Inhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries

Article abstract of DOI:10.1016/j.bmc.2009.05.070

VIM-2 is an Ambler class B metallo-β-lactamase (MBL) capable of hydrolyzing a broad-spectrum of β-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule in

Full text of DOI:10.1016/j.bmc.2009.05.070

Bioorganic & Medicinal Chemistry 17 (2009) 5027–5037
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitors of VIM-2 by screening pharmacologically active and
click-chemistry compound libraries
Dmitriy Minond a, S. Adrian Saldanha a, Prem Subramaniam a, Michael Spaargaren a, Timothy Spicer a,
Joseph R. Fotsing b, , Timo Weide b, Valery V. Fokin b, K. Barry Sharpless e, Moreno Galleni c,
Carine Bebrone c, Patricia Lassaux c, Peter Hodder a,d,
*
a Lead Identification, Translational Research Institute, The Scripps Research Institute, Scripps Florida, 130 Scripps Way #1A1, Jupiter, 33458 FL, USA
b Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037 CA, USA
c Laboratoire d’Enzymologie & Centre d’Ingénierie des Protéines, Institut de Chimie, B6 Sart Tilman, Université de Liège, B-4000 Liège, Belgium
d Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, 130 Scripps Way #1A1, Jupiter, 33458 FL, USA
e Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037 CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
VIM-2 is an Ambler class B metallo-b-lactamase (MBL) capable of hydrolyzing a broad-spectrum of b-lactam
antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active
research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. In the pre-
sented research, a compound library screening approach was used to identify and characterize VIM-2 inhib-
itors from a library of pharmacologically active compounds as well as a focused ‘click’ chemistry library. The
four most potent VIM-2 inhibitors resulting from a VIM-2 screen were characterized by kinetic studies in
order to determine Ki and mechanism of enzyme inhibition. As a result, two previously described pharma
cologic agents, mitoxantrone(1,4-dihydroxy-5,8-bis([2-([2-hydroxyethyl]amino)ethyl]amino)-9,10-anthra-
cenedione)and4-chloromercuribenzoicacid(pCMB)werefoundtobeactive,theformerasanon-competitive
Received 13 April 2009
Revised 22 May 2009
Accepted 27 May 2009
Available online 22 June 2009
Keywords:
VIM-2
Inhibitors
b-Lactamase
inhibitor (Ki = K0 = 1.5 0.2
lM) and the latter as a slowly reversible or irreversible inhibitor. Additionally,
i
two novel sulfonyl-triazole analogs from the click library were identified as potent, competitive VIM-2
inhibitors: N-((4-((but-3-ynyloxy)methyl)-1H-1,2,3-triazol-5-yl)methyl)-4-iodobenzenesulfonamide (1,
Ki = 0.41 0.03
lM) and 4-iodo-N-((4-(methoxymethyl)-1H-1,2,3-triazol-5-yl)methyl)benzenesulfonamide
(2, Ki = 1.4 0.10
lM). Mitoxantrone and pCMB were also found to potentiate imipenem efficacy in MIC and
synergy assays employing Escherichia coli. Taken together, all four compounds represent useful chemical
probes to further investigate mechanisms of VIM-2 inhibition in biochemical and microbiology-based assays.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
more clinically relevant MBLs.8–12 Pathogenic clinical isolates
containing VIM-2 have been found in Europe, Asia, and the
Bacterial infections that demonstrate antibiotic resistance are
a major public health concern.1–3 One well-known mechanism of
resistance, particularly prevalent in Gram-negative bacteria, in-
volves endogenous b-lactamase enzymes. These enzymes cata-
lyze hydrolysis of b-lactam antibiotics, rendering them
inactive.4,5 Metallo-b-lactamases (MBLs) are Ambler class ‘B’
zinc-dependent enzymes capable of hydrolyzing a broad-spec-
trum of clinically relevant b-lactam classes, such as penicillins,
cephalosporins, and carbapenems.4 Due to an increase in the rate
of infections attributed to bacteria harboring MBLs, there is con-
cern of their growing clinical threat.6,7 Along with IMP-1, an-
other subclass B1 MBL, VIM-2 is considered to be one of the
Americas.8
High-throughput screening (HTS) is an effective tool for rap-
idly identifying novel scaffolds for drug discovery, and more re-
cently aiding chemical probe development.13 Presented here is
the execution of a screening-based effort to discover selective
VIM-2 inhibitors. This effort entailed the development and exe-
cution of an HTS-compatible VIM-2 nitrocefin assay. To further
confirm VIM-2 activity and also identify non-selective com-
pounds, a VIM-2 fluorescence-resonance energy transfer (FRET)
inhibition assay and an IMP-1 inhibition assay were employed.
Additionally, antibiotic potentiation studies were conducted in
Escherichia coli to determine efficacy of four compounds demon-
strating biochemical potency. The success of this effort at identi-
fying and characterizing these inhibitors, including the molecular
modeling of the competitive inhibitors in the VIM-2 active site,
is presented.
* Corresponding author. Tel.: +1 561 228 2100; fax: +1 561 228 3054.
E-mail address: hodderp@scripps.edu (P. Hodder).
Present address: Senomyx, Inc., 4767 Nexus Center Dr., San Diego, 92121 CA, USA.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.070

5028
D. Minond et al. / Bioorg. Med. Chem. 17 (2009) 5027–5037
(i.e., 10
l
M). All enzymatic assays were configured to run as end-
M)
2. Results
point assays in the presence of high concentrations of zinc (50
l
and stopped by the addition of EDTA at <50% substrate turnover. In
these assay conditions, the well-characterized metallo-b-lactamase
inhibitor 2-(2-chlorobenzyl) succinic acid (NSC 20707) inhibited
2.1. VIM-2 and IMP-1 enzyme inhibition assays
Three enzymatic assays were executed to identify and charac-
terize VIM-2 inhibitors. The VIM-2 nitrocefin assay (Fig. 1a)14
was screened against a library of pharmacologically active com-
pounds (LOPAC, n = 1280 compounds) as well as a novel click-
chemistry library enriched in metalloenzyme inhibitors (n = 267
compounds). To confirm the activity of VIM-2 inhibitors found
via the nitrocefin assay, a FRET-based CCF2 substrate assay was
employed15 (Fig. 1b). Finally, an IMP-1 nitrocefin assay was used
to determine the selectivity of potent compounds identified from
the screening effort.
The enzymology for each assay was optimized to enable its com-
patibility with high-throughput screening (HTS) techniques;16 final
conditions of all assays are presented in Table 1. In summary, it was
found that an enzyme concentration of ꢀ0.1 nM yielded an optimal
assay signal in 25 min. At this enzyme concentration, the nitrocefin
assay window was acceptable when assayed at substrate concentra-
IMP-1 with a Ki value (1.6 0.3
the literature (3.3 1.7
M17), and also exhibited modest potency
against VIM-2 (IC50 = 33 M) Table 1.
lM) comparable to that found in
l
9
l
2.2. LOPAC and click-chemistry library screening assays
The LOPAC was tested at a single concentration (14 M) in trip-
l
licate, while click-chemistry test compounds were screened as 10-
point dose–response curves in triplicate. Two different criteria
were used to determine activity: in the case of the LOPAC library,
a standard inhibition cutoff parameter was calculated (see Section
5); any compound that yielded an average percent inhibition great-
er than this cutoff parameter, that is, P8.82% inhibition, was de-
clared active and advanced to dose–response experiments. For
the click-chemistry library screen a compound that yielded an
IC50 value less than 10
two separate artifact assays proved that the inhibitors declared ac-
lM was declared active. The results of
tions of ꢀ2KM (i.e., 60
lM). The CCF2 assay was able to achieve a
comparable assay window at a substrate concentration of ꢀ1/2KM
Figure 1. (a) Nitrocefin assay principle. A change in absorbance at k = 495 nm is measured after the cephalosporin core of nitrocefin is hydrolyzed by b-lactamase, (b) CCF2
(FRET) assay principle. In the intact CCF2 substrate, coumarin moiety’s donor FRET resulting from k = 409 nm excitation is efficiently quenched by the acceptor fluorescein
moiety; hydrolysis of the b-lactam leads to the increase of a donor fluorescence measured at 447 nm and simultaneous decrease of an acceptor fluorescence at measured at
520 nm.
Table 1
Summary of nitrocefin and CCF2 assay parameters, including IC50 values for the positive control inhibitor 2-(2-chlorobenzyl) succinic acid (NSC-20707)
a
b
d
Enzyme
Format
KM
(
l
M)
[Enzyme] (nM)
[Substrate] (
lM)
NSC 20707 IC50
33
1.6 0.3
50 18
(lM)
S/Bc
Z0
VIM-2
IMP-1
VIM-2
Nitrocefin
Nitrocefin
CCF2
30
29
21
9
4
7
0.13
0.1
0.1
60
60
10
9
3.2 0.1
3.5 0.1
4.0 0.1
0.75 0.1
0.81 0.03
0.79 0.09
a
KM values calculated for IMP-1 and VIM-2 assays error reported as the standard deviation of 3 replicates. Reported value for VIM-2 with Nitrocefin is 18
M.55
IC50 error reported as the standard deviation of 16 replicates.
S/B error reported as the standard deviation of 24 replicates. The signal to basal (S/B) is defined as the ratio of raw signals from wells with positive and negative controls.
Z0 error reported as the standard deviation of 24 replicates. Z0-factor is a measure of assay quality and was calculated according to Ref. 32.
l
M11 and that
for IMP-1 with Nitrocefin is 27
l
b
c
d

D. Minond et al. / Bioorg. Med. Chem. 17 (2009) 5027–5037
5029
tive in the screening assays could not be attributed to intrinsic
compound absorbance (see Section 5.14).
ference with the fluorescence emission of CCF2 at 535 nm (see Sec-
tion 5.15). The potency of both compounds was not significantly
affected by the absence of zinc in the assay buffer (data not
shown).
2.3. Determination of VIM-2 inhibitor potency, Ki and
mechanism
Two click-chemistry library compounds were identified from the
VIM-2 nitrocefin screen. These sulfonamide-1,2,3-triazole analogs
(sulfonyl-triazoles), N-((4-((but-3-ynyloxy)methyl)-1H-1,2,3-tria-
zol-5-yl)methyl)-4-iodobenzenesulfonamide (compound 1) and 4-
iodo-N-((4-(methoxymethyl)-1H-1,2,3-triazol-5-yl)methyl)benzen
esulfonamide (compound2) werefound to be competitiveinhibitors
of VIM-2 (Table 2). They also demonstrated a comparable potency in
the VIM-2 nitrocefin and CCF2 assays and their potency was not sig-
nificantly affected by the absence of zinc in the assay buffer. Both
compounds were found to be inactive in the IMP-1 inhibition assay.
All four metallo-b-lactamase inhibitors were tested for potency
against serine b-lactamases from Ambler class A (TEM-1) and C
(AmpC). None of the tested compounds exhibited inhibitory activ-
ity against either TEM-1 or AmpC at the highest concentration
The potency of any compound found active in the LOPAC and
click-chemistry library screens was determined in the VIM-2 nitr-
ocefin and CCF2 assay formats. The Ki values and mechanism of ac-
tion for the four most potent compounds (two from LOPAC and two
from the click library) were also measured (Fig. 2). From the LOPAC
screen, mitoxantrone, an anthracenedione antibiotic and antineo-
plastic, was found to be a pure non-competitive inhibitor of VIM-
2 with Ki = K0 = 1.5 0.2
lM. The sulfhydryl reagent 4-chloromer-
i
curibenzoic acid (pCMB)18,19 was the only other inhibitor of VIM-
2 from the LOPAC screen. Rapid dilution experiments with VIM-2
were performed as a first step to characterize its mode of action.20
The recovery of VIM-2 activity after dilution of pCMB was gradual
and only 16.5% of activity was recovered after 15 min (data not
shown). The near linearity of enzyme recovery over this time per-
iod would suggest that this compound acts as a slowly reversible
or irreversible inhibitor. Also pCMB was found to have modest po-
tency in the IMP-1 nitrocefin assay (82% inhibition was achieved
tested 25 lM (Table 2).
2.4. MIC assays
Mitoxantrone, pCMB, as well as compounds 1 and 2 were fur-
ther tested for their inherent antimicrobial activity. Minimal inhib-
itory concentration (MIC) values were measured in both resistant
(BL21/VIM-2) and non-resistant (BL21) E. coli, with imipenem as
a positive control. The sulfonyl-triazoles, 1 and 2, exhibited no effi-
after 15 min of preincubation with IMP-1 and 140 lM pCMB)
whereas mitoxantrone was inactive at the range of concentrations
tested (Table 2). In contrast to pCMB, mitoxantrone could not be
established as an inhibitor in the VIM-2 CCF2 assay due to its inter-
cacy at the highest concentrations tested (60
lg/mL). In contrast,
mitoxantrone exhibited antibacterial activity (MIC = 8.4
lg/mL)
against both resistant (BL21/VIM-2) and non-resistant (BL21)
E. coli (Table 3). Similarly, pCMB demonstrated antibacterial activ-
ity (MIC = 17.9 lg/mL) against resistant (BL21/VIM-2) and non-
resistant (BL21) E. coli. For comparison, the MIC of imipenem
against resistant (BL21/VIM-2) and non-resistant (BL21) E. coli
was 1.9 lg/mL and 0.2 lg/mL, respectively.
H
N
OH
OH
O
O
HN
HN
OH
OH
O
OH
Cl Hg
pCMB
N
H
Mitoxantrone
2.5. Inhibitor/antibiotic synergy assays
O
S
O
S
O
O
I
CH
I
The four compounds were also tested for their ability to reverse
the resistance of VIM-2 expressing E. coli to imipenem challenge. In
N
H
O
N
O
H
CH3
HN
N
HN
N
non-resistant E. coli (BL21) the MIC for imipenem was 0.2
in resistant E. coli (BL21/VIM-2) the MIC was ꢀ9-fold less potent
(i.e., 1.9 g/mL). To assess potentiation of imipenem efficacy by a
single dose of inhibitor, imipenem MIC values were calculated in
the presence of 50 M of each of the four inhibitors. Mitoxantrone
lg/mL;
N
N
1
2
l
Figure 2. Structures of the four VIM-2 inhibitors found from HTS efforts. Mitoxan-
trone and pCMB were found active in LOPAC screening. Compounds 1 and 2 are
sulfonyl-triazoles found from click-chemistry library screening.
l
and pCMB were able to completely reverse the effects of VIM-2 on
Table 2
Results of potency and kinetic assays for different VIM-2 inhibitors
b
Compound
name
VIM-2 Kia
inhibition)
(
lM) (mechanism of
IC50 or max inhibition @ tested concentrationc
VIM-2 (Nitrocefin)
VIM-2 (CCF2)
IMP-1 Nitrocefin
TEM-1
AmpC
Nitrocefin
Nitrocefin
Mitoxantrone
1.5 + 0.2 (noncompetitive)
(Slowly reversible or irreversible)
0.41 0.03 (competitive)
0.63 0.04
(67% @ 44
NAe
l
M
NDd
NA
(83% @ 36
1.3 0.1
(76% @ 44
4.1 0.3
(90% @ 44
>56
(0% @ 56
NA
(82% @ 140
>56
(0% @ 56
>56
>25
(0% @ 25
>25
(0% @ 25
>25
(0% @ 25
>25
>25
(0% @ 25
>25
(0% @ 25
>25
(0% @ 25
>25
l
M)
M)
M)
M)
l
M)
M)
l
M)
M)
M)
M)
l
M)
M)
M)
M)
pCMB
(80% @ 15
l
l
M
l
M)
M)
M)
l
l
l
l
l
l
l
1
2
3.3 0.4
(79% @ 56
7.3 1.9
(74% @ 56
l
M
l
l
l
M)
M)
1.41 0.12 (competitive)
l
M
lM
l
l
(0% @ 56
l
(0% @ 25
(0% @ 25
a
Ki error reported as the standard deviation of 4 replicates.
IC50 error reported as the standard deviation of 3 replicates.
In parentheses maximum inhibition achieved at the indicated concentration.
ND—Not determined due to assay artifact.
NA—Not applicable due to the nature of inhibition.
b
c
d
e

5030
D. Minond et al. / Bioorg. Med. Chem. 17 (2009) 5027–5037
Table 3
Results of VIM-2 inhibitor MIC and inhibitor plus imipenem MIC potentiation assays
Compound name
Compound MIC
g/mL)
Imipenem MIC @ 50 lM
(
l
of compound (lg/mL)
BL21
BL21+VIM-2
BL21+VIM-2
Mitoxantrone
pCMB
1
2
8.4
8.4
0.2
0.2
1.9
1.9
—
17.9
>60
>60
0.2
17.9
>60
>60
1.9
Imipenem
All experiments were repeated at least on three different days and MIC values were
determined as per CLSI guidelines51. The E. coli strain ATCC 25922 was used as a
quality control reference.
imipenem potency, while the click-chemistry sulfonyl-triazoles
did not demonstrate potentiation of imipenem (Table 3).
Mitoxantrone and pCMB were further tested against resistant
E. coli (BL21/VIM-2) using a checkerboard microdilution meth-
od.21–23 Ranges of pCMB and mitoxantrone concentrations that
encompassed their respective MICs were tested for the potentia-
tion of imipenem efficacy. As determined by the methods de-
scribed previously23 both pCMB and mitoxantrone exhibited
synergy with imipenem when present at concentrations as low
Figure 3. (a) Proposed mode of binding of sulfonyl-triazole compound 1 to VIM-2.
VIM-2 is depicted in ribbon form and the two loops that make key interactions with
active site binders are indicated. Compound 1 is positioned as a binder of the
binuclear zinc cluster through the sulfonyl group and shown as a stick model, with
colors as follows: Carbon is yellow, oxygen is red, nitrogen is blue, iodine is purple,
and sulfur is yellow. The sulfonamide group is surface exposed, whilst the propargyl
group (R) buries into a hydrophobic pocket. Highlighted residues are implicated in
key binding interactions with compound 1. (b) Click sulfonyl-triazole chemotype
identified as selective VIM-2 inhibitors. The chemotype is discussed in text.
as 2.2 lg/mL and 2.1 lg/mL, respectively (Table 4).
2.6. Determination of bacteriostatic/bactericidal properties of
pCMB and mitoxantrone
conformation for the active site. The ligand induced structure of
VIM-2 with bound inhibitor phenylC3SH (PDB 2YZ3) was used
for modeling comparison. To interrogate inhibitor binding require-
ments in the VIM-2 active site, attempts were made to dock all four
compounds into the VIM-2 active site from the native (PDB 1KO3)
and inhibitor bound (PDB 2YZ3) structures. The inhibitors from the
LOPAC screen failed to dock rationally into either structure. How-
ever, docking studies with the two inhibitors from the click-chem-
istry library proved more fruitful: both compounds were predicted
to bind to the VIM-2 active site through the sulfonyl group as a zinc
binding group (Fig. 3).
‘Time kill’ experiments were performed to assess bacteriostatic/
bactericidal properties of pCMB and mitoxantrone.23 Both pCMB
and mitoxantrone were tested against resistant (BL21/VIM-2)
and non-resistant (BL21) E. coli over a period of 24 h at concentra-
tions of 2.2 and 2.1 lg/mL, respectively. The effect of 2.1 lg/mL
mitoxantrone was virtually indistinguishable from that of the
uninhibited growth control in both imipenem-resistant and non-
resistant E. coli (data not shown). Although 2.2 lg/mL of pCMB
exhibited slight growth inhibition for the first 6 h of the experi-
ment, this was followed by growth to the level of the uninhibited
control.
3. Discussion
2.7. Modeling and docking with VIM-2
3.1. Screening for VIM-2 inhibitors
Docking studies were performed in an attempt to bring insight
into the mechanism of VIM-2 inhibition (Fig. 3). The PDB structure
1KO3 for VIM-2 was used for the modeling studies, since this struc-
ture was of high resolution (1.9 Å) and provided the most open
Several structural and phylogeny studies show that the MBLs
are members of an ancient metalloenzyme superfamily.12 The
active site for this enzyme class is formed by a shallow cleft con-
taining one or two Zn2+ cofactor ions and two flexible loops.24–26
The plasticity of these loops is thought to enable this enzyme
class to hydrolyze a broad-spectrum of b-lactam antibiotic sub-
strates. Recently, the importance of these loops for inhibitor rec-
ognition in VIM-2 was demonstrated by solving the structure of
Table 4
Activity of imipenem in combination with pCMB and mitoxantrone versus BL21/VIM2
E. coli
this enzyme with
a
potent mercaptocarboxylate inhibitor26
(PDB 2YZ3). The first loop (Loop1: Phe61 to Ala64) makes key
aromatic and hydrophobic interactions with the bound inhibitor
through residues Phe61 and Tyr67, while the other loop (Loop2:
Ile223-Trp242) undergoes a ligand-induced side chain reorienta-
tion of Asn233 to form a salt bridge with the carboxylate of the
inhibitor.
The potency of the well-characterized and potent IMP-1 inhib-
itor NSC20707 in the VIM-2 assay serves as an example of how
compounds bearing zinc-binding moieties can be useful MBL
inhibitors. Although the VIM-2 mercaptocarboxylate inhibitor de-
scribed above was not available for this study, it was anticipated
that NSC20707 (which has a zinc-chelating succinate moiety)
would inhibit VIM-2 activity. Since its succinate group binds to
Results obtained from checkerboard microdilution experiment. Shaded values are
combination of imipenem and inhibitor that were found to be synergistic.

D. Minond et al. / Bioorg. Med. Chem. 17 (2009) 5027–5037
5031
the binuclear zinc cluster in IMP-1,27 and IMP-1 and VIM-2 share
this zinc cluster, it was not surprising that NSC20707 demon-
Unfortunately, previously described VIM-2 inhibitors were not
available for this study;31 however, the IMP-1 inhibitor
NSC20707 served as a useful positive control for both the VIM-2
and IMP-1 assays, allowing the evaluation of assay performance
(i.e., Z-factor32).
strated modest potency in both the nitrocefin (IC50 = 33
9 lM)
and CCF2 (IC50 = 50 18 M) VIM-2 enzymatic assays (Table 1).
l
3.2. Click-chemistry library composition
3.4. Choice of CCF2 VIM-2 assay as a counterscreen
The principles and benefits of click chemistry are described in
detail elsewhere.28 The goal of click chemistry is to use robust, high
yielding, and clean reactions to access diverse compound libraries.
This rapid and reliable approach, in turn, reduces the amount of
time taken to produce biologically active molecules. The click
chemistry library employed here was specifically designed to
probe the active site of VIM-2 via compounds containing moieties
that have demonstrated activity against a variety of metalloen-
zymes (Fig. 4). These include known and putative zinc-binding
fragments, such as hydroxamates, amides, ureas, thiadiazoles, car-
boxylates, and the sulfonyl-triazoles 1 and 2. The sulfonamide moi-
ety of inhibitors 1 and 2 is known to be a zinc binding group29 and
has been used in the design of inhibitors of the metalloenzyme,
carbonic anhydrase.30 However, in order to avoid the discovery
of non-specific zinc chelators, the VIM-2 nitrocefin screens were
Although more commonly used for mammalian reporter gene
assays,15 the use of the CCF2 substrate in microbiology applications
has been previously described.33,34 In the present study, the KM va-
lue of CCF2 for VIM-2 was determined to be 21
is comparable to that previously measured for the enzyme in the
nitrocefin format (18
M).11 Additionally the VIM-2 CCF2 assay
7 lM. This result
l
yielded reproducible, high quality data that were comparable to
those obtained from the nitrocefin assay (Table 1). However, the
CCF2 assay is prone to FRET artifact, as evidenced by its inability
to detect inhibition of VIM-2 by the colored compound mitoxan-
trone. Taken together, these results suggest that the CCF2 and nitr-
ocefin substrates may complement each other for inhibition
studies.
conducted in the presence of high concentrations (50
l
M) of zinc.
3.5. The LOPAC library gauges assay compatibility for HTS
Consequently, this high concentration of zinc served to abrogate
inhibition derived solely from zinc-binding. Despite the presence
of zinc binding groups, the activity of 1 and 2 appears independent
of the concentration of zinc in the assay buffer; this pharmacology
will be further characterized in future studies.
The LOPAC library was screened primarily to validate the com-
patibility of the VIM-2 nitrocefin assay to a larger HTS effort. In this
regard the VIM-2 nitrocefin assay appears to be HTS-compatible as
demonstrated by high assay Z-factor (i.e., >0.5) and a low percent-
age of potent compounds (ꢀ0.1%, i.e., <1% overall) resulting from
the LOPAC screening effort.32 Also important to note is that none
of the compounds that appeared active in LOPAC screening could
be attributed to their interference with the measured absorbance;
this result signifies that this type of absorbance artifact may be
negligible in a larger-scale HTS campaign. Although the CCF2
VIM-2 and nitrocefin IMP-1 assays were not subjected to LOPAC
screening for this study, their high Z0-factor suggests that they
may also be suitable for HTS.
3.3. Assay development
Typical of screening assay development described elsewhere16
the enzymatic assays described here were optimized to meet
HTS-specific criteria. Since HTS assays are usually run against large
compound libraries (n = 105–106 compounds), they should be ame-
nable to automation, economically feasible and robust without
sacrificing the desired pharmacology being measured. Therefore,
all enzymatic assays were miniaturized and run in 1536-well
microtiter plates as homogeneous, endpoint (rather than kinetic)
experiments and configured to use sparing amounts of reagents.
Since the LOPAC library consists of a diverse set of 1280 well-
known chemical probes to a variety of eukaryotic and prokaryotic
targets, it is reasonable to assume that a small number of these
Figure 4. Click-chemistry strategy to synthesize selective VIM-2 inhibitors. Representative N-substituted and N-unsubstituted 1,2,3-triazoles from alkyne and azide
fragments with known or putative zinc binding groups are shown including hydroxamates, amides, ureas, thiadiazoles, carboxylate, and sulfonamides.

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D. Minond et al. / Bioorg. Med. Chem. 17 (2009) 5027–5037
compounds would be found active in any given screening effort. In
the context of the enzymatic assays presented here, the confirma-
tion of pCMB as an inhibitor of VIM-2 and IMP-1 represents an
example of this utility. In contrast, mitoxantrone was not expected
to be a potent inhibitor of VIM-2. Although its exact mechanism of
action at this point is unclear its identification confirms another
utility of the LOPAC, viz. to probe well-characterized compounds
for activity in an alternative pharmacology.
Mitoxantrone is a well-characterized chemotherapeutic43 and is
known to have antibacterial and antiviral activity.44 This has been
attributed to its affinity toward double and single-stranded RNA
and DNA.45 Similarly, the antibacterial action of pCMB may be
attributed to its ability to modify functionally important cysteine
residues in many proteins. For example, pCMB was shown to irre-
versibly dissociate bacterial ribosomes, thereby inhibiting protein
synthesis.36
At concentrations below the MIC for these compounds checker-
3.6. Characterization of the VIM-2 competitive inhibitors
board experiments showed that mitoxantrone at 2.1
lg/mL or
pCMB at 2.2 g/mL can restore the potency of imipenem against
l
The primary purpose of the VIM-2 CCF2 and IMP-1 nitrocefin
assays was to confirm VIM-2 inhibition and selectivity of com-
pounds found active in the VIM-2 nitrocefin assay, respectively.
Their utility has been demonstrated here by characterizing a novel
class of selective click-chemistry derived sulfonyl-triazole VIM-2
inhibitors. In particular, compound 1 has comparable potency (Ta-
resistant E. coli (BL21/VIM-2) (Table 4). At these lower concentra-
tions no toxicity to E. coli cells was observed. However, as MIC
experiments measure a single timepoint, time-kill experiments
were performed to study the kinetics of growth inhibition induced
by these compounds. Although at 2.2 lg/mL pCMB slightly inhib-
ited the growth at 6 h, the reduction of colony forming units as
compared to the negative control was less than three log units,
which is not sufficient to classify this compound as being bacteri-
ble 2, Ki = 0.41 0.03 lM) to a previously reported mercaptocarb-
oxylate class of VIM-2 inhibitor, that is, rac-2-
mercaptopropionic acid (PhenylC3SH, Ki = 0.22
x
l
-phenylpropyl-3-
M).31 In contrast
cidal or bacteriostatic. Conversely, mitoxantrone at 2.1 lg/mL had
to the mercaptocarboxylates, the two sulfonyl-triazoles described
no effect on E. coli growth. As a result, time-kill experiments
here demonstrate complete selectivity for VIM-2.
showed conclusively that pCMB and mitoxantrone are not bacteri-
Although the sulfonyl-triazoles are potent in vitro, the MIC re-
sults show that they do not have intrinsic antibacterial properties
nor potentiate the in situ potency of imipenem. From the analysis
of the MIC studies it can be assumed that the lack of efficacy for
sulfonyl-triazoles is due to their inability to reach and/or remain
in the E. coli periplasm in sufficient concentration to abrogate imip-
enemase activity. It is currently unclear whether this is because of
a bacterial resistance mechanism or whether they are perhaps
interacting with off-target entities. Future studies with hyperper-
meable and efflux deficient strains will address this issue.
cidal or bacteriostatic at 2.2 and 2.1 lg/mL, respectively, and there-
fore in synergy experiments must act primarily by inhibiting VIM-
2 in situ.
3.9. Docking studies with competitive inhibitors
To interrogate inhibitor binding requirements in the VIM-2 ac-
tive site, attempts were made to dock all four compounds into the
active site of VIM-2 from the native (PDB 1KO3) and inhibitor
bound (PDB 2YZ3) structures. Important to note, the active site
from the inhibitor-bound structure (PDB 2YZ3) is too small to
accommodate these compounds since Asn233, the residue that
forms a salt bridge with the inhibitor, occludes part of this hydro-
phobic cavity. This orientation of Asn233 and surrounding residues
on Loop2 determine the size and shape of the hydrophobic cavity.
The inhibitors from the LOPAC screen failed to dock rationally
into either structure. Although mitoxantrone has the ability to
complex zinc through its alcohol, carbonyl, or amine groups, a real-
istic docking result was not achieved. This is consistent with its
mode of action as a non-competitive VIM-2 inhibitor. Although
pCMB is small and fits easily into the VIM-2 active site its mecha-
nism of action is believed to be through the formation of a covalent
or slowly reversible electrostatic bond with sulfhydryl groups. As
such, modeling studies with standard docking protocols would
not be expected to identify a realistic ligand–protein docking pose
for this class of inhibitor.
Docking studies with the two inhibitors from the click-chemis-
try library proved more fruitful (Fig. 3). Compounds 1 and 2 are
predicted to bind to VIM-2 by exploiting the sulfonyl moiety as a
binder of the active site binuclear zinc cluster. One zinc atom is
chelated by both oxygen atoms, with distances of 2.5 Å and 2.9 Å
away from the metal. Additionally, one oxygen of the sulfonyl
group is located between the zinc cluster with distances of 2.5 Å
and 3.0 Å from the metals. As comparison, the free thiol of the
VIM-2 inhibitor PhenylC3SH, in PDB 2YZ3, is 2.2 Å away from
one zinc in the binuclear cluster. The oxygen to zinc binding dis-
tances observed in the current docking studies is longer than ex-
pected (<2.5 Å), but this is likely a consequence of the slight loss
in atomic detail when performing molecular docking. The pre-
dicted mode of binding for 1 orients the molecule such that the
arylsulfonamide is solvent exposed while the alkoxyl group (R)
buries into a hydrophobic subpocket at the base of the active site.
From these studies, it appears that compound 1, which displays the
larger propargyl group, best exploits this hydrophobic cavity. In
3.7. Biochemical characterization of VIM-2 LOPAC inhibitors
Mitoxantrone was found to be a potent, non-competitive inhib-
itor of VIM-2 with a Ki of 1.5 0.2 lM. When tested with IMP-1,
TEM-1, and AmpC it did not exhibit any inhibitory activity, (Table
2) which indicates that its interaction with VIM-2 is specific. In the
absence of structural studies it is difficult to predict the mode of
binding to VIM-2 by this compound. Further structural and mech-
anistic investigations, such as mutual exclusivity studies,20 will be
required to pinpoint the mode of binding.
In contrast to mitoxantrone, pCMB demonstrated potency
against IMP-1 (Table 2). This cysteine-reactive reagent, which
forms covalent or slowly reversible electrostatic bonds with sulf-
hydryl groups, is widely used for enzyme characterization, includ-
ing b-lactamases.19,35–38 With this in mind, a mechanism of action
can be proposed for VIM-2 and IMP-1 inhibition. Both enzymes
share conserved binuclear zinc binding motifs, whereby three his-
tidine residues coordinate one zinc ion; cysteine (i.e., Cys221),
aspartate and another histidine coordinate the other ion.10 It has
been shown12 that once oxidized, Cys221 loses its ability to partic-
ipate in coordination of the second zinc ion, which deleteriously af-
fects enzyme activity.24,39 Therefore it is reasonable to assume that
the inhibition of VIM-2 and IMP-1 occurs as a result of pCMB react-
ing with the corresponding cysteine residues. Thus the fact that
pCMB exhibited no inhibitory activity against TEM-1 and AmpC
(Table 2) is in agreement with the literature findings that cysteine
residues are not involved in catalysis by these enzymes.40–42
3.8. Bacterial characterization of VIM-2 LOPAC inhibitors
When tested alone mitoxantrone and pCMB exhibited antibac-
terial activity against resistant (BL21/VIM-2) and non-resistant
(BL21) E. coli (MIC = 8.4 and 17.9 lg/mL, respectively, Table 3).

D. Minond et al. / Bioorg. Med. Chem. 17 (2009) 5027–5037
5033
the docking model the propargyl is 3.3 Å away from Tyr224, but it
is likely that the aromatic side chain of this residue will orient to
form a better hydrophobic interaction with the propargyl. Further-
more the ether oxygen of 1 is 2.3 Å from the backbone amide
hydrogen of Asn233 and likely forms a hydrogen bond.
on its alkoxyl group. Compound 3 is closely related but displays a
bulkier alkoxy group. Presumably, the larger size of this dioxolane
moiety is detrimental to the inhibition of VIM-2. Compounds 4 to 6
display even bulkier R groups which is consistent with them being
inactive against VIM-2.
Modeling studies also implicate the arylsulfonamide and tria-
zole moieties as potentially being involved in aromatic interactions
with four residues that ring the active site, namely His118, Trp87
and Phe61 and Tyr67 in Loop1. Both arylsulfonamides46 and tria-
zoles47 can make strong interactions with aromatic groups through
pi-stacking (edge or face) as well as other hydrophobic modes of
binding. The triazole moiety is 2.7 Å from Trp87, while the benzyl
group of the sulfonamide is 3.0 Å from His118. Similarly, the tria-
zole group is 3.2 Å from Tyr67 and 5.6 Å from Phe61. However,
as compounds were docked into a rigid representation of the na-
tive structure the importance of all four aromatic residues for
inhibitor binding cannot be fully appreciated, since ligand-induced
changes will be required for the correct orientation of these resi-
dues. This is particularly relevant for Phe61 and Tyr67 as the loop
to which they belong is known to be highly flexible.26
As represented in Figures 2 and 3b and Table 5, the sulfonyl-tri-
azole class represents a novel scaffold for further development of
more potent and/or selective VIM-2 inhibitors. Furthermore they
contain ‘click chemistry’ moieties. Since click-chemistry com-
pounds (by definition) have chemical features which facilitate ra-
pid study of SAR,28,48 our research has been directed to the
improvement of sulfonyl-triazole potency and efficacy. Based on
the results of the studies presented here, a medicinal chemistry ef-
fort is being pursued to improve the potency for this chemotype
against VIM-2 by further elaborating structure–activity relation-
ships (SARs) around the arylsulfonamide and R substituents. In
particular, the importance of the substitution pattern around the
arylsulfonamide (which is predicted to be solvent exposed) as well
as the hydrophobicity of the R position (which is predicted to bind
in a deeper hydrophobic cavity) merits further exploration.
3.10. SAR of the sulfonyl-triazole scaffold
4. Conclusion
In order to investigate structure–activity relationship (SAR)
amongst the sulfonyl-triazoles, an in silico study was performed
by retrieving VIM-2 screening results of click-chemistry library
compounds containing this scaffold (Table 5). Among the group
of structural analogs, some modest inhibition was observed for
three of the compounds at the highest test concentration
In conclusion, a repertoire of HTS-compatible assays was used
to aid the identification of VIM-2 inhibitors. These assays identified
a novel class of competitive and selective VIM-2 inhibitors, repre-
sented here by click-chemistry library compounds 1 and 2. The
same assays were also used to identify and characterize two
well-known pharmacologic agents from the LOPAC library,
mitoxantrone and pCMB, which appear to be non-competitive
and irreversible/slowly reversible VIM-2 inhibitors, respectively.
The click-chemistry library inhibitors did not show efficacy in
MIC assays, whereas the LOPAC inhibitors were able to restore
(56 lM). Of course, only 1 and 2 demonstrated potency against
VIM-2; however, the inspection of all compounds may suggest that
steric hindrance prevents inhibition of the VIM-2 active site. For
example, compound 1 differs from 2 by the presence of an alkyne
Table 5
SAR of sulfonyl-triazoles within the click-chemistry library
ID
Scaffold
R
IC50 or max inhibitiona
CH
1
3.3 0.4
7.3 1.9
l
M (79% @ 56
M (74% @ 56
l
M)
M)
O
*
CH3
2
3
4
5
6
l
l
O
O
*
*
O
S
O
I
O
N
H
R
CH3
>56
>56
>56
>56
l
l
l
l
M (31% @ 56
M (11% @ 56
l
l
M)
M)
O
CH3
HN
N
N
*
*
N
CH3
N
N
N
Cl
M (5% @ 56
M (0% @ 56
lM)
Cl
Cl
H3C
lM)
N
*
a
IC50 error reported as the standard deviation of 3 replicates or in parentheses maximum inhibition achieved at the indicated concentration in the nitrocefin assay for
VIM-2.

5034
D. Minond et al. / Bioorg. Med. Chem. 17 (2009) 5027–5037
efficacy of b-lactam antibiotic imipenem via VIM-2 inhibition in
situ. Further studies will lead to a better understanding of their ex-
act mechanism of action, and aim to improve the efficacy of the
competitive click-chemistry derived inhibitors described here.
5.4. Synthesis of the azide derivatives from the corresponding
chlorides
To a solution of N-(4-chlorobut-2-ynyl)arylsulfonamide in DMF
(2 mL/mmol of chloride) was added NaN3 (2.0 equiv). The resulting
suspension was stirred at room temperature for 12 h before being
quenched by the addition of saturated NaCl (10 mL/mmol of chlo-
ride). The reaction mixture was extracted twice with Et2O (10 mL/
mmol of chloride) and the combined organic layers were dried over
MgSO4 before being concentrated under reduced pressure at room
temperature to furnish the crude product. The crude product was
used for the next reaction without further purification.
5. Experimental
5.1. Sources of screening compounds
The inhibition control compound NSC 20707 (2-(2-chloroben-
zyl) succinic acid) was obtained from the Drug Synthesis and
Chemistry Branch of National Cancer Institute (MD, USA). The li-
brary of pharmacologically active compounds (LOPAC) used for
screening was purchased from Sigma–Aldrich (St. Louis, MO). The
267-membered click-chemistry library was from an in-house
compound collection. The methods for click-chemistry syntheses
presented below represent generalized protocols and more
detailed syntheses will be presented in a future publication.
5.5. Thermally induced Banert Cascade; procedure for the
synthesis of NH-triazole-arylsulfonamides
To a solution of the azide (1 mL/mmol of azide) in dioxane was
added the nucleophile (1 mL/mmol of azide; 2.5 equiv for a solid
nucleophile). The reaction mixture was stirred at 75 °C for 4 h. Sub-
sequently the reaction was cooled to room temperature and the
solvent was concentrated under reduced pressure to furnish the
crude product. The title compounds were purified by flushing
through a short pad of silica gel (50–100% EtOAc in hexanes to
10% MeOH in EtOAc with 0.5% NEt3) and obtained as yellow oils.
Solid compounds were purified by crystallization from Et2O/
hexane.
5.2. General synthetic procedures for ‘click-chemistry’
compounds
Reagents and solvents were purchased from commercial
sources and were used as received. Reaction progress was moni-
tored by TLC using Merck Silica Gel 60 F-254 with detection by
UV. Silica Gel 60 (Merck 40–63
lm) was used for column chroma-
tography. 1H NMR and 13C NMR spectra were recorded with Bruker
AMX-300 spectrometers. Proton magnetic resonance (1H NMR)
spectra were recorded at 300 MHz. Data are presented as follows:
chemical shift (ppm), multiplicity (s = singlet, d = doublet, t = trip-
let, q = quartet, quin = quintet, sep = septet, m = multiplet,
b = broad), coupling constant, J (Hz) and integration. Carbon mag-
netic resonance (13C NMR) spectra were recorded at 75 MHz. Data
for 13C NMR are reported in terms of chemical shifts (ppm). HPLC
homogeneities were determined using an Agilent 1100 LC/MSD
with an Agilent 1100 SL mass spectrometer. System A: Zorbax
4.6 mm ꢁ 30 mm, SB-C18 reverse phase column, preceded by a
Phenomenex C18 guard column, eluting with 10–100% MeCN
(+0.05% TFA) in 0.05% TFA, linear gradient over 10 min then iso-
cratic for 5 min, 0.5 mL/min flow rate with UV detection at
254 nm. System B: Zorbax 4.6 mm ꢁ 150 mm, SB-C18 reverse
phase column, preceded by a Phenomenex C18 guard column, elut-
ing with 10–100% MeOH (+0.05% TFA) in 0.05% TFA, linear gradient
over 10 min then isocratic for 10 min, 0.5 mL/min flow rate with
UV detection at 254 nm.
5.5.1. 4-Iodo-N-((4-((but-3-ynyloxy)methyl)-1H-1,2,3-triazol-5-
yl)methyl)benzenesulfonamide (1)
1H NMR (300 MHz, DMSO-d6): d = 8.17 (s, 1H), 7.96 (d, J = 8.1,
2H), 7.52 (d, J = 8.1, 2H), 4.47 (s, 2H), 4.08 (s, 2H), 3.44 (t, J = 6.9,
2H), 2.77 (t, J = 2.4, 1H), 2.37 (dt, J = 6.9, 2.4, 2H); 13C NMR
(75 MHz, DMSO-d6): d = 139.9, 137.9, 100.4, 81.7, 71.9, 67.7, 19.0;
LC–MS m/z (%): 447.2 (100) [M+H]+ (calcd 447.00), 469.1 (55)
[M+Na]+ (calcd 468.98); Yield: 92%.
5.5.2. 4-Iodo-N-((4-(methoxymethyl)-1H-1,2,3-triazol-5-
yl)methyl)benzenesulfonamide (2)
1H NMR (300 MHz, DMSO-d6): d = 8.25 (s, 1H), 7.95 (d, J = 8.4,
2H), 7.53 (d, J = 8.4, 2H), 4.33 (s, 2H), 4.01 (s, 2H), 3.15 (s, 3H);
13C NMR (75 MHz, DMSO-d6): d = 139.9, 137.9, 128.2, 100.3, 63.4,
57.4, 45.5; LC–MS m/z (%): 409.1 (100) [M+H]+ (calcd 408.98),
431.0 (40) [M+Na]+ (calcd 430.97), 839.0 (15) [2M+Na]+ (calcd
838.94); Yield: 94%.
5.5.3. 4-Iodo-N-((4-(((2,2-dimethyl-1,3-dioxolan-4-
yl)methoxy)methyl)-1H-1,2,3-triazol-5-
5.3. Synthesis of the sulfonamide derivatives from the
corresponding sulfonylchlorides
yl)methyl)benzenesulfonamide (3)
1H NMR (300 MHz, DMSO-d6): d = 7.95 (d, J = 8.4, 2H), 7.53 (d,
J = 8.4, 2H), 4.78 (s, 1H), 4.01–3.94 (m, 5H), 3.65–3.60 (m, 2H),
1.30 (s, 3H), 1.25 (s, 3H); LC–MS m/z (%): 509.1 (100) [M+H]+ (calcd
509.04), 431.0 (55) [M+Na]+ (calcd 531.02); Yield: 74%.
To a solution of the sulfonylchloride in THF/H2O (5 mL/mmol of
sulfonylchloride) was added K2CO3 (3.0 equiv) followed by 4-chlo-
robut-2-yn-1-amine hydrochloride (1.3 equiv) at 0 °C. The reaction
mixture was stirred at 0 °C for 1 h before allowed to warm up to
room temperature. The reaction mixture was diluted with EtOAc
(5 mL/mmol of acid) and washed twice with 1 M HCl (20 mL/mmol
of acid), NaHCO3 (6 mL/mmol of acid), and brine (6 mL mmol of
acid). The organic portion was dried over MgSO4 before being con-
centrated under reduced pressure to furnish the crude product.
Purification by crystallization from Hex/EtOAc afforded the corre-
sponding sulfonamide in almost quantitative yield as white solids.
5.5.4. 4-Iodo-N-((4-((1-methyl-1H-indol-3-yl)methyl)-1H-1,2,3-
triazol-5-yl)methyl)benzenesulfonamide (4)
1H NMR (300 MHz, DMSO-d6): d = 8.17 (t, J = 5.4, 1H), 7.91 (d,
J = 8.1, 2H), 7.52–7.34 (m, 4H), 7.14–6.95 (m, 3H), 3.99–3.95 (m,
4H), 3.78 (s, 3H); LC–MS m/z (%): 508.1 (100) [M+H]+ (calcd
508.03), 530.1 (55) [M+Na]+ (calcd 530.01); Yield: 65%.
5.5.5. N-((4-((4-(3,4-Dichlorophenyl)piperazin-1-yl)methyl)-
1H-1,2,3-triazol-5-yl)methyl)-4-iodobenzenesulfonamide (5)
1H NMR (300 MHz, DMSO-d6): d = 8.30 (s, 1H), 7.95 (d, J = 8.4,
2H), 7.54 (d, J = 8.4, 2H), 7.25 (d, J = 9.0, 1H), 7.10 (d, J = 3.0, 1H),
6.91 (dd, J1 = 9.0, J2 = 3.0, 1H), 4.11 (s, 2H), 3.53 (s, 2H), 3.10
5.3.1. 4-Iodo-N-(4-chlorobut-2-ynyl)benzenesulfonamide
1H NMR (300 MHz, DMSO-d6): d = 7.96 (d, J = 8.7, 2H), 7.52 (d,
J = 8.7, 2H), 4.22 (t, J = 6.0, 1H), 3.80 (dt, J = 6.0, 1.8, 2H), 3.70 (s,
2H); LC–MS m/z (%): 391.9 (100) [M+Na]+ (calcd 391.90).

D. Minond et al. / Bioorg. Med. Chem. 17 (2009) 5027–5037
5035
(s, 4H), 2.41 (s, 4H); LC–MS m/z (%): 607.2 (100) [M+H]+ (calcd
606.99); Yield: 69%.
VIM-2; and ‘Negative Control’ is defined as the absorbance mea-
sured from wells containing nitrocefin alone.
5.5.6. N-((4-((4-(5-Chloro-2-methylphenyl)piperazin-1-
yl)methyl)-1H-1,2,3-triazol-5-yl)methyl)-4-
5.9. Determination of kinetic parameters
iodobenzenesulfonamide (6)
Initial velocities were determined from linear portions of plots
of fluorescence at 447 nm or absorbance at 495 nm versus time.
Initial velocities were used to determine kinetic parameters utiliz-
ing GraphPad Prism version 5.01 (GraphPad Software, Inc., La Jolla,
CA). KM values were determined by non-linear regression analysis
using the one site hyperbolic binding model49 and additionally
evaluated by linear analysis. All Ki values were determined by
non-linear regression (hyperbolic equation) analysis using the
mixed inhibition model which allows for simultaneous determina-
tion of mechanism of inhibition.49 Mechanism of inhibition was
determined using the ‘alpha’ parameter derived from a mixed-
model inhibition by GraphPad Prism. Kinetic responses over a
range of substrate concentrations and with several inhibitor con-
centrations were analyzed with Hanes–Woolf reciprocal plots.
The mechanism of inhibition determined by this analysis (see Sup-
plementary data) agreed with non-linear regression (hyperbolic
equation) analysis using the mixed inhibition model in GraphPad
Prism.
1H NMR (300 MHz, DMSO-d6): d = 8.34 (s, 1H), 7.96 (d, J = 8.7,
2H), 7.55 (d, J = 8.7, 2H), 7.17 (d, J = 8.4, 1H), 7.01–6.95 (m, 2H),
4.12 (s, 2H), 3.55 (s, 2H), 2.78 (s, 4H), 2.44 (s, 4H), 2.19 (s, 3H);
LC–MS m/z (%): 587.1 (100) [M+H]+ (calcd 587.05); Yield: 68%.
5.6. IMP-1 and VIM-2 nitrocefin kinetic assays
All reagents were obtained from Invitrogen (USA) unless noted
below. IMP-1, VIM-2, and nitrocefin (BD Diagnostic Systems, USA)
working solutions were prepared in buffer containing 50 mM
HEPES, 50
Sigma–Aldrich (St. Louis, MO). Kinetic assays were conducted by
incubating the range of substrate concentrations (10–100 M)
lM ZnSO4 Sigma–Aldrich (St. Louis, MO), 0.05% Brij 35
l
with 0.1 nM enzyme at room temperature. Determinations of inhi-
bition constants and modalities were conducted by incubating the
range of substrate concentrations (10–100 lM) with 0.1 nM en-
zyme at room temperature in the presence of varying concentra-
tions of inhibitors. Absorbance was measured by a Tecan Safire2
monochromator microplate reader at 495 nm. Initial velocities
were obtained from plots of absorbance at 495 nm versus time,
using data points from only the linear portion of the hydrolysis
curve. Substrate hydrolysis was continuously monitored.
5.10. Nitrocefin microtiter plate assays
Thenitrocefinassay began by dispensing 2.5 lL of 0.26 nMVIM-2
(or 0.2 nM IMP-1) to the appropriate wells of a 1536-well microtiter
plate (Greiner, USA). Following enzyme addition, 28 nL of controls or
test compounds was added to the appropriate wells. The plates were
then incubated at room temperature for 15 min. To start the reac-
5.7. VIM-2 CCF2 kinetic assays
tion, 2.5
tions of 60
(VIM-2) or 0.1 nM (IMP-1). The plate was then incubated for
25 min at rt and the reaction stopped by the addition of 5 L of
500 mM EDTA (Invitrogen, USA). Immediately after EDTA addition
absorbance readings at 495 nm were performed using a Viewlux
multipurpose plate reader (Perkin–Elmer, Finland).
l
L of 120
lM nitrocefin was dispensed. Final concentra-
VIM-2 and CCF-2 (Invitrogen, USA) working solutions were pre-
l
M for substrate and enzyme were either 0.13 nM
pared in buffer containing 50 mM HEPES, 50
35, pH 7.1. Kinetic assays were conducted by incubating the range
of substrate concentrations (5–50 M) with 0.1 nM enzyme at
lM ZnSO4, 0.05% Brij
l
l
room temperature. Fluorescence-resonance energy transfer (FRET)
was measured by a Safire2 (Tecan, USA) using excitation = 409 nm
and emission = 447 nm. Initial velocities were obtained from plots
of fluorescence at 447 nm versus time, using data points from only
the linear portion of the hydrolysis curve. Substrate hydrolysis was
continuously monitored.
5.11. FRET (CCF2) microtiter plate assays
The FRET assay began by dispensing 1.25 lL of 0.2 nM VIM-2 to
5.8. Rapid dilution experiments
the appropriate wells of a 1536-well microtiter plate (Greiner,
USA). Following enzyme addition, 11 nL of positive control or test
compound was added. The plates were then incubated at room
The rapid dilution technique is described in detail elsewhere.20
VIM-2 was incubated for 30 min at room temperature with or
without 24 lM pCMB. This high concentration of pCMB corre-
temperature for 15 min. To start the reaction, 1.25
CCF2-FA substrate (Invitrogen, USA) was dispensed. Final concen-
trations of substrate and enzyme were 10 M and 0.1 nM, respec-
tively. The plate was then incubated for 25 min at rt and the
reaction stopped by the addition of 2.5 L of 500 mM EDTA. Imme-
lL of 20 lM
sponds to 10ꢁ of its IC50 in the VIM-2 nitrocefin assay (Table 2)
l
and resulted in 90% inhibition. A rapid 1:100 dilution was made di-
rectly into 60 lM nitrocefin solution (the solution’s composition
l
was identical to the VIM-2 nitrocefin microtiter plate assay de-
scribed below). Absorbance readings were initiated immediately
after the addition of corresponding high and low concentration
mixtures. Absorbance was measured on a Safire2 (Tecan, USA)
monochromator microplate reader at 495 nm for 30 min at 1 min
intervals. Absorbance at 495 nm was plotted versus time. Percent
activity in test wells was calculated according to the following
equation:
diately after EDTA addition, fluorescence readings were performed
in ratiometric mode using the Viewlux (Perkin-Elmer, Finland)
plate reader with an excitation wavelength of 409 nm and emis-
sion wavelengths of 460 nm and 535 nm.
5.12. Assay quality control
Three quality control parameters were calculated during the
screening on a per-plate basis: (a) the signal-to-basal ratio (S/B);
(b) the coefficient for variation [CV; CV = (standard deviation/
mean) ꢁ 100] for all compound test wells; and (c) Z- or Z0-factor
% Activity ¼ 100 ꢁ ðTest ꢂ Negative ControlÞ=ðPositive Control
ꢂ Negative ControlÞ
[Z0-factor = 1 ꢂ [[3 ꢁ (rp
+
r
n)]/(lp
ꢂ
ln)], where r is the standard
where ‘Test’ is defined as the absorbance measured from wells con-
taining nitrocefin, VIM-2, and pCMB; ‘Positive Control’ is defined as
the absorbance measured from wells containing nitrocefin and
deviation and
controls].32
l is the mean for positive (p) and negative (n)

5036
D. Minond et al. / Bioorg. Med. Chem. 17 (2009) 5027–5037
5.13. Screening assay protocols
(TEM-1) or 2.0 nM (AmpC), respectively. The plate was then incu-
bated for 25 min at rt and the reaction stopped by the addition of
10 L of 50 M potassium clavulanate in assay buffer (PBS pH 7.4,
0.05% Brij) in case of TEM-1 assay. In case of the AmpC assay reac-
tion was not quenched. Absorbance readings at 495 nm were per-
formed using Envision multipurpose plate reader (Perkin–Elmer,
Finland).
The nitrocefin LOPAC HTS assays were performed identical to
the nitrocefin microtiter-plate based assays described above. All
compounds were tested at 14 lM final concentration in triplicate.
Potency (dose–response) screening protocols were identical to the
corresponding microtiter plate assays described above, except that
each test compound was assayed in triplicate using 10 1:3 serial
l
l
dilutions, starting at a nominal test concentration of 56
l
M. For
5.17. Transformation of E. coli with VIM-2 plasmid
each compound, either raw absorbance (nitrocefin assay) or ratio
(CCF2 assay) data were fitted with a four parameter equation
describing a sigmoidal dose–response curve with adjustable base-
line using GraphPad Prism version 5.01 suite of programs. The IC50
values were generated from fitted curves by solving for X-intercept
at the 50% inhibition level of Y-intercept. Assays were run in the
The plasmid (pET-9a-based) containing the VIM-2 gene has
been described in detail elsewhere.11 Competent E. coli BL21
(DE3) (Novagen, USA) was transformed with the plasmid using
standard techniques and was selected on LB agar + kanamycin
(30 lg/mL). Transformed bacteria were aliquoted as frozen glyc-
presence and absence of 50
l
M ZnSO4.
erol stocks at ꢂ80 °C and were used directly in experiments by
inoculating a starter culture in LB or Iso-Sensitest broth. The stock
vials were discarded after inoculation.
A standard mathematical algorithm50 was used to determine
inhibitory compounds (‘hits’) in the VIM-2 nitrocefin assay. Two
values were calculated: (a) the average percent inhibition of all
compounds tested; and (b) three times their standard deviation.
The sum of these two values was used as a cutoff parameter, that
is, any compound that exhibited a % inhibition greater than the
cutoff parameter was declared active.
5.18. MIC assays
Minimal inhibitory concentration (MIC) assays were conducted
using twofold serial broth dilution method as recommended by
Clinical and Laboratory Standards Institute (CLSI).51 All testing
was performed in 14 mL tubes (BD, USA) in 2 mL final volume.
E. coli BL21 were grown in LB broth (Fisher Scientific, USA) with
(transformed BL21-VIM-2 control strain) or without (untrans-
5.14. Absorbance artifact assays
Two variants of this assay were executed. In the first variant,
the nitrocefin screening protocol was followed with one excep-
tion: compounds were added after the reaction was quenched
formed BL21 control strain) kanamycin (30 lg/mL) at 37 °C for
5 h. Imipenem (Fisher Scientific, USA) or inhibitor was titrated in
Iso-Sensitest broth (Oxoid, UK) using 10 point twofold serial dilu-
tions immediately prior to testing. Each tube was inoculated with
1 mL of bacterial inoculums of 5 ꢁ 105 CFU/mL. The tubes were
shaken for at least 18 h at 37 °C under aerobic conditions. MIC
was determined as per CLSI.52 The E. coli strain ATCC 25922 was
used as a quality control reference.
by the addition of 500 mM EDTA. In the second variant, 5.0
of buffer containing 50 mM HEPES, 50 M ZnSO4, 0.05% Brij 35,
pH 7.1 and 5.0 L of 500 mM EDTA was added to each well of a
lL
l
l
1536-well clear-bottom black plate; 28 nL of test compounds
was added next. Absorbance was measured at 495 nm using the
Viewlux plate reader (Perkin–Elmer, Finland). Any compound that
exhibited a raw absorbance value greater than the average absor-
bance of all compounds tested was considered an absorbance
artifact.
5.19. Inhibitor/antibiotic synergy testing
The combined effect of simultaneous application of inhibitor
and Imipenem was determined using a ‘checkerboard’ method.21
All testing was performed in non-treated clear 96 well plates
(Corning, USA) in 0.2 mL final volume. E. coli were grown in LB
broth with (transformed BL21-VIM-2 control strain) or without
5.15. FRET artifact assays
Two variants of this assay were executed. In the first variant, the
CCF2 screening protocol was followed with one exception: com-
pounds were added after reaction was quenched by the addition
(untransformed BL21 control strain) kanamycin (30 lg/mL) at
of EDTA. In the second variant, 2.5
l
L of buffer containing 50 mM
37 °C for 5 h. Imipenem was titrated in Iso-Sensitest broth using
8 point twofold serial dilutions immediately prior to testing.
Inhibitor was titrated in Iso-Sensitest broth using 9 point two-
fold serial dilutions. Imipenem and inhibitor were serially di-
luted down columns and rows, respectively. This resulted in
concentrations of each agent that ranged from at least 4ꢁ down
to 0.25ꢁ of respective MIC values. Each well was then inoculated
with 0.1 mL of bacterial inoculums of 1 ꢁ 106 CFU/mL. The plates
were shaken for at least 18 h at 37 °C under aerobic conditions.
MIC was determined as per CLSI.52 The b-lactam resistant
E. coli strain ATCC 35218 was used as a quality control reference.
HEPES, 50 M ZnSO4, 0.05% Brij 35, pH 7.1 and 2.5
l
l
L of 500 mM
EDTA was added to each well of 1536-well solid white plate;
11 nL of test compound was added next. Fluorescence readings
were performed in ratiometric mode using the Viewlux plate read-
er with an excitation wavelength of 409 nm and emission wave-
lengths of 460 nm and 535 nm. Any compound that exhibited a
460 nm/535 nm ratio value greater than the average ratio of all
compounds tested was considered FRET assay artifact.
5.16. TEM-1 and AmpC nitrocefin assays
Results of synergy testing were interpreted using a Total Frac-
P
AmpC (Cephalosporinase, from Enterobacter cloacae) was
obtained from Sigma (USA). The nitrocefin assay began by dispens-
ing 20 lL of 0.3 nM TEM-1 in assay buffer (or 4.0 nM AmpC) (PBS
pH 7.4, 0.05% Brij or PBS pH 7.4, 5% glycerol, for TEM-1 and AmpC,
respectively) to the appropriate wells of a 384-well microtiter
plate (Greiner, USA). Following enzyme addition, 100 nL of controls
or test compounds was added to the appropriate wells. The plates
were then incubated at room temperature for 15 min. To start the
tional Inhibitory Concentration ( FIC) method as described pre-
viously,23,53 where:
X
FIC ¼ FIC A þ FIC B
ð1Þ
ð2Þ
ð3Þ
FIC A ¼ MIC AB combination=MIC A alone
FIC B ¼ MIC AB combination=MIC B alone
The effect of combined imipenem/inhibitor application was con-
sidered synergistic when the FIC was 60.5, indifferent when the
FIC was >0.5 and <2, and antagonistic when the FIC was P2.
P
reaction, 20
l
L of 200
l
M nitrocefin was dispensed. Final concen-
M and 0.15 nM
P
P
trations of substrate and enzyme were 100
l

D. Minond et al. / Bioorg. Med. Chem. 17 (2009) 5027–5037
5037
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(Becton-Dickinson, USA) along with the appropriate test and con-
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We thank the National Institute of Neurological Disorders and
Stroke, National Institute of Health (NS059451, P.H.), the Skaggs
Institute for Chemical Biology (K.B.S.), and the W.M. Keck Founda-
tion (K.B.S.) for the support of this work. Mr. Pierre Baillargeon and
Mrs. Lina DeLuca (Lead Identification Division, Translational Re-
search Institute, Scripps Florida) are thanked for their assistance
with compound management. Professor Hugh Rosen (The Scripps
Research Institute), Jean-Marie Frère (d’Enzymologie & Centre
d’Ingénierie des Protéines, Institut de Chimie) and Lynn L. Silver,
Ph.D. (LL Silver Consulting, LLC) are thanked for their helpful
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Supplementary data
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