 50 mM KCl, 10 mM
MgCl2, 0.5 mM DTT, 0.05 % Triton-X, 0.1 mg/mL BSA and 5 % glycerol. Reactions were
incubated at 37 °C for 20 minutes with the indicated concentrations of D-captopril, L-
captopril, D-MBP, or L-MBP (see Figure S5 in the supplemental material) and quenched by
addition of 2 μL of 80 % formamide/ 10 mM EDTA and heating at 95 °C for 5 min.
Following separation on a 20 % polyacrylamide/ 7 M urea denaturing gel, substrate and
product bands were visualised by a Typoon Trio+ Variable Model Imager (excitation at 488
nm with blue laser at 400 V).
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MIC determinations
The bacteria used were non-clonal international isolates where the mechanisms of
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carbapenem resistance have been genetically defined by sequencing. As a negative control E.
coli ATCC 25922 was used. MBL genes were obtained by PCR using standard procedures
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and inserted into a pK18 vector which was used to transform E. coli J53(34). Transconjugates
were MBL genes carried on native wild-type plasmids conjugated into J53 (35). Both
transformants and transconjugants were verified by DNA sequencing and for MBL
production by using the MTS MBL strip (Liofilchem, Roseto, Italy). Additionally, five
Escherichia coli (NDM-1), 10 Klebsiella pneumoniae (5 NDM-1, 3 VIM-4 and 2 IMP-4),
one Serratia macescens (IMP-4), two Pseudomonas aeruginosa (VIM-2, AIM-1) and E. coli
ATCC 25922 were used in the test panel. MICs were determined using microbroth dilution
method according to CLSI guidelines. Strains were cultured and tested in cation adjusted
Muller-Hinton agar and broth (Beckton Dickinson, USA).
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164 Results
MBL inhibition by Captopril stereoisomers
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We first synthesised the four possible captopril stereoisomers (D-, L-, epi-D- and epi-L-
captopril, see Scheme S1 and S2 and Experimental section in the supplemental material) and
tested them as inhibitors against BcII and clinically relevant MBLs from the B1 subclass
(IMP-1, VIM-2, SPM-1 and NDM-1) (Table 1 and Figure 3) (9). Comparing the previously
reported D-captopril and L-captopril inhibition values to our results for NDM-1, IMP-1 and
BcII reveals relatively small differences (Table 1), likely due, at least in part, to different
assay conditions (for D-captopril: NDM-1 IC50 20.1 µM vs 7.9 µM (36); BcII IC50 10.7 µM
vs 45 µM (Ki) (17) and for L-captopril: NDM-1 IC50 157.4 µM vs 202 µM (36); IMP-1 IC50
7.2 µM vs 12.5 µM (Ki) (37) and BcII IC50 80.4 µM vs 65 µM (Ki) (17)). In all cases D-
captopril was the most potent of the four possible captopril stereoisomers (NDM-1 IC50
20.1±1.5 µM, IMP-1 IC50 7.2±1.2 µM, VIM-2 IC50 0.072±0.010 µM, SPM-1 IC50 261.8±1.3
µM and BcII IC50 10.7±1.2 µM) (Table 1). D-Captopril was consistently more potent than L-
captopril (~7 fold for NDM-1, ~3 fold for IMP-1, ~60 fold for VIM-2, ~2 fold for SPM-1 and
~8 fold for BcII) (Table 1). Both epi-L- and epi-D-captopril were poor inhibitors of BcII and
SPM-1 (IC50 values all ≥ 500 μM); whereas for NDM-1 and IMP-1, unlike epi-L-captopril,
epi-D-captopril showed some activity (NDM-1 IC50 64 μM and IMP-1 IC50 173 μM).
Relatively potent IC50 values were observed for both epi-L- and epi-D-captopril against VIM-
2 (IC50 5.5 μM). The captopril derivatives, D- and L-MBP, were less potent (IC50 > 500 µM)
against all MBLs when compared to D- and L-captopril (Table 1).
A key issue in work towards obtaining clinically relevant MBL inhibitors is the degree of
selectivity of the bacterial MBLs over that of human metallo-enzymes, including MBL-fold
enzymes. Although L-captopril is a well-studied ACE-2 inhibitor, there are no reports of its
selectivity versus human-MBL-fold enzymes. We tested L- and D-captopril, as well as D- and
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L-MBP against the human MBL fold enzymes DCLRE1A and DCLRE1B (33), hHAGH and
hACE-2 (zinc dependent human metallo enzyme); no inhibition was observed at 100 μM
under our standard assay conditions (see Figure S5 in the supplemental material).
Pathogen susceptibility to D – and L-captopril
Since D- and L-captopril captopril were consistently the most potent stereoisomers against the
tested MBLs (Table 2), we tested them against non-clonal multidrug-resistant bacteria
expressing various MBLs. We used a variety of cloned MBLs, conjugant, and wild-type
clinical isolates. The panel of strains were tested for meropenem/ceftazidime MICs with and
without L- or D-captopril at 8 mg/L. (Table 2). In order to correlate with the cellular studies
(see below), we determined the IC50 values for D-and L-captopril against VIM-4 (VIM-4 IC50
1.7±0.4 μM for D-captopril and IC50 3.9±0.5 μM for L-captoril). Pathogenic strains from
different geographical origins (e.g. Greece (A-33, VIM-4) or India (IR 60, NDM-1)) (38),
and display resistance against BLs as well as fluoroquinoline and aminoglycoside antibiotics
were selected for MIC tests (Table 2). Whilst L-captopril showed potentiation with cloned
and transconjugated MBLs, against wild-type strains there was little synergy observed and
generally less than D-captopril (Table 2). The addition of D-captopril potentiates the efficacy
of meropenem against most of the VIM-2, VIM-4, IMP-4 and NDM-1 producing strains
tested including E. coli, K. pneumoniae, S. marascens, and P. aeruginosa (Table 2).
Structural analysis of captopril binding to IMP-1, VIM-2 and BcII
We then investigated the mode of binding of the captopril stereoisomers to MBLs by
crystallography. We determined high-resolution crystal structures for D- or L-captopril in
complex with IMP-1 (1.71 and 2.01 Å resolution, respectively), VIM-2 (1.40 and 1.20 Å
resolution, respectively) and BcII (1.18 and 1.10 Å resolution, respectively). For comparison,
structures of di-Zn(II)-VIM-2 and di-Zn(II)-BcII without inhibitors were also determined, to
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1.20 and 1.30 Å resolution, respectively. For all structures, the ‘crystal systems’ were similar
to those previously reported (VIM-2 (7), IMP-1 (39) and BcII (40)). (Note, we use the
standard numbering scheme for class B β-lactamases (BBL numbering (41)).
As anticipated, in all cases the overall protein folds observed were the characteristic αβ/βα
MBL sandwich fold (2 β-sheets sandwiched with 2 helices buttressed against each external
face of the sandwich) (8). The active sites, which are located at one end of the two β-sheets in
a groove surrounded by several loops, were in all cases occupied by two zinc ions as
expected for B1 subclass MBLs. The L3 and L10 loops which flank the active site are located
opposite each other and are involved in substrate binding (42). The L3 loop (residues 61–66
(BBL numbering)) is located between strands β3 and β4 and the L10 loop (residues 223–241)
is located between strand β11 and helix α4, which includes Lys224/Arg228 and Asn233, the
side chains of which are directly involved in substrate and inhibitor binding (41, 43).
The electron density maps for the ligands in the various MBL:captopril complexes suggested
variable ligand occupancies and were carefully analysed between rounds of refinement (see
example Figure S6 in the supplemental material). For BcII, both D- and L-captopril were
modelled and refined with 70% occupancy. For the VIM-2:D-captopril complex structure, the
ligands were modelled and refined with 100% occupancy and for the VIM-2:L-captopril
complex with 80% occupancy. In the IMP-1:D-captopril and IMP-1:L-captopril structures the
ligands were modelled and refined with 100% occupancy in chain A, but the residual density
present in chain B was interpreted as too weak (< 50% occupancy) to include in the model.
Preliminary structural analysis indicated partial oxidation of the metal binding cysteine
(Cys221) had occurred in both the crystallised BcII and VIM-2 proteins, in similar manner to
that observed in previous crystallographic studies on these B1 MBLs (40, 44). Due to the
likelihood of active site cysteine oxidation (Cys221) interfering with the active site Zn(II)
chemistry and hence with our analysis of ligand binding, we worked to minimise Cys221
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oxidation. In all cases, cysteine oxidation during crystallisation could be prevented by the
addition of tris(2-carboxyethyl)phosphine (TCEP) (40) (except for IMP-1 where cysteine
oxidation in the absence of TCEP was not observed).
Our MBL:captopril complex structures show a similar overall captopril binding mode to
those previously observed for NDM-1:L-captopril (21), BlaB:D-captopril (22) and L1:D-
captopril (15), but differ significantly from the FEZ-1:D-captopril (23) and CphA: D-captopril
complex structures (Figure 3; see Figure S2 in the supplemental material). Our MBL:L-
captopril structures are most similar to the reported NDM-1:L-captopril complex structure
(21) and our MBL:D-captopril structures are most similar to the L1:D-captopril complex
structure (15) (see Figure S2 in the supplemental material). Comparison of the MBL:captopril
complex structures with the active site of apo-MBLs reveals that several water molecules are
displaced upon the binding of the D- or L-captopril (see Figure S7 in the supplemental
material), these displacements likely contribute to the strength of inhibitor binding.
Captopril has distinct features that enable metallo-protein binding. The thiol acts as a metal
binding ligand that displaces the proposed ‘hydrolytic’ water molecule (or hydroxide) that
bridges the two active site metal ions. A carbonyl group leads to the conformationally
constrained prolyl-ring and a methyl group extends from the carbon bonded to the thiol. Both
the L- and D-captopril diastereoisomers present two distinct binding faces (Figure 4). One
face is hydrophobic and is formed by the methyl group and the proline ring methylenes; the
hydrophobic face interacts with residues from the L3 loop (Trp87BcII; Trp64IMP-1 and
Val61IMP-1; and Trp87VIM-2, Phe61VIM-2 and Tyr67VIM-2) (Figure 4). Such interactions are in
agreement with the proposed role of the ‘mobile’ L3 loop in interacting with the hydrophobic
N-acyl substituents of cephalosporin and penicillin MBL substrates (45). The other face of
captopril is more hydrophilic and is positioned to form hydrogen bonds to residues in the L10
loop. In all our MBL:D-captopril structures, the D-captopril carboxylate is positioned to form
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electrostatic and hydrogen-bonding interactions distances ranging from 2.9 Å for BcII and
VIM-2 to 2.3 Å for IMP-1 with a conserved positively charged residue (Lys224IMP-1,BcII or
Arg228VIM-2), which is predicted to be involved in binding the β-lactam substrate carboxylate
(46, 47) (Figure 4 and Figure S4 in the supplemental material). As observed for our MBL:D-
captopril structures, the IMP-1:L-captopril structure also has the inhibitor carboxylate
positioned to form an electrostatic interaction with the conserved basic residue, i.e. Lys224.
In contrast, in the BcII:L-captopril and VIM-2:L-captopril structures, the captopril
carboxylate is orientated away from the conserved positively charged Lys/Arg residue
(Lys224/Arg228). In all MBL:L-captopril structures (including for IMP-1), the carboxylate is
positioned to interact with the conserved asparagines (Asn233) from the L10 loop. IMP-1 is
thus apparently special case, i.e. where both L- and D-captopril bind in similar modes with the
inhibitor carboxylate positioned to interact with Lys224IMP-1. In the structures of BcII and
VIM-2 complexed with D-captopril and of VIM-2 with L-captopril, the captopril amide
carbonyl oxygen is positioned to interact with the conserved asparagine (Asn233) from the
L10 loop (Figure 4 and see Figure S4 in the supplemental material). In all cases the L3 and
L10 loops were observed to move slightly towards the inhibitors relative to their positions in
the absence of inhibitor, consistent with an induced fit mechanism during substrate binding.
This is most clearly observed in the case of VIM-2 (Figure 4).
The observed binding modes for the D-captopril carboxylate in our structures differ from the
binding mode observed in the reported BlaB D-captopril structure (22) despite a similar
binding mode of the captopril thiol(ate) to the zinc ions. In the case of BlaB the D-captopril
carboxylate is rotated by ~180,° elative to the thiolate mode of binding in our structures, such
that it does not interact with the conserved Lys224BlaB, but binds to Lys167BlaB from the L10
loop. The conserved Asn233BBL in the L10 loop is replaced by tyrosine in BlaB (22); this
Asn-Tyr substitution likely contributes to the different D-captopril binding mode in BlaB
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compared to our structures. Thus, D-captopril is likely to bind to NDM-1, which contains an
asparagine (Asn233BBL) rather than a tyrosine at this position, in a similar manner to that
observed for BcII, IMP-1, and VIM-2 (rather than the BlaB binding mode) (21).
In all of our MBL:captopril structures, the distance from the Zn(II) atoms to the bridging
thiolate sulphur atom is ~2.3 Å. Similar distances are reported for the BlaB (B1) and L1 (B3)
D-captopril structures (PDB IDs: 1M2X and 2FU8) with a slightly smaller distance for the L-
captopril NDM-1 structure (PDB ID: 4EXS) (21). Different values have been observed for
the MBL inter-metal distance in the absence of exogenous ligands, with reported values
ranging from 2.5 to 4.5 Å (48), which may in part be dependent on the actual metals bound,
which is not always possible to assign based on diffraction data alone (48). An inter-zinc
distance of 3.5 Å was observed for both our di-Zn(II)-BcII and di-Zn(II)-VIM-2 apo
structures, where, as previously observed, a bridging water was present. The inter-zinc
distances increase to 3.8 Å and 3.7 Å upon D- or L-captopril binding to BcII and VIM-2,
respectively, with the binding thiolate. This observation is consistent with the different van
der Waals atomic radii for sulphur (1.8 Å) and oxygen (1.5 Å). For IMP-1 the observed Zn-
Zn distance (3.5 Å (PDB ID: 1DDK) (39), as in our BcII and VIM-2 structures) increased to
3.7 Å on binding of either the L- or D-captopril stereoisomers.
14

307 Discussion
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
Although once of little clinical relevance, MBLs are now of increasing importance (6). The
VIM type B1 MBLs are a major problem in parts of Asia, being present in up to 99% of MBL
positive multidrug resistant strains (49). Thus, there is a genuine need for a response to MBL-
mediated resistance. The finding that D-captopril, a stereoisomer of the clinically used L-
captopril, is consistently the most potent inhibitor of the captopril stereoisomers against
MBLs and can potentiate meropenem against VIM-2 and other MBL expressing pathogens is
of interest.
Although several crystal structures of MBLs in combination with either D- or L-captopril
have been reported, structures of the same MBL in complex with D- and L-captopril isomers
have not been reported previously. The captopril isomer binding modes that we observe are
related in that they all involve thiol(ate) zinc chelation, as in most of the previously reported
structures. An exception is the reported FEZ-1:D-captopril structure (23), which, due to its
relatively poor quality, may not be representative of binding in solution.
Correlations between the observed binding modes and the potency of inhibition can be made.
More potent inhibition was always observed when an interaction between the captopril
carboxylate and the conserved basic Lys/Arg (Lys224BBL) involved in substrate binding is
observed in the crystal structures. In all cases, VIM-2 manifested lower IC50 values compared
to the other tested MBLs - this correlates with the additional interactions observed between
Arg228VIM-2 with the captopril carboxylate, as well as additional interactions of the VIM-2 L3
loop with the hydrophobic face of captopril. Secondly, the observation of a decreased number
of hydrogen bonding/electrostatic interactions for L- over D-captopril generally reflects
weaker inhibition. L-Captopril was observed to be more potent in the case of IMP-1 than for
the other MBLs tested and its IC50 value was only 3-fold higher than D-captopril. This
observation of relative potent inhibition for IMP-1 by L- captopril correlates with the
15

333
334
observation that for IMP-1, but none of the other MBLs, the L-captopril carboxylate favours
binding to Lys224IMP-1
.
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
Product inhibition is commonly observed for MBLs (16). Comparison of our MBL:captopril
structural complexes with the MBL:β-lactam product complexes (PDB ID: 4HL2) (Figure 1),
shows that the most potent of the captopril isomers, D-captopril, has the most similar mode of
binding to hydrolysed β-lactams (50), especially penicillins, consistent with D-captopril being
the most potent inhibitor (Table 1). However, D-captopril was significantly less potent (> 20
fold) against SPM-1 than for all other MBLs tested; the other captopril stereoisomers did not
inhibit SPM-1 (IC50 > 500 µM). This difference may reflect the unusual nature of SPM-1 as a
proposed B1/B2 ‘hybrid’ MBL.(42) A challenge in MBL inhibition is to obtain the breadth of
selectivity towards the majority of prokaryotic MBLs, often with relatively low sequence
similarity (~30% for the MBLs we used) (see Table S1 in the supplemental material), without
inhibiting the related human MBL-fold enzymes. The combined structural and inhibition
results reveal that captopril stereoisomers can potently inhibit B1 MBLs via related, but
sometimes different binding modes. These observations may be important in developing
potent inhibitors with the required breadth of selectivity against different subtypes of MBLs,
i.e. medicinal chemists may specifically aim to identify single compounds that bind
differently to different MBL subtypes.
352
Acknowledgements
353
354
355
356
We thank the Medical Research Council (MRC)/Canadian Grant G1100135 and
MR/L007665/1 grant for support of J.B, S.Y.B, P.J.M and C.J.S. Cancer Research UK
(CRUK) is kindly acknowledged for the support of S.S.v.B and C.J.S and the Biotechnology
and Biological Sciences Research Council (BBSRC) for the support of C.J.S.
16

357
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504
23

505
506
Table 1. IC50 values for the four captopril stereoisomers and the derivatives of captopril (MBP) against different
MBLs. The IC50 values are reported in µM.
Entry
BcII
IMP-1
7.2±1.2
VIM-2
SPM-1
261.8±1.3
>500
NDM-1
20.1±1.5(7.9b)
157.4±1.3(202b)
64.6±1.4
>500
D-captopril
L-captopril
epi-D-captopril
epi-L-captopril
D-MBP
10.7±1.2 (45a)
80.4±1.1 (65a)
>500
0.072±0.01
23.3±1.3(12.5a) 4.4±0.8
173.2±1.2
436±1.1
>500
5.5±0.7
5.5±1.5
>500
>500
423.8±1.5
>500
>500
>500
>500
L-MBP
>500
>500
>500
>500
>500
507
508
509
a Ki values from the literature;b IC50 values from the literature. All experiments were performed in triplicate or
more. Nonlinear regression analysis was used to calculate the IC50 values and their corresponding 95%
confidence intervals (GraphPad Prism). Error bars represent standard deviations.
510
24

511
512
Table 2. Minimum inhibitory concentrations (MICs) of meropenem (MEM) or ceftazidime (CFZ) with and
without D- and L-captopril (D- or L-CAP) versus various Gram negative bacteria.
Strain / Inhibitor
Genotype
MEM
MIC
MEM MIC MEM MIC
CFZ
MIC
CFZ MIC
+ D-Cap
(8mg/L)
CFZ MIC
+ L-CAP
(8mg/L)
+ D-CAP
(8mg/L)
+ L -CAP
(8mg/L)
E. coli 25922
-
<0.125 <0.125
<0.125
8
0.125 <0.125
<0.125
32
E. coli J53 +
blaNDM-1
blaNDM-1
blaVIM-2
blaVIM-2
64
128
8
8
512
512
32
8
NDM-1 clone
E. coli J53 +
NDM-1
16
2
16
4
16
8
32
16
8
transconjugate
E. coli J53 +
VIM-2 clone
E. coli J53 +
VIM-2
2
1
1
32
4
transconjugate
E. coli J53 +
blaIMP-1
1
0.25
0.5
128
128
32
32
IMP-1 clone
blaDHA-1, blaCTX-
K. pneumoniae
15, blaTEM-1
,
128
8
-
-
-
IR16 (NDM-1)
blaSHV-1, blaNDM-1
blaDHA-1, blaCTX-
E. coli IR10
15, blaTEM-1,
64
2
64
-
-
-
(NDM-1)
blaOXA-1, blaNDM-
1
25

blaDHA-1, blaCTX-
15, blaTEM-1
K. pneumoniae
,
16
2
16
-
-
-
-
-
-
-
-
-
-
-
IR8 (NDM-1)
blaSHV-1, blaNDM-1
blaDHA-1, blaCTX-
E. coli IR15
15, blaTEM-1,
8
0.25
8
(NDM-1)
blaOXA-1, blaNDM-
1
blaDHA-1, blaCTX-
K. pneumoniae
15, blaTEM-1
,
8
1
8
IR19 (NDM-1)
blaSHV-1, blaNDM-1
blaDHA-1, blaCTX-
E. coli IR24
15, blaTEM-1,
512
32
512
(NDM-1)
blaOXA-1, blaNDM-
1
blaDHA-1, blaCTX-
E. coli IR60
(NDM-1)
15, blaTEM-1,
128
32
128
-
-
blaOXA-1, blaNDM-
1
blaDHA-1, blaCTX-
K. pneumoniae
15, blaTEM-1,
64
4
64
-
-
HR8 (NDM-1)
blaOXA-1, blaSHV-
1, blaNDM-1
blaDHA-1, blaCTX-
K. pneumoniae
15, blaTEM-1
,
32
8
4
1
16
16
-
-
-
-
N16 (NDM-1)
blaSHV-1, blaNDM-1
K. pneumoniae
blaCTX-15, blaTEM-
26

A33 (VIM-4)
1, blaSHV-12a,
blaVIM-4
blaCTX-15, blaTEM-
K. pneumoniae
1, blaSHV-12a
,
8
1
4
1
1
2
8
-
-
-
-
-
-
-
-
-
-
A34 (VIM-4)
blaVIM-4
blaCTX-15, blaTEM-
K. pneumoniae
1, blaSHV-12a
,
16
16
A35 (VIM-4)
blaVIM-4
blaCTX-15, blaTEM-
1, blaSHV-12a
blaIMP-4. blaOXA-1
K. pneumoniae
,
8
4
B12 (IMP-4)
blaDHA-1
,
S. marcescens
blaAMPC, blaTEM-
1, blaIMP-4
4
4
B13 (IMP-4)
blaCTX-15, blaTEM-
K. pneumoniae
1, blaSHV-12a
,
32
16
B19 (IMP-4)
blaIMP-4
P. aeruginosa
blaAMPC, blaVIM-2 512
512
512
512
512
4470 (VIM-2)
P. aeruginosa
blaAMPC, blaAIM-1 512
(AIM-1)
513
514
27

515
516
517
518
519
520
521
522
Figure 1. Captopril has structural similarity to the hydrolysed penicillin product of metallo-β-
lactamase catalysis (penicilinoic acid). A) Outline mode of action of metallo-β-lactamases
(MBLs); B) Structures of the 4 captopril stereoisomers (2S,2S, 2S,2R, 2R,2S, and 2R,2R), D-
and L-MBP; C) Binding mode of hydrolysed ampicillin with NDM-1 (PDB code: 4HL2); D)
Binding mode of D-captopril with IMP-1 (PDB ID: 4C1G – described in this study).
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Figure 2. Crystallographic analysis reveal different binding modes for D- or L-captopril.
Preliminary crystal structures of BlaB, NDM-1, CphA, FEZ-1 and L1 complexed with L- and
D-captopril (PDB IDs :1M2X (1.50 Å), 4EXS (2.40 Å), 2QDS (1.66 Å), 1JT1 (1.65 Å) and
2FU8 (1.80 Å)). Zinc ions are represented by pink spheres, D- and L-captopril ligands are in
magenta, and the amino acid residues interacting with captopril are depicted as grey stick
models. Hydrogen bonds, zinc coordination bonds and hydrophobic interactions are shown as
thin black dashes.
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Figure 3. IC50 curves of all captopril stereoisomers tested against (A) BcII, (B) IMP-1 and
(C) VIM-2 reveal different potencies. The L- and epi-L-captopril stereoisomers are
represented with solid lines and the D- and epi-D-captopril stereoisomers are depicted with
dash lines.
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