ChemBioChem
10.1002/cbic.201600078
COMMUNICATION
Negative controls were monitored in the absence of itaconic acid
or active compound, both with and without DMSO. Positive
controls were acquired using kanamycin (40 µM) or ampicillin
(40 µM). Visible light absorption by the growth medium and by
DMSO were subtracted from the readings. All data points are
means from triplicate measurements.
Note: In the case of casamino acids and thiamine supplemented
medium, the plates incubation time was reduced to 24 hrs which
was enough to reach a stationary state.
Acknowledgements
This work was supported by research grants from the Canadian
Institute of Health Research (CIHR) and the Natural Sciences
and Engineering Research Council of Canada (NSERC) to K.A.
CIHR also provided funding as part of the Drug Development
Training Program graduate scholarship (to J.H.C.).
Keywords: antibiotic, intracellular pathogen, itaconate
degradation, medicinal chemistry, pantothenate
aMIC Determination
[1]
[2]
C. F. Nathan, J. Clin. Invest. 1987, 79, 319–326.
Bacteria were allowed to grow overnight on agar at 37°C, before
inoculation into liquid NB or LB and further growth at 37°C and
250 rpm until stationary growth was reached (~24 hrs). An
aliquot (100 µL) of this culture was then transferred into M9A
medium (10 mL). The suspension was incubated at 37°C and
250 rpm until OD reached 0.6 (between 16 and 26 hrs) at which
point aliquots (10 µL) were used to inoculate each well of two
96-well microplates (flat colorless bottom) containing M9A,
itaconic acid (0, 0.04, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5, 5, 10, 20
and 40 mM) and compound 1a (0, 0.7 and 1.4 mM from a 140
mM solution in DMSO). The plate was covered with a gas-
permeable moisture barrier adhesive seal (purchased from
4titude, UK) and incubated for 48 hrs at 37°C and 350 rpm.
Negative controls were included on each plate, for growth in the
absence of itaconic acid or active compound, both with and
without DMSO. Positive controls were acquired with kanamycin
(40 µM) or ampicillin (40 µM). Visible light absorption by the
growth medium and by DMSO were subtracted from the
readings. All data points are means from triplicate
measurements.
M. Sugimoto, H. Sakagami, Y. Yokote, H. Onuma, M. Kaneko, M.
Mori, Y. Sakaguchi, T. Soga, M. Tomita, Metabolomics 2012, 8,
624–633.
[3]
[4]
A. Michelucci, T. Cordes, J. Ghelfi, A. Pailot, N. Reiling, O.
Goldmann, T. Binz, A. Wegner, A. Tallam, A. Rausell, et al., Proc.
Natl. Acad. Sci. U. S. A. 2013, 110, 7820–7825.
K. Höner Zu Bentrup, A. Miczak, D. L. Swenson, D. G. Russell, J.
Bacteriol. 1999, 181, 7161–7167.
[5]
[6]
H. L. Kornberg, Biochem. J. 1966, 99, 1–11.
P. S. Solomon, R. C. Lee, T. J. G. Wilson, R. P. Oliver, Mol.
Microbiol. 2004, 53, 1065–73.
[7]
[8]
M. C. Lorenz, G. R. Fink, Nature 2001, 412, 83–6.
I. Olivas, M. Royuela, B. Romero, M. C. Monteiro, J. M. Mínguez, F.
Laborda, J. R. De Lucas, Fungal Genet. Biol. 2008, 45, 45–60.
[9]
L. S. Derengowski, A. H. Tavares, S. Silva, L. S. Procópio, M. S. S.
Felipe, I. Silva-Pereira, Med. Mycol. 2008, 46, 125–34.
[10]
J. D. McKinney, K. Höner zu Bentrup, E. J. Muñoz-Elías, A. Miczak,
B. Chen, W. T. Chan, D. Swenson, J. C. Sacchettini, W. R. Jacobs,
D. G. Russell, Nature 2000, 406, 735–738.
[11]
[12]
J. Sasikaran, M. Ziemski, P. K. Zadora, A. Fleig, I. A. Berg, Nat.
Chem. Biol. 2014, 10, 371–377.
Biosynthetic In Vitro Assay
E. coli PanK, PPAT and DPCK were expressed and purified
following existing literature.[24] Each reaction mixture (500 μL)
contained ATP (5.0 mM), KCl (20 mM), MgCl 2 (10 mM), DTT
(2.0 mM), PanK (5 μg, 278 nM), PPAT (5 μg, 500 nM), and
DPCK (5 μg, 454 nM) in Tris-Cl Buffer (50 mM, pH 7.6). The
reaction was initiated with the addition of pantetheine (5.0 mM)
for comparison, water as a negative control, or compounds
1a,g,i,j (5.0 mM). Reactions were incubated for 3 h at RT and
then stopped by heating the mixture to 95°C for 5 min. The
precipitated protein was removed by centrifugation (13,000 rpm
for 5 min), and the supernatant was analyzed by LC−MS.
Reversed-phase analytical HPLC was performed with an
analytical 4.60 × 250 mm, SYNERGI 4 μm Hydro-RP 80A
(Phenomenex) column coupled to an Agilent 6120 Quadrupole
LC−MS system for ESI-MS analysis. The HPLCconditions had
the sample eluted at a flow rate of 0.5 mL min−1 using a
combination of mobile phase A (H2O) and mobile phase B
(acetonitrile). Elution conditions are as follows: isocratic 1%
phase B from 0 to 3 min; followed by the following linear
gradients of phase B: 1−10% from 3−5 min; 10−15% from 5−10
min; 15−30% from 10−13 min; and finally isocratic phase B at
30% from 13−26 min. The detector was set to 214 nm. These
experiments were run in duplicates.
W. R. Martin, F. Frigan, E. H. Bergman, J. Bacteriol. 1961, 82, 905–
908.
[13]
[14]
R. A. Cooper, H. L. Kornberg, Biochem. J. 1964, 91, 82–91.
C. Pujol, J. P. Grabenstein, R. D. Perry, J. B. Bliska, Proc. Natl.
Acad. Sci. U. S. A. 2005, 102, 12909–12914.
[15]
[16]
Y. Zhao, R. Jansen, W. Gaastra, G. Arkesteijn, B. A. M. van der
Zeijst, J. P. M. van Putten, Infect. Immun. 2002, 70, 5319–5321.
C. A. Santiviago, M. M. Reynolds, S. Porwollik, S.-H. Choi, F. Long,
H. L. Andrews-Polymenis, M. McClelland, PLoS Pathog. 2009, 5,
e1000477.
[17]
[18]
K. Vong, I. S. Tam, X. Yan, K. Auclair, ACS Chem. Biol. 2012, 7,
470–475.
R. Leonardi, Y.-M. Zhang, C. O. Rock, S. Jackowski, Prog. Lipid
Res. 2005, 44, 125–153.
[19]
[20]
S. Jackowski, C. O. Rock, J. Bacteriol. 1984, 158, 115–120.
K. Yang, Y. Eyobo, L. A. Brand, D. Martynowski, D. Tomchick, E.
Strauss, H. Zhang, J. Bacteriol. 2006, 188, 5532–5540.
[21]
[22]
T. Artemova, Y. Gerardin, C. Dudley, N. M. Vega, J. Gore, Mol. Syst.
Biol. 2015, 11, 1–11.
C. L. Strelko, W. Lu, F. J. Dufort, T. N. Seyfried, T. C. Chiles, J. D.
Rabinowitz, M. F. Roberts, J. Am. Chem. Soc. 2011, 133, 16386–
16389.
[23]
[24]
R. C. Allen, R. Popat, S. P. Diggle, S. P. Brown, Nat. Rev. Microbiol.
2014, 12, 300–308.
I. Nazi, K. P. Koteva, G. D. Wright, Anal. Biochem. 2004, 324, 100–