Assay platform for clinically relevant metallo-β-lactamases

Article abstract of DOI:10.1021/jm400769b

Metallo-β-lactamases (MBLs) are a growing threat to the use of almost all clinically used β-lactam antibiotics. The identification of broad-spectrum MBL inhibitors is hampered by the lack of a suitable screening platform, consisting of appropriate substrates and a set of clinically relevant MBLs. We report procedures for the preparation of a set of clinically relevant metallo-β-lactamases (i.e., NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (Sa?o Paulo MBL), and VIM-2 (Verona integron-encoded MBL)) and the identification of suitable fluorogenic substrates (umbelliferone-derived cephalosporins). The fluorogenic substrates were compared to chromogenic substrates (CENTA, nitrocefin, and imipenem), showing improved sensitivity and kinetic parameters. The efficiency of the fluorogenic substrates was exemplified by inhibitor screening, identifying 4-chloroisoquinolinols as potential pan MBL inhibitors.

Full text of DOI:10.1021/jm400769b

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Article
An Assay Platform for Clinically Relevant Metallo-#-Lactamases
Sander S van Berkel, Jürgen Brem, Anna M. Rydzik, Ramya Salimraj, Ricky Cain, Anil Verma,
Raymond J. Owens, Colin W. G. Fishwick, James Spencer, and Christopher J. Schofield
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400769b • Publication Date (Web): 30 Jul 2013
Downloaded from http://pubs.acs.org on August 4, 2013
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An Assay Platform for Clinically Relevant Metalloꢀβꢀ
Lactamases
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Sander S. van Berkel,#,‡ Jürgen Brem,#,‡ Anna M. Rydzik,# Ramya Salimraj, Ricky Cain,ꢀ Anil
Verma,†,§ Raymond J. Owens,†,§ Colin W. G. Fishwick,ꢀ James Spencer, Christopher J. Schofield*,#
# Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
School of Cellular and Molecular Medicine, University of Bristol, Medical Sciences Building, Bristol,
BS8 1TD, UK.
† Oxford Protein Production Facility UK, The Research Complex at Harwell, Rutherford Appleton
Laboratory Harwell Science and Innovation Campus, Oxfordshire OX11 0FA, UK
§ Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, Oxford, OX3 7BN,
UK
ꢁ School of Chemistry, University of Leeds, Leeds, LS2 9JT, UK
KEYWORDS: Fluorogenic substrates, Metalloꢀβꢀlactamases, Assay platform, Panꢀinhibitors.
ABSTRACT: Metalloꢀβꢀlactamases (MBLs) are a growing threat to the use of almost all clinically used
βꢀlactam antibiotics. The identification of broad spectrum MBL inhibitors is hampered by the lack of a
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suitable screening platform, consisting of appropriate substrates and a set of clinically relevant MBLs.
We report procedures for the preparation of a set of clinically relevant metalloꢀbetaꢀlactamases (i.e.
NDMꢀ1, IMPꢀ1, SPMꢀ1, and VIMꢀ2) and the identification of suitable fluorogenic substrates
(umbelliferoneꢀderived cephalosporins). The fluorogenic substrates were compared to chromogenic
substrates (CENTA, nitrocefin and imipenem), showing improved sensitivity and kinetic parameters.
The efficiency of the fluorogenic substrates was exemplified by inhibitor screening, identifying 4ꢀ
chloroisoquinolinols as potential pan MBL inhibitors.
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INTRODUCTION
Selection pressure caused by frequent use of βꢀlactam antibiotics, including penicillins,
cephalosporins, carbapenems and monobactams, has resulted in the emergence and dissemination of
highly efficient resistance mechanisms in clinically relevant bacterial pathogens. This rapid adaptation
of bacterial strains to commonly used antibiotics is an increasing global threat to public healthcare.1,2 An
important mechanism of resistance to βꢀlactam antibiotics, including clinically challenging Gramꢀ
negative organisms, involves βꢀlactamase catalysis. βꢀLactamases can be classified into those
employing a nucleophilic serinylꢀresidue at their active site (serineꢀβꢀlactamases, SBLs, classes A, C,
and D) and those employing one or two zinc ions to promote hydrolysis (metalloꢀβꢀlactamases, MBLs,
Class B).3 The combination of a penicillin antibiotic and an SBL inhibitor has substantially extended the
utility of the former, as, for example, in the case of amoxicillin and clavulanic acid (Augmentin).4 In
addition to clavulanic acid, two other Class A (penicillinase) βꢀlactamase inhibitors are used,
Tazobactam and Sulbactam; all three inhibitors are themselves βꢀlactams though in contrast to
penicillins they react with SBLs to form relatively stable acylꢀenzyme complexes.5 These SBL
inhibitors, however, appear to have no effect on MBLs.6 βꢀLactam antibiotics have been developed that
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possess resistance to SBLs or, as in the case of carbapenems, to be inhibitors or relatively poor
substrates.7 However, since the pioneering introduction of the SBL targeting inhibitors, progress in the
development of clinically useful, βꢀlactamase specific inhibitors has been limited. In the case of SBLs,
at least one compound, Avibactam is currently in clinical trials as a combined Class A and Class C SBL
inhibitor.8 MBLs, which, unlike the SBLs, are not structurally or mechanistically related to the
penicillinꢀbinding proteins (PBPs), were first identified approximately 50 years ago.9
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Figure 1. Substrates for metalloꢀβꢀlactamase activity measurements.
However, as a result of their apparently restricted distribution to the chromosomes of less pathogenic
species, they were long not considered to represent a significant threat to the clinical effectiveness of βꢀ
lactams. The emergence of horizontally acquired MBLs, i.e. horizontal gene transfer whereby genetic
material can be transferred between individual bacteria of the same species or between different species,
has led to their extensive dissemination across geographical and species boundaries and now threatens
the effectiveness of almost all βꢀlactams, including carbapenems (i.e. “last resort” antibiotics);10 the
monobactam Aztreonam is a current exception. MBLs identified on transferrable plasmids (e.g. the
NDM, IMP, VIM, GIM, SPM type enzymes) are considered a prevalent and immediate clinical
problem.11 Various structurally diverse types of MBL inhibitors have been described, including
carboxylic/succinic acids,12 triazoles/tetrazoles,13 thiols,14 trifluoromethyl ketones,15 and others.16 To
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date, no clinically useful MBL inhibitor has been reported. This, in part, may reflect the technical and
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scientific challenges in the development of an MBL–βꢀlactamꢀbased combination therapy. There is also
extensive structural diversity in the MBL family, particularly in the proposed mobile loop regions
around the active site.17 In order for an MBL inhibitor to be clinically useful it is possible that more than
one MBL will need to be inhibited. In an effort to help enable work that will lead to the development of
useful MBL inhibitors we here describe an assay platform for clinically relevant MBLs, including
protein production procedures and assay conditions using both chromogenic and fluorogenic substrates.
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RESULTS AND DISCUSSION
Several screening methods for the inꢁvitro and inꢁvivo detection of βꢀlactamases have been reported.18
Representative substrates currently in use for βꢀlactamases include; chromogenic cephalosporinꢀbased
substrates, such as CENTA,19 PADAC20 and nitrocefin;21 cephalosporinꢀbased fluorogenic substrates,22
bioluminescent probes23 and FRETꢀbased substrates.24 These substrates have mainly been applied in
research centered around SBLs, and only in one case on MBLs (L1 and CcrA).22c The large scale
application of these type of compounds in automated HTS screening for MBL inhibitors is, however,
largely hampered by their lengthy and often difficult synthesis and the high costs associated with these
substrates. More importantly, these substrates suffer from poor substrate recognition by MBLs as a
result of the high diversity of this enzyme family varying significantly in sequence, structure and
substrate specificity, thus making it hard to use a single substrate for broad MBL activity screening. The
development of new assays for broad range MBL activity screening, based on hydrolysis of
chromogenic or fluorogenic βꢀlactams would, however, significantly facilitate inhibitor identification.
Not unimportantly, large scale inhibitor screening against different MBLs needs an inexpensive
substrate, thus requiring a simple, scalable and costꢀefficient synthetic route. To develop assay
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procedures suitable for a range of MBLs, we targeted four clinically relevant enzymes as well as the
MBL from Bacillus cereus (BcII) which has been extensively used as a model enzyme. The clinically
relevant enzymes chosen were NDMꢀ1 (New Delhi MBL), IMPꢀ1 (Imipenemase), SPMꢀ1 (Sao Paulo
MBL), VIMꢀ2 (Verona integron–encoded MBL). To identify MBL substrates useful for efficient
inhibitor screening, we prepared and tested substrates on the above mentioned panel of MBLs. These
substrates included three chromogenic substrates, i.e. imipenem, nitrocefin and CENTA and five
coumarinꢀlinked substrates, i.e. FC1ꢀ5 (Figure 1). Fluorescent substrates FC1 and FC2 release
7ꢀmercaptoꢀ4ꢀmethylꢀcoumarin upon hydrolysis, resulting in a decrease of fluorescence. Conversely,
fluorogenic substrates FC3ꢀFC5 liberate 7ꢀhydroxyꢀcoumarin upon hydrolysis of the lactam ring,
producing an increased fluorescence signal (Figure 2).
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Figure 2. Hydrolysis of substrates by MBLs resulting either in an increase or decrease of fluorescence.
Protein production. Details are provided in the Supplementary Information. For the production of
recombinant BcII, IMPꢀ1, and NDMꢀ1 MBLs in Escherichia coli vectors that have been previously
described, were used, i.e. the pET9aꢀBcII, pETꢀ26b IMPꢀ1, and pOPINF NDMꢀ1 plasmids.25 In the case
of the MBLs VIMꢀ2 and SPMꢀ1, new pTriExꢀbased pOPINF vectors were constructed,26 These vectors
encode the mature VIMꢀ2 and SPMꢀ1 sequences (i.e. with the periplasmic export sequences removed)
fused to Nꢀterminal hexahistidineꢀtags, cleavable with 3C protease. The VIMꢀ2 and SPMꢀ1 genes were
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codon optimized for E. coli (Genscript, Piscataway, NJ 08854, USA) and inserted into the pOPINF
vector. All the MBLs were produced using inꢀhouse constructed E. coli BL21(DE3) pLyS cells, which
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were grown in 2TY medium. For the protein purifications standard procedures were used. The purity of
the MBLs of the protein was ascertained to >95% by SDSꢀPAGE analysis (see Supp. Info. Fig.SI_4).
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Synthesis of substrates. CENTA was prepared using a modified literature procedure.27 Cleavage of
5,5’ꢀdithioꢀbisꢀ(2ꢀnitrobenzoic acid) by dithiolthreitol (DTT) yielded 3ꢀcarboxylꢀ4ꢀnitrothiophenol
which was subsequently used to convert commercially available cephalothin into CENTA by heating
(65 °C, 3 hours). Use of chromatographically purified 3ꢀcarboxylꢀ4ꢀnitrothiophenol gave CENTA in
high purity and satisfactory yield after a single acid/base extraction.28
Scheme 1. Synthesis of FC1ꢀ5; A) thiaꢀcoumarin cephalosporin; B) hydroxylꢀcoumarin
cephalosporins. Reagents and conditions: a) MMC, DiPEA, DMF, r.t. 2h, b) TFA/anisole (5:1), 0 °C, 30
min., c) mCPBA (1 eq.), CH2Cl2, 0 °C, 1h, d) NaI (10 eq.), acetone, r.t., 2h, e) 7ꢀHC, K2CO3, MeCN,
r.t., 4h, f) mCPBA (2 eq.), CH2Cl2, 0 °C, 3h.
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Fluorogenic cephalosporin 1 (FC1) was prepared from commercially available paraꢀmethoxy benzyl
(PMB) esterꢀprotected chloroꢀcephalosporin (CC) via substitution with 7ꢀmercaptoꢀ4ꢀmethylꢀcoumarin
(MMC), followed by acidꢀmediated PMBꢀdeprotection (Scheme 1A). Treatment of CC with MMC
resulted in the formation of both the ꢁ2ꢀ and ꢁ3ꢀalkene isomers of compound 1. Subjecting the ꢁ2–ꢁ3ꢀ
isomer mixture to acidꢀmediated PMB deprotection conditions led to complete deprotection within 1
hour, generating FC1 (ꢁ2–ꢁ3 mix). To circumvent the ꢁ2–ꢁ3 isomerization issue, oxidation of the
thiazine sulfur atom with mCPBA was performed prior to alkylation (Scheme 1A).29 The ꢁ3ꢀalkene (S)ꢀ
sulfoxide 2 was obtained in good yield (72%); subsequent alkylation with MMC in the presence of
N,N’ꢀdiisopropylethylamine gave 3 as a single isolate isomer in reasonable yield (49%). Acidꢀmediated
PMBꢀdeprotection yielded FC2 in good yield (88%). Notably, MMC appears to be nonꢀfluorescent in
its free form, whereas the thioether cephalosporin derivatives (i.e. 1 and 3) showed significant
fluorescence. While this was not our objective, FC2 has potential as an `inverse fluorogenic’ substrate
(for spectroscopic data see Supp. Info. Fig. SI_1).
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With the aim of developing a fluorogenic MBL substrate we focused on 7ꢀhydroxyꢀcoumarin (7ꢀHC)
cephalosporin derivatives; in the knowledge that umbelliferone, shows strong fluorescence in its free
form whilst being near nonꢀfluorescent when etherꢀderivatized.30 During the course of our investigations
Rao and coworkers reported the synthesis and application of FC3 and FC4 as SBL fluorogenic
substrates (specific for BalC over TEMꢀ1).31 Their reported procedure (with minimal ꢁ2–ꢁ3ꢀ
isomerisation) for the synthesis of FC3 and FC4 proved to be efficient and yielding the desired
fluorogenic probes (FC3 and FC4) in satisfactory yields (Scheme 1B). Besides substrates FC3 (sulfide)
and FC4 (Sꢀsulfoxide) we also prepared substrate FC5 (sulfone). Treatment of compound 4 with two
equivalents of mCPBA, gave clean conversion to compound 6, which upon PMBꢀdeprotection yielded
substrate FC5 in good yield.
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Table 1. Kinetic data chromogenic and fluorogenic substrates on MBLs
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Literature data
Entry
Enzyme
NDMꢀ1
Substrate
[E]a
KM
kcat
(sꢀ1)
kcat/KM
(ꢃMꢀ1ꢀsꢀ1)
kcat/KM
Ref
(pM)
(ꢂM)
(ꢃMꢀ1ꢀsꢀ1)
1
Nitrocefin
CENTA
Imipenem
FC3
1000
1000
250
50
8.8 ± 1.1
34.6 ± 6.6
111.2 ± 11.7
17.6 ± 1.7
4.0 ± 0.8
25.3
92.5
398.5
102.6
125.7
298
2.9
2.7
4.1
ND
0.5
ꢀ
33
ND
33
ꢀ
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2
3
3.6
4
5.8
5
FC4
50
32.0
124.1
31.2
1.8
ꢀ
ꢀ
6
FC5
50
2.4 ± 0.2
ꢀ
ꢀ
7
VIMꢀ2
IMPꢀ1
SPMꢀ1
BcII
Nitrocefin
CENTA
Imipenem
FC4
100
1000
1000
50
7.2 ± 0.6
225.6
48.2
41.6
125.5
291.2
2794
431.9
1200
12200
8706
0.7
42.7
ND
3.8
ꢀ
34
ND
34
ꢀ
8
26.1 ± 3.1
37.8 ± 11.5
6.3 ± 0.4
9
1.1
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21
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25
26
20.0
19.1
50.2
25.3
28.1
807
517
0.04
0.2
FC5
100
50
15.2 ± 1.2
55.7 ± 4.8
17.1 ± 2.0
42.7 ± 6.7
15.2 ± 0.7
16.8 ± 1.9
16.0 ± 3.7b
25.3 ± 6.1
330.9 ± 35.5
2.5 ± 0.2
ꢀ
ꢀ
Nitrocefin
CENTA
Imipenem
FC4
2.3
2.0
1.2
ꢀ
35
27
35
ꢀ
100
50
1
FC5
5
ꢀ
ꢀ
Nitrocefin
CENTA
Imipenem
FC4
50ꢀ103
10ꢀ103
10ꢀ103
500
1000
1000
1000
1000
1000
1000
0.12
ND
1.0
ꢀ
36
ND
36
ꢀ
6.1
37.2
8.7
0.1
3.5
FC5
2.7 ± 0.2
27.38
14.9
8.9
10.3
1.8
ꢀ
ꢀ
Nitrocefin
CENTA
Imipenem
FC4
8.1 ± 1.0
0.64
0.15
0.13
ꢀ
37
27
38
ꢀ
135.9 ± 16.4
>1000
0.06
0.4
583
24.4 ± 1.7
40.4 ± 3.3
17.1
151.1
0.7
FC5
3.7
ꢀ
ꢀ
a) The enzyme concentrations of the purified proteins were determined using a NanoDrop spectrometer;
concentrations of diluted solutions used in the assay were calculated from the original concentration; b) The
following apparent Ki value for substrate/product inhibition was determined; Ki = 36.6 ± 15.2. ND = Not
determined.
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Metalloꢀβꢀlactamase assays. Initially timeꢀdependent changes in absorbance and/or emission spectra
upon hydrolysis of the potential MBL substrates were recorded to investigate the optimal wavelengths
for analysis (see Supp. Info. Fig. SI_2ꢀ3). For the frequently used chromogenic substrates, i.e. imipenem
(carbapenem) and nitrocefin and CENTA (cephalosporins) this resulted in preferred readꢀout absorption
wavelengths of 300, 405 and 492 nm, respectively. For fluorogenic substrates FC3ꢀFC5 the absorption
and emission spectra of umbelliferon in HEPES assay buffer (pH = 7.5) were measured and their
corresponding optimal wavelengths applied in the fluorescenceꢀbased assay (λex = 380 nm and λem =
460 nm).32
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We then determined kinetic parameters for different MBL–substrate combinations. The data for the
chromogenic substrates (Table 1) are generally in good agreement with previously reported data,27,39
with the only substantial dissimilarity being the lower KM values obtained by us in the case of CENTA,
possibly because of increased purity of the substrate. Direct quantitative comparison of the obtained
data with reported values is, however, difficult as a result of variations in protein production conditions,
concentration of metalꢀions added, etc. The improved stability of CENTA compared to imipenem
renders it an attractive substrate for inhibitor identification. However, MBL inhibition by the hydrolyzed
cephalosporin products (for BcII,40 CphA,41 and Sfhꢀ142) or the liberated thiophenol from CENTA (for
IMPꢀ1) could complicate interpretation of the results. Examination of the data in Table 1 reveals the
high sensitivity of the fluorogenic MBL substrates, which enable the use of enzyme concentrations
(generally << 1 nM) well below those used with chromogenic substrates (generally 1 – 50 nM).
In the case of the combination NDMꢀ1 and FC3ꢀFC5, up to 5ꢀ20 times lower enzyme concentration
was used as compared to enzyme concentrations required for imipenem and nitrocefin. Further testing
of NDMꢀ1 with the three fluorogenic substrates FC3ꢀFC5, with different sulfur oxidation states,
disclosed significantly different kcat/KM (up to 20ꢀfold) implying catalytic efficiency is altered by the
oxidation state of the thiazine sulfur atom. We observed that the stability of FC3 in aqueous solution is
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substantially lower than that of FC4 and FC5. Autohydrolysis studies in buffer at room temperature
showed substantial hydrolysis of FC3 after 3 hours, while both FC4 and FC5 were found to be stable
for at least 8 hours. Solutions of FC4 and FC5 could be used for 1ꢀ2 days when stored on ice.
Moreover, DMSO stock solutions, stored at ꢀ20 °C, could be used several months after preparation
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without any detectable decomposition. Storage of compounds FC4 and FC5 as solids at ꢀ20 °C allowed
for the use of these compound over a prolonged period of time (i.e. months). As a result FC4 and FC5
were tested on the other MBLs.
For nearly all the clinically important MBLs, FC4 and FC5 showed higher kcat/KM values than the
chromogenic substrates (with VIMꢀ2 being the only exception). On analysis of the VIMꢀ2 results we
found that FC5 is preferred over FC4 and performs equally well as nitrocefin, having similar kcat values
though slightly different KM values (7.2 ꢂM vs 15.2 ꢂM, respectively) and consequently different
kcat/KM values (31.2 ꢃMꢀ1.sꢀ1 vs 19.1 ꢃMꢀ1.sꢀ1).
The kinetic data of the five substrates for IMPꢀ1 reveal that FC4 outperforms all other substrates
mainly as a result of its high kcat value (i.e. 807 ꢃMꢀ1.sꢀ1). As depicted in Figure 2A substrate
concentrations up to 100 ꢃM could be applied without perturbing the measurement. Although FC4 has a
low KM compared to the other substrates tested on IMPꢀ1, its high sensitivity allows for accurate
measurement as indicted by the low interꢀexperimental error (Figure 3A, experiment performed in
triplicate).43 As a result of the high sensitivity of both FC4 and FC5 a 20 fold lower IMPꢀ1
concentration could be used (i.e. pM enzyme concentration range) while retaining a KM value in the ꢂM
range, thus allowing for the detection of weak binding fragments or slowꢀbinding inhibitors.44
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Figure 3. Top: IMPꢀ1 with FC4; bottom: SPMꢀ1 with nitrocefin (substrate or product inhibition). Errors
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are reported as standard error, n = 3.
In the case of SPMꢀ1 both FC4 and FC5 outperform all the tested chromogenic substrates.
Interestingly, we found that SPMꢀ1 is inhibited by nitrocefin/nitrocefinꢀderived species at a
concentration of 36 ꢃM (see Figure 3B and Table 1). Although βꢀlactams can act as inhibitors of MBLs,
as is apparent for nitrocefin and imipenem at high concentrations (e.g. nitrocefin for SPMꢀ1 – this paper,
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and for CphꢀA and Sfhꢀ1 see refs 45 and 42, respectively); the high sensitivity of the here reported
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fluorogenic substrates (FC4 and FC5) allows for the use of extremely low substrate concentrations.
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In silico studies. In an attempt to investigate the different substrate binding constants of nitrocefin,
imipenem, CENTA and FC4 for the different MBLs with the structures of the preꢀhydrolysis
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complexes, we performed in silico docking studies. Docking of nitrocefin, imipenem, CENTA and FC4
in IMPꢀ1, NDMꢀ1 and VIMꢀ2 (PDBꢀID: 1JJT,12b 3Q6X,46 and 1KO3,47 respectively) was performed
using AutoDock 4,48 and SPROUT software.49 After analysis of the obtained substrateꢀenzyme models
(see Supp. Info.) the different docking poses, calculated by Autodock 4 and SPROUT, were scored and
compared to the substrate binding constants (KM) (Table SI_1). Assessment of the SPROUT docking
scores generally reveals a better correlation with the kinetic parameters obtained in vitro (specifically
KM values in the cases of NDMꢀ1 and VIMꢀ2) compared to the docking scores generated by Autodock
4. However, for all three enzymes, the highest ranking substrate in terms of SPROUT and Autodock
score is FC4 which was also measured to have the highest binding affinity for all three enzymes.
Although inꢀdepth structural analyses are required, these modeling results also suggest that the
cephalosporins may bind to the active site in more than one orientation, including nonꢀcatalytically
productive ones. Overall, these results emphasize the importance of experimentally determining optimal
MBLꢀsubstrate combinations, and the need for structural analyses on MBLꢀsubstrate complexes.
Inhibitor Screening. In order to achieve clinical utility, MBL inhibitors may need to act on more
than one MBL subfamily. This is potentially challenging because of the generally low sequence
similarity between MBLs of different subfamilies (B1, B2, B3). Most clinically relevant MBLs are from
the B1 subfamily, however, SPMꢀ1 presents a high similarity with both the B1 and B2 subfamilies,50
hence was used as starting point for proof of principle inhibitor screening. To validate our screening
method we screened a series of 4ꢀchloroisoquinolinol derivatives (7aꢀ7g)28 against SPMꢀ1 using FC4 as
a reporter substrate (Table 2). This set of compounds was selected as a result of their previously
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reported inhibition of metalꢀbinding oxygenases.51 Screening for residual activity of compounds 7aꢀ7g,
at concentrations of 200 ꢃM and 1 mM, revealed that the 4ꢀchloroisoquinolinols bearing a Pheꢀ, Tyrꢀ, or
Valꢀbased sideꢀchain, were poor inhibitors. In contrast, the compounds derivatized with a Trp, Leu, Asp
or Glu residue inhibited SPMꢀ1, with the tryptophan functionalized chloroisoquinolinols Sꢀ7d and Rꢀ7d
giving the most promising results (3% and 8% residual activity (RA) at 200 ꢃM).
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Table 2. Residual activities (RA) on SPMꢀ1 at 1 mM and 200 ꢀM a) b)
RA
1 mM
(%)
RA
200 ꢀM
(%)
βꢀResorcylic acid
Salicylic acid
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89
ꢀ
ꢀ
R = (S)ꢀCH2ꢀ4ꢀOHꢀPh (Sꢀ7a)
R = (R)ꢀCH2ꢀ4ꢀOHꢀPh (Rꢀ7a)
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ꢀ
ꢀ
R = (S)ꢀCH2Ph (Sꢀ7b)
R = (R)ꢀCH2Ph (Rꢀ7b)
<5
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ꢀ
ꢀ
R = (S)ꢀiPr (Sꢀ7c)
R = (R)ꢀiPr (Rꢀ7c)
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ꢀ
ꢀ
R = (S)ꢀ3ꢀmethyleneꢀ1Hꢀindole (Sꢀ7d)
R = (R)ꢀ3ꢀmethyleneꢀ1Hꢀindole (Rꢀ7d)
<5
<5
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8
R = (S)ꢀiBu (Sꢀ7e)
R = (R)ꢀiBu (Rꢀ7e)
<5
<5
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R = (S)ꢀCH2CO2H (Sꢀ7f)
R = (R)ꢀCH2CO2H (Rꢀ7f)
<5
<5
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R = (S)ꢀ(CH2)2CO2H (Sꢀ7g)
<5
<5
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R = (R)ꢀ(CH2)2CO2H (Rꢀ7g)
a) For the synthesis of compound 7aꢀ7g see Supp. Info.
b) The remaining activity was measured by applying a 10 min. preincubation time
used in previous MBL work, see refs 18a and 44
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Following from the positive screening results obtained with the SPMꢀ1/FC4 pair, IC50 values were
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determined for Sꢀ7d and Rꢀ7d against our MBL panel using FC4 and the conditions reported in Table 1;
the FC4 concentrations correspond to the experimentally determined KM values (Table 1). The IC50
values (Table 3) reveal that both Sꢀ7d and Rꢀ7d are relatively weak, but broad spectrum MBL inhibitors
with IC50 values ranging from 20ꢀ130 ꢃM. Since the inhibitor activity of these compounds is in the low
ꢃM range, they may serve as good starting points for further optimization.
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Table 3. IC50 values for a panel of metalloꢀbetaꢀlactamasesa (in ꢃM)
MBLs
SPMꢀ1
IMPꢀ1
NDMꢀ1
VIMꢀ2
Sꢀ7d
Rꢀ7d
23.2 ± 1.1
75.6 ± 1.5
61.4 ± 1.3
54.7 ± 1.4
61.3 ± 1.3
46.6 ± 1.1
74.1 ± 1.6
47.1 ± 1.1
55.3 ± 1.3
132.4 ± 1.3
Bc II
a
IC50 determinations were performed in duplicate over a range
of 0.25 ꢀ200 ꢃM
To further validate our assay platform we measured the IC50 value of the reported inhibitor L
ꢀcaptopril52
against NDMꢀ1. Measuring at the KM value of FC4 on NDMꢀ1 we obtained an IC50 value of 10.0 ±1.9
ꢀcaptopril.53 Using imipenem as reported substrate, an IC50 of 13.2 ± 1.5
ꢀM and a Ki of 5.0 ꢀM for
L
ꢀM and a Ki of 6.6 ꢃM was determined. Thus, under the same assay conditions , we found that the
fluorogenic substrate (FC4) and the chromogenic substrate (imipenem) produced similar IC50 and Ki
values. However, our values are lower than the previously reported IC50 and Ki values for Lꢀcaptopril on
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NDMꢀ1 using imipenem as reported substrate (202 ꢀM54 and 39 ꢀM,52b respectively), possibly
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reflecting differences in assay conditions and protein constructs and enzyme production procedures.
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CONCLUSIONS
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The development of broad screen MBL inhibitors has been hampered by the lack of an appropriate,
efficient screening platform for multiple MBLs. The development of such a screening platform, has
been hindered by the lack of suitable detection substrates. The results of our comparative study,
employing 5 substrates and 5 MBLs, reveal striking differences in the kinetic parameters for different
enzymeꢀsubstrate combinations. Fluorogenic substrates FC4 and FC5 are highly sensitive and efficient
substrates for the clinically relevant MBLs, with one or both of them having kcat/KM values of >10 ꢃMꢀ
1ꢀsꢀ1. The suitability of FC4 for inhibitor screening was demonstrated by testing a set of 4ꢀ
chloroisoquinolinols as MBL inhibitors. This work yielded two compounds (Sꢀ7d and Rꢀ7d) that
displayed inhibitory activity against our panel of MBLs, albeit at moderate inhibition concentrations
(ꢃM); nonetheless, the results reveal the feasibility of broad spectrum MBL inhibition.
EXPERIMENTAL SECTION
The supplementary information contains a complete general experimental section, including all
procedures and equipment used. In general, chemicals were purchased from commonly used suppliers
(Aldrich, Acros, Alfa Aesar, and TCI) and were used without further purification. (6R,7R)ꢀ4ꢀ
Methoxybenzyl 3ꢀ(chloromethyl)ꢀ8ꢀoxoꢀ7ꢀ(2ꢀphenylacetamido)ꢀ5ꢀthiaꢀ1ꢀazaꢀbicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀ
carboxylate (PMBꢀprotected CepꢀCl) was obtained from Activate Scientific (Prien, Germany).
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(6R,7R)ꢀ4ꢀmethoxybenzyl 8ꢀoxoꢀ3ꢀ(((2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)methyl)ꢀ7ꢀ(2ꢀphenylacetamido)ꢀ5ꢀ
thiaꢀ1ꢀazabicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀcarboxylate (4)
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To a suspension of ClꢀCepꢀOPMB ester (680 mg, 1.4 mmol) in acetone (10 mL) was added NaI (2.10
g, 14.0 mmol, 10 eq.). The reaction was stirred at r.t. for 2 hours after which the solvent was removed in
vacuo. The crude mixture was partitioned between H2O (10 mL) and EtOAc (10 mL) and the layers
separated. The H2Oꢀlayer was extracted with EtOAc (2 x 15 mL) after which the combined organic
layers were washed with 5% NaS2O3 aq. solution (2 x 20 mL) and brine (20 mL). The organic layer was
dried over Na2SO4, filtered, and concentrated in vacuo. The crude product dissolved in MeCN (15 mL)
and 7ꢀhydroxyꢀcoumarin (454 mg, 2.8 mmol, 2 eq.) and K2CO3 (4.2 mmol, 4 eq.) were added. The
conversion of the reaction was monitored by TLC analysis (cHex/EtOAc 1:1) and completion was
reached after 4 hours. The solvent was evaporated in vacuo and the resulting crude mixture was
partitioned between H2O (10 mL) and EtOAc (10 mL). After separation of the layers, the H2Oꢀlayer was
extracted with EtOAc (2 x 15 mL), after which the combined organic layers were washed with 5%
NaS2O3 aq. solution (2 x 20 mL) and brine (20 mL). The organic layer was dried over Na2SO4, filtered,
and concentrated in vacuo. The crude product was purified by column chromatography (cHex/EtOAc
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1
1:1) to yield compound 4 as a light brown solid (392 mg, 46%). RF = 0.30 (cHex/EtOAc 1:1). H NMR
(400 MHz, DMSOꢀd6) δ = 9.20 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 9.5 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H),
7.17 ꢀ 7.33 (m, 8H), 6.92 (d, J = 1.5 Hz, 2H), 6.87 (dd, J = 8.5, 2.5 Hz, 1H), 6.79 (d, J = 8.5 Hz, 2H),
6.30 (d, J = 9.5 Hz, 1H), 5.44 (dd, J = 7.5, 4.0 Hz, 1H), 5.11 ꢀ 5.19 (m, 2H), 5.01 ꢀ 5.10 (m, app. d, 2H),
4.64 ꢀ 4.76 (m, app. q, 2H), 3.69 (s, 3H), 3.47 ꢀ 3.57 (m, 2H) ppm. 13C NMR (101 MHz, DMSOꢀd6) δ =
170.9, 167.0, 163.8, 160.9, 160.3, 159.3, 155.2, 144.3, 135.7, 130.0 (2C), 129.5, 129.1 (2C), 128.2 (2C),
126.9, 126.5, 122.4, 118.8, 113.7 (2C), 112.9, 112.7 (2C), 101.4, 69.8, 67.2, 60.8, 55.0, 52.9, 49.7, 41.6
ppm. LRMS calcd. for C33H27N2O8S, MꢀH = 611.15, mass found; MꢀH = 611.0.
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(6R,7R)ꢀ4ꢀmethoxybenzyl 8ꢀoxoꢀ3ꢀ(((2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)methyl)ꢀ7ꢀ(2ꢀphenylacetamido)ꢀ5ꢀ
thiaꢀ1ꢀazabicycloꢀ[4.2.0]octꢀ2ꢀeneꢀ2ꢀcarboxylate 5ꢀoxide (5)
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To a suspension of compound 4 (122 mg, 0.2 mmol) in dry CH2Cl2 (5 mL) cooled to 0 °C was added
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mCPBA (45 mg, 0.2 mmol, 1 eq.). The reaction was stirred at 0 °C for 30 min. followed by an
additional hour at r.t. (a white precipitate was formed). The crude product was dryꢀloaded onto silica
and purified by column chromatography (CH2Cl2/EtOAc, 8:2) to yield compound 5 as cream white solid
(60 mg, 48%) RF = 0.80 (CH2Cl2/MeOH, 9:1). 1H NMR (400 MHz CDCl3) δ = 7.64 (d, J = 9.5 Hz, 1H),
7.27 ꢀ 7.40 (m, 8H), 6.91 (d, J = 8.5 Hz, 2H), 6.74 ꢀ 6.78 (m, 2H), 6.67 (d, J = 10.0 Hz, 1 H), 6.30 (d, J
= 9.5 Hz, 1 H), 6.10 (dd, J = 10.0, 5.0 Hz, 1H), 5.22 ꢀ 5.35 (m, app. mix of d and q, 3H), 4.80 (d, J =
13.5 Hz, 1H), 4.45 (dd, J = 5.0, 1.5 Hz, 1H), 3.99 (d, J = 19.0 Hz, 1H), 3.81 (s, 3H) 3.65 (m, app d, J =
5.5 Hz, 2H), 3.28 (d, J = 19.0 Hz, 1H) ppm. 13CꢀNMR: (101 MHz, DMSOꢀd6) δ = 171.1, 164.5, 160.7
(2C), 160.2, 159.4, 155.2, 144.3, 135.8, 130.4 (2C), 129.5, 129.1 (2C), 128.3 (2C), 126.8, 126.6, 125.0,
119.6, 113.7 (2C), 112.8, 101.6, 67.5, 67.3, 66.5, 58.4, 55.1, 45.4, 41.4 ppm. LRMS calcd. for
C33H27N2O9S, MꢀH = 627.14, mass found; MꢀH = 627.0.
(6R,7R)ꢀ4ꢀmethoxybenzyl 8ꢀoxoꢀ3ꢀ(((2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)methyl)ꢀ7ꢀ(2ꢀphenylacetamido)ꢀ5ꢀ
thiaꢀ1ꢀazabicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀcarboxylate 5,5ꢀdioxide (6)
To a suspension of compound 4 (100 mg, 0.16 mmol) in dry CH2Cl2 (10 mL) cooled to 0 °C was
added mCPBA (74 mg, 0.35 mmol, 2 eq.). The reaction was stirred at 0 °C for 30 min. (formation of
sulfoxide observed) after which the reaction was warmed to r.t. and stirred overnight. Upon completion
of the reaction, the crude product was dryꢀloaded on silica and purified by column chromatography
(CH2Cl2/MeOH 9:1) to yield the desired product as a white solid (35 mg, 34%). RF = 0.85
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(CH2Cl2/MeOH 9:1). 1H NMR (400 MHz, DMSOꢀd6) (small amount of mCBA present) δ = 8.93 (d, J =
8.5 Hz, 1H), 8.01 (d, J = 9.5 Hz, 1H), 7.87 ꢀ 7.93 (m, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.18 ꢀ 7.34 (m, 7H),
6.94 ꢀ 6.88 (m, 2H, 2 signals overlapping), 6.81 ꢀ 6.86 (m, app. d, 2H), 6.33 (d, J = 9.5 Hz, 1H), 6.01
(dd, J = 8.5, 5.0 Hz, 1H), 5.43 (d, J = 4.5 Hz, 1H), 5.22 (s, 2H), 4.89 ꢀ 4.84 (m, app. d, 2H), 4.46 (d, J =
18.5 Hz, 1H)(part of ABꢀsystem), 4.23 (d, J = 18.5 Hz, 1H)(part of ABꢀsystem), 3.70 (s, 3H), 3.60 (d, J
= 5.0 Hz, 2H) ppm. 13CꢀNMR: (101 MHz, DMSOꢀd6) δ = 170.9, 164.5, 160.6 (2C), 160.2, 159.4, 155.2,
144.3, 135.6, 130.4 (2C), 129.5, 129.2 (2C), 128.2 (2C), 126.6, 126.5, 124.6, 124.0, 113.7 (2C), 112.9,
112.8, 101.6, 67.8, 66.8, 66.2, 58.4, 55.1, 50.9, 41.2 ppm. LRMS calcd. for C33H27N2O10S, MꢀH =
643.14, mass found; MꢀH = 643.0.
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(6R,7R)ꢀ8ꢀoxoꢀ3ꢀ(((2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)methyl)ꢀ7ꢀ(2ꢀphenylacetamido)ꢀ5ꢀthiaꢀ1ꢀ
azabicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀcarboxylic acid 5ꢀoxide (FC4)
Compound 5 (50 mg, 81.6 ꢃmol) was cooled to 0 °C prior to the addition of TFA/anisole (5:1, 3 mL).
The resulting reaction mixture was stirred at 0 °C for 30 min. with constant monitoring of the
conversion by TLC analysis (CH2Cl2/EtOAC, 8:2). Upon completion of the reaction cold Et2O (5 mL)
was added which resulted in the formation of a precipitate. The solid material was filteredꢀoff and
washed with Et2O (2 x 5 mL) and subsequently dried under high vacuum to yield the desired product as
an offꢀwhite solid (15 mg, 37%). RF = 0.10 (CH2Cl2/MeOH +AcOH, 9:1). 1H NMR (400 MHz, DMSOꢀ
d6) δ = 8.45 (d, J = 8.5 Hz, 1H), 8.00 (d, J = 9.5 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.27 ꢀ 7.32 (m, 4H),
7.24 (td, J = 8.5, 4.0 Hz, 1H), 6.93 ꢀ 7.01 (m, 1H), 6.31 (d, J = 9.5 Hz, 1H), 5.81 (dd, J = 8.0, 4.5 Hz,
1H), 5.14 (d, J = 12.5 Hz, 1H), 4.87 ꢀ 4.93 (m, 1H), 3.98 (d, J = 18.0 Hz, 1H), 3.70 (d, J = 14.0 Hz, 1H)
(part of ABꢀsystem), 3.62 (d, J = 18.5 Hz, 1H), 3.54 (d, J = 14.0 Hz, 1H) (part of ABꢀsystem) ppm. 13Cꢀ
NMR: (101 MHz, DMSOꢀd6) δ = 171.1, 164.2, 162.2, 161.1, 155.3, 144.3, 135.8, 129.6, 129.1 (2C),
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128.3 (2C), 126.6, 112.8 (2C), 101.6, 67.5, 66.3, 58.3, 45.3, 41.5 ppm. LRMS calcd. for C25H19N2O8S,
MꢀH = 507.09, mass found; MꢀH = 507.0. Retention time = 7.15 min. (purity = 99%).
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(6R,7R)ꢀ8ꢀoxoꢀ3ꢀ(((2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)methyl)ꢀ7ꢀ(2ꢀphenylacetamido)ꢀ5ꢀthiaꢀ1ꢀ
azabicyclo[4.2.0]octꢀ2ꢀeneꢀ2ꢀcarboxylic acid 5,5ꢀdioxide (FC5)
Compound 6 (20 mg, 0.03 mmol) was cooled to 0 °C prior to the addition of TFA/anisole (5:1, 1.2
mL). The reaction was stirred at 0 °C for 30 min followed by 30 min at r.t. with constant monitoring of
the conversion by TLC analysis (CH2Cl2/MeOH, 9:1). Upon completion of the reaction cold Et2O (5
mL) was added which resulted in the formation of a precipitate. The solid material was filteredꢀoff and
washed with Et2O (2 x 3 mL) and subsequently dried under high vacuum to yield the desired product as
an offꢀwhite solid (10 mg, 63%). RF = 0.10 (CH2Cl2/MeOH +AcOH, 9:1). 1H NMR (400 MHz, DMSOꢀ
d6) δ = 8.92 (d, J = 9.0 Hz, 1H), 8.01 (d, J = 9.5 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.19 ꢀ 7.31 (m, 5H)
7.04 (d, J = 2.5 Hz, 1H), 6.99 (dd, J = 8.5, 2.5 Hz, 1H), 6.32 (d, J = 9.5 Hz, 1H), 5.97 (dd, J = 8.5, 4.5
Hz, 1H), 5.42 (d, J = 4.5 Hz, 1H), 4.90 ꢀ 5.00 (m, app. q., 2H), 4.41 (d, J = 18.0 Hz, 1H), 4.19 (d, J =
18.0 Hz, 1H), 3.54 ꢀ 3.65 (m, app. q., 2H) ppm. 1H NMR (101 MHz, DMSOꢀd6) δ = 170.9, 164.3, 162.1,
160.8, 160.2, 155.2, 144.3, 135.6, 129.6, 129.2 (2C), 128.2 (2C), 126.5, 125.2, 123.8, 112.9, 112.9,
101.7, 66.8, 66.3, 58.3, 50.8, 41.2 ppm. LRMS calcd. for C25H19N2O8S, MꢀH = 507.09, mass found; Mꢀ
H = 507.0. Retention time = 8.98 min. (purity = 95%).
ASSOCIATED CONTENT
Supporting Information. Experimental procedures, characterization of intermediates and target
compounds, description of protein product and purification, biological assays, determination of residual
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activity measurements and IC50 values as well as in silico docking data. This material is available free of
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AUTHOR INFORMATION
Corresponding Author
To whom correspondence should be addressed. Tel: +44 (0) 1865 275625;
email: christopher.schofield@chem.ox.ac.uk
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank the Medical Research Council (MRC)/Canadian grant G1100135, Biotechnology and
Biological Sciences Research Council (BBSRC) for support of J.B., R.S., R.C. and A.V. The Cancer
Research UK (CRUK) is kindly acknowledged for the support of S.S.v.B. The OPPFꢀUK is supported
by the MRC and BBSRC.
ABBREVIATIONS
MBL = metalloꢀβꢀlactamase, SBL = serineꢀβꢀlactamase, FC = fluorogenic cephalosporin, DiPEA =
N,N’ꢀdiisopropylethylamine; DMF = dimethylformamide; 7ꢀHC = 7ꢀhydroxy coumarin; MeCN =
acetonitril; mCPBA = metaꢀchloro perbenzoic acid; MMC = 7ꢀmercaptoꢀ4ꢀmethyl coumarin; PMB =
paraꢀmethoxy benzyl; AcOH = acetic acid; TFA = trifluoroacetic acid.
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to detect potential slow binding inhibitors as for example in the case of NDMꢀ1 / nitrocefin, where the
substrate presented a low ꢂM range KM with the enzyme concentration being in the same ꢂM range
(also see ref 39).
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