6,11-Dioxobenzo[ f]pyrido[1,2- a]indoles Kill Mycobacterium tuberculosis by Targeting Iron-Sulfur Protein Rv0338c (IspQ), A Putative Redox Sensor

Article abstract of DOI:10.1021/acsinfecdis.0c00531

Screening of a diversity-oriented compound library led to the identification of two 6,11-dioxobenzo[f]pyrido[1,2-a]indoles (DBPI) that displayed low micromolar bactericidal activity against the Erdman strain of Mycobacterium tuberculosis in vitro. The activity of these hit compounds was limited to tubercle bacilli, including the nonreplicating form, and to Mycobacterium marinum. On hit expansion and investigation of the structure activity relationship, selected modifications to the dioxo moiety of the DBPI scaffold were either neutral or led to reduction or abolition of antimycobacterial activity. To find the target, DBPI-resistant mutants of M. tuberculosis Erdman were raised and characterized first microbiologically and then by whole genome sequencing. Four different mutations, all affecting highly conserved residues, were uncovered in the essential gene rv0338c (ispQ) that encodes a membrane-bound protein, named IspQ, with 2Fe-2S and 4Fe-4S centers and putative iron-sulfur-binding reductase activity. With the help of a structural model, two of the mutations were localized close to the 2Fe-2S domain in IspQ and another in transmembrane segment 3. The mutant genes were recessive to the wild type in complementation experiments and further confirmation of the hit-target relationship was obtained using a conditional knockdown mutant of rv0338c in M. tuberculosis H37Rv. More mechanistic insight was obtained from transcriptome analysis, following exposure of M. tuberculosis to two different DBPI; this revealed strong upregulation of the redox-sensitive SigK regulon and genes induced by oxidative and thiol-stress. The findings of this investigation pharmacologically validate a novel target in tubercle bacilli and open a new vista for tuberculosis drug discovery.

Full text of DOI:10.1021/acsinfecdis.0c00531

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Article
6,11-Dioxobenzo[f]pyrido[1,2-a]indoles kill Mycobacterium tuberculosis
by targeting iron-sulfur protein Rv0338c (IspQ) a putative redox sensor
Rita Szekely, Monica Rengifo-Gonzalez, Vinayak Singh, Olga Riabova,
Andrej Benjak, Jeremie Piton, Mena Cimino, Etienne Kornobis, Valerie
Mizrahi, Kai Johnsson, Giulia Manina, Vadim Makarov, and Stewart T. Cole
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.0c00531 • Publication Date (Web): 15 Sep 2020
Downloaded from pubs.acs.org on September 15, 2020
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6,11-Dioxobenzo[f]pyrido[1,2-a]indoles kill Mycobacterium
tuberculosis by targeting iron-sulfur protein Rv0338c (IspQ) a
putative redox sensor
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Rita Székely†,1,x, Monica Rengifo-Gonzalez‡,2,x, Vinayak Singh∥,3,4, Olga Riabova§,
Andrej Benjak†,5, Jérémie Piton†,1, Mena Cimino#,6, Etienne Kornobis#,7,8, Valerie
Mizrahi∥, Kai Johnsson‡,9, Giulia Manina#,6, Vadim Makarov§, and Stewart T. Cole†,#*
†
Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne,
Switzerland
‡ Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne,
CH-1015 Lausanne, Switzerland
∥ MRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of
Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular
Medicine & Department of Pathology, University of Cape Town, Anzio Road, Observatory
7925, Cape Town, South Africa
§ FRC Fundamentals of Biotechnology, Russian Academy of Science, 119071 Moscow,
Russian Federation
# Institut Pasteur, 75015 Paris, France
Current addresses
1 FIND, Campus Biotech, Chemin des Mines 9, 1202 Geneva, Switzerland
2 Department of Fundamental Microbiology, University of Lausanne, CH-1015 Lausanne,
Switzerland
3 Drug Discovery and Development Centre (H3D), University of Cape Town, Rondebosch
7701, South Africa
4 South African Medical Research Council Drug Discovery and Development Research Unit,
Department of Chemistry and Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, Rondebosch 7701, South Africa
5 Department for BioMedical Research, University of Bern, 3012 Bern, Switzerland
6 Microbial Individuality and Infection, Institut Pasteur, 75015 Paris, France
7 Biomics, C2RT, Institut Pasteur, 75015 Paris, France
8 Hub Bioinformatique et Biostatistique, USR 3756 CNRS, Institut Pasteur, 75015 Paris,
France
9 Max Planck Institute for Medical Research, Department of Chemical Biology, 69120,
Heidelberg, Germany
X These authors contributed equally
* Corresponding author: stewart.cole@pasteur.fr
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ABSTRACT: Screening of a diversity-oriented compound library led to the
identification of two 6,11-dioxobenzo[f]pyrido[1,2-a]indoles (DBPI) that displayed
low micromolar bactericidal activity against the Erdman strain of Mycobacterium
tuberculosis in vitro. The activity of these hit compounds was limited to tubercle bacilli,
including the non-replicating form, and to Mycobacterium marinum. On hit expansion
and investigation of the structure activity relationship, selected modifications to the
dioxo moiety of the DBPI scaffold were either neutral or led to reduction or abolition
of antimycobacterial activity. To find the target, DBPI-resistant mutants of M.
tuberculosis Erdman were raised and characterized first microbiologically and then by
whole genome sequencing. Four different mutations, all affecting highly conserved
residues, were uncovered in the essential gene rv0338c (ispQ) that encodes a
membrane-bound protein, named IspQ, with 2Fe-2S and 4Fe-4S centres and putative
iron-sulfur-binding reductase activity. With the help of a structural model, two of the
mutations were localized close to the 2Fe-2S domain in IspQ and another in
transmembrane segment 3. The mutant genes were recessive to the wild type in
complementation experiments and further confirmation of the hit-target relationship
was obtained using a conditional knock-down mutant of rv0338c in M. tuberculosis
H37Rv. More mechanistic insight was obtained from transcriptome analysis, following
exposure of M. tuberculosis to two different DBPI; this revealed strong upregulation of
the redox-sensitive SigK regulon and genes induced by oxidative and thiol-stress. The
findings of this investigation pharmacologically validate a novel target in tubercle
bacilli and open a new vista for tuberculosis drug discovery.
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KEYWORDS: Tuberculosis, drug discovery, chemical genomics, iron-sulfur-binding
reductase, 6,11-dioxobenzo[f]pyrido[1,2-a]indoles
Tuberculosis remains a global health problem for which new interventions and
therapeutic strategies are sorely needed 1. Despite intensive efforts, progress towards a
broadly efficacious new vaccine remains disappointing and this reflects not only the
magnitude of the challenge but also the complexity of protecting against and killing an
intracellular pathogen 2. For over 60 years we have been able to curb the etiologic agent
Mycobacterium tuberculosis by means of short course chemotherapy involving 6-
months of combination therapy 3-4. However, resistance to two or more of the four front-
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line agents on which it relies is increasingly common 1, as is resistance to the second-
line drugs which are much less effective and more toxic than their frontline counterparts
5. This has led to renewed interest in discovering new chemical entities (NCE) to
develop as leads for the next generation of tuberculosis drugs and a variety of
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approaches have been undertaken to do so . The most effective means of lead
generation has been by phenotypic screening of compound libraries for activity against
M. tuberculosis growing in vitro 6 or more recently using host cell-based screens 7.
The first new TB drug candidates, bedaquiline 8, pretomanid 9 and delamanid 10
were discovered in the 2000s and are now nearing the end of phase 3 clinical trials 11.
All three new drugs have been approved for the treatment of multidrug resistant
tuberculosis (MDR-TB). In the past decade many additional phenotypic screens were
conducted and these also generated new leads some of which are currently in early stage
11
clinical trials . However, while there have been some impressive successes such as
12
the discovery and clinical development of telacebec (Q203) , the number of NCE
remains relatively limited and many of them target the same functions in the pathogen.
This has led to the notion of the existence of “promiscuous targets” such as DprE1
(decaprenylphosphoryl-β-d-ribose oxidase), MmpL3 (a mycolic acid transporter) and
QcrB (the B-subunit of respiratory cytochrome bc1 complex) that are each inhibited by
a wide variety of pharmacophores 13. Such promiscuity may reflect the bacterial growth
conditions used in the screening assays, the limited chemical diversity in the compound
13
collections or the periplasmic accessibility of the active sites of the targets . In an
attempt to explore more chemical space, Switzerland’s National Centre of Competence
in Research (NCCR) in Chemical Biology established a diversity-oriented library from
many different commercial sources to aid the identification of bioactive molecules 14.
In this investigation, we screened the NCCR library and uncovered a new scaffold for
drug development, the dioxo-dihydrobenzo-pyridoindoles, and demonstrate that these
compounds target an essential, yet previously uncharacterized, membrane-bound redox
enzyme that is likely involved in energy metabolism or redox sensing.
RESULTS AND DISCUSSION
Screening diversity-oriented compound library. The NCCR (National
Centre of Competence in Research) library of 67,393 commercially available
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compounds was tested for growth inhibition of the Erdman strain of Mycobacterium
tuberculosis (Mtb) using the resazurin reduction microtitre assay (REMA) in a single
point (10 µM) screen 15. This led to the identification of 340 molecules inhibiting
growth by >60 %. The 66 most active compounds were purchased afresh for hit
confirmation and further investigation. Hit confirmation was performed using both the
Erdman and H37Rv strains of Mtb by the BacTiter-GloTM (Promega) and REMA assays
in order to compare their performance in viability testing. The results of both assays
showed good correlation and most of the MICs determined with Erdman were close to
those determined with strain H37Rv. Hits were also tested using the following
algorithm: REMA with drug susceptible (DS), drug resistant (DR) and multidrug
resistant (MDR) clinical and laboratory isolates; REMA with the streptomycin-starved
18b (SS18b) strain of M. tuberculosis, a model to find drugs active against non-
replicating bacteria 16; growth inhibition activity against a range of bacteria and yeast;
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17
cytotoxicity (TD50) for HepG2 cells; mutagenicity using the SOS-chromotest ; and
finally, microsomal stability.
Among the hits, compound F1414-1444 was considered particularly interesting
for two reasons. First, a close analog, F1414-1438, was also found in the primary screen
thus providing the basis for a structure activity relationship (SAR) analysis and second,
because the compound appears to inhibit a new, previously uncharacterized target, as
will be described below. Details of the other hits and their targets will be presented
elsewhere.
Initial microbiological characterization of F1414-1444 and F1414-1438.
The two 6,11-dioxobenzo[f]pyrido[1,2-a]indoles (DBPI) analogs, active against the
Erdman strain of M. tuberculosis in the phenotypic screen of the NCCR compound
library, were further characterized by a range of microbiologic and cell-based tests.
From Table 1 it can be seen that both DBPI compounds display low micromolar activity
in two different assays against the Erdman and H37Rv strains of M. tuberculosis, and
this activity decreased in the absence of Tween 80, which is known to increase
susceptibility to some drugs, such as RIF 18.
Table 1. Structure and MIC of selected DBPI on M. tuberculosis Erdman and
H37Rv
MIC99 (µM) by REMA or BacTiter Glo
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Cmpd
Structure
Erdman Erdman H37Rv BacTiter
NO
6
7
8
9
Tween
19.5
O
O
F1414-1444
0.85
1.1
1.09
2.5
4.68
3.2
CH3
N
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O
O
CH3
H3C
O
O
F1414-1438
8.44
N
O
O
MIC99 is the concentration where 99% of bacterial growth is inhibited.
In order to investigate whether F1414-1444 is bactericidal we determined its MBC99 by
incubating bacteria with concentrations of the compound ranging from 0.5 - 16X MIC
for 7 and 14 d in a REMA assay. Dilutions were then spread on 7H10 plates to obtain
single colonies. After 3 weeks incubation colonies were counted and compared to
controls. The MBC99 value of F1414-1444 was 1.56 µM, which is close to its MIC99
thus indicating that the compound has bactericidal activity. Furthermore, cidality was
found to be time-dependent (supporting information Figure S1).
Activity against non-replicating tubercle bacilli, clinical isolates and drug-
resistant laboratory strains. Strong activity was detected against the streptomycin-
starved 18b (SS18b) strain, a model to test drugs against non-replicating bacteria
(Table 2) indicating that DBPI should be active against latent tuberculosis. However,
while the inhibition levels obtained were comparable to that seen with the control
drug, RIF, higher concentrations were required.
Table 2. Activity against SS18
Cmpd
SS18b
Imax (%)
Conc. (µM) of
Imax
23.6
6.4
F1414-1444
F1414-1438
54
70
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RIF
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0.03
Imax value represents the highest % of inhibition.
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The susceptibility of five clinical isolates to DBPI compounds was then
evaluated by REMA in the presence of Tween. The collection included a fully DS
isolate, three DR-strains and an MDR-strain (Table 3). Both compounds were active
against all DR-isolates and the MIC values were in line with those determined using
the reference strains (Table 1).
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Table 3. Activity against clinical isolates and drug-resistant mutants of M.
tuberculosis
MIC99 (µM)
-----------------------------------
Isolate/strain
Description,
genotype
Drug
resistance
1414-1444
1414-1438
ABG
clinical isolate,
pan-susceptible
-
0.79
4.4
3.0
6.5
CHUV80045776 clinical isolate, INH
2.5
katG(S315T)
clinical isolate, INH
inhA promoter
HUG.MB.3649
2.2
c(-15)t
HUG.MI.1020
MDR-TB
clinical isolate, INH, STR
katG(S315T)
clinical isolate, INH, RIF
rpoB(S531L),
2.3
2.5
2.7
3.47
katG(S315T)
Lsr1
H37Rv
qcrB(L176P)
H37Rv
qcrB(T313A)
H37Rv
Lansoprazole
Q203
3.0
2.4
1.1
2.3
2.5
2.2
Q203-R
DR3
BM212
mmpL3(V681I,
S87P)
DR5
DR6
H37Rv mmpL3 BM212
(G253E,
D46G)
H37Rv mmpL3 BM212
(V681I, M492T,
V564A)
1.5
1
5.1
1.2
DR7
DR8
H37Rv
mmpL3(Q40R)
H37Rv mmpL3 BM212
BM212
0.7
0.7
1.1
1.2
(G253E, I516T)
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NTB1
H37Rv
dprE1(C391S)
H37Rv
BTZ043
7.2
2.4
7.4
2.9
H37Rv CFZ-R
BDQ/CFZ
rv0678(S63R)
7
The table is a composite and presents data obtained on different dates. All clinical isolates were tested
together, the other strains were tested later and on several occasions.
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To test the possibility that DBPI compounds act on one of the promiscuous
targets introduced above, their activity was tested against a panel of drug-resistant
mutants of strain H37Rv harboring different mutations in dprE1, mmpL3 and qcrB. In
virtually all cases, the DBPI-susceptibility of the mutants was close to that of the
parental strain H37Rv thus indicating a new mechanism of action (Table 3).
Furthermore, DBPI compounds were also active against a BDQ/CFZ-resistant mutant
that overproduces the efflux pump MmpL5 due to a mutation in gene rv0678 (mmpR5).
Activity against other bacteria and yeast. To establish whether F1414-1444
has broad spectrum antimicrobial activity a collection of various pathogenic and non-
pathogenic Gram-positive and Gram-negative bacteria, and yeast was assembled, then
tested by REMA for DBPI susceptibility (Supporting information Table S1). Activity
was restricted to the M. tuberculosis complex and its ancestor Mycobacterium
marinum. No activity was detected against Mycobacterium smegmatis, selected non-
tuberculous mycobacteria (NTMs) and Corynebacteria. The combined results indicate
that DBPI compounds have a very narrow range of activity.
Target identification. Mutants of the Erdman strain resistant to F1414-1444
were raised in vitro and characterized by MIC determination and whole genome
sequencing (WGS) in an attempt to find the likely target (Table 4). At 3xMIC, mutants
arose at a frequency of 3.3x10-7. Irrespective of the level of F1414-1444 used for their
selection all of the mutants were highly resistant displaying an increase in MIC of 90
to >125-fold (Table 5). Furthermore, all mutants displayed cross-resistance to F1414-
1438 thereby indicating that the compounds share the same mechanism of action.
All nine mutants were subjected to WGS and bioinformatic analysis and found to
harbor polymorphisms in the ERDMAN_0374 gene, which is identical to rv0338c in
strain H37Rv, except for a single nucleotide polymorphism (SNP) in codon 502 that
changes Leu to Phe, L502F. The corresponding protein will thus be referred to as
Rv0338c or IspQ (iron-sulfur protein Q) henceforth.
From Table 4 it may be seen that two of the mutations (Arg641Gly and
Asp695Gly) occurred several times whereas the others were singletons. To exclude the
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possibility that these mutations were not DBPI-related the rv0338c sequences in ~1,500
M. tuberculosis genomes were inspected 19. None of the above mutations, nor the
Erdman polymorphism, was found but two SNP were detected, namely, G403980A
(c.1862C>T) which occurred in ~99% of the genomes and T404326C (c.1516A>G)
occurring in 92% of the cases. These SNP cause the non-synonymous substitutions
Ala621Val and Arg506Gly, respectively. In this set, the Ala621 polymorphism is
restricted to 13 strains and isolates, all derived from H37, the parent of H37Rv.
Furthermore, Arg506 occurs in 123 genomes, that all cluster on the H37Rv "sub-
branch" of the branch of M. tuberculosis lineage 4. The ancestor of the tubercle bacilli,
M. canettii has the Val621/Gly506 duet (see supporting information). These amino acid
substitutions do not affect susceptibility to DBPI as indicated by the data reported here,
for example the lineage 2 strain, 18b tested above, has the Val621/Gly506 duet as do
92% of the other M. tuberculosis complex members.
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The rv0338c gene has been reported to be essential on the basis of Tn saturation
mutagenesis 20 and to encode a putative iron-sulfur containing heterodisulfide reductase
21, that we have called IspQ for iron-sulfur protein Q.
Table 4. WGS analysis of F1414-1444-resistant mutants
Number of Selected at
Relevant
SNPs
Mutant used
elsewhere
Domain
location
Interpro
mutants
X MIC
Domain*
sequenced
3
1
3
3
Asp695Gly
DBPI2
End Cys-rich IPR004017
MOBIDB_LITE
Pro775_Val7 DBPI1
76fs
Disordered
1
4
10
20
Gly125Val
Arg641Gly
DBPI3
DBPI4
TM3
TMHELIX
IPR004017
Cys-rich

Interpro domains from https://www.ebi.ac.uk/interpro/
Target validation. In order to confirm that the protein encoded by rv0338c
plays a role in the mechanism of action of DBPI compounds a genetic approach was
applied. The empty pMV261 vector and its pMVE_0374 construct (i.e. pMV261
carrying the ERDMAN_0374 gene, equivalent to rv0338c) were transformed into the
parental Erdman strain and its four representative DBPI-resistant mutants (Table 4).
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When the parent strain was diploid for rv0338c, no change in MIC was observed.
However, in the case of all four resistant mutants, complementation with the wild type
gene restored full susceptibility to F1414-1444 (Table 5). In control experiments,
transformation of the resistant mutants with the pMV261 vector led to no change in the
MIC for F1414-1444. Taken together, the findings provide strong evidence for
causality and indicate that the resistant phenotype is recessive when the wild type gene
is present.
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Table 5. Complementation analysis of F1414-1444-resistant mutants
Strain
MIC (µM)
0.8
Erdman
DBPI1
100
75
DBPI2
DBPI3
>100
100
0.8
DBPI4
Erdman::pMVE_0374
DBPI1::pMVE_0374
DBPI2::pMVE_0374
DBPI3::pMVE_0374
DBPI4::pMVE_0374
Erdman::pMV261
DBPI1::pMV261
0.8
0.8
0.8
0.8
0.8
100
Construction and use of conditional knock-down mutants. To further
investigate the hit-target relationship we exploited the Tet-Off system as done
previously with success for GuaB2, another new drug target in M. tuberculosis 22. To
investigate whether F1414-1444 retains target selectivity for IspQ (Rv0338c) in live
cells, we asked whether conditional depletion of the protein would sensitize M.
tuberculosis H37Rv to the growth inhibitory effects of the DBPI compounds. For this
purpose, we constructed anhydrotetracycline (ATc)-regulated rv0338c conditional
knockdown mutants (hypomorphs) in the Tet-OFF configuration, as described recently
22. The rv0338c promoter region was replaced by the ATc-regulated promoter-operator
element, Pmyc1tetO, by a single-crossover (SCO) homologous recombination event to
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generate the promoter-replacement mutant, rv0338c-SCO (supporting information
Figure S2). A construct bearing the tetracycline repressor was then introduced into
rv0338c-SCO to obtain a conditional Tet-OFF mutant. Since rv0337c, the gene situated
31 bp downstream of rv0338c, is also essential care was taken to avoid potential polar
effects on gene expression. Measurement of rv0337c mRNA levels by q-PCR in the
resistant mutants DBPI1 and DBPI2 (Table 4) revealed these to be about 40 - 50% of
that of the parental strain. Consequently, a second copy of rv0337c was introduced into
the attB site of rv0338c-SCO and the resultant strain Rv0338c_Tet-OFF::attB_rv0337c
used for further investigation.
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Growth of Rv0338c_Tet-OFF::attB_rv0337c in 7H9 medium was inhibited in
a dose-dependent manner by ATc thereby confirming the essentiality of rv0338c
(supporting information Figure S3 and S4) in vitro. Next, the conditional knockdown
mutant was tested in the checkerboard assay by simultaneously varying the
concentrations of ATc and F1414-1444. The results are displayed in Figure 1A where
they are compared to those obtained with an unrelated drug INH (Figure 1B). In the
presence of ATc alone at a concentration of 6.25 µg/ml, growth of Rv0338c_Tet-
OFF::attB_rv0337c was reduced to about 50% of the ATc-free level. Addition of
increasing amounts of F1414-1444 or F1414-1438 (supporting information Figure S4)
led to even slower growth culminating in >90% inhibition (Figure 1A). A similar, but
more pronounced trend, was observed with 3.12 µg/ml ATc alone, a level that does not
impact the growth of the hypomorph as evidenced by its growth curve in the presence
of INH (Figure 1B). To illustrate this point, 1 µM F1414-1444 inhibits growth by ~50%
in the presence of 3.12 µg/ml ATc but has no noticeable impact in its absence. The
combined data show that as the IspQ level declines less DBPI is required to inhibit
growth leading us to conclude that IspQ (Rv0338c) is a major DBPI target.
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Figure 1. ATc dose-dependent modulation of growth of Rv0338c hypomorph (Rv0338c_Tet-
OFF::attB_rv0337c) as a function of DBPI concentration. For control purposes H37Rv was included and
grown in the presence or absence of ATc; note the lack of growth inhibition. The conditional knockdown
mutant was grown at the ATc concentrations indicated by the color coding in the presence of increasing
amounts of (A). F1414-1444, (B) Isoniazid, INH. The data represents Mean ± SEM, obtained from at
least three biological replicates.
Datamining, structural modeling and function prediction. The
Rv0338c/IspQ protein comprises 882 amino acids and is likely to participate in energy
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production and conversion according to the TubercuList
,
Interpro
(https://www.ebi.ac.uk/interpro/) and COG databases 23. The N-terminal domain
(amino acid residues 1 – 231), comprising six transmembrane segments (Figure 2,
supporting information Figure S5), is followed by a [4Fe-4S] ferredoxin-type, iron-
sulfur binding domain (residues 279 – 420, IPR009051), which, in turn, is followed by
two cysteine-rich [2Fe-2S] domains (residues 477 – 568, 607 – 693). The bulk of the
protein (residues 232 - 882) is predicted to be located in the periplasmic space. Three
of the polymorphisms associated with F1414-1444 resistance are non-synonymous
mutations that cause amino acid substitutions whereas the fourth is a frameshift
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mutation that will truncate the protein by ~100 residues (Table 4). The C-terminal
domain following the frameshift mutation is proline-alanine-rich, repetitive and of low
complexity and predicted to be intrinsically disordered (Figure 2).
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To glean insight into the impact of the four F1414-1444 resistance-associated
mutations on IspQ function, a structural model of the protein was built using the web
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server phyre2 and appropriate templates from sequence-related proteins of known
structure present in PDB. See legend to Figure 2 for details and additional information.
Figure 2. Structural model of IspQ (Rv0338c). (A) The transmembrane domain was modeled using the
web server phyre2 24 and the structure of the transmembrane domain of Escherichia coli nitrate reductase
A subunit as template 25. Likewise, the Fe-S binding domains were modeled using as template the
structures of the HdrC and HdrB subunits of the heterodisulfide reductase of the archea
Methanothermococcus thermolithotrophicus 26. (B) The orientation and position of the Fe-S domains
with respect to the transmembrane domain were arbitrarily chosen in the graphical representation and
the C-terminal domain, which could not be modeled is represented by a grey-dashed circle connected to
the C-terminal end of Fe-S domain. (C) Close-up of the entry pocket for the 2Fe-2S site. The side chain
of Aspartate 695 is located near the entry of the pocket situated close to the 2Fe-S site (12 Å). The C-
terminal part after Pro775 should be located close to this pocket and might impact its structure.
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On inspection of multisequence alignments of IspQ orthologs from various
tubercle bacilli, M. marinum, M. smegmatis and NTMs positions Gly125, Arg641,
Arg695 and Pro775 were all found to be invariant implying that they may be
functionally important. Orthologs of IspQ are also present in the leprosy bacilli, M.
leprae and M. lepromatosis with their downsized genomes 27-28, and again this is
indicative of the biological importance of the protein. However, in the leprosy bacilli,
Gly125 has been replaced by Ser but the other three residues were conserved. In all
cases the most divergent part of the protein is the C-terminal stretch after Pro775 as this
appears to be composed of simple sequence repeats that vary in copy number between
mycobacterial species (see supporting information Figures S3, S4). Compared to its
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counterparts in tubercle bacilli, the C-terminal domain of MMAR_0611 , the IspQ
ortholog of the DBPI-susceptible M strain of M. marinum is over 100 amino acids
longer due to the expansion of tandem repetitive motifs (see supporting information
Figure S6).
While the precise biological role of IspQ remains to be elucidated some
information is available about its subcellular location and the regulation of its gene. The
protein has been identified in the membrane fraction of M. tuberculosis H37Rv by
different investigators using mass-spectrometry based proteomic techniques and
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notably in Triton X-114 extracts that contain solubilized membrane proteins
.
Being in the plasma membrane IspQ is ideally located to interact with components of
the electron transport chain and lipophilic electron carriers. Consistent with its role as
an iron-sulfur protein, expression of rv0338c is induced by iron and controlled by IdeR,
an iron-dependent regulator 32.
Transcriptome analysis. Identifying transcripts whose levels change following
exposure of M. tuberculosis to drugs or inhibitors is a well-established means of gaining
insight into mechanisms of action or resistance 33. RNA was thus extracted from strain
H37Rv that had been exposed to two different concentrations of F1414-1444 or a new
analog 11626157 (see next section for details). The latter generally proved the more
active. Transcripts whose expression was up- or down-regulated by both compounds
were identified by RNA-Seq and bioinformatics was then used to glean functional
insight (Supporting Table S2). There were many more up- than down-regulated genes
(Figure 3A) and expression of rv0338c was not notably affected by DBPI exposure
(Figure 3B).
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In both cases, genes associated with the following broad functions were shared:
redox reactions, oxidative and thiol stress, and drug efflux (Figure 3B). The most highly
induced gene was mpt70 (rv2875), which, together with the linked genes, mpt83, dipZ,
rv2876 and rv2877c, is part of the sigma factor K regulon 34. Expression of other SigK-
regulated genes, namely rv0449c, rv0447c, rv0448c, located adjacent to those for SigK
and its anti-sigma factor, RskA, were also upregulated by 11626157. The basal
expression of mpt70 and mpt83 in M. tuberculosis is low and strongly induced after
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infection of macrophages and, under reducing conditions SigK, dissociates from
RskA, to transcribe genes involved in the regulation of redox homeostasis and the
serodominant antigens Mpt70 and Mpt83 36.
Exposure to the two DBPI compounds also led to induction of sets of
37
functionally unrelated genes required for arginine biosynthesis (argBCDFGJR)
,
38
electron transfer and energy production (cydBCD)
and metallation of secreted
39 , 40
proteins (CtpC), such as the manganese-requiring superoxide dismutase, SodA
.
All of these functions are induced by oxidative stress, as is the RND protein, MmpL6
that seemingly enables the more virulent, modern epidemic strains of M. tuberculosis
to resist such stress 41-42. A second RND protein, MmpL5, and its partner MmpS5, were
also upregulated by DBPI, but since MmpL5 exports many drugs this is most likely a
resistance mechanism 43 , 44 , 45. From Table 3, it may be seen that the MmpL5
overproducer, strain H37Rv CFZ-R, is susceptible to both DBPI compounds tested.
MmpL6 expression is upregulated by triclosan and plumbagin, which cause oxidative
stress, and presence of the complete mmpS6-mmpL6 locus can also confer INH and
rifampin tolerance to some strains of M. tuberculosis 42.
Another highly upregulated gene that has been reported to be involved in drug
resistance is rv0560c, encoding a methyltransferase, which N-methylates pyrido-
benzimidazoles thus abrogating their bactericidal activity 46. The neighboring gene
rv0559c was also upregulated by 11626157 and this encodes a secreted protein of
unknown function. Overproduction of Rv0560c occurs following exposure of M.
tuberculosis to a range of drugs and inhibitors, including salicylate and para-amino-
salicylate 47, membrane depolarizers (carbonyl cyanide m-chlorophenyl hydrazone,
valinomycin, or dinitrophenol), the respiratory inhibitors chlorpromazine and
thioridazine and the detergent SDS 46, 47
.
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Expression of a cluster of three genes (rv3093c, rv3094c and rv3095) was
downregulated by both DBPI compounds and these code for an oxidoreductase, a
protein of unknown function and an HTH transcriptional regulator (Figure 3B).
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Figure 3. Gene expression profiling in response to F1414-1444 and 11626157. (A)
Venn diagrams showing the number of genes significantly up- or down-regulated between two
different concentrations of the two DBPI compounds (color-coded) and DMSO. (B) The heat map
shows genes whose expression was up- or down-regulated in red and blue, respectively, according
to a log2 scale. Grey bars indicate singletons that were induced by one or other DBPI but not by
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both. Preference is given to genes whose expression was most strongly affected by both DBPI or
that are functionally related. Functional information is from http://tuberculist.epfl.ch/.
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Hit expansion and structure activity relationship (SAR) exploration. To
probe the structure activity relationship, thirteen new DBPI analogs of F1414-1444
were synthesized by medicinal chemistry. For the SAR we explored position 12 of the
benzo[f]pyrido[1,2-a]indole scaffold and also changed protons in positions 2 and 3 to
methyl groups. Scheme 1 outlines the synthetic route and the structures of the new
analogs and details of their antitubercular activity are presented in the supporting
information Table S3. We found that esters generally have stronger antitubercular
activity than acids. Changing oxygen in the carboxylic moiety to nitrogen (11626154)
or elimination of this oxygen from the structure leads to complete loss of activity
probably due to formation of a strong hydrogen bond between the NH proton and 11-
oxygen. The sequential introduction of Me (11626150), Et (11626149) and i-Pr
(11626157) in the ester moiety revealed a good correlation between antibacterial
activity and the alkyl size with the best activity found for the compound with the i-Pr
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substitution.
Interaction of the Me group in positions 2 or 3 of 6,11-
dioxobenzo[f]pyrido[1,2-a]indoles did not lead to a significant change in activity
(Table S3).
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R3
R1
O
O
O
O
R2
O
Cl
Cl
KOH
R3
+
+
EtOH
a)
R1
H3C
i-PrOH
N
N
b)
R2
R1=R2=H, R3=COOEt, COOMe, COMe, COPh
R1=Me, R2=H, R3=COOEt, COOMe, COMe
R1=H,R2=Me, R3=COOEt, COMe
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R1=H, Me
R2=H,
O
O
O
COOH
COCl
SOCl2
DMF
R3=COOEt
R1
N
N
CCl4
O
c)
R1=H, Me
O
O
Nu
H-Nu
Nu=NHCH2Ph, O-i-Pr
CH3CN
d)
N
O
Scheme 1. Synthetic route for 12-R-benzo[f]pyrido[1,2-a]indole-6,11-dione
derivatives
Data presented in Table 6 show that the two best characterized new derivatives
also act by inhibition of IspQ (Rv0338c) as all four DBPI-resistant mutants of M.
tuberculosis remained resistant but susceptibility could be restored on complementing
with the wild type gene.
Table 6. MIC determination and susceptibility to F1414-1444 analogs
MIC99 (µM)
---------------------------------------------------------------------------
Strain name
F1414-1438
11626157
11626159
O
O
O
N
N
CH3
N
CH3
O
O
O
O
O
O
O
O
O
CH3
H3C
Erdman
DBPI1
DBPI2
1.56
20.2
15.3
1.56
10.2
12.5
5.1
20.1
22.3
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DBPI3
15.5
40.2
1.7
12.2
12.6
1.54
22.2
21.4
5.6
DBPI4
DBPI4::pMVE_037
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Drug-like properties. To establish the drug-like properties of selected DBPI
derivatives prior to testing in animal models we performed a series of in vitro ADME/T
tests. Clearance was measured using the microsomal stability assay with a pool of
human microsomes from 50 donors. Under these controlled conditions, clearance was
found to be low to medium (Table 7). Furthermore, none of the DBPI compounds
showed significant toxicity for HepG2 cells and all were found to be non-mutagenic
using the SOS-chromotest. Consistent with this neither F1414-1444 nor 1438 were
found to intercalate into DNA using electrophoretic mobility shift assays.
Table 7. Microsomal stability assay and cytotoxicity
Compound
F1414-1444
F1414-1438
11626157
11626159
CBZ
Clearance category
Medium
Medium
Low
HepG2 TD50 (µM)
CLint (L/min/mg)
5.50
5.36
44.3
40.0
NT
1.59
14.78*
4.82
Medium
Low
NT
NT
NIF
44.79
High
NT
CBZ, carbamazepine (low clearance control); NIF, nifedipine (high clearance control). *With
this molecule depletion was observed after 60 min also in the control tubes containing
microsomes but not containing the NADPH regeneration solution which was not taken into
consideration during the evaluation of the CLint value. NT, not tested.
CONCLUDING REMARKS
The aim of exploring new chemical space for tuberculosis drug discovery was
successfully met thanks to the use of the NCCR diversity-oriented compound library
for phenotypic screening. This strategy uncovered many hits that will be described
elsewhere and the DBPI series, which to our knowledge, has never been associated with
antitubercular activity in previous screens. The best DBPI compounds show a number
of attractive features as their MIC is close to their MBC, they retain activity against
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non-replicating tubercle bacilli, and act in the single digit micromolar range.
Furthermore, although orthologs of their target, IspQ, are present in numerous
actinobacteria, the range of activity of DBPI is restricted to tubercle bacilli, which is a
desirable feature. The early ADME/T properties encourage further testing in zebrafish
or murine models of tuberculosis and synthesis is currently being scaled up for this
purpose. On investigating the structure activity relationship, it was observed that the
dioxo moiety was critical for activity thus directing future SAR modifications to other
components of the DBPI scaffold.
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We present strong evidence for IspQ, a membrane-bound, putative iron-sulfur-
binding reductase being the primary target of DBPI compounds. More than 20 years
after the publication of the genome sequence of M. tuberculosis H37Rv 48 we provide
proof for an essential role for this protein and the first clue to its role by means of
chemical biology. Its precise function remains unclear but there are strong hints from
the structural model, sequence similarities, gene expression profiling and regulation
studies that the protein, with its two Fe-S centres, might serve as a heterodisulfide
reductase and a role in electron transport is to be expected. From the drug development
standpoint this is encouraging because some of the most impressive progress in clinical
trials has been made with drug candidates that inhibit ATP and energy production as
exemplified by bedaquiline 8 and telacebec 12. An additional desirable aspect for drug
development is the prediction that the active site of the IspQ protein is predicted to be
located in the periplasm so inhibitors will not need to cross the plasma membrane to
achieve their therapeutic effect. It is noteworthy that the active sites of three
promiscuous tuberculosis drug targets, DprE1, MmpL3 and QcrB are all directly
accessible from the periplasm 13, 49
.
Finally, a membrane-associated oxidoreductase complex, comprising the
superoxide-detoxifier, SodA, the integral membrane protein, DoxX, and the thiol-
oxidoreductase, SseA, was recently found to link radical detoxification with cytosolic
40
thiol homeostasis . It is conceivable that IspQ could play a similar role by sensing
redox changes or perturbation of the activity of electron transport chains, but more work
is required to test this hypothesis.
METHODS
NCCR library
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The NCCR library screened in this work is composed of 67,393 commercially available
molecules ordered from Life Chemicals, Enamine and ChemDiv. The biggest set in the
library with 53,620 molecules comprises diverse representatives of chemical space. On
average six molecules represent one cluster enriched with 3D structures and sp3
centres. The rest of the library is composed of synthetic molecules designed and
selected by using all commercially available natural products and derivatives as
reference.
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Bacterial culture condition
Culture conditions of M. tuberculosis strains such as Erdman, H37Rv (ATCC25618),
clinical isolates and other laboratory strains are described in the Supporting
Information.
Screening the NCCR library
Primary screening and dose-response determination were carried out in 384-well plates
in duplicate using the resazurin reduction microtiter assay (REMA) as previously
described 15. Description of the instruments, material, concentrations and volumes used
in the HTS screening are detailed in the Supporting Information.
Toxicity assay
HepG2 human hepatocarcinoma cells were used for studying the toxicity of the
investigated molecules. Supporting Information contains more details.
Genotoxicity assay
The genotoxicity of the compounds was evaluated by the SOS chromotest on LB-agar
plates 17.
Determination of Minimal Bactericidal Concentration (MBC)
In order to determine the minimal bactericidal concentration (MBC99) of F1414-1444
Mtb Erdman cells (100 µL of OD600 0.0001 in complete 7H9 medium) were treated
with different compound concentrations (1/2XMIC-16XMIC). Treated and untreated
bacteria were incubated at 37°C during 7 and 14 d. After this time, 50 µL of undiluted
and serially diluted cells were plated on 7H10 agar plates and incubated for 3 weeks at
37°C and the number of CFU determined for each plate where growth was observed.
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The compound concentration, where the CFU was lower than or equal to 99% of the
CFU for the non-treated control, was considered as the MBC99.
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Resistant mutant isolation and genomics
In order to isolate resistant mutants Mtb Erdman (3 x 107 bacteria) were plated on
Middlebrook 7H10 plates containing compounds at concentrations of 3X, 6X, 10X,
20X and 50X of the MIC previously determined in liquid medium. Selected individual
colonies were resuspended in 20 µL of 7H9 and re-streaked on 7H10 plates containing
the same concentration of compound from which they were isolated. Two to four
colonies from each parent colony were then resuspended in 1 mL of complete 7H9 and
incubated one week at 37°C without shaking. Then the suspension was transferred to 5
mL of complete 7H9 medium and the bacteria were grown to logarithmic phase to
perform the REMA assay to test their dose-response to the compound. The
susceptibility pattern of individual colonies was compared to that of the Erdman WT
strain. Genomic DNA of the WT and mutant strains was sequenced on Illumina
MiSeq/HiSeq instruments and reads processed as detailed in the Supporting
Information.
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Overexpression
In order to validate the putative drug target of the compound, the empty pMV261 vector
and its pMVE_0374 construct were both transformed into the parental Erdman strain
and its four representative DBPI-resistant mutants. The open reading frame of
ERDMAN_0374 was amplified from Mtb Erdman genomic DNA by PCR using a
primer pair Rv0338c-FWD-BamHI 5´TTAGGATCCAGTGACCACGCAAACGCTC
3´and Rv0338c-REV-HpaI2 5´AATGTTAACTCAGCGTTTGCCCGGTGGACG 3´
to clone them into pMV261 50. The constructs were transformed into Erdman WT and
respective mutant strains and transformants were selected on 7H10 plates containing
25 µg/mL kanamycin. The total RNA was extracted by standard methods and the
overexpression of the genes was verified by real-time reverse transcription-PCR (RT-
PCR) using the primer pairs Rv0338c_qPCR_F 5´TTGTGGTCATGTGTGACCTG
3´and Rv0338c_qPCR_R 5´TTCTTGAACAGCACCGACAG 3´. The dose-response
to the compound was tested for at least two clones overexpressing each of the genes of
interest, in comparison with the parent WT or mutant strains alone and carrying the
empty pMV261 plasmid.
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Transcriptomics
Briefly, M. tuberculosis H37Rv was cultured to exponential phase (OD600nm 0.5), and
exposed for 6 h to DMSO, as a control, and to either F1414-1444 or 11626157 (10 µM
and 30 µM). Each sample was independently prepared in triplicate. Total RNA was
extracted using hot-phenol and RNA integrity evaluated by Bioanalyzer profiling.
Ribosomal RNA was depleted using the QIAseq FastSelect -5S/16S/23S QIAGEN kit,
and libraries were prepared using the TruSeq Stranded mRNA Library Preparation kit
(Illumina). Sequencing was performed on the NextSeq500 WGS system at the Biomics
platform (Institut Pasteur). Data were processed and alignment and annotation done
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using Bowtie ; the quality control (QC) report; the count expression table and the
differential analysis were obtained using DESeq2 and SARTools (1.7.0). A more
extensive account of the methods used may be found in the Supporting Information.
Metabolic stability assay (human microsomal CLint)
To study the metabolic stability of the molecule, human liver microsomes pooled from
50 donors were used as described in the Supporting Information.
ASSOCIATED CONTENT
Supporting Information is available and comprises details of methods used, three tables, and six figures. This
information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
*E-mail: stewart.cole@pasteur.fr
ORCID: 0000-0003-1400-5585
Author Contributions
x
R.S. and M.R. contributed equally and executed most of the experiments. R.S., V.S., G.M., K.J., V.Mi.,
V.Ma. and S.T.C. conceived and designed the experiments. O.R. synthesized compounds. V.S.
constructed and analyzed cKD mutants. A.B. performed genome analysis and provided bioinformatics
support. J.P. constructed structural models. M.C., E.K. and G.M. performed RNA-Seq analysis. R.S. and
S.T.C. wrote the manuscript, to which all authors contributed comments before approval of the final
version.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
ADME/T, Absorption Distribution Metabolism Excretion/Toxicity; ATc, anhydrotetracycline; BDQ,
bedaquiline; CBZ, carbamazepine; CFZ, clofazimine; CFU, colony-forming units; DBPI, dioxo-
dihydrobenzo-pyridoindole; INH, isoniazid; MIC99, minimal inhibitory concentration leading to 99%
growth inhibition; NIF, nifedipine; NTM, nontuberculous mycobacterial; REMA, resazurin microtiter
assay; SNP, single nucleotide polymorphisms; STR, streptomycin; WGS, whole genome sequencing.
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ACKNOWLEDGMENTS
We thank Julien Bortoli Chapalay, Damiano Banfi, Antoine Gibelin and Gerardo Turcatti from EPFL’s
Biomolecular Screening Facility for help with screening and compound management, Valérie Briolat
and Marc Monot, from the Institut Pasteur Biomics Platform for RNA-Seq support and Priscille Brodin,
Tony Maxwell, Claudia Sala and Anthony Vocat of the MM4TB consortium for advice and technical
support. This work was funded by the European Community’s Seventh Framework Programme
(MM4TB Grant 260872); the European Commission Marie Curie Fellowship (PIEF-GA-2012-327219
to R.S.); the Ministry of Education and Science of the Russian Federation (Agreement №
14.616.21.0065; unique identifier RFMEFI61616X006.); grant VEGA (1/0284/15) and France
Génomique (ANR-10-INBS-09-09).
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
28
ACS Paragon Plus Environment