Targeting Mycolic Acid Transport by Indole-2-carboxamides for the Treatment of Mycobacterium abscessus Infections

Article abstract of DOI:10.1021/acs.jmedchem.7b00582

Mycobacterium abscessus is a fast-growing, multidrug-resistant organism that has emerged as a clinically significant pathogen in cystic fibrosis (CF) patients. The intrinsic resistance of M. abscessus to most commonly available antibiotics seriously restricts chemotherapeutic options. Herein, we report the potent activity of a series of indolecarboxamides against M. abscessus. The lead compounds, 6 and 12, exhibited strong activity in vitro against a wide panel of M. abscessus isolates and in infected macrophages. High resistance levels to the indolecarboxamides appear to be associated with an A309P mutation in the mycolic acid transporter MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis remained unaffected, the indolecarboxamides strongly inhibited the transport of trehalose monomycolate, resulting in the loss of trehalose dimycolate production and abrogating mycolylation of arabinogalactan. Our data introduce a hereto unexploited chemical structure class active against M. abscessus infections with promising translational development possibilities for the treatment of CF patients.

Full text of DOI:10.1021/acs.jmedchem.7b00582

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Article
Targeting Mycolic Acid Transport by Indole-2-carboxamides
for the Treatment of Mycobacterium abscessus Infections
Alan P. Kozikowski, Oluseye K Onajole, Jozef Stec, Christian Dupont, Albertus Viljoen, Matthias Richard,
Tridib Chaira, Shichun Lun, William R. Bishai, V. Samuel Raj, Diane Ordway, and Laurent Kremer
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017
Downloaded from http://pubs.acs.org on June 2, 2017
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Targeting Mycolic Acid Transport by Indoleꢀ2ꢀcarboxamides for
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the Treatment of Mycobacterium abscessus Infections
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Alan P. Kozikowskia*, Oluseye K. Onajolea,b,#, Jozef Stecc,d,#, Christian Duponte,#, Albertus Viljoene,
Matthias Richarde, Tridib Chairaf,g, Shichun Lunh, William Bishaih, V. Samuel Rajf, Diane Ordwayi,
and Laurent Kremere,j
aDrug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, College of
Pharmacy, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612, USA.
bDepartment of Biological, Chemical and Physical Sciences, Roosevelt University, 425 S. Wabash
Avenue, Chicago, IL 60605, USA.
cDepartment of Pharmaceutical Sciences, College of Pharmacy, Chicago State University, 9501 S.
King Drive, Chicago, IL 60628, USA.
dDepartment of Pharmaceutical Sciences, College of Pharmacy, Marshall B. Ketchum University,
2575 Yorba Linda Boulevard, Fullerton, CA 92831, USA.
eInstitut de Recherche en Infectiologie (IRIM), CNRS, UMR 9004,Université
de Montpellier, France.
fCentre for Drug Design Discovery and Development (C4D), SRM University, DelhiꢀNCR, Rajiv
Gandhi Education City, Sonepat – 131 029, Haryana, India
gDaiichi Sankyo India Pharma Private Limited, Sector – 18, Gurgaon, 122 015, Haryana, India
hJohns Hopkins School of Medicine, JHU Center for TB Research, 1550 Orleans St., Baltimore,
MD, USA.
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iColorado State University, Department of Microbiology, Immunology & Pathology, Mycobacteria
Research Laboratory, Fort Collins, CO 80523 USA
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jINSERM, IRIM, 34293 Montpellier, France.
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KEYWORDS: Mycobacterium abscessus, pathogen, indoleꢀ2ꢀcarboxamides, mycolic acid
transporter, antimicrobial agent, cystic fibrosis.
ABSTRACT
Mycobacterium abscessus is a fastꢀgrowing, multidrugꢀresistant organism that has emerged as a
clinically significant pathogen in cystic fibrosis (CF) patients. The intrinsic resistance of M.
abscessus to most commonly available antibiotics seriously restricts chemotherapeutic options.
Herein, we report the potent activity of a series of indolecarboxamides against M. abscessus. The
lead compounds, 6 and 12, exhibited strong activity in vitro against a wide panel of M. abscessus
isolates and in infected macrophages. High resistance levels to the indolecarboxamides appear to be
associated with an A309P mutation in the mycolic acid transporter MmpL3. Biochemical analyses
demonstrated that while de novo mycolic acid synthesis remained unaffected, the
indolecarboxamides strongly inhibited the transport of trehalose monomycolate, resulting in the
loss of trehalose dimycolate production and abrogating mycolylation of arabinogalactan. Our data
introduce a hereto unexploited chemical structure class active against M. abscessus infections with
promising translational development possibilities for the treatment of CF patients.
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INTRODUCTION
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Fighting multidrugꢀresistant bacteria requires repurposing of existing drugs or the development of
new more rapidly acting and cost effective antibiotics. Chronic obstructive pulmonary disease
(COPD) and cystic fibrosis (CF) are associated with unresolved therapeutic needs, resulting from
severe and even fatal infections by multidrugꢀresistant bacteria. Lung infections in CF patients
represent the most frequent and most serious manifestations, since they are responsible for more
than 90% of CF patient deaths.1 In the context of CF and COPD, Mycobacterium abscessus, a fastꢀ
growing mycobacterial species, has emerged in recent years as an important opportunistic pathogen
increasingly responsible for mortality.2 The incidence of M. abscessus infection in the CF
population rises, suggesting a potential link between a genetic defect in the Cystic Fibrosis
Transmembrane Conductance Regulator (CFTR) and M. abscessus lung infection. In this
population, M. abscessus accelerates inflammatory lung damage, leading to increased morbidity
and mortality.3 A recent genomic study of a global collection of clinical isolates indicated that the
majority of M. abscessus infections are acquired through aerosol transmission of recently emerged
dominant circulating clones.4
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Although a rapid grower, M. abscessus shares important physiopathologic features with
pathogenic slowꢀgrowing mycobacteria such as the ability to persist silently for years and even
decades5 in the human host and to induce lung disease associated with caseous lesions and
granuloma formation in lung parenchyma.6, 7 Infections caused by M. abscessus are difficult to treat
and to eradicate because M. abscessus is naturally resistant to most antibiotics, including nearly all
current antitubercular drugs.8 The recommended treatment for pulmonary infections includes a
combination of a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and
an intravenous βꢀlactam (cefoxitin or imipenem) to be taken at least for 12 months9, 10 (Figure 1).
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The cure rate is estimated to only 25ꢀ40% in the case of resistance to macrolides, which appears in
40ꢀ60% of the isolates.11 In addition, unsuccessful eradication of M. abscessus represents a
contraindication for lung transplantation by several CF centers, leaving patients without therapeutic
options. Therefore, more specific, active, and at the same time less toxic antimicrobial agents are
urgently needed.
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Despite being a formidable respiratory mycobacterial pathogen, little is known with respect to
the molecular and cellular mechanisms leading to immunopathogenesis and infection. However,
recent genomic data highlighted the considerable sequence similarity between Mycobacterium
tuberculosis and M. abscessus12, 13, suggesting that these species may share common biochemical
pathways. From this, it can be inferred that compounds inhibiting biosynthetic pathways in M.
tuberculosis may also be active against M. abscessus, and that already existing data generated
during previous tuberculosis (TB) drug discovery programs would be useful to identify new
chemotypes with strong activity against M. abscessus, an approach that would obviate the need to
initiate chemical screens de novo. Indeed, we have recently validated this approach and undertaken
an M. abscessus “crossꢀscreen” with a confirmed chemical series arising from a known set of potent
nonꢀtoxic antitubercular hits.14 This led to the discovery of a new piperidinolꢀbased compound 1
(PIPD1) (Figure 1), showing selective activity against M. abscessus both in vitro and in vivo.15
This success prompted us to further explore this strategy by testing a small library of indoleꢀ2ꢀ
carboxamide derivatives, previously shown to exhibit strong activity against M. tuberculosis.16ꢀ18
In the recent past, we have designed a series of indolecarboxamides with potent activity against
both drugꢀsusceptible and drugꢀresistant strains of M. tuberculosis by targeting the putative mycolic
acid transporter MmpL3.18 We identified a single point mutation in MmpL3, which confers high
resistance to the indolecarboxamide class, while remaining susceptible to currently used firstꢀ and
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secondꢀline TB drugs, signifying a lack of crossꢀresistance. The lead compound 2 (Figure 1) was
found to be active against M. abscessus, thus prompting further investigation of other
indolecarboxamide analogs. The bioavailability of the indolecarboxamides, combined with their
ability to kill tubercle bacilli as well as nonꢀtubercular mycobacteria (NTM), indicates great
potential for the translational development of this structural class for the treatment of M. abscessus
infections in CF patients. Herein, we describe SAR studies of a series of indolecarboxamide
analogs against M. abscessus and provide biological evidence that they are working through
inhibition of mycolic acid transport.
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(Figure 1)
RESULTS
The synthesis of compounds 2ꢀ29 was carried out by following procedures reported previously.16ꢀ18
Briefly, starting from the appropriate indoleꢀ2ꢀcarboxylic acid, a standard amide coupling protocol
(Scheme 1) was performed to furnish the amides 2ꢀ23 and 25ꢀ28. Compound 24 was obtained by
LiAlH4 reduction of the amide 2, whereas compound 29 was prepared by coupling intermediate 45
with cycloheptanecarbonyl chloride.
(Scheme 1)
Structureꢀactivity relationship of new indoleꢀ2ꢀcarboxamides against M. abscessus.
The indoles 2ꢀ29 were initially screened in vitro against the M. tuberculosis16, 17 and M. abscessus
strains to obtain their respective MIC values (Table 1). The lead compound 2 with a cyclooctyl
group displayed excellent activity against both M. tuberculosis and M. abscessus. Presence of only
a single methyl at positions 4, 5, or 6 of the indole ring (compounds 3ꢀ5) resulted in a twoꢀfold
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decrease in activity against M. abscessus in comparison to 2. A similar trend in activity was
observed when these compounds were screened against M. tuberculosis16, thus indicating that the
presence of two nonꢀpolar substituents in the indole ring helps to improve activity. Likewise,
compound 6 that possesses a lipophilic bromine atom at position 6 displayed a twoꢀfold increase in
activity when compared to its 6ꢀmethyl counterpart, compound 5. Compound 7 bearing an Nꢀ
adamantyl group was nearly 130ꢀfold less active than compounds 6 and 8. These findings further
support the notion that the activity of these compounds is driven by their lipophilicity, and that the
tolerance for steric bulk at the amide N is fairly high but limited. Additionally, the effect of a single
methyl group at position 7, as in compound 9, was evaluated. Compound 9 showed a large loss in
activity against M. tuberculosis and M. abscessus as compared to other monoꢀsubstituted
compounds with methyl groups at positions 4, 5, or 6 (3ꢀ5), indicating that substitution at this
position is unfavorable. Next, we investigated the effect of diꢀsubstitution at other positions of the
indole ring. The 5,7ꢀdimethylindole 10 showed 16ꢀ and 2ꢀfold loss of activity against the M.
tuberculosis and M. abscessus strains, respectively, as compared to its 4,6ꢀdimethyl counterpart 2.
In this case, both molecules have equal CLogP values, and the differential activities observed are
thus a consequence of the substitution pattern in the indole ring.
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(Table 1)
After finding that substituents at the 4 and 6ꢀpositions of the indole ring were optimal for activity,
other 4,6ꢀdisubstituted derivatives, such as the 4,6ꢀdimethoxy, 4,6ꢀdichloro, 4,6ꢀdifluoro, and 4,6ꢀ
bisꢀ(trifluoro)methyl analogs, were investigated for their activity against M. abscessus. Compound
11 bearing the 4,6ꢀdimethoxyindole moiety displayed an 8ꢀfold loss of activity compared to
compound 2 (4,6ꢀdimethyl substitution), suggesting that the more polar methoxy substituents are
less favorable compared to methyl due to their reduced lipophilicity. Next, we replaced the two
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methyl groups of 2 with more lipophilic and metabolically stable halogen atoms to afford
compounds 12 (4,6ꢀdichloroindole analog; CLogP = 6.16) and 13 (4,6ꢀdifluoroindole analog;
CLogP = 5.02). Both of these compounds showed activities identical (0.12 ꢁg/mL) to that of the
lead compound 2 against M. abscessus. In the case of M. tuberculosis, compound 12 displayed the
same activity as did the lead 2, while compound 13 was approximately 8ꢀfold less active than
compound 2. Of interest is the fact that while compound 12 is more lipophilic than 13, this property
did not enhance its activity in the M. abscessus screening assay in contrast to the result that was
observed in the case of the M. tuberculosis assay. Both compounds 14 and 15 were inactive against
M. abscessus while retaining activity against M. tuberculosis.
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Next, the activities of the 4,6ꢀdichloroindole analogs 16–22 bearing different substituents on the
amide nitrogen were explored. Compounds 16 (containing a Nꢀadamantyl group) and 17
(containing a Nꢀ(R)ꢀ(+)ꢀbornyl group) showed poor activities (MIC > 32 ꢁg/mL) against M.
abscessus. These modifications thus resulted in an at least 256ꢀfold decrease in activity of 16 and
17 as compared to lead compound 2. Introduction of a simple nꢀoctyl group or a transꢀgeranyl
group on the amide nitrogen yielded the compounds 18 and 19. Again, both of these compounds
were found to be inactive (MIC > 32 ꢁg/mL) against M. abscessus, suggesting that these lipophilic
alkyl and alkenyl chains are likely unable to bind to the target site. The analog 20 bearing an αꢀ
branched cyclohexylmethyl group (MIC = 0.5 ꢁg/mL) was moderately active whereas compound
21 bearing the indanyl group proved to be inactive against M. abscessus. The spiro compound 22
(CLogP = 7.04) proved to be the most active compound against M. tuberculosis (MIC = 0.00098
ꢁg/mL) in the in vitro assay; disappointingly, this excellent activity was not duplicated in the M.
abscessus screening (MIC > 32 ꢁg/mL). Furthermore, the reverse amide derivative 23 was inactive
against M. abscessus, while the amine 24 exhibited a 4ꢀfold drop in activity compared to 2.
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Compound 25 containing a 4,6ꢀbis(trifluoromethyl)indole, and the benzofuran derivatives 26ꢀ2817
showed MIC values between 0.5 and 1.0 µg/mL, while the benzothiazole derivative 29 was inactive
against the susceptible strain of M. abscessus.
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Overall, the SAR of these indoleꢀ2ꢀcarboxamides reveals some parallels in their activity against
M. tuberculosis and M. abscessus, but this is not strictly true for all of the analogs that were studied.
In most cases the indole analogs reported herein are more active against M. tuberculosis than
against M. abscessus. Also, it is apparent that, based on the limited number of compounds studied
so far, the cyclooctyl group is preferred for activity against M. abscessus and that in a few cases
these cyclooctyl analogs are more active against M. abscessus than M. tuberculosis. We believe that
the present data provide fertile ground to inspire the generation of a host of improved analogs that
may show even better activity against M. abscessus.
Activity of compounds 12 and 6 against M. abscessus clinical isolates.
The two lead compounds, 12 and 6, were further evaluated in in vitro and in vivo assays. Both
exhibited an MIC of 0.125 µg/mL against the smooth (S) as well as the rough (R) variants of the
reference M. abscessus strain CIP104536 (Table 2). Notably, both compounds were similarly
active against 9 different clinical strains isolated from CF and nonꢀCF patients. The M. abscessus
complex is classified into three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp.
bolletii, and M. abscessus subsp. massiliense19, 20, and the distinction between these subspecies is
clinically relevant because they can respond differently to antibiotics.20 We found that 30 clinical
isolates comprising 11 isolates of M. abscessus subsp. abscessus, 11 of M. abscessus subsp.
massiliense, and 8 of M. abscessus subsp. bolletii with different susceptibility profiles to amikacin,
were equally sensitive to 12 and 6, generally exhibiting MICs of 0.125 µg/mL (Table 2). The lack
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of crossꢀresistance between the indolecarboxamides and amikacin suggests that these compounds
inhibit a different biological function, which is not targeted by amikacin.
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To further understand the properties of the indolecarboxamides, exposure of M. abscessus
104536 (S) in exponential growth phase to 0.125 µg/mL of compound 12, corresponding to 1 x
MIC, resulted in a notable reduction in viable colonyꢀforming units (CFU) of about 2 orders of
magnitude in 3 days as compared to the initial inoculum. This in vitro killing effect of compound
12 was even more pronounced than the one obtained when exposing the cultures to 8 µg/mL
imipenem (2 x MIC) (Figure 2A). A comparable killing effect was obtained when cultures were
treated with the same dose of compound 6 (Figure 2A).
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(Figure 2 and Table 2)
Activity of compound 12 against intracellular M. abscessus.
The intracellular killing activity of 12 was next assessed after infection of PMAꢀactivated THPꢀ1
macrophages. To avoid growth of extracellular mycobacteria, cells were extensively washed and
treated with amikacin. When left untreated for 4 days, the number of intracellular M. abscessus was
increased by more than 2 orders of magnitude, while exposure to compound 12 (3 µg/mL, 24 x
MIC) prevented any multiplication within the macrophages (Figure 2B). Treatment with imipenem
(96 µg/mL, 24 x MIC) also significantly reduced the intracellular M. abscessus load, similar to
compound 12. The intracellular activity of both compounds was further confirmed by counting the
number of macrophages infected with M. abscessus expressing tdTomato at day 4 postꢀtreatment. A
significant decrease in the percentage of infected cells treated with either imipenem or compound
12 was found (Figure 2C). Overall, these results clearly indicate that compound 12 is able to enter
human macrophages and to stop bacterial replication.
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The A309P mutation in MAB_4508 confers high resistance levels to compound 12.
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To search for the molecular mechanism of action of the inhibitor, we first selected a spontaneous
mutant of M. abscessus resistant to compound 12, designated CIP_OK4ꢀ10R, which was
subsequently found to exhibit high resistance levels to both compounds 12 and 6 (MIC values of 32
µg/mL) (Table 3). Gene sequencing identified a single g925c single nucleotide polymorphism in
MAB_4508, encoding MmpL3, which results in an amino acid replacement at position 309
(A309P). Interestingly, we had previously identified the same mutation in a compound 1ꢀresistant
mutant (CIP_PIPD1R),15 which showed crossꢀresistance with compounds 12 and 6. As anticipated,
the A309P mutant selected on compound 12 was found to be resistant to compound 1 (Table 3).
To further validate the A309P mutation as the mechanistic basis for resistance to the
indolecarboxamides, both the wildꢀtype MAB_4508 gene and the MAB_4508 allele harboring the
A309P mutation were cloned in pMV261 under the control of the constitutive hsp60 promoter to
allow overexpression of the wildꢀtype and mutated versions of the protein in M. abscessus.
Whereas overproduction of the wildꢀtype protein failed to result in increased resistance to
compounds 6 or 12 as compared to the control drugꢀsusceptible strain, overexpression of the
mutated protein resulted in high resistance levels when plated on 7H10OADC media (Figure 3). This
indicates that transferring the single point mutation identified in the spontaneously resistant mutant
to a susceptible strain is sufficient to confer resistance to compounds 6 and 12.
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Together, these results implicate the A309P mutation as critical in conferring high resistance
levels to indoleꢀ2ꢀcarboxamides in M. abscessus and present evidence for the involvement of
MmpL3 in the mode of action of this family of inhibitors.
(Figure 3)
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Lead compound 12 inhibits mycolic acid transport in M. abscessus.
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Indolecarboxamides have previously been proposed to target the mycolic acid transporter MmpL3
in M. tuberculosis18, and it has been reported that a mutation in mmpL3 correlates with resistance in
M. abscessus. To explore whether compound 12 may alter this pathway in M. abscessus, untreated
and 12ꢀtreated cultures were metabolically labeled with [14C]acetic acid to monitor (glyco)lipid
synthesis. When applied at concentrations of up to 50 x MIC (6.2 µg/mL), compound 12 had no
effect on de novo mycolic acid biosynthesis (Figure 4A). However, following apolar lipid
extraction and their subsequent separation by thin layer chromatography (TLC), a dramatic
inhibition of trehalose dimycolate (TDM) synthesis was observed concomitantly with an
accumulation of trehalose monomycolate (TMM) (Figure 4B and 4D), the latter being the substrate
of MmpL3 for the transport of mycolic acids onto the mycobacterial surface where TDM is formed.
To test whether treatment with compound 12 also impacts mycolylation of arabinogalactan (AG),
radiolabeled mycolic acids were extracted from delipidated bacteria.15, 21 The doseꢀdependent
inhibition of [14C]acetic acid incorporation suggests that compound 12 inhibits AG mycolylation
with a pronounced decrease of cell wallꢀbound αꢀ and α’ꢀ mycolic acids (Figure 4C and 4D). The
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two types of mycolic acids (i.e., α and α’) correspond to longꢀchain (C77ꢀ79) and shortꢀchain (C62ꢀ64
)
mycolic acids, respectively, as reported earlier.22 Indeed, compound 12 behaved similarly to
compound 1, an unrelated compound that inhibits the transport of mycolic acid transport in M.
abscessus by targeting MmpL3.15 Importantly, the mutant strain carrying the MAB_4508(A309P)
allele was found to be totally refractory to the inhibition of TMM transport and to obstruction of
AG mycolylation as compared to the parental strain, even in the presence of high concentrations of
compound 12 (Figure 4EꢀG). In addition, very similar TLC profiles were obtained when treating
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M. abscessus cultures with increasing doses of compound 6 (Supporting Information, Figure S1),
indicating that both analogs target the same biosynthetic pathway. To exclude the possibility that
compounds 12 and 6 may inhibit the synthesis of TDM and mycolylated arabinogalactan directly
rather than the mycolic acid transport by targeting the mycolyltransferase activity of the antigen 85
complex23, both compounds were tested for their potential inhibitory activity in an in vitro
enzymatic assay using purified Ag85C24. This assay is based on a fluorescent probe (resorufin
butyrate) that produces a direct measurement of the Ag85C enzymatic activity. Concentrations
ranging from 2.5 to 50 µM of compounds 6 or 12 failed to inhibit the Ag85C activity (Supporting
Information, Figure S2). In sharp contrast, ebselen inhibited the activity of Ag85C in a doseꢀ
dependent manner, as reported previously24.
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Taken collectively, these biochemical data underline a mode of action that results in the
abolition of mycolic acid translocation from the cytoplasm where they are synthesized, to the
periplasmic side of the plasma membrane for subsequent use in the biogenesis of TDM and transfer
onto the essential AG component of the mycobacterial cell wall.
(Figure 4)
Preliminary ADME studies on compound 12.
Based on the encouraging biological results described above, selected ADME studies were
conducted on compound 12 to assess its drugꢀlike properties (Table 4). The cell permeability of
compound 12 was measured in the PAMPA cell line assay system. Compound 12 showed a low
permeability with 0.2 x 10ꢀ6 cm/sec at pH 7.4 and 0.3 x 10ꢀ6 cm/sec at pH 5.0, respectively. In
mouse and rat liver microsomes, compound 12 showed a high intrinsic clearance of 4.8 mL/min/g
liver and 6.3 mL/min/g liver, respectively. Compound 12 showed a low propensity to inhibit CYP
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1A2, 2C8 2C9, 2C19, 2D6, and 3A4 with an IC50 value of ≤ 20 ꢁM. The compound exhibited high
plasma protein binding in mouse (98.9%) and rat (99.2%). Based on the AMES mutagenicity assay,
compound 12 showed no mutagenic potential in the histidine auxotrophic strains of S. typhimurium
TA98 and TA100 at 0.24–500 ꢁg/well. Since the compound showed less than a 2ꢀfold increase in
the number of revertant colonies compared to the negative controls in either the presence or absence
of S9, it can be inferred that it has no mutagenic potential under the test conditions. The compound
showed precipitation at 500 and 250 ꢁg/well.
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DISCUSSION AND CONCLUSIONS
M. abscessus is an emerging causative agent of chronic lung disease, particularly in patients with
altered host defenses or disrupted airway clearance mechanisms, such as in CF. M. abscessus
infection represents a threat to patients with CF with increased prevalence in recent years.25,26
Among mycobacterial species, M. abscessus is regarded as one of the most drugꢀresistant species,
and chemotherapeutic options are very limited.8 The fact that most available treatments are often
unsuccessful or poorly tolerated by patients, emphasizes the urgent need for developing more active
and betterꢀtolerated drugs. As originally conjectured, the notion of screening drugs that had
previously demonstrated activity against M. tuberculosis for possible efficacy against M. abscessus
has been shown to provide good lead candidates for combating M. abscessus infections.15, 27
Following this crossꢀscreen approach, we have screened a library of indoleꢀ2ꢀcarboxamides,
reported to be highly active against M. tuberculosis.16, 17 This allowed us to define a new structural
class of compounds exhibiting promising activity against M. abscessus, further validating the value
of utilizing data obtained from prior TB screens to directly identify new chemotypes with strong
activity against M. abscessus. Our lead compounds 6 and 12 display very low MIC values against a
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vast panel of clinical strains with different susceptibility profiles to antibiotics, regardless of
whether they were isolated from CF or nonꢀCF patients. Moreover, the efficacy of compounds 6
and 12 was not dependent on the smooth or rough morphotype of the M. abscessus subspecies,
which is of particular interest since M. abscessus subsp. abscessus, M. abscessus subsp.
massiliense, and M. abscessus subsp. bolletii infections can present various drug sensitivity test
results, responses to antibiotics, and clinical symptoms.28, 29 Additionally, compound 12 exhibited
appreciable activity in infected macrophages.
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To pinpoint the possible molecular mechanism of action of these inhibitors, we identified a
spontaneous mutant which exhibited a high resistance phenotype to both lead compounds. Gene
sequencing identified a single g925c single nucleotide polymorphism in MAB_4508, encoding
MmpL3. This mutation resulted in an A309P amino acid replacement also previously identified in a
compound 1ꢀresistant mutant,15 which itself showed crossꢀresistance to compounds 6 and 12.
Consistent with these findings, compounds 6 and 12 inhibited the translocation of TMM, resulting
in the abolition of both TDM formation and mycolylation of AG. Our analyses also revealed that
compounds 6 and 12 were not capable to inhibit the activity of Ag85C in vitro. Together, our
combined genetic and biochemical data reflect the in accessibility of the intracellularly generated
TMM to the Ag85 family of mycolyltransferases,23 similarly to other MmpL3 inhibitors, such as
compound 1,15 the adamantylꢀurea AU1235,21,30 or NꢀgeranylꢀN'ꢀ(2ꢀadamantyl)ethaneꢀ1,2ꢀdiamine
(SQ109).30
Furthermore, sequence analysis indicates that the MmpL3 protein of M. abscessus (MAB_4508)
and its counterpart in M. tuberculosis (Rv0206c)18,31 show high similarity in multiple aspects. In
addition to similar gene sizes, 3006 and 2835 base pairs, respectively, amino acid sequence blasting
also revealed 56% identity and 69% similarity. Bioinformatic analysis indicates that both
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MAB_4508 and Rv0206c are transmembrane proteins comprising 12 transmembrane helices.
Currently, MmpL3 appears to be one of the most promising antimycobacterial pharmacological
targets as multiple chemical entities have recently been shown to inhibit MmpL3 activity not only
in M. tuberculosis but also in M. abscessus, thus opening a new field centered on the inhibition of
mycolic acid transport.15, 18, 21, 30ꢀ34 Interestingly, recent findings indicate that MmpL3 inhibitors can
also act synergistically with other antiꢀtubercular drugs, which is of interest to efforts aiming at
reducing the length of treatments.35 Whether indoleꢀ2ꢀcaboxamides may also act in synergy with
drugs used for the treatment of M. abscessus infections remains to be investigated.
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Tetrahydropyrazo[1,5ꢀa]pyrimidineꢀ3ꢀcarboxamide (THPP) has recently been reported to
inhibit the biogenesis of mycolic acids by targeting the enoylꢀcoenzyme A (CoA) hydratase
EchA6 rather than the previously assumed MmpL3 target,36 even though several mutations
identified in mmpL3 had been proposed to confer resistance to THPP37. It was subsequently
demonstrated that THPP competes with CoAꢀbinding so as to arrest mycolic acid production,
leading to the conclusion that spontaneous resistanceꢀconferring mutations in mmpL3 can
potentially obscure the actual target identification of small inhibitors. Our results failed to show an
inhibition of mycolic acid biosynthesis, even in the presence of high concentrations of compounds
6 or 12, and clearly demonstrated a doseꢀdependent inhibition of TDM synthesis with a
concomitant accumulation of TMM. In addition, overexpression of the MmpL3(A309P) variant in
a genetically susceptible strain correlated with high resistance levels to compounds 6 and 12 and
rendered the strain refractory to TMM transport inhibition. Together, these results extend the
results obtained previously in M. tuberculosis that indoleꢀ2ꢀcarboxamides act by altering the
transport of TMM across the membrane in pathogenic, fastꢀgrowing nonꢀtubercular mycobacteria.
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How compound 1 and our indoleꢀ2ꢀcarboxamides inhibit MmpL3, and whether they have access
to the same or different binding cavities in MmpL3, remains to be established. The design of further
improved indolecarboxamides targeting mycolic acid transport would also greatly benefit from
future structural and functional studies on MmpL3. Moreover, details as to whether the activity of
the indolecarboxamides results from specific inhibition of the MmpL3 transporter functions or
through indirect mechanisms, for example involving the dissipation of the protonꢀmotive force as
recently suggested38, require additional investigations. In summary, our indoleꢀ2ꢀcarboxamides
represent a very promising chemotype that is able to alter the mycolic acid profile in M. abscessus,
a species that is naturally resistant to most mycolic acid biosynthesis inhibitors. Of particular
interest for medicinal chemistry and drug development, these indole analogs are easy to prepare and
generally show reasonably good ADME properties as reported earlier16 and as further investigated
herein for compound 12. This work, along with previous findings using compound 1, support the
view that targeting the transport of mycolic acids rather than their biosynthesis, has significant
translational potential for development of these chemotypes into real drugs for M. abscessus
treatment and control. Research is currently underway to assess their efficacy in M. abscessus
animal models.
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EXPERIMENTAL SECTION
Chemistry.
General information. The tested 28 compounds were synthesized and characterized as reported in
literature.16, 17 Purities of final compounds were confirmed to be ≥ 95% by analytical HPLC, which
was carried out using an Agilent 1100 HPLC system with a Synergi 4 ꢁm HydroꢀRP 80A column,
and a variable wavelength detector G1314A; method 1, flow rate 1.4 mL/min, gradient elution over
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20 min, from 30% MeOH–H2O to 100% MeOH with 0.05% TFA added to both solvents; method 2,
flow rate 1.4 mL/min, gradient elution over 20 min, from 50% MeOH–H2O to 70% MeOH–H2O
with 0.05% TFA added to both solvents.
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Bacterial strains.
M. abscessus subsp. abscessus CIP104536T, M. abscessus subsp. bolletii CIP108541T, and M.
abscessus subsp. massiliense CIP108297T reference strains were used along with a series of clinical
isolates as reported previously.27 Strains were routinely grown and maintained at 30 °C in
Middlebrook 7H9 broth (BD Difco) supplemented with 0.05% Tween 80 (SigmaꢀAldrich) and 10%
oleic acid, albumin, dextrose, and catalase (OADC enrichment; BD Difco) (7H9T/OADC), or on
Middlebrook 7H10 agar (BD Difco) containing 10% OADC enrichment (7H10OADC), and in the
presence of antibiotics when required. For drug susceptibility testing, bacteria were grown in
cationꢀadjusted MuellerꢀHinton broth (CaMHB; SigmaꢀAldrich).
Drug susceptibility testing.
The minimal inhibitory concentrations (MIC) were determined according to the Clinical and
Laboratory Standards Institute (CLSI) guidelines.39 The broth microdilution method was used in
CaMHB with an inoculum of 5x106 CFU/mL in the exponential growth phase. Briefly, the bacterial
suspension was seeded in 100 µL volumes in all of the wells of a 96ꢀwell plate, except for the first
column to which 198 µL of the bacterial suspension was added to each well. In the first column, 2
µL of compound at its highest concentration was added in six wells (the solvent used to dissolve the
compound was added to the two outermost wells as a control). Twoꢀfold serial dilutions were then
carried out by transferring 100 µL from the wells in the first column to the next column and
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repeating this for each successive column. Plates were subsequently incubated at 30 °C for 3ꢀ5
days. MICs were recorded by visual inspection and by absorbance at 560 nm to confirm visual
recordings. Experiments were done in triplicate on three independent occasions.
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Timeꢀkill assay.
Microtiter plates were set up as for the MIC determination. Serial dilutions of the bacterial
suspensions from these microtiter plates were plated after 0, 24, 48, 72, and 96 h of exposure to
different drug concentrations. Colonyꢀforming units (CFU) were counted after 4 days of incubation
at 30 °C.
Intramacrophage killing assay.
THPꢀ1 cells were grown in RPMI medium containing 10% fetal bovine serum (RPMIFBS) and
differentiated with 20 ng/mL phorbol myristate acetate (PMA) in 12ꢀwell flatꢀbottom tissue culture
microplates (1x105 cells/well) and incubated for 48 h at 37 °C. Cells were infected with a
multiplicity of infection (MOI) of 1:2 and incubated at 37 °C in the presence of 5% CO2 for 2 h.
Wells were carefully washed three times with 1 x PBS, refed with RPMIFBS supplemented with 200
µg/mL amikacin, reꢀincubated at 37 °C in the presence of 5% CO2 for 1 h, and washed again three
times with PBS prior to the addition of 1 mL RPMIFBS supplemented with either 3 µg/mL
compound 12 or 96 µg/mL imipenem. Media were changed on a daily basis with fresh drug
preparations. At various time points (0, 48, and 96 h), cells were washed three times with PBS and
lysed by adding 100 µL 1% Triton X100. Cell lysis was stopped after 2 min by adding 900 µL PBS,
and serial dilutions were plated to monitor the intracellular bacterial counts. Experiments were done
two times independently. For determining the percentage of infected macrophages, THPꢀ1 cells
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were prepared and infected with M. abscessus expressing tdTomato and treated with drugs, as
described above. Untreated and drugꢀtreated infected cells (96 h time point) were observed under a
fluorescence microscope to count the number of bacteriaꢀcontaining macrophages, as previously
described.15
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Selection of a compound 12ꢀresistant mutant.
Exponentially growing M. abscessus cultures were plated on 7H10OADC containing 6 µg/mL of
compound 12. After one week of incubation at 30 °C, a single colony was selected and grown in
liquid medium, assessed by MIC determination, and subsequently scored for resistance to
compound 12. Identification of putative SNPs in the MAB_4508 (MmpL3) gene was carried out by
PCR amplification using the MAB_4508seq1 (5‘ꢀGGCCACCGTCTTTACCAATAꢀ3’) and
MAB_4508seq7 (5’ꢀ GTGCCGTACCTGAATGTCCTꢀ3’) primers to produce a 3549 bp amplicon
to gain full coverage sequencing of MAB_4508, as done previously.15 The SNP was confirmed by at
least two independent sequencing reactions. In order to overꢀexpress the MAB_4508 A309P allele,
the MAB_4508 open reading frame was PCRꢀamplified using as template purified genomic DNA of
CIP_OK4ꢀ10R, Phusion DNA polymerase (Finnzymes, Finland) and the primers MAB_4508f and
MAB_4508r, as previously described.15 The obtained PCR product was subsequently digested with
HindIII and ligated to MscI/HindIII linearised pMV261.
Wholeꢀcell radiolabeling experiments and lipid analysis.
For visualizing drugꢀinduced changes in the lipid profile, increasing concentrations of compound
12 were added to exponentiallyꢀgrowing cultures for 1 h, and metabolic labeling of lipids was
performed by adding 1 ꢀCi/mL of [14C]acetate (56 mCi/mmol) for an additional 2 h at 37 °C. Cells
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were harvested and either delipidated or used immediately to extract mycolic acids, as previously
described.40,22 The apolar lipid fraction containing trehalose monomycolate (TMM) and trehalose
dimycolate (TDM) was separated on a oneꢀdimensional thin layer chromatography (TLC) plate
using chloroform/methanol/water (40:8:1, v/v/v) and revealed after exposure to a film. Delipidated
cells were further processed to extract the mycolic acids41, which were analyzed by
TLC/autoradiography using petroleum ether/acetone (95:5, v/v) and exposure to a film to reveal
14Cꢀlabeled mycolic acid methyl esters (MAME).
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Cell Permeability.
The permeability of compound 12 was measured at two pH values (pH 7.4 and pH 5.0) in PrismaTM
HT aqueous buffer (pION Inc.). The pH was adjusted with 0.5 N NaOH. The test compound in
buffer solutions (25 ꢁM) was added to the PAMPA donor plate (in triplicate). The acceptor plate
was painted with 4 ꢁL of GITꢀ0 lipid solution (pION Inc.), and ASB (acceptor sink buffer, pION
Inc.) was added. The acceptor and donor plates were stacked, sealed with a rubber plate, and
incubated for 4 h at 25 ± 2 °C. The donor and acceptor plate solutions were analyzed by an LCꢀMS
(UPLC) system.
In vitro study ꢀ CYP inhibition.
Inhibition of CYP by compound 12 was evaluated using pooled human liver microsomes (Cat#
452118, BDꢀBiosciences). 10 ꢁM of compound 12 or the positive control was preincubated with
human liver microsomes for 5 min at 37 °C. The reaction was initiated by addition of an NADPH
regenerating system and incubated at 37 °C with gentle agitation for the specified time
recommended for each of the CYP’s (5 min for CYP3A4, 10 min for CYP1A2, 2C8, 2C9, and 2D6,
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and 40 min for CYP2C19). The samples were vacuumꢀfiltered using Captiva 96 well plates
followed by LCꢀMS/MS analysis. The formation of standard metabolite (concentration/peak area
ratio) was estimated. The percentage of inhibition of the test compound and the positive control in
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comparison to a negative control (DMSO) were determined using the equation:
Average metabolite concentration of test compound/positive control
% Inhibition = 100 − ꢀꢁ
ꢂ × 100ꢃ
Average metabolite concentration of no inhibitor control
Plasma Protein Binding.
Plasma protein binding studies were performed using an ultracentrifuge method. A 5 ꢁM solution
of compound 12 was prepared in mouse or rat plasma using 500 ꢁM stock solution (in DMSO) and
incubated for 10 min at 37 °C. For determination of total plasma concentration, 5 ꢁL of incubated
plasma was mixed thoroughly with 50 ꢁL of saline, 50 ꢁL of acetonitrile, and 100 ꢁL of 1 ꢁM
niflumic acid. For determination of unbound concentrations, the incubated plasma sample was
centrifuged at 200,000 x g for 4.5 h, and 50 ꢁL of supernatant from the middle layer was taken after
removing the upper layer with a Beckmann coulter slicer. To this solution, 5 ꢁL blank plasma, 50
ꢁL acetonitrile, and 100 ꢁL of 1 ꢁM niflumic acid were added. The samples were filtered into a 96ꢀ
deep well storage plate with a Captiva 0.45 ꢁm 96ꢀwell filter plate, and appropriate volumes of all
samples were analyzed by LCꢀMS/MS.
Metabolic stability.
Compound 12 (0.5 ꢁM) was incubated in a reaction mixture consisting of mouse or rat liver
microsomes and an NADPH regenerating system. Aliquots were withdrawn at 3 min intervals for
30 min and analyzed for the parent compound by LCꢀMS/MS. The loss of parent was expressed as
the percentage of the test compound remaining, and the rate of decay was estimated by monoꢀ
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exponential decay kinetics. The rate of decay was normalized to microsomal protein expressed as
mL/min/g liver.
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AMES mutagenicity assay.
Compound 12 was evaluated for its mutagenic potential in the presence and absence of the S9
fraction by investigating its ability to induce reverse mutations at the histidine locus in the genome
of Salmonella typhimurium strains. Two histidine auxotrophic strains of Salmonella typhimurium
were used, namely the TA98 strain carrying a frame shift mutation, and the TA100 strain carrying a
baseꢀpair substitution. Plates were incubated at 37 ± 2 °C for 72 h, and the experiments were
carried out in triplicates. DMSO was used as vehicle control. Sodium azide, 2ꢀnitrofluorene (2NF),
and 2ꢀaminoanthracene (2AA) were used as positive controls. A compound was considered positive
if the number of revertants increased by ≥ 2ꢀfold over the negative control in a doseꢀdependent
manner.
ASSOCIATED CONTENT
SUPPORTING INFORMATION
The supporting information contains 2 supplementary figures. Material is available free of charge
via the Internet at http://pubs.acs.org. Molecular formula strings and some data (CSV) are also
available as supporting information.
AUTHOR INFORMATION
#These authors contributed equally
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Corresponding Authors: *To whom correspondence should be addressed. Laurent Kremer: Eꢀ
mail: laurent.kremer@irim.cnrs.fr (biological screening); Alan P. Kozikowski: Eꢀmail:
alankozikowski@gmail.com (medicinal chemistry).
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ACKNOWLEDGMENTS
The authors wish to thank F. RoquetꢀBanères for technical assistance. We are indebted to the
Howard Hughes foundation for providing funds that allowed us to initiate this work. OK
acknowledges Roosevelt University for generous financial support. JS acknowledges Chicago State
University (CSU) College of Pharmacy and CSU Center for Teaching and Research Excellence
(Faculty Seed Grant) for financial support. A Faculty Development Stipend (JS) from the Marshall
B. Ketchum University College of Pharmacy is also acknowledged. The authors wish to thank the
Association Gregory Lemarchal and Vaincre La Mucoviscidose (RF20130500835) for funding CD.
LK also acknowledges the support by the Fondation pour la Recherche Médicale (FRM)
(DEQ20150331719). This work was funded in whole or in part with federal funds from the
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of
Health and Human Services, AIO99534ꢀ02, NIH/NIAID services under contracts
HHSN272201000009I/HHSN27200001 and HHSN272201000009I/HHSN27200005, principal
investigator Dr. A.J. Lenaerts, coꢀprincipal investigator, D. O.
The authors declare no conflict of interest.
ABBREVIATIONS USED:
CF, cystic fibrosis; COPD, chronic obstructive pulmonary disease; CFTR, cystic fibrosis
transmembrane conductance regulator; TB, tuberculosis; NTM, nonꢀtubercular mycobacteria;
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CaMHB, cationꢀadjusted MuellerꢀHinton broth; MIC, minimal inhibitory concentration; CLSI,
Clinical Laboratory Standards Institute; CFU, colonyꢀforming units; PMA, phorbol myristate
acetate; SAR, structural activity relationship; TMM, trehalose monomycolate; TDM, trehalose
dimycolate; TLC, thin layer chromatography; MAME, mycolic acid methyl ester; DMSO, dimethyl
sulfoxide; 2NF, 2ꢀnitrofluorene; 2AA, 2ꢀaminoanthracene; AG, arabinogalactan; THPP,
tetrahydropyrazo[1,5ꢀa]pyrimidineꢀ3ꢀcarboxamide.
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