Discovery of inhibitors of Burkholderia pseudomallei methionine aminopeptidase with antibacterial activity

Article abstract of DOI:10.1021/ml400034m

Evaluation of a series of MetAP inhibitors in an in vitro enzyme activity assay led to the first identification of potent molecules that show significant growth inhibition against Burkholderia pseudomallei. Nitroxoline analogues show excellent inhibition potency in the BpMetAP1 enzyme activity assay with the lowest IC₅₀ of 30 nM and inhibit the growth of B. pseudomallei and B. thailandensis at concentrations ≥31 μM.

Full text of DOI:10.1021/ml400034m

Letter
pubs.acs.org/acsmedchemlett
Discovery of Inhibitors of Burkholderia pseudomallei Methionine
Aminopeptidase with Antibacterial Activity
Phumvadee Wangtrakuldee,† Matthew S. Byrd,‡ Cristine G. Campos,‡ Michael W. Henderson,‡
Zheng Zhang,† Michael Clare,§ Ali Masoudi,∥ Peter J. Myler,∥ James R. Horn,† Peggy A. Cotter,‡
and Timothy J. Hagen*,†
†Department of Chemistry and Biochemistry, Northern Illinois University, 1425 West Lincoln Highway, DeKalb, Illinois 60115-2828,
United States
‡Department of Microbiology and Immunology, University of North Carolina, 116 Manning Drive, Chapel Hill, North Carolina
27599, United States
§Clare Associates LLC, 5154 West Brown Street, Skokie, Illinois 60077, United States
∥Seattle Biomedical Research Institute, 307 Westlake Avenue N, Seattle, Washington 98109-5219, United States, and Department of
Global Health and Department of Biomedical Informatics and Medical Education, University of Washington, Seattle, Washington
98195, United States
S
* Supporting Information
ABSTRACT: Evaluation of a series of MetAP inhibitors in an in vitro
enzyme activity assay led to the first identification of potent molecules
that show significant growth inhibition against Burkholderia pseudo-
mallei. Nitroxoline analogues show excellent inhibition potency in the
BpMetAP1 enzyme activity assay with the lowest IC50 of 30 nM and
inhibit the growth of B. pseudomallei and B. thailandensis at
concentrations ≥31 μM.
KEYWORDS: BpMetAP1, methionine aminopeptidase inhibitor, melioidosis, Burkholderia pseudomallei
Methionine aminopeptidase (MetAP) is a dinuclearmetallo-
protease that removes the N-terminal methionine from nascent
proteins.6 MetAP is conserved in all life forms from bacteria to
humans. Genetic studies have shown that deleting the MetAP-
encoding gene in numerous prokaryotes results in either a slow-
growth or lethal phenotype.6 These studies reveal the critical
role of MetAPs in prokaryote survival and suggest that these
enzymes may be promising targets for the discovery of new
antibiotics.
Early studies by Lu et al. suggested inhibitors of M.
tuberculosis MetAP enzymes (MtMetAP1a and MtMetAP1c)
inhibited the growth of mycobacteria in culture.7 Similarly,
potent inhibitors of human MetAP2 were shown to exhibit
activities against HMVEC proliferation.8 While possessing only
limited amino acid sequence similarity to Mt-MetAP1 (37%
identity/46% similarity) and human MetAP2 (12% identity/
18% similarity), B. pseudomallei MetAP1 contains significant
similarity in the active site (Supplemental Figure 1). This
observation suggests that inhibitors effective against MetAP1
from both M. tuberculosis and humans might also be effective
inhibitors of B. pseudomallei MetAP1.
Burkholderia pseudomallei is the causative agent of melioidosis, a
severe and often fatal infection that manifests as pneumonia or
septicemia.1 Melioidosis is endemic to Southeast Asia and
Northern Australia, where it is a significant cause of morbidity
and mortality and can also be found in other tropical regions.2
The Centers for Disease Control and Prevention consider B.
pseudomallei to be a bioterrorism risk and have thus listed the
organism as a Tier 1 select agent that could potentially cause a
large-scale public health crisis if used in an attack (www.
selectagents.gov). Although it is critical to identify antimicrobial
agents active against B. pseudomallei, the process of drug
discovery has been challenging. The current standard treatment
for melioidosis is intensive administration of the third-
generation cephalosporin antibiotic ceftazidime followed by a
regimen of trimethoprim-sulfamethoxazole or amoxicillin-
clavulanic acid for 3−6 months.2 Ceftazidime is highly active
in vitro and was shown to be safe for clinical use; however,
resistance to this drug correlating with clinical use has been
documented.2−4 In addition, B. pseudomallei is intrinsically
resistant to several classes of antibiotics, including aminoglyco-
sides and macrolides, due to expression of resistance
determinants such as beta-lactamase and multidrug efflux
pumps.5 This intrinsic resistance creates an even greater
challenge for the scientific community to find alternative
treatments that act via novel mechanisms.
Received: January 23, 2013
Accepted: July 1, 2013
© XXXX American Chemical Society
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Several inhibitors of MetAP enzymes have been reported
over the past several years. Triazole compounds were reported
to be potent MetAP inhibitors, demonstrating activity in many
organisms through the interaction of each of the triazole
nitrogen atoms in positions 1 and 2 with divalent cobalt ions.9
Furan-type compounds displayed submicromolar inhibitory
activity against Escherichia coli methionine aminopeptidase.10
The carboxyl group in these compounds coordinates with the
metal ions at the enzyme active site pocket. Derivatives of
anthranilic acid sulfonamide were reported to have potent
MetAP inhibition activities against human MetAP2.8 Chlor-
osubstitution at the para-position on the sulfonyl phenyl was
observed to enhance inhibition. Nitroxoline, or 5-nitro-8-
hydroxyquinoline, exhibited activity against human MetAP2 in
vitro in the nanomolar range.11 The phenolic group at the 8-
position dissociates to O− at physiological pH allowing the O−
and the nitrogen atom at the 1 position to participate in
binding to the active site metal ion.12 Here, representative
compounds from each chemical series of MetAP inhibitors
were generated and characterized to determine their inhibition
potencies against BpMetAP1 (Figure 1 and Scheme 1).
Scheme 1. Syntheses of MetAP Inhibitors and BpMetAP1
IC50 Data
a
a
Reagents and conditions: (a) CH2O, EtOH, R7H, 80 °C 24 h. (b)
b
NaOH, EtOH, 70 °C, 20 min. (c) 1 M Na2CO3, pH 8. BpMetAP1
IC50 values in μM with standard error obtained from nonlinear
Figure 1. Commercial compounds purchased from Sigma-Aldrich.
aBpMetAP1 IC50 values in μM with standard error obtained from
nonlinear regression analysis of the average observed activity (in
triplicate) versus inhibitor concentration data. bIn cases where analysis
is limited by compound solubility, the minimum estimate of IC50 is
provided.
regression analysis of the average observed activity (in triplicate)
versus inhibitor concentration data. In cases where analysis is limited
by compound solubility, the minimum estimate of IC50 is provided.
c
amino-5-thio-1,2,4-triazole with the respective benzyl bromide.
Compounds 16−21 were produced with yields between 47 and
92% following recrystallization. Compound 24 was synthesized
from 2-amino-5-bromobenzoic acid and p-toluenesulfonyl
chloride, providing the product in a 76% yield. Following a
similar procedure, compound 25 was synthesized using 4-
chlorobenzenesulfonyl chloride as a starting material, with a
yield of 92%
Seven compounds (1−4, 9, and 22−23) were purchased
from Sigma-Aldrich (Figure 1). Compounds 5−8 were
synthesized by Mannich reaction of 5-chloroquinolin-8-ol
with formaldehyde and secondary amine. The synthesis of
each of these compounds is shown in Scheme 1. Compounds
5−8 were produced with yields between 68 and 95% after
recrystallization. Compounds 10−15 were synthesized similarly
using 5-nitroquinolin-8-ol as the starting material, with similar
yields. Compounds 16−21 were synthesized by alkylation of 3-
The four chemical series of compounds (1−25) were
profiled for in vitro BpMetAP1 activity (Figure 1 and Scheme
1) using an established activity inhibition assay.13 Overall,
B
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compounds across the four different series exhibited a wide
range of inhibition potencies.
determinants for the nitroxline-based compounds within
BpMetAP1 and hMetAP2 enzymes. In addition, the lack of a
differentiation between BpMetAP1 and human MetAP
(particularly hMetAP2) may reflect the relatively small
scaffolding and/or similarities across the three different
MetAP active sites.
Structural studies were explored to better understand the
nature of inhibitor interactions within the BpMetAP1 active
site. Because of poorly diffracting BpMetAP1 crystals, a
homology model of the BpMetAP1 was constructed from
MtMetAP1 (PDB code: 3IU9). Using the Sybyl docking suite,
compounds 9−15 and 18−20 were docked into the active site
of BpMeAP1. The 8-hydroxyquinoline ligand within the
Aeromonasproteolytica aminopeptidase binding pocket (PDB
code: 3VH9) served as a structural constraint for docking
compounds 9−15. A full negative charge was assigned to the
oxygen atom in each compound due to the phenolic group
donating its proton at physiological pH. Docking produced
ligand poses where the negative charge from the oxygen and
the nitrogen atom at the 1 position was centered on the two
active site metal ions (Figure 2a). Functional groups extended
It was shown that oxine derivatives (compounds 1−15)
generally did not exhibit significant inhibition if the nitro group
is replaced in the 5-position (compounds 1−8). The inhibition
potencies of compounds 1 through 8 ranged from low
micromolar to more than 250 μM (Figure 1 and Scheme 1).
The addition of functional groups on the 7-position of the 5-
chloroquinolin-8-ol generally did not increase the potency as
shown by compounds 6−8. However, the Mannich product 5,
bearing a piperidyl group, displayed increased potency (9 μM).
Nitroxoline derivatives were found to effectively inhibit
BpMetAP1 activity compared to other oxines, with IC50 values
ranging from low micromolar to 30 nM. The addition of
functional groups on the 5-position was shown to increase the
inhibitory potencies. The dimethylamino and pyrrolidyl
Mannich products (11 and 12) displayed increased potency,
while the piperidyl, dimethylamino, morpholino, thiomorpho-
lino, and N-methylpiperazino groups (10, 13, 14, and 15)
displayed decreased potency. The triazole-type compounds 16,
17, and 18 demonstrated inhibition of BpMetAP1 in the low
micromolar range. Different substituents at the C2 and C4
position on the phenyl ring did not alter the inhibitory effect of
these compounds. Addition of bulky functional groups, such as
tert-butyl and isopropyl group at the C4 position, was shown to
decrease the potency of the triazole compounds against
BpMetAP1. Compounds 19−21 displayed an increase in IC50
values compared to triazole compounds with smaller functional
group at the C4 position (16, 17, and 18). Furan-type
compounds (22 and 23) and sulfonamide compounds (24 and
25) displayed IC50 values of more than 250 μM, indicating that
they are not potent inhibitors of BpMetAP1.
The selectivity of several oxine derivatives (compounds 1−6
and 8−9) was evaluated through comparisons to published IC50
values from human MetAP1 and MetAP2 enzymes (Table 1).14
Overall, most compounds display a lack of apparent selectivity
across the three MetAP enzymes. While several compounds
display lower potency against BpMetAP1, compounds 5 and 9
possess higher potency toward BpMetAP1 versus hMetAP1,
but similar potency when compared to published IC50 values
for hMetAP2. These results may indicate similar binding
Figure 2. Proposed binding modes for 15 (a) and 18 (b) in the
BpMetAP1 binding site.
from position 7, are observed to reside within a binding pocket
(Tyr202, Cys203, Gly204, His205, His212, and Met240).
Analysis of triazole-type compound 18 suggested that its
inhibitory effect is dependent upon the presence of nitrogen
atoms at positions 1 and 2 (Figure 2b). These nitrogen atoms
coordinate with the metal ions in a manner similar to other
triazole-type compounds that bind at the active site of MetAP.9
The phenyl group of compound 18 fits in to a hydrophobic
binding pocket (Tyr27, Pro94, Tyr97, Cys105, Thr 133, and
Phe211). The presence of bulky functional groups extending
from the C4 position (e.g., 20) appear to clash into the side of
the pocket, which is consistent with the observed loss of
inhibitory potency. There are several differences in the binding
pockets of hMetAP2 and BpMetAP1. In particular, Phe366/
Met240, Leu328/Tyr202, and Asn329/Cys203 represent
several residue differences (hMetAP2/BpMetAP1) that reside
within the active site. These different residues may allow for
introduction of larger substituents, or introduction of functional
groups to hydrogen bond may result in increased selectivity.
To determine if inhibitors of BpMetAP1 were effective
against Burkholderia in vivo, compounds 1−25 were evaluated
for the ability to inhibit the growth of Burkholderia thailandensis.
B. thailandensis is closely related to B. pseudomallei, displaying
98% sequence identity at the nucleotide level. However, B.
thailandensis is not a select agent pathogen, which allows for its
Table 1. Comparison IC50 Values of Compounds against
BpMetAP1 and Human MetAP Enzymes, hMetAP1 and
a
hMetAP2
b
d
d
#
BpMetAP1
213 118
hMetAP1
hMetAP2
1
>15
>15
>15
>15
c
2
>250
>15
3
>250
2.03 0.3
1.27 0.6
7.68 1.2
2.66 0.2
c
4
>250
12.9 1.0
>50
5
9
6
c
6
>250
>250
>50
c
8
>50
3.81 0.74
9
0.06 0.03
>250
>15
0.055 0.02
e
24
25
ND
0.009
e
>250
ND
11
a
b
IC50 values in μM. IC50 values and standard error obtained from
nonlinear regression analysis of the average observed activity (in
c
triplicate) versus inhibitor concentration data. In cases where analysis
is limited by compound solubility, the minimum estimate of IC50 is
d
e
provided. Values from Bhat et al.14 unless otherwise noted. Values
from Wang et al.15
C
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use as a model without the requirement of a BSL-3 facility.16
Cells were grown in the presence of 1 mM inhibitor, 0.5 mM
kanamycin (Km), or DMSO alone for approximately 24 h.
Treatment with Km at 0.5 mM results in complete killing of
both B. thailandensis and B. pseudomallei in vitro and thus serves
as a positive control for growth inhibition in our assay.5,17−19
Growth was monitored by reading the absorbance at 600 nm
every 15 min, and the area under the curve was calculated for
cells grown in each condition (Supplemental Table 1).
Treatment with the majority of oxine derivatives (compounds
2−8) resulted in greater than 50% growth inhibition compared
to the DMSO control and similar to the Km control, while
compound 1 showed minimal inhibition of bacterial growth
(approximately 9%; Supplemental Table 1 and Figure 3a).
compounds showed growth inhibition of B. thailandensis greater
than 50%. Only five compounds were active against both
purified BpMetAP1 and B. thailandensis, and of these, four are
nitroxoline or its derivatives. Compounds 5, 9, 11, 12, and 15
displayed inhibition of cell growth and MetAP inhibition. Six
compounds 2, 3, 4, 6, 7, and 8 inhibited cell growth but had
IC50 values greater than 250 uM in the MetAP assay. The
compounds 4, 6, 7, and 8 had limited solubility in the enzyme
inhibition assay, which may explain the discrepancy between
the enzyme inhibition data and the antibacterial data.
Compounds 4, 7, 9, 11, and 12 showed inhibition of growth
but had high background due to solubility limitations.
Compound 10 was inactive in the cell growth assay but had
an IC50 of 100 nM in the MetAP inhibition assay. Our results
highlight the fact that in vitro and in vivo assays are required to
sufficiently evaluate the antimicrobial potential of small
molecule inhibitors. The observation that oxine derivatives 2
and 3 were inactive against purified BpMetAP1, yet inhibited
growth of B. thailandensis, suggest that these oxine compounds
may have a target other than MetAp.
Following this initial assessment of potential inhibitors
against B. thailandensis, we evaluated nitroxoline (compound
9) and a nitroxoline derivative (compound 15) at different
concentrations to determine the minimum inhibitory concen-
tration (MIC) that would prevent growth and the minimum
bactericidal concentration (MBC) that would result in cell
death. Using the cell growth inhibition assay, we observed an
MIC of 31 μM (approximately 6 μg/mL) for compound 9,
which is within the range of MIC values reported for
ceftazidime against clinical B. pseudomallei isolates (1−6 μg/
mL).4 For compound 15, we found the MIC to be 500 μM.
The MBC of compound 9 was 250 μM; however, we did not
reach the MBC of compound 15 as growth was observed at the
highest concentration tested (1 mM). Growth curves used to
determine the MIC values can be found in the Supporting
Information.
Although testing these compounds in B. thailandensis is a
simple and effective process, it is possible that B. pseudomallei-
specific resistance mechanisms may reduce or eliminate the
effectiveness of nitroxoline in the growth inhibition assay.
Therefore, we tested nitroxoline against the virulent B.
pseudomallei strain Bp340, a derivative of the clinical
melioidosis isolate 1026b that lacks an aminoglycoside/
macrolide-specific multidrug efflux pump (to facilitate antibiotic
selection in the laboratory).5 Treatment with nitroxoline
resulted in the complete growth inhibition of B. pseudomallei
Bp340 at the lowest concentration tested (31.25 μM; Figure
3b). While we did not determine the MIC of nitroxoline against
B. pseudomallei, our data indicate that it is at least as low as for
B. thailandensis. A subsequent test of nitroxoline against B.
pseudomallei 1026b revealed no difference in inhibition
compared to Bp340, suggesting that the lack of the multidrug
efflux pump does not affect the activity of this compound (data
not shown).
In conclusion, we evaluated a set of small molecule inhibitors
of B. pseudomallei MetAP1 in an enzyme activity assay. These
compounds displayed a range of MetAP1 inhibition. A
homology model of the BpMetAP1 was generated from
MtMetAP1, and the proposed binding mechanisms of two of
the most potent compounds from the in vitro assay were
evaluated through molecular docking. Assessment of the
antibacterial cell-growth inhibition reveals that five of the
compounds that display MetAP1 inhibition had modest to high
Figure 3. (a) Growth inhibition of B. thailandensis by indicated
compounds and (b) growth inhibition of B. pseudomallei by compound
9.
Nitroxoline (compound 9) and three nitroxoline derivatives
(compounds 11, 12, and 15) were potent growth inhibitors,
while the remaining nitroxoline derivatives exhibited weak to
moderate growth inhibition (Supplemental Table 1). None of
the triazole compounds exhibited growth inhibition greater
than 26%, consistent with the moderate to high IC50 values
observed in the activity inhibition assay. It was previously
reported that triazole-derived compounds do not exhibit
antibacterial activity, and our results support this observation.8
Neither the furan (22 and 23) nor the sulfonamide (24 and 25)
derivatives demonstrated significant growth inhibition (Supple-
mental Table 1), consistent with the low enzyme inhibition
potencies. In fact, the furan compounds displayed essentially no
ability to inhibit bacterial growth and represent the weakest
class of compounds tested.
Ten compounds demonstrated activity in the enzyme
inhibition assay with IC50 values lower than 60 μM, while 11
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Development of Ceftazidime Resistance in an Acute Burkholderia
pseudomallei Infection. Infect. Drug Resist. 2012, 5, 129−132.
(5) Schweizer, H. P. Mechanisms of Antimicrobial Resistance in
Burkholderia pseudomallei: Implications for Treatment of Melioidosis.
Future Microbiol. 2012, 7, 1389−1399.
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Methionine Excision. Cell. Mol. Life Sci. 2004, 61, 1455−1474.
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cell growth inhibitory effects, while others had minimal effects.
Compound 15 is a potent inhibitor in the enzyme activity assay
and shows nearly complete cell growth inhibition in vivo. The
efficiency of this molecule in inhibiting BpMetAP1 activity and
in arresting cell growth suggests that nitroxoline-derived
compounds may be useful candidates for potential melioidosis
therapeutics. While there was little selectivity observed between
BpMetAP1 and hMetAP2, homology modeling suggests there
are several difference in the binding pocket that may be
targeted in future design efforts.
(8) Kawai, M.; BaMaung, N. Y.; Fidanze, S. D.; Erickson, S. A.;
Comess, K. M.; Kalvin, D.; Wang, J.; Zhang, Q.; Lou, P.; Tucker-
Garcia, L.; Bouska, J.; Bell, R. L.; Lesniewski, R.; Henkin, J.; Sheppard,
G. S. Development of Sulfonamide Compounds As Potent Methionine
Aminopeptidase Type II Inhibitors with Antiproliferative Properties.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for the synthesis and characterization
of the compounds, the in vitro activity assay, the in vivo
(9) Oefner, C.; Douangamath, A.; D’Arcy, A.; Hafeli, S.; Mareque, D.;
̈
1
antibacterial assay, and the H NMR and 13C NMR spectra of
Sweeney, A. M.; Padilla, J.; Pierau, S.; Schulz, H.; Thormann, M.;
Wadman, S.; Dale, G. E. The 1.15 Å Crystal Structure of the
Staphylococcus aureus Methionyl-Aminopeptidae and Complexes with
Triazole Based Inhibitors. J. Mol. Biol. 2003, 332, 13−21.
(10) Huang, Q.; Huang, M.; Nan, F.; Ye, Q. Metalloform-Selective
Inhibition: Synthesis and Structure−Activity Analysis of Mn(II)-Form-
Selective Inhibitors of Escherichia coli Methionine Aminopeptidase.
Bioorg. Med. Chem. Lett. 2005, 15, 5386−5391.
the reported compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(T.J.H.) E-mail: thagen@niu.edu.
(11) Shim, J. S.; Matsui, Y.; Bhat, S.; Nacev, B. A.; Xu, J.; Bhange, H.
C.; Dhara, S.; Han, K. C.; Chong, C. R.; Pomper, M. G.; So, A.; Liu, J.
O. Effect of Nitrooxline on Angiogenesis and Growth of Human
Bladder Cancer. J. Natl. Cancer Inst. 2010, 102, 1855−1873.
(12) Pelletier, C.; Prognon, P.; Bourlioux, P. Roles of Divalent
Cations and pH in Mechanism of Action of Nitroxoline against
Escherichia coli Strains. Antimicrob. Agents Chemother. 1995, 39, 707−
712.
(13) Zhou, Y.; Guo, X.; Yi, T.; Yoshimoto, T.; Pei, D. Two
Continuous Spectrophotometric Assays for Methionine Amino-
peptidase. Anal. Biochem. 2000, 280, 159−165.
(14) Bhat, S.; Shim, J. S.; Zhang, F.; Chong, C. R.; Liu, J. O.
Substituted Oxines Inhibit Endothelial Cell Proliferation and Angio-
genesis. Org. Biomol. Chem. 2012, 10, 2979−2992.
(15) Wang, G. T.; Mantei, R. A.; Kawai, M.; Tedrow, J. S.; Barnes, D.
M.; Wang, J.; Zhang, Q.; Lou, P.; Garcia, L. A.; Bouska, J.; Yates, M.;
Park, C.; Judge, R. A.; Lesniewski, R.; Sheppard, G. S.; Bell, R. L. Lead
Optimization of Methionine Aminopeptidase-2 (MetAP2) Inhibitiors
Containing Sulfonamides of 5,6-Disubstituted Anthranilic Acids.
Bioorg. Med. Chem. Lett. 2007, 17, 2817−1822.
(16) Yu, Y.; Kim, H. S.; Chua, H. H.; Lin, C. H.; Sim, S. H.; Lin, D.;
Derr, A.; Engels, R.; DeShazer, D.; Birren, B.; Nierman, W. C.; Tan, P.
Genomic Patterns of Pathogen Evolution Revealed by Comparison of
Burkholderia pseudomallei, the Causative Agent of Melioidosis, to
Avirulent Burkholderia thailandensis. BMC Microbiol. 2006, 6, 46.
(17) Anderson, M. S.; Garcia, E. C.; Cotter, P. A. The Burkholderia
bcpAIOB Genes Define Unique Classes of Two-Partner Secretion and
Contact Dependent Growth Inhibition Systems. PLoS Genet. 2012, 8,
e1002877.
Funding
We acknowledge Northern Illinois University for supporting
this work. This project has been funded in part with funds from
the National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Department of Health and
Human Services, under Contract Nos. HHSN272200700057C
and HHSN272201200025C.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We appreciate comments and advice regarding homology
model and docking studies from Dr. Michael Clare. We also
thank Mr. Sriram Jakkaraju for technical assistance regarding
surface plasmon resonance work. Dr. Herbert Schweizer kindly
provided Burkholderia strains.
ABBREVIATIONS
■
BpMetAP1, Burkholderia pseudomallei methionine aminopepti-
dase 1; DMSO, dimethyl sulfoxide; HMVEC, human micro-
vascular endothelial cells; Km, kanamycin; MetAP, methionine
aminopeptidase; Met-Gly-Pro-AMC, methionine-glycine-pro-
line-7-amino-4-methylcoumarin; rhDPPIV, human DPPIV/
CD26
(18) Schweizer, H. P.; Peacock, S. J. Antimicrobial Drug-Selection
Markers for Burkholderia pseudomallei and B. mallei. Emerging Infect.
Dis. 2008, 14, 1689−1692.
(19) Mima, T.; Schweizer, H. P. The BpeAB-OprB Efflux Pump of
Burkholderia pseudomallei 1026b Does Not Play a Role in Quorum
Sensing, Virulence Factor Production, or Extrusion of Aminoglyco-
sides but Is a Broad-Spectrum Drug Efflux System. Antimicrob. Agents
Chemother. 2010, 54, 3113−3120.
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