Phenylboronic Acid Derivatives as Validated Leads Active in Clinical Strains Overexpressing KPC-2: A Step against Bacterial Resistance

Article abstract of DOI:10.1002/cmdc.201700788

The emergence and dissemination of multidrug resistant (MDR) pathogens resistant to nearly all available antibiotics poses a significant threat in clinical therapy. Among them, Klebsiella pneumoniae clinical isolates overexpressing KPC-2 carbapenemase are

Full text of DOI:10.1002/cmdc.201700788

Accepted Article
Title: Phenyl boronic acids development led to validated leads active
in clinical strains overexpressing KPC-2: a step against bacterial
resistance.
Authors: Giuseppe Celenza, Mattia Vicario, Pierangelo Bellio, Linciano
Pasquale, Mariagrazia Perilli, Antonio Oliver, Jesus Blazquez,
Laura Cendron, and Donatella Tondi
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ChemMedChem
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“
Phenyl boronic acids development led to validated leads active
in clinical strains overexpressing KPC-2:
a step against bacterial resistance”
[
a]
[b]
[a]
[c]
Giuseppe Celenza , Mattia Vicario , Pierangelo Bellio , Pasquale Linciano ,
[
a]
[d]
[e]
[b]*
[c]*
Mariagrazia Perilli , Antonio Oliver , Jesús Blazquez , Laura Cendron and Donatella Tondi
[
a]
Dr. G. Celenza, https://orcid.org/0000-0003-2796-9228, Dr. P. Bellio, Prof. M. Perilli
Dipartimento di Scienze Cliniche Applicate e Biotecnologie.
Università dell’Aquila,
Via Vetoio, 1, 67100 L’Aquila, Italy
[
[
[
[
b]
c]
d]
e]
Dr. M. Vicario, Dr. L. Cendron, https://orcid.org/0000-0002-0125-0461, E-mail: laura.cendron@unipd.it
Dipartimento di Biologia, Department
Università di Padova,
Viale G. Colombo 3, 35121, Padova, Italy
Dr. P. Linciano, Dr. D. Tondi, https://orcid.org/0000-0002-5195-5531, E-mail: tondid@unimore.it
Dipartimento di Scienze della Vita,
Università di Modena e Reggio Emilia,
Via Campi 103, 41100, Modena, Italy.
Dr. A. Oliver
Servicio de Microbiología and Unidad de Investigación.
Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Mallorca,
Palma de Mallorca, Spain
Dr. J. Blazquez
Department of Microbial Biotechnology, National Center for Biotechnology,
Consejo Superior de Investigaciones Científicas (CSIC),
C/ Darwin, 3, Campus de la Universidad Autonoma-Cantoblanco, 28049-Madrid, Spain
Supporting information for this article is given via a link at the end of the document.
Abstract: The emergence and dissemination of multi drug resistant
MDR) pathogens resistant to near all available antibiotics poses a
resistance, the rapid evolution and spread of carbapenemases,
β-lactamases (BLs) with versatile hydrolytic capacities able to
inactivate last resort carbapenems, represents a menace in
(
significant threat in clinical therapy. Among them, Klebsiella
pneumoniae clinical isolates overexpressing KPC-2 carbapenemase
are the most worrisome, extending bacterial resistance to last resort
carbapenems. In this study we investigate the molecular recognition
requirements in KPC-2 active site by small phenyl boronic acid
derivatives. Four new phenyl boronic acid derivatives were designed
and tested vs KPC-2. For the most active, despite their simple
chemical structures, nanomolar affinity was achieved. New
derivatives restored susceptibility to meropenem in clinical strains
overexpressing KPC-2. Moreover no cytotoxicity was detected in cell
viability assays, further validating the designed leads. Two
crystallographic binary complexes of best inhibitors binding KPC-2
[
1–7]
treating highly resistant infections.
Carbapenem-resistant Enterobacteriaceae (CREs), reported
with increased frequency, are progressively spreading
[
1,2,5,6,8]
throughout the world thus leaving no effective antibiotics.
In particular CREs possessing blaKPC-2 (e.g. Klebsiella
pneumoniae) are high-priority target pathogens for the
development of novel antibacterials: KPC-producing organisms
confer resistance not only to all available β-lactams antibiotics
(including aminothiazoleoxime cephalosporins such as
cefotaxime) but also to other antimicrobial classes such as
[
9–11]
fluoroquinolones and aminoglycosides.
Moreover KPCs are
were obtained at high resolution. Kinetic descriptions of slow binding, weakly inhibited by therapeutically available β-lactamase
[
12]
time dependent inhibition and interactions geometries in KPC-2 were
fully investigated. This study will ultimately lead toward optimization
and development of more effective KPC-2 inhibitors.
inhibitors (i.e. clavulanic acid, Figure 1a). As a consequence,
KPCs mediated infections are difficult to treat and often
associated with high therapeutic failure and high mortality rates,
[
13,14]
making KPCs a major global health threat.
Among developed carbapenemases inhibitors, novel chemical
entities not resembling the β-lactam ring are rare, with few
exceptions such as the recent approved avibactam (Figure
Introduction
[
15]
1
b).
This non β-lactam (BL) inhibitor, able to restore
The emergence of bacterial strains resistant to available
antibiotic armamentarium is nowadays a pressing issue in
clinical therapy. Among the several mechanisms of bacterial
susceptibility to β-lactams when administered in combination, is
however slowly hydrolyzed by KPC-2, suggesting peculiarities in
[15]
the catalytic mechanism for class A carbapenemase.
1
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In the de novo BL inhibitors scenario, boronic acid derivatives
play an interesting role: they possess a non β-lactam moiety and
act as competitive inhibitors of BLs, covalently, but reversibly,
binding the catalytic serine of BLs belonging to class A, C and
While the presence of the boronic acid group guarantees a
covalent linking to Ser-70, sinking de facto the inhibitor in the
active site, a proper linker could correctly orient the lateral
carboxylic chain in the targeted site, without preventing other
classical interactions between the ligand and residues Thr-237,
Glu-166 and Asn-170, as seen in the KPC-2-3-nitro-phenyl
[
16–21]
D.
Very recently boronic acids have also been reported to
inhibit zinc-dependent BLs, opening the way for the
[
22-23]
[26]
development of dual action inhibitors.
RPX7009, a highly
boronic complex.
[
24]
potent inhibitor of KPC is currently in development.
With the purpose of mimicking distance, chemistry, and
interactions of C3(4)′ carboxylate of β-lactam antibiotics,
structural considerations suggested to introduce a carboxylic
moiety, properly spaced by a two atomic vinyl chain, in the ortho
position, adjacently to boronic group of compound 1. Therefore,
compound [2-(2-carboxyvinyl)phenyl]boronic acid (2) was
selected as a promising compound 1 candidate derivative (Table
At present few crystal structures of KPC-2 in its apo and
complexed form have been disclosed making KPC-2 an
attractive target for structure based drug design: there is still
much to be done to fully understand KPC-2 catalysis machinery
[
2]
and its ability to overcome carbapenems action.
We report here the rational optimization of phenyl boronic acid
derivatives acting as novel, non β-lactam like inhibitors via a
time dependent mechanism of inhibition (Figure 1c). For the two
most active compounds, X-ray crystallographic binary
complexes were determined, confirming that designed
molecules take advantage of critical consensus binding sites,
peculiar of KPC-2 and other class A carbapenemases. The best
inhibitor, in light of its nanomolar affinity for the enzyme, its
1).
Moreover
its
ethyl
derivatives
[2-(2-
carboxyethyl)phenyl]boronic acid (4) was synthesized ad hoc to
introduce flexibility on the lateral chain of 2 with the aim to
evaluate its contribution in overall binding and optimal
rearrangement. For both derivatives the corresponding
analogues, carrying the lateral carboxylic chain in meta (3 and 5)
were selected as non-proficuous probes and evaluated for their
affinity to KPC-2. Compounds 3 and 5, carrying a non-optimally
oriented lateral chain, could support the validation of 2 and 4
while investigating enzyme plasticity.
–
1
ligand efficiency of 0.62 kcal mol , its ability to synergize in
clinical isolates last resort carbapenem meropenem and its no-
[
25]
cytotoxicity, was validated as lead-like molecule.
Table 1. Phenyl boronic acid 1 and its optimized derivatives: improving
binding affinity vs KPC-2.
[
a]
Code
Structure
IC50 (µM)
i
K (µM)
[
b]
1
31.9
7.64
[
c]
2
10.1
2.43
Figure 1. Clavulanic acid, a β-lactam base inhibitor (a); Avibactam, a KPC-2
de novo inhibitor (b); phenyl boronic acid (c)
[
b]
3
4
130.1
31.17
Results and Discussion
[c]
4
.72
1.13
Design and synthesis
5
[b]
2
21.3
53.02
Starting from 1 (Table 1), a micromolar inhibitor of KPC-2 (K
i
7
.64 µM) with detectable synergic activity towards organisms
[
a]
Determined vs KPC-2 (3.1 nM), Phosphate buffer 50 mM, pH 7.0,
overexpressing KPC-2, we were interested in obtaining
a
[
b]
reporter substrate nitrocefin.
Estimated from IC50 as per competitive
validated lead with improved affinity to KPC-2 carbapenemase
and most importantly able to significantly restore susceptibility to
carbapenems in pathogen overexpressing KPC-2 (Table 1).
Taking into account specific binding requirements in KPC-2, to
localize the most appropriate point of derivatization in 1 and to
rationally select proficuous chemical groups suitable for
derivatization, we referred to available X-ray structures of KPC-2,
binding respectively the 3-nitro-phenyl boronic, a close analogue
inhibitor from Cheng-Prusoff equation. Reporter substrate Nitrocefin at
[
c]
1
15 µM.
Estimated from IC50 as per competitive inhibitor from Cheng-
Prusoff equation. Reporter substrate Nitrocefin at 300 µM. All the
experiments were performed in triplicate. Experimental error never
exceeds 5%.
[
15,26]
BL Inhibition Studies and Susceptibility Assays.
of compound 1, and the antibiotic avibactam (Figure 1b).
In order to investigate the inhibitory activity of the phenyl boronic
acid derivatives, KPC-2 full-length and KPC-2 C-terminus
In a possible derivatization of phenyl boronic acid 1, we
reasoned that the introduction of a carboxylic group mimicking
the C3(4)′ carboxylate of β-lactam antibiotics, a key recognition
feature in BLs, might improve KCP-2 binding via a large
interaction network with Arg-220, Ser-130, Thr-237 and Thr-235.
All mentioned residues are located in the β-lactam carboxylate-
binding pocket where the sulfate group of avibactam, as well as
the carboxylic group of other known class A inhibitors, are
deletion were overexpressed and purified in
a
single
chromatographic step, yielding more than 150 mg of protein per
liter of culture, with a purity degree higher than 99%. In our
M
experimental conditions, the K for nitrocefin measured for KPC-
[
26,28,29]
2
full-length has been determined as 36.0 µM.
i
values for derivatives 1-5 were obtained from IC50 as per
K
[
17,27]
competitive inhibition (Cheng-Prusoff equation), in agreement
with the inhibition patterns in this series of compounds and with
known to bind.
2
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experiments investigating the effect of increasing substrate
concentrations (Table 1).
respect to 7.64 µM of 1, 18 and 55 fold improvement
respectively) thus corresponding to an increase of at least 2.65
Kcal/mol, coherent with the designed additional H-bond
interaction (ΔΔG=RTln(Ki compound 2/Ki compound 1)).
Binding reversibility was determined, for all derivatives, by
measuring the recovery of the enzymatic activity after a rapid
and large dilution of the enzyme-inhibitor complex. While 1, 3
and 5 behave as rapid fully-reversible inhibitors, 2 and 4 exhibit
slow reversibility, as observed from the peculiar shape of the
[
30]
time-course curve (Figure 2).
Figure 2. Recovery of KPC-2 activity after rapid dilution of the enzyme-
inhibitor complex. The curve in gray refers to the control sample pre-incubated
and diluted in the absence of inhibitor. Curves in magenta, orange and blue
refer to 1, 3 and 5 respectively at 40 µM, 150 µM and 250 µM. These
compounds behave as rapidly reversible inhibitors. The curve in green
describes the time-course of the slowly reversible compound 4 at 12 µM.
Specifically the slow reversibility is visible in the first 2 minutes where the
shape of the curve is indicative of a measurable recovery of enzymatic activity.
The curve in purple is the time-course of compound 2 at 6 µM, acting as a very
slowly reversible inhibitor.
Figure 3. Kinetics characterization of (A) 2 and (B) 4. Plots of kobs as a
function of the slow binding inhibitor conforming to the two-step mechanism
(equation A.2, see methods). Experimental data fitted to equation A.3 shows a
hyperbolic function of inhibitor concentration [I]. The intercept of the curve on
the y-axis can be used to estimate the k-2 value, while the horizontal
asymptote at infinite inhibitor concentration (maximum value of kobs
)
represents the sum of k+2 and k-2 values. Concentration-response plots for the
1
*app
final inhibited states of (C) 2 and (D) 4 . K
i
can be calculated as the half-
maximal concentration of the isotherm of the fractional velocity vss/v (where v
0
0
For 2 and 4, behaving as slowly reversible inhibitors, the
apparent and true constants of inhibition were therefore
determined, as well as the rate constants of the isomerization
is the initial rate of the uninhibited reaction) as a function of the decimal
logarithm of the inhibitor concentrations. All the experiments were performed
in triplicate. Experimental error never exceeds 5%.
[
31,32]
steps (k+2 and k-2) (Figure 3A-D, Table 2).
As usual for this mechanism of inhibition, the k+2 rate constant
resulted higher than the k-2 rate constant for both analyzed
Table 2. Compounds 2 and 4 time dependent, slow reversible kinetics
parameters.
compounds.
*
The high affinity inhibition constants K
i
for 2 and 4 are
*app
*
app
-
1
-1
-1
K
i
K
i
K
i
K
i
(µM)
#
k
max (s
)
k
+2 (s
)
k-2 (s )
respectively 6-fold and 8-fold lower than those calculated for the
initial encounter complex (Table 2).
(µM)
(µM)
(µM)
-2
-2
-3
2
2.92×10
2.89×10
2.40×10
2.53×10
5.22×10
3.59×10
2.86
0.43
14.57
2.43
Both compounds behave as competitive inhibitors as
demonstrated by the hyperbolic decrease of the kobs value at
increasing reporter substrate concentrations (Figure 4A and 4B).
Affinity data for designed ortho derivatives 2 and 4 show, as
-
2
-2
-3
4
3.2
0.14
6.79
1.13
expected,
a
potency improvement vs KPC-2 (3-7 fold,
considering the initial encounter complex) with respect to 1,
while the derivatization in meta in 3 and 5 was detrimental (4
and 7-fold loss in activity respectively). The meta substitution in
fact does not allow an optimal orientation of the lateral
carboxylated chain and reasonably affect negatively phenyl
boronic overall binding orientation. They served as non-
proficuous ligands highlighting the importance of correctly
predicting and orienting new binding interactions without
perturbing preexistent binding geometry (Table 1).
When comparing binding affinity for compounds 2 and 4, acting
as slowly reversible inhibitors, however, the true constants of
M
Figure 4. Effect of reporter substrate concentration (relative to K ) on kobs for 2
at 12 µM (A) and 4 at 6 µM (B). For both compounds the values of kobs diminish
hyperbolically with increasing substrate concentration as per competitive
model of inhibition.
i
inhibition K* must be considered. As a consequence the
improvement of potency for 2 and 4 largely exceeded one order
of magnitude with respect to 1 (0.43 µM and 0.14 µM for 2 and 4
3
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Compounds 2 and 4 confirmed our hypothesis about the
importance of the carboxylate moiety in modulating affinity and
potency to KPC-2 specifically with respect to other related
carbapenemases (unpublished). Their ability to interact with the
carboxylate-binding pocket defined by Arg-220, Ser-130, Thr-
fractional inhibitory concentration index (FICI) were determined
against E.coli-blaKPC-2 (Table 3) and six clinical isolates of
Klebsiella pneumoniae expressing (4 isolates) or not (2 isolates)
KPC-2 (Table 4). Noteworthy, none of the tested compounds
had intrinsic antibiotic activity (MIC >256 µg/mL) against
[
17, 22]
2
37 and Thr-235 renders them stronger inhibitors compared to
employed strains, including the susceptible ones.
lead 1.
Obtained results confirm synergistic activity and quantify the
synergistic effect when present, thus further validating our
optimized leads (Table 3, Table 4).
To investigate the ability of designed derivatives 2 and 4 to cross
the outer membrane reaching the periplasmic space, where
KPC-2 is secreted and confined in Gram negative bacteria, thus
synergically preventing β-lactam antibiotics hydrolysis by KPC-2,
double disc interaction assays were carried out on E.coli
overexpressing blaKPC-2 gene. An E.coli BL21(DE3) strain, not
harboring blaKPC-2 gene, therefore susceptible, was used as
control (Figure 5). The synergic effect of 2 and 4 was tested in
combination with the antibiotics amoxicillin (AMX), its
combination with clavulanic acid (AMC), third generation
cephalosporin cefotaxime (CTX), monobactam, aztreonam
Results against E.coli-blaKPC-2 show, first of all, that our phenyl
boronic acids act with a synergic mechanism, with only two
exceptions, represented by 1- and 4-CTX combination having a
FIC index slight above 0.5 (indifference) (Table 3).
As far as potency is concerned, 2 and 4 always exert a stronger
synergistic activity compared to starting compound 1.
While for 1 the MIC improvements, for compound concentration
ranging from 128 to 16 µg/mL, resulted between 2 and 32 fold in
the best case (i.e. in combination with MEM, 32 fold
improvement at 16 µg/mL), for 2 and 4 MIC reduction was more
dramatic and at concentrations ranging from 32 down to 0.25
µg/mL for 2 and from 4 to 0.5 µg/mL for 4 (Table 3).
(
(
ATM) and the carbapenems imipenem (IMP) and meropenem
MEM) (see supplemental material).
Compound
2 reduces MICs by a factor of 32 when in
combination with AMC and AMX at a concentration as low as
0
3
.25 µg/mL. In combination with ATM the reduction is >16 fold at
2 µg/mL and, very promising, by a factor of 16 when in
AMX
2
AMC
2
CTX
2
ATM
30 µg
2
IPM
2
MEM
2
association with last resort carbapenem MEM at just 0.25 µg/mL.
Moreover compound 2 resulted synergic also in combination
with CTX, while 1 and 4 did not.
2
5 µg
400 µg 30 µg
400 µg 30 µg
400 µg
400 µg 10 µg
400 µg 10 µg
400 µg
Results for 4 were very promising as well. Compound 4 reduces
MICs by 64 fold when in combination with AMC and AMX at 2
and 4 µg/mL concentrations respectively. Moreover it reduces
MIC by a factor of 32 when in combination with MEM at 0.5
µg/mL concentration, showing comparable potency as 2 when in
association with last resort carbapenem (Table 3).
AMX
4
AMC
4
CTX
4
ATM
4
IPM
10 µg
4
MEM
4
2
5 µg
400 µg 30 µg
400 µg 30 µg
400 µg 30 µg
400 µg
400 µg 10 µg
400 µg
Figure 5. Disk Diffusion Susceptibility Tests for compounds 2 and 4 in
combination with AMX, AMC, CTX, ATM, IMP and MEM. Synergic activity can
be ascertained when the inhibition zones around any of the antibiotic discs are
augmented in the direction of the disc containing the compounds of interest.
Steaming from these encouraging results compounds 1, 2 and 4
were further tested for their synergistic activity against a set of
KPC-2 overproducing clinical isolates. MEM was chosen as the
antibiotic considering the synergistic effects exerted by all
compounds when in combination with it (Table 3).
The E.coli overexpressing blaKPC-2 gene was not susceptible to
AMX and AMC while a certain antibiotic effects was observed
with CTX, MEM, AZT and IMP (see supplemental information).
However, in combination with the above antibiotics, 2 and 4
synergized with all tested antibiotics in the E.coli strain
producing KPC-2 while 1 did not exert any synergistic effect
when tested in association with antibiotics AMX and AMC, at
least at the concentration employed in the disk diffusion tests
The six K. pneumoniae clinical strains used in this study were
isolated from different patients at the Hospital Universitario Son
Espases, Palma de Mallorca, Spain. Two out of six clinical
strains were not KPC-2 producers and were susceptible to
meropenem (strains 1-2; MIC <0.25 ug/mL). In turn four strains
harbored blaKPC-2 gene and were resistant to MEM (strains 3-6)
(
Table 4).
(
Figure 5; for additional information see supplemental material).
In their combination with AMX, AMC, CTX, ATM, IMP and MEM,
and 4 potentiate antimicrobial activity as demonstrated by the
All compounds showed clear synergisms with MEM against the
four resistant strains, except compound 1 against strain 6. The
obtained results were very encouraging: while all compounds
were capable of reducing MIC when in association with MEM,
including starting compound 1 (MIC reduction between 16 to 64
2
increasing in inhibition zones between antibiotics and
compounds. The addressed synergistic effect confirms the
ability for 2 and 4 to reach the periplasmic space of Gram (-)
bacteria, preventing β-lactam antibiotics degradation by KPC-2.
The synergic effect of 2 and 4 with AMX and AMC is much more
evident than with other antibiotics. As a matter of fact, KPCs
hydrolyze carbapenems, cefotaxime and aztreonam less
fold at
concentration as low as 1 µg/mL reducing MIC in most cases by
024 fold, thus restoring bacterial susceptibility to meropenem
MEM) (Table 4).
4 µg/mL), compounds 2 and 4 were active at
1
(
efficiently
than
penicillins
and
narrow-spectrum
[
12]
cephalosporins.
For 1, 2 and 4 MICs by a two-dimensional checkerboard
microdilution assay and drug interactions model via the
4
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Table 3. In vitro interaction between antibiotics and boronic compounds determined by FICI vs E.coli-blaKPC-2
.
Checkerboard microdilution assays and Drug interaction model
[
a]
[a]
MIC
antibiotic
µg/mL)
MIC
Inhibitor
concentration
(µg/mL)
[b]
FIC Index
Compound
Antibiotic
combination
ΣFICmin
INT
(
(µg/mL)
1
6/8
/4
32
64
<0.1875
<0.1875
SYN
SYN
AMC
AMX
ATM
128/64
>16384
>64
8
512
32
<0.0938
SYN
1
32
64
<0.3750
<0.3750
SYN
SYN
1
6
128
CTX
MEM
AMC
AMX
ATM
CTX
MEM
AMC
AMX
ATM
CTX
MEM
512
2
256
0.0625
2/1
16
16
<0.5313
<0.0626
<0.0318
<0.1255
<0.0938
<0.5
IND
SYN
SYN
SYN
SYN
SYN
SYN
SYN
SYN
SYN
IND
64/32
8192
>64
256
1
0.25
0.25
32
256
2
4
64
128
0.25
4
0.0625
2/1
<0.0630
<0.0234
<0.0195
<0.0166
<0.5010
<0.0323
128/64
16384
>64
512
2
256
2
4
64
0.5
0.5
0.5
256
0.0625
SYN
[
a]
[b]
MIC values were determined as the median of three independent experiments.
INT, interpretation; IND, indifference; SYN, synergy; ANT, antagonism.
Synergy is defined when the FICI is ≤0.5, antagonism when the FICI is >4, and indifference when the FICI is >0.5 and ≤4.
[
28]
Cell viability Assays
already reported, a minor truncation of the C-terminus of KPC-
2 was implemented, resulting in improved crystal growth and
diffraction quality.
Cell viability against mouse embryonic fibroblasts (MEFs) and
human CCD-Lu34 (CCD) cells was determined for compound 1
(
data not shown) and for most active compounds 2 and 4 via a
The structure of the EcKpc-2:2 complex was determined to a
resolution of 1.4 Å. In addition to 2, 154 water molecules and 3
ethylene glycol molecules were included and the final model
refined to an R (work) value of 17.7% and an R (free) value of
18.5%. Analogously, high resolution structure of EcKpc-2:4
complex was obtained by soaking techniques (1.5 Å). The final
model was refined to 15.9% R(work) value and 18.3% R(free)
including 4 and 384 water molecules. In both cases, the electron
density clearly shows the presence of the inhibitor trapped into
the active site, covalently bound to catalytic Ser-70.
[
33]
tetrazolium salt (MTT) assay reduction assay (Figure 6).
After a 24-h exposure to compound 2 no general cytotoxicity
was detected at 50 µM (116 fold the K) against both tested
i
cellular lines: 86% and 88% of cell viability against MEFs and
human CCD-Lu34 cells respectively. For compound 4 after a 24-
h exposure no general cytotoxicity was detected at 25 µM (176
i
fold the K): 93% and 97% of cell viability against MEFs and
human CCD-Lu34 cells respectively. For compound 2 cell
survival gets down to 37% for MEFs and to 54% for CDD only
when compound reached a concentration 700 fold the Ki; while
First of all, 2 and 4 form a covalent bond, via its boron atom,
with the Oγatom of Ser-70. Both boronate oxygens interact
extensively: boronic acid O2 oxygen establishes multiple
hydrogen bonds with the catalytic base Glu-166, backbone NH
of catalytic Ser-70 and Asn-170 sidechain, while boronic acid O1
oxygen hydrogen binds backbone CO and NH of Thr-237 and
backbone NH of catalytic Ser-70 as well. Overall, derivative 2
as well as derivative 4 adopts a deacylation transition-state
analogue conformation with inverted boron configuration. In
such a conformation, previous described for other class A BLs,
the positions of the boronic acid oxygens are the following: one
is located in the oxyanion hole and the other one displaces the
i
for compound 4 at 150 µM (over 1000 fold the K) cell survival
was still stable at 75 % respect to non-exposed cells (Figure 6).
In conclusion cell viability assays did not show a significant
decrease in viability for cell lines grown in the presence of either
compound 2 or 4 while viability was strongly affected by DMSO
used as positive control.
Crystal structures of KPC-2_compound
_compound 4 complexes.
Compounds 2 ([2-(2-carboxyvinyl)phenyl]boronic acid) and 4 ([2-
2-carboxyethyl)phenyl]boronic acid) binding orientation in KPC-
were experimentally determined via X-ray crystallography. As
2
and KPC-
2
(
2
5
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Table 4. In vitro interaction between antibiotics and boronic compounds determined by FICI vs K. pneumoniae clinical strains.
Checkerboard microdilution assays and Drug interaction model vs Klebsiella pneumoniae clinical strains
[
a]
[a]
[a]
MIC
MIC
MIC
Inhibitor
concentration
(µg/mL)
[
b]
Clinical
Strains
FIC Index
INT
Compound
MEM
compound
combination
ΣFICmin
(
µg/mL)
(µg/mL)
(µg/mL)
Strain 3
Strain 4
Strain 5
Strain 6
256
256
≥256
≥256
≥256
≥256
4
16
4
4
SYN
SYN
SYN
IND
<
0,023
<0,0937
<0,0234
1
256
4
4
≥256
≥256
256
<
1,5
Strain 3
Strain 4
Strain 5
Strain 6
Strain 3
Strain 4
Strain 5
Strain 6
256
256
≥256
≥256
2
1
1
1
8
1
1
1
8
<0,0117
<0,0015
<0,0015
<0,0156
<0,0195
<0,0015
<0,0015
<0,0234
SYN
SYN
SYN
SYN
SYN
SYN
SYN
SYN
0.25
0.25
4
2
256
≥256
≥256
≥256
≥256
≥256
≥256
≥256
256
4/2
256
0.25
0.25
8/4
4
256
≥256
[
a]
[b]
MIC values were determined as the median of two independent experiments, INT, interpretation; IND, indifference; SYN, synergy; ANT, antagonism.
Synergy is defined when the FICI is ≤0.5, antagonism when the FICI is >4, and indifference when the FICI is >0.5 and ≤4.
A
deacylation water normally positioned between Glu-166 and
1
1
50
00
[
27,34]
MEF
CCD
Asn-170.
Compound 2 orients its phenyl ring against one hydrophobic
region defined by Trp-105 and Leu-167 in the proximity of the
opening of the binding site. The phenyl ring of 2 is involved in
cation-π and π-π interactions with Asn-132 and Trp-105,
5
0
0
respectively. Finally, as expected,
2
orients its ortho
l
o
carboxyvinyl lateral chain towards the C3(4′) carboxylate binding
pocket, highly conserved in all serine-BLs. In this site, the
carboxylate group is trapped in a network of hydrogen bonds
tr
n
10µM
50µM
MSO
D
o
100µM
300µM
C
ꢀ
B
1
50
00
50
0
MEF
CCD
with Thr-237, Thr-235, Ser-130 sidechains and
a highly
conserved water molecule (Wat 99#, Figures 7A-B and Figure 8).
Despite the higher degree of freedom of the carboxyethyl ortho
substituent (4), such inhibitor assume a conformation closely
overlapping that described above for 2: the 2-carboxyethyl
lateral chain occupies the targeted binding pocket of β-lactam
antibiotics C3(4)′ carboxylate with similar orientation. The highly
conserved water molecule (Wat 105#) is also present in the site
contributing to the carboxylate chain binding geometry
1
l
o
ntr
5µM
MSO
D
o
25µM
50µM
150µM
C
ꢀ
Figure 6. Cell proliferation graphics determined by MTT assay after 24 hours
incubation with compounds 2 (A) and 4 (B).
[
35]
optimization (Figures 9A, 9B and Figure 10 ).
The overall EcKpc-2 protein structure well superposes to the C-
term truncated KPC-2 structure in complex with citric acid and to
[
28]
the KPC-2 apoprotein (PDB id 3C5A and 3DW0) with r.m.s.d.
and points towards 2 in the active site of a symmetry-related
protein molecule. Indeed, Arg-256 orients its cationic sidechain
toward such an adjacent active site and establishes interactions
with the 2 phenyl ring (cation-π interaction) and the main chain
carbonyl of Thr-237 (H-bond). Analogous considerations can be
deduced for the EcKpc2-4 complex.
of 0.45 and 0.21 Å over 264 aligned Cα atoms, respectively
[
36]
(
software Gesamt).
The only significant shift (r.m.s.d. higher
than 2 Å) pertains a short loop, from Thr-254 to Arg-256, and it
is due to interactions promoted by the crystal packing. Indeed,
Arg-256 side chain is clearly rotated if compared to apoprotein
6
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Figure 7. Crystal structure of KPC-2 in complex with 2. (A) omit map (phenix.composite_omit_map) is shown (8σ contour level). (B) ligand binding mode and the
main residues involved in the ligand-protein interactions are shown as sticks (carbon atoms in green for 2 and in gray for EcKpc-2, oxygen atoms in red, nitrogen
in blue, boron atom in pink, water oxygen in cyan). Pictures were prepared using Pymol (The PyMOL Molecular Graphics System, version 1.5.0.4; Schrödinger
LLC: New York, 2010; http://www.pymol.org).
represents the inhibition constant of the high affinity complex.
*
The determined values of K
i
for 2 and its close analogue 4 are
*
very similar (K
i
0.43 and 0.14 µM respectively) as well as their
[
31,32]
binding orientations in KPC-2 (Figures 7B and 9B).
The meta isomers 3 and 5 behaved differently. As confirmed by
the kinetics of inhibition, they act as fully reversible inhibitors,
characterised by the only inhibition constant of the Michaelis
complex EI. It is clear that they do not meet the requirements for
the formation of a measurable and stable transition-state, since
their mechanism of inhibition conforms to a simple reversible
[
31]
scheme (E+I=EI).
Figure 8. Key Interactions KPC-2 and the ligand 2. Distances between 2.5
and 3.6 Å have been selected for non-covalent bonds and listed. Atoms
identification codes are defined in the Ligand binding site plot (Ligplus
Conclusions
Starting from a known phenyl boronic acid with low synergic
activity towards organisms overexpressing KPC-2
carbapenemase and through multidisciplinary approach
[
35]
software).
a
As previously mentioned, the presence of a covalent bond
between the boronic group of 2 (and 4) and the catalytic serine
of KPC-2 is clearly visible in the electron density.
including design, synthesis, kinetic and X-ray crystallography we
identified and validated two nanomolar KPC-2 inhibitors.
Their kinetic description as derivatives acting as time dependent,
slow reversible inhibitor of KPC-2 has been deeply investigated.
In the derivatization of the phenyl boronic acid, the introduction
The crystallographic data above described are consistent with
the kinetics of inhibition where the enzyme isomerization has
been experimentally determined by the direct observation of the
in ortho position of
a carboxylated lateral chain strongly
distinctive saturable function of
concentration.
kobs versus the inhibitor
improved affinity to KCP-2 (2 and 4) while for the meta
derivatives, (3,5) the lateral chain was detrimental for activity
underlying the importance of an optimal rearrangement of the
carboxylate moiety. Undoubtedly, in derivatives 2 and 4, the
improved affinity to KPC-2 is completely attributable to the
additional hydrogen bond in the β-lactam carboxylate binding
The boronic derivatives 2 and 4 lead to the formation of a
relatively stable transition-state analogue passing through an
initial encounter complex.
The slow-reversible nature of the inhibition for 2 and 4 is
therefore, the consequence of the non-zero value of the rate
constant k-2, while the time-dependence is due to the low value
of k+2 rate constant, which is in turn, the consequence of the
isomerization step. All these features contribute, de facto, to the
[
17,37,38]
pocket as confirmed by X-ray crystallography.
Moreover, respect to the starting derivative 1, in the designed
ortho derivatives 2 and 4 we have clearly potentiate the in vitro
antimicrobial activity of a number of β -lactam antibiotics,
including the last resort meropenem. Noteworthy, in clinical
isolates, derivatives 2 and 4 at 1 µg/mL in combination with
meropenem were able to reduce MIC by 1024 fold down to 0.25
formation of two inhibitor-enzyme complexes characterised by
*
two different inhibition constants, K
i
and K
i
. The last one
7
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Figure 9. Crystal structure of KPC-2 in complex with 4. (A) omit map (phenix.composite_omit_map) is shown (8σ contour level). (B) ligand binding mode and the
main residues involved in the ligand-protein interactions are shown as sticks (carbon atoms in yellow for the 4 and in pale orange for the protein, oxygen atoms in
red, nitrogen in blue, boron atom in pink, water oxygen in cyan). Pictures were prepared using Pymol (The PyMOL Molecular Graphics System, version 1.5.0.4;
Schrödinger LLC: New York, 2010; http://www.pymol.org).
µg/mL, restoring completely susceptibility. In turn no cytotoxicity Experimental Section
was detected in cell viability assays.
Our small boronic acid derivatives have been clear validated as
lead-like KPC-2 inhibitors and undoubtedly the high-resolution
structures of KPC-2 in complex with 2 and 4 represent a starting
point for future drug design and optimization efforts.
Chemistry: Reagents were purchased from Sigma-Aldrich and were of
reagent grade. Phenyl boronic acid (1) was purchased from Sigma-
Aldrich while [2-(2-carboxyvinyl)phenyl]boronic acid (2) and [3-(2-
carboxyvinyl)phenyl]boronic acid (3) were purchased from Alfa Aesar
(
Table 1). [2-(2-carboxyethyl)phenyl]boronic acid (4) and [3-(2-
carboxyethyl)phenyl]boronic acid (5) were synthesized starting from [2-
2-carboxyvinyl)phenyl]boronic acid (2) and [3-(2-
carboxyvinyl)phenyl]boronic acid (3) respectively through catalytic
hydrogenation under H (21 psi) in EtOH using Pd-C 10% as catalyst.
In the here-exploited lead identification and validation we chose
to focus on carbapenemases inhibitors, taking advantage of
critical consensus binding sites shared by other therapeutically
important groups of carbapenemases and BLs: by targeting
common hot spots our inhibitors could be further developed as
broad-spectrum inhibitors. Further studies are under way with
the aim to optimize affinity and spectrum of action vs other
serine based carbapenemases.
(
2
Catalytic hydrogenation was conducted in a Parr stirred reactor at room
temperature for 5 hours. Reactions progress was monitored by TLC on
precoated silica gel 60 F254 plates (Merck). The catalyst was removed
by filtration and the solvent removed under vacuum and residue washed
with diethyl ether, producing 4 and 5 as white crystalline solid (Table 1).
All compounds, both commercially available and in house synthesized
(
Table 1) were characterized through monodimensional NMR, HR-ESI
monoisotopic MS analysis and melting point. NMR spectra were
1
13
recorded on a Bruker 400 spectrometer with H at 400.134 MHz, C at
1
1
1
00.62 MHz and B at 128 MHz. Proton chemical shift was referenced to
the TMS internal standard. Chemical shifts are reported in parts per
million (ppm, δ units). Mass spectra were obtained on a 6520 Accurate-
Mass Q-TOF LC/MS and 6310A Ion TrapLC-MS(n). The melting points
were recorded on a Stuart, SMP3 (Barloworld Scientific Limited Stone,
Staffordshire, UK) and were uncorrected. The structures of all
compounds were consistent with their analytical and spectroscopic data
(
see supplemental information, Table S1, NMR spectra).
Cloning of blaKPC-2: The blaKPC-2 gene was kindly provided by Prof.
Sergei Vakulenko (University of Notre Dame du Lac, Indiana, USA). The
gene was amplified by PCR using the reported oligonucleotides (see
supplemental information, Table S2). The gene encoding the full-length
protein has been used for kinetic and antimicrobial susceptibility assays,
Figure 10. Key Interactions KPC-2 and the ligand 4. Distances between 2.5
and 3.6 Å have been selected for non covalent bonds and listed. Atom
identification codes are defined in the Ligand binding site plot (Ligplus
[
35]
software).
[
28]
while the C-terminus deleted form
has been used for crystallographic
studies. KPC-2_for (BamHI restriction site) and KPC-2_rev (HindII
restriction site) were used to clone the full-length gene into the pET-24a
vector (Novagen). The truncated form of KPC-2 was cloned as the full-
length using KPC-2_rev_del containing HindIII restriction site and a stop
[
28]
codon, 12 nucleotides upstream the natural end of blaKPC-2 gene.
The
PCR products were double digested by BamHI and HindIII (Biolabs, New
8
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ChemMedChem
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England), ligated and subsequently transformed into competent
Escherichia coli HB101. Transformed colonies were selected on Luria
Bertani (LB) agar plate (50 mg/mL kanamycin). Clones were confirmed
by colony PCR and DNA sequencing using T7_for and T7_term
oligonucleotides.
the inhibitory mechanism involving enzyme isomerization step. In the
latter case the scheme of reaction is:
ꢀ
!!
ꢀ
!!
∗
ꢀ
+ ꢀ ⇌ ꢀꢁ ⇌ ꢀꢁ
ꢀ!!
(A.2)
ꢀ!!
Expression and purification of recombinant KPC-2: Recombinant
plasmids were transformed into competent E.coli BL21 (DE3) cells for
protein expression. 50 mL of Tryptic Soy Broth (TSB) (50 mg/L
kanamycin) were inoculated with fresh colonies and grown at 37°C. 4 mL
of the overnight culture were used to inoculate 1.3 L of TSB (50 mg/L
kanamycin) grown at 37°C with shaking 0.5 optical density at 600 nm.
Expression of recombinant bla gene was induced by adding 1.0 mM IPTG
The scheme describes a two-step binding mechanism where a first step
is characterized by a rapid equilibrium binding of the inhibitor to the
enzyme to form the encounter complex (EI) and
a second step
characterized by a slow isomerization of the enzyme to form the high
affinity complex (EI*).
The value of kobs for this mechanism is a saturable function of inhibitor
concentration (Figures 3A and 3B) and can be describe by the following
equation:
(
isopropyl-D-thiogalactopyranoside) and the cells were again allowed to
grow at 20 °C overnight. Bacteria were harvested by centrifugation at
000 rpm for 20 minutes. The pelleted cells were resuspended in Tris-
HCl 50 mM pH 7.4-7.5. Periplasmatic proteins were extracted as reported
in pET System Manual (TB055 10th Edition Rev. 0403) and
4
!
!""
!![!]
ꢀ
!"# = ꢀ!!
+
(A.3)
!!
![!]
B
subsequently dialyzed in sodium acetate buffer (50 mM, pH 5.0). Proteins
were conveniently purified in a single step using a Macro-Prep High S
resin and eluted using sodium acetate 50 mM pH 5.0 and a sodium
chloride (NaCl) linear gradient from 100 to 500 mM. Purified proteins
k
-2 can be estimated as the intercept of the hyperbolic curve when the
concentration of the inhibitor is equal to zero. The horizontal asymptote
of the curve represents the maximum value of kobs (kmax) which is:
[
29]
were dialyzed overnight in sodium phosphate buffer 50 mM, pH 7.0.
ꢀ!"#
= ꢀ!! + ꢀ!!
(A.4)
Steady state enzyme kinetic measurements and determination of
inhibition constants: Kinetic parameters of full-length KPC-2 enzyme
against the reporter substrate nitrocefin were determined by monitoring
the variation in the absorbance at 482 nm in 50 mM phosphate buffer
For the enzyme isomerization mechanism, as described in equation A.2,
app
two apparent constants of inhibition can be defined: K
i
for the initial
*
app
inhibitor encounter complex (EI), and K
i
for the final high-affinity
conformation (EI*).
(
PB) (pH 7.0) at 25°C. All measurements were carried on Lambda 2
app
The value of K
i
is calculated as the concentration of the inhibitor
spectrophotometer (Perkin Elmer). The steady-state kinetic parameter K
M
yielding the half value of kmax.
was determined by non-linear least-square fit of the Michaelis – Menten
equation using OriginPro 8.5.1.
When the inhibitor behaves as competitive, the values of
calculated by Cheng-Prusoff equations:
i
K are
Determination of IC50 and inhibitor reversibility: The half maximal
inhibitory concentration (IC50) of boronic acid derivatives was determined
as follows. Boronic acids were dissolved in dimethyl sulfoxide (DMSO) to
a concentration of 10 mM and stored at -20°C. Each compound was
tested at concentrations ranging from 1 µM to 500 µM for inhibitory
activity vs full-length KPC-2 enzyme (3.1 nM) in 50 mM of PB at pH 7.0 at
!
""
!
ꢀ!
= ꢀ! 1 + !
(A.5)
!
*
app
K
i
can be obtained as the half-maximal concentration of the plot vss/v
0
(where v is the initial rate of the uninhibited reaction) as a function of the
0
*
decimal logarithm of the inhibitor concentrations (Figures 3C and 3D). K
i
2
5°C in the presence of nitrocefin as the reported substrate. Nitrocefin
can be obtained by the following equation:
hydrolysis was followed at 482 nm. All the experiments were performed
in triplicate. Experimental error never exceeds 5% (Table 1). The
reversibility of inhibition for each derivative was determined as well. KPC-
!!
∗
ꢀ!
= !! !!!
ꢀꢀ
(A.6)
!!!
2
was incubated at 25°C for 10 minutes at a concentration of 100-fold
over the concentration required for the activity assay, with
concentration of inhibitor about 5-fold the estimated from the
All the experiments were performed at 25°C in PB 50 mM, pH 7.4 using
nitrocefin at 180 µM (5-fold the K value).
a
M
K
i
The mechanism of inhibition for the time-dependent inhibitors can be
determined by measuring the effects of reporter substrate concentration
on kobs value at fixed inhibitor concentration. For a competitive inhibitor
the value of kobs will decrease hyperbolically with increasing substrate
previously determined IC50 values. After the pre-incubation time, the
recovery of the enzymatic activity was determined after rapid and large
dilution, (100-fold) of the enzyme-inhibitor complex, with the reaction
buffer (PB 50 mM, pH 7.0) containing the reporter substrate nitrocefin at
[
31,32]
[
30]
concentration (Figure 4A and 4B).
1
80 µM. The time-course of nitrocefin hydrolysis was followed at 482
The experiment was conducted in PB 50 mM, pH 7.4 at 25°C using
nitrocefin as the reporter substrate at concentrations ranging from 10 to
nm (Figure 2).
3
4
K
M
50 µM (about 0.3-fold to 10-fold the [S]/K ratio) and compounds 2 and
Time dependent inhibitors: For compounds behaving as reversible
time-dependent inhibitors (2 and 4) the time course curve, for reactions
monitored at fixed concentrations of enzyme, reporter substrate and slow
binding inhibitor, is described by the following equation:
at fixed concentration of 12 µM and 6 µM, respectively (about 5-fold the
i
values).
All the experiments were performed in triplicate. Experimental error never
exceeds 5%. All the kinetic parameters and the non-linear fitting were
calculated using the software OriginPro 8.5.1.
!
!!!!!
ꢀ
= ꢀ ꢀ +
(1 − ꢀ!!!"#!
!"#
)
(A.1)
!
!
!
Double Disk diffusion assay: The antimicrobial susceptibility of E.coli
BL21
(DE3)
pET-24a-blaKPC-2
versus
amoxicillin
(AMX),
where vss is the steady-state velocity, v
i
the initial velocity and kobs the
amoxicillin/clavulanic acid (AMC), cefotaxime (CTX), aztreonam (AMT),
and meropenem (MEM) (Oxoid) in combination with 1, 2 and 4 were
investigated by double disc diffusion assay (Figure 5). 400 µg of each
boronic acid derivative were dispensed individually on sterile blank filter
paper disc (Oxoid). The maximum amount of DMSO (dimethyl sulfoxide)
used to dissolve the phenyl boronic acid derivatives (26.86 µg) was also
tested for control (see supplemental information). Discs were placed on
rate constant for conversion from the initial to the steady-state velocity.
Fitting of a progress curve to equation A.1, allows the estimation of v
i
, vss
and kobs
.
The plot showing the dependence of kobs on inhibitor concentration is
useful to distinguish between simple reversible slow binding inhibitor and
9
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ChemMedChem
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FULL PAPER
5
MH Agar plates inoculated with 5×10 CFU/mL of bacterial suspension at
a distance equal to the half measured inhibition diameter for each drug
alone. E.coli (DE3) pET-24a, without blaKPC-2 gene, was used as control.
The plates were incubated 18 hours at 37°C and the inhibition diameters,
in millimeters, were measured. Additional data and figure are available as
supplemental information.
Crystallization, Data Collection and Structures determination of
EcKPC-2 in complex with 2 and 4: For crystallization purposes, KPC-2
was further purified using
a Superose 12 10/300GL column (GE
Helthcare) equilibrated in 40 mM Bis-Tris pH 5.9 and subsequently
concentrated by ultrafiltration to 20 mg/mL in the same buffer. EcKPC-2:2
and EcKPC-2:4 complex crystals were obtained by soaking techniques.
The apo EcKPC-2 crystals were grown in 15% polyethylene glycol 6000
(
PEG 6000) in 200 mM potassium thiocyanate (KSCN) and 100 mM
Checkerboard microdilution assay: The in vitro interactions between
sodium citrate (pH 4) at 20°C using the vapor diffusion hanging drop
method. Crystals appeared after one week. KPC-2 crystals were soaked
for 2-3 hours at 20°C with 2 or 4 (3 mM) dissolved into reservoir solution.
The compound solution was prepared by mixing reservoir solution and a
the antibiotics used for the double disk assay and the boronic
compounds were investigated by
a two-dimensional checkerboard
microdilution assay, using a 96-well microtitration plates as previously
[
39]
described. Assays were conducted against the E.coli BL21 (DE3) pET-
4a-blaKPC-2 and against six K. pneumoniae clinical strains isolated from
5
0 mM stock solution of 2 or 4 in DMSO. For data collection, the crystals
2
were back washed in cryoprotectant solution (reservoir solution
supplemented with 20% ethylene glycol), left equilibrating for 30 seconds
and flash-frozen in liquid nitrogen. X-ray diffraction data were collected
at the beamline id23eh1 and id23eh2 of the European Synchrotron
Radiation Facility (ESRF, Grenoble). EcKPC-2:2 and EcKPC-2:4 crystals
diffracted up to 1.4 and 1.5 Å maximum resolution, respectively. Data
sets were integrated, scaled and truncated through the automated
different patients at the Hospital Universitario Son Espases, Palma de
Mallorca, Spain. The microtiter plates were incubated at 37°C for 18
hours. The growth in each well was quantified spectrophotometrically at
5
95 nm by
a microplate reader iMark, BioRad (Milan, Italy). The
percentage of growth in each well was calculated as:
ꢀ
ꢁ!"#$ !"#$%&'(%"& !"## – ꢀꢁ!"#$%&'()*
ꢁ!"#$ !"## !"## − ꢀꢁ!"#$%&'()*
processing pipeline feasible at ESRF MX beamlines and further
ꢀ
[42,43]
evaluated by data reduction software.
The phase problem was
[
44]
solved by molecular replacement with PHASER 2.6.0.
E. coli KPC-2
[
28]
structure as template (PDB id 3C5A).
refinement cycles using Refmac5 and Refine,
model rebuilding with Coot molecular graphic software
Final structure was obtained by
where the background was obtained from the microorganism-free plates,
processed as the inoculated plates. The MICs for each combination of
drugs were defined as the concentration of drug that reduced growth by
[
45]
alternated with manual
[
46]
through the
CCP4i2 interface (CCP4-7.0). In both cases, after few refinement cycles,
a clear electron density in the difference maps was observed as a
continuous surrounding the catalytic Ser-70 and could be clearly
interpreted as the inhibitor molecule, covalently bound to the side chain
oxygen of Ser-70. The ligand molecule was built and optimized with
8
0% compared to that of organisms grown in the absence of drug.
Drug interaction models: To assess the nature of the in vivo
interactions between the compounds and antibiotics against E.coli BL21
(
DE3) pET-24a-blaKPC-2 and six K. pneumoniae clinical strains, the data
[
47]
Elbow,
manually fitted in the difference maps using Coot molecular
obtained from the checkerboard assay were analyzed in order to
[
45]
[
40]
graphic tools and further refined using Refmac5 and Refine.
Five
calculate the fractional inhibitory concentration index.
percent of the X-ray data were used for R-free cross-validation. Pymol
and CCP4mg Molecular Graphics software were used to generate
illustrations, while composite omit maps with simulated annealing were
The fractional inhibitory concentration index (FICI), is the mathematical
expression of the effect of the combination of antibacterial agents
expressed as:
calculated
phenix.composite_omit_map). LIGPLOT/GIANT were used to analyse
and visualize protein-ligand interactions (additional data and refinement
using
Phenix
environment
maps
options
[
48]
(
ꢀ
ꢁꢂ!"
ꢀꢁꢂ!"
ꢀ
ꢁꢂꢃ = ꢀꢁꢂ! + ꢀꢁꢂ!
=
+
ꢀ
ꢁꢂ!
ꢀꢁꢂ!
[35]
statistics are available as supplemental information, Table S3.
The
crystallographic data for the binary complexes of KPC-2 with 2 and 4
were deposited in the PDB (accession code: 5LL7 for 2 and 5MGI for 4).
where MIC
A
B
and MIC are the MICs of drugs A and B when acting alone
and MICAB and MICBA are the MICs of drugs A and B when acting in
combination. Among all ΣFICs calculated for each microplate, the FICI
was determined as the lowest Σ FIC ( Σ FICmin
)
when synergy is
Founding
supposed, or the highest ΣFIC (ΣFICmax) when antagonism is evident.
Since in MIC determination, the variation in a single result places a MIC
value in a three-dilution range (±1 dilution), the reproducibility errors in
MIC checkerboard assays are considerable.
This work was supported by grant FAR2014 (Finanziamento di
Ateneo per la Ricerca) from University of Modena and Reggio
Emilia to DT supporting independent research on
Carbapenemases.
For that reason, the interpretation of FICI data should be done taking into
consideration values well below or above the theoretical cut-off (1.0)
defined by Berenbaum. Synergy was, therefore, defined when FICI ≤ 0.5,
while antagonism was defined when FICI >4. A FIC index between 0.5
Acknowledgments
[
41]
and 4 (0.5< FICI ≤4) was considered indifferent.
The blaKPC-2 gene was kindly provided by Prof. Sergei Vakulenko
of the Notre Dame University of Indiana, USA. The authors
thank the Centro Interdipartimentale Grandi Strumenti (CIGS) of
Modena for access to its facilities and analytical support. JB and
AO were funded by Instituto de Salud Carlos III, Subdirección
General de Redes y Centros de Investigación Cooperativa,
Ministerio de Economía y Competitividad, the Spanish Network
for Research in Infectious Diseases (REIPI RD12/0015) co-
financed by European Development Regional Fund ‘A way to
achieve Europe’ ERDF.
Cell viability assay: Cell viability was determined with the 3-[4,.5-
dimethylthiazol-2-yl]- 2,.5- diphenyltetrazolium bromide (MTT) reduction
assay. Mouse embryonic fibroblasts (MEFs) and human CCD-Lu34 cells
4
[33]
were seeded (8x10 ) in 96-well plates and allowed to attach overnight.
The day after, cells were treated with the indicate concentrations of
compounds 2 and 4 for 24 hours. At different time points, cells were
incubated for 3 hours at 37 °C with MTT (0.5 mg/mL in phosphate-
buffered saline (PBS)). The formazan crystals were dissolved by adding
1
00 µL of stop solution (90% 2-propanol, 10% Dimethyl sulfoxide
(
DMSO)) to each well followed by quick mixing. After 15 min, the
absorbance at 595 nm was estimated using a plate reader (Tecan
Infinite® M200 PRO). Cells not treated with the compounds were
considered as negative control while cells treated with 15% DMSO were
used as positive control of cytotoxicity.
1
0
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[
[
19] D. Tondi, S. Calo, B. K. Shoichet, M. P. Costi, Bioorg. Med. Chem. Lett.
Data Availability
2
010, 20, 3416–3419.
20] L. J. Rojas, M. A. Taracila, K. M. Papp-Wallace, C. R. Bethel, E. Caselli,
C. Romagnoli, M. L. Winkler, B. Spellberg, F. Prati, R. A. Bonomo,
Antimicrob. Agents Chemother. 2016, 60, 1751–1759.
The coordinates and structure factors for the KPC-2-compound
2
and the KPC-2-compound 4 complexes were deposited in the
Protein Data Bank (PDB identifiers 5LL7 and 5MGI,
[21] J. P. Werner, J. M. Mitchell, M. A. Taracila, R. A. Bonomo, R. A.
respectively).
Powers, Protein Sci. 2017, 26, 515–526.
,
[
22] M. Santucci, F.Spyrakis, S.Cross, A. Quotadamo D. Farina, D. Tondi, F.
De Luca, J. D., Docquier, A. Prieto, C. Ibacache, J. Blázquez, A.
Supplementary data
.
Venturelli, G.Cruciani, MP. Costi Scientific Reports 2017, 7, (1),
Article number: 17716. doi:10.1038/s41598-017-17399-7
Supplementary data associated with this article can be found, in
the online version, at -----, Table S1. Chemical characterization
data for boronic acids derivatives 1-5. Table S2,
Oligonucleotides used for cloning blaKPC-2 gene. Table S3 and
S4 Double Disk Synergy Assays Interpretative Information.
Table S5. Data Collection and Refinement Statistic for binaries
complexes 5LL7 and 5MGI. Table S6. In silico properties and
ligand efficiencies for starting compound 1 and leads 2 and 4.
[23] J. Brem, R. Cain, S. Cahill, M. A. McDonough, I. J. Clifton, J.-C.
Jiménez-Castellanos, M. B. Avison, J. Spencer, C. W. G. Fishwick, C. J.
Schofield, Nature Communications. 2016, 7, 12406.
[
[
[
24] D. M. Livermore, S. Mushtaq, J. Antimicrob. Chemother. 2013, 68,
825–1831.
1
25] A. L. Hopkins, G. M. Keserü, P. D. Leeson, D. C. Rees, C. H. Reynolds,
Nat. Rev. Drug Discov. 2014, 13, 105–121.
26] W. Ke, C. R. Bethel, K. M. Papp-Wallace, S. R. R. Pagadala, M.
Nottingham, D. Fernandez, J. D. Buynak, R. A. Bonomo, F. van den
Akker, Antimicrob. Agents Chemother. 2012, 56, 2713–2718.
27] N. Q. Nguyen, N. P. Krishnan, L. J. Rojas, F. Prati, E. Caselli, C.
Romagnoli, R. A. Bonomo, F. van den Akker, Antimicrob. Agents
Chemother. 2016, 60,3,1760-1766.
1
13
H-NMR and C-NMR spectra. Double Disk Synergy Assays
[
[
pictures.
Conflict of interest
28] S. Petrella, N. Ziental-Gelus, C. Mayer, M. Renard, V. Jarlier, W.
Sougakoff, Antimicrob. Agents Chemother. 2008, 52, 3725–3736.
The authors declare no conflict of interest.
[29] R. Crompton, H. Williams, D. Ansell, L. Campbell, K. Holden, S.
Cruickshank, M. J. Hardman, Lab. Invest. 2016, 96, 439–449.
[
[
30] G. Celenza, C. Luzi, M. Aschi, B. Segatore, D. Setacci, C. Pellegrini, C.
Keywords: Bacterial Resistance • Klebsiella Pneumoniae KPC-
Forcella, G. Amicosante, M. Perilli, J. Antimicrob. Chemother. 2008, 62,
2
• X-ray diffraction • clinical strains • cell viability
9
91–997.
31] R. A. Copeland, Enzyme:
A
Pratical Introduction to Structure,
Mechanism and Data Analysis, Wiley, New York, 2000.
References:
[
[
[
32] W. X. Tian, C. L. Tsou, Biochemistry 1982, 21, 1028–1032.
33] T. Mosmann. Journal of Immunological Methods. 1983, 65 (1–2): 55–63.
34] N. C. J. Strynadka, M. Eisenstein, E. Katchalski-Katzir, B. K. Shoichet, I.
D. Kuntz, R. Abagyan, M. Totrov, J. Janin, J. Cherfils, F. Zimmerman,
et al., Nat Struct Mol Biol 1996, 3, 233–239.
[
[
1]
2]
[1]
K. M. Papp-Wallace, A. Endimiani, M. A. Taracila, R. A. Bonomo,
Antimicrob. Agents Chemother. 2011, 55, 4943–4960.
D. Tondi, S. Cross, A. Venturelli, M. P. Costi, G. Cruciani, F. Spyrakis,
Curr. Drug Targets 2016, 17, 983–1005.
[
35] A. C. Wallace, R. A. Laskowski, J. M. Thornton, Protein Eng. 1995, 8,
[
[
3]
4]
D. M. Livermore, N. Woodford, Trends Microbiol. 2006, 14, 413–420.
D. Farina, F. Spyrakis, A. Venturelli, S. Cross, D. Tondi, M. Paola Costi,
Curr. Med. Chem. 2014, 21, 1405–1434.
1
27–134.
[
[
36] E. Krissinel, J. Mol. Biochem. 2012, 1, 76–85.
37] D. C. Marciano, N. G. Brown, T. Palzkill, Protein Sci. 2009, 18, 2080–
[
5]
J.-M. Frère, E. Sauvage, F. Kerff, Curr. Drug Targets. 2016, 17, 974–
2
089.
9
82.
[
[
38] N. C. Strynadka, H. Adachi, S. E. Jensen, K. Johns, A. Sielecki, C.
Betzel, K. Sutoh, M. N. James, Nature 1992, 359, 700–705.
[
[
6]
T. Naas, L. Dortet, B. I. Iorga, Curr. Drug Targets. 2016, 17, 1006–1028.
D. Schillaci, V. Spanò, B. Parrino, A. Carbone, A. Montalbano, P.
Barraja, P. Diana, G. Cirrincione, S. Cascioferro. Journal of Medicinal
Chemistry. 2017, 60 (20), 8268-8297
7 ]
39] P. Bellio, B. Segatore, A. Mancini, L. Di Pietro, C. Bottoni, A. Sabatini, F.
Brisdelli, M. Piovano, M. Nicoletti, G. Amicosante, et al., Phytomedicine
2
015, 22, 223–230.
40] W. R. Greco, G. Bravo, J. C. Parsons, Pharmacol. Rev. 1995, 47, 331–
85.
[
8]
9]
F.Spyrakis, G. Celenza, F, Marcoccia, M. Santucci, S.Cross, P. Bellio,
L. Cendron, M. Perilli, D. Tondi. ACS Med. Chem. Lett. 2018, 9 (1), 45–
[
3
5
0
[
[
41] F. C. Odds, J. Antimicrob. Chemother. 2003, 52, 1.
[
[
H. Giamarellou, Int. J. Antimicrob. Agents 2010, 36 Suppl 2, S50-4.
42] S. Monaco, E. Gordon, M. W. Bowler, S. Delageniere, M. Guijarro, D.
Spruce, O. Svensson, S. M. McSweeney, A. A. McCarthy, G. Leonard,
et al., J. Appl. Crystallogr. 2013, 46, 804–810.
10] D. M. Livermore, S. Mushtaq, M. Warner, J.-C. Zhang, S. Maharjan, M.
Doumith, N. Woodford, J. Antimicrob. Chemother. 2011, 66, 48–53.
11] F. Perez, A. Endimiani, A. J. Ray, B. K. Decker, C. J. Wallace, K. M.
Hujer, D. J. Ecker, M. D. Adams, P. Toltzis, M. J. Dul, et al., J.
Antimicrob. Chemother. 2010, 65, 1807–1818.
[
[
[
[
43] P. R. Evans, G. N. Murshudov, Acta Crystallogr. D. Biol. Crystallogr.
2
013, 69, 1204–1214.
44] A. J. McCoy, R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C.
Storoni, R. J. Read, J. Appl. Crystallogr. 2007, 40, 658–674.
45] G. N. Murshudov, P. Skubák, A. A. Lebedev, N. S. Pannu, R. A. Steiner,
R. A. Nicholls, M. D. Winn, F. Long, A. A. Vagin, Acta Crystallogr. Sect.
D 2011, 67, 355–367.
[
[
[
12] P. Nordmann, G. Cuzon, T. Naas, Lancet. Infect. Dis. 2009, 9, 228–236.
13] O. Blennow, P. Ljungman, Br. J. Haematol. 2016, 172, 497–511.
14] E. Cerceo, S. B. Deitelzweig, B. M. Sherman, A. N. Amin, Microb. Drug
Resist. 2016, 22, 412–431.
[
[
[
[
15] N. P. Krishnan, N. Q. Nguyen, K. M. Papp-Wallace, R. A. Bonomo, F.
van den Akker, PLoS One 2015, 10, e0136813.
[
[
[
46] P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Acta Crystallogr. Sect.
D 2010, 66, 486–501.
16] D. Tondi, R. A. Powers, E. Caselli, M. C. Negri, J. Blazquez, M. P. Costi,
B. K. Shoichet, Chem. Biol. 2001, 8, 593–611.
47] N. W. Moriarty, R. W. Grosse-Kunstleve, P. D. Adams, Acta Crystallogr.
D. Biol. Crystallogr. 2009, 65, 1074–1080.
17] D. Tondi, A. Venturelli, R. Bonnet, C. Pozzi, B. K. Shoichet, M. P. Costi,
J. Med. Chem. 2014, 57, 5449–5458.
48] P. V Afonine, R. W. Grosse-Kunstleve, N. Echols, J. J. Headd, N. W.
Moriarty, M. Mustyakimov, T. C. Terwilliger, A. Urzhumtsev, P. H. Zwart,
P. D. Adams, Acta Crystallogr. D. Biol. Crystallogr. 2012, 68, 352–367.
18] A. Venturelli, D. Tondi, L. Cancian, F. Morandi, G. Cannazza, B.
Segatore, F. Prati, G. Amicosante, B. K. Shoichet, M. P. Costi, J. Med.
Chem. 2007, 50, 5644–5654.
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Entry for the Table of Contents
This study focuses on the design of new phenyl boronic acid derivatives active vs KPC-2 carbapenemase with nanomolar affinity.
New derivatives were able to restore susceptibility to meropenem in clinical strains overexpressing KPC-2 and resulted not cytotoxic
against human cells. The crystallographic binary complexes of best inhibitors binding KPC-2 were obtained at high resolution. Kinetic
descriptions of slow binding, time dependent inhibition and interactions geometries in KPC-2 were fully investigated.
1
2
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