Antituberculosis Activity of the Antimalaria Cytochrome bcc Oxidase Inhibitor SCR0911

Article abstract of DOI:10.1021/acsinfecdis.9b00408

The ability to respire and generate adenosine triphosphate (ATP) is essential for the physiology, persistence, and pathogenicity of Mycobacterium tuberculosis, which causes tuberculosis. By employing a lead repurposing strategy, the malarial cytochrome bc

Full text of DOI:10.1021/acsinfecdis.9b00408

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Article
Anti-tuberculosis activity of the anti-malaria
cytochrome bcc oxidase inhibitor SCR0911
Sherilyn Chong, Malathy Sony Subramanian Manimekalai, Jickky Palmae Sarathy, Zoe C. Williams, Liam
K. Harold, Gregory M Cook, Thomas Dick, Kevin Pethe, Roderick Wayland Bates, and Gerhard Grüber
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00408 • Publication Date (Web): 24 Feb 2020
Downloaded from pubs.acs.org on February 25, 2020
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Anti-tuberculosis activity of the anti-malaria cytochrome bcc oxidase inhibitor
SCR0911
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Chong Shi Min Sherilyn1,2,3, Malathy Sony Subramanian Manimekalai1, Jickky Palmae
Sarathy4, Zoe C. Williams5, Liam K. Harold5, Gregory M. Cook5, Thomas Dick4,6, Kevin
Pethe1,7, Roderick W. Bates2, and Gerhard Grüber1,*
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1School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,
Singapore 637551, Republic of Singapore
2School of Physical and Mathematical Sciences, Nanyang Technological University, 21
Nanyang Link, Singapore 637371, Republic of Singapore
3Nanyang Institute of Technology in Health and Medicine, Interdisciplinary Graduate School,
Nanyang Technological University, Republic of Singapore
4Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National
University of Singapore, 14 Medical Drive, Singapore 117599, Republic of Singapore
5Department of Microbiology and Immunology, School of Biomedical Sciences, University
of Otago, Dunedin 9054, New Zealand
6Center for Discovery and Innovation, Hackensack Meridian Health, 340 Kingsland Street
Building 102, Nutley, NJ 07110, USA
7Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore,
636921, Singapore
*To whom correspondence should be addressed: Prof. Dr. Gerhard Grüber, Tel.: (65) 6316
2989; E-mail: ggrueber@ntu.edu.sg
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The ability to respire and generate ATP is essential for the physiology, persistence and
pathogenicity of Mycobacterium tuberculosis, which causes Tuberculosis. By employing a
lead repurposing strategy, the malarial cytochrome bc1 inhibitor SCR0911 was tested against
mycobacteria. Docking studies were carried out to reveal potential binding and to understand
the binding interactions with the target, cytochrome bcc. Whole cell-based and in vitro assays
demonstrated the potency of SCR0911 by inhibiting cell growth and ATP synthesis in both
the fast- and slow- growing M. smegmatis and M. bovis bacillus Calmette–Guérin,
respectively. The variety of biochemical assays and the use of a cytochrome bcc-deficient
mutant strain validated the cytochrome bcc oxidase as the direct target of the drug. The data
demonstrate the broad-spectrum activity of SCR0911 and opens the door for structure-
activity relationship studies to improve the potency of new mycobacteria specific SCR0911
analogs.
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Key Words: Tuberculosis, Mycobacteria, OXPHOS pathway, Multi Drug Resistance,
Extremely Drug Resistance, Telacebec, Q203
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Tuberculosis (TB) is one of the deadliest contagious diseases. In 2018, there were 1.4
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million deaths due to TB.2 The current treatment of TB mainly utilizes a combination of
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antibiotics that requires long-term therapy.
This results in the emergence of multidrug-
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resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), which are difficult to
manage and treat.6-8
There has been a recent resurgence of activity with a number of new compounds in the
TB-drug pipeline, including promises of respiration by the electron transport chain (ETC) and
ATP synthesis (oxidative phosphorylation) as new target spaces for drug development since
the generation of ATP is essential for the bacterial pathogen’s physiology, persistence and
pathogenicity.9 For example, clofazimine, which affects non-proton translocating NADH
dehydrogenase (type II) of the ETC, is an antimycobacterial agent with good in vitro
activity.10 Telacebec (Q203), a cytochrome bcc-aa3 terminal oxidase complex inhibitor is a
bacteriostatic drug against Mtb.11 The concept that oxidative phosphorylation inhibitors could
shorten the time of therapy for drug-resistant tuberculosis is supported by the clinical
development of Sirturo® (bedaquiline, BDQ), a drug targeting the Mtb F1FO ATP synthase. 12,
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The cyt-bcc-aa3 is required for optimum mycobacterial growth but is not strictly
essential.14 However, given the safety profile of the most advanced inhibitors demonstrated in
human clinical trial phase 1 and 2, the synergy between Telacebec and inhibitors of the
oxidative phosphorylation pathway,15-17 and the synthetic lethal interaction between cyt-bcc-
aa3 and the cytochrome bd oxidase (cyt-bd),15, 18 drugs targeting the cyt-bcc-aa3 probably
have their place in a rational drug combination for drug-resistant tuberculosis.
Mycobacteria harbour a number of primary dehydrogenases to fuel the ETC, and two
terminal respiratory oxidases, an aa3-type cytochrome c oxidase (cyt-bcc-aa3) and a
bacterial-specific cytochrome bd-type menaquinol oxidase (cyt-bd). These are present for
dioxygen reduction coupled to the generation of a proton motive force (PMF, the sum of
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proton-gradient and the membrane potential), which is finally used for the condensation of
ADP + Pi to form ATP by the engine F1FO-ATP synthase (F-ATP synthase).19 Cyt-bcc,
complex III of the ETC, aids in the transfer of electrons from menaquinol to cytochrome aa3,
known as the complex IV of the ETC, in which reduction of oxygen to water occurs.
Cytochrome bcc consists of a Qo and Qi binding pocket, menaquinol is oxidized to
menaquinone in the Qo site, releasing protons which contribute to the generation of the PMF.
Menaquinone binds to the Qi site and becomes reduced in the process, to regenerate
menaquinol.20, 21 Besides cytochrome bcc, Mtb also possesses a cyt-bd, which is as a non-
proton translocating terminal oxidase with functional importance under hypoxia in vitro and
in vivo. 22-26
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There is growing evidence for the potential to repurpose drugs originally developed for
the treatment of other diseases, for example clofazimine, for use against drug-resistant TB.27,
28
Evidence for successful repurposing of antimalaria compounds for treating TB has been
seen previously with triclosan, effective against wild-type and drug-resistant strains of Mtb
and Plasmodium falciparum, the causative agent of malaria, by inhibiting the enoyl acyl
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carrier protein reductase . Previously, it was uncovered that 4-(1H)- quinolone, such as
SCR0911, are potent antimalarial compounds that target the cytochrome bc1 of P.
falciparum.30, 31 The same authors have prepared a homology model of the P. falciparum cyt-
bc1, proposing SCR0911-binding and its derivatives to the Qi site of the complex (Fig. 1). 30,
31 Recently, a crystal structure of the bovine cytochrome bc1 confirmed binding of SCR0911
in the Qi site, although differences were noted in the orientation and amino acids involved
compared to the one within the P. falciparum complex (Fig. 1B), which may also reflect the
lower potency of SCR0911 against the human enzyme.30, 32
SCR0911 has an encouraging anti-malarial activity of 12 nM, with a low inhibition to
bovine heart bc1, supporting the lower potency of SCR0911 against human enzyme.30
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Although the P. falciparum and human cyt-bc1 amino acids, which are proposed to be
involved in SCR0911-binding, are not conserved in Mycobacterium species, except for
residue G38 (Fig. 1B), with the low potency of SCR0911, along with the cryo-EM and crystal
structure of SCR0911 with bovine cytochrome bc1, we investigated herein whether SCR0911
could bind to the mycobacterial cyt-bcc complex, and whether such interactions would have
sufficient potency to inhibit oxidative phosphorylation and bacterial growth.
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RESULTS AND DISCUSSION
Docking of SCR0911 with Mtb cyt-bcc complex
In order to study the potential binding site and binding mode of SCR0911 in Mtb cyt-bcc,
docking studies were carried out. Since the structure of Mtb cyt-bcc complex was not
available, a homology model was generated based on the M. smegmatis Electron Microscopy
(EM) structure (PDB ID: 6ADQ) as a template as described in Material and Methods.21 The
sequence identity between Mtb and M. smegmatis is high (81% for QcrA, 83% for QcrB and
80% for QcrC), therefore, a good model could be predicted with a sequence coverage of
around 90%, 97% and 80%, and a GMQE (Global Model Quality Estimation) of around 0.76,
0.90 and 0.68 for QcrA, B and C, respectively. The Mtb model subunits are structurally very
similar to the template M. smegmatis structures with r.m.s. deviation of 0.061, 0.063 and
0.069Å for QcrA, QcrB and QcrC, respectively. The individual subunits were assembled, and
a dimer model was generated based on the M. smegmatis structure, which was then used to
identify the binding region of SCR0911 (Supplementary Figure S1A and Fig. 2A-D). As the
binding region of SCR0911 in Mtb cyt-bcc is unknown, the whole model was used for
searching. 100 protein-ligand complex configurations were generated and analyzed.
Clustering analysis revealed multiple binding sites of the compound in the Mtb cyt-bcc model
with major and minor populations. Overlapping of the two monomers of the dimer revealed
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two major and two minor binding sites with populations of 33% and 21% as well as 13% and
8%, respectively. Multiple site binding of the compound is expected, since the substrate
menaquinone has three more binding sites in addition to the known two Q-sites in the M.
smegmatis cyt-bcc structure.21 The first major binding site with 33% population of the Mtb
cyt-bcc-SCR0911 complex configuration lies on both QcrB and QcrC subunits through
hydrophobic interactions, which were largely contributed from QcrB (Fig. 2A). This complex
configuration has a free energy of binding of -7.01 kcal/mol with a theoretical inhibition
constant (Ki) of 7.26 μM. In this complex configuration, SCR0911 is closer to high-spin
heme (7.1 Å) and binds near the Qo-site, the closest distance being 3.6 Å with F308 (Fig. 2A).
The quinolone moiety of SCR0911 has a π-π interaction with W262 of QcrA and is stabilized
by hydrophobic interactions with M259 of QcrA and Y389 and M390 of QcrB. F308 of QcrB
stabilizes the methoxy group and M126 of QcrB stabilizes the pyridine ring. L349 and I386
of QcrB stabilizes the trifluoromethoxyphenyl group through hydrophobic interactions. Since
SCR0911 binds in such a way that it covers the Qo-site from behind, we assume that it affects
the region near the Qo-site. As it is closer to the heme, it might also indirectly influence the
electron transfer pathway between the heme, thereby contributing to the inhibition of the
enzyme (Fig. 2B). Moreover, the compound binds exactly at the menaquinone-binding site,
although this position is reported to be a functionally irrelevant region. The large population
of SCR0911 binding to this pocket might indicate that this binding region is significant,
which needs to be evaluated in the future (Fig. 2B).21 The second major binding site with a
population of 21%, has interactions with all three subunits, viz. QcrA, B and C, with residues
from subunits B and C contributing more to the interaction (Supplementary Figure 1B).
Although largely hydrophobic interactions are observed, a hydrogen bond with S133 of QcrA
is noted (Supplementary Figure 1C). The free energy of binding is -5.54 kcal/mol with a
theoretical Ki of 86.78 μM. In this position, the compound is farther away from all the heme
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molecules in the complex (distance from low and high spin heme in QcrB is 17.5 and 29.6 Å,
respectively, and heme cD2 in QcrC is 17.2 Å away) and also from the Qi and Qo-site (21.0 Å
and 25.6 Å, respectively). SCR0911 is not close to any of the menaquinone-binding sites,
therefore, the importance of this position and the possible mechanism of action could not be
deduced.
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Of the two minor binding sites, the first one has a population of 13% and interacts
through hydrophobic interactions with subunits QcrA and B and has a free energy of binding
of -5.94 kcal/mol with the theoretical Ki of 44.40 μM (Supplementary Figure 1D, E). While
the compound is not close to any of the heme molecules or to the Q-sites or the menaquinone
sites, its proximity to the iron-sulfur (2Fe-2S) cluster in subunit QcrA (10.9 Å) might be
relevant, as binding of SCR0911 at this position may affect the residues close to this cluster
and thereby indirectly provide inhibition to the complex (Supplementary Figure 1F). The
second minor binding site with a population of 8% is an important site as SCR0911 binds
exactly in the Qi-site (Fig. 2C). It has a free energy of binding of -5.75 kcal/mol with a
theoretical Ki of 61.10 μM. The compound is closer to a high-spin heme (3.2 Å) and has
hydrophobic interactions with residues of subunit QcrB (Fig. 2D). The oxygen atom of the
quinolone moiety has a weak hydrogen bond interaction with Y53 and the methoxy group is
stabilized by hydrophobic interaction with L52, H240 and F45. The pyridine ring has face-to-
face π-interaction with W236 and the trifluoromethoxyphenyl group is stabilized by L31, L35
and Q34. Hence in this complex configuration, binding of SCR0911 in the Qi-site and near
the heme affects both menaquinone binding and influences the electron transfer pathway,
thereby leading to inhibition of the complex (Fig. 2C & D). This complex configuration
position is similar to the binding of the compound in the structure of bovine cyt-bc1 complex
(Fig. 2E).32 However, the trifluoromethoxyphenyl pyridine moiety orients in a different
direction although the quinolone moiety binds to a similar position. This differential binding
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could be caused by the structural variations near the Qi-site and the changes near the N-
terminal helices of the Mtb and bovine qcrB subunit (Fig. 2E). The helix holding W236 of the
Qi-site, is moved inward, which pushes the 2nd helix from the N-terminus down in Mtb QcrB
subunit. These structural variations create a wide space near the Qi-site and allow a larger
movement and the differential binding of the compound. The differential binding mode is not
uncommon, as it has previously been reported that in the structure of bovine cyt-bc1 complex
the two potent antimalarial agents in vivo, SCR0911 and GSK932121, bind in the same Qi-
site but with a different binding mode.31, 32
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Antimycobacterial activity of SCR0911
An alternative synthesis protocol for SCR0911 was established (Material and Methods
and Fig. 3) to study the effect of the compound on mycobacterial growth and energetics. The
modified scheme allows the direct synthesis of an oxazole directly from a carboxylic acid.30
In the synthesis scheme by Charoensutthivarakul et al. (2015), an isatoic anhydride was first
prepared from the carboxylic acid 6 using triphosgene, which will in turn be used to
synthesize the oxazole 4 (Fig. 3). With the direct synthesis of the oxazole 4 directly from a
carboxylic acid 6, the synthesis was shortened and the use of triphosgene eliminated.
To gain a preliminary indication of the effectiveness of SCR0911 on mycobacteria, a
growth inhibitory assay was carried out on both M. bovis BCG and M. smegmatis by
assessing MIC values. As shown in Figure 4A, the MIC50 of BDQ for M. smegmatis was
comparable with those reported (17 ± 2.3 nM), and an MIC50 of 188 ± 14 nM was found for
M. bovis BCG (Fig. 4B).15, 16, 33 The MIC50 of SCR0911 was determined to be 272 ± 41 μM
and 107 ± 5.8 μM against M. smegmatis (Fig. 4A) and M. bovis BCG (Fig. 4B), respectively.
As SCR0911 is proposed to bind to the cytochrome bcc, the M. smegmatis Δbcc mutant strain
ZCW111 was studied with BDQ as a negative control. The MIC50 of BDQ was 16 nM ± 1.9
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(Fig. 4C) which is similar to the MIC50 of BDQ in WT M. smegmatis (17 nM ± 2.3). Like the
mycobacterial bcc-inhibitor Telacebec. SCR0911 was unable to inhibit the growth of the
bcc mutant strains at the highest tested concentration (800 μM)(Fig. 4C), indicating that the
compound may inhibit cell growth by interferring with the function of the cytochrome bcc.
To further determine if SCR0911 targets the respiratory chain specifically, in-vitro assays
were carried out.
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SCR0911 triggers a rapid intracellular ATP depletion in mycobacteria.
To determine if the anti-mycobacterial activity is due to oxidative phosphorylation
inhibition, an intracellular ATP synthesis assay was carried out on M. bovis BCG (Fig 4D).
The IC50 of Telacebec and BDQ was found to be 0.5 ± 0.03 nM and 29 ± 2.7 nM,
respectively, similar to the reported IC50 values in Mtb H37Rv.11 Under similar conditions,
SCR0911 had an effect on ATP levels at an IC50 of 50 ± 6.7 μM, indicating its ability to
inhibit ATP synthesis.
To gain more insights into the inhibition of ATP synthesis, and to uncover the target of
SCR0911, ATP synthesis was measured in inverted membrane vesicles (IMVs) in the
presence of the electron donor NADH. The IC50 values obtained from the IMVs of M.
smegmatis and M. bovis BCG were 7.6 ± 0.4 μM and 6.9 ± 1.4 μM, respectively (Fig. 5A-B).
These IC50 values for both strains indicate its ability to bind to its target the cytochrome bcc
complex in the oxidative phosphorylation pathway, which ultimately reduces ATP synthesis.
Interestingly, the higher IC50 (IC50 = 50 ± 6.7 μM) observed for the intracelluar ATP
synthesis inhibition by SCR0911 above might reflect a phenomenon observed for BDQ and
Telacebec, whose inhibition of the F-ATP synthase and cytbcc-aa3 oxidase, respectively,
causes an activation of the respiration of M. tuberculosis and M. smegmatis leading to
dysregulation of mycobacterial metabolism.5, 25, 34
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The results obtained from the IMVs motivated the use of purer IMVs, which were
generated according to a recent protocol including two additional ultracentrifugation steps.35
ATP synthesis initiated with NADH were carried out on these PMVs, and the IC50 obtained
(3.8 ± 0.3 μM) was comparable to that of the IMVs (Fig. 5C). To determine if SCR0911 may
affect the NADH-dehydrogenase or the succinate-dehydrogenase, a similar experiment was
carried out using succinate instead of NADH, as succinate is taken up directly by complex II
of the ETC. Telacebec was employed in this experiment as a positive control as it binds to the
cytochrome bcc complex.11 The IC50 obtained for Telacebec with NADH (1.1 ± 0.8 nM) and
succinate (2.0 ± 0.1 nM) as the electron donor was comparable (Fig. 5C and D). Similarly for
SCR0911, the IC50 was 3.4 ± 0.8 μM with succinate, alike to using NADH as a substrate (Fig.
5D). Hence, it can be inferred that SCR0911 does not inhibit the NADH-dehydrogenase nor
the succinate dehydrogenase.
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In parallel, a possible effect of SCR0911 on the respiratory cytochrome oxidases, and
thereby on oxygen utilization by these oxidases was tested by using methylene blue as an
oxygen probe. In the presence of oxygen, methylene blue becomes oxidized to cause a liquid
solution to turn blue. However, in an actively respiring bacterial culture where oxygen is
consumed, methylene blue is not readily oxidized and hence it decolourises. The effect of
SCR0911 on bacterial oxygen consumption was observed through its effect on methylene
blue decolourisation.
Thioridazine (THZ) is known to directly affect respiration, and is used as a positive
control in the methylene blue assay.36 As visualized in Figure 6A, 2.5x MIC50 of SCR0911
inhibited methylene blue decolourisation, indicating that oxygen consumption was inhibited,
similar to the positive control of THZ. This is reflected in the calculated decolourization
percentage which was 0% at 2.5x MIC50 of SCR0911 (Fig 6B). Similar observations were
made at 5x and 10x MIC50 of SCR0911 (Fig 6A). However, the solubility of SCR0911 at
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these higher concentrations could have affected the absorbance readings of the methylene
blue decolourisation in the samples and consequently caused aberrant calculated
decolourization percentage values (Fig 6B). Collectively, these results establish that
SCR0911 effectively affects the electron flow from menaquinol to the final electron acceptor
oxygen within the cyt-bcc-aa3 super-complex, providing hints to its binding target.
Interestingly, in case of the cytochrome bcc inhibitor Q203, which does not cause oxygen
consumption inhibition in Mtb H37Rv strains, it has been predicted that Q203 binds to the
Qo-site with the binding region spanning from residues 307DFYMMWTEGLA317.11, 15 Out of
the four Mtb cyt-bc1-SCR0911 complex configurations studied, only the first major binding
site is closer to the Qo-site and it is behind the Qo-site. Therefore, it can be speculated that
SCR0911 might have a different mode of action than Q203.
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Since BDQ inhibition in the F-ATP synthase has been shown to work also as an
uncoupler, and to test whether SCR0911 might reveal any undefined uncoupling effects, an
ATP-driving proton translocation assay was carried out. At the start of the experiment, 9-
amino-6-chloro-2-methoxyacridine (ACMA) was added to IMVs, which were prepared
according to Kamariah et al. (2019), and fluorescence was observed.35 With the addition of
NADH, an electron transfer was initiated, followed by the formation of a proton gradient,
which caused quenching of the fluorescence dye and reduction in intensity. When SCR0911
was added, the fluoresence changed only weakly, while when the protonophore SF6847 was
used as a positive control, the pH gradient collapsed, indicating that SCR0911 is not an
uncoupler (Fig. 7).
CONCLUSION
One of the strategies to discover new drugs is to repurpose existing compounds, thus
reducing the cost and time of drug discovery and / or development. The synthesis and
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biological testing of SCR0911 revealed that the antimalarial compound is a potent inhibitor
of M. smegmatis and M. bovis BCG growth. Effective reduction of ATP synthesis, in both the
mycobacterial IMVs and whole cell assays, demonstrated that SCR0911 disrupts oxidative
phosphorylation of mycobacteria. The use of the electron donor succinate excluded NADH-
dehydrogenase and the succinate-dehydrogenase as direct target, while the loss of SCR0911
potency in the M. smegmatis Δbcc mutant strain supported that cytochrome bcc is the direct
target of the compound. The reduction in oxygen consumption confirms the interruption of
electron flow within the cyt-bcc-aa3 super-complex. The docking studies presented, confirm
the whole cell-based and in vitro studies and described the ability of SCR0911 to bind to the
Qi site of the mycobacterial cyt-bcc, similar to the region modelled for the compound inside
the P. falciparum oxidase.32 Despite having a similar binding region, the interacting residues
differ vastly, indicating SCR0911 adopts a different conformation in the mycobacterial cyt-
bcc complex, and that SCR0911 is surrounded by mycobacterial specific residues
(Supplementary Figure 1; Fig. 2E). Opening the door for the design and structure-activity
relationship studies to improve the potency of new mycobacteria specific SCR0911 analogs
as well as to avoid toxicity in compounds targeting the human cytochrome bc1.
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MATERIALS AND METHODS
Homology modelling of Mtb cyt-bcc
The Mtb cyt-bcc-model was generated using homology modelling within the SWISS-
MODEL server.37 The program accepts the target amino acid sequence an. searches for
evolutionary related protein structures against the template library SMTL using BLAST and
HHblits.38, 39 The templates were then ranked based on global model quality estimate (GMQE)
and quaternary structure quality estimate (QSQE). Finally, the 3D model of the Mtb cyt-bcc
was generated with the M. smegmatis EM structure (PDB ID: 6ADQ) as a template and using
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ProMod3 modelling engine that first transfers conserved atom coordinates defined by the
target-template alignment.21 Afterwards, insertions/deletions were constructed using loop
modelling. The heme groups were manually modelled into the Mtb complex, the individual
subunits were assembled to generate a monomer, and a dimer model was created based on the
M. smegmatis EM structure (PDB ID: 6ADQ).
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Docking studies of SCR0911 with Mtb cyt-bcc
Docking studies were carried out for SCR0911 with the homology model of Mtb cyt-bcc.
The three-dimensional structure of SCR0911 was retrieved from the crystal structure of
bovine cytochrome bc1 complex (PDB ID: 5OKD).32 The SCR0911 was docked into the
model of Mtb cyt-bcc -using AutoDock 4.2.40 Autodock is a computational docking program
which is based on the Lamarckian Genetic Algorithm search method and an empirical free
energy scoring function. The free-energy scoring function is based on a linear regression
analysis, the AMBER force field, and a larger set of diverse protein-ligand complexes with
known inhibition constants. The program consists of two parts, AutoDock for docking of the
ligand to a set of grids describing the target protein; and AutoGrid for pre-calculating these
grids. The 100 protein-ligand complex models scored based on the weighted sum of steric
interactions, hydrophobic interaction, and hydrogen bonding were analyzed. The complex
configurations were then clustered based on the results by conformational similarity,
visualizing conformations, visualizing interactions between ligands and proteins, and
visualizing the affinity potentials created by AutoGrid. The best model with the highest score
from each cluster was selected for further analysis. The model was visualized using PyMOL,
and the interactions profile was represented using LIGPLOT.41, 42
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Synthesis of SCR0911
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The synthesis of SCR0911 was carried out following the procedure by
Charoensutthivarakul et al. (2015) with several modifications to the synthesis scheme.30
4,4,5,5-Tetramethyl-2-(4-(trifluoromethoxy)phenyl)-1,3,2-dioxaborolane (2)43
A mixture of bis(pinacolato)diboron (0.658 g, 2.59 mmol), compound 7 (0.500 g, 2.07
mmol), potassium acetate (0.311 g, 3.17 mmol) and Pd(dppf)Cl2 in tetrahydrofuran (THF) (5
ml) was heated at reflux overnight under nitrogen before cooling to room temperature. Ethyl
acetate (15 ml) was added, and the mixture was filtered through a pad of Celite (R). The
filtrate was washed with water (10 ml x 2) and concentrated in vacuo. The residue was
purified by flash chromatography (10 – 25% ethyl acetate:hexane) to afford the title
compound as a colourless solid (0.39 g, 1.35 mmol, 65%). δH (400 MHz, CDCl3) 7.84 (d, J =
8.6 Hz, 2H), 7.20 (d, J = 8.7 Hz, 2H), 1.34 (s, 12H) ppm.44
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5-Bromo-N-methoxy-N-methylnicotinamide (9)
4- (Dimethylamino)pyridine (DMAP) (0.242 g, 1.98 mmol) and N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI) (2.85 g, 14.9 mmol) were
added to a solution of compound 8 (2 g, 9.90 mmol) and N, O-dimethylhydroxylamine
hydrochloride (1.45 g, 14.9 mol) in dichloromethane (36 ml). The mixture was placed under
nitrogen and triethylamine (2.0 ml, 14.8 mmol) was added dropwise. The mixture was stirred
overnight at room temperature. Water (50 ml) was added, followed by extraction with
dichloromethane (3 x 30 ml). The combined organic layers were washed with brine (50 ml),
dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by flash
chromatography (50% ethyl acetate: hexane) to afford the title compound as a yellow solid
(2.10 g, 8.55 mmol, 86%). δH (400 MHz, CDCl3) 8.87 (d, J = 1.8 Hz, 1H), 8.75 (d, J = 2.3 Hz,
1H)8.17 (t, J = 2.0 Hz, 1H), 3.57 (s, 3H), 3.39 (s, 3H) ppm.45
1-(5-Bromopyridin-3-yl)propan-1-one (3)46
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Ethyl magnesium bromide (34.2 mmol, 4.0 eq) was added dropwise to a solution of
amide 9 (2.11 g, 8.61 mmol) in THF (85 ml) under nitrogen at 0 °C. The solution was stirred
for 30 minutes at 0 °C, then quenched with saturated NH4Cl (50 ml) before extraction with
diethyl ether (3 x 50 ml). The combined organic layers were washed with brine (50 ml), dried
over MgSO4 and concentrated in vacuo. The residue was purified by flash chromatography
(50% ethyl acetate: hexane) to afford the title compound as a yellow solid (0.655 g, 3.06
mmol, 36%). δH (400 MHz, CDCl3) 9.07 (d, J = 1.7 Hz, 1H), 8.84 (d, J = 2.1 Hz, 1H), 8.40 –
8.33 (m, 1H), 3.01 (q, J = 7.2 Hz, 2H), 1.25 (t, J = 7.2 Hz, 3H) ppm; HRMS (EI) 213.9849
[M+H]+.
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1-(5-(4-(Trifluoromethoxy)phenyl)pyridin-3-yl)propan-1-one (1)30
Compound 2 (0.321 g, 1.12 mmol) was added to a stirred solution of compound 3 (0.218
g, 1.02 mmol), Pd(dppf)Cl2 (0.059 g, 0.081 mmol) and potassium carbonate (0.463 g, 3.37
mmol) in THF: H2O (16 ml: 6.4ml) under nitrogen. The mixture was heated at reflux
overnight before cooling to room temperature. Water (10 ml) was added, and the organic
layer was extracted with ethyl acetate (3 x 15 ml). The combined organic layers were washed
with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by flash
chromatography (30% ethyl acetate: hexane) to afford the title compound as a yellow solid
(0.284 g, 0.96 mmol, 47%). δH (400 MHz, CDCl3) 9.16 (d, J = 2.0 Hz, 1H), 8.98 (d, J = 2.3
Hz, 1H), 8.40 (t, J = 2.2 Hz, 1H), 7.67 – 7.62 (m, 2H), 7.36 (d, J = 7.9 Hz, 2H), 3.09 (q, J =
7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H) ppm.30
2-Iodo-5-methoxyaniline (11)47
N-Iodosuccinimide (0.995 g, 4.06 mmol) was added to a solution of 3-methoxyaniline
(0.46 ml, 4.06 mmol) in dichloromethane (100 ml) and acetic acid (0.25 ml, 4.25 mmol). The
mixture was stirred overnight at room temperature. Aqueous NaHCO3 (100 ml) was added,
and the organic layer was extracted with ethyl acetate (3 x 40 ml). The combined organic
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layers were washed with brine, dried over MgSO4 and concentrated in vacuo. The residue
was purified by flash chromatography (ethyl acetate: hexane) to afford the title compound as
a brown oil (0.530 g, 2.13 mmol, 52%). δH (400 MHz, CDCl3) 7.48 (d, J = 8.7 Hz, 1H), 6.33
(d, J = 2.8 Hz, 1H), 6.14 (dd, J = 8.7, 2.8 Hz, 1H), 4.07 (s, 2H), 3.74 (s, 3H) ppm.47
Methyl 2-amino-4-methoxybenzoate (12)
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A solution of compound 11 (0.123 g, 0.440 mmol), and Pd(dppf)Cl2 (0.0129 g, 0.0176
mmol) in methanol (2 ml) was placed under an atmosphere of carbon monoxide (balloon).
Triethylamine (0.06 ml, 0.474 mmol) was added to the reaction mixture and carbon
monoxide was bubbled through the mixture for 30 seconds. The mixture was heated at reflux
overnight. Ethyl acetate (10 ml) was added and the mixture was filtered through a pad of
Celite(R), before concentration in vacuo. The residue was purified by flash chromatography
(10 – 25% ethyl acetate:hexane) to afford the title compound as a brown solid (0.07 g, 0.384
mmol, 87%). δH (400 MHz, CDCl3) 7.79 (d, J = 9.0 Hz, 1H), 6.23 (dd, J = 9.0, 2.5 Hz, 1H),
6.11 (d, J = 2.5 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H) ppm.48
2-Amino-4-methoxybenzoic acid (6)
Lithium hydroxide (0.032 g, 0.77 mmol) was added to a solution of compound 12 (0.070 g,
0.38 mmol) in THF:H2O (1 ml :1ml). The mixture was heated at reflux for 5 hours. The
mixture was allowed to cool to room temperature and the aqueous layer was collected. 2 M
HCl was added dropwise until the pH reached to 3. The aqueous solution was extracted with
EtOAc (3 x 10 ml) before concentrating in vacuo to afford a white solid (0.057 g, 0.343
mmol, 89%). δH (400 MHz, (CD3)2SO) 7.60 (d, J = 8.9 Hz, 1H), 6.22 (d, J = 2.5 Hz, 1H),
6.09 (dd, J = 8.9, 2.5 Hz, 1H), 3.71 (s, 3H) ppm.49
2-(4,4-Dimethyl-4,5-dihydrooxazol-2-yl)-5-methoxyaniline (4)
Triphenylphosphine (0.474 g, 1.81 mmol), triethylamine (0.25 ml, 1.81 mmol), CBr4
(0.599 g, 1.81 mmol), aromatic acid 6 (0.100 g, 0.602 mmol), and 2-amino-2-methylpropan-
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1-ol (0.07 ml, 0.704 mmol) in toluene (3 ml) was stirred under nitrogen for 20 minutes at
room temperature before being heated at 90 °C overnight. The solvent was evaporated under
reduced pressure, and the residue was diluted with diethyl ether and filtered. The residual
solid was washed with ether three times. The filtrate was concentrated in vacuo and purified
by flash chromatography (5% -20% ethyl acetate:hexane) to afford the title compound as a
white solid (0.0256 g, 0.116 mmol, 19%). δH (400 MHz, CDCl3) δ 7.59 (d, J = 8.8 Hz, 1H),
6.83 (s, 1H), 6.24 (dd, J = 8.8, 2.5 Hz, 1H), 6.17 (d, J = 2.5 Hz, 1H), 3.96 (s, 2H), 3.78 (s,
3H), 1.35 (s, 6H) ppm.30
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7-methoxy-3-methyl-2-(5-(4-(trifluoromethoxy)phenyl)pyridin-3-yl)quinolin-4(1H)-one
(SCR0911)
Trifluoromethane sulfonic acid (0.006 ml, 0.067 mmol) was added to a solution of
oxazoline 1 (0.0806 g, 0.366 mmol) and ketone 4 (0.983, 0.33 mmol) in dry n-butanol (5.5
ml). The mixture was heated at reflux for 24 hours. Saturated sodium carbonate solution (10
ml) was added. The aqueous solution was extracted with ethyl acetate (3 x 15 ml) and
combined organic layers were washed with brine (50 ml), dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash chromatography (5 – 20% ethyl
acetate: hexane) to afford the title compound as a brown solid (0.062 g, 0.155 mmol, 42%).
δH (400 MHz, DMSO) 11.56 (s, 1H), 9.10 (d, J = 2.2 Hz, 1H), 8.79 (d, J = 2.0 Hz, 1H), 8.34
(t, J = 2.2 Hz, 1H), 8.03 (dd, J = 18.1, 8.9 Hz, 4H), 7.54 (d, J = 7.9 Hz, 3H), 1.94 (s, 3H) ppm;
HRMS (EI) 427.1288 [M+H]+ 30
.
Minimum inhibitory concentration determination
The growth inhibition dose-response assay was carried out using the broth microdilution
method, as described previously.34, 50 Briefly, clear 96-well flat-bottom Costar cell culture
plates (Corning) were filled with 100 μl of complete 7H9 medium supplemented with
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albumin–dextrose–catalase (ADC), without glycerol, in each well. Each compound was
added to the first well in each row to create two times the desired highest final concentration.
Subsequently, a 10-point 2-fold serial dilution was carried out starting from the first well in
each row. M. bovis bacillus Calmette–Guérin (BCG), M. smegmatis mc2155- and the isogenic
M. smegmatis strain ZCW111 Δbcc mutant used for the assays were grown to mid-log phase
in complete 7H9 medium. The cells were washed with glycerol-free 7H9 media and
subsequently diluted to an OD600 of 0.1 and 0.01, respectively. 100 μl of the diluted culture
was added to each well to create a final OD600 of 0.05 and 0.005 for M. smegmatis and M.
bovis Bacillus Calmette-Guérin (BCG) respectively, in all wells. For M. smegmatis strains,
the plates were incubated at 37 °C on an orbital shaker set at 180 rpm for 24 hours. For M.
bovis BCG strains, the plates were incubated at 37 °C without shaking for 7 days. At the end
of the incubation period, the culture in all wells was manually resuspended, and the OD600
was read using a TECAN Infinite Pro 200 plate reader. The MIC50 reported represents the
concentration that inhibits 50% of growth compared to the untreated culture.
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Markerless mutant construction of the Δbcc mutant
In brief, to generate the M. smegmatis Δbcc mutant, two homologous regions either side
of the target gene (primers ZW1, ZW2, ZW3, ZW4) (Supplementary Table 1) were cloned
into pX33 using NEBuilder® HiFi DNA Assembly (NEB) as per the manufacturer’s
instructions with the SpeI restriction enzyme (NEB). The pX33 constructs with homologous
regions (pX33qcr) were electroporated into M. smegmatis mc2155 and grown at 28°C to
ensure permissibility of the pX33 temperature-sensitive plasmid. Confirmation of the plasmid
transformation was achieved by treating colonies with 250 mM catechol. Colonies containing
the plasmid turn yellow owing to the catechol dioxygenase produced by pX33. Following
plasmid confirmation, the cells were grown on liquid media at 28°C and then plated at 40°C
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to select integration events based on the homology of the gene flanking regions. Integrants
were confirmed through catechol screening (as above). Integrants were spread-plated on
sucrose (10% w/v) containing media to force recombination to either the wildtype genotype
or the markerless deletion mutant.51 The Δbcc mutant was confirmed using PCR (primer pair,
ZW5, ZW6) and whole-genome sequencing (WGS) and aligned using to wildtype to confirm
that the gene was deleted using geneious (Supplementary Figure S2A and S2B). The resulting
bcc deletion mutant was designated ZCW111.
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Complementation of M. smegmatis strain ZCW111 bcc mutant
The construct for the strain ZCW111 Δbcc complementation was made using the pMV361
vector backbone. The qcrCAB genes were amplified using LH1 and LH2 primer pairs
(Supplementary Table 1). The respective product was then digested with HindIII and HpaI
restriction enzymes before ligation into the pMV361 vector. The resulting plasmid is termed
pMV361qcr. Functionality of the pMV361qcr vector for complementation was tested through
the growth of Δbcc and Δbcc + pMV361qcr on HdeB agar with 50 mM succinate as the sole
carbon and energy source (Supplementary Figure S2C).
DNA manipulation and cloning
All molecular biology techniques were carried out according to standard procedures. All
restriction enzymes and DNA modifying enzymes used were from New England Biolabs
(NEB) and were in their appropriate buffers. All constructs were confirmed using either PCR
or restriction digests followed by sequencing. PCR for construct creation was done using
Thermo Fisher Phusion polymerase following the recommended conditions with appropriate
annealing temperatures and extension time.
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Intracellular ATP synthesis assay
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The intracellular ATP synthesis assay was carried out using the broth microdilution
method as described previously, using the BacTiter-GloTM Microbial Cell Viability Assay kit
(Promega) to quantify the intracellular ATP.34, 50, 52 Clear 96-well flat-bottom Costar cell
culture plates (Corning) were filled with 100 μl of complete 7H9 medium supplemented with
ADC, in each well. Each compound was added to the first well in each row to create two
times the desired highest final concentration. Subsequently, a 10-point 2-fold serial dilution
was carried out starting from the first well in each row. M. bovis BCG stains were grown in
complete 7H9 medium to mid-log phase and subsequently diluted to an OD600 of 0.1. 100 μl
of the diluted culture was added to each well to create a final OD600 of 0.05 in all wells. The
plates were incubated at 37 °C on an orbital shaker set at 110 rpm for 24 hours. 50 μl of
samples (total culture) were mixed with an equal volume of freshly prepared BacTiter-GloTM
reagent in white flat bottomed 96 well plates (Corning) and lysis was carried out for 10 min
at room temperature with shaking at an amplitude of 3 mm inside the TECAN Infinite Pro
200 plate reader. Emitted luminescence was displayed as relative light units (RLU).
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Production of M. smegmatis inverted membrane vesicles (IMVs)
Preparation of the IMVs were carried out as previously described by Hotra et al., 2016
and Kamariah et al., 2019.53 35
ATP synthesis assay using IMVs
The ATP synthesis assay was quantified with the CellTiter-Glo® Luminescent Cell
Viability Assay kit (Promega). ATP synthesis measurement was carried out on white flat-
bottomed 96-well microtiter plates (Corning). 2.5 μl of compounds at varying concentration
was spotted on the individual wells. This was followed by the addition of 25 μl of PMVs or
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IMVs (5 μg/ml) and 25 μL of assay buffer (50 mm MOPS, pH 7.5, 10 mM MgCl2),
containing 10 μM ADP, 250 μM Pi and 1 mM NADH. The plates were incubated at room
temperature for 30 minutes before the addition of 25 μl of CellTiter-Glo® reagent. The plates
were incubated for another 10 min in the dark at room temperature. The luminescence was
measured by a TECAN Infinite Pro 200 plate reader.
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Oxygen consumption assay
Oxygen comsumption assay was carried out using methylene blue as previously
described by Li et al., 2017.54.The percentage of methylene blue decolourization in the
samples was determined using the methylene blue absorbance readings and the equation
below as described previously by Benetoli et al., where Ao represents the absorbance of the
7H9 sample, while As represents the absorbance of the sample of interest 55.
퐴표 ― 퐴푠
퐷% =
× 100
퐴표
Assay for ATP‐driven proton translocation
ATP‐driven proton translocation of plasma membrane vesicles of M. smegmatis were
measured on the basis of a decrease in ACMA fluorescence using a Cary Eclipse
fluorescence spectrophotometer (Varian Inc., Palo Alto, CA) as described previously.56 IMVs
or PMVs (0.18 mg/mL) were pre‐incubated at 37 °C in 10 mM HEPES/KOH (pH 7.5), 100
mM KCl, 5 mM MgCl2 containing 2 μM ACMA, 50 μM ADP and 5 mM Pi. A baseline was
obtained by monitoring for 2 min. The reaction was started by adding 0.5 mM NADH or
succinate as electron donors. After approximately another 2 to 4 min when the florescence
intensity was quenched completely, 15 μM of the test compound was added. After another 2
minutes, the proton gradient was collapsed completely via the addition of 2 μM of the
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uncoupler SF6847 (Alexis Corporation, Lausen, Switzerland). The excitation and emission
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Acknowledgements:
This research was supported by the National Research Foundation (NRF) Singapore, NRF
Competitive Research Programme (CRP), Grant Award Number NRF–CRP18–2017–01). C.
S. M. Sherilyn is grateful to receive an NTU research scholarship at Nanyang Technological
University Singapore.
Conflict of interest: The authors declare that they have no conflicts of interest with the
contents of this article.
Author contributions: C.S.M.S., M.S.S.M., G.M.C., T.D., K.P, and G.G. conceived and
designed the experiments. C.S.M.S., M.S.S.M., J.P.S., Z.C.W., and L.K.H. performed the
experiments and analyzed the data. C.S.M.S., M.S.S.M., and G.G. wrote the paper with
contribution from other authors.
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Figure 1. (A) Chemical structure of SCR0911.30 The main difference lies in the position of
the (trifluoromethoxy)benzene group in respect to the pyridine ring. (B) Sequence alignment
of the cytochrome b of bovine, P. falciparum, M. smegmatis, M. bovis, and M. tuberculosis.
The orange boxes indicate the residues that are significant in the P. falciparum homology
model with the binding of the analog of SCR0911. The green boxes indicate the residues that
are significant in the bovine crystal structure with the binding of SCR0911. The numbers on
the boxes refers to the residue number with respect to P. falciparum.
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Figure 2. Docking studies of SCR0911 with Mtb cyt-bcc complex. (A) Cartoon
representation of the 1st major docked complex configuration of Mtb cyt-bcc with SCR0911.
Only a monomer is shown for clarity. Subunit qcrA is highlighted in red, qcrB in magenta
and subunit qcrC in yellow. The heme groups are shown in sphere representation and
SCR0911 in stick (green). The iron-sulfur cluster in subunit qcrA is shown as red sphere.
(inset) The interaction profile of the SCR0911 with the Mtb cyt-bcc complex residues. (B)
Zoomed in view of 1st major docked complex configuration of Mtb cyt-bcc with SCR0911
showing the proximity of the compound to the Qo-site. The menaquinones are shown as line
representation with the functional ones near Qo-and Qi-site in red and the others three in
green. SCR0911 falls in the position of one of the menaquinone positions. The low and high
spin hemes are marked. (C) Cartoon representation of the 2nd minor docked complex
configuration of Mtb cyt-bcc with SCR0911. (inset) The interaction profile of the SCR0911
with the Mtb cyt-bcc complex residues. (D) Zoomed in view of 2nd minor docked complex
configuration of Mtb cyt-bcc with SCR0911 showing the compound binding on the Qi-site.
The menaquinones are shown as line representation with the functional ones near Qo-and Qi-
site in red and the others three in green. SCR0911 binds in the similar position as that of the
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menaquinone in the Qi-site. (E) Structural overlap of qcrB subunits of Mtb (magenta) and
bovine (grey) cyt-bcc complex. The SCR0911 in the 2nd minor complex configuration of Mtb
cyt-bcc is shown in green and the bound SCR0911 in the bovine cyt-bcc structure is shown as
grey stick representation. Although the quinolone moiety binds at similar position, the
trifluoromethoxyphenyl pyridine moiety orient in different direction showing a diffential
binding.
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Figure 3. Modified synthesis scheme for SCR0911.
Figure 4. Growth inhibition of BDQ (black) and SCR0911 (blue) in M. smegmatis (A), M.
bovis BCG (B), and M. smegmatis Δbcc mutant (C). The MIC50 values for BDQ in M.
smegmatis, M. bovis BCG, and M. smegmatis Δbcc mutant are 17 nM, 188 nM and 16 nM,
respectively, while the MIC50 values for SCR0911 in M. smegmatis and M. bovis BCG are
272 μM and 107 μM. A growth curve of the M. smegmatis Δbcc mutant in the presence of
increasing amounts of Telacebec (Q203) is shown in red. (D) Intracellular ATP synthesis
assay of BDQ (black), Telacebec (Q203) (red) and SCR0911 (blue) in M. bovis BCG
indicates an IC50 of 29 nM, 0.5 nM and 50 μM, respectively. Error bars represents the
standard deviations. The experiment has been carried out on two biological replicates.
Figure 5. Inhibition of ATP synthesis by SCR0911 in IMVs from M. smegmatis
demonstrates an IC50 of 7.6 μM (A) and 6.9 μM in M. bovis BCG (B). Inhibition of ATP
synthesis by SCR0911 and Telacebec (Q203) in PMVs from M. smegmatis using NADH as a
donor (C) portrays an IC50 of 3.8 μM and 1.1 nM, respectively, while the PMVs from M.
smegmatis using succinate as a donor (D) portrays an IC50 of 3.4 μM and 2.0 nM,
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respectively. Error bars represents the standard deviations. The experiment has been carried
out on two biological replicates.
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Figure 6. Oxygen consumption assay with methylene blue as an indicator with M. smegmatis
in the presence of SCR0911. The 7H9 sample contains only the 7H9 media and served as a
blank. The drug free (DF) sample, containing the untreated M. smegmatis cells, served as the
negative control, while THZ was used as positive control. (A) Visual representation of one
representative replicate of the tubes at the various conditions was shown. (B) A bar chart of
percent decolorization was plotted for M. smegmatis, indicating the effectiveness of
SCR0911 in inhibiting oxygen respiration. Error bars represents the standard deviation. Error
bars represents the standard deviation. The experiment has been carried out on two biological
replicates.
Figure 7. Substrate-driven proton pumping in WT M. smegmatis PMVs with NADH as the
substrate, demonstrating no major uncoupling effect with 15 uM of SCR0911. The
experiment has been carried out on three biological replicates.
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