E. Romero, S. Oueslati, M. Benchekroun et al.
European Journal of Medicinal Chemistry 219 (2021) 113418
the mature proteins: NDM-1 (from amino 29 to 293), OXA-48 (from
amino 23 to 261), KPC-2 (from amino 30 to 293) and deleted of the
stop codon, were amplified by PCR using primers including NdeI
and XhoI restriction sites. Then, PCR product was cloned into
pET41b vector (Invitrogen®, Life Technologies, Cergy-Pontoise,
France), using NdeI and XhoI restriction enzymes, to obtain a C-
Term His8-tag. The accuracy of the recombinant plasmid was
verified by sequencing, using a T7 promoter and T7 terminator with
an ABI Prism 3100 automated sequencer (Applied Biosystems,
Thermo Fisher Scientific, Les Ulis, France). The nucleotide se-
4.5. Minimal inhibition concentrations
MIC values were determined by broth microdilution, in tripli-
guidelines. The enterobacterial clinical strain E. coli NDM-1 GUE
expressing the carbapenemase NDM-1 was used [56]. Experiments
were performed in microtiter plates containing the medium with
imipenem and inhibitors (dissolved in DMSO). Three inhibitor
concentrations were tested: 50, 100 and 200
mM. Plates were
ꢀ
incubated overnight at 37 C for 18e24 h. In all MIC assays, a
negative control (bacteria incubated with different concentrations
of compound) was performed. No antibiotic effect was observed,
for all the compounds, even with the highest concentration tested
4.3. Protein expression and purification for NDM-1, OXA-48 and
(200 mM).
KPC-2
The recombinant plasmids harboring b-lactamase genes were
transformed into E. coli BL21 (DE3), and an overnight culture was
4.6. Incubations in hepatic microsomes
Compounds (5 M) were incubated in 0.5 mg/mL of pooled male
used to inoculate 2 L of Luria Bertani medium broth containing
ꢀ
5
0
m
g/mL of kanamycin. Bacteria were cultured at 37 C, until an OD
m
of 0.6 at 600 nm was reached. The expression of the
b
-lactamase
mouse liver microsomes (from Biopredic, France), in 0.1 M phos-
phate buffer at pH 7.4 at 37 C. After prewarming the mixture for
ꢀ
ꢀ
genes was carried out at 37 C for 3 h, with 1 mM of IPTG as an
inducer. Cells were pelleted by centrifugation, at 6000 g for 15 min,
then resuspended with 25 mM of phosphate sodium pH 7.4,
5 min, reactions were initiated by the addition of NADPH (1 mM).
ꢀ
Incubations (400
m
L) were performed at 37 C for 0, 5, 15, 30 and
3
00 mM of K
2
SO
4
(for OXA-48) or NaCl (for NDM-1 and KPC-2), and
45 min in duplicate and the reaction immediately terminated by
adding 200 L of cold acetonitrile. Samples (including the control to
10 mM of imidazole. Bacterial cells were disrupted by sonication,
m
and the bacteria debris was removed by 2 centrifugations: first at
evaluate non NADPH-dependent stability) were centrifuged and
the supernatant fractions analyzed by UPLC-MS/MS with multiple
reaction monitoring (MRM). Diphenhydramine was used as posi-
tive control in the mouse liver microsomes tests. The MRM area
response of the analyte was set to 100% with the T0 incubation, the
relative decrease in MRM area ratio intensity over time against that
of the control (percent parent decrease) was used to determine the
half-life (t1/2) of compounds in the incubation. Half-life values
were calculated from the relationship:
ꢀ
1
0,000 g for 1 h at 4 C, and then the supernatants obtained were
ꢀ
centrifuged at 96,000 g for 1 h at 4 C. The soluble fraction was
filtered and then passed through a HisTrap HP column (GE
TM
Healthcare®, Velizy-Villacoublay, France). The recombinant pro-
teins were eluted, using elution buffer (25 mM of phosphate so-
dium pH 7.4, 300 mM of K SO or NaCl, and 500 mM of imidazole).
2 4
The eluted proteins were concentrated by using Vivaspin 20 (10000
MWCOPES Sartorius®, Aubagne, France), dialyzed against 0.1 M of
HEPES (pH 7.5) buffer and 150 mM K
2
SO
4
or NaCl and then loaded
T1/2 (min) ¼ 0.693/k, where k is the slope of the Ln concen-
onto a HiLoad 16/600 Superdex 75 pg (GE Healthcare). The protein
purity, estimated by SDSePAGE (see Figures S4eS6 in the Supple-
mentary Information), was more than 99%, and the pooled fractions
were dialyzed against 100 mM sodium phosphate buffer pH 7 (and
tration vs time curve. The intrinsic clearance (CLint) was calculated
as: CLint ¼ (0.693 x incubation volume (
mL))/(t (min) x mg of
microsomal protein).
5
4
0 mM ZnSO for NDM-1) and concentrated, using Vivaspin col-
umns. Protein concentration was determined by Bradford Protein
assay (Bio-Rad, Marnes-La-Coquette, France).
4.7. Cell culture and proliferation assay
4
.4. In vitro
b
-lactamase inhibition assay
Assays were carried out at the Institut de Chimie des Substances
Naturelles by the CIBI screening platform. Cell lines were obtained
from the American Type Culture Collection (Rockville, USA) and
were cultured according to the supplier’s instructions. Briefly, hu-
man MRC-5 cells were grown in DMEM supplemented with 10%
fetal calf serum (FCS) and 1% glutamine and HCT116 colorectal
carcinoma cells were grown in RPMI 1640 containing 10% FCS and
Spectrophotometric assays were performed using ULTROSPEC
000 UV spectrophotometer and the SWIFT II software (GE
2
Healthcare, Velizy-Villacoublay, France). Assay conditions were as
follows: 100 mM phosphate buffer, pH 7 (supplemented with
5
0
mM ZnSO
4
when testing NDM-1, and with 50 mM NaHCO
M imipenem (Sigma-Aldrich,
3
ꢀ
[
60,61] when testing OXA-48), 100
m
1% glutamine. All cell lines were maintained at 37 C in a humidi-
Saint-Quentin Fallavier, France). The reaction was monitored at
2
fied atmosphere containing 5% CO . Cell growth inhibition was
determined by an MTS assay according to the manufacturer’s in-
structions (Promega, Madison, WI, USA). Briefly, the cells were
ꢀ
2
97 nm, time course 600 s at 25 C with 3 min of incubation
(compound/carbapenemase). Enzyme concentrations were: 1 nM
for NDM-1, 1.7 nM for KPC-2 and 5.8 nM for OXA-48. Initial steady-
state velocities from imipenem hydrolysis were measured at a
wavelength of 297 nm. IC50 values for each inhibitor compound
were assayed at seven different concentrations, and performed in
seeded in 96-well plates (2.5 ꢂ 103 cells/well) containing 200
mL of
growth medium. After 24 h of culture, the cells were treated with
the test compounds at different final concentrations. After 72 h of
incubation, 40 mL of resazurin was added for 2 h before recording
triplicate. The K
i
of each inhibitor was determined according to the
absorbance at 490 nm with a spectrophotometric plate reader. The
IC50 value corresponded to the concentration of compound
inducing a decrease of 50% in absorbance of drug-treated cells
compared with untreated cells. Experiments were performed in
triplicate. Paclitaxel was used as the reference compound.
ChengꢁPrusoff equation: K ¼ IC50/(1 þ [S]/Km), where [S] is the
i
imipenem concentration and Km is the Michaelis constant of the
enzymes [62]. Percentage of inhibition was determined when the
IC50 of the compound was lower than 10 mM.
11