ACS Medicinal Chemistry Letters
Letter
labeled lipids derived from M. bovis BCG treated with test
compounds (16, 33, 47), BM212 (MmpL3 inhibitor, positive
control), and ethambutol (inhibitor of arabinogalactan syn-
thesis, negative control) is depicted in Figure 2A. Quantifica-
tion of TDM and TMM levels by densitometry revealed a
time-dependent suppression of TDM that coincided with the
accumulation of TMM in lipids extracted from cultures treated
with 16, 33, 47, and BM212 but not ethambutol (Figure
2B,C). This pattern of change was consistent with MmpL3
inhibition.
Next, the experiment was repeated on one of the M. bovis
BCG mutants and here the strain B47-4 was selected because
its mutation C1932G/F644L was the only recurring poly-
morphism among the mutants raised against the test
compounds. Consistent with the aberration in the mmpL3
gene sequence, the anticipated changes in TDM and TMM
levels were not observed in lipids extracted from B47-4
exposed to BM212, 16, 33, and 47 (Figure 2D−F). Similar
findings were obtained with 41, 53, and 59 (Figure S3). These
findings duly recapitulate the MmpL3 inhibitory activities of
these compounds.
The ability of MmpL3 to transfer mycolates across the
mycobacterial inner membrane is dependent on a functioning
proton motive force (PMF), but curiously, inhibition of
MmpL3 is not consistently linked to a loss in the PMF.17−19 In
general, direct inhibitors bind to MmpL3 without dissipating
the electrical or proton gradient that contributes to the PMF.
Indirect MmpL3 inhibitors dissipate the PMF, but the causality
of these events remains to be resolved. Having confirmed
MmpL3 as a putative target of the short-listed compounds, we
proceeded to determine if inhibition was accompanied by a
loss in the membrane potential (ΔΨ) or proton gradient
(ΔpH). Changes in ΔΨ were monitored in M. bovis BCG
cultures using the fluorescence probe DiOC2. Briefly, DiOC2
fluorescence would change from red to green in cells with
depolarized membranes, and declining red/green fluorescence
ratios were indicative of losses in ΔΨ. Here we observed a slow
decline in the fluorescence ratio of treated cells over time, with
pronounced losses observed at time points (≥12 h) that
coincided with cell death. Hence, it was unlikely that these
compounds disrupted the mycobacterial ΔΨ (Figure S4). To
determine if the compounds abolished the proton gradient
(ΔpH), we used the radiometric pH indicator BCECF-AM to
monitor changes in the intracellular pH of spheroplasts
prepared from M. smegmatis.17 Briefly, disruption of ΔpH
would acidify the intracellular space within the spheroplasts.
This was duly observed in the presence of the protonophore
CCCP but not BM212, which inhibited MmpL3 without
disrupting the PMF (Table S4).17 No acidification was also
observed in spheroplasts treated with the test compounds.
Hence, we concluded that these compounds inhibited MmpL3
without disrupting the PMF.
susceptible to microsomal breakdown than the indolecarbox-
amides. Third, the remarkable activity advantage of the N-
cyclooctyl ring was consistently observed in the carboxamides
of all scaffolds, but it was only evident when attached to the
methylamine N of indole. Lastly, the interception of MmpL3
was consistently observed among potent analogs drawn from
the different heteroaromatic rings.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
■
sı
Synthesis, characterization, and purity determinations of
synthesized compounds, NMR spectra of 16 and 33,
MIC, MBC, Vero cell viability, PAMPA Pe, solubility,
microsomal stability, and PMF determinations, mutant
selection, and lipid profiling (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Mei-Lin Go − Department of Pharmacy, National University
Yulin Lam − Department of Chemistry, National University of
Authors
Yu Jia Tan − Department of Chemistry, National University of
Singapore, 117543, Singapore
Ming Li − Department of Pharmacy, National University of
Singapore, 117543, Singapore
Gregory Adrian Gunawan − Department of Chemistry,
National University of Singapore, 117543, Singapore
Samuel Agyei Nyantakyi − Department of Pharmacy,
National University of Singapore, 117543, Singapore
Thomas Dick − Center for Discovery and Innovation,
Hackensack Meridian Health, Hackensack Meridian School
of Medicine, Nutley, New Jersey 07110, United States;
Department of Microbiology and Immunology, Georgetown
University, Washington, D.C. 20057, United States
Complete contact information is available at:
Author Contributions
‡Y.J.T. and M.L. are joint first authors. All authors made equal
contributions.
Notes
The authors declare no competing financial interest.
The incipient indole-2-carboxamide scaffold has yielded
several potent bactericidal lead compounds. Here, we showed
the feasibility of an amide−amine replacement in retaining
potency, as exemplified by the indolylmethylamine 33
(MIC90Mtb 0.13 μM), and importantly, the striking improve-
ment in solubility elicited by this modification. The solubility
advantage persisted even when indole was replaced by the
more lipophilic benzothiophene and benzoselenophene
scaffolds, but overall only the indoles displayed compelling
potencies. Second, the indolylmethylamines displayed favor-
able solubility and PAMPA Pe profiles but were more
ACKNOWLEDGMENTS
■
This work was funded by the Ministry of Education (MoE)
Academic Research Fund Grants R148000234114 and
R148000286114 to M.L.G., Ministry of Health National
Medical Research Council Grant NMRC/TCR/011-NUHS/
2014 (Singapore), and the National Institute of Allergy and
Infectious Diseases of the National Institutes of Health (USA)
Grant R01AI132374 to T.D. The content is solely the
responsibility of the authors and does not necessarily represent
the official views of the NIH. The authors gratefully
H
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX