TBAJ-876, a 3,5-dialkoxypyridine analogue of bedaquiline, is active against Mycobacterium abscessus

Article abstract of DOI:10.1128/AAC.02404-19

Lung disease caused by Mycobacterium abscessus is very difficult to cure, and treatment failure rates are high. The antituberculosis drug bedaquiline (BDQ) is used as salvage therapy against this dreadful disease. However, BDQ is highly lipophilic, displays a long terminal half-life, and presents a cardiotoxicity liability associated with QT interval prolongation. Recent medicinal chemistry campaigns resulted in the discovery of 3,5-dialkoxypyridine analogues of BDQ which are less lipophilic, have higher clearance, and display lower cardiotoxic potential. TBAJ-876, a clinical development candidate of this series, shows attractive in vitro antitubercular activity and efficacy in a murine tuberculosis model. Here, we asked whether TBAJ-876 is active against M. abscessus. TBAJ-876 displayed submicromolar in vitro activity against reference strains representing the three subspecies of M. abscessus and against a collection of clinical isolates. Drug-drug potency interaction studies with commonly used anti-M. abscessus antibiotics showed no antagonistic effects, suggesting that TBAJ-876 could be coadministered with currently used drugs. Efficacy studies, employing a mouse model of M. abscessus infection, demonstrated potent activity in vivo. In summary, we demonstrate that TBAJ-876 shows attractive in vitro and in vivo activities against M. abscessus, similar to its BDQ parent. This suggests that next-generation BDQ, with improved tolerability and pharmacological profiles, may be useful for the treatment of M. abscessus lung disease in addition to the treatment of tuberculosis.

Full text of DOI:10.1128/AAC.02404-19

EXPERIMENTAL THERAPEUTICS
crossm
TBAJ-876, a 3,5-Dialkoxypyridine Analogue of Bedaquiline, Is
Active against Mycobacterium abscessus
Jickky Palmae Sarathy,a Uday S. Ganapathy,b Matthew D. Zimmerman,b Véronique Dartois,b,c Martin Gengenbacher,b,c
Thomas Dickb,c
aDepartment of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
bCenter for Discovery and Innovation, Hackensack Meridian Health, Nutley, New Jersey, USA
cDepartment of Medical Sciences, Hackensack Meridian School of Medicine, Seton Hall University, Nutley, New Jersey, USA
ABSTRACT Lung disease caused by Mycobacterium abscessus is very difficult to
cure, and treatment failure rates are high. The antituberculosis drug bedaquiline
(BDQ) is used as salvage therapy against this dreadful disease. However, BDQ is
highly lipophilic, displays a long terminal half-life, and presents a cardiotoxicity liabil-
ity associated with QT interval prolongation. Recent medicinal chemistry campaigns
resulted in the discovery of 3,5-dialkoxypyridine analogues of BDQ which are less li-
pophilic, have higher clearance, and display lower cardiotoxic potential. TBAJ-876, a
clinical development candidate of this series, shows attractive in vitro antitubercular
activity and efficacy in a murine tuberculosis model. Here, we asked whether TBAJ-
876 is active against M. abscessus. TBAJ-876 displayed submicromolar in vitro activity
against reference strains representing the three subspecies of M. abscessus and
against a collection of clinical isolates. Drug-drug potency interaction studies with
commonly used anti-M. abscessus antibiotics showed no antagonistic effects, sug-
gesting that TBAJ-876 could be coadministered with currently used drugs. Efficacy
studies, employing a mouse model of M. abscessus infection, demonstrated potent
activity in vivo. In summary, we demonstrate that TBAJ-876 shows attractive in vitro
and in vivo activities against M. abscessus, similar to its BDQ parent. This suggests
that next-generation BDQ, with improved tolerability and pharmacological profiles,
may be useful for the treatment of M. abscessus lung disease in addition to the
treatment of tuberculosis.
KEYWORDS TBAJ-876, bedaquiline, Mycobacterium abscessus, NTM, nontuberculous
mycobacteria
Citation Sarathy JP, Ganapathy US,
Zimmerman MD, Dartois V, Gengenbacher M,
Dick T. 2020. TBAJ-876, a 3,5-dialkoxypyridine
analogue of bedaquiline, is active against
Mycobacterium abscessus. Antimicrob Agents
Chemother 64:e02404-19. https://doi.org/10
.1128/AAC.02404-19.
ontuberculous mycobacteria (NTM) are environmental mycobacteria that can
N
cause pulmonary disease (NTM-PD). The disease occurs mostly in patients with
immunodeficiencies or preexisting pulmonary conditions such as cystic fibrosis or
bronchiectasis (1–4). Infection typically occurs through contact with contaminated
household or municipal water, which are habitats for NTM (5, 6). A recent study
proposed that human-to-human transmission may also occur (7). NTM-PD is an emerg-
ing global health concern given that the number of cases is increasing worldwide (8, 9).
The disease is prevalent in the United States, the Middle East, and Asia (10–13).
Mycobacterium abscessus is one of the common NTM species identified in patients with
NTM-PD (10, 13). M. abscessus presents as a complex of three closely related subtaxa,
M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp.
massiliense (14).
Copyright © 2020 Sarathy et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Thomas Dick,
thomas.dick@hmh-cdi.org.
Received 5 December 2019
Returned for modification 3 January 2020
Accepted 13 January 2020
Accepted manuscript posted online 21
January 2020
Published 24 March 2020
Treatment of M. abscessus infections is largely based on recommendations derived
from retrospective studies (15). The regimen typically consists of a macrolide (e.g.,
clarithromycin) combined with an aminoglycoside (amikacin) and a ␤-lactam (imi-
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FIG 1 Structures of BDQ and TBAJ-876. TBAJ-876 was described as “compound 46” previously (45). The
compound retains BDQ’s quinoline (A) and dimethylamino (D) groups. BDQ’s phenyl (B) and naphthalene
(C) groups are replaced by the 2,3,5-trimethoxypyridin-4-yl and 3,5-dimethoxypyridin-4-yl groups,
respectively.
penem or cefoxitin) (1, 16, 17). Chemotherapy can be long-lasting, as negative sputum
cultures for 1 year are recommended before cure can be declared (1, 17). Despite such
stringent clinical practices, success rates are reported to be only 25 to 58%, disease
often reoccurs (18–23), and adverse side effects such as gastrointestinal distress,
ototoxicity, and nephrotoxicity are commonly experienced by patients (23). In addition,
there is a shortage of alternative treatments when regimens fail, as M. abscessus is
intrinsically resistant to many antibiotics (16, 24, 25). Treatment failure is often associ-
ated with inducible clarithromycin resistance (25), which is mediated by the erm(41)
gene. Functional erm(41) can be present in M. abscessus subsp. abscessus and subsp.
bolletii but is typically absent in M. abscessus subsp. massiliense (26, 27). Collectively, the
issues associated with M. abscessus treatment highlight the need to develop new drugs
or reposition existing antibiotics (28, 29).
Bedaquiline (BDQ) (Sirturo) (Fig. 1) is a first-in-class diarylquinoline (DARQ) (30) used
for the treatment of multidrug-resistant tuberculosis (31, 32). This highly potent anti-
tuberculosis drug (30) exerts its antibacterial activity by inhibiting Mycobacterium
tuberculosis F-ATP synthase (30, 33). Interestingly, BDQ is active against the M. abscessus
complex (34–37) and has been utilized as salvage therapy for patients with persistent
M. abscessus infection (38). However, BDQ has several pharmacological and toxicolog-
ical liabilities. The drug is highly lipophilic (cLogP [logarithm of the drug’s partition
coefficient between n-octanol and water] ϭ 7.25), has a long terminal half-life, and
accumulates in tissues (39, 40). Furthermore, the drug causes prolongation of the QT
interval due to inhibition of cardiac human ether-a-go-go-related gene (hERG) potas-
sium ion channels (39, 40). A recent medicinal chemistry campaign, conducted to
address these issues, led to the discovery of 3,5-dialkoxypyridine analogues of BDQ
(41–46). TBAJ-876 (Fig. 1) is a clinical development candidate that emerged from this
series. It displays lower inhibition of hERG channels and higher clearance and retains in
vivo efficacy against M. tuberculosis (45). We have shown that TBAJ-876 is bactericidal
and as potent as BDQ against M. tuberculosis and retains BDQ’s on-target activity via
binding to the c and ␧ subunits of the mycobacterial F-ATP synthase (47, 48).
TBAJ-876 is active not only against M. tuberculosis but also against the fast-growing
saprophytic mycobacterial model organism Mycobacterium smegmatis (47), suggesting
that the compound may display broad-spectrum antimycobacterial activity. The goal of
the present work was to evaluate the clinical potential of TBAJ-876 against M. abscessus
lung disease. In vitro activities of TBAJ-876 against reference strains and clinical isolates
of the M. abscessus complex were measured, and the in vivo efficacy of this new
compound was determined in a mouse model of M. abscessus infection.
RESULTS
TBAJ-876 is active against reference strains of the M. abscessus complex. To
determine whether TBAJ-876 is active against the three subspecies that form the M.
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TABLE 1 Growth-inhibitory potency of TBAJ-876 against reference strains representing the subspecies of the M. abscessus complex in
7H9 and CAMH mediaa
MIC against strain (␮M)
Type of
medium
M. abscessus subsp. abscessus
ATCC 19977
M. abscessus subsp. bolletii
CCUG 50184-T
M. abscessus subsp. massiliense
CCUG 48898-T
DARQ
TBAJ-876
7H9
CAMH
0.48
1.05
0.53
0.40
0.42
1.10
BDQ
7H9
CAMH
0.56
1.20
0.76
0.50
0.64
0.84
aThe experiment was carried out three times independently, and the MICs are displayed as mean values. BDQ was used as a positive control. DARQ, diarylquinoline.
abscessus complex, each subspecies reference strain was tested for its susceptibility to
the compound in complete Middlebrook 7H9 broth. TBAJ-876 displayed high potency,
with MICs ranging from 0.42 to 0.53 ␮M across all subspecies, within 2-fold of BDQ’s
MIC (Table 1). These results suggest that TBAJ-876 is active against the M. abscessus
complex, displaying a potency similar to that of the parent drug against all subspecies.
Since drug susceptibility testing for M. abscessus is often carried out in cation-
adjusted Mueller-Hinton (CAMH) medium (49, 50), the potency of TBAJ-876 and BDQ
was measured in this medium, revealing a minor increase in the MICs against M.
abscessus subsp. abscessus ATCC 19977 and M. abscessus subsp. massiliense CCUG
48898-T for both compounds (Table 1). Thus, TBAJ-876 and BDQ exert similar potencies
against reference strains of the M. abscessus complex in Middlebrook 7H9 and CAMH
media.
TBAJ-876 is active against clinical isolates of the M. abscessus complex. Next,
we determined whether this potent activity holds true against a panel of clinical
isolates. This collection included M. abscessus Bamboo, an isolate previously sequenced
(51) and used in drug screening campaigns by our group (52–54). TBAJ-876 was
uniformly potent against M. abscessus Bamboo and other clinical isolates, with MICs
ranging from 0.14 to 0.45 ␮M (Table 2), similar to the MICs observed for the subspecies
reference strains (Table 1). Thus, TBAJ-876 is active in vitro against clinical isolates of the
M. abscessus complex, including those resistant to clarithromycin, the pillar of M.
abscessus treatment.
TBAJ-876 is bacteriostatic against M. abscessus in vitro. To determine whether
TBAJ-876 is bacteriostatic or bactericidal against M. abscessus, we measured the survival
of M. abscessus Bamboo upon drug exposure for 5 days at concentrations ranging from
3- to 300-fold the MIC. TBAJ-876 effectively suppressed bacterial growth at all concen-
trations tested but did not reduce the bacterial burden below the initial inoculum,
TABLE 2 Growth-inhibitory potency of TBAJ-876 against clinical isolates of the M. abscessus complexa
MIC (␮M)
Isolateb
M. abscessus subsp.
erm(41) sequevarc
Clarithromycin susceptibilityc
TBAJ-876
BDQ
Bamboo
M9
abscessus
abscessus
massiliense
abscessus
bolletii
abscessus
abscessus
abscessus
bolletii
C28
T28
Deletion
T28
T28
T28
T28
T28
C28
Sensitive
Resistant
Sensitive
Resistant
Resistant
Resistant
Resistant
Resistant
Sensitive
0.46
0.30
0.30
0.43
0.45
0.31
0.14
0.30
0.30
0.55
0.36
0.31
0.55
0.56
0.44
0.15
0.28
0.42
M111
M199
M232
M337
M421
M422
M506
aThe experiment was carried out three times independently, and the MICs are displayed as mean values. BDQ was used as a positive control.
bM. abscessus Bamboo is a clinical isolate whose genome characterization was described previously (51). The other clinical isolates were identified to the species level
and characterized as described previously (52).
cerm(41) is the methylase gene responsible for inducible clarithromycin resistance. The “C28” and “deletion” sequevars confer susceptibility to clarithromycin, while the
“T28” sequevar confers inducible resistance against clarithromycin (26, 27).
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FIG 2 Bacteriostatic activity of TBAJ-876 against M. abscessus Bamboo in vitro. Shown is the growth and survival
of M. abscessus Bamboo treated with 3-, 30-, and 300-fold the MIC of TBAJ-876 (A) or the positive-control BDQ (B)
over a period of 5 days. The MICs of TBAJ-876 and BDQ are 0.46 ␮M and 0.55 ␮M, respectively (Table 2). The
experiment was carried out three times independently, and the results are represented as mean values, with the
standard deviations displayed as error bars. The graphs were generated using GraphPad Prism 5 software.
similar to BDQ (Fig. 2), which was previously shown to be highly bacteriostatic against
M. abscessus in vitro (36).
TBAJ-876 does not antagonize the activity of commonly used anti-M. abscessus
antibiotics. Since treatment of M. abscessus infections requires drug combinations, we
investigated pharmacodynamic interactions of TBAJ-876 with standard-of-care anti-M.
abscessus antibiotics. We measured the growth inhibition activity of TBAJ-876 com-
bined with clarithromycin, amikacin, cefoxitin, or imipenem against M. abscessus Bam-
boo, using the “checkerboard titration” assay. TBAJ-876 was also combined with
rifabutin, which was recently shown to be active against M. abscessus and a potential
repurposing candidate (52, 55–58). The fractional inhibitory concentration index (FICI)
was calculated to characterize the interaction between TBAJ-876 and each of the test
drugs (59). All TBAJ-876 combinations were largely additive, similar to BDQ observa-
tions (Table 3). Thus, TBAJ-876 does not exert any observable antagonism with major
antibiotic classes used to treat M. abscessus disease.
TBAJ-876 is active in a mouse model of M. abscessus infection. To determine
whether TBAJ-876’s attractive in vitro potency against M. abscessus translates into in
vivo efficacy, NOD SCID mice were infected intranasally (58) and received daily oral
doses of 10 and 30 mg/kg of body weight (the projected human efficacious doses) of
TABLE 3 In vitro interaction of TBAJ-876 with selected drugs against M. abscessus Bambooa
MIC (␮M) of:
Combination with
DARQ (drug B)
Drug A
alone
Drug A in
combination
Drug B
alone
Drug B in
combination
Drug A (class)
FICIb
Outcomeb
Clarithromycin (macrolide)
TBAJ-876
BDQ
0.53
0.24
0.13
0.46
0.55
0.13
0.22
0.74
0.65
Additivity
Additivity
Amikacin (aminoglycoside)
Cefoxitin (␤-lactam)
TBAJ-876
BDQ
44
9.6
14.7
0.46
0.55
0.09
0.07
0.41
0.46
Synergy
Synergy
TBAJ-876
BDQ
43
9.7
12
0.46
0.55
0.09
0.11
0.42
0.48
Synergy
Synergy
Imipenem (␤-lactam)
Rifabutin (rifamycin)
TBAJ-876
BDQ
360
4.2
32
56
0.46
0.55
0.21
0.21
0.55
0.54
Additivity
Additivity
TBAJ-876
BDQ
2.4
1.0
0.46
0.55
0.05
0.20
0.68
0.60
Additivity
Additivity
aThe experiment was carried out three times independently, and the MICs are displayed as mean values. BDQ was used as a positive control.
bThe FICI was calculated as (MIC of drug A in combination/MIC of drug A alone) ϩ (MIC of DARQ B in combination/MIC of DARQ B alone) (71). A FICI of Յ0.5
indicates synergy, a FICI of Ͼ0.5 to 4 indicates additivity (no interaction), and a FICI of Ͼ4 indicates antagonism (59).
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FIG 3 In vivo activity of TBAJ-876 against M. abscessus in NOD SCID mice. Shown are the effects of
TBAJ-876 and the positive controls, clarithromycin (CLR) and BDQ, on bacterial loads in lungs (A) and
spleen (B) of infected mice. M. abscessus subsp. abscessus K21 was used for the infection studies, as
described previously (58). Mice, which were intranasally infected with ϳ106 CFU for 1 day, underwent
drug treatment for 10 consecutive days. TBAJ-876 (10 or 30 mg/kg), clarithromycin (250 mg/kg), and BDQ
(20 mg/kg) were administered once daily by oral gavage to groups of 6 mice per study arm. At 11 days
postinfection, organ homogenates were plated on agar to determine the bacterial load. Results were
analyzed using one-way analysis of variance (ANOVA) multicomparison and Tukey’s posttest (*, P Ͻ 0.05;
**, P Ͻ 0.01; ***, P Ͻ 0.001; ****, P Ͻ 0.0001). The MICs of TBAJ-876 and BDQ against the strain were
0.09 ␮M and 0.3 ␮M, respectively. The experiment was carried out twice, and one representative data set
is shown. The results are represented as mean values, with the standard deviations displayed as error
bars.
TBAJ-876, starting at 1 day postinfection and for 10 consecutive days. Drug efficacy was
defined as a statistically significant reduction of CFU in a study group relative to the
vehicle control at the end of the experiment. The macrolide clarithromycin and BDQ,
serving as positive controls at human-equivalent doses, significantly reduced lung
bacterial loads by approximately 1 log CFU (Fig. 3A). At 10 mg/kg, TBAJ-876 was as
efficacious as BDQ. Increasing the dose of TBAJ-876 to 30 mg/kg resulted in increased
efficacy in both lungs and spleen, although the difference was not statistically signif-
icant (Fig. 3A and B). Thus, TBAJ-876 reduces lung and spleen M. abscessus burdens in
vivo with an efficacy similar to that of BDQ.
DISCUSSION
TBAJ-876 is a 3,5-dialkoxypyridine BDQ analogue discovered through a recent
medicinal chemistry campaign (45) and currently developed against tuberculosis.
Compared to BDQ, this new compound displays low hERG channel inhibition and less
pronounced accumulation in tissues while retaining similar efficacy in mice infected
with M. tuberculosis (45). Hence, TBAJ-876 presents itself as a potential next-generation
BDQ with improved pharmacokinetic and tolerability profiles for the treatment of
tuberculosis. Since BDQ is active against M. abscessus, we determined whether this
holds true for TBAJ-876. Here, we show that TBAJ-876 and BDQ are similarly active
against M. abscessus in vitro. TBAJ-876 exhibits similar potencies against reference
strains of the M. abscessus complex and against a collection of clinical isolates repre-
senting the three subspecies of M. abscessus. Importantly, TBAJ-876’s in vitro activity
translates into in vivo efficacy, as demonstrated in a mouse model of M. abscessus
infection. Hence, we provide convincing preclinical evidence that TBAJ-876 may be
useful not only for the treatment of tuberculosis but also for the treatment of lung
disease caused by M. abscessus.
Furthermore, we show that TBAJ-876, similar to BDQ (60), does not antagonize the
activity of drugs commonly used for the treatment of M. abscessus infection and could
thus be safely coadministered with the standard of care, provided that there is an
absence of pharmacokinetic drug-drug interactions. In the treatment of tuberculosis,
BDQ is not administered concomitantly with rifampin because it is a substrate of
CYP3A4, induced by rifampin, thus causing drug-drug interactions (61). Whether TBAJ-
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876 is metabolized by CYP3A4 in patients remains to be determined. Regardless,
rifabutin has a much reduced CYP3A4 induction activity compared to rifampin (62),
making it the rifamycin of choice in patient populations receiving antiretroviral thera-
pies that include CYP3A4 substrates (63).
Given its ability to inhibit energy metabolism via inhibition of the mycobacterial
F-ATP synthase (47), TBAJ-876 could also potentiate the activity of several other
antibiotics through the depletion of intrabacterial ATP, which in turn would affect the
activity of ATP-binding cassette (ABC) transporters (64, 65) acting as membrane-bound
efflux pumps (66). Inhibition of these pumps via ATP starvation could impede drug
efflux, which has emerged as an important determinant of drug resistance in M.
abscessus (28).
While we show that TBAJ-876 does not antagonize growth inhibition by imipenem
or cefoxitin, a recent study suggests antagonism between BDQ and ␤-lactams when the
readout is bactericidal activity (67). BDQ was shown to block a lethal ATP burst induced
by imipenem and cefoxitin, thus eliminating the bactericidal activity of these cell wall
synthesis inhibitors against M. abscessus (67). Hence, caution may have to be exercised
regarding the coadministration of TBAJ-876 with ␤-lactams in the clinical setting.
In conclusion, this study has shown that the new BDQ analogue TBAJ-876 is active
in vitro and in vivo against the M. abscessus complex. This compound represents a
potential alternative to BDQ, with improved pharmacokinetic and tolerability profiles,
for the treatment of M. abscessus lung disease.
MATERIALS AND METHODS
Bacterial strains, culture media, and chemicals. The reference strains of the subspecies of the M.
abscessus complex were purchased from the American Type Culture Collection (ATCC) and Culture
Collection University of Goteborg (CCUG). M. abscessus subsp. abscessus ATCC 19977 and M. abscessus
subsp. bolletii CCUG 50184-T harbor the inducible clarithromycin resistance-conferring erm(41) T28
sequevar (65, 68). M. abscessus subsp. massiliense CCUG 48898-T harbors the nonfunctional erm(41)
deletion sequevar, and hence, it is susceptible to clarithromycin (69).
M. abscessus Bamboo is a clinical isolate from a patient with amyotrophic lateral sclerosis and
bronchiectasis. The strain was provided by Wei Chang Huang (Taichung Veterans General Hospital,
Taichung, Taiwan). This strain was previously whole-genome sequenced, which showed that it belongs
to M. abscessus subsp. abscessus and harbors the inactive, clarithromycin-sensitive erm(41) C28 sequevar
(GenBank accession no. MVDX00000000) (51).
The other eight M. abscessus clinical isolates (M9, M111, M199, M232, M337, M421, M422, and M506),
used for in vitro characterization of TBAJ-876’s activity, were provided by Jeanette W. P. Teo (Department
of Laboratory Medicine, National University Hospital, Singapore). The subspecies and erm(41) sequevars
of these isolates were determined previously (52).
For the in vivo efficacy study, the clinical isolate M. abscessus subsp. abscessus K21 was used. This
strain harbors the inactive, clarithromycin-sensitive erm(41) C28 sequevar as determined previously (58).
M. abscessus K21 was isolated from a patient and provided by Won-Jung Koh (Division of Pulmonary and
Critical Care Medicine, Samsung Medical Center, Seoul, South Korea).
All M. abscessus strains were maintained in Middlebrook 7H9 medium (BD Difco) supplemented with
0.2% (vol/vol) glycerol (Fisher Scientific), 0.05% (vol/vol) Tween 80 (Sigma-Aldrich), and 10% (vol/vol)
Middlebrook albumin-dextrose-catalase (ADC) (BD Difco). Cation-adjusted Mueller-Hinton (CAMH) broth
(BD Difco) was also used to culture M. abscessus strains and was prepared according to the manufac-
turer’s instructions.
TBAJ-876 was synthesized by Bioduro LLC (Beijing, China) as described in the supplemental material,
while BDQ was purchased from MedChem Express. Both TBAJ-876 and BDQ were dissolved in 100%
dimethyl sulfoxide (DMSO) (MP Biomedicals) and sterilized using 0.2-␮m polytetrafluoroethylene (PTFE)
membrane filters (Acrodisc; Pall) before use.
Growth inhibition dose-response assay. The broth microdilution method (70) was utilized for
carrying out a growth inhibition dose-response assay. Briefly, each well of clear 96-well flat-bottom
Costar cell culture plates (Corning) was filled with 100 ␮l of liquid medium (complete 7H9 medium or
CAMH broth). TBAJ-876 or BDQ was added to the first well in each row of the plate to create two times
the desired highest final concentration. A 10-point 2-fold serial dilution of TBAJ-876/BDQ was carried out
starting from this first well. The M. abscessus strains used for this assay were grown to mid-exponential
phase and then diluted to an optical density at 600 nm (OD600) value of 0.1 using the same liquid
medium used to run the assay. One hundred microliters of the diluted culture was added to each well
to create a final OD600 value of 0.05 in each well. The plates were incubated for 3 days at 37°C, with
shaking at 110 rpm using an orbital shaker. After the incubation period, the cultures in all wells were
manually resuspended, and the OD600 of each well was read using a Tecan Infinite Pro 200 plate reader.
The reported MIC values represent the concentration that inhibits 90% of bacterial growth compared to
the untreated culture.
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Measurement of growth and survival of M. abscessus Bamboo under TBAJ-876 or BDQ treat-
ment. M. abscessus Bamboo was cultured in complete 7H9 medium and grown to mid-exponential
phase. Subsequently, the culture was diluted to ϳ106 CFU/ml using complete 7H9 medium. Two
milliliters of the diluted culture was transferred to each 14-ml round-bottom tube (SPL Life Sciences).
TBAJ-876 and BDQ were added to their respective tubes to achieve final concentrations of 3-, 30-, or
300-fold their MICs. The tubes were incubated at 37°C under shaking at 160 rpm for 5 days. At the
indicated time points, 10 ␮l of each sample was taken and diluted. For drug-free samples, 10 ␮l of 10Ϫ3
to 10Ϫ7 dilutions of each sample was plated out on Middlebrook 7H10 agar (BD Difco) supplemented
with 0.2% (vol/vol) glycerol and 10% (vol/vol) Middlebrook oleic acid-albumin-dextrose-catalase (OADC)
(BD Difco). For TBAJ-876- or BDQ-treated samples, 10 ␮l of 10Ϫ3 to 10Ϫ5 dilutions of each sample was
plated out. The agar plates were incubated for 4 days at 37°C, and subsequently, CFU were determined
by counting the colonies. Graphs were generated using GraphPad Prism 5 software.
Checkerboard titration assay for assessing in vitro interaction of TBAJ-876 or BDQ with other
drugs. A checkerboard titration assay was carried out as described previously (71, 72). Briefly, TBAJ-876
or BDQ and one drug (clarithromycin, amikacin, cefoxitin, imipenem, or rifabutin) were added to
complete 7H9 medium-containing 96-well flat-bottom Costar cell culture plates. Twofold serial dilutions
were done to allow 7 different concentrations of TBAJ-876 or BDQ (0 ␮M to 2 ␮M) to be tested for
interaction with 10 different concentrations of the drugs (0 ␮M to 4 ␮M for clarithromycin, 0 ␮M to
100 ␮M for amikacin, 0 ␮M to 90 ␮M for cefoxitin, 0 ␮M to 400 ␮M for imipenem, and 0 ␮M to 80 ␮M for
rifabutin). Hence, a total of 70 different concentration combinations were tested for each combination
between TBAJ-876 or BDQ and a drug. Each 96-well plate had a 7H9 medium-only control well and a
drug-free bacterial culture control well. M. abscessus Bamboo was cultured in complete 7H9 medium and
grown to mid-exponential phase. Subsequently, the culture was diluted to an OD600 of 0.1 using
complete 7H9 medium and added to each well in the 96-well plate to create a final OD600 value of 0.05.
The plates were incubated for 3 days at 37°C, under shaking at 110 rpm on an orbital shaker. After the
incubation period, the culture in each 96-well plate was manually resuspended, and the OD600 of each
well was read using a Tecan Infinite Pro 200 plate reader. Calculation of the fractional inhibitory
concentration index (FICI) was done to analyze the results. The FICI is calculated as (MIC of drug A in
combination/MIC of drug A alone) ϩ (MIC of DARQ B in combination/MIC of DARQ B alone) (71). This
calculation was done only for wells which showed 90% inhibition of bacterial culture growth compared
to drug-free bacterial culture wells. A FICI of Յ0.5 indicates synergy, a FICI of Ͼ0.5 to 4 indicates
additivity (no interaction), and a FICI of Ͼ4 indicates antagonism (59).
Determination of in vivo activity of TBAJ-876. In vivo efficacy determinations were carried out as
described previously, using 8-week-old female NOD.CB17-Prkdcscid/NCrCrl (NOD SCID) mice (Charles River
Laboratories) and the M. abscessus subsp. abscessus K21 strain (58). Briefly, anesthetized animals were
infected by intranasal delivery of ϳ106 CFU of M. abscessus subsp. abscessus K21. Acute infection was
achieved within 1 day. Drugs or the vehicle control was administered once daily for 10 consecutive days
by oral gavage to the mice, starting from 1 day postinfection. Clarithromycin was formulated in 0.5%
carboxymethyl cellulose–0.5% Tween 80–sterile water and administered at a dose of 250 mg/kg.
TBAJ-876 and BDQ (as the free-base form) were formulated in 20% (2-hydroxypropyl)-␤-cyclodextrin and
administered at doses of 10 mg/kg and 30 mg/kg (TBAJ-876) or 20 mg/kg (BDQ). The BDQ dose of
20 mg/kg was selected to achieve efficacious exposure (area under the concentration-time curve
[AUC]/MIC) comparable to that of 30 mg/kg TBAJ-876, based on the previously reported pharmacokinetic
properties of both antibiotics (45). All mice were euthanized 24 h after the last dose (11 days postinfec-
tion), and their lungs and spleen were aseptically removed prior to homogenization. The bacterial load
in these organs was determined by plating serial dilutions of the organ homogenates onto Middlebrook
7H11 agar (BD Difco) supplemented with 0.2% (vol/vol) glycerol and 10% (vol/vol) OADC. The agar plates
were incubated for 5 days at 37°C prior to counting of colonies. All experiments involving live animals
were approved by the Center for Discovery and Innovation Institutional Animal Care and Use Committee.
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 0.1 MB.
ACKNOWLEDGMENTS
We thank Ding Wang and his team at Bioduro LLC (Beijing, China) for the synthesis
of TBAJ-876. We are grateful to Wei Chang Huang (Taichung Veterans General Hospital,
Taichung, Taiwan) for providing the M. abscessus Bamboo strain, Jeanette W. P. Teo
(Department of Laboratory Medicine, National University Hospital, Singapore) for pro-
viding the collection of M. abscessus clinical isolates for in vitro profiling, and Won-Jung
Koh (Division of Pulmonary and Critical Care Medicine, Samsung Medical Center, Seoul,
South Korea) for providing the M. abscessus K21 strain for in vivo studies.
J.P.S. receives a research scholarship from the Yong Loo Lin School of Medicine,
National University of Singapore. T.D. holds a Toh Chin Chye Visiting Professorship at
the Department of Microbiology and Immunology, National University of Singapore.
The research reported in this publication is supported by the National Institute of
April 2020 Volume 64 Issue 4 e02404-19
aac.asm.org 7

Sarathy et al.
Antimicrobial Agents and Chemotherapy
Allergy and Infectious Diseases of the National Institutes of Health under award no.
R01AI132374. The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of Health.
Conceptualization, J.P.S., V.D., M.G., and T.D.; investigation, J.P.S., U.S.G., M.D.Z., and
M.G.; writing—original draft, J.P.S. and T.D.; writing—review and editing, all authors;
funding acquisition, T.D.; supervision, T.D.
We declare no commercial or financial relationships that could be construed as a
potential conflict of interest.
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