Mce3R Stress-Resistance Pathway Is Vulnerable to Small-Molecule Targeting That Improves Tuberculosis Drug Activities

Article abstract of DOI:10.1021/acsinfecdis.9b00099

One-third of the world's population carries Mycobacterium tuberculosis (Mtb), the infectious agent that causes tuberculosis (TB), and every 17 s someone dies of TB. After infection, Mtb can live dormant for decades in a granuloma structure arising from the host immune response, and cholesterol is important for this persistence of Mtb. Current treatments require long-duration drug regimens with many associated toxicities, which are compounded by the high doses required. We phenotypically screened 35 6-azasteroid analogues against Mtb and found that, at low micromolar concentrations, a subset of the analogues sensitized Mtb to multiple TB drugs. Two analogues were selected for further study to characterize the bactericidal activity of bedaquiline and isoniazid under normoxic and low-oxygen conditions. These two 6-azasteroids showed strong synergy with bedaquiline (fractional inhibitory concentration index = 0.21, bedaquiline minimal inhibitory concentration = 16 nM at 1 μM 6-azasteroid). The rate at which spontaneous resistance to one of the 6-azasteroids arose in the presence of bedaquiline was approximately 10^-9, and the 6-azasteroid-resistant mutants retained their isoniazid and bedaquiline sensitivity. Genes in the cholesterol-regulated Mce3R regulon were required for 6-azasteroid activity, whereas genes in the cholesterol catabolism pathway were not. Expression of a subset of Mce3R genes was down-regulated upon 6-azasteroid treatment. The Mce3R regulon is implicated in stress resistance and is absent in saprophytic mycobacteria. This regulon encodes a cholesterol-regulated stress-resistance pathway that we conclude is important for pathogenesis and contributes to drug tolerance, and this pathway is vulnerable to small-molecule targeting in live mycobacteria.

Full text of DOI:10.1021/acsinfecdis.9b00099

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Article
The Mce3R stress-resistance pathway is vulnerable to small
molecule targeting that improves tuberculosis drug activities
Xinxin Yang, Tianao Yuan, Rui Ma, Kieran Chacko, Melissa Smith, Gintaras Deikus, Robert
Sebra, Andrew Kasarkskis, Harm van Bakel, Scott G. Franzblau, and Nicole S Sampson
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00099 • Publication Date (Web): 23 Apr 2019
Downloaded from http://pubs.acs.org on April 24, 2019
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The Mce3R stress-resistance pathway is vulnerable to small-molecule targeting that
improves tuberculosis drug activities
Xinxin Yang,a,1 Tianao Yuan,a,1 Rui Ma,b Kieran I. Chacko,c Melissa Smith,c,d Gintaras Deikus,c,d Robert
Sebra,c,d Andrew Kasarskis,c,d Harm van Bakel,c,d Scott G. Franzblau,b Nicole S. Sampsona,e,f,2
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aDepartment of Chemistry, 100 John S. Toll Drive, Stony Brook University, Stony Brook, NY 11794-
3400
bInstitute for Tuberculosis Research, 833 South Wood Street, 425 PHARM, University of Illinois at
Chicago, Chicago, Illinois 60612-7231
cDepartment of Genetics and Genomic Sciences, One Gustave L. Levy Place - Box 1498, Icahn School of
Medicine at Mount Sinai, New York City, NY, USA, 10029
dIcahn Institute for Genomics and Multiscale Biology, One Gustave L. Levy Place - Box 1498, Icahn
School of Medicine at Mount Sinai, New York City, NY, USA, 10029-6574
eInstitute of Chemical Biology and Drug Discovery, 100 John S. Toll Drive, Stony Brook University,
Stony Brook, NY 11794-3400
fStellenbosch Institute for Advanced Study (STIAS), Wallenberg Research Centre at Stellenbosch
University, 10 Marais Street, Stellenbosch 7600, South Africa
Running title: 6-Azasteroids improve TB drug activities
Keywords: cholesterol, co-drug, low oxygen
1Contributed equally to this work.
2To whom correspondence may be addressed. Email: nicole.sampson@stonybrook.edu
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One-third of the world’s population carries Mycobacterium tuberculosis (Mtb), the infectious agent that
causes tuberculosis (TB), and every 17 seconds someone dies of TB. After infection, Mtb can live
dormant for decades in a granuloma structure arising from the host immune response; and cholesterol is
important for this persistence of Mtb. Current treatments require long-duration drug regimens with many
associated toxicities, which are compounded by the high doses required. We phenotypically screened 35
6-azasteroid analogues against Mtb and found that at low micromolar concentrations, a subset of the
analogues sensitized Mtb to multiple TB drugs. Two analogues were selected for further study to
characterize the bactericidal activity of bedaquiline and isoniazid under normoxic and low-oxygen
conditions. These two 6-azasteroids showed strong synergy with bedaquiline (fractional inhibitory
concentration index = 0.21, bedaquiline minimal inhibitory concentration = 16 nM at 1 µM 6-azasteroid).
The rate at which spontaneous resistance to one of the 6-azasteroids arose in the presence of bedaquiline
was approximately 10−9, and the 6-azasteroid-resistant mutants retained their isoniazid and bedaquiline
sensitivity. Genes in the cholesterol-regulated Mce3R regulon were required for 6-azasteroid activity,
whereas genes in the cholesterol catabolism pathway were not. Expression of a subset of Mce3R genes
was down-regulated upon 6-azasteroid treatment. The Mce3R regulon is implicated in stress resistance
and is absent in saprophytic mycobacteria. This regulon encodes a cholesterol-regulated stress-resistance
pathway that we conclude is important for pathogenesis and contributes to drug tolerance, and that this
pathway is vulnerable to small-molecule targeting in live mycobacteria.
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Cholesterol metabolism plays an important role in the persistence, virulence, and intracellular survival of
Mycobacterium tuberculosis (Mtb).1-5 Mtb is able to survive and replicate inside macrophages6 by
utilizing host-derived nutrients, including cholesterol.1-3, 7 Mtb catabolism of cholesterol provides a source
of acetyl-coenzyme A (CoA), pyruvate, and propionyl-CoA, which can be utilized for energy production
and as lipid precursors.8 Catabolism proceeds through b-oxidation of the side chain, and oxidative
cleavage of the sterol rings.9 Comparison of transcriptional profiles of Mtb cultured with and without
cholesterol identified over 200 genes that are regulated by cholesterol.2 At least 52 cholesterol-regulated
genes are clustered within the Mtb genome and encode the enzymes needed for catabolism.2, 8 Two TetR
family regulators, KstR1 and KstR2, control transcription of the majority of these catabolism genes10-11
and are de-repressed by CoA metabolites of the catabolism pathway.12-13
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Owing to the large number of potential targets in the Mtb cholesterol metabolism pathway, the protein
target that would be the most vulnerable to inhibition is not clear.14 Therefore, we undertook whole-cell
phenotypic screening to identify inhibitors of Mtb growth. We reasoned that use of a steroid scaffold
would bias the screen to target cholesterol metabolism. We further sought a scaffold with
pharmacokinetic properties that would be advantageous for future in vivo experimentation.
For this purpose, we chose the 6-azasteroid scaffold (Figure 1). The 6-azasteroids were developed by
Glaxo-Wellcome as part of a 5α-reductase inhibitor program for treatment of benign prostatic hyperplasia,
but they were subsequently abandoned in favor of 4-azasteroids.15-18 Several 6-azasteroids were found to
be orally bioavailable in rats, dogs, or both and to have low in vivo toxicity.16 Because of the
pharmacokinetic properties of 6-azasteroids and their structural similarity to cholesterol, we chose to
screen them as possible inhibitors of cholesterol metabolism in Mtb.
Previously, we demonstrated that 6-azasteroids with a large, hydrophobic R1 side chain and an
unsubstituted N6 atom (e.g., 4a, Figure 1) are competitive inhibitors of 3β-hydroxysteroid
dehydrogenase.19 However, because this enzyme is not essential for Mtb survival in the mouse or guinea
pig models of infection, its relevance as a drug target was questionable.20 We reasoned that other enzymes
in the Mtb cholesterol metabolism pathway might also be susceptible to inhibition by compounds with the
6-azasteroid scaffold. In the present study, we identified several 6-azasteroids with anti-mycobacterial
activity that improve the activity of existing anti-TB drugs, and we established a relationship between
activity and side-chain structure. By investigating the mechanism of action and the target of two of the
active 6-azasteroids in Mtb, we identified a connection between stress resistance and cholesterol-regulated
genes that reside outside the cholesterol catabolic pathway. The inhibitors described herein offer a
strategy for combating innate drug tolerance in Mtb and highlight the complex role of cholesterol-
regulated genes in Mtb infection.
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O
O
O
OMe
1. LiOH
NHR1
NHR1
Dioxane/H2O
60 oC, 18 h
2. HCl
1. TFA
DCM, 20 oC
2 h
O
N
O
N
O
N
H
2. NaHCO3
3. SOCl2, pyridine, DMF
Toluene, 0 oC, 1 h
CO2-t-Bu
CO2-t-Bu
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3a-3v
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4a-4v
DCM, 16 h
O
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NHR1
NaH, iodomethane
DMF, 20 oC, 5 h
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5a: R2 = methyl
6a, 6b: R2 = n-propyl
7a: R2 = n-hexyl
R1=
n:
CO2Me
a: -(2,5-di-t-butyl)phenyl
b: -(2-t-butyl, 5-trifluoromethyl)phenyl
c: -(2,5-di-trifluoromethyl)phenyl
d: -(3,5-di-t-butyl)phenyl
o:
p:
q:
r:
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t:
SMe
CO2Et
OH
e: -(2-t-butyl)phenyl
f: -(3-t-butyl)phenyl
g: -(4-t-butyl)phenyl
OMe
CF3
h: -(2-trifluoromethoxy, 4-fluoro)phenyl
i: -(2-trifluoromethoxy, 4-chloro)phenyl
j: -(2-trifluoromethoxy, 4-bromo)phenyl
k: -(2-trifluoromethoxy, 4-iodo)phenyl
l: -(2-trifluoromethoxy, 3-trifluoromethyl)phenyl
m: benzyl
HN
O
S
u:
v:
N
N
Figure 1. Structures and synthesis of 6-azasteroids. Key intermediate 1 was prepared as described
in the literature16-18 and subjected to further elaboration as shown.
RESULTS
6-Azasteroids were prepared from intermediate 1. Following a previously established synthetic route,15-18
we prepared a library of 6-azasteroids from key intermediate 1 (Figure 1), which was prepared on a 500 g
scale. Briefly, hydrolysis of the methyl ester group of 1 followed by activation of the resulting carboxylic
acid and substitution reactions with various primary amines provided amides 3 with more than 20
different substituents at R1. Subsequent deprotection of the ring nitrogen provided compounds 4a–4v.
Alkylation of 4a or 4b at N6 provided compounds 5a–7a and 6b. Treatment of 4a with an excess of
sodium hydride and iodomethane yielded 8a, which has a methyl group both on the ring nitrogen and at
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C2 All compounds were purified by column chromatography before testing. Yields from intermediate 1
2
ranged from 60 to 90%.
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6-Azasteroids inhibit Mtb growth in combination with isoniazid. We initially screened the synthesized 6-
azasteroids (Figure 1) to determine their minimal inhibitory concentrations (MICs) in a Mtb growth assay.
As a carbon source, we used cholesterol solubilized in tyloxapol detergent micelles to ensure that we
could detect activity against the cholesterol catabolism pathway. The 6-azasteroid concentrations were
varied from 0.15 to 40 µM. We were unable to obtain accurate MICs, but we estimated the values to
exceed 40 µM for the compounds that showed at least some activity.
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Despite the lack of MICs for most of the 6-azasteroids, we recognized that they might show synergistic
activity when combined with isoniazid (INH), a frontline TB drug. Therefore, we screened for inhibition
of Mtb growth at a fixed concentration of 6-azasteroid (20 µM) while varying the INH concentration. The
potentiation potency was recorded as a fold change of the INH MIC. Of the 35 6-azasteroids tested in
combination with INH, seven (4a, 4d, 5a–8a, and 3c) improved the INH MIC at least 16-fold (Figure 2).
In addition, compound 6b showed moderate potentiation, with a 4-fold decrease in the INH MIC. We also
tested two 4-azasteroids, finasteride and dutasteride, which inhibit human 5α-reductase and are FDA-
approved for treatment of benign prostatic hyperplasia. Neither of the 4-azasteroids showed inhibitory
activity, either alone or in combination with INH.
1.2
1
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0.6
0.4
0.2
0
Figure 2. 6-Azasteroids improve the efficacy of INH. CDC1551 Mtb in 7H9 medium with cholesterol
as a carbon source was incubated with 20 µM 6-azasteroid, finasteride, dutasteride, or DMSO control
and varying concentrations of INH (2-fold dilutions) for 14 days at 37 °C. The INH concentration at which
no growth occurred was recorded as the MIC. Results are shown as the ratio of the INH MIC in the
presence of azasteroid or DMSO to the MIC of INH alone (0.2–0.4 μM). A ratio of 0.5–1 is considered to
indicate no change in the INH MIC. The data shown are representative of at least two experiments.
A clear structure–activity relationship was observed for the 6-azasteroids. Among the compounds with
R2 = H (4), an aniline side chain at R1 was required. Compounds with side chains derived from aliphatic
amines (4n, 4o, 4p, 4r, 4s) or aliphatic amines linked to aromatic groups (4m, 4q, 4t) were inactive.
Furthermore, compounds with aniline side chains bearing a single substituent (4e, 4f, 4g), compounds
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with aniline side chains bearing polar substituents (4b, 4c, 4h, 4i, 4j, 4k, 4l), and compounds with
pyridyl-based side chains (4u and 4v) were inactive. Only 4a and 4d, the (2,5-di-t-butyl)phenyl and the
(3,5-di-t-butyl)phenyl compounds, improved INH activity. The activity of parent compound 4a was
retained upon installation of an alkyl R2 substituent on the ring nitrogen (5a, 6a, 7a). Interestingly,
moderate potentiation was observed upon conversion of the inactive 2-t-butyl-5-trifluoromethylaniline-
derived compound 4b to N-propyl derivative 6b. Likewise, compound 3c, which has a t-
butyloxycarbamate-protected ring nitrogen, was active, whereas the corresponding unprotected compound
(4c) was not. Thus, derivatization of the aza ring nitrogen was well tolerated and, in some cases, could
convert inactive compounds into potentiators of INH activity.
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Figure 3. Azasteroids 4a and 6a inhibit Mtb growth and are bactericidal in combination with INH or
BDQ. (A) CDC1551 Mtb was cultured in 7H9 medium with glycerol as a carbon source and was diluted to
an OD (600 nm) of 0.2. These cultures were incubated with 10 μM 4a or 6a or with DMSO as a control
(two replicates), and the growth of Mtb was monitored by OD (600 nm) for 15 days. Data are
representative of at least two experiments. (B) H37Rv(mlux) Mtb was cultured in 7H9/glycerol medium,
and diluted to approximately 5–10 RLU per well. These cultures were incubated with INH, BDQ, and/or
4a at the indicated concentrations or with DMSO as a control, and the growth of Mtb was monitored by
autoluminescence for 9 days. (C) H37Rv(mlux) Mtb was cultured in 7H9/glycerol medium, and diluted to
approximately 5–10 RLU per well. These cultures were incubated with INH, BDQ, and/or 6a at the
indicated concentrations or with DMSO as a control, and the growth of Mtb was monitored by
autoluminescence for 9 days. (D) CDC1551 Mtb was cultured in 7H9/glycerol medium, and diluted to an
OD (600 nm) of approximately 0.002. These cultures were incubated with BDQ and/or 6a at the indicated
concentrations or with DMSO as a control. After 2 or 7 days, the cultures were diluted, and the CFUs
were determined by serial dilution onto agar plates. Error bars in panels B–D indicate SDs (n = 2). The
limit of detection is 10 CFU/mL.
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6-Azasteroid activity is not specific for cholesterol catabolism. Next, we assessed the specificity of the 6-
azasteroid scaffold for cholesterol catabolism. We found that when glycerol was used as the carbon
source, the seven active 6-azasteroids retained their ability to improve the efficacy of INH. As a negative
control, we tested compound 4g, which was inactive with cholesterol as the carbon source, and found it
remained inactive in glycerol (Figure S1). Thus, the activity of 6-azasteroids was not carbon-source-
specific.
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Two of the active compounds, 4a and 6a, were selected for further study. Their effect on the rates of
CDC1551 Mtb growth was monitored by optical density (OD). As expected from our initial MIC screen,
10 µM 4a or 6a did not inhibit the growth of Mtb on glycerol as a carbon source over the course of 15
days (Figure 3A).
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6-Azasteroids improve the activity of other TB drugs and drug candidates. Compounds 4a and 6a were
tested as potentiators of the following additional TB drugs and drug candidates: rifampin, pretomanid,
bedaquiline (BDQ), clofazimine, pyrazinamide, moxifloxacin, linezolid, and ethionamide. We found that
these two 6-azasteroids improved the activity of at least five of the TB drugs in vitro: rifampin,
pretomanid, BDQ, pyrazinamide, and ethionamide (Tables 1 and S1A). The 6-azasteroids did not appear
to improve the activity of clofazimine, linezolid, or moxifloxacin. BDQ, in addition to INH, was selected
for further study because it is used for the treatment of multidrug-resistant Mtb infection.
Table 1. 6-Azasteroids 4a and 6a improve the efficacy of multiple TB drugs.a
Drug MIC
Drug
Drug target
Aloneb
(μM)
With
4a
With
6a
INH
cell-wall synthesis
RNA polymerase
cell-wall synthesis
ATP synthase
unknown
0.4
Rifampinc
Pretomanid
BDQ
0.025
0.05
0.4
Clofazimined
25
Pyrazinamidec unknown
20,000
0.4
Moxifloxacin
Linezolid
DNA synthesis
protein synthesis
cell-wall synthesis
0.8
Ethionamidec
6.25
aCDC1551 Mtb or H37Rv(mlux) Mtb in 7H9 medium with glycerol as a carbon
source was incubated at 37 °C with 20 µM 4a or 6a or with DMSO control and
one of the listed drugs (2-fold dilution) at various concentrations for 14 days
(CDC1551) or 6 days (H37Rv(mlux)). The drug concentration at which no growth
occurred was recorded as the drug MIC. The ratio of the drug MIC in the
presence of azasteroid to the MIC of the drug alone was determined. Ratios of
0.0625–0.125 are indicated by green shading and are considered to reflect
potentiation. Ratios of 0.5–1 are indicated by red shading and are considered to
reflect no change in MIC. Data are representative of at least two independent
biological replicate experiments, which were performed in technical triplicate.
MIC values are listed in Table S1A. bValues in this column are MICs for
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CDC1551 Mtb except for clofazimine. cExperiment performed only with
CDC1551 Mtb. dExperiment performed only with H37Rv(mlux) Mtb.
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6-Azasteroids show synergism with INH or BDQ. Using the H37Rv(mlux) strain, we carried out a
checkerboard growth-inhibition assay at various 4a or 6a and ΙΝΗ or BDQ concentrations. As expected,
neither 10 nor 20 µM 4a inhibited the growth of H37Rv(mlux) (Figure 3B). A sub-MIC concentration of
INH (0.032 µM) also failed to inhibit growth. However, when Mtb was treated simultaneously with 20
µM 4a and 0.032 µM INH, growth was inhibited. Similarly, at a low concentration (4 µM), BDQ alone
inhibited growth by approximately 50%. Importantly, when BDQ was used in combination with 4a,
complete inhibition was observed. Likewise, combinations of 6a and 0.032 µM INH or 4 µM BDQ
resulted in complete inhibition (Figure 3C). Growth readings at day 9 were used to prepare an
isobolographic plot for INH and 6-azasteroid (Figure S2). Although an accurate 6-azasteroid MIC could
not be determined from the plots, the combinations of 4a or 6a and INH were clearly synergistic.
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6-Azasteroids improve bactericidal activity. Combining 6a with BDQ (1.8 µM) improved the bactericidal
activity of BDQ. Specifically, the combination of 10 µM 6a and BDQ resulted in a 2 log10 CFU
improvement in bacterial kill at day 7 of treatment. However, the rate of bacterial kill by BDQ was not
increased, as evidence by the 2-day time points (Figures 3D and S3A). The combination of 4a and INH
was also bactericidal (Figure S3B).
6-Azasteroids are more potent under low-oxygen conditions than under normoxic conditions. We also
conducted a checkerboard low-oxygen recovery assay (LORA) of H37Rv(mlux) growth inhibition by 4a
or 6a and ΙΝΗ or BDQ at various concentrations (Figure 4). Both 4a and 6a were active in the LORA,
showing MICs of 11 and 13 µM, respectively. Although INH was not active under low-oxygen conditions
(MIC » 400 µM), treatment with INH plus 4a or 6a reduced the MIC at least 2-fold (Figure 4A). The
LORA fractional inhibitory concentration (FIC) indices for INH/4a and INH/6a were 0.75 and 0.69,
respectively. BDQ had an MIC of 160 nM in the LORA. Compound 4a or 6a improved BDQ activity in
the LORA (Figure 4B). At 4.4 µM, 4a reduced the BDQ MIC approximately 50-fold (to 3 nM), and 6a
reduced the BDQ MIC to 23 nM. The FIC index for both BDQ/4a and BDQ/6a was 0.21, indicating
strong synergy.
6-Azasteroids improve the bactericidal activity of BDQ under low-oxygen conditions. The bactericidal
activities of 4a and 6a were determined under low-oxygen conditions. The minimum bactericidal
concentration of 4a was 80 µM, and that of 6a was higher than 80 µM (80% kill). To assess the
bactericidal activity of combinations of 6-azasteroids and BDQ, we performed a time-kill assay with 10
µM 4a or 6a in combination with BDQ at concentrations below the minimum bactericidal concentration
(Figure 4C). At day 10 of treatment, the combination of 10 µM 4a and 0.15 µM BDQ resulted in a 3.2
log10 CFU reduction in bacterial kill relative to that in a no-drug control group. In addition, this same
combination (10 µM 4a plus 0.15 µM BDQ) resulted in a 1.4 log10 CFU reduction in bacterial kill at day
10 relative to that in the BDQ-only group at a slightly higher BDQ concentration (0.25 µM); that is 10
µM 4a plus 0.15 µM BDQ was more potent than 1–0.25 µM BDQ alone (Figure 4C).
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BDQ (μM)
0
0
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0.5 0.25
0.15 0.15
6-aza (10 μM)
–
+
+
–
–
–
+ +
Figure 4. 6-Azasteroids and INH or BDQ interact under low-oxygen conditions. Isobolographic
analysis of co-drug LORA MIC in H37Rv(mlux) Mtb: (A) INH and 4a or 6a and (B) BDQ and 4a or 6a.
The lines connecting 400 μM INH or 0.21 μM BDQ and 13 μM 4a or 6a denote lines of additivity.
H37Rv(mlux) Mtb was cultured and diluted to approximately 5–10 RLU per well. These cultures were
incubated with INH or BDQ and 4a or 6a at a checkerboard of concentrations (3-fold dilutions) or with
DMSO as a control under hypoxic conditions for 10 days. The RLU values for H37Rv(mlux) Mtb after 28
h of normoxic recovery were recorded. The drug concentrations in wells with at least 90% inhibition of
growth relative to that in the no-drug control are plotted. Plotted values are averages (n = 3). (C)
H37Rv(mlux) Mtb was cultured at approximately 1 × 105 CFU/mL. These cultures were incubated with
BDQ, 4a (10 μM), or 6a (10 μM), alone or in combination, at the indicated concentrations under hypoxic
conditions for 10 days. The cultures were diluted, and CFUs were determined by serial dilution onto agar
plates. Plotted values are averages with SDs (n = 3); *p < 0.05. The limit of detection is 10 CFU/mL.
6-Azasteroids retain potentiation activity in cholesterol catabolism mutants. Because 6-azasteroids are
cholesterol analogues, we determined whether the cholesterol catabolism pathway was required for
azasteroid activity. For this purpose, we tested the activity of 6-azasteroids in combination with INH in
mutant Mtb strains with disrupted cholesterol catabolism genes. Specifically, we selected strains with
mutations in fadA5, chsE4 (fadE26), fadE31, and fadE33 (the transcription of which is repressed by the
two main transcriptional regulators of cholesterol catabolism, KstR1 and KstR2)10-11 and in hsd (which
encodes the first enzyme in the pathway).21 KstR1 regulates the transcription of genes encoding side-
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chain- and A/B-ring-degrading enzymes, whereas KstR2 regulates the transcription of genes encoding
C/D-ring-degrading enzymes. These two regulators are de-repressed by CoA metabolites in the
pathway.12-13 We found that none of these five genes were required for 4a to improve INH activity against
Mtb grown with either cholesterol or glycerol as the carbon source (Tables 2 and S1B).
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8
9
Table 2. The Mce3R regulon is required for azasteroid 4a activity.a
Chold
Glycd
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Regulon
Mutated Mtb
geneb
None (WT-
CDC1551)
None (WT-
H37Rv)
hsd
KstR1
KstR2
Mce3R
fadA5
chsE4 (fadE26)
fadE31
fadE33
echA13
fadE18
melF
melH
fadE18::pfadE18
Mce3R
Speciesc
INHd
BDQd
not conserved M. avium
M. smegmatis
M. marinum
Mtb
conserved
aThe data shown are representative of at least two
independent biological replicates and were performed in
technical triplicate. bWith cholesterol or glycerol as a
carbon source, the indicated strain of Mtb in 7H9 medium
was incubated with 20 μM 4a or DMSO control and INH at
various concentrations (2-fold dilutions) for 14 days at
37 °C. cWith glycerol as a carbon source, the indicated
species of mycobacteria in 7H9 medium was incubated
with 20 μM 4a or DMSO control and INH or BDQ at
various concentrations (2-fold dilutions) for 4–7 days at
37 °C. dThe drug concentration at which no growth
occurred was recorded as the drug MIC. MIC values are
shown in Table S1B. The ratio of the drug MIC in the
presence of 4a to the MIC of INH or BDQ alone was
determined. Ratios of 0.0625–0.125 are indicated with
green shading and reflect potentiation. Ratios of >0.125
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and ≤0.5 are indicated with orange shading and reflect low
potentiation due to low activity of INH against M. marinum.
Ratios >0.5 are indicated with red shading and reflect no
change in MIC.
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The Mce3R regulon is required for potentiation of INH by 6-azasteroids. Previously, we discovered that
the Mce3R regulon encodes FadE17-FadE18, an unusual heterotetrameric acyl-CoA dehydrogenase that
is associated with cholesterol metabolism.22 Moreover, Mce3R regulates the mel2 operon, which is
implicated in Mtb persistence in macrophages23 and in resistance to oxidative stress24 that is associated
with lipid metabolism.25-26 The regulon is de-repressed upon treatment with cholesterol, most likely by a
cholesterol metabolite,2 and expression of the regulon is up-regulated during hypoxia and in the stationary
phase.27 We found that azasteroid 4a no longer improved INH activity when tested against Mtb Mce3R
regulon mutants fadE18, melF, and melH (Table 2), and this loss of activity was independent of carbon
source.
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The cholesterol catabolism pathway regulated by KstR1 and KstR2 is found in both saprophytic and
pathogenic mycobacteria.28 In contrast, the Mce3R regulon is conserved in M. marinum and Mtb but not
in other pathogens (e.g., M. avium) or in saprophytic mycobacteria (e.g., M. smegmatis). In results
consistent with our mutant potentiation data, we found that 6-azasteroid 4a did not improve INH or BDQ
activity when tested against M. smegmatis or M. avium grown with glycerol as a carbon source (Table 2).
However, inhibition of M. marinum growth by BDQ and INH was increased by addition of 4a.
Spontaneous resistance to 6a is Mce3R-independent. We examined the frequency with which
spontaneous resistance to one of the azasteroids arose (Table 3). Specifically, resistant mutants were
raised against 6a alone and in combination with BDQ. The reported frequency of in vitro resistance to 20
µM BDQ ranges from 9 × 10–9 to 5 × 10−7 mutations per cell division, and mutations arise predominately
in the atpE gene.29 We found similar frequencies of BDQ resistance in this study. The frequencies of
resistant mutant formation upon treatment with 6a in combination with BDQ were comparable to the
frequencies of BDQ resistance. Treatment with 6a alone resulted in an approximately 5–10-fold higher
frequency of resistance. All the resistant mutants that formed upon treatment of 6a alone or in
combination with BDQ had significant alterations in colony morphology. The wild-type colonies were
thick, rough, irregular, and yellow, whereas the mutant colonies were thin, small, round, and almost
transparent (Figure S4). Twenty mutants were selected, and six of these grew in liquid culture. The
mutant colonies unable to grow in liquid culture were restreaked on agar plates, and one more mutant
grew. All the resistant mutants grew slowly in liquid culture or on agar plates. The 6a, BDQ, and INH
MICs against these seven mutants were determined (Figure S1C). Against three of the mutants (ASR1,
ASR4, and ASR5), the MIC of 6a increased more than 4-fold with respect to the MIC against WT-
CDC1551, whereas 2-4 fold increase was observed for the remainder of the mutants. The INH and BDQ
MICs against all the 6a-selected mutants were unchanged (1-2 fold increase) compared to their MICs
against WT-CDC1551.
DNA from six of the resistant mutants was sequenced. Only three mutants (ASR1, ASR2, and ASR3)
produced high-quality data; the DNA from the other three mutants was highly fragmented. Twenty-nine
nonsynonymous single nucleotide polymorphisms (SNPs), occurring in 25 genes, were identified and
confirmed by at least 10 Illumina reads (Figure S5). Of the 25 genes, 6 belong to the PE/PPE family and 3
encode enzymes involved in PDIM biosynthesis.
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Because mutant ASR2 showed no significant increase in the 6a MIC, we focused on analyzing mutants
ASR1 and ASR3, in which we identified, respectively, 21 and 11 nonsynonymous SNPs, with 3 SNPs
that were identical in both mutants. A stop gain occurred at S154 in Rv2940c, which encodes a
mycocerosic acid synthase involved in PDIM biosynthesis. In addition, we identified a frameshift
insertion at P188 in Rv3467, a non-essential gene, and a recombination of PPE19. No Mce3R gene
mutations were identified in the resistant mutants.
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9
As for individual nonsynonymous mutations, we determined that ASR1 carried a nonsynonymous SNP in
guaB3 (Rv3410c), which is an essential gene annotated as an inosine-5'-monophosphate dehydrogenase
(Figure S5). However, GuaB3 does not perform this dehydrogenase enzymatic function.30 The guaB3
gene resides in a three-gene operon Rv3409c-guaB3-guaB2, and Rv3409c is required for bacterial survival
and growth in macrophages.31 We also identified frameshift deletions in Rv0678, Rv2552c, and Rv3267
from ASR1. These genes are involved in the regulation of a cell efflux system, aromatic amino acid
synthesis, and cell-wall synthesis, respectively. A frameshift deletion was also identified in Rv0204c from
ASR3. The disruptions in genes involved in mycobacterial cell-wall biosynthesis are consistent with the
phenotypic changes in colony morphology that we observed.
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Table 3. Frequencies of in vitro spontaneous resistance to 6a and BDQ.a
Frequency of resistanceb
BDQ
BDQ
BDQ
(20 μM)
(10 μM)
(0 μM)
6a (400 μM)
6a (200 μM)
6a (0 μM)
7.8 × 10-8
ND
5.5 × 10-9 3.4 × 10-7
ND
2 × 10-7
1.1 × 10-7
3.6 × 10-6
–
aCDC1551 Mtb was plated on 7H11 medium supplemented with
10% OADC containing the indicated concentration of drug(s).
Resistant mutants were counted after 3–4 weeks. ND = not
detected. bMutations per cell division.
6-Azasteroids suppress the transcription of Mtb stress-response genes including the Mce3R regulon.
RNA-seq gene expression profiles of the wild-type CDC1551 Mtb and the resistant mutants ASR1 and
ASR2 were generated after 6 h of exposure to 4a or 6a. Upon treatment of the wild-type with 4a or 6a,
genes involved in lipid metabolism, cell-wall synthesis and cell processes, PE/PPE, and information
pathways (TubercuList functional classes) were generally down-regulated (>2-fold). The global
expression profiles for 4a and 6a treatment were like the profile for BDQ treatment, with >80% of the
genes that were down-regulated by BDQ (>1.5-fold) also being down-regulated (>1.5-fold) by 4a.32 In the
absence of any of the test compounds, resistant mutants ASR1 and ASR2 had a common set of 49 up-
regulated (>2-fold) genes, including stress-response genes regulated by DosR, PhoP/R, MprA, Crp, and
WhiB3.33
In addition to the observed general response to drug treatment, six sets of genes that appeared to be
specific to the 6-azasteroid mechanism of action were up- or down-regulated by treatment of wild-type
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CDC1551 Mtb with 4a or 6a (Figures 5 and S6). The magnitude of differential expression of these
specific genes in response to treatment with 4a or 6a was much lower in ASR1 than in the wild-type, a
result that is consistent with the increased drug MIC.
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KstR1_CholCat
Mce3R
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Mce1R
KstR2_CholCat
KstR1_mce4R
Mel1
Figure 5. Differentially expressed transcripts as analyzed by RNA-seq are visualized in heatmaps.
The transcriptional profiles of Mtb strains CDC1551 (WT), ASR1, and ASR2 exposed for 6 h to 4a (30
μM) or 6a (30 μM) were compared with the profile of the corresponding untreated group. Gene names
are indicated to the right of the heatmap, and strains and treatment conditions are shown on the bottom.
The color scales represent log2-fold changes in gene expression in treated groups relative to expression
in untreated groups; each colored cell represents the mean of biological triplicates. Red, blue, and black
lines denote the echA13-fadE17-fadE18, mel2, and mce3 operons, respectively.
In the wild-type CDC1551 Mtb, the KstR1 and KstR2 regulon genes that encode cholesterol catabolism
enzymes were either up-regulated or unaffected (Figure 5). In contrast, KstR1 genes not directly related to
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cholesterol catabolism were greatly down-regulated (up to 25-fold, not shown). Additionally, KstR1-
regulated genes in the mce4 operon, which encodes the cholesterol transport system, were down-regulated
by 4a or 6a. Genes in the mce1R operon, which is associated with mycolic acid transport, and in the mel1
operon, which encodes putative membrane protein and transglutaminases thought to be important for
infection,23 were greatly down-regulated (>5-fold). In the wild-type, the Mce3R-regulated echA13-
fadE17-fadE18 operon was down-regulated by treatment with 4a or 6a, whereas the other two Mce3R-
regulated operons were unaffected. In addition, guaB2, which is in the Rv3409c-guaB3-guaB2 operon,
was up-regulated by 4a or 6a (Table S4). Gene expression of representative genes from Mce3R, KstR1
and KstR2 regulons and mel1 operon in Mtb CDC1551 exposed to 4a, was confirmed by qRT-PCR
(Figure S6).
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DISCUSSION
We tested 37 azasteroids in MIC assays and identified a set of eight 6-azasteroids that clearly sensitized
Mtb to existing TB drugs (Figure 2). We observed a clear structure–activity relationship, indicating that
the compounds acted on a specific target or targets. The TB agents that exhibited synergy with these 6-
azasteroids utilize a wide array of killing mechanisms, disrupting processes ranging from cell-wall
biosynthesis to ATP biosynthesis (Table 1).
We found that genes involved in cholesterol catabolism were not required for the activity of 6-azasteroid
4a (Table 2), despite our previous work demonstrating that 6-azasteroids, including 4a, inhibit the first
enzyme in the catabolic pathway, 3β-hydroxysteroid dehydrogenase (hsd),19 and a body of evidence
indicating that cholesterol catabolism is important for Mtb persistence and survival in a mouse model of
infection.1-2 Moreover, the 6-azasteroids were active against Mtb grown on a sugar carbon source,
glycerol, as well as against Mtb grown on a cholesterol carbon source (Figure 2). Importantly, 6-
azasteroids 4a and 6a were active under low-oxygen growth conditions. Taken together, the evidence
suggests that 6-azasteroids act on a target that contributes to drug tolerance regardless of carbon source or
oxygen level.
Transcriptional,2 phenotypic,34 and biochemical profiling22 have identified genes outside the Mtb
cholesterol catabolism gene cluster that are regulated by cholesterol, that are important for growth on
cholesterol in vitro, or that have cholesterol-specific structural motifs. Several of these genes are found
within a TetR-like transcriptional regulon controlled by the Mce3R repressor.26-27 Transposon disruption
of mce3R significantly inhibits growth of Mtb on cholesterol.34 However, the genes within the Mce3R
regulon are not required for catabolism of cholesterol,8-9 suggesting that these genes have an alternate role
in Mtb survival. Therefore, we investigated the importance of four genes in the Mce3R regulon (fadE18,
echA13, melF, and melH) in 6-azasteroid activity. Intriguingly, fadE18, melF, and melH were required for
6-azasteroid potentiation of INH activity. Although echA13, annotated to encode an enoyl-CoA hydratase,
was not required, the Mtb genome encodes many other enoyl-CoA hydratases that might compensate for
disruption of this gene.
The precise biochemical functions of the proteins encoded in the Mce3R regulon have yet to be
established. However, bioinformatics and phenotypic assays allow for tentative assignment of their
function. The fadE17, fadE18, and mel2 genes are important contributors to Mtb resistance to various
cellular stresses. The entire Mce3R locus is present in M. marinum and provides resistance to reactive
oxygen species and reactive nitrogen species.24 Significantly, the Mce3R regulon is absent in saprophytic
mycobacteria,28 which suggests that the primary role of the regulon is mediating host-derived stresses,
such as those encountered by Mtb in activated macrophages. Consistent with this observation, the mel2
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operon is required for persistence and dissemination of Mtb infection in mice.35
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The fadE17 and fadE18 genes encode an acyl-CoA dehydrogenase with an unusual α2β2 heterotetrameric
that is characteristic of acyl-CoA dehydrogenases that oxidize cholesterol-derived substrates.22 The melF
and melH genes reside in a single operon (Rv1936-Rv1941) of the mel2 locus, which encodes homologs of
the Lux luminescence system. This system catalyzes formation of a fatty acid aldehyde by means of an
ATP-dependent process.
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The mel2 operon is thought to provide resistance to oxidative stress, which is consistent with the idea that
the operon encodes catalytic machinery to generate a fatty acid aldehyde that can remove oxidizing
species from the cellular milieu.24 When Mtb production of ergothioneine, which is a redox couple in Mtb,
is disrupted, fadE18 is up-regulated.36 When Mtb is exposed to a lysosomal soluble fraction prepared from
activated macrophages, both fadE17 and fadE18 are induced.37 In addition, a recent study has shown that
cysteine in combination with INH or rifampin can enhance oxygen consumption by Mtb, thereby leading
to increased production of reactive oxygen species and sterilization of Mtb cultures.38 Upon addition of
cysteine to INH-treated Mtb cultures, the transcription level of fadE18 increases nearly 4-fold. Treatment
of Mtb with BDQ for 48–96 h results in up-regulation of mel2 genes, especially melF, which is up-
regulated about 8-fold.32 These responses indicate that the Mce3R regulon plays a critical role in the Mtb
response to oxidative, host-induced, or drug-induced stress.
In contrast to treatment with INH, rifampin, or BDQ, treatment with 6-azasteroid 4a or 6a for 6 h resulted
in 3- to 4-fold down-regulation of the Mce3R-regulated echA13-fadE17-fadE18 operon. Although the
transcription levels of mel2 genes were unchanged or only marginally changed after 6 h of 6-azasteroid
treatment, the response of the mel2 gene to BDQ treatment is likely to be indirect, given the long post-
BDQ treatment time (48–96 h) at which up-regulation is observed.32 Together with our mutant experiment
in which fadE18 was required for 6-azasteroid potentiation, the evidence supports a mechanism in which
the 6-azasteroids suppress fadE18/Mce3R-dependent activation of a drug-induced stress-resistance
pathway.
Mtb spontaneous resistant mutants that were raised against 6a showed a thinner, almost transparent cell-
wall phenotype and were difficult to culture. Importantly, we found that Mce3R genes were not directly
involved in spontaneous resistance to the 6-azasteroid. Among the genes where nonsynonymous
mutations occurred in the resistant mutants, PE/PPE genes were not investigated further as candidates for
involvement in the azasteroid-resistance phenotype, because of their high variability in Mtb. In addition, it
is unlikely that genes involved in PDIM biosynthesis are directly involved in the azasteroid-resistance
phenotype, because the loss of PDIM production is commonly observed during in vitro propagation of
Mtb cultures.39 The remainder of the mutations identified were in genes involved in mycobacterial cell-
wall synthesis and efflux systems, which is consistent with a mechanism of resistance that decreases 6-
azasteroid uptake and simultaneously the growth fitness of Mtb is reduced.
In addition to suppressing Mce3R genes, 6-azasteroids also suppressed the expression of genes in the
DosR,40 PhoP/R,33 and SigB regulons41 and in the icl1 operon.42 All these genes are involved in Mtb stress
resistance and establishment of persistence. There is evidence that inhibitors of DosR genes also increase
the activity of INH,43 although the increase is not as dramatic as that observed for 6-azasteroids. This
enhancement of INH activity by the 6-azasteroids indicates that they inhibit a network of Mtb stress
resistance and that the disruption of this network leads to the potentiation of TB drugs.
Co-drug sensitization is emerging as a useful strategy for treating TB, including multidrug-resistant TB.44-
47 Variations on this strategy include targeting specific drug-activation pathways,46 as well as more
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general drug-desensitization targets such as the DosRST regulon43 and thiol stress.47 Thus, these drug-
2
sensitization strategies are a powerful tool for killing resilient and/or nonreplicating Mtb.
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In summary, our work further supports the idea that Mce3R-regulated genes are important for managing
the Mtb cellular response to drug-induced stress (Figure 6). Moreover, our findings suggest that Mtb
depends on cholesterol or its metabolites for activation of stress resistance through the Mce3R regulon
(Figure 6). Our discoveries that 6-azasteroid suppression of drug tolerance depends on Mce3R-regulated
genes and occurs under low-oxygen conditions present an intriguing avenue for further development of
co-drugs that can improve the efficacy of existing TB drugs in vivo.
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Figure 6. The efficacy of 6-azasteroids requires Mce3R-regulated genes. Genes in the Mce3R
regulon are required for 6-azasteroid anti-mycobacterial activity. Cholesterol transcriptionally regulates
the Mce3R genes,2 and this regulon is repressed by Mce3R, a tetR-like repressor protein.
MATERIALS AND METHODS
Synthesis of 6-azasteroid precursor 1 and the corresponding 17β-carboxylic acid. 17β-Carbomethoxy-6-
t-butoxycarbonyl-6-azaandrost-4-en-3-one (1) was synthesized as reported previously16-18 and then
converted to 17β-carboxy-6-t-butoxycarbonyl-6-azaandrost-4-en-3-one as described in the literature.17
Both compounds were assessed to be greater than 95% pure by 1H NMR spectroscopy.
General procedure for coupling amines to the 17β-carboxylic acid and subsequent deprotection of the
ring nitrogen.17 17β-Carboxy-6-t-butoxycarbonyl-6-azaandrost-4-en-3-one (30 mg) was suspended in 1
mL of toluene. One drop of DMF and 15 µL of pyridine were added to the solution, which was then
cooled in an ice bath, treated with 10 µL of thionyl chloride, and stirred for 1 h. The solution was filtered,
and the filtrate was concentrated. The resulting acid chloride residue was dissolved in DCM (5 mL) and
treated with the desired amine to give amide 3. Amide 3 was dissolved in DCM (1 mL) and treated with 2
mL of TFA at 20 °C to remove the BOC protecting group from the ring nitrogen. After 2 h, the reaction
mixture was poured into saturated NaHCO3 solution, the layers were separated, and the organic layer was
washed with brine and dried over Na2SO4. Chromatography using 5% (v/v) MeOH in DCM provided
deprotected amide 4.
Procedure for N6 alkylation of deprotected amides 4a and 4b.8, 18 Amides 4a and 4b (30 mg) were
dissolved separately in THF (5 mL) and treated with 1.2 equiv of NaH. After stirring for 30 min at 20 °C,
the reaction mixture was treated with the desired iodoalkane (1 equiv) for a further 30 min. When the
reaction was complete as judged by TLC (approx. 5 h), 30 mL of EtOAc was added, and the resulting
solution was washed with water and brine (3× each) and dried over Na2SO4. Chromatography using 5%
(v/v) MeOH in DCM provided N6-alkylated amides 5a, 6a, 6b, and 7a. For the synthesis of 8a, amide 4a
(30 mg) was dissolved in DMF and treated with 3 equiv of NaH. After 30 min at 20 °C, 5 equiv of
iodomethane was added into the reaction, followed by stirring at 20 °C for 2 h to give compound 8a.
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Active azasteroids 4a, 5a, 6a, 8a, and 3c were resynthesized for verification and were determined to be
greater than 99% pure by LC-MS.
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Bacterial strains and culture conditions. Mutant and complemented strains used in this study and their
sources are listed in Table S2. Mtb strains CDC1551, H37Rv, and H37Rv(mlux), also known as
H37Rv(pFCA-luxABCDE), were used as wild-type strains for this study. Mtb strains were cultured at
37 °C either in Middlebrook 7H9 medium (broth) supplemented with 0.2% glycerol or 0.1 mM
cholesterol, 0.5% BSA, 0.08% NaCl, and 0.05% (v/v) tyloxapol (7H9/glycerol or 7H9/cholesterol
medium) or on 7H11 medium (agar) supplemented with 10% oleate-albumin-dextrose-NaCl (OADC),
0.5% glycerol, and 0.05% Tween 80.
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Determination of MICs. MICs were determined by means of a broth microdilution assay.48 Briefly, cells
were grown to mid-log phase and then diluted 1000-fold in defined media. Cell suspension was added to a
96-well plate containing compound dilutions to obtain a final volume of 100 µL. Plates were incubated at
37 °C for 14 days, and MICs were determined as the lowest concentration that resulted in complete
inhibition of growth by visual inspection or luminescence readouts (mlux). For isobolograms, 5-fold serial
dilutions of INH or BDQ were used so that the concentration range in a single plate was sufficient. If the
90% reduction in RLU could not be determined directly, a linear interpolation between two RLU values
was used to determine the concentration at 90% inhibition.
LORA.49 A low-oxygen-adapted culture of H37Rv(mlux) Mtb expressing a luciferase was used for the
LORA. H37Rv(mlux) Mtb stored at –80 °C (1 × 105 CFU/mL) was thawed and exposed to 2-fold or 3-
fold serial dilutions of test compound diluted in 7H12 broth in black 96-well plates, which were incubated
for 10 days at 37 °C in an anaerobic jar under hypoxic conditions created with an Anoxomat system
(MART Microbiology). Luminescence readouts were obtained after 28 h of normoxic recovery in 5%
CO2. To calculate the MIC, the dose response curve was plotted as percentage growth and fitted to the
Gompertz model. The MIC was defined as the lowest concentration inhibiting recovery of the
luminescence signal by 90% relative to bacteria-only controls. FIC is defined as the ratio of the MIC of
the inhibitor when used in combination to the MIC of the inhibitor alone. FIC index is the sum of the FICs
for the two drugs used. The minimum concentrations that worked in combination were used. The
determination of drug MICs for rifampin, INH, linezolid, pretomanid, and BDQ were conducted as
positive controls.
LORA time-kill assay. The low-oxygen-adapted Mtb strain H37Rv(mlux) was cultured as described above
for the LORA. Compounds 4a and 6a were tested at 10 µM in combination with BDQ at 0.5, 0.25, 0.15,
0.0625, and 0.05 µM. At 0, 7, and 10 days, cultures were recovered in an aerobic environment (5% CO2)
for 28 h and then were serially diluted in 7H9 broth and plated on 7H11/OADC agar plates. Drug
concentration is diluted at least 300–fold. Plates were incubated at 37 °C, and CFUs were counted after 3–
4 weeks. The limit of detection is 10 CFU/mL. Statistical analysis was done using an unpaired Student’s
t-test (GraphPad Prism 4).
Aerobic time-kill assay. Mtb was grown at 37 °C to mid-log phase and then diluted in fresh medium to 5
× 105 CFU/mL. Test compounds were added at defined concentrations. Aliquots of cultures were
withdrawn at specified time points, and either OD (600 nm) (CDC1551) or luminescence (mlux) was
recorded. At 0, 2, and 7 days, aliquots of cultures were serially diluted in 7H9 broth and plated on
7H11/OADC agar plates. Drug concentration is diluted at least 300–fold. Plates were incubated at 37 °C,
and CFUs were counted after 3–4 weeks. The limit of detection is 10 CFU/mL.
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Frequency of resistance. Mtb mutants resistant to 6a were isolated by means of the procedure reported by
Ioerger et al.50 H37Rv Mtb bacteria were grown at 37 °C to mid-log phase and then diluted in fresh
Middlebrook 7H9 medium containing ADC-Tween 80 to 5 × 108 CFU/mL. Middlebrook 7H11/OADC
agar plates with 6a at 200 or 400 µM with or without 10 or 20 µM BDQ were inoculated with 108, 107,
106, and 105 CFU/plate, and the plates were incubated at 37 °C for 3–4 weeks. Resistance was tested by
measuring the MICs of 6a, BDQ, and INH. The frequency of the appearance of resistant mutants was
calculated.
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Whole genome sequencing. Mtb strains CDC1551, ASR1, ASR2, and ASR3 were grown to log phase,
and their genomes were extracted with cetyltrimethylammonium bromide and lysozyme as described in
the literature.51 Whole genome preparation, sequencing, assembly, and pairwise analysis were performed
as previously described.52 Briefly, DNA was sheared into ~20,000 bp fragments using Covaris G-tube
spin columns and was end-repaired before being ligated to SMRTbell adapters (Pacific Biosciences). The
resulting library was treated with an exonuclease cocktail to remove unligated DNA fragments and was
size-selected on a Sage Science BluePippin system to obtain fragments of ≥7000 bp. The P5-C3
sequencing enzyme and chemistry were used to sequence the resulting libraries on the Pacific Biosciences
(PacBio) RS II platform. Resulting PacBio sequencing data were assembled using HGAP3 (ver. 2.2.0).
For Illumina sequencing, genomic DNA (1 µg) was sheared to achieve ~200 bp fragments using a
Bioruptor Pico sonicator (Diagenode). Library preparation was performed using the end repair, A-tailing,
and adaptor ligation NEBNext DNA library prep modules for Illumina from New England Biolabs,
according to the manufacturer’s protocol. The resulting libraries were sequenced on the Illumina HiSeq
2500 platform. Illumina reads were then mapped to the curated PacBio assemblies using samtools
mpileup53 to correct PacBio sequencing errors. Genome circularization, curation, and annotation were
performed with a custom postassembly pipeline (https://github.com/powerpak/pathogendb-pipeline).52
Finally, NUCmer (ver. 3.1)54 was used for aligning mutant genome strains to the PS00103 reference
genome to identify genetic variants. Whole genome sequencing data are available in the NCBI BioProject
database under accession number PRJNA482894.
RNA-seq transcriptional profiling and qRT-PCR. Mtb strains CDC1551, ASR1, and ASR2 were grown
to OD ~0.6 and treated with 30 µM 4a or 6a or with vehicle control for 6 h in Middlebrook 7H9 medium
supplemented with 0.2% glycerol, 0.5% BSA, 0.08% NaCl, and 0.05% (v/v) tyloxapol. Total RNA was
extracted using TriZol, with chloroform back extraction and 70% ethanol precipitation. RNA was purified
with an RNeasy kit (Qiagen) with DNAse treatment. Total RNA was processed for ribosomal reduction
library construction. Libraries were sequenced as single-end 75 bp reads on an Illumina NextSeq500
sequencer following the manufacturer's protocols (Cofactor Genomics, Inc.). The sequence reads were
aligned to the CDC1551 Mtb complete genome (The National Center for Biotechnology Information
Database) using STAR (https://github.com/alexdobin/STAR). For each transcript or patch, the reads per
kilobase million (RPKM) expression value was calculated for each sample. For each replicate group, the
mean and coefficient of variation for each transcript or patch were calculated across the expression values
for the samples in that group. These means were considered to be the expression values for the replicate
group. P-values were calculated for comparisons between the means of each pair of replicate groups using
a Welch’s t-test corrected for false discovery rate by means of the Benjamini–Hochberg procedure. For
each comparison, differentially expressed genes were identified as genes with an average normalized
count of >100, differential gene expression of >2-fold, and a P-value of <0.05. Three biological replicates
were performed. RNA-seq data are available in the NCBI GEO database under accession number
GSE118482. Total RNA was reverse-transcribed using PrimeScript RT Master Mix (TaKaRa Bio). The
resulting cDNA was used for PCR amplification using iTaq Universal SYBR Green Supermix (Bio-Rad).
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Relative level of gene expression was calculated by the DDCq method with 16s rRNA as an internal
control. Primers are listed in Table S3.
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ACKNOWLEDGMENTS
Research reported in this publication was supported by the National Heart, Lung, and Blood Institute and
the National Institute of Allergy and Infectious Disease of the National Institutes of Health (award nos.
U01HL127522, R01AI134054, and HHSN272201100009I) and through BEI Resources. The content is
solely the responsibility of the authors and does not necessarily represent the official views of the
National Institutes of Health. Additional support was provided by the Center for Biotechnology, a New
York State Center for Advanced Technology; Stony Brook University; Cold Spring Harbor Laboratory;
Brookhaven National Laboratory; the Feinstein Institute for Medical Research; and the New York State
Department of Economic Development under Contract C14051. Shearson Editorial Services (Cornwall,
NY, USA) provided English-language editing of the text of this paper.
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ABBREVIATIONS
BDQ, bedaquiline; CFU, colony forming unit; DMF, dimethylformamide; DMSO, dimethyl sulfoxide;
FIC, fractional inhibitory concentration; INH, isoniazid; LC-MS, liquid chromatograph-mass
spectrometry; LORA, low oxygen recovery assay; MIC, minimum inhibitory concentration; Mtb,
Mycobacterium tuberculosis; PDIM, phthiocerol dimycocerosates; PRKM, reads per kilobase million;
qRT-PCR, quantitative reverse transcriptase real-time polymerase chain reaction; TB, tuberculosis; TLC,
thin-layer chromatography; THF, tetrahydrofuran; WT, wild-type.
SUPPORTING INFORMATION
Supporting Tables S1-S4, Figures S1-S6 and spectral data for synthesized compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR CONTRIBUTIONS
Designed research: X.Y., T.Y., N.S.S.
Performed research: X.Y., T.Y., R.M., K.I.C.
Whole Genome Sequencing: M.S., G.D., R.S., A.K.
Analyzed data: X.Y., T.Y., R.M., K.I.C., H.v.B., S.F., N.S.S.
Wrote the paper: X.Y., T.Y., N.S.S.
Conflict of interest statement: X.Y., T.Y., and N.S.S. are named inventors on patents and patent
applications related to this article.
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