Characterization of a novel pyranopyridine inhibitor of the AcrAB efflux pump of Escherichia coli

Article abstract of DOI:10.1128/AAC.01866-13

Members of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC of Escherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of the Enterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versus E. coli AB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 μM) in combination with 0.016 μg/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) of E. coli AB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 μg/ml CIP alone. In contrast, phenyl-arginine-β-naphthylamide (PAβN), a known EPI, did not increase the bactericidal activity of 0.016 μg/ml CIP at concentrations as high as 100 μM. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active against Enterobacteriaceae species and Pseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens. Copyright

Full text of DOI:10.1128/AAC.01866-13

Characterization of a Novel Pyranopyridine
Inhibitor of the AcrAB Efflux Pump of
Escherichia coli
Timothy J. Opperman, Steven M. Kwasny, Hong-Suk Kim,
Son T. Nguyen, Chad Houseweart, Sanjay D'Souza, Graham
C. Walker, Norton P. Peet, Hiroshi Nikaido and Terry L.
Bowlin
Antimicrob. Agents Chemother. 2014, 58(2):722. DOI:
10.1128/AAC.01866-13.
Published Ahead of Print 18 November 2013.
Updated information and services can be found at:
http://aac.asm.org/content/58/2/722
These include:
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Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB
Efflux Pump of Escherichia coli
Timothy J. Opperman,a Steven M. Kwasny,a Hong-Suk Kim,b Son T. Nguyen,a Chad Houseweart,a† Sanjay D’Souza,c
Graham C. Walker,c Norton P. Peet,a Hiroshi Nikaido,b Terry L. Bowlina
Microbiotix, Inc., Worcester, Massachusetts, USAa; Department of Molecular and Cell Biology, University of California, Berkeley, California, USAb; Department of Biology,
Massachusetts Institute of Technology, Cambridge, Massachusetts, USAc
Members of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC of Escherichia coli, play ma-
jor roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump
inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI
with potent activity against RND efflux pumps of the Enterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP),
levofloxacin, and piperacillin versus E. coli AB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity
alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 ␮M) in combination with 0.016 ␮g/ml CIP (min-
imally bactericidal) decreased the viability (CFU/ml) of E. coli AB1157 by 10,000-fold after 4 h of exposure, in comparison with
0.016 ␮g/ml CIP alone. In contrast, phenyl-arginine-␤-naphthylamide (PA␤N), a known EPI, did not increase the bactericidal
activity of 0.016 ␮g/ml CIP at concentrations as high as 100 ␮M. MBX2319 increased intracellular accumulation of the fluores-
cent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradi-
ent. MBX2319 was broadly active against Enterobacteriaceae species and Pseudomonas aeruginosa. MBX2319 is a potent EPI
with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens.
ultidrug resistance (MDR) in Gram-negative pathogens, in- to the exterior (8). These RND family pumps not only produce
M
cluding Enterobacteriaceae, Pseudomonas aeruginosa, Acin-
intrinsic levels of resistance to antibacterial agents, including the
fluoroquinolones (FQs) (e.g., ciprofloxacin [CIP] and levofloxa-
cin [LVX]), ␤-lactams (e.g., piperacillin [PIP], meropenem, and
aztreonam) (9), and ␤-lactamase inhibitors (e.g., clavulanate and
sulbactam) (10, 11), but also produce an MDR phenotype when
overproduced (12). In addition, elimination of RND pumps in P.
aeruginosa by genetic deletion (13) or inhibition with a potent
efflux pump inhibitor (EPI) (14) decreases the frequency of resis-
tance to levofloxacin. In E. coli, a functional AcrAB-TolC is re-
quired for selection of mutations in the targets of FQs (gyrA and
gyrB) that give rise to FQ resistance (15). Furthermore, RND
pumps have been shown to play a role in the virulence of the
enteric pathogen Salmonella enterica serovar Typhimurium (16),
and EPIs that target RND pumps have been shown to inhibit bio-
film formation in E. coli and Klebsiella pneumoniae (17). There-
fore, EPIs could be used as adjunctive therapies with an FQ or
␤-lactam antibiotic to improve antibacterial potency at low anti-
biotic concentrations, to reduce the emergence of resistance, to
inhibit biofilm formation, and to decrease the virulence of enteric
pathogens.
etobacter spp., and Stenotrophomonas maltophilia, poses a signifi-
cant threat to the effective treatment of infections caused by these
organisms (1–4). The MDR threat has been exacerbated by the
recent decrease in commercial efforts to discover and develop new
antibacterial agents. In addition, antibacterial agents that have
been introduced recently into the clinic or are in development,
such as daptomycin, gemifloxacin, telithromycin, and telavancin,
are not active against Gram-negative pathogens. Recently FDA-
approved agents with activity against Gram-negative bacteria in-
clude tigecycline and doripenem. While tigecycline is active
against bacteria producing a tetracycline-specific pump in vitro, it
is pumped out rapidly by the ubiquitous multidrug pumps, and its
pharmacokinetic properties limit its use for treating urinary tract
infections (UTIs) and bloodstream infections (5), as will the evo-
lution of resistance during therapy (6). Clearly, novel strategies for
effectively treating infections caused by MDR Gram-negative
pathogens are urgently needed.
The MDR phenotype has been attributed to both acquired and
intrinsic mechanisms of resistance. However, the resistance-nod-
ulation-division (RND) efflux pumps of Gram-negative bacteria
play a major role in MDR. Because of their broad substrate spec-
ificity, overexpression of these efflux pumps results in decreased
susceptibility to a diverse array of antibacterial agents and biocides
(7). The major efflux pump of Escherichia coli is a typical resis-
tance-nodulation-division (RND) pump, which is a tripartite
structure consisting of an integral membrane efflux transporter
with broad substrate specificity (AcrB), an outer membrane chan-
nel (TolC), and a periplasmic protein adapter (AcrA). Antibiotics
enter the periplasmic space through a porin or by diffusion
through the lipid bilayer, where they interact with the substrate-
binding pocket of AcrB. The AcrB transporter uses the proton
motive force to extrude the compound into the TolC channel and
Several potent efflux pump inhibitors have been described in
Received 26 August 2013 Returned for modification 21 September 2013
Accepted 9 November 2013
Published ahead of print 18 November 2013
Address correspondence to Timothy J. Opperman, topperman@microbiotix.com.
† Deceased.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.01866-13.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01866-13
722 aac.asm.org
Antimicrobial Agents and Chemotherapy p. 722–733
February 2014 Volume 58 Number 2

Novel Pyranopyridine Efflux Pump Inhibitor
TABLE 1 Bacterial strains used in this study
Organism
Strain
Genotype/description
Reference or source
Escherichia coli
AB1157
thr-1 araC14 leuB6(Am) ⌬(gpt-proA)62 lacY1 tsx-33 qsr=-0
glnV44(AS) galK2(Oc) ␭Ϫ rac-0 hisG4(Oc) rfbC1 mgl-
51 rpoS396(Am) rpsL31(Strr) kdgK51 xylA5 mtl-1
argE3(Oc) thi-1
51
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
⌬tolC mutant
⌬acrB mutant
⌬acrF mutant
⌬macB mutant
⌬emrB mutant
285
287
331
ATCC 25922
HN1157
AB1157 ⌬tolC::kan
AB1157 ⌬acrB::kan
AB1157 ⌬acrF::kan
AB1157 ⌬macB::kan
This study
This study
This study
This study
This study
This study
This study
Baylor College of Medicine
ATCCb
AB1157 ⌬emrB::kan
AB1157 CIPr, overexpresses effluxa
AB1157 CIPr, overexpresses effluxa
CIPr, UTI isolate
F= araD139 ⌬(argF-lac)U169 rpsL150 rel-I flb-5301 ptsF25
deoCI thi-J ⌬lamB106 ⌬ompF80 zei06::Tn10 ompCI24
acrR::kan
35
Escherichia coli
HN1159
HN1157 ⌬acrAB::spc
35
Escherichia coli
NCM3722
E. coli K-12 prototroph
52
Enterobacter cloacae
Enterobacter aerogenes
Klebsiella pneumoniae
Klebsiella pneumoniae
Shigella flexneri
Salmonella enterica (typhimurium)
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Proteus mirabilis
ATCC 13047
ATCC 13048
ATCC 700603
ATCC 13882
ATCC 12022
ATCC 14028
PAO1
ATCC 27853
ATCC 25933
BAA-856
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
53
ATCC
ATCC
ATCC
Proteus mirabilis
UTI clinical isolate
a Isolated as a ciprofloxacin-resistant mutant during serial passage with subinhibitory levels of ciprofloxacin.
b ATCC, American Type Culture Collection.
the literature (18); however, none has reached clinical develop- lin, chloramphenicol, tetracycline, ethidium bromide, gentamicin, crystal
violet, cephalexin, amoxicillin, rifampin, cefotaxime, carbenicillin, novo-
biocin, erythromycin, linezolid, acriflavine, chlorhexidine, benzalkonium
chloride, and cetylpyridinium chloride. ␤-D-[methyl-3H]Thiogalactopy-
ranoside ([3H]TMG) (1 mCi/ml, 7 Ci/mmol) was obtained from
Moravek Biochemicals (Brea, CA). Luria broth (LB) (Miller) and agar
were purchased as prepared dehydrated media from Becton Dickinson
(Franklin Lakes, NJ). The compound libraries used in high-throughput
screens were purchased from ChemBridge (San Diego, CA), ChemDiv
(San Diego, CA), and TimTec (Newark, DE).
ment. A family of peptidomimetics, including phenyl-arginine-␤-
naphthylamide (PA␤N) (MC-207 110), exhibiting potent inhibi-
tion of efflux pumps in P. aeruginosa, has been developed for use
as adjunctive therapy (14, 19–23). Some of these inhibitors were
validated using in vivo infection models (20, 21, 23); however, they
were abandoned because of toxicity (24). In addition, a series of
pyridopyrimidine EPIs specific for the MexAB efflux pump of P.
aeruginosa advanced to the preclinical stage (12, 25–30). In this
paper, we describe the discovery and in vitro characterization of
MBX2319, a novel pyranopyridine inhibitor of the RND class
AcrAB-TolC efflux pump in E. coli and other pathogens of the
Enterobacteriaceae.
Chemistry. The synthesis of MBX2319 is described in the supplemen-
tal material.
Antibacterial activity assays. The MICs of antibacterial agents and
biocides were determined using the broth microdilution method, essen-
tially as described in CLSI protocol M7-A7 (32) with the following excep-
tions. LB was used instead of Mueller-Hinton broth. Serial 2-fold dilu-
tions of test compounds were made in dimethyl sulfoxide (DMSO) at
concentrations 50-fold higher than the final concentration; the diluted
compounds were added to the assay plates, and 100 ␮l of the bacterial
culture was added to each well. The final concentration of DMSO in each
assay was 2%. Where indicated, MIC assays were performed in the pres-
ence of an efflux pump inhibitor (EPI) at a final concentration of 25 ␮M.
MIC assays were performed in triplicate, and the geometric mean was
calculated. Checkerboard MIC assays using an EPI and an antibacterial
agent were performed essentially as described previously (33), with the
same modifications as used for the MIC assays described above.
Time-kill assays. Killing curve assays were performed essentially as
described previously (33). Exponential bacterial cultures grown in LB
were diluted to a cell density of ϳ1 ϫ 107 cells/ml in LB, followed by
MATERIALS AND METHODS
Strains and reagents. The strains used in this study are listed in Table 1.
The following strains were obtained from the Keio collection (31):
JW0451 (⌬acrB::kan), JW5503 (⌬tolC::kan), JW3234 (⌬acrF::kan),
JW2661 (⌬emrB::kan), and JW0863 (⌬macB::kan). The deletion muta-
tions in each of these strains were transferred to AB1157 using P1 phage
transduction (Table 1). The construction of the E. coli cell-based reporter
strain (SOS-1) that was used for high-throughput screening will be pub-
lished elsewhere. Ciprofloxacin was purchased from ICN Biomedicals
(Aurora, OH). Triclosan (Irgasan) was a generous gift from Ciba Specialty
Chemicals, Inc. (High Point, NC). Hoechst 33342 (H33342) was pur-
chased from Molecular Probes (Eugene, OR). The following reagents were
purchased from Sigma-Aldrich (St. Louis, MO): phenyl-arginine-␤-
naphthylamide (PA␤N), cyanide-m-chlorophenyl hydrazone (CCCP),
levofloxacin, norfloxacin, nalidixic acid, piperacillin, cloxacillin, oxacil- addition of CIP and/or an EPI. Viability was monitored over 2 to 4 h by
February 2014 Volume 58 Number 2
aac.asm.org 723

Opperman et al.
making serial dilutions in saline solution and spotting 5 ␮l of each dilution
onto the surface of an LB agar plate in triplicate. Colonies were counted
after incubation of the plates at 37°C for 16 to 18 h, CFU/ml values were
calculated, and the average and standard deviation for the three replicates
were determined. For treatments that decreased CFU/ml values below the
limit of detection for the spot plating method, the 100-␮l samples were
diluted into 5 ml LB top agar, poured onto LB agar plates, and incubated
at 37°C for 18 h. To calculate the fraction of the control for each sample,
the average CFU/ml values for treated samples were divided by values for
the same sample at 0 h (time 0). Each experiment was repeated at least
three times, and a representative experiment is shown.
H33342 accumulation assay. The H33342 accumulation assay was
used to evaluate the effect of EPIs on the activity of the AcrAB-TolC efflux
pump in several bacterial species, essentially as described previously (34).
Briefly, bacterial cultures were grown overnight in LB (Miller) with aera-
tion at 37°C and were used to inoculate fresh cultures (1:100 dilution),
which were grown in LB (Miller) with aeration until an optical density at
600 nm (OD600) of 0.4 to 0.6 was reached. Bacterial cells were harvested by
centrifugation, and the cell pellet was washed with a volume of phosphate-
buffered saline (PBS) containing 1 mM MgSO4 and 20 mM glucose
(PBSMϩG) equivalent to the original volume of the culture. After cen-
trifugation, the cell pellets were resuspended in PBSMϩG, and the OD600
of each suspension was adjusted to 0.2. Aliquots of 190 ␮l were transferred
to the wells of a 96-well assay plate (flat-bottom black plate, no. 3515;
Costar, Corning, NY). Various concentrations of test compounds dis-
solved in DMSO or an equivalent volume of solvent alone were added to
a total of 8 assay wells (one column of wells) for each condition tested. The
final concentration of DMSO in all assays was 2%. The assay plates were
incubated at 37°C for 15 min, and 10 ␮l of a solution of 50 ␮M H33342 in
PBSMϩG was added to each assay well, resulting in a final dye concentra-
tion of 2.5 ␮M. The fluorescence (excitation and emission filters of 355
and 460 nm, respectively) of each well was measured at 37°C every 5 min
for 30 min, using a Victor2 V 1420 Multilabel HTS counter (PerkinElmer,
Waltham, MA). The average values and standard deviations for the eight
replicates for each condition were calculated using Microsoft Excel. Each
experiment was repeated at least three times, and a representative exper-
iment is shown.
FIG 1 Structures of the efflux pump inhibitors MBX2319 and PA␤N.
itive control) was added and preincubated for 10 min at room tempera-
ture. Then, an aliquot of 250 ␮l of cells was removed and added to 5 ␮l of
a 5 mM [3H]TMG solution (final concentration, 0.1 mM; 10 ␮Ci/␮mol).
After further incubation for 10 min, aliquots (200 ␮l) were removed and
filtered with a 0.45-␮m HA filter (diameter, 25 mm; Millipore, Billerica,
MA). The filters were washed two times with 5 ml of PBB and counted
with 5 ml of EcoLume scintillation cocktail (ICN Biomedicals, Costa
Mesa, CA) in a Delta 300 liquid scintillation counting system (model
6891; TM Analytic, Elk Grove Village, IL).
Outer membrane integrity assay. HN1159 cells were grown in mod-
ified LB broth with 5 mM MgSO4, diluted 500-fold in LB, and incubated at
30°C, with shaking, until the OD600 reached 0.65. The cells were har-
vested, washed twice (50 mM potassium phosphate buffer [pH 7.0]), and
resuspended in the same buffer at an OD600 of 0.65. The cell suspension
was transferred to the wells of an assay plate containing compound or
solvent, nitrocefin was added at a final concentration of 75 ␮M, OD486
was measured over 20 min, and the velocity of hydrolysis (Vh) was calcu-
lated.
Kinetics of nitrocefin efflux by AcrAB-TolC in E. coli. The effects of
EPIs on the kinetic parameters of AcrAB-TolC in E. coli were estimated as
described previously (35). Briefly, HN1157 was grown in modified LB
broth, diluted 100-fold in fresh medium, and incubated at 30°C with
shaking until the OD600 reached 0.65. The cells were harvested, washed
twice (50 mM potassium phosphate buffer [pH 7.0], 5 mM MgCl2), and
resuspended in the same buffer at an OD600 of 0.8 (corresponding to 0.24
mg dry weight/ml). Nitrocefin was added at the desired final concentra-
tion, and the mixture was incubated at 25°C while the OD486 was mea-
sured over 30 min. The nitrocefin concentration in the periplasm (Cp) was
Cytotoxicity assay. The cytotoxicity of MBX2319 against a mamma-
lian cell line (HeLa, ATCC CCL-2) was determined as described previ-
ously (37). The compound concentration that inhibited the conversion of
the vital stain MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tet-
razolium bromide] to formazan by 50% (CC50), in comparison with an
calculated from the rate of hydrolysis (Vh) by the periplasmic ␤-lactamase untreated control, was determined using the 4-parameter curve-fitting
and the kinetic constants of the ␤-lactamase, as described previously (35). program contained in GraphPad Prism version 5.04. The assay was per-
The kinetic constants were derived by curve fitting of the velocity of AcrB formed in triplicate in at least two separate experiments.
(Ve) versus Cp values using GraphPad Prism version 5.04 (GraphPad),
RESULTS
with the Michaelis-Menten equation. Each assay was repeated at least
three times, and representative data are presented.
Identification of MBX2319. To identify compounds that act syn-
ergistically with ciprofloxacin (CIP), we developed a cell-based
reporter assay that reports on both SOS-1 cell induction and via-
bility. Using the cell-based reporter assay, we screened 183,400
compounds and identified 1,782 primary hits, which were evalu-
ated in a panel of secondary assays for prioritization based on
potency and specificity. The details of the screening and secondary
assays will be published elsewhere. Based on the results of these
analyses, MBX2319 (Fig. 1) was chosen for further study.
Uptake of [3H]TMG by the LacY permease. To estimate the effects of
EPIs on the proton motive force in E. coli HN1157, the accumulation of
[3H]TMG by the LacY permease was measured essentially as described
previously (36). Briefly, a culture of NCM3722 cells (K-12 wild type
[WT]) was grown at 37°C, with shaking, in LB containing 1 mM isopropyl
␤-D-1-thiogalactopyranoside (IPTG), was harvested at an OD600 of ϳ0.8,
and was washed two times with 50 mM KHPO4 buffer (pH 7.0), 5 mM
MgCl2 (PB). The cells were resuspended in PB, and the OD600 was ad-
justed to 0.8. The cell suspension was used immediately, without the fur-
ther addition of an energy source. To 1 ml of cell suspension, 5 ␮l of test
compound solution (final concentrations of 0.2, 2, and 20 ␮M), 20 mM
MBX2319 potentiates the antibacterial activity of fluoro-
quinolone and ␤-lactam antibiotics against E. coli. We utilized a
CCCP (final concentration of 100 ␮M; negative control), or DMSO (pos- checkerboard assay to determine whether MBX2319 potentiates
724 aac.asm.org
Antimicrobial Agents and Chemotherapy

Novel Pyranopyridine Efflux Pump Inhibitor
TABLE 2 MBX2319 potentiates the antibacterial activity of fluoroquinolone and ␤-lactam antibiotics against E. coli strains that are efflux proficient
and efflux overexpressers but not against ⌬acrB or ⌬tolC mutants
MIC (␮g/ml) for MBX2319 and a drug concentration of:
MIC ratio for:
MBX2319b
MBX2319
MIC (␮M)
Strain
Druga
0 ␮M
1.56 ␮M
3.13 ␮M
6.25 ␮M
12.5 ␮M
25 ␮M
50 ␮M
Mutantc
AB1157 (WT)
Ն100
Ն100
Ն100
Ն100
Ն100
CIP
LVX
PIP
CIP
LVX
PIP
CIP
LVX
PIP
CIP
LVX
PIP
CIP
LVX
PIP
CTX
CIP
LVX
PIP
CTX
CIP
LVX
PIP
CTX
CIP
LVX
PIP
0.016
0.063
4
0.008
0.031
2
0.008
0.016
1
0.008
0.016
1
0.008
0.016
0.5
0.008
0.016
0.5
0.008
0.016
0.5
2
4
8
1
2
1
1
1
2
2
4
8
8
8
4
8
1
1
1
1
4
8
8
4
1
1
1
1
1
1
1
4
4
32
4
4
16
1
1
1
0.016
0.031
0.5
0.25
0.25
1
⌬tolC mutant
⌬acrB mutant
⌬acrF mutant
285 (EOE)d
0.004
0.016
0.125
0.004
0.016
0.250
0.016
0.063
4
0.004
0.016
0.125
0.004
0.016
0.125
0.008
0.031
2
0.25
0.5
4
0.125
0.063
0.063
0.25
0.031
0.5
0.004
0.016
0.125
0.004
0.016
0.125
0.008
0.031
1
0.125
0.25
2
0.063
0.063
0.063
0.25
0.031
0.25
0.5
0.004
0.008
0.125
0.004
0.016
0.125
0.008
0.016
0.5
0.125
0.25
2
0.063
0.063
0.063
0.25
0.031
0.25
0.5
0.004
0.008
0.125
0.004
0.016
0.125
0.008
0.016
0.5
0.125
0.25
2
0.063
0.063
0.063
0.25
0.031
0.25
0.004
0.008
0.125
0.004
0.016
0.125
0.008
0.016
0.5
0.125
0.25
2
0.031
0.063
0.063
0.25
0.031
0.25
0.004
0.008
0.125
0.004
0.016
0.125
0.008
0.016
0.5
0.125
0.25
2
0.031
0.063
0.063
0.25
0.031
0.25
1
2
8
0.25
0.063
0.063
0.25
0.031
1
285 ⌬tolC
287 (EOE)
287 ⌬tolC
Ն100
Ն100
Ն100
16
2
0.016
0.031
0.25
0.25
0.125
0.5
16
2
16
0.5
8
0.5
2
0.063
0.125
0.125
0.25
0.25
2
0.063
0.125
0.125
0.25
0.25
2
0.063
0.125
0.125
0.25
4
4
0.25
0.125
0.125
0.25
0.015
0.125
0.125
0.125
0.25
0.015
0.125
0.125
0.125
0.25
0.015
0.063
0.125
0.125
0.25
0.015
CTX
0.015
0.015
0.015
4
a CIP, ciprofloxacin; LVX, levofloxacin; PIP, piperacillin; CTX, cefotaxime.
b Highest ratio of MIC with no compound to MIC with MBX2319.
c MIC for WT/MIC for mutant in the absence of an EPI.
d EOE, efflux overexpresser.
the activity of two fluoroquinolones, ciprofloxacin (CIP) and tivity of fluoroquinolones and ␤-lactams is through inhibition of
levofloxacin (LVX), and a ␤-lactam, piperacillin (PIP), against E. efflux, we determined whether MBX2319 potentiated the antibac-
coli AB1157. The results, shown in Table 2, demonstrate that terial activity of CIP, LVX, and PIP against a panel of efflux-defec-
MBX2319 at 12.5 ␮M decreased the MICs of CIP, LVX, and PIP by tive mutants of E. coli AB1157. We reasoned that the antibiotic
2-, 4-, and 8-fold, respectively. MBX2319 alone did not exhibit sensitivity of mutants lacking the target of MBX2319 would not be
antibacterial activity (MIC, Ն100 ␮M). In addition, MBX2319 affected by the compound. The results of a checkerboard assay,
increased the bactericidal activity of 0.016 ␮g/ml CIP (1ϫ MIC), shown in Table 2, demonstrate that the MICs for the ⌬tolC and
which is minimally bactericidal against E. coli AB1157, in a dose- ⌬acrB mutants were not affected by MBX2319, whereas mutants
dependent manner (Fig. 2A). The highest concentration of defective in other pumps that interact with TolC, such as AcrF
MBX2319 (3.13 ␮M) decreased viability (CFU/ml) by 10,000-fold (Table 2), MacB, and EmrB (data not shown), exhibited MIC
after 4 h of exposure, in comparison with CIP alone at 1ϫ MIC. In shifts similar to those of the WT (AB1157). Similarly, MBX2319
contrast, MBX2319 alone at concentrations up to 50 ␮M did not potentiated the bactericidal activity of CIP against the ⌬acrF strain
affect growth. For comparison, we measured the effects of various but not against the ⌬tolC and ⌬acrB strains (Fig. 2C). Finally,
concentrations of phenyl-arginine-␤-naphthylamide (PA␤N), a MBX2319 potentiated the antibacterial activity of CIP, LVX, and
known EPI (14), in combination with 0.016 ␮g/ml CIP in the PIP by 4- to 8-fold against E. coli strains 285 and 287 (Table 2),
time-kill assay. The results of the assay are shown in Fig. 2B and which are CIP-resistant mutants of E. coli AB1157 that were se-
demonstrate that PA␤N at concentrations as high as 100 ␮M did lected during serial passage with subinhibitory concentrations of
not increase the bactericidal activity of 0.016 ␮g/ml CIP. This CIP and exhibit increased efflux activity (see the supplemental
finding is consistent with a previous report that showed that material). Significantly, MBX2319 reduced the MICs against E.
PA␤N at a concentration of 25 mg/ml (48 ␮M) had a limited effect coli strains 285 and 287 to levels comparable to those obtained
on the antibacterial activity of fluoroquinolones against Entero- against isogenic efflux-defective mutants (⌬tolC). These findings
bacteriaceae (38).
indicate that the AcrAB-TolC efflux pump, which is the major
MBX2319 is an efflux pump inhibitor. To verify that the efflux pump in E. coli (39), is a target of MBX2319. However,
mechanism by which MBX2319 potentiates the antibacterial ac- because deletion mutations of acrF, acrD, macB, and emrB do not
February 2014 Volume 58 Number 2
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Opperman et al.
FIG 2 Effects of the efflux pump inhibitors MBX2319 and PA␤N on the bactericidal activity of ciprofloxacin (CIP) in time-kill assays. (A) Bactericidal activity
of varying concentrations of MBX2319 (0.19 to 3.13 ␮M) combined with a bacteriostatic concentration of CIP (1ϫ MIC, 0.01 ␮g/ml) against E. coli AB1157. (B)
Bactericidal activity of varying concentrations of PA␤N (6.25 to 100 ␮M) combined with a minimally bactericidal concentration of CIP (1ϫ MIC, 0.01 ␮g/ml)
against E. coli AB1157. (C) Bactericidal activity of 25 ␮M MBX2319 combined with a minimally bactericidal concentration of CIP against E. coli AB1157 and
isogenic efflux-defective mutants after 2 h of exposure. Black bars, CIP alone (1ϫ MIC, 0.01 ␮g/ml); gray bars, CIP plus 25 ␮M MBX2319. (D) Bactericidal
activity of 25 ␮M MBX2319 combined with a minimally bactericidal concentration of CIP against E. coli AB1157 (a), E. coli ATCC 25922 (b), K. pneumoniae
ATCC 700603 (c), S. flexneri ATCC 12022 (d), S. enterica ATCC 14028 (e), and E. aerogenes ATCC 13048 (f).
affect susceptibility to the antibiotics used in this study, our results and PA␤N increased H33342 accumulation to levels that were
do not rule out the possibility that MBX2319 also inhibits the about 45% and 52%, respectively, of those of the ⌬acrB strain.
efflux pumps encoded by those genes.
MBX2319 was more effective in this assay at lower concentra-
To confirm that MBX2319 directly inhibits efflux, we used an tions (3.1 to 12.5 ␮M) than was PA␤N. Both compounds in-
assay that measures accumulation of the fluorescent DNA-bind- creased H33342 accumulation in the hyperefflux strains 285
ing dye Hoechst 33342 (H33342), which is a substrate of the and 287 (Fig. 3D).
AcrAB-TolC pump, in E. coli AB1157 (34). This assay has been
Finally, we measured the effects of MBX2319 and PA␤N on the
used to estimate efflux activity in E. coli and S. enterica (34). When real-time efflux of nitrocefin by the AcrB efflux pump in intact
H33342 enters the cell, it binds to the DNA minor groove, be- cells (35). This assay measures the rate of nitrocefin hydrolysis by
comes fluorescent, and can be detected using a fluorescent plate the AmpC ␤-lactamase as a function of the external nitrocefin
reader (with excitation at 355 nm and emission at 460 nm). Efflux- concentration, to estimate the periplasmic concentration (Cp) of
competent cells extrude H33342 and accumulate the dye at a rel- nitrocefin, which can be used to estimate the Km and the maxi-
atively low rate, resulting in low levels of fluorescence. Conversely, mum rate of metabolism (Vmax) for the AcrAB-TolC efflux pump.
efflux-defective cells accumulate H33342 at a higher rate, resulting The assay was repeated 12 times with and without 0.2 ␮M
in increased levels of fluorescence. The results of the H33342 ac- MBX2319, and clear inhibition of efflux was seen in all cases ex-
cumulation assay are shown in Fig. 3A and B. The ⌬acrB strain was cept one. Typical data are shown in Fig. 4A, in which curve-fitting
used as a positive control, indicating the maximum levels of suggests that most of the inhibition occurred through a large (4.4-
H33342 accumulation possible. MBX2319 (Fig. 3A) and PA␤N fold) increase in the Km. Although the curve-fitting also suggested
(Fig. 3B) increased accumulation of H33342, in comparison with that there was a marginal (2-fold) increase in the Vmax, this is of
the untreated control, in a dose-dependent manner, although not uncertain significance, because the fitting had to be done in a
linearly for MBX2319 at higher concentrations (25 to 50 ␮M) that quasi-linear portion of the plot. In most other assays, MBX2319
approached the aqueous solubility limit. MBX2319 did not in- caused little change in the Vmax (not shown). These data are con-
crease accumulation of H33342 in the ⌬acrB and ⌬tolC strains sistent with the notion that MBX2319 competes with nitrocefin for
(Fig. 3C), which is consistent with the hypothesis that the binding site or decreases access to the binding site. Higher con-
AcrAB-TolC is the target. At a concentration of 25 ␮M, MBX2319 centrations of MBX2319 (1 to 10 ␮M) completely inhibited nitro-
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Novel Pyranopyridine Efflux Pump Inhibitor
FIG 3 (A and B) Effects of MBX2319 (A) and PA␤N (B) on accumulation of the fluorescent DNA-binding dye H33342, an AcrAB efflux pump substrate, in E.
coli AB1157. (C) Effects of MBX2319 on H33342 accumulation in E. coli AB1157 and isogenic efflux-defective mutants. (D) Effects of MBX2319 on H33342
accumulation in E. coli AB1157 and isogenic mutants (MUT) 285 and 287, which exhibit reduced susceptibility to ciprofloxacin due to overexpression of efflux
pumps. RFU, relative fluorescence units.
cefin efflux and prevented kinetic analyses. In contrast, PA␤N at con-
centrations ranging from 0.2 to 10 ␮M had no effect on the efflux of
nitrocefin, suggesting that this compound does not directly affect
AcrB function in this range of concentrations. A representative result
for an assay performed using 1 ␮M PA␤N is shown in Fig. 4B.
MBX2319 potentiates multiple antibiotics and biocides. Ef-
flux pump inhibitors are known to increase the antibacterial ac-
tivity of a diverse group of antibiotics and biocides. To test this
prediction, we measured the ability of MBX2319 to increase the
susceptibility of E. coli AB1157 to a broad spectrum of antibiotics
and biocides. The data, shown in Table 3, demonstrate that
MBX2319 increased susceptibility to several known AcrAB-TolC
substrates, including CIP, LVX, nalidixic acid, PIP, oxacillin, and
chloramphenicol, but not to gentamicin and carbenicillin, which
FIG 4 Effects of MBX2319 and PA␤N on the kinetic parameters (Km and
are not substrates (40). In general, the MIC shifts produced by
MBX2319 were smaller than those of the ⌬acrB and ⌬tolC strains
but were similar to those produced by PA␤N for fluoroquinolone
Vmax) of the AcrAB-TolC efflux pump in intact E. coli HN1157 cells using the
nitrocefin efflux assay. (A) E. coli HN1157 cells treated with 0.2 ␮M MBX2319
(2319). (B) E. coli HN1157 cells treated with 1 ␮M PA␤N.
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Opperman et al.
TABLE 3 MBX2319 potentiation of the antibacterial activity of diverse antibacterial agents and biocides
MIC (␮g/ml) forb:
WT with:
MIC ratio for:
⌬acrB mutant,
no EPI
⌬tolC mutant,
no EPI
⌬acrB
mutante
⌬tolC
mutantf
Compounda
No EPI
MBX2319
PA␤N
MBX2319c
PA␤Nd
CIP
LVX
Norfloxacin
Nalidixic acid
PIP
Cloxacillin
Oxacillin
Carbenicillin
Chloramphenicol
Tetracycline
Gentamicin
Rifampin
Novobiocin
Erythromycin
Linezolid
EtBr
0.016
0.063
0.063
16
0.008
0.016
0.031
4
0.5
64
64
8
2
1
8
8
128
16
60
256
8
0.25
32
1
0.031
0.031
0.125
2
0.008
0.016
0.031
2
0.25
4
4
2
2
0.5
8
8
16
2
15
32
1
0.125
4
1
2
2
0.004
0.016
0.031
1
0.25
1
1
1
1
0.5
4
8
2
2
15
8
2
4
2
4
8
8
8
0.5
4
2
1
2
1
8
4
1
4
2
1
1
2
1
0.5
2
0.5
8
0.5
2
4
0.25
4
1
2
4
1
32
2
1
4
2
1
1
4
2
4
2
8
16
128
128
2
4
4
1
2
8
64
16
8
32
4
4
4
2
16
16
512
512
4
8
4
2
2
64
64
16
32
32
32
8
1
16
2
4
8
512
512
4
8
2
256
128
16
2
2
4
4
128
4
120
256
8
0.25
32
1
8
16
128
128
240
256
32
0.5
32
1
CV
IRG
Acriflavine
Chlorhexidine
BAC
1
0.016
4
1
2
2
8
1
16
2
32
4
16
4
8
2
CPC
2
a CIP, ciprofloxacin; LVX, levofloxacin; PIP, piperacillin; EtBr, ethidium bromide; CV, crystal violet; IRG, Irgasan (triclosan); BAC, benzalkonium chloride; CPC, cetylpyridinium
chloride.
b Geometric means of MICs from at least three replicate experiments are presented. The final concentration of the EPIs MBX2319 and PA␤N was 25 ␮M.
c MIC with no compound/MIC with 25 ␮M MBX2319.
d MIC with no compound/MIC with 25 ␮M PA␤N.
e MIC for WT/MIC for ⌬acrB strain.
f MIC for WT/MIC for ⌬tolC strain.
and ␤-lactam antibiotics. Interestingly, the MIC shift produced by requires a proton motive force (42). The results are shown in Table 4.
PA␤N for rifampin was greater than that of the ⌬acrB strain, sug-
gesting an additional mechanism of action for PA␤N.
MBX2319 did not significantly inhibit uptake and accumulation of
[3H]TMG at concentrations up to ϳ20 ␮M and thus does not inhibit
efflux indirectly by perturbing the proton gradient.
MBX2319 does not perturb bacterial membranes. Because
the AcrAB-TolC efflux pump utilizes the proton motive force
(41), compounds that perturb the proton gradient across the cyto-
plasmic membrane can inhibit efflux through an indirect mecha-
nism. To determine whether MBX2319 perturbs the transmembrane
proton gradient, we measured uptake and accumulation of
[3H]TMG in the presence of MBX2319 by the LacY permease, which
PA␤N has been shown to affect the integrity of the outer mem-
brane, which is predicted to increase the rate of permeation of anti-
biotics into the periplasm (14). To determine whether MBX2319 af-
fectsthepermeabilityoftheoutermembrane, wemeasuredtheinflux
of nitrocefin in a strain deficient in AcrAB (HN1159). The data
shown in Table S3 in the supplemental material demonstrate that 20
␮M MBX2319 does not increase the rate of nitrocefin influx, indicat-
ing no effect on outer membrane permeability.
TABLE 4 TMG accumulation in E. coli NCM3722 in the presence
Spectrum of activity. To determine whether MBX2319 in-
hibits the AcrAB-TolC orthologs of other Gram-negative
pathogens, we measured the antibacterial activity of MBX2319
in combination with several antibiotics using three assays.
First, we measured the MICs of several antibiotics, alone or in
combination with MBX2319 or PA␤N at a concentration of 25
␮M, against several Gram-negative pathogens. The data are
shown in Table 5. MBX2319 increased significantly the activity
of CIP and LVX against the majority of the organisms tested,
whereas PA␤N (25 ␮M) did not significantly affect the MICs.
MBX2319 and PA␤N increased the activity of PIP and cefo-
taxime against the majority of organisms tested; however,
MBX2319 was active against more organisms than was PA␤N.
of MBX2319
TMG accumulation
(mean Ϯ SD)
(nmol/mg dry wt)
(n ϭ 3)
Concentration
(␮M)
Compound
% control
None
0
0
100
0.2
2
20b
4.4 Ϯ 0.4
0.1 Ϯ 0.0
0.2 Ϯ 0.0
4.4 Ϯ 0.4
4.5 Ϯ 0.1
4.3 Ϯ 0.2
100
3
5
100
103
96
None (ϪIPTG)a
CCCP
MBX2319
MBX2319
MBX2319
a TMG accumulation in cells grown in the absence of IPTG.
b The compound was slightly above the solubility limit in the assay buffer.
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TABLE 5 Spectrum of activity of MBX2319
MIC (␮g/ml) forb:
MIC ratio for:
Organism
Druga
No EPI
MBX2319
PA␤N
MBX2319c
PA␤Nd
Escherichia coli AB1157
CIP
LVX
PIP
0.016
0.031
4
0.008
0.016
0.707
0.022
0.031
0.031
4
2
2
5.7
4.8
0.5
1
1
CTX
0.1
0.25
0.42
Escherichia coli ATCC 25922
Escherichia coli 331
CIP
LVX
PIP
0.016
0.031
2.83
0.005
0.013
1.68
0.022
0.022
4
2.8
2.4
1.7
1.2
0.7
1.4
0.7
0.3
CTX
0.074
0.063
0.25
CIP
LVX
PIP
128
64
4
32
128
32
4
4
1
2
1
11.3
0.707
0.031
5.6
5.6
4
CTX
0.125
0.353
0.353
Salmonella enterica ATCC 14028
Shigella flexneri ATCC 12022
Enterobacter aerogenes ATCC 13048
Klebsiella pneumoniae ATCC 700603
P. aeruginosa PAO1
CIP
LVX
PIP
0.031
0.062
2.4
0.008
0.016
0.5
0.044
0.044
4
4
4
4.8
3.4
0.7
1.4
0.6
0.7
CTX
0.21
0.063
0.297
CIP
LVX
PIP
0.031
0.063
1
0.008
0.016
0.25
0.031
0.031
1
4
4
4
2
1
2
1
1
CTX
0.063
0.031
0.063
CIP
LVX
PIP
0.037
0.177
4
0.008
0.044
1.4
0.031
0.063
16
4.8
4
2.8
0.8
1.2
2.8
0.2
0.3
CTX
2
2.4
6.7
CIP
LVX
PIP
0.29
0.71
113
8
0.088
0.149
113
8
0.25
0.5
113
8
3.4
4.7
1
1.2
1.4
1
CTX
1
1
CIP
LVX
PIP
0.22
1.6
4
0.28
0.14
0.89
4
0.8
0.8
2
1.6
1.8
1
2
2
4
CTX
8
8
2
1
P. aeruginosa ATCC 27853
CIP
LVX
PIP
0.21
1
16
0.125
1
19
8
0.149
0.5
19
1.7
1
0.84
6.7
1.4
2
0.8
2.8
CTX
53.8
19
a CIP, ciprofloxacin; LVX, levofloxacin; PIP, piperacillin; CTX, cefotaxime.
b Geometric means of MICs from at least three replicate experiments are presented. The final concentration of the EPIs MBX2319 and PA␤N was 25 ␮M.
c MIC with no compound/MIC with 25 ␮M MBX2319.
d MIC with no compound/MIC with 25 ␮M PA␤N.
In addition, MBX2319 increased the activity of CIP and LVX tive against any of the strains tested in this assay. Third, the
against E. coli 331, which is resistant to fluoroquinolones. H33342 accumulation assay was used to verify that MBX2319 in-
MBX2319 was not effective against Proteus mirabilis or any of the hibits efflux in other Gram-negative pathogens. The results of this
antibiotics tested (data not shown).
assay are shown in Fig. 5A to H. MBX2319 (25 ␮M) increased
Second, a time-kill assay was used to verify the potentiating H33342 accumulation in the majority of organisms tested, includ-
activity of 25 ␮M MBX2319 against Gram-negative pathogens ing Shigella flexneri, K. pneumoniae, S. enterica, and Enterobacter
(Fig. 2D). The combination of MBX2319 and a minimally bacte- cloacae, and showed weak activity against E. coli 331 (CIP resis-
ricidal concentration of CIP (0.5ϫ or 1ϫ the MIC) decreased the tant), P. mirabilis, and P. aeruginosa.
viability of Shigella flexneri, Salmonella enterica, Enterobacter aero-
MBX2319 increases the antibacterial activity of levofloxacin
genes, and Klebsiella pneumoniae by 100- to 1,000-fold, in com- and piperacillin against a diverse panel of E. coli strains. To
parison with CIP alone. In contrast, 25 ␮M PA␤N was not effec- determine whether MBX2319 increases the antibacterial activity
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Opperman et al.
FIG 5 Effects of MBX2319 and PA␤N on the accumulation of H33342 in various Gram-negative organisms. (A) E. coli AB1157. (B) E. coli 331. (C) Shigella
flexneri ATCC 12022. (D) Klebsiella pneumoniae ATCC 13882. (E) Salmonella enterica (serovar Typhimurium) ATCC 14028. (F) Enterobacter cloacae subsp.
cloacae ATCC 13047. (G) Proteus mirabilis ATCC 25933. (H) Pseudomonas aeruginosa ATCC 27835.
of LVX and PIP against a diverse panel of E. coli strains, we mea- was determined as described previously (37). The concentration
sured MICs for LVX and PIP in the absence and presence of 25 of compound that reduced cell viability by 50%, the CC50, was
␮M compound. The panel of 24 strains (see Table S4 in the sup- determined using a four-parameter curve-fitting algorithm
plemental material) is composed of strains that were publicly (GraphPad Prism). The CC50 for MBX2319 against HeLa cells was
available clinical isolates; however, none of the strains was resis- Ն100 ␮M; however, it is possible that the apparent lack of cyto-
tant to high levels of fluoroquinolones. Because the spectrum of toxicity is due, at least in part, to the relatively low aqueous solu-
activity includes the pathogens that are prevalent in urinary tract bility of this compound.
infections (UTIs), the E. coli panel included several strains isolated
from UTIs. MBX2319 decreased the LVX MIC50 and MIC90 (the
DISCUSSION
concentrations of LVX that inhibit growth of 50% and 90% of the In this report, we describe the preliminary in vitro characterization
strains, respectively) by 4-fold (Fig. 6). In contrast, MBX2319 did of MBX2319, a novel inhibitor of the RND class AcrAB-TolC ef-
not have a significant effect on the PIP MIC50 or MIC90, probably flux pump, which is the major efflux pump in E. coli and other
because ϳ20% of the strains appeared to be resistant to PIP, as Enterobacteriaceae and plays a major role in the MDR phenotype
evidenced by the plateau in the cumulative percentage susceptible of these pathogens (7). Our results demonstrate that MBX2319
at ϳ80%, which is likely to be the result of ␤-lactamase expression. exhibits the following characteristics of an EPI, as described by
Cytotoxicity. The cytotoxicity of MBX2319 against HeLa cells Lomovskaya et al. (14): (i) it potentiates the antibacterial activity
FIG 6 Cumulative MICs for levofloxacin (LEV) and piperacillin against a panel of 25 strains of E. coli, in the absence and presence of 25 ␮M MBX2319.
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Novel Pyranopyridine Efflux Pump Inhibitor
of diverse agents that are substrates of AcrAB-TolC, (ii) it does not but it did not overcome resistance caused by mutations in the FQ
potentiate the antibacterial activity of agents that are not sub- target. These results indicate a potential for broad-spectrum ac-
strates, (iii) it does not exhibit activity against mutants lacking tivity against pathogens of the Enterobacteriaceae family. The ma-
functional AcrAB-TolC pumps, (iv) it inhibits the extrusion or jority of published EPIs are not active against the MexAB-OprM
accumulation of AcrAB-TolC substrates, and (v) it does not affect efflux pump of P. aeruginosa (48), with the notable exceptions of
the energy source of AcrAB-TolC (proton gradient).
PA␤N (14) and DS-9001 (12), which exhibit activity against this
The most likely mechanism of inhibition of MBX2319 is pump. However, MBX2319 exhibited limited, but significant, ac-
through competitive inhibition and/or blockage of access to the tivity against P. aeruginosa, suggesting that it may be possible to
substrate binding site of AcrB. However, our data do not rule out develop analogs with an extended spectrum of activity. AcrB and
the possibility that MBX2319 inhibits other RND pumps, such as MexB are very similar in both primary structure (69.8% identity
AcrF. The substrate binding pocket of AcrB is large, flexible, and and 83.2% similarity) and three-dimensional structure (root
rich in phenylalanine residues that define the binding pocket and mean standard deviation of 1.4 Å) (49). However, subtle differ-
interact with substrate molecules via hydrophobic and ring-stack- ences in primary and tertiary structures appear to underlie the
ing interactions (43, 44). Docking studies suggested that different observed differences in substrate specificity (50). Because of the
pump substrates bind to distinct sites in the substrate binding similarities that exist in the remainder of the substrate binding
pocket (45). However, it was unclear how inhibitors such as PA␤N pocket, it may be possible to design analogs of MBX2319 with
or 1-(1-naphthylmethyl)piperazine (NMP) could inhibit the ex- improved potency against MexB. Attempts to identify the poten-
trusion of multiple chemically distinct substrates. The results of tial sites of interaction of MBX2319 are under way.
molecular dynamics simulations (46) suggested that PA␤N and
Recent reports have underlined the importance of RND family
NMP interact with the “G-loop” (also known as the switch loop), efflux pumps in the MDR phenotype (7) and in resistance to fluo-
which separates the access site and the substrate binding pocket. roquinolones and ␤-lactams in particular. MBX2319 potentiated
The G-loop is predicted to move substrates to the distal binding the activity of fluoroquinolone and ␤-lactam antibiotics against E.
pocket through peristaltic action. Mutations that are predicted to coli and other important enterobacterial pathogens, such as Shi-
prevent movement of the loop (G614P plus G621P and G616P gella flexneri, Salmonella enterica (serovar Typhimurium), Entero-
plus G619P) abolished the efflux of doxorubicin and increased the bacter aerogenes, E. cloacae (data not shown), and Klebsiella pneu-
sensitivity of the mutant strain to erythromycin (47). Therefore, moniae, that utilize RND family pumps. In addition, MBX2319
efflux pump inhibitors could inhibit the efflux of diverse sub- potentiated the activity of CIP and LVX against P. aeruginosa
strates by AcrB through binding to the G-loop. However, differ- PAO1, a laboratory strain, but not against the reference strain
ences in the spectrum of compounds potentiated by MBX2319 ATCC 27854. However, the MIC of cefotaxime against ATCC
versus PA␤N suggest that MBX2319 binds to a different site in the 27854 was decreased significantly (ϳ6-fold) by MBX2319. Taken
AcrB binding pocket. Experiments that will further define the together, the data suggest that MBX2319 has the potential to be
mechanism of MBX2319 are under way in our laboratories.
active against important Gram-negative pathogens of the Entero-
The pyranopyridine MBX2319 appears to be a novel EPI, as it is bacteriaceae and P. aeruginosa. Based on the spectrum of activity
not structurally similar to any of the EPIs that have been described versus Gram-negative pathogens and the classes of antibiotics that
previously. However, a comparison of the structures of MBX2319 are potentiated by MBX2319, it could be useful as an adjunctive
and other EPIs, such as the peptidomimetic PA␤N and the pyri- therapy in combination with a fluoroquinolone, ␤-lactam, or
dopyrimidine D13-9001 (12), reveals that these potent EPIs con- ␤-lactam/␤-lactamase inhibitor.
tain at least two hydrophobic ring systems, which presumably
ACKNOWLEDGMENTS
interact with hydrophobic residues in the substrate binding site.
The activity exhibited by MBX2319 in in vitro assays was compa-
rable or superior to that of PA␤N at the same concentrations.
Unlike PA␤N, the activity of MBX2319 was not dependent on the
presence of primary amines, which were required for activity and
were responsible for the toxicity of PA␤N and later analogs (24).
The mechanism of action of MBX2319 appears to be through the
exclusive inhibition of AcrAB-TolC, in contrast to PA␤N, which
also increases the permeability of the outer membrane (14). This
additional mechanism was apparent in the potentiation of rifam-
pin to levels greater than those of an ⌬acrB strain (Table 3). Fi-
nally, MBX2319 possesses several drug-like properties, including a
molecular weight of 409.54, a calculated logP value of 4.03, five
hydrogen bond acceptors, zero hydrogen bond donors, a polar
surface area of 45.49 Å2, and two rotatable bonds. We are currently
exploring the structure-activity relationship of this novel scaffold
to develop analogs with improved activity.
This paper is dedicated to the memory of our late colleague Chad House-
weart.
The research reported in this article was supported by the National
Institute of Allergy and Infectious Diseases of the National Institutes of
Health (grants R43 AI074116 and R43 AI100332). G.C.W. is an American
Cancer Society Professor and is supported by NIH grant R01 CA021615.
The content of this article is solely the responsibility of the authors and
does not necessarily represent the official views of the National Institutes
of Health.
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