Structural studies of triazole inhibitors with promising inhibitor effects against antibiotic resistance metallo-β-lactamases

Article abstract of DOI:10.1016/j.bmc.2020.115598

Metallo-β-lactamases (MBLs) are an emerging cause of bacterial antibiotic resistance by hydrolysing all classes of β-lactams except monobactams, and the MBLs are not inhibited by clinically available serine-β-lactamase inhibitors. Two of the most commonly encountered MBLs in clinical isolates worldwide – the New Delhi metallo-β-lactamase (NDM-1) and the Verona integron-encoded metallo-β-lactamase (VIM-2) – are included in this study. A series of several NH-1,2,3-triazoles was prepared by a three-step protocol utilizing Banert cascade reaction as the key step. The inhibitor properties were evaluated in biochemical assays against the MBLs VIM-2, NDM-1 and GIM-1, and VIM-2 showed IC₅₀ values down to nanomolar range. High-resolution crystal structures of four inhibitors in complex with VIM-2 revealed hydrogen bonds from the triazole inhibitors to Arg228 and to the backbone of Ala231 or Asn233, along with hydrophobic interactions to Trp87, Phe61 and Tyr67. The inhibitors show reduced MIC in synergy assays with Pseudomonas aeruginosa and Escherichia coli strains harbouring VIM enzymes. The obtained results will be useful for further structural guided design of MBL inhibitors.

Full text of DOI:10.1016/j.bmc.2020.115598

Journal Pre-proofs
Structural studies of triazole inhibitors with promising inhibitor effects against
antibiotic resistance metallo-β-lactamases
Zeeshan Muhammad, Susann Skagseth, Marc Boomgaren, Sundus Akhter,
Christopher Fröhlich, Aya Ismael, Tony Christopeit, Annette Bayer, Hanna-
Kirsti S. Leiros
PII:
DOI:
Reference:
S0968-0896(20)30428-4
https://doi.org/10.1016/j.bmc.2020.115598
BMC 115598
To appear in:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
8 January 2020
26 May 2020
9 June 2020
Please cite this article as: Muhammad, Z., Skagseth, S., Boomgaren, M., Akhter, S., Fröhlich, C., Ismael, A.,
Christopeit, T., Bayer, A., Leiros, H.S., Structural studies of triazole inhibitors with promising inhibitor effects
against antibiotic resistance metallo-β-lactamases, Bioorganic & Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.bmc.2020.115598
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Structural studies of triazole inhibitors with
promising inhibitor effects against antibiotic
resistance metallo--lactamases
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Zeeshan Muhammad1, Susann Skagseth2, Marc Boomgaren1, SundusAkhter1, Christopher Fröhlich2, Aya
Ismael1, Tony Christopeit2, Annette Bayer1,*, Hanna-Kirsti S. Leiros2,*
1
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Department of Chemistry, Faculty of Science and Technology, UiT The Arctic University of Norway, N-9037
Tromsø, Norway.
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The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and
Technology, UiT The Arctic University of Norway, N-9037 Tromsø, Norway.
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Corresponding authors: Annette Bayer, E-mail: annette.bayer@uit.no , Phone +47 77 64 40 69;
Hanna-Kirsti S. Leiros, E-mail: hanna-kirsti.leiros@uit.no , Phone +47 77 64 57 06.
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Keywords:
Metallo-β-lactamase inhibitor, NH-triazole, inhibition properties, crystal structure,
structural guided design.
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Abbreviations: DMSO, dimethyl sufoxide; GIM, German imipenemase; IC50, half maximal inhibitory
concentration; IPM, imipenem; MBL, metallo-β-lactamase; NCF, nitrocefin; NDM, New Delhi metallo-
β-lactamase; VIM-2, Verona integron-encoded metallo-β-lactamase.
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Running Title: Triazole inhibitors hitting MBLs
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Abstract
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Metallo-β-lactamases (MBLs) are an emerging cause of bacterial antibiotic resistance by hydrolysing
all classes of β-lactams except monobactams, and the MBLs are not inhibited by clinically available
serine-β-lactamase inhibitors. Two of the most commonly encountered MBLs in clinical isolates
worldwide - the New Delhi metallo-β-lactamase (NDM-1) and the Verona integron-encoded metallo-
β-lactamase (VIM-2) - are included in this study.
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A series of several NH-1,2,3-triazoles was prepared by a three-step protocol utilizing Banert cascade
reaction as the key step. The inhibitor properties were evaluated in biochemical assays against the
MBLs VIM-2, NDM-1 and GIM-1, and the former show IC50 values down to nanomolar range were
confirmed. High-resolution crystal structures of four inhibitors in complex with VIM-2 revealed
hydrogen bonds from the triazole inhibitors to Arg228 and to the backbone of Ala231 or Asn233, along
with hydrophobic interactions to Trp87, Phe61 and Tyr67. The inhibitors show reduced MIC in synergy
assays with Pseudomonas aeruginosa and Escherichia coli strains harbouring VIM enzymes. The
obtained results will be useful for further structural guided design of MBL inhibitors.
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1 Introduction
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The emergence and spread of antibiotic resistant bacteria are defined as a global health problem by
the World Health Organization (WHO).1 The increase in Gram-negative antibiotic resistance bacteria is
particularly worrisome. Pan-resistance or extreme drug resistance are now commonly used terms to
describe clinically important isolates of Pseudomonas aeruginosa, Acinetobacter baumannii and
Enterobacteriaceae that are resistant to virtually all antibiotics2.
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There are several causes for antibiotic resistance but the most common mechanism for -lactam
resistance is the presence of -lactamases enzymes that cleave the -lactam ring rendering the drug
inactive.3-5 Drug treatment using -lactamase inhibitors (BLI) as adjuvants to re-potentiate antibiotics
is already in clinical use, e.g. the new serine-BLI avibactam has been approved in USA as a combination
treatment with ceftazidime against complicated urinary tract infections and intra-abdominal
infections.5 Other examples in the clinic are the -lactam-BLI combinations amoxicillin-clavulanate,
6
ticarcillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam.5, These BLIs inactivate
primarily class A serine β-lactamase (SBL) enzymes, and avibactam also inhibits class C and some class
D SBL enzymes.5, 6
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For class B metallo-β-lactamases (MBLs) no clinically approved BLIs are available. Lately, interesting
results have been reported for the thiazole-4-carboxylic acid analogue ANT431 showing promising
results against NDM-1 (Ki = 0.29 M) and VIM-2 (Ki = 0.19 M) including in vivo inhibitor efficacy.7
Other recently described inhibitors include the natural product aspergillomarasmine,8 which showed
in vivo inhibitor efficacy against NDM-1, azolylthioacetamides9 with Ki (NDM-1) = 0.43 M,
bisthiazolidines10 with Ki (NDM-1) = 7-19 M and 1,2,4-triazole-3-thiones with Ki (NDM-1) = 0.72 M.11,
12 However, the clinical need for an MBL inhibitor is still outstanding.
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Fokin and coworkers reported that 1,2,3-NH-triazoles of the general formula 1 were promising VIM-2
inhibitors.13, 14 In their study, the best compounds (1cc`and 1cd`) showed sub-micromolar activity (IC50
= 0.07 μM; Ki = 0.02 μM) against VIM-2, and were able to re-potentiate the -lactam antibiotic
imipenem in VIM-2 producing Escherichia coli (BL21) cells when tested at 150 μM concentration.13
Based on docking studies, they predicted 1 to bind to the VIM-2 active site through the sulfonyl group
as a zinc binding group. We became interested to gain insight into the inhibitory action of the 1,2,3-
NH-triazoles 1 in order to further explore the potential of this scaffold as MBL inhibitors. With our
experience in crystallization of inhibitors with MBLs15-17 we aimed for crystallographic studies of
enzyme–inhibitor complexes to understand the mode of binding and low IC50 values exhibited by the
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1,2,3-NH-triazoles 1. The exact interactions involved in the substrate binding of VIM- 2 are not clear,
since no crystal structure in complex with a substrate has been reported.
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Here, we report the synthesis of a small focussed library of new and reported13, 14 (1cc', 1cd', 1dg',
1dh') analogues of 1,2,3-NH-triazoles and our investigation of their potential as inhibitors of the
previously reported VIM-2, and additionally NDM-1, a clinically important MBL, and GIM-1. The
compounds were evaluated in biochemical and cell-based assays, and for the most promising
compounds the inhibitory effect in synergy with meropenem was tested against clinical strains of P.
aeruginosa (VIM-2), K. pneumoniae (NDM-1) and E. coli (VIM-29). We obtained crystal structures of
four inhibitors in complex with VIM-2. In addition, a structure activity relationship (SAR) analysis of the
observed inhibition patterns is provided using reported crystal structures of the three target enzymes
(NDM-1 complex with hydrolysed ampicillin 18, VIM-2 in complex with fragments 17 and wild type GIM-1
19).
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2 Results and discussion
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2.1 Synthesis of NH-1,2,3-triazole inhibitors
A small library of NH-1,2,3-triazoles 1aa'–1dj' were synthesized as shown in Scheme 1A.13 Treatment
of 4-chlorobutyneamine 2 with sulfonyl chlorides and base (K2CO3) provided the chlorosulfonamides
3a-d, which were converted to the respective azidosulfonamides 4a-d. The crude azides 4a–d
containing different sulphonamide groups (R1 in red) underwent the Banert cascade20-22 to NH-triazole
sulphonamides 1aa'-di' in the presence of a range of nucleophiles (R2 in blue). For a mechanistic
proposal of the Banert cascade see Scheme 1B.21, 23, 24 The acetate substituted triazole 1dj' was
prepared by acetylation of the corresponding alcohol obtained from cyclisation with water as
nucleophile.
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Scheme 1. Synthesis and structures of NH-1,2,3-triazole based inhibitors.
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2.2 Characterization of inhibitor properties against VIM-2, GIM-1 and NDM-1
The inhibitory activities of the NH-1,2,3-triazoles 1aa'–1dj' against the MBLs NDM-1, VIM-2 and GIM-
1 were evaluated as the half maximal inhibitory concentration (IC50) values in biochemical competition
assays (Table 1). For VIM-2 and GIM-1, the IC50 values were measured using nitrocefin as a reporter
substrate, while IC50 values for NDM-1 were measured with imipenem as reporter substrate. Nitrocefin
is hydrolysed by NDM-1 with a too high catalytic efficiency and is unsuitable as a reporter substrate
for NDM-1.25 The sequence identity between the three MBL enzymes used is 28% for VIM-2 versus
GIM-1, 32% between VIM-2 and NDM-1 and 24% between NDM-1 and GIM-1.15
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Compounds 1cc', 1cd', 1dg' and 1dh' have been evaluated as VIM-2 inhibitors by Fokin and coworkers
resulting in IC50 values of 0.07 µM for 1cc', 1cd' and 1dh' and 7.3 µM for 1dg',13 which were similar to
our IC50 values of 0.23 (1cc'), 0.12 (1cd'), 0.53 (1dh') and 15 (1dg') µM (Table 1). When these triazoles
were investigated against GIM-1, the inhibition was poor for 1dg' (IC50 = 169 µM) and 1dh' (IC50 = 193
µM) and no inhibition was observed for 1cc' and 1cd' and none of the four triazoles (1cc', 1cd', 1dg'
and 1dh') were active against NDM-1.
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Investigation of an extended library containing triazoles 1aa'-dj' confirmed the observation that the
compounds with the general structure of 1 were selective VIM-2 inhibitors with IC50s from 0.07–23
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µM, while inhibition of GIM-1 and NDM-1 was generally weaker. GIM-1 was inhibited by 9 compounds
(IC50s from 18–353 µM) with 1dd' (IC50 = 18 µM) being the best inhibitor. The reason for the weak
inhibition of GIM-1 is not obvious and other inhibitor classes showed good inhibition towards GIM-1
in this assay.15 Only 7 compounds were active against NDM-1 (IC50s from 81–231 µM) with 1de' (IC50 =
81 µM) being the most active compound.
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The most potent VIM-2 inhibitors 1cc' and 1cd' (R1 = 2,5-dichlorophenyl and 1dc' and 1dd' (R1 = 4-
iodophenyl) showed high nanomolar inhibition (IC50 = 0.067–0.23 µM). These compounds are
structurally similar as they contain halogenated aromatic R1 substituents although with different
spatial arrangement (2,5-substitution compared to 4-substitution) and cyclic alkyl amino groups as R2
substituents (cyclohexyl or adamantly), which may explain the similar inhibition properties.
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The library contained two series of compounds containing identical R2 groups; one consisting of 1bc'
(IC50 = 1.5 µM), 1cc' (IC50 = 0.23 µM) and 1dc' (IC50 = 0.067 µM) with R2 like cyclohexylamino and the
other consisting of 1bd' (IC50 = 2.3 µM), 1cd' (IC50 = 0.12 µM), and 1dd' (IC50 = 0.16 µM) with R2 like
adamantylamino. Comparison of the compounds in a series provided an indication that the
halogenated R1 groups of compounds 1cc'/1cd' (R1 = 2,5-dichlorophenyl) and 1dc'/1dd' (R1 = 4-
iodophenyl) were slightly advantageous (a 5–10-fold reduction in IC50s) over the hydrocarbon based R1
group of compounds 1bc'/1bd' (R1 = 4-methylphenyl).
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A wide range of R2 substituents were investigated in the 1d series (Table 1). The inhibition of VIM-2
varied from IC50 of 0.07 µM for 1dc' (R2 = cyclohexylamino) to IC50 > 250 µM for 1de' with the very
bulky triphenylmethylamino group as R2 substituent. In the middle range (IC50 = 15–21 µM), we found
inhibitors 1df' (R2 = 4-methoxybenzylsulfide), 1dg' (R2 = iso-propoxy) and 1dj' (R2 = acetate) with
structurally very different R2 substituents. Unfortunately, inhibitor 1di' with benzylamine as R2
substituent resulted in precipitation.
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2.3 Evaluation of inhibitors in bacterial cell assays, with whole E. coli cells and synergy assays
To investigate the inhibitory activity against MBLs in bacterial cells, two different assays were used.
The first was E. coli SNO3 cells transformed with blaVIM-2, blaGIM-1 or blaNDM-1 (Table 1). The enzyme
production was induced by addition of IPTG. The inhibitory activity was measured as the difference in
speed of hydrolysis of the reporter substrate between the presence and absence of inhibitor, according
to equation 1. A high degree of inhibition then indicate that the inhibitor prevents the MBL hydrolytic
activity of breaking down the reporter substrate, but works as an inhibitor in a cell.
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The inhibitory activities against VIM-2 in bacterial cells of 1aa'-1dj' (Table 1) varied from 95% inhibition
to inactive and were in good agreement with the inhibition determined in the biochemical assay. The
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most active inhibitors (1bb', 1bc', 1cc', 1cd', 1dc' and 1dd') determined in the biochemical assay (IC50
from 0.07–2.3 µM) gave 82–96% inhibition in the cell based assay, except for 1cd', where no inhibition
was found in the whole cell assay. This indicates that the inhibitors cross the outer E. coli membrane
and hit VIM-2 localized in the periplasmic space. The reason for 1cd' only being active towards purified
VIM-2 (IC50=0.12 µM) and not in the whole cell assay (with VIM-2), can be that this inhibitor did not
cross the outer E. coli membrane which is different from the most active inhibitors.
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For GIM-1 producing E. coli SNO3 cells, we observed much lower levels of percent inhibition for
inhibitors 1aa'-1dj' with 3-25% in agreement with the higher IC50 values observed against this enzyme
compared to VIM-2 (Table 1). The highest percent inhibition was obtained for 1df' with 33%. In the
NDM-1 whole cell assay, the percent inhibition was further decreased with most of the investigated
compounds showing no inhibition (Table 1). Only inhibitors 1dd' and 1df' showed inhibitor properties
with 17% and 34% inhibition, respectively.
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Additionally a second cell based assay was performed where the inhibitory effect in synergy with
meropenem of 1cc', 1dc' and 1dj' was tested against clinical strains of P. aeruginosa (VIM-2), K.
pneumoniae (NDM-1) and E. coli (VIM-29) (Table 2). Not surprising, none of the tested inhibitors
affected the NDM-1 producing K. pneumoniae strain. Inhibitor 1cc' gave a reduced MIC from 64 to 8
mg/L in VIM-2 producing P. aeruginosa, but did not affect the E. coli strain. Our most promising hit is
1dj', which at low inhibitor concentration (50 mM) lowered the MIC from 64 to 1 mg/L for VIM-2
producing P. aeruginosa and from 16 to 1 mg/L for VIM-29 producing E. coli. VIM-2 and VIM-29 have
90% sequence identity, thus it is likely that an E. coli producing VIM-29 could also be inhibited and give
a reduce MIC. In the synergy assay meropenem was the reporter substrate. The own effect from the
inhibitors (>500 µM) did not show any toxicity (data not shown).
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2.4 Triazole inhibitors bound to VIM-2 in crystal structure complexes
Crystal structures of VIM-2 in complex with the inhibitors 1cc', 1dh', 1di' and 1dj' were used to
investigate the interactions involved in the binding of the inhibitors. For the inhibitors 1cc', 1dh' and
16
1di', the DMSO-free co-crystallization method was applied to obtain complex structures, whereas
the inhibitor 1dj' was soaked into native VIM-2 crystals.
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The complex structures with the inhibitors 1cc' and 1dh' crystallized in the space group P21212 with
one protein molecule in the asymmetric unit. The two other complexes structures crystallized in the
space group C2 with two protein molecules in the asymmetric unit. Space group C2 has previously
been reported for VIM-2 17, but space group P21212 has not been observed before. The resolution of
the obtained structures was ≤ 1.5 Å, with the complex structure VIM-2_1cc' showing the best
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resolution of 1.07 Å. To our knowledge, this is the highest resolution structure so far reported for VIM-
2. Details on the statistics for the data collection and the refinement are shown in Table 3 and Table
4.
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Overall the protein structures as well as active site conformations of the obtained VIM-2 complexes
were all in accordance with previously reported structures and no major differences were noticed. 15-
17, 26-28 The crystal structures with the inhibitors 1dh' and 1cc' showed unassigned electron density in
the active site of the enzyme, clearly corresponding to the inhibitors (Figure 2A and D). Also, the
structures with inhibitors 1di' and 1dj' showed unassigned electron density in the active site of one or
both VIM-2 protein molecules, respectively. However, this electron density was less well defined and
more ambitious to interpret, reflecting a lower occupancy and a higher degree of disorder of the bound
inhibitors (Figure 2G and K). Several of the structures showed radiation damages,29-31 caused to
interactions between the bright synchrotron X-ray beam and the protein in the crystal. This is seen as
positive peaks in the difference Fourier electron density maps. Upon radiation damage, the absorbed
energy is dissipated as covalent bond breakage and heat resulting in higher thermal vibration. In the
complex structure with the inhibitor 1dh' and 1cc', the Cys221 was partially oxidized to the cysteine
sulfonate, a radiation damage previously observed for VIM-217, 28. Furthermore, radiation damage in
the iodine-carbon bound in the inhibitors 1dh', 1di' and 1dj' was observed, most likely due to electron
capture. 30
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The position of the four inhibitors in the active site of VIM-2 and the interactions with the protein are
shown in Figure 2. A common feature in the binding mode of all inhibitors was the orientation of the
NH-1,2,3-triazole moiety and the sulfonamide group. The triazole moiety directly interacted with one
of the two zinc ions in the active site and with the bridging hydroxide ion. In addition, one of the
nitrogen atoms in the triazole ring formed an interaction with Arg228 through a hydrogen bond. The
sulfonamide group occupied slightly different positions in the complex structures of 1cc', 1dh', 1di' and
1dj' (conformation A). However, the main orientation was similar and allowed hydrogen bond
interactions with the protein backbone of Ala231 and/or Asn233. Previously reported docking studies
with arylsulfonamid-NH-123-triazoles suggested that either the sulfonamide group13, 14 or the triazole
ring32 interact with the zinc ions in the active site. Furthermore, in all docking studies the hydroxide
ion bridging the two zinc ions was replaced by the inhibitor. In contrast, our results clearly show that
the hydroxide ion is not replaced by the inhibitors and that the triazole moiety interacts only with one
of the two zinc ions (Zn2), whereas the sulfonamide group does not participate in the zinc binding.
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The inhibitors 1dh', 1di' and 1dj' have a 4-iodophenyl group in the R2 position towards His263 and
Arg228. In all complex structures with these inhibitors, the iodine-carbon bond was radiation damaged
and the electron density map clearly showed that the iodine was separated from the inhibitor (Figure
2D, G and K). These radiation damages most likely induced changes in the orientation of the benzene
ring as well as the iodine, as also observed for a brominated DNA/RNA hybrid.30 Hence, the
interpretation of the interactions with the iodophenyl group was difficult. However, the results
indicate that the moiety forms hydrophobic interaction with Tyr67 and His263. Furthermore, the
benzene ring might form a cation- stacking with Arg228. In the inhibitor 1cc', the iodobenzene moiety
in position R2 is replaced by a dichlorobenzene, which adopts two different conformation. In both
conformations, the benzene ring forms hydrophobic interactions with the Tyr67, with conformation A
adopting an orientation better suited for a - stacking. In addition, one of the chlorine ions interacts
in both conformations with Arg228 and in conformation B with the backbone of Ala231.
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All four inhibitors have different substituents in the R1 position towards Trp87. The inhibitor 1cc' has a
cyclohexylamine moiety at this position. The cyclohexyl ring of the moiety interacts with His118, Trp87
and Phe61, and the amine nitrogen interacts with the hydroxide ion located between the two active
site Zn ions (Figure 2B, C) through a hydrogen bond. Similar interactions were observed both for
inhibitor 1dh' between the catalytic hydroxide ion and the oxygen of the isopropoxy moiety (Figure
2E, F) and for inhibitor 1di' involving the nitrogen of the benzylamino group (Figure 2H, I). In the latter
complex structure, the benzene ring of the benzylamino group additionally seems to interact with
Trp87, Phe61 and Tyr67. However, for the benzylamino group of 1di' and the methyl acetate moiety
of 1dj', only weak electron densities were observed. Hence, the interpretation of the exact orientations
and the interactions with the protein were difficult. The low electron density and the disordered
structure might indicate that these moieties do not form strong interactions with the protein.
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The binding interactions identified in the above VIM-2 inhibitor complexes may explain the reduced
inhibitor activity of the NH-triazoles towards NDM-1 and GIM-1. The important reside determinants in
the NDM-1 binding site are Phe63, Lys224 and Ala228 (see e.g. 15). In the VIM-2 inhibitor complexes
we observed hydrophobic interactions with Tyr67 and cation- stacking interactions with Arg228,
which both are not possible with the corresponding Val67 and Lys224, respectively, in NDM-1. On the
other hand, the hydrogen bonding interaction of the NH-triazole to Arg228 in VIM-2 may be possible
with the adjacent Lys224 in NDM-1. The GIM-1 the binding site includes Tyr64, Val76, Arg224 and
Trp228. For GIM-1, the guanidino group of Arg224 overlaps with the corresponding group of Arg228
in VIM-2 making hydrogen bonding to the NH-triazole possible. However, Tyr64 in GIM-1 is too far
away to replace the nice - stacking involving Tyr67 in VIM-2, thus might explain the lower inhibitor
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potential with high IC50 values for GIM-1. Moreover, VIM-29 (UniProt J7HGI2), present in the E. coli
strain used for the synergy assay, carries Tyr67, His224 and Ser228, so the polar His228 and aromatic
Tyr67 could allow for good inhibitor binding and support the low MIC in the synergy assays (Table 2).
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3 Conclusion
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Inspired by encouraging results on using triazoles as MBL inhibitors, we prepared a small focused
chemical library with 15 NH-1,2,3-triazole molecules, which was tested for inhibitory against VIM-2,
GIM-1 and NDM-1.
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We found that VIM-2 was inhibited by several NH-1,2,3-triazole and the new inhibitor 1dc' gave nano-
molar affinity with IC50 of 0.067 µM. This inhibitor also affected GIM-1 (IC50=69 µM) and NDM-1
(IC50=148 µM), but to a much lower extent.
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The most promising inhibitor was 1dj' with moderate IC50 values of 23 µM (VIM-2), 48 µM (GIM-1) and
231 µM (NDM-1). More interestingly, the synergy assay found 1dj' to effects two clinical isolates. One
P. aeruginosa producing VIM-2 reduced the MIC from 64 mg/L with only meropenem to only 1 mg/L
for meropenem and 1dj'; and an E. coli producing VIM-29 showed reduced MIC from 16 mg/L (only
meropenem) to 1 mg/L when combining meropenem and 1dj'. The 1.50 Å complex structure of VIM-
2_1dj' show tight aromatic - stacking to Tyr67, binding from the NH-1,2,3-triazole group to Arg228
and the active site Zn2 ion, and the presence of the hydroxyl ion between the two zinc ions. Our new
complex structure of VIM-2_1dj' is therefore a valuable starting point for structure guided inhibitor
design of a new inhibitor targeting several MBL enzymes simultaneously.
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Tables and figures:
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Table 1. The molecular structures of the synthesized inhibitors with measured inhibition concentrations
(IC50) against pure VIM-2, GIM-1 and NDM-1 enzymes; followed by %inhibition (equation 1) in E. coli
SNO3 bacterial whole cell experiments with blaVIM-2, blaGIM-1 or blaNDM-1.
VIM-2 a
GIM-1 a
NDM-1 b
IC50
%
IC50
%
IC50
%
inhib a
inhib a
inhib
R1
R2
(M) a
(M) a
(M)
1aa'
1bb'
23
29
NI
NI
NI
NI
7.2
60
128
4.5
142
NI
1bc'
1bd'
1.5
2.3
82
84
NI
2.3
NI
144
ND
NI
NI
83 (67)
1cc'*
1cd'
0.23
0.12
94
NI
NI
7
98
NI
NI
P (7.7)
21
ND
1dc'
0.067
0.16
95
96
69
18
3
148
ND
NI
1dd´
22
17
1de'
>250
19
353
3
81
NI
1df'
21
45
227
33
ND
34
1dg'
15
51
85
169
193
11
11
NI
NI
NI
NI
1dh'*
0.53
- 11 -

1di'*
1dj'*
P
ND
ND
ND
48
ND
ND
ND
ND
ND
23
231
b
260
261
262
a the reported substrate was nitrocefin; the reported substrate was imipenem; NI: no observable
inhibition; ND: not determined; P: precipitated. * A VIM-2 complex structure is reported here.
263
- 12 -

264
265
266
Table 2. Synergy test of selected inhibitors against clinical strains containing VIM-2, NDM-1 or VIM-29
and meropenem (MEM) or MEM and inhibitor to determine the MIC in mg/L. The inhibitor
concentration was 50 µM (1cc', 1dj') or 125 µM (1dc').
Ref. no
Species
MBL
K34-7
K66-45
50639799
P. aeruginosa
K. pneumoniae
E. coli
VIM-2
NDM-1
VIM-29
MIC (mg/L)
MIC (mg/L)
MIC (mg/L)
MEM
64
8
32-64
32
32
64
P
16
16
nd
nd
P
MEM+1cc'*
MEM+1dd'
MEM+1dc'
MEM+1dh'*
MEM+1di'*
MEM+1dj'*
16
8
P
P
P
P
1
64
1
267
268
269
* a VIM-2 complex structure is reported here. nd: not determined.
P: precipitated
- 13 -

270
271
Table 3. X-ray data collection statistics for VIM-2 in complex with compound 1cc', 1dh', 1di' and 1dj'.
Values in parenthesis are for the highest resolution shell.
VIM-2_1cc'
ID23-1, ESRF
0.97625
VIM-2_1dh'
ID23-1, ESRF
0.97625
-173
VIM-2_1di'
ID29, ESRF
0.983998
-173
VIM-2_1dj'
ID29, ESRF
0.983998
-173
Diffraction source
Wavelength (Å)
Temperature (°C)
-173
Crystal-detector distance (mm) 158.67
201.24
0.1
275.00
0.05
275.00
0.1
Rotation range per image (°)
Total rotation range (°)
Exposure time per image (s)
Space group
0.15
135
130
130
180
0.037
P 21212
0.037
0.04
0.037
P 21221
C2
C2
98.35,
44.34,
60.84
90.00
90.75,
45.81,
63.93
90.00
100.59,
79.03,
67.24
101.28,
79.27,
67.69
a, b, c (Å)
γ (°)
130.09
130.35
38.26-1.07
(1.10-1.07)
52.26-1.40
(1.43-1.40)
31.33-1.50
(1.52-1.50)
39.63-1.50
(1.52-1.50)
Resolution range (Å)
No. of unique reflections
Multiplicity
115206
52181
61683
64863
4.8 (4.5)
97.8 (93.6)
6.4 (100)
11.4 (1.5)
4.7 (4.6)
98.3 (87.4)
6.3 (10.5)
13.0 (1.5)
2.5 (2.5)
95.8 (98.0)
4.8 (27.9)
11.5 (2.7)
3.4 (3.3)
99.5 (100)
7.2 (87.9)
9.4 (1.2)
Completeness (%)
Rmerge (%)
Mean 〈 I/σ(I)〉
Overall B-factor from Wilson
10.41
17.31
13.60
16.38
plot (Å2)
272
273
- 14 -

274
275
Table 4. Crystallographic refinement statistics for VIM-2 in complex with compounds 1dd', 1ei', 1ej' and
1ek'.
VIM-2_1cc'
6TM9
VIM-2_1dh'
6TMC
VIM-2_1di'
6TMB
VIM-2_1dj'
6TMA
PDB entry
Final Rwork (%)
Final Rfree (%)
11.01
13.16
13.87
15.74
12.50
15.80
16.42
19.54
Molecules in
asymmetric unit
1
1
2
2
No. of non-H atoms
Protein
1989
1888
3676
3600
2 Zn2+, 1 Cl-, 1
OH-
2 Zn2+, 1 Cl-, 1 OH-
6 Zn2+, 4 Cl-, 2 OH- 6 Zn2+, 4 Cl-, 2 OH-
Ions
52 (2
conformations)
86 (2
conformations)
Ligand
23
26
Water
526
344
662
627
R.m.s. deviations
Bonds (Å)
0.010
1.217
0.012
1.005
0.010
1.016
0.005
0.748
Angles (°)
Average B factors (Å2)
Protein
13.87
20.9
17.32
22.72
Ion
10.94
17.3
21.80
27.94
Ligand (occupancy)
Water
14.56 (0.6/0.3)
33.16
22.9 (0.86)
38.6
46.61 (0.78)
34.46
64.31 (0.51/0.49)
37.53
Ramachandran plot
Most favoured (%)
Allowed (%)
97.12
2.06
96.9
2.2
97.75
2.25
97.93
1.61
- 15 -

277
278
- 16 -

279
280
281
282
283
284
285
286
287
288
Figure 2. Final elections density maps (A, D, G and K), crystal structures (B, E, H and L) and interaction plots (C,
F, I and M) for the inhibitor 1cc' (A, B and C), 1dh' (D, E, and F), 1di' (G, H and I) and 1dj' (K, L and M). The 2F0-FC
maps are shown in blue at 1.0 for 1cc' and 1dh' and at 0.9 for 1di' and 1dj'. The FC-F0 maps are shown at 4
(green) and -4 (red). In the crystal structures, carbon atoms of the inhibitors are depicted in cyan and protein
carbon atoms in salmon. For the interaction plots, crystal structures were analysed using LIGPLOT 33. Hydrogen
bonds are shown as green dashed lines and hydrophobic interactions by red arcs. All distances are given in Å.
Distances for alternative conformation are given in brackets. For inhibitor 1dj', the figures are only shown for
the VIM-2 molecule with higher ligand occupancy. In panel G, two water molecules are shown (red sphere),
which are assumed to be artefacts from the native structure without bound ligand.
- 17 -

289
4 Materials and Methods
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
4.1 Organic Synthesis
All reagents and solvents were purchased from commercial sources and used as supplied unless
otherwise stated. Compounds 3a, 3c, 3d, 4a, 4c, 4d, 1cc', 1cd', 1dg' and 1dh' were prepared according
to the literature.13 Reactions were monitored by thin-layer chromatography (TLC) with Merck pre-
coated silica gel plates (60 F254). Visualization was accomplished with either UV light or by immersion
in potassium permanganate or phosphomolybdic acid (PMA) followed by light heating with a heating
gun. Purification of reactions was carried out by flash column chromatography using silica gel from
Merck (Silica gel 60, 0.040 - 0.063 mm). Purity analysis was carried out on Waters Acquity UPLC® BEH
C18 (1.7 μm, 2.1 × 100 mm) column on a Waters Acquity I-class UPLC with Photodiode Array Detector.
NMR spectra were obtained on a 400 MHz Bruker Avance III HD equipped with a 5 mm SmartProbe
BB/1H (BB = 19F, 31P-15N). Data are represented as follows: chemical shift, multiplicity (s = singlet, d
= doublet, t = triplet, q = quartet, m = multiplet), coupling constant (J, Hz) and integration. Chemical
shifts (δ) are reported in ppm relative to the residual solvent peak (CDCl3: δH 7.26 and δC 77.16;
Methanol-d4: δH 3.31 and δC 49.00). Positive ion electrospray ionization mass spectrometry was
conducted on a Thermo electron LTQ Orbitrap XL spectrometer.
305
306
4.1.1 Synthesis of the sulfonamide derivatives from the corresponding sulfonylchlorides
The sulfonamides were prepared following the a procedure described by Weide et al.13
307
308
309
310
311
312
313
314
N-(4-chlorobut-2-ynyl)benzenesulfonamide (3a)13
Benzenesulfonylchloride (500 mg, 2.8 mmol, 1.0 equiv), 4-chlorobut-2-yn-1-amine hydrochloride 2
(515.26 mg, 3.68 mmol, 1.3 equiv), and K2CO3 (1.161 g, 8.4 mmol, 3.0 equiv), in THF/H2O (14 mL, 1:1)
gave compound 3a (650 mg, 72%) as a colorless solid. Rf = 0.37 (hexane/ethyl acetate, 65:35).
Analytical data were in accordance with literature.13 1H NMR (400 MHz, CDCl3) δ 8.00 – 7.87 (m, 2H),
7.70 – 7.50 (m, 3H), 3.95 (t, J = 2.1 Hz, 2H), 3.89 (t, J = 2.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 139.4,
132.8, 128.9, 127.2, 80.3, 79.4, 77.2, 32.9, 29.6. HRMS (ESI): Calcd. for C10H10O2NClNaS [M+H]+
266.0012; found 266.0013.
315
316
317
318
319
N-(4-chlorobut-2-ynyl)-4-methylbenzenesulfonamide (3b)
Toluene-4-sulfonylchloride (500 mg, 1.9 mmol, 1.0 equiv), 4-chlorobut-2-yn-1-amine hydrochloride 2
(477.42 mg, 2.50 mmol, 1.3 equiv), and K2CO3 (1.087 g, 7.5 mmol, 3.0 equiv), in THF/H2O (12 mL, 1:1)
gave compound 3c (550 mg, 85%) as a colorless solid. Rf = 0.46 (hexane/ethyl acetate, 35:65). 1H NMR
(400 MHz, CDCl3): δ 7.77 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 3.88 (d, J = 2.1 Hz, 4H), 2.43 (s, 3H).
- 18 -

320
321
13C NMR (101 MHz, CDCl3): δ 143.9, 136.6, 129.7, 129.5, 127.5, 80.7, 79.5, 33.1, 29.9, 29.4, 21.6. HRMS
(ESI): Calcd. for C11H12O2NClNaS [M+H]+ 280.0169; found 280.0169.
322
323
324
325
326
327
328
329
2,5-dichloro-N-(4-chlorobut-2-ynyl)benzenesulfonamide (3c)13
2,5-Dichlorobenzenesulfonylchloride (1000 mg, 4.1 mmol, 1.0 equiv), 4-chlorobut-2-yn-1-amine
hydrochloride 2 (741.4 mg, 5.3 mmol, 1.3 equiv), and K2CO3 (1.687 g, 12.21 mmol, 3.0 equiv), in
THF/H2O (21 mL, 1:1) gave compound 3d (1.084 mg, 87%) as a colorless solid. Rf = 0.40 (hexane/ethyl
acetate, 35:65). Analytical data were in accordance with literature.13 1H NMR (400 MHz, CDCl3): δ 8.09
(d, J = 2.2 Hz, 1H), 7.52 – 7.48 (m, 2H), 5.33 (t, J = 6.5 Hz, 1H), 3.96 (d, J = 6.3 Hz, 2H), 3.82 (s, 2H). 13
C
NMR (101 MHz, CDCl3): δ 138.6, 133.6, 133.3, 132.4, 130.9, 129.8, 79.7, 79.4, 77.2, 33.1, 29.3. HRMS
(ESI): Calcd. for C10H8O2NCl3S [M-H]- 311.9233; found 311.9230.
330
331
332
333
334
335
336
4-Iodo-N-(4-chlorobut-2-ynyl)-benzenesulfonamide (3d)13
4-Iodobenzensulfonylchloride (1000 mg, 3.3 mmol, 1.0 equiv), 4-chlorobut-2-yn-1-amine
hydrochloride 2 (602 mg, 4.3 mmol, 1.3 equiv), and K2CO3 (1.368 g, 9.9 mmol, 3.0 equiv), in THF/H2O
(18 mL, 1:1) gave compound 3e (880 mg, 72%) as a colorless solid. Rf = 0.41 (hexane/ethyl acetate,
1
35:65). H NMR (400 MHz, CDCl3): δ 7.96 – 7.83 (m, 2H), 7.61 (d, J = 8.6 Hz, 2H), 4.67 (s, 1H), 4.01 –
3.76 (m, 4H). 13C NMR (101 MHz, CDCl3): δ 176.2, 139.5, 138.4, 128.8, 100.5, 80.3, 79.8, 33.1, 29.7.
HRMS (ESI): Calcd. for C10H8O2NClIS [M-H]- 367.9022; found 367.9003.
337
338
4.1.2 Synthesis of the azide derivatives from the corresponding chlorides
The azides were prepared following modified procedure based on Weide et al.13
339
340
341
342
343
344
345
N-(4-azidobut-2-ynyl)benzenesulfonamide (4a)
N-(4-chlorobut-2-ynyl)benzenesulfonamide (3a) (100 mg, 0.41 mmol, 1.0 equiv), sodium azide (31.98
mg, 0.492 mmol, 1.2 equiv), and NH4Cl (5.5 mg, 0.1 mmol, 0.25 equiv), in DMF (5 mL) gave compound
1
4a (66.3 mg, 66%) as a colorless solid. Rf = 0.33 (hexane/ethyl acetate, 65:35). H NMR (400 MHz,
CDCl3): δ 7.91 (d, J = 7.0 Hz, 2H), 7.66 – 7.46 (m, 3H), 5.12 (s, 1H), 3.92 (s, 2H), 3.64 (s, 2H). 13C NMR
(101 MHz, CDCl3): δ 139.5, 132.7, 128.9, 127.1, 81.2, 76.9, 39.4, 32.8. HRMS (ESI): Calcd. for
C10H10O2N4NaS [M+Na]+ 273.0419; found 273.0417.
346
347
348
349
350
N-(4-azidobut-2-ynyl)-4-methylbenzenesulfonamide (4b)
N-(4-chlorobut-2-ynyl)-4-methylbenzenesulfonamide (3b) (100 mg, 0.39 mmol, 1.0 equiv), sodium
azide (30.0 mg, 0.47 mmol, 1.2 equiv), and NH4Cl (5.2 mg, 0.09 mmol, 0.25 equiv), in DMF (5 mL) gave
compound 4b (80 mg, 84%) as a colorless solid. Rf = 0.43 (hexane/ethyl acetate, 65:35). 1H NMR (400
MHz, CDCl3): δ 7.78 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 5.03 (t, J = 5.8 Hz, 1H), 3.89 (d, J = 6.1 Hz,
- 19 -

351
352
2H), 3.67 (s, 2H), 2.42 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 144.3, 137.0, 130.1, 127.8, 82.0, 77.4, 40.1,
33.4, 21.9. HRMS (ESI): Calcd. for C11H12O2N4NaS [M+Na]+ 287.0576; found 287.0573.
353
354
355
356
357
358
359
360
N-(4-azidobut-2-ynyl)-2,5-dichlorobenzenesulfonamide (4c)
2,5-Dichloro-N-(4-chlorobut-2-ynyl)benzenesulfonamide 3c (1000 mg, 3.2 mmol, 1.0 equiv), sodium
azide (250.8 mg, 3.85 mmol, 1.2 equiv), and NH4Cl (42.4 mg, 0.8 mmol, 0.25 equiv), in DMF (49 mL)
gave compound 4c (980 mg, 96%) was obtained as a colorless solid. Rf = 0.41 (hexane/ethyl acetate,
65:35). 1H NMR (400 MHz, CDCl3): δ 8.09 (d, J = 2.2 Hz, 1H), 7.48 (s, 2H), 5.43 (t, J = 6.2 Hz, 1H), 3.97
(dd, J = 6.3, 0.7 Hz, 2H), 3.63 (t, J = 2.0 Hz, 2H). 13C NMR (101 MHz, CDCl3): δ 138.9, 133.92, 133.90,
133.6, 132.6, 131.2, 130.1, 80.6, 77.5, 77.2, 39.6, 33.2. HRMS (ESI): Calcd. for C10H7N4O2Cl2S [M-H]-
316.9670; found 316.9661.
361
362
363
364
365
366
367
N-(4-azidobut-2-ynyl)-4-iodobenzenesulfonamide (4d)
N-(4-chlorobut-2-ynyl)-4-iodobenzenesulfonamide 3d (850 mg, 2.3 mmol, 1.0 equiv), sodium azide
(179.7 mg, 2.76 mmol, 1.2 equiv), and NH4Cl (36.6 mg, 0.69 mmol, 0.25 equiv), in DMF (30 mL) gave
compound 4d (780 mg, 90%) was obtained as a colorless solid. Rf = 0.37 (hexane/ethyl acetate, 65:35).
1H NMR (400 MHz, CDCl3): δ 7.88 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H), 5.19 (s, 1H), 3.92 (s, 2H),
3.68 (d, J = 2.0 Hz, 2H). 13C NMR (101 MHz, CDCl3): δ 139.5, 138.46, 138.45, 138.4, 128.9, 128.8, 100.6,
81.3, 77.4, 77.2, 39.8, 33.1. HRMS (ESI): Calcd. for C10H9IN4O2S [M-H]- 374.9412; found 374.9407.
368
369
370
371
372
373
4.1.3 General Procedure for the synthesis of NH-triazole-arylsulfonamides
To azide 4a–d (1 equiv) as solution in CH2Cl2 or neat was added the nucleophile (1-5 equiv.). The
reaction mixture was stirred at 60 oC for 16 h. The reaction was concentrated under reduced pressure
to give the crude product. All triazoles were purified by flash column chromatography on silica gel (50–
100% EtOAc in hexanes to 10% MeOH in EtOAc ). Repeated column chromatography provided most
compounds with purity greater than 90% as determined by HPLC.
374
375
376
377
378
379
N-((5-(aminomethyl)-1H-1,2,3-triazol-4-yl)methyl)benzenesulfonamide (1aa’)
N-(4-azidobut-2-ynyl)benzenesulfonamide 4a (68 mg, 0.27 mmol, 1.0 equiv) and ammonium hydroxide
solution (28% NH3 in H2O, 2 mL) gave 1aa’ (47 mg, 65%) as a pale yellow oil. 1H NMR (400 MHz, DMSO-
d6): δ 7.79 (s, 2H), 7.71 – 7.38 (m, 3H), 4.06 (s, 2H), 3.70 (s, 2H). 13C NMR (101 MHz, DMSO-d6): δ 143.3,
140.6, 139.2, 132.8, 129.5, 129.5, 126.9, 37.7, 35.6. HRMS (ESI): Calcd. for C10H14O2N5S [M+H]+
268.0862; found 268.0863. HPLC purity: 91%
380
381
382
N-((5-(tert-butoxymethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-methylbenzenesulfonamide (1bb’)
N-(4-azidobut-2-ynyl)-4-methylbenzenesulfonamide 4b (66 mg, 0.25 mmol, 1.0 equiv) in CH2Cl2 (2 mL)
and 2-methyl-2-propanol (74 mg, 1.0 mmol, 4.0 equiv) gave 1bb’ (55 mg, 65%) as colorless oil. 1H NMR
- 20 -

383
384
385
386
(400 MHz, CD3OD): δ 7.69 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 4.50 (s, 2H), 4.18 (s, 2H), 2.42 (s,
3H), 1.26 (s, 9H). 13C NMR (101 MHz, CD3OD): δ 144.8, 144.2 (determined from HMBC), 143.8
(determined from HMBC), 138.5, 130.7, 128.2, 75.5, 56.2, 38.6, 27.7, 21.4. HRMS (ESI): Calcd. for
C15H22O3N4NaS [M+Na]+ 361.1300; found 361.1305. HPLC purity: 87%
387
388
389
390
391
392
393
N-((5-((cyclohexylamino)methyl)-1H-1,2,3-triazol-4-yl)methyl)-4-methylbenzenesulfonamide (1bc')
N-(4-azidobut-2-yny)-4-methylbenzenesulfonamide 4b (78 mg, 0.29 mmol, 1.0 equiv) and
1
cyclohexylamine (115 mg, 1.16 mmol, 4.0 equiv) gave 1bc’ (75 mg, 71%) as colorless solid. H NMR
(400 MHz, CD3OD) δ 7.78 – 7.69 (m, 2H), 7.43 – 7.34 (m, 2H), 4.15 (s, 2H), 3.98 (s, 2H), 2.72 – 2.60 (m,
1H), 2.43 (s, 3H), 2.07 – 1.98 (m, 2H), 1.84 – 1.75 (m, 2H), 1.71 – 1.63 (m, 1H), 1.38 – 1.12 (m, 5H). 13C
NMR (101 MHz, CD3OD) δ 144.9, 140.9, 140.1, 138.4, 130.8, 128.2, 57.4, 40.3, 38.7, 32.6, 26.8, 25.9,
21.5. HRMS (ESI): Calcd. for C17H26O2N5S [M+H]+ 364.1798; found 364.1802.
394
395
396
397
398
399
400
N-((5-((adamantylamino)methyl)-1H-1,2,3-triazol-4-yl)methyl)-4-methylbenzenesulfonamide (1bd')
N-(4-azidobut-2-ynyl)-4-methylbenzenesulfonamide 4b (20 mg, 0.07 mmol, 1.0 equiv) in CH2Cl2 (2 mL)
and 1-adamantylamine (34.2 mg, 0.226 mmol, 3.0 equiv) gave 1bd' (24 mg, 83%) as colorless solid. 1H
NMR (400 MHz, CD3OD) δ 7.75 – 7.68 (m, 2H), 7.39 – 7.33 (m, 2H), 4.14 (s, 2H), 4.01 (s, 2H), 2.41 (s,
3H), 2.17 – 2.11 (m, 3H), 1.85 (d, J = 2.9 Hz, 6H), 1.79 – 1.66 (m, 6H). 13C NMR (101 MHz, CD3OD) δ
144.9, 141.0, 139.5, 138.3, 130.8, 128.1, 55.6, 41.1, 38.7, 37.1, 35.2, 30.8, 21.5. HRMS (ESI): Calcd. for
C21H28O2N5S [M-H]- 414.1970; found 414.1965. HPLC purity: 89%
401
402
403
404
405
406
407
408
2,5-Dichloro-N-((5-((cyclohexylamino)methyl)-1H-1,2,3-triazol-4-yl)methyl)benzenesulfonamide (1cc')13
N-(4-azidobut-2-ynyl)-2,5-dichlorobenzenesulfonamide 4c (93 mg, 0.29 mmol, 1.0 equiv) in CH2Cl2 (4
mL) and cyclohexylamine (86.1 mg, 0.87 mmol, 3 equiv) gave 1cc’ (88 mg, 72%) as colorless solid. 1H
NMR (400 MHz, CD3OD): δ 7.96 (d, J = 2.4 Hz, 1H), 7.57 (ddAB, J = 8.5, 2.4 Hz, 1H), 7.53 (dAB, J = 8.5 Hz,
1H), 4.27 (s, 2H), 3.99 (s, 2H), 2.74 – 2.62 (m, 1H), 2.05 – 2.02 (m, 2H), 1.82 – 1.78 (m, 2H), 1.69 – 1.65
(m, J = 12.5 Hz, 1H), 1.37 – 1.16 (m, 5H). 13C NMR (101 MHz, CD3OD): δ 140.8, 140.7, 140.1, 134.6,
134.3, 134.1, 131.7, 131.3, 57.5, 40.2, 38.3, 32.6, 26.8, 25.9. HRMS (ESI): Calcd. for C16H22O2N5Cl2S
[M+H]+ 418.0861; found 418.0866. HPLC purity: 95%
409
410
411
412
413
414
2,5-Dichloro-N-((5-((adamantylamino)methyl)-1H-1,2,3-triazol-4-yl)methyl)benzenesulfonamide
(1cd')13
N-(4-azidobut-2-ynyl)-2,5-dichlorobenzenesulfonamide 4c (102 mg, 0.32 mmol, 1.0 equiv), 1-
adamentylamine (58.2 mg, 0.38 mmol, 1.2 equiv) in CH2Cl2 (4 mL) gave 1cd' (111 mg, 73%) as colorless
solid. 1H NMR (400 MHz, CD3OD) δ 8.00 – 7.95 (m, 1H), 7.62 – 7.51 (m, 2H), 4.28 (s, 2H), 3.97 (s, 2H),
2.16 (s, 3H), 1.86 (d, J = 2.9 Hz, 6H), 1.82 – 1.69 (m, 6H). 13C NMR (101 MHz, CD3OD) δ 140.9, 140.6,
- 21 -

415
416
140.1, 134.6, 134.3, 134.2, 131.7, 131.3, 54.7, 41.6, 38.4, 37.3, 35.2, 30.9. HRMS (ESI): Calcd. for
C20H26O2N5ClS [M+H]+ 470.1182; found 470.1179. HPLC purity: 94%
417
418
419
420
421
422
423
N-((5-((cyclohexylamino)methyl)-1H-1,2,3-triazol-4-yl)methyl)-4-iodobenzenesulfonamide (1dc')
N-(4-azidobut-2-ynyl)-4-iodobenzenesulfonamide 4d (86 mg, 0.228 mmol, 1.0 equiv), cyclohexylamine
(83.9 mg, 0.845 mmol, 4.0 equiv) in CH2Cl2 (2 mL) gave 1dc' (99 mg, 86%) as colorless oil. 1H NMR (400
MHz, CDCl3): δ 7.82 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 4.08 (s, 2H), 3.99 (s, 2H), 2.80 (s, 1H),
2.13 – 2.01 (m, 2H), 1.76 (d, J = 7.1 Hz, 2H), 1.64 (d, J = 12.4 Hz, 1H), 1.34 – 1.11 (m, 6H). 13C NMR (101
MHz, CDCl3): δ 139.9, 139.6, 138.3, 136.5, 128.5, 99.8, 57.5, 39.8, 38.4, 31.5, 25.5, 24.9. HRMS (ESI):
Calcd. for C16H23O2N5SI [M+H]+ 476.0612; found 476.0612. HPLC purity: 99%
424
425
426
427
428
429
430
431
N-((5-((adamantylamino)methyl)-1H-1,2,3-triazol-4-yl)methyl)-4-iodobenzenesulfonamide (1dd')
N-(4-azidobut-2-ynyl)-4-iodobenzenesulfonamide 4d (77 mg, 0.20 mmol, 1.0 equiv), 1-
adamantylamine (37.2 mg, 0.226 mmol, 1.2 equiv) in CH2Cl2 (4 mL) gave 1dd' (102 mg, 96%) as colorless
1
solid. H NMR (400 MHz, DMSO-d6): δ 8.01 – 7.89 (m, 2H), 7.55 – 7.50 (m, 2H), 5.78 – 5.73 (m, 1H),
4.14 – 4.05 (m, 2H), 3.70 – 3.63 (m, 2H), 3.23 – 3.12 (m, 1H), 2.00 (s, 3H), 1.65 – 1.48 (m, 12H). 13C NMR
(101 MHz, DMSO-d6): δ δ 142.2 (determined by HMBC), 139.9, 139.3 (determined by HMBC), 138.0,
128.3, 100.4, 50.4, 41.5, 37.6, 36.2, 34.2, 28.9. HRMS (ESI): Calcd. for C20H27O2N5SI [M+H]+ 528.0925;
found 528.0925. HPLC purity: 97%
432
433
434
435
436
437
438
N-((5-((tritylamino)methyl)-1H-1,2,3-triazol-4-yl)methyl)-4-iodo-benzenesulfonamide (1de')
N-(4-azidobut-2-ynyl)-4-iodobenzenesulfonamide 4d (100 mg, 0.26 mmol, 1.0 equiv) in CH2Cl2 (5 mL)
and triphenylmethanamine (82.2 mg, 0.319 mmol, 1.2 equiv) gave 1de' (63 mg, 93%) as colorless solid.
1H NMR (400 MHz, CDCl3): δ 7.69 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.1 Hz, 5H), 7.37 – 7.20 (m, 12H), 4.25
(s, 2H), 3.42 (s, 2H). 13C NMR (101 MHz, CDCl3): δ 145.0, 139.6, 138.4, 128.7, 128.6, 128.31, 128.27,
128.1, 127.0, 100.1, 71.4, 38.4, 37.8. HRMS (ESI): Calcd. for C29H26O2N5SI [M+Na]+ 658.0748; found
658.0744. HPLC purity: 81%
439
440
441
442
443
444
445
446
N-((5-((4-methoxybenzylthio)methyl)-1H-1,2,3-triazol-4-yl)methyl)-4-iodo-benzenesulfonamide (1df')
N-(4-azidobut-2-ynyl)-4-iodobenzenesulfonamide 4d (80 mg, 0.21 mmol, 1.0 equiv) in CH2Cl2 (5 mL)
and 4-methoxybenzyl mercaptan (161.9 mg, 1.05 mmol, 5 equiv) gave 1df' (70 mg, 63%) as dark brown
oil. 1H NMR (400 MHz, CDCl3): δ 7.77 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H),
6.81 (d, J = 8.5 Hz, 2H), 6.35 (s, 1H), 4.22 (s, 2H), 3.77 (s, 3H), 3.60 (s, 2H), 3.55 (s, 2H). 13C NMR (101
MHz, CD3OD): δ 160.2, 141.7 (determined by HMBC), 141.4, 140.4 (determined by HMBC), 139.5,
131.1, 129.6, 114.9, 100.5, 55.7, 38.3, 36.2, 25.1. HRMS (ESI): Calcd. for C18H18O3N4IS2 [M-H]- 528.9876;
found 528.9835. HPLC purity: 96%
- 22 -

447
448
449
450
451
452
4-Iodo-N-((5-(methoxymethyl)-1H-1,2,3-triazol-4-yl)methyl)benzenesulfonamide (1dg')14
N-(4-azidobut-2-ynyl)-4-iodobenzenesulfonamide 4d (106 mg, 0.28 mmol, 1.0 equiv) in methanol (2
mL) gave 1dg' (101 mg, 88%) as colorless oil. 1H NMR (400 MHz, CD3OD) δ 7.90 (d, J = 8.5 Hz, 2H), 7.54
(d, J = 8.5 Hz, 2H), 4.48 (s, 2H), 4.22 (s, 2H), 3.33 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 143.1 (determined
from HMBC), 142.7 (determined from HMBC), 141.5, 139.5, 129.6, 100.4, 65.5, 58.7, 38.1. HRMS (ESI):
Calcd. for C11H13O3N4IS [M+H]+ 409.9861; found 409.9848. HPLC purity: 98%
453
454
455
456
457
458
459
4-Iodo-N-((5-(isopropoxymethyl)-1H-1,2,3-triazol-4-yl)methyl)benzenesulfonamide (1dh’)13
N-(4-azidobut-2-ynyl)-4-iodobenzenesulfonamide 4d (80 mg, 0.212 mmol, 1.0 equiv), in isopropanol (2
mL) gave 1dh’ (99 mg, 86%) as colorless oil. 1H NMR (400 MHz, CD3OD): δ 7.95 – 7.89 (m, 3H), 7.55 (d,
J = 8.1 Hz, 2H), 4.55 (s, 2H), 4.23 (s, 2H), 3.76 – 3.61 (m, 1H), 1.18 (d, J = 6.1 Hz, 6H). 13C NMR (101 MHz,
CD3OD): δ 141.4, 140.8 (2 × triazole C, determined from HMBC), 139.5, 129.6, 100.5, 73.2, 60.9, 49.0,
38.0, 22.2. HRMS (ESI): Calcd. for C13H17O3N4SINa [M+Na]+ 458.9958; found 458.9958. HPLC purity:
95%
460
461
462
463
464
465
466
N-((5-((benzylamino)methyl)-1H-1,2,3-triazol-4-yl)methyl)-4-iodobenzenesulfonamide (1di')
N-(4-azidobut-2-ynyl)-4-iodobenzenesulfonamide 4d (100 mg, 0.26 mmol, 1.0 equiv),
phenylmethanamine (139.0 mg, 1.3 mmol, 3 equiv) in CH2Cl2 (5 mL) gave 1di' (95 mg, 72%) as colorless
1
oil. H NMR (400 MHz, CDCl3): δ 7.74 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.39 – 7.26 (m, 5H),
4.15 (s, 2H), 3.86 (d, J = 8.2 Hz, 4H). 13C NMR (101 MHz, CDCl3): δ 140.9 (triazole, determined by HMBC),
140.6 (triazole, determined by HMBC), 139.7, 138.4, 137.1, 128.9, 128.6, 128.1, 100.0, 53.1, 42.5, 38.1.
HRMS (ESI): Calcd. for C20H23O2N2IS [M-H]- 482.0146; found 482.0519.
467
468
469
470
471
472
473
474
475
476
477
478
(4-((4-iodophenylsulfonamido)methyl)-1H-1,2,3-triazol-5-yl)methyl acetate (1dj’)
N-(4-azidobut-2-ynyl)-4-iodobenzenesulfonamide 4d (300 mg, 0.80 mmol, 1.0 equiv) and H2O (2 mL)
were heated at 60oC overnight. The reaction mixture was evaporated to yield crude 5-hydroxymethyl
triazole. A small portion of the crude was taken and acylated as follows. 5-Hydroxymethyl triazole (50
mg, 0.13 mmol, 1.0 equiv), Et3N (55 L, 0.39 mmol, 3.0 equiv), DMAP (1.5 mg, 0.013 mmol, 10 mol%)
and acetic anhydride (12 L, 0.13 mmol,1.0 equiv) were stirred in CH2Cl2 (20 mL/mmol of triazole) for
15–30 min (reaction was monitored by TLC). The reaction mixture was evaporated and purified by
column chromatography to yield 1dj’ (31mg, 54%) as colorless solid. 1H NMR (400 MHz, CD3OD): δ 7.98
– 7.84 (m, 2H), 7.62 – 7.49 (m, 2H), 5.12 (s, 2H), 4.24 (s, 2H), 2.04 (s, 3H). 13C NMR (101 MHz, CD3OD):
δ 172.3, 141.5 (2*C; triazole, determined by HMBC), 140.8 (triazole, determined by HMBC), 139.5,
129.5, 100.5, 57.1, 37.9, 20.6. HRMS (ESI): Calcd. for C12H14O4N4SI [M+H]+ 436.9775; found 436.9775.
HPLC purity: 89%
- 23 -

479
4.2 Biological activity
480
481
482
483
484
485
486
487
488
4.2.1 Gene Constructs of VIM-2, NDM-1 and GIM-1
In this study two types of gene constructs were used. The first included the native leader sequence to
allow the proteins to be transported to the periplasm, for the three enzymes VIM-2 from Pseudomonas
aeruginosa strain 301-5473 (GenBank no Q9K2N0), GIM-1 from P. aeruginosa (GenBank no Q704V1;
19, 34) and NDM-1 (GenBank no. E9NWK5, e.g 35, 36), where the latter blaNDM-1 gene is reported from
several organisms. Cloning of blaNDM-1 or blaGIM-1 genes into the Escherichia coli pET26b(+) vector
(Novagen) was performed using the Primers listed in Table S3, and by restriction cutting as described
for VIM-26.37 Cloning of blaVIM-2 into pET26b(+) is described previously.38 The obtained E. coli pET-
26b(+) MBL constructs were further used in the in the whole cell-based inhibitor assays.
489
490
491
492
493
494
495
In the second set of gene constructs used for the recombinant gene expression, the native leader
sequence was removed and replaced with a hexa-His tag and a TEV cleavage site as reported earlier
for VIM-2 (residues V27-E268;17) and GIM-1 (residues Q19-D250;19) both in pDEST14. NDM-1 used a
codon optimized synthetic gene (Life Technologies, Thermo Fisher Scientific), with a TEV cleavage site
with sequence ENLYFQG and residues G36-R280 in NDM-1 transformed in pDONR™221, and further
sub cloned into pDEST17 with carries an N-terminal hexa His-tag, yielding pDest17-NDM-1 construct.
Herein the residue numbering is the class B β-lactamase numbering scheme will be applied.39
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
4.2.2 Recombinant protein expression and purification of VIM-2, GIM-1 and NDM-1
The proteins were expressed and purified following this protocol. pDest17-NDM-1 was transformed
into E. coli BL21 Star (DE3) pLysS (Invitrogen), and pDEST14 plasmids with VIM-2 or GIM-1 were
transformed into in-house modified E. coli BL21 Star (DE3) pLysS (Invitrogen) cells with the pRARE
plasmid (Novagen) in order to allow expression of genes encoding tRNAs for rare codons.40 Precultures
grown in Terrific Broth (TB) media with 100 µg/ml ampicillin (Sigma-Aldrich) and 34 µg/ml
chloramphenicol (Sigma-Aldrich). The precultures were inoculated to 2 L Luria-Bertani (LB) media
containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol and grown at 37 °C to reach an optical
density (OD600 nm) of 0.5-1.0 before induced expression with 0.4 mM isopropyl β-D-1-
thigalactopyranoside (IPTG; Sigma-Aldrich). The induced cultures were grown overnight at 20°C before
collecting the cells by centrifugation (8,900 X g, 30 min, 4 °C). Buffer A containing 50 mM HEPES pH
7.2, 100 µM ZnCl2 and 150 mM NaCl was used to resuspend the cell pellets, following sonication and
collecting the supernatants by centrifugation (3000 g, 40 min, 4°C). The recombinant proteins were
affinity purified using a 1 ml or 5 ml His-Trap HP column (GE Healthcare) in buffer A washed with 5%
buffer B (50 mM HEPES pH 7.2, 100 µM ZnCl2, 150 mM NaCl and 1 M imidazole), before eluted in a
gradient of 5 to 100 % buffer B. Peak fractions were investigated using 4-20% sodium dodecyl sulfate
- 24 -

512
513
514
515
516
517
polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad) 41. The fractions containing MBL protein was
added in-house-made His-tagged TEV protease in a 1:100 mg ratio of TEV:protein and dialyzed at 4 °C
overnight using 10-kDa cutoff Snakeskin (Pierce) in buffer C (50 mM HEPES pH 7.2, 150 mM NaCl, 1
mM EDTA and 1 mM β-mercaptoethanol). To remove uncleaved protein and TEV protease a second
His-Trap purification was performed. SDS-PAGE analysis was used to estimate a purity of ~95% of the
fractions containing protein, which then were pooled and dialyzed in buffer A overnight.
518
519
520
521
522
523
524
525
526
527
528
529
4.2.3 Dose Rate Inhibition Studies for IC50 Determination
The half-maximal inhibitory concentration (IC50) against the VIM-2, NDM-1 and GIM-1 enzymes were
determined by using sixteen different concentration of inhibitor compounds ranging from 0 µM to 250
µM. A 100 µl solution with 50 mM HEPES buffer (pH 7.2), 100 µM ZnCl2), purified enzyme (1 nM VIM-
2, NDM-1 or GIM-1) and 2.5-0 mM inhibitor was incubated in a 96 well plate at 25 °C for 5 min. In
addition, the enzyme buffer contained 400 µg/ml Bovine Serum Albumin (BSA) to prevent protein
unfolding and loss of activity due to low concentrations 42, 43. 100 µM of the reporter substrate
nitrocefin (VIM-2, GIM-1) or imipenem (NDM-1) was added to the enzyme-inhibitor solution and the
increase in absorbance at 482 nm (nitrocefin) or 300 nm (imipenem) was recorded on a Spectramax
M2e spectrophotometer (Molecular Devices). Each data point was performed in triplicates and the
initial velocity for each inhibitor concentration was analysed by a log [inhibitor] vs. response curve
fitting to calculate IC50 in GraphPad Prism 5.0 software.
530
531
532
533
534
535
536
537
538
539
540
541
542
4.2.4 Cell-based screening assay of the inhibition potential
The inhibitory activity of the inhibitors was investigated in a cell-based assay using a β-lactamase-
negative E. coli SNO3 (ampA1 ampC8 pyrB recA rpsL)44 transformed with pET26b(+) containing blaVIM-
2, blaGIM-1 or blaNDM-1. The screen was conducted in 96-well plates (Corning) in duplicates. 50 µl
overnight culture (adjusted to an OD600 of 1 in LB broth) of E. coli SNO3 containing one of the MBLs,
inhibitor (with a final concentration of 250 µM), 0.8 mM (final concentration) Isopropyl ß-D-1-
thiogalactopyranoside (Sigma, IPTG) and 50 µl LB media, were added to each well. The plate was
incubated at 37 °C for 20 min with shaking to induce the expression of the MBL. Subsequently, 50 µl
nitrocefin (diluted in 50 mM HEPES pH 7.2 and 100 µM ZnCl2 to give a final concentration of 1.6 mM in
the assay) was added. Nitrocefin hydrolysis was measured at OD482 every minute for 3 hours with
shaking (47 seconds) in between reads using a Spectramax M2e spectrophotometer (Molecular
Devices). EDTA (250 µM concentration) was used as positive control and wells containing no inhibitor
as negative controls. The percent inhibition was calculated according to equation 1.
543
- 25 -

% inhibition = Slope (No inhibitor)– Slope (Inhibitor)
544
545
X 100%
equation (1)
Slope (No inhibitor)
546
547
548
549
550
551
552
553
554
555
556
557
The synergistic effect of the inhibitors with meropenem was tested against selected clinical bacterial
strains containing MBLs. The bacterial strains were plated on lactose agar plates with 100 mg/L
ampicillin and lactose agar and incubated overnight at 37°C. The inhibitors were diluted to a final
concentration of 50 µM (1cc', 1dj') or 125 µM (1dc') in Mueller Hinton (MH) broth. In order to monitor
the effect of the DMSO in the assay, a DMSO control was included with a concentration of 5%.
Meropenem was diluted in MH broth in a 2-fold dilution series with final concentrations of 256 µg/mL
- 0.0625 µg/mL. The microtiter plates were inoculated with a 0.5 McFarland suspension of the bacterial
strain in 0.85% NaCl, which were diluted in MH broth. A quality check of bacterial suspension in 0.85%
NaCl in a 1:100 ratio was incubated on MH agar plates overnight at 37°C. The final CFU/mL inoculum
were calculated and compared to a standard. The microtiter plates were incubated for 20 hours at
37°C. The minimum inhibitory concentrations (MIC) were detected by visual inspection of the plates
the next day.
558
559
560
561
562
563
564
565
4.3 Crystallization, X-ray data collection and data analysis
The DMSO-free co-crystallization method45 was used to crystallize VIM-2 in complex with the inhibitor
1cc´, 1dh´ and 1di´. In brief, the inhibitors were dissolved in DMSO and used to pre-coat the reservoir
wells of an MRC-96-well crystallization plate (Molecular Dimensions) by DMSO evaporating. Reservoir
solution consisting of 22-27% polyethylene glycol (PEG) 3350 and 0.2 M magnesium formate was
added to every well and incubated for 24 h. The reservoir solution was mixed with the protein solution
(9.4 mg/ml) in a 1:1 ratio and used for sitting-drop experiments. Protein crystals were harvested after
1-2 weeks.
566
567
568
569
570
Soaking of native VIM-2 crystals was used for the crystallization of VIM-2 in complex with the inhibitor
1dj´ 17.The native VIM-2 crystals were grown using the hanging drop-method and reservoir solution
consisting of 22-27% PEG 3350 and 0.2 M magnesium formate. 1dj´ was dissolved in DMSO at a
concentration of 100 mM. The native VIM-2 crystals were transferred to reservoir solution containing
2.5 mM 1dj´and harvested after 12 h.
571
572
573
574
575
After harvesting, all crystals were transferred to 25% PEG 3350, 0.2 M MgCl2, 15% ethylene glycol, 50
mM HEPES pH 7.2 and flash cooled in liquid nitrogen. The collection of the X-ray data was carried out
at the ID23-1 or ID-29 at the European Synchrotron Radioation Facility (ESRF) in Grenoble, France. The
data sets were integrated, scaled and truncated using XDS 46, POINTLESS and AIMLESS 47, 48. Molecular
49
replacement was carried out using PHASER with a previously published VIM-2 structure (PDB:
- 26 -

576
577
578
579
580
581
582
583
1KO3)28. Several refinement cycles in PHENIX 50 and molecular modeling in WinCoot 51 according to the
2Fo-Fc and Fo-Fc map were used to obtain the final structure. For the complex structures with the
inhibitors 1dh´ and 1cc´, all atoms except water were refined with anisotropic B-factors. For the
complex structures with 1di´ and 1dj´, TLS parameters and anisotropic B-factors refinement for Zn2+
and Cl- were applied. R-free cross validation was done with 5% of the data. Conditions for the data
collection and refinement statistics are shown in Table 1. The PyMOL Molecular Graphic System,
33
version 1.4.1. (Schrödinger), and LIGPLOT
interactions.
were used to generate illustrations and visualize
584
585
4.3.1 PDB accession codes.
Coordinates and structure factors of have all been deposited in the Protein Data Bank with accession
586
587
numbers 6TMC (VIM-2_1dh´), 6TM9 (VIM-2_1cc´), 6TMB (VIM-2_1di´) and 6TMA (VIM-2_1dj´).
Acknowledgements
588
589
590
591
592
This work was supported by the Tromsø Research Foundation and the Research Council of Norway
(project numbers 218539, SYNKNOYT 2011 and 213808, FRIMEDBIO 2011), and this is highly
acknowledged. The provision of beam time at ID23-1 and ID29, ESRF, Grenoble are highly valued. We
acknowledge Trine Josefine O. Carlsen for purification and help with crystallization of proteins. Silje
Lauksund and Ørjan Samuelsen are both acknowledged for synergy testing.
593
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