Dual mechanism of action of 5-Nitro-1,10-phenanthroline against mycobacterium tuberculosis

Article abstract of DOI:10.1128/AAC.00969-17

New chemotherapeutic agents with novel mechanisms of action are urgently required to combat the challenge imposed by the emergence of drug-resistant mycobacteria. In this study, a phenotypic whole-cell screen identified 5-nitro-1,10-phenanthroline (5NP) as a lead compound. 5NP-resistant isolates harbored mutations that were mapped to fbiB and were also resistant to the bicyclic nitroimidazole PA-824. Mechanistic studies confirmed that 5NP is activated in an F₄₂₀-dependent manner, resulting in the formation of 1,10-phenanthroline and 1,10-phenanthrolin-5-amine as major metabolites in bacteria. Interestingly, 5NP also killed naturally resistant intracellular bacteria by inducing autophagy in macrophages. Structure-activity relationship studies revealed the essentiality of the nitro group for in vitro activity, and an analog, 3-methyl-6-nitro-1,10-phenanthroline, that had improved in vitro activity and in vivo efficacy in mice compared with that of 5NP was designed. These findings demonstrate that, in addition to a direct mechanism of action against Mycobacterium tuberculosis, 5NP also modulates the host machinery to kill intracellular pathogens.

Full text of DOI:10.1128/AAC.00969-17

AAC Accepted Manuscript Posted Online 11 September 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.00969-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
The dual mechanism of action of 5-Nitro-1,10-Phenanthroline against
Mycobacterium tuberculosis
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
#
1
#2,3
4
1
Saqib Kidwai , Chan-Yong Park , Shradha Mawatwal , Prabhakar Tiwari , Myung Geun
2
1
5
5
6
Jung , Tannu Priya Gosain , Pradeep Kumar , David Alland , Sandeep Kumar , Avinash
Bajaj , Yun-Kyung Hwang , Chang Sik Song , Rohan Dhiman , Ill Young Lee * and
Ramandeep Singh *
6
2
3
4
2
1
1
Vaccine and Infectious Disease Research Centre, Translational Health Science and
Technology Institute, NCR Biotech Science Cluster, PO Box # 4, Faridabad, Haryana, India.
Eco-Friendly New Materials Research Centre, Korean Research Institute of Chemical
Technology, Daejeon, Korea.
Department of Chemistry, Sung Kyun Kwan University, Suwon, Korea.
Laboratory of Mycobacterial Immunology, Department of Life Science, National Institute
of Technology, Rourkela, Odisha, India.
Division of Infectious Disease, Department of Medicine and the Ruy V. Lourenco Centre
2
3
4
5
for the Study of Emerging and Reemerging Pathogens, Rutgers University-New Jersey
Medical School, Newark, NJ, USA.
Laboratory of Nanotechnology and Chemical Biology, Regional Centre for Biotechnology,
6
NCR Biotech Science Cluster, PO Box # 4, Faridabad, Haryana, India.
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Running title: Deciphering the mechanism of Mtb killing by 5-nitro-1,10-phenanthroline.
# These authors contributed equal to the study.
* Correspondence: ramandeep@thsti.res.in (RS) or iylee@krict.re.kr (IYL)
Keywords: Autophagy/
F
420
dependent/ phenotypic screening/Mycobacterium
tuberculosis/5-nitro-1,10-phenanthrolines
1

4
5
5
9
0
1
Abstract
New chemotherapeutics agents with novel mechanism of action are urgently required to
combat the challenge imposed by the emergence of drug resistant mycobacteria. In this study,
a phenotypic whole cell screen identified 5-nitro-1,10-phenanthroline (5NP) as a lead
compound. 5NP resistant isolates harbored mutations mapped to fbiB and were also resistant
to bicyclic nitroimidazole, PA-824. Mechanistic studies confirmed that 5NP is activated in
52
53
54
55
56
57
58
59
60
61
62
63
F420 dependent manner resulting in formation of 1,10-phenanthroline and 1,10-phenanthrolin-
5-amine as major metabolites in bacteria. Interestingly, 5NP also killed naturally resistant
intracellular bacteria by inducing autophagy in macrophages. Structure activity relationship
studies revealed the essentiality of nitro group for in vitro activity and an analog, 3-methyl-6-
nitro-1,10-phenanthroline was designed resulting in improved in vitro activity as well as in
vivo efficacy in mice. These findings demonstrate that in addition to direct mechanism of
action against Mtb, 5NP also modulated host machinery to kill intracellular pathogen.
2

64
Introduction
Tuberculosis (TB) continues to be one of the major cause of unnatural morbidity and
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
mortality affecting the world today (1). The outcome of TB treatment is hampered due to the
ability of Mycobacterium tuberculosis (Mtb) to switch to a phenotypically drug-tolerant
dormant state (2). This situation is further aggravated due to emergence of multi-drug
resistant (MDR) and extensively drug resistant (XDR) Mtb strains (3, 4). Approximately, five
percent of TB cases across the globe are estimated to be MDR-TB, which includes 5% of
newly diagnosed TB cases and 20.5% of previously treated TB cases. Recently, few reports
have also suggested emergence of totally drug-resistant (TDR) Mtb strains with very limited
chance of successful treatment (5, 6). The current WHO recommended regimen for drug-
susceptible and resistant bacteria lasts for 6 months or 18-24 months, respectively. This
lengthy duration is probably required due to inherently slow bacterial growth rates and poor
penetration of drugs in TB lesions. In order to tackle this situation, there is an urgent need to
develop a regimen that i) includes drugs with novel mechanism of action ii) targets bacteria
in different metabolic state iii) is able to shorten duration of treatment and (iv) is effective
against MDR and XDR-TB.
80
81
82
83
84
85
86
87
88
Phenotypic or target based screening have led to identification of various scaffolds,
that possess activity against drug-resistant TB (7). Target based platforms rely on protein
essentiality in vivo, however, translation of in vitro potency into whole cell activity has been
a major challenge. Unlike target based screens, cell based screens results in identification of
scaffolds that are able to penetrate the cell membrane and inhibit their intracellular target.
Phenotypic screening in association with next-gen sequencing platform has led to
identification of various pro-drugs and scaffolds with novel mechanism of action. FDA has
approved administration of bedaquiline (TMC-207) and delamanid (OPC-67683) for
treatment of individuals with MDR-TB (8-10). TMC-207 inhibits ATP synthase enzyme and
3

8
9
9
9
9
9
9
9
9
9
9
9
0
1
2
3
4
5
6
7
8
9
is active against both replicating and non-replicating bacteria (11, 12). Bicyclic
nitroimidazoles (OPC-67683 and PA-824) are pro-drugs activated by Rv3547 (deazaflavin
dependent nitroreductase, Ddn) in a F420 dependent manner resulting in formation of des-nitro
metabolite and generation of reactive nitrogen intermediate (13-15). Phenotypic screens have
also led to identification of structurally diverse compounds that target either MmpL3, a
lipoprotein involved in transport of mycobacterial trehalose monomycolate (TMM) into Mtb
cell wall or DprE1, an enzyme involved in synthesis of cell wall arabinans or QcrB, a
component of the cytochrome bc1 complex of respiratory chain (7, 16, 17). These screens
have also resulted in identification of newer scaffolds targeting already validated drug targets
such as InhA or KasA (18, 19). Additionally, various repurposed drugs are being evaluated in
various clinical trials in an effort to decrease duration of chemotherapy for treatment of
individuals with MDR-TB (20-24).
1
00
101
102
103
104
105
106
107
108
109
110
In this study, we have performed phenotypic whole cell screening using M. bovis
BCG as surrogate host and identified 5-nitro-1,10-phenanthroline (5NP) as one of the lead
compound. We demonstrate that similar to PA-824 and 5-nitrothiophenes, 5NP are activated
in F420 dependent manner but clear difference exists in the nitroreductase mediating this
activation. Unlike PA-824, 5NP is able to induce autophagy in macrophages, a phenomenon
reversed in the presence of 3-methyl adenine. We report that the nitro group is essential for
both in vitro activity and induction of autophagy in macrophages. The structural analog with
methyl substitution at 3’ position possessed better in vitro activity and showed in vivo
potency. These observations demonstrate that 5NP exhibits dual mechanism of action against
Mtb by inhibiting mycolic acid biosynthesis and augmenting host anti-microbial pathways.
4

111
Results
112
113
114
115
116
117
118
119
120
121
122
123
124
125
Phenotypic whole cell based screening to identify Mtb growth inhibitors: M. bovis BCG
due to its higher degree of relatedness and lack of pathogenicity was used as a surrogate host
for phenotypic screening of small molecule library. This small molecule library consisting of
2300 compounds was procured from National Cancer Institute-Developmental Therapeutic
Program (NCI-DTP, http://nci.nih.gov/repositories.html). For preliminary screening, all
compounds were evaluated for their ability to inhibit growth of M. bovis BCG at a single
concentration of 50 M. We observed that out of 2300 compounds, 52 compounds possessed
anti-mycobacterial activity at the tested concentration. These primary hits were further
rescreened for activity against either M. bovis BCG or isoniazid-sensitive or isoniazid-
resistant Mtb strains in a dose response assay determining their minimum inhibitory
concentration (MIC). We observed that 35 scaffolds displayed similar activity against both
isoniazid-sensitive and isoniazid-resistant Mtb strains suggesting a mechanism of action
unlike that of isoniazid (Table 1, Fig. 1A). As shown in Table 1, most of these compounds
displayed comparable in vitro activity against both M. bovis BCG and Mtb.
126
127
128
129
130
131
132
133
134
Next, we determined the cellular cytotoxicity of these hits against THP-1
macrophages and observed that 24 of these primary hits were non-cytotoxic even at 25 M
concentration (Table 1). Few of these non-cytotoxic scaffolds were further evaluated for their
in vitro mode of activity (bactericidal or bacteriostatic) and ability to inhibit growth of
intracellular M. bovis BCG (Fig. 1A). In our in vitro growth inhibition experiments, we
observed that exposure of M. bovis BCG to either NSC 125531 or NSC 94945 or NSC 60339
or NSC 228155 resulted up to 60.0 – 100.0 fold reduction in bacterial counts in comparison
to untreated cultures (Fig. 1B, *P<0.05). As shown in Fig. 1B, approximately 8.0 fold
reduction in viable bacterial counts was observed in NSC 4263 exposed M. bovis BCG (Fig.
5

135
136
137
138
139
140
141
142
143
144
145
146
147
148
1B, *P<0.05). The exposure of M. bovis BCG to either NSC 26371 or NSC 163501 or NSC
149286 resulted in bacteriostatic effect in liquid cultures (Fig. 1B). In THP-1 macrophages,
we did not observe any intracellular killing of M. bovis BCG after 4 days exposure to either
NSC 228155 or NSC 60339 or NSC 26371 (Fig. 1C). In concordance with in vitro results,
bacteriostatic effect was observed in macrophages exposed to NSC 163501 (Fig. 1C). As
shown in Fig. 1C, exposure to either NSC 125531 or NSC 94945 or NSC 4263 resulted in
approximately 20.0 fold, 5.0 fold and 8.0 fold reduction in intracellular bacterial counts,
respectively, in comparison to untreated wells (Fig. 1C, **P<0.01). These lead compounds
NSC 125531, NSC 94945 and NSC 4263 identified in our screen were also able to inhibit
growth of intracellular Mtb in THP-1 macrophages (Fig. 1D, **P<0.01). These three
scaffolds displayed MIC in the range of 0.78 – 1.56 M against slow growing mycobacteria.
These three scaffolds NSC 125531, NSC 9495 and NSC 4263 lacked activity against E. coli
and displayed MIC values of >50 M, 50 M and 25 M, respectively against M.
smegmatis.
149
150
151
152
153
154
155
156
157
158
We next determined the activity of lead compounds against both drug sensitive and
drug resistant clinical strains (25) (Table 2). These drug-resistant clinical strains were as
sensitive as the drug-susceptible strain to these scaffolds indicating lack of cross-resistance to
known TB drugs (Table 2). Additionally, a number of scaffolds have recently been shown to
target Mtb by inhibiting MmpL3, therefore, we also tested these lead compounds for cross-
resistance to MmpL3 inhibitors using Mtb strains resistant to SQ109 and DA8. As shown in
Table 2, these strains were found susceptible to the identified lead compounds ruling out
MmpL3 as their putative target. This data suggests a novel mechanism of action for these
scaffolds and underscore developmental potential of these compounds for use against drug-
resistant TB.
6

159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
Generation and characterization of drug specific spontaneous resistant mutants. In
order to gain insight into their mechanism of action and identify the cellular targets for lead
compounds, we attempted to raise spontaneous drug-resistant mutants. We successfully
raised such mutants for NSC 4263, but repeated efforts to raise such mutants for NSC 94945
and NSC 125531 were futile, indicating an unusually high barrier to genetic resistance or
non-specific mechanism of action or the mutations in targets were not well tolerated. M.
bovis BCG resistant raised against NSC 4263 (5-nitro-1,10-phenanthroline, 5NP) displayed
>8.0 fold increase in MIC to the parent compound in comparison to the wild type strain
-6
(Table 2). The in vitro frequency of 5NP resistance in M. bovis BCG is approximately 10 ,
10 fold higher in comparison to the frequency of resistant mutations for PA-824 and
isoniazid. These resistant strains displayed consistent resistance to 5NP but remained
susceptible to standard anti-tubercular drugs (Table 2, Supplementary Table 1). For target
identification, genomic DNA was isolated from both wild type, 5NP resistant strain and
subjected to whole genome sequencing. Detailed analysis of sequencing data revealed an
inactivating frameshift mutation in fbiB gene that encodes coenzyme F420 L-glutamate ligase,
.
an essential enzyme involved in F420 biosynthesis Sanger sequencing of additional five
independent isolates also revealed mutations in fbiB confirming role of FbiB in resistance to
this compound. Complementation of resistant strains with a functional FbiB expressed from
episomal plasmid under constitutive hsp60 promoter restored their sensitivity to 5NP without
affecting susceptibilities to standard TB drugs such as isoniazid and levofloxacin (Table 2).
179
180
181
182
183
F420 (8-hydroxy-5-deazaflavin), a two electron transfer cofactor has been implicated
in redox balance, methane biosynthesis and activation of bicyclic nitroimidazoles (26-28).
The mycobacterial strains deficient in F420 biosynthesis are also resistant to other nitro
containing drugs such as 5-nitrothiophenes and bicyclic nitroimidazoles (13, 14, 29).
Therefore, we next investigated and observed that our resistant strains were also resistant to
7

184
185
186
187
188
189
190
191
192
killing by PA-824, a bicyclic nitroimidazole (Table 2). PA-824 resistant mutants are
characterized by mutations in either i) FGD-1, an enzyme involved in regeneration of
reduced F420 or ii) FbiA or FbiB or FbiC, enzymes involved in F420 biosynthesis or iii) Ddn,
nitroreductase involved in PA-824 activation (13). As shown in Table 2, we observed that
loss of functional FGD-1 (T3 mutant) or F420 biosynthetic enzymes (FbiC, 5A1 mutant or
+
+
FbiB, 7A2 mutant) also resulted in cross-resistance to 5NP. However, FGD F420 PA-824
resistant strain harboring mutation in Ddn was as sensitive as wild type strain to 5NP. These
results suggest the presence of a discrete F420 dependent nitroreductase that might activate
5NP in mycobacteria.
193
194
195
196
197
198
199
200
201
202
In vitro metabolite profiling of 5NP by LC/MS. It is well known that activation of bicyclic
nitroimidazoles such as PA-824 and OPC-67683 by Ddn results in formation of des-nitro
metabolite in bacteria (14, 15). Based on our observation that killing activity of 5NP is F420
dependent, we hypothesized that 5NP is a prodrug and its activation results in formation of
des-nitro product in bacteria. In support of our hypothesis, we observed two major peaks with
m/z corresponding to 181.09 and 195.10 in M. bovis BCG exposed to 5NP (Fig. 2A). We
next performed a detailed analytical characterization of synthetic des-nitro metabolite and
confirmed that peak with m/z of 181.09 was indeed that of 1,10-phenanthroline. As shown in
Fig. 2B, we did not observe any endogenous peaks corresponding to 181.09 m/z and 195.10
m/z in samples prepared from untreated M. bovis BCG.
203
204
205
206
207
Based on these observations, we propose the following mechanism for 5NP
activation in mycobacteria (Fig. 2C). The first step of activation involves hydride transfer
2
from H F420 to 4-position of 5NP resulting in formation of nitronic acid intermediate. This is
followed by protonation of nitronic acid resulting in formation of des-nitro metabolite
(metabolite 1, m/z-181.09 and 182.10 (isotope)) and release of nitrous acid (Fig. 2C).
8

208
209
210
211
212
213
214
215
Alternatively, reduction of nitronic acid intermediate leads to formation of hydroxylamine
intermediate, which upon further reduction results in formation of 1,10-phenanthrolin-5-
amine (metabolite 2, 195.10 and 196.11 (13)). We observed metabolite peaks corresponding
to metabolite 1 and metabolite 2 in our LC/MS experiments (Fig. 2A). In our MIC
determination assays, synthetically derived 1,10-phenanthroline (2) and 1,10-phenanthrolin-
5-amine (3) displayed MIC value of 12.5 M against Mtb (Supplementary Text, Table 3).
These findings suggest that antimycobacterial component of 5NP mediated Mtb killing
occurred during the process of its activation.
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
5NP inhibition of Mtb growth is independent of chelation of metal ions. 1,10-
phenanthroline series of compounds act as powerful chelating agents by virtue of their ability
to form thermodynamically stable metal ion complexes. These phenanthroline-metal
complexes have been demonstrated to inhibit growth of drug resistant Pseudomonas
aeruginosa and Clostridium difficile (30, 31) and also biofilm formation by P. aeruginosa
and Enterococcus faecalis (31, 32). 1,10-phenanthroline also possess fungistatic activity by
inhibiting secretory metallopeptidase of Phialophora verrucosa (33). Therefore, we next
investigated whether 5NP inhibits Mtb growth by chelating intracellular metal ions. To
determine this, we measured MIC of 5NP against M. bovis BCG both in the presence or
absence of various metal ions (Supplementary Table 2). As shown in Supplementary Table 2,
the addition of metal ions such as MgCl
did not alter MIC values for 5NP against M. bovis BCG. As shown in Supplementary Table 2,
we observed that addition of 200 g/ml and 1 g/ml ZnCl in liquid medium completely
2 2 2 2 4 3
or ZnCl or CaCl or Fe (SO ) in liquid medium
2
inhibited growth of M. bovis BCG. These results suggested that metal chelation is not the
plausible mechanism by which 5NP inhibited mycobacterial growth.
9

231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
5NP inhibits mycolic acid biosynthesis of Mtb. Bicyclic nitroimidazoles are known to
inhibit mycolic acid biosynthesis, therefore we next investigated the effect of 5NP exposure
on Mtb mycolic acid biosynthesis. The analysis of extracted polar lipids revealed that
exposure of Mtb to 5NP results in inhibition of trehalose monomycolates (TMM) and
trehalose dimycolates (TDM) biosynthesis, a phenomenon similar to that observed in the case
of INH exposed bacteria (Supplementary Fig. 1). TMMs/TDMs were identified by exposing
Mtb to DA5 that inhibits MmpL3 dependent transport of TMM across the membrane thereby
resulting in accumulation of TMM and reduction of TDM levels (Supplementary Fig. 1) (34).
We did not observe inhibition of biosynthesis of TDM and TMM in Mtb exposed to either
NSC 125531 or NSC 94945 (Supplementary Fig. 1). In order to confirm inhibition of
mycolic acid biosynthesis in 5NP treated samples, we analysed total mycolates by preparing
mycolic acid methyl esters (MAMEs) and fatty acid methyl esters (FAMEs). Consistent with
our observation of reduced levels of TDM and TMM in 5NP treated bacteria, we noticed
dose dependent reduction in MAMEs and FAMEs (Fig. 3A and 3B). Similar reduction in
levels of MAMEs was also noticed with INH treatment, a known inhibitor of FAS-II. This
observed reduction in FAMEs indicates that FAS-I pathway might be inhibited in 5NP
treated Mtb. As reported earlier, we also observed inhibition of MAMEs and FAMEs in PA-
824 treated Mtb.
Structure activity relationship studies of 5NP. We initially synthesized the parent
compound, 5NP (compound 1) and evaluated its activity against Mtb. The synthesized lead
compound 5NP (1) exhibited MIC value of 0.78 M against Mtb similar to that observed in
our phenotypic whole cell screen. We next started medicinal chemistry approach to design,
synthesize and evaluate the activity of 5NP structural analogs against Mtb (Table 3). The
detailed synthesis and analytical characterization for these structural analogs has been
included as Supplementary information (Supplementary Text). We observed that removal of
10

256
257
258
259
260
261
262
263
264
265
266
267
nitro group (2) reduced the activity of parent compound against Mtb. The des-nitro derivative
(2) exhibited approximately 10.0 fold less activity against Mtb (MIC 12.5 M). In order to
further evaluate the role of nitro group on anti-mycobacterial activity of this series, we
synthesized various compounds with modifications at R4 position. As shown in Table 3,
substitution of nitro group with either electron donating groups such as amino (3), methoxy
(4), methyl (5), carbamoyl (6) or electron withdrawing groups such as bromo (7), chloro (8)
and cyano (9) was unfavorable, as these analogs possessed reduced activity against both M.
bovis BCG and Mtb in comparison to the parent compound (5NP, 1, Table 3). These results
demonstrate that the nitro group at R4 position is important for the anti-mycobacterial
activity of 5NP. We also observed that M. bovis BCG 5NP resistant strain was as sensitive as
the parental strain to these structural analogs indicating that their anti-microbial activity is
independent of F420 levels in bacteria (Table 3).
268
269
270
271
272
273
274
275
276
277
278
279
Next, we synthesized various compounds to determine effect of R2 position
substitution on the anti-tubercular activity of 5NP (Table 3). We observed that the addition of
electron donating groups such as methyl (10) and ethyl (11) at R2 position increased the
potency of parent compound by 4.0 fold (Table 3, Fig. 3C). Furthermore, replacement of
methyl group with other functional groups such as vinyl (12), phenyl (13), substituted phenyl
(14-19), thiophen-2-yl (20), 3-(6-fluoro pyridin-3-yl, 21) was unfavorable as these
substitutions reduced the anti-mycobacterial activity of 10 (Table 3). In vitro MIC
determination experiments revealed that derivatives with halogen substitution such as bromo
(22) or iodo (23) were more potent in comparison to analogs with cyano (24) substitution
(Table 3). Therefore, structure activity relationship studies led to identification of 3-methyl-6-
nitro-1,10-phenanthroline (10) and 3-ethyl-6-nitro-1,10-phenanthroline (11) as the most
active compounds with a MIC value of 0.195 M against both M. bovis BCG and Mtb. As
11

2
80
81
expected, in comparison to wild type strain, M. bovis BCG 5NP resistant strain displayed
2
>4.0 fold increase in MIC to most of these analogs (Table 3).
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
In vivo activity of 3-methyl-6-nitro-1,10-phenanthroline in murine model of
tuberculosis. The lead compound (10) and positive control, rifampicin were further
evaluated for efficacy in chronic model of infection. For animal experiments, female BALB/c
mice were infected via aerosol route with low dose ~ 100 bacilli of Mtb H37Rv. Following 4
weeks of infection, mice were treated by gavaging continuously with either 25 or 100 mg/kg
compound 10 or 10 mg/kg Rif daily for 4 weeks. We observed that treatment with 25 mg/kg
of compound 10 did not result in reduction of bacterial loads in both spleens and lungs of
animals at both 14 days and 28 days post-treatment (Fig. 3D and 3E). As expected, daily
administration of Rif at 10 mg/kg reduced the bacterial loads by 1.6 log10 counts in both
lungs and spleens of infected animals at 4 weeks post-treatment (Fig. 3E, ***P<0.001). As
shown in Fig. 3E, we also noticed that treatment with 100 mg/kg of compound 10 resulted in
approximately 9.0 and 60.0 folds reduction in lungs and splenic bacterial counts, respectively
(**P<0.01 and ***P<0.001 in the case of lungs and spleens, respectively). In the case of 10
mg/kg Rif or 100 mg/kg compound 10 treated animals, the splenic bacterial counts were
below the limit of detection. We also observed treatment with 10 mg/kg of Rif or 100 mg/kg
of compound 10 resulted in 9.0 and 5.0 fold reduction, respectively in lung bacillary counts at
2 weeks post-treatment (Fig. 3D, **P<0.01 in the case of 10 mg/kg Rif and *P<0.05 in the
case of 100 mg/kg compound 10). To the best of our knowledge, this is the first study where
we report that this series possess anti-mycobacterial activity in mice and underscore their
potential for further development.
3
02
03
3
12

304
5NP induces autophagy in differentiated THP-1 macrophages.
305
306
307
308
309
310
311
312
313
314
315
316
317
318
Numerous host pathways such as generation of reactive oxygen species, phagosome
lysosome fusion, autophagy pathways have been demonstrated to control growth of
intracellular pathogens (35, 36). Therefore, we next investigated whether 5NP is able to
induce autophagy, thereby resulting in killing of intracellular bacteria. We compared the
ability of 5NP (compound 1), 3-methyl-6-nitro-1,10-phenanthrolie (compound 10), 1,10-
phenanthroline (compound 2) and PA-824 to induce autophagy in macrophages by
quantifying expression of Atg-3, Atg-7, Beclin-1 and conversion of LC3-I to LC3-II. As
shown in Fig. 4A and Fig. 4B, exposure to compound 1 and 10 resulted in significant
induction of Beclin-1, Atg-7 and Atg-3 expression at 24 hours post-treatment (*P<0.05,
**P<0.01). Interestingly, we did not observe any significant increase in expression of these
proteins in macrophages treated with either compound 2 or PA-824 (Fig. 4A and Fig. 4B). As
expected, dose dependent increase in expression of Beclin-1 and Atg-3 was observed in
macrophages treated with compound 1 (Fig. 4C). These observations implicated that the nitro
group is essential for induction of host autophagy pathways by 5NP.
319
320
321
322
323
324
325
326
During the process of autophagy, cytosolic form of LC3-I undergoes complex C-
terminal proteolytic and lipid modification to generate LC3-II, which is then translocated to
autophagosomal membranes (37). In concordance with earlier observations, we noticed
increased conversion of LC3-I to LC3-II in THP-1 macrophages treated with either
compound 1 or 10 in comparison to compound 2 or PA-824 treated macrophages (Fig. 4A
and Fig. 4B, *P<0.05). In concordance, higher LC3 puncta formation was observed in THP-1
macrophages treated with either compound 1 or 10 in comparison to DMSO treated
macrophages (Fig. 4D). A detailed quantitative analysis of confocal microscopy images also
13

327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
revealed that LC3 puncta formation was significantly increased in compound 1 or 10 treated
macrophages (Fig. 4E, *P<0.05).
We next combined 3-methyl adenine (3MA) an inhibitor of autophagosome
formation with compound 1 to quantify expression levels of various autophagic markers in
THP-1 macrophages. As shown in Fig. 5A and Fig. 5B, addition of 3MA abrogated the
ability of compound 1 to induce protein expression of Beclin-1, Atg-3 or Atg-7 or conversion
of LC3-I to LC3-II in THP-1 macrophages. As expected, pre-treatment with 3-MA reduced
LC3 puncta formation in THP-1 macrophages treated with compound 1 (Supplementary Fig.
2, *P<0.05). Taken together, these observations demonstrate that 5NP has the property to
induce autophagy in THP-1 macrophages. Further, we also noticed significantly higher LC3
puncta formation in THP-1 macrophages treated with other nitro containing 5 NP analogs (18
or 22 or 24) in comparison to non-nitro containing derivatives (2 or 3 or 4 or 5 or 7 or 8,
Supplementary Fig. 3, ***P<0.001, **P<0.01, *P<0.05 and data not shown). We also
investigated the ability of NSC 94945 and NSC 125531 to induce autophagy in macrophages.
As shown in Supplementary Fig. 4A and 4B, exposure to NSC 125531 also resulted in
upregulation of expression of Beclin-1, Atg-3 and Atg-7 or conversion of LC3-I to LC3-II in
THP-1 macrophages (*P<0.05, **P<0.01).
Host directed therapies (HDTs) have recently emerged as a promising intervention
strategy to tackle the problem of drug resistant TB. Several reports have demonstrated that
small molecules modulating host anti-microbial pathways inhibit intracellular growth of both
drug susceptible and resistant Mtb. In this study we demonstrate that unlike PA-824, 5NP is
able to induce autophagy in THP-1 macrophages. Therefore, we next investigated whether
5NP is able to inhibit intracellular growth of resistant mycobacteria (5NP resistant strain and
M. smegmatis). In support of our hypothesis we observed that treatment with 5NP resulted in
significant inhibition of intracellular growth of both 5NP resistant strain and M. smegmatis in
14

352
353
354
355
356
357
comparison to untreated macrophages (Fig. 5C and 5D, *P<0.05). We also demonstrate that
pre-incubation of macrophages with 3-MA reduces the killing activity of 5NP against
intracellular resistant strains (Fig. 5C and 5D, *P<0.05, **P<0.01). As expected, PA-824 was
unable to inhibit growth of M. smegmatis in THP-1 macrophages (Fig. 5C). To the best of our
knowledge, this is the first study where we demonstrate that 5NP requires a F420 dependent
nitroreductase for activation and upregulates autophagy in host macrophages (Fig. 6).
358
15

359
Discussion
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
Due to questionable long-term protective efficacy afforded by BCG vaccination,
control of TB infections is primarily dependent on 6-24 months of chemotherapy. However,
the rapid emergence of drug-resistant Mtb strains, HIV-TB nexus and Mtb ability to persist in
non-replicating stage poses a serious challenge to combat TB. Therefore, it is of utmost
importance to identify new scaffolds with novel mechanism of action that target both drug-
resistant and metabolically less active bacteria. The combination of phenotypic whole cell
screens along with activity against intracellular bacteria led to identification of NSC 125531,
NSC 94945 and 5NP as lead compounds. These identified hits were active against both drug-
susceptible and multi-drug resistant clinical strains indicating that they inhibit a novel
bacterial pathway. In concordance with previous reports we observed good correlation
between activities of these primary hits against M. bovis BCG and Mtb. The lead compounds
were comparably less active against M. smegmatis which might be due to variations in i)
essentiality of targets of both strains ii) affinity of drug-targets for these identified hits or iii)
relative intracellular concentration due to enhanced efflux or a defect in cellular uptake. 5NP
has also been earlier reported to inhibit Mtb growth in vitro and in macrophages but its
mechanism of killing was still unknown (38).
376
377
378
379
380
381
382
Identification of small molecules by phenotypic screening is followed by next gen
sequencing of genomic DNA isolated from drug-specific revertant strains to identify single
nucleotide polymorphisms (SNPs) associated with their mechanism. Using a combination of
genetic and biochemical tools we were able to decipher mechanism of Mtb killing by 5NP.
Using whole genome sequencing, we identified a frame shift mutation that results in FbiB
inactivation, an enzyme involved in F420 biosynthesis in resistant strain and complementation
with functional FbiB restored its susceptibility to 5NP. These findings confirmed that
16

383
384
385
386
387
388
389
390
391
392
393
394
depletion of F420 levels is associated with resistance to 5NP as also reported in the case of 5-
nitrothiophenes and bicyclic nitroimidazoles (13, 14, 29). The mechanism was unequivocally
confirmed by PA-824 cross-resistance studies using Mtb strains harboring mutations in either
FGD-1 or F420 biosynthetic enzymes (13). Surprisingly, Mtb strains with a defect in Ddn
enzyme activity were fully sensitive to 5NP (13). These observations suggest that 5NP is
likely to be activated by an unknown nitroreductase in an F420 dependent manner. Similar to
bicyclic nitroimidazoles, we observed that 1,10 phenanthroline (des-nitro) and 1,10-
phenanthrolin-5-amine were the major cellular metabolites of 5NP in M. bovis BCG (15).
The proposed mechanism for 5NP activation in mycobacteria would result in generation of
reactive nitrogen intermediate, which inhibits bacterial growth by affecting electron transport
chain. Similar to PA-824, exposure of Mtb to 5NP also inhibited Mtb FAS-I/FAS-II
pathways, thereby, resulting in depletion of MAMEs and FAMEs.
395
396
397
398
399
400
401
402
403
404
405
406
407
1,10 phenanthroline have been shown to mediate microbial killing by virtue of its
ability to either bind DNA or chelate metal ions or inhibit acetyl-CoA synthesis (30, 39-41).
However, in this study we demonstrate that chelation of metal ions is not the mechanism by
which 5NP inhibited Mtb growth in vitro. A detailed structure activity relationship approach
revealed that i) nitro group at R4 position is important for anti-mycobacterial activity ii)
addition of methyl and ethyl group at 3’ position increased the activity of parent compound.
We also show that treatment of mice with 100 mg/kg of 3-methyl-6-nitro-1,10-
phenanthroline for 28 days results in approximately 10.0 and 60.0 folds reduction of bacterial
counts in lungs and spleens of Mtb infected animals, respectively. Here, we also demonstrate
that 5NP is able to activate autophagy in THP-1 macrophages in a dose dependent manner
and nitro group is essential for this host modulating activity. This observed induction in
autophagy could be due to higher rates of formation of autophagosomes and was reversed in
the presence of 3-MA, a known inhibitor of autophagosome formation.
17

408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
Several studies have shown that treatment of mice with immunomodulatory agents
in conjunction with current TB drugs results in faster clearance of MDR-TB. HDT’s possess
several advantages such as i) inability of pathogen to acquire resistance against them ii)
ability to clear intracellular drug resistant and non-replicating bacilli and iii) can lead to
shortening of duration of chemotherapy. Autophagy is regulated by mTOR and is an essential
component of host anti-microbial machinery (35, 42). Polymorphisms in the IRGM1
(LRG47) has been shown to be associated with susceptibility to TB in different geographical
regions (43-45). Upregulation of autophagy related genes like Beclin-1, P2X7, VDR, NOD2,
TLR8 have been reported as biomarkers to identify TB infected individuals (46-48). Several
molecules such as rapamycin, nortriptyline, PE and tat-beclin-1 peptide also induce
autophagy and possess anti-bacterial and anti-viral activities (35, 49-51). In addition to these,
several other immunomodulatory agents such as metformin, imatinib, ibuprofen, zileuton and
vorinostat are also being currently evaluated for treatment of individuals with MDR-TB (52,
53). Here, we show that both PA-824 and 5NP are activated in a F420 dependent manner but
unlike PA-824, 5NP is able to induce autophagy. This 5NP dependent modulation of host
pathway also resulted in clearance of resistant bacteria by macrophages.
424
425
426
427
428
429
430
431
In conclusion, 5NP represents a promising scaffold that possesses activity against
both multi-drug-resistant and susceptible Mtb. Mechanistic studies revealed that 5NP is a
prodrug requiring F420 for activation, a process independent of Ddn. We also showed that 3-
methyl-6-nitro-1,10-phenanthroline killed Mtb in mice tissues by targeting both host and
pathogen. To the best of our knowledge, this is the first study where we report that 5NP has a
dual mode of action against Mtb by inhibiting mycolic acid biosynthesis and activating host-
autophagy pathways. Future experiments would involve detailed mechanistic studies for the
remaining identified scaffolds and synthesis of 5NP structural analogs with better in vivo and
18

4
32
33
pharmacokinetic and pharmacodynamic properties. These findings should lead the way for
further discovery and development of scaffolds with similar anti-bacterial properties.
4
434
19

435
Materials and Methods
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
Strains, culture conditions and chemicals: M. smegmatis, M. bovis BCG and Mtb H37Rv
were cultured in Middlebrook (MB) 7H9 broth supplemented with 10% albumin dextrose
saline, 0.2% glycerol, 0.05% tween-80 or MB 7H11 agar supplemented with 10% OADS as
per standard protocols. E. coli culturing was performed in Luria-Bertani (LB) broth or agar at
o
37 C, 200 rpm. The chemical library consisting of approximately 2300 compounds was
obtained from National Cancer Institute-Development Therapeutic Program (NCI-DTP,
http://nci.nih.gov/repositories.html). The compounds in this library belonged to either
diversity set or mechanistic set or natural product set. All other chemicals used in this study
unless mentioned were procured from Sigma Aldrich (St. Louis, MO, USA). For MIC
determination assays, compounds were prepared as 50 mM stocks in DMSO and evaluated
for antimycobacterial activity in concentration ranging from 50 to 0.05 M. For these assays,
various strains were grown in MB 7H9 medium till OD600nm of 0.2, diluted 1000x and added
to 96 well plates containing drugs. MIC99 value was defined as the drug concentration at
which no visible bacterial growth was observed. For MIC determination, 96-well plates were
incubated either for 1 day in the case of E. coli or 2 days in the case of M. smegmatis or 14
days in the case of M. bovis BCG and Mtb as per standard protocols.
4
4
4
4
52
53
54
55
Cell cytotoxicity assays: Cell viability assays were performed using MTT cell viability kit as
4
per manufacturer’s recommendations (Sigma Aldrich, MO, USA). Briefly, 2.5 x 10 THP-1
cells were seeded in triplicate wells in 96 well plates. Next day, macrophages were washed
and overlaid with medium containing drugs in the concentration ranging from (25 M to 0.05
M) for 96 hours. In order to determine cell viability, 10 l of MTT reagent was added in
each well, formazan crystals were dissolved in 100 l of lysis solution. The absorbance was
measured at 562 nm in a plate reader and cell viability was calculated according to following
456
457
458
20

4
59
60
formula: % cell viability = (OD562nm of test sample/ OD562nm of control) x 100. CC50 was
defined as the concentration of drug at which 50% of cells were viable.
4
461
462
463
464
465
Antimycobacterial activity in vitro and in macrophages: For in vitro experiments, M.
bovis BCG cultures were grown till OD600nm of 0.2 in MB 7H9 medium and subsequently
exposed to various drugs. At 7 days post-exposure, bacterial cultures were harvested, washed
once with 1x phosphate buffered saline (PBS) and bacterial counts were enumerated by
o
plating 100 l of 10 fold serial dilutions on MB7H11 plates at 37 C for 3-4 weeks.
466
467
468
469
470
471
472
473
474
475
476
For intracellular killing experiments, THP-1 macrophages were seeded at a density of
5
5
2 x 10 (for M. bovis BCG experiment) or 5 x 10 (for M. smegmatis experiment) in 12-well
plate and differentiated by the addition of PMA. Subsequently, macrophages were infected
with either M. bovis BCG or M. smegmatis at a MOI of 1:10 or 1:1, respectively. Following
3-4 hours post-infection, extracellular bacteria were removed by overlaying macrophages
with RPMI medium containing 200 g/ml amikacin. After 2 hours, infected macrophages
were overlaid with RPMI medium. Next day, macrophages were washed twice with 1x PBS
and overlaid with RPMI medium containing drugs. At designated time points (72 hours and
96 hours in the case of M. smegmatis and M. bovis BCG, respectively), macrophages were
lysed in 1x PBS-0.1% triton X-100 and 100 l of 10-fold serial dilutions were plated on MB
o
7H11 plates at 37 C for 3-4 weeks.
477
478
479
480
481
482
Generation of spontaneous resistant mutant and complementation strains: For
generation of drug-specific spontaneous resistant mutant strains, mid-log phase cultures of M.
bovis BCG were plated on MB 7H11 plates containing drugs at concentration of 10x MIC.
The colonies appearing on these drug-containing plates were grown till OD600nm of 0.2 – 0.3
and MIC values were determined as described above. For target identification, genomic DNA
was isolated from both wild type and resistant mutant strains and subjected to Next-gen
21

483
484
485
486
sequencing (Edge-Bio, Gaithersberg, MD). For complementation analysis, fbiB was PCR
amplified and cloned into mycobacterial expression vector, pVV16. The complemented strain
was constructed by electroporation of pVV16-FbiB in the 5NP resistant strain and
transformants were selected on MB 7H11 plates with 25 g/ml kanamycin.
487
488
489
490
491
492
493
494
495
496
497
498
499
500
Lipid analysis of mycolic acids from Mtb: Mtb cultures were grown till OD600nm of 0.3 –
0.4 and 5 ml cultures were treated with various drugs. Following 1 hour post-incubation, Mtb
1
4
was labeled using 1 Ci/ml of C-acetate (56 mCi/mmol, Perkin Elmer, USA) for 4 hours.
The labeled bacilli were harvested, pellets were hydrolysed by addition of 2 ml tetra-n-butyl
o
ammonium hydroxide (TBAH) and incubated overnight at 100 C. The fatty acids were
esterified by addition of 4 ml of dichloromethane, 300 l of methyl iodide and 2 ml of water
followed by end to end mixing for 1 hour at room temperature. The phases were separated by
centrifugation, lower phase was washed twice with distilled water, dried under stream of air,
resuspended in 3 ml of diethyl ether and centrifuged. The solvent excluding insoluble
material was transferred to new glass tubes, dried and resuspended in 200 l
dichloromethane. Equal volumes were loaded on a silica gel 60 F254 thin layer
chromatography (TLC) plates and resolved using hexane/ethyl acetate (19:1, v/v, 2 runs). The
bands corresponding to FAMEs and MAMEs were visualized by phosphorimaging and
quantified using Image quant 5.2 software (GE health care life science, UK).
501
502
503
504
505
506
For analysis of TDM and TMM levels, Mtb cells were treated and labeled as
described above. The labeled bacilli were harvested, resuspended in chloroform/methanol
(2:1, v/v) and incubated overnight at 55°C. The samples were centrifuged, supernatants were
dried in new glass tubes and resuspended in 200 l of chloroform/methanol (2:1, v/v). Equal
volumes of various samples were spotted on normal phase TLC plates, resolved using
chloroform/methanol/water (62:24:4, v/v/v) and radioactive spots were visualized by
22

5
07
08
phosphorimaging. The radioactive spots corresponding to TDM and TMM were quantified
using Imagequant 5.2 software (GE healthcare Life Sciences, UK).
5
509
510
511
512
513
514
Intracellular metabolite analysis for M. bovis BCG treated with 5NP: For metabolite
o
analysis, mid-log phase cultures of M. bovis BCG were incubated with 20 M of 5NP at 37 C
with end to end mixing. Following 8 hours of incubation, bacterial cells were harvested, lysed
by addition of acetonitrile, clarified lysates were prepared by centrifugation and filtered using
0.22
-spec analysis. Mass spectra were
recorded with AB SCIEX Triple TOF 5600 system.
515
516
517
518
519
520
521
522
523
524
Synthesis of small molecule: All reagents for chemical synthesis of 5NP structural analogs
were of ACS grade and purchased from either Sigma Aldrich or TCI or Alfa Aesar. The
detailed protocol for chemical synthesis of these structural analogs has been described in
Supplementary text. The chemical reactions were monitored by thin layer chromatography
using F254 glass backed silica plates. The plates were visualized by short wave ultra violet
light (254 nm) and stained with p-anisaldehyde and phosphomolybdic acid. The final
compounds were dissolved in either CdCl
3
or d
6
3
-DMSO or Cd Od for nuclear-magnetic
1
13
resonance (NMR) spectra. NMR spectra ( H-NMR, C-NMR) were recorded using Varian
Gemini 300 MHz, Bruker DPX-300MHz and 500 MHz spectrometer. High-resolution Mass
spectrometry (TOF MS ES or ESI) was performed using a JOEL JMS-700.
525
526
527
528
529
Animal ethics statement: All animal experimental procedures were reviewed and approved
by Institutional animal ethics committee of Translational Health Science and Technology
Institute and International Centre for Genetic Engineering and Biotechnology (ICGEB). Mice
efficacy experiments were performed at Tuberculosis Aerosol Challenge Facility according
to the protocol of ICGEB animal care committee.
23

530
531
532
533
534
535
536
537
538
In vivo mouse efficacy experiments: Female BALB/c mice were procured from National
Institute of Nutrition, Hyderabad, India. In vivo mouse efficacy experiments were performed
after aerosol infection of BALB/c mice with approximately 100 CFU of Mtb. At 4 weeks
post-infection, mice were treated with either 25 mg/kg or 100 mg/kg 3-methyl-6-nitro-1,10-
phenanthroline or 10 mg/kg rifampicin for 14 days or 28 days. These compounds were daily
orally administered in 1% carboxy-methyl cellulose formulations. The bacterial loads were
determined in lungs and spleens of infected animals (5 animals per group) at both 2 weeks
and 4 weeks post-treatment. For bacterial enumeration, tissues were homogenized in saline
o
and 100 l of 10-fold serial dilutions were plated on MB 7H11 plates at 37 C for 3-4 weeks.
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
Autophagy experiments and immunoblot analysis: For autophagy experiments, THP-1
6
cells were differentiated by the addition of 50 ng/ml PMA and 1.5 x 10 THP-1 were seeded
per well in 12 well plates. Next day, differentiated THP-1 cells were treated with various
drugs and clarified lysates were prepared by lysing macrophages in RIPA (50 mM Tris-Cl,
pH-7.4, 150 mM NaCl, 5 mM EDTA, 1% triton X-100, 0.5% sodium deoxycholate, 0.1%
SDS) buffer containing protease inhibitor cocktail (Sigma Aldrich, MO, USA). The amount
of protein in cell lysates was quantified using BCA kit (Sigma Aldrich, MO, USA), 50 g of
protein was electrophoresed on 15% SDS-PAGE and subjected to immunoblot analysis. The
nitrocellulose membrane after protein transfer was blocked in 2% skimmed milk and probed
overnight with either anti-Beclin-1 or anti-Atg-3 or anti-Atg-7 or anti-LC3 (Cell Signaling
Technology, MA, USA) antibodies at 4 degree. This was followed by incubation of the
nitrocellulose membrane with secondary antibody for 45 minutes at room temperature and
protein bands were visualized using enhanced chemiluminiscence kit (GE healthcare Life
Science, UK). In order to determine equal loading among various lanes, blots were stripped
and probed with -actin antibody (Cell signaling Technology, MA, USA) following the same
protocol as described above.
24

555
556
557
558
559
560
561
562
563
564
565
Immunofluorescence Detection of LC3
5
For confocal microscopy experiment, 5 x 10 THP-1 cells were differentiated by PMA
addition on 18 mm glass-cover slips in 12-well plate. Next day, differentiated THP-1 cells
were treated with various drugs for 24 hours, washed two times with 1x PBS and fixed in 4%
paraformaldehyde. The fixed cells were blocked in buffer containing 2% BSA and 0.3% NP-
40 for 1 hour and incubated overnight with anti-human LC3 antibody (Cell Signaling
0
Technology, MA, USA) at 4 C. The labeled cells were subsequently washed and incubated
with goat anti-rabbit IgG Alexa Fluor 568 antibody (Thermo Fisher Scientific, IL, USA) for 1
hour. The cells were then washed three times with 1x PBS, coverslips were mounted on glass
slides with fluoromount aqueous mounting medium (Sigma-Aldrich, USA) and observed
under confocal scanning laser microscope (CSLM) (Leica Microsystems, Wetzlar, Germany).
566
567
568
569
For quantitative analysis, images were taken randomly and numbers of LC3 puncta per cell were
counted in all the fields by an observer who did not have any information about the experimental set
up.
Statistical analysis: Prism 5 software (Version 5.01; GraphPad software Inc, CA, USA) was
570
571
572
used for statistical analysis and the generation of graphs. For normally distributed data,
comparisons were performed by a paired (two-tailed) t – test. Differences between groups
were considered significant at P<0.05.
573
25

574
Funding
575
576
577
578
579
580
581
582
RS acknowledges the financial support received from THSTI and Department of
Biotechnology (BT/COE/34/15219/2015). RS acknowledges DBT for National Bioscience
Award
(BT/HRD/35/02/18/2009). RD is thankful to Department of Science and Technology, Govt.
of India for financial support (DST/INSPIRE/Faculty
(BT/HRD/NBA/37/01/2014)
and
Ramalingaswami
fellowship
award/2014/DST/INSPIRE/04/2014/01662; EMR/2016/000048). IYL acknowledges the
funding received through the National Research Foundation of Korea from Ministry of
Science, ICT and the Korean Research Institute of Chemical Technology.
583
Acknowledgements
584
585
586
587
588
589
590
591
592
The authors are also thankful to Prof. Anil K Tyagi for access to BSL-3 facility. We thank the
technical staff of Tuberculosis aerosol challenge facility, ICGEB for their help during animal
experiments. TP and SM are thankful to DBT and MHRD, Govt. of India for their respective
fellowship. The authors are grateful to Dr. Clifton E Barry and Dr. Bill Jacobs for sharing
Mtb PA-824 resistant and INH resistant Mtb strains, respectively. Mr. Sushanth Pradhan
(Biotechnology and Medical Engineering department, NIT Rourkela) is gratefully
acknowledged for technical assistance in confocal microscopy experiment. The technical help
provided by Mr. Rajesh and Mr. Surender is highly acknowledged.
Competing financial interests. The authors declare no competing financial interests.
593
594
595
596
Author contributions. RS conceived the idea. SK, PT, TPG, PK and RS conducted
microbiology and animal experiments. CYP, MGJ, YKH, CSS, AB and IYL performed
chemical synthesis. SM and RD conducted macrophage experiments. RS, IYL and RD
supervised the study. RS and RD wrote the manuscript with inputs from PK, IYL and AB.
26

597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
References
1.
2.
Daniel TM. 2006. The history of tuberculosis. Respiratory medicine 100:1862-
1870.
Barry CE, 3rd, Boshoff HI, Dartois V, Dick T, Ehrt S, Flynn J, Schnappinger D,
Wilkinson RJ, Young D. 2009. The spectrum of latent tuberculosis: rethinking
the biology and intervention strategies. Nature reviews. Microbiology 7:845-855.
Zumla A, Nahid P, Cole ST. 2013. Advances in the development of new
tuberculosis drugs and treatment regimens. Nature reviews. Drug discovery
12:388-404.
3.
4.
5.
6.
Zumla A, Raviglione M, Hafner R, von Reyn CF. 2013. Tuberculosis. The New
England journal of medicine 368:745-755.
Udwadia ZF. 2012. MDR, XDR, TDR tuberculosis: ominous progression. Thorax
67:286-288.
Velayati AA, Farnia P, Masjedi MR, Ibrahim TA, Tabarsi P, Haroun RZ, Kuan
HO, Ghanavi J, Farnia P, Varahram M. 2009. Totally drug-resistant tuberculosis
strains: evidence of adaptation at the cellular level. The European respiratory
journal 34:1202-1203.
7.
8.
Mdluli K, Kaneko T, Upton A. 2015. The tuberculosis drug discovery and
development pipeline and emerging drug targets. Cold Spring Harbor
perspectives in medicine 5.
Pym AS, Diacon AH, Tang SJ, Conradie F, Danilovits M, Chuchottaworn C,
Vasilyeva I, Andries K, Bakare N, De Marez T, Haxaire-Theeuwes M, Lounis
N, Meyvisch P, Van Baelen B, van Heeswijk RP, Dannemann B, Group TCS.
2016. Bedaquiline in the treatment of multidrug- and extensively drug-resistant
tuberculosis. The European respiratory journal 47:564-574.
9.
Gler MT, Skripconoka V, Sanchez-Garavito E, Xiao H, Cabrera-Rivero JL,
Vargas-Vasquez DE, Gao M, Awad M, Park SK, Shim TS, Suh GY, Danilovits M,
Ogata H, Kurve A, Chang J, Suzuki K, Tupasi T, Koh WJ, Seaworth B, Geiter
LJ, Wells CD. 2012. Delamanid for multidrug-resistant pulmonary tuberculosis.
The New England journal of medicine 366:2151-2160.
10.
11.
Diacon AH, Lounis N, Dannemann B. 2014. Multidrug-resistant tuberculosis
and bedaquiline. The New England journal of medicine 371:2436.
Andries K, Verhasselt P, Guillemont J, Gohlmann HW, Neefs JM, Winkler H,
Van Gestel J, Timmerman P, Zhu M, Lee E, Williams P, de Chaffoy D, Huitric
E, Hoffner S, Cambau E, Truffot-Pernot C, Lounis N, Jarlier V. 2005. A
diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis.
Science 307:223-227.
12.
13.
Koul A, Dendouga N, Vergauwen K, Molenberghs B, Vranckx L, Willebrords
R, Ristic Z, Lill H, Dorange I, Guillemont J, Bald D, Andries K. 2007.
Diarylquinolines target subunit c of mycobacterial ATP synthase. Nature
chemical biology 3:323-324.
Manjunatha UH, Boshoff H, Dowd CS, Zhang L, Albert TJ, Norton JE, Daniels
L, Dick T, Pang SS, Barry CE, 3rd. 2006. Identification of a nitroimidazo-
oxazine-specific protein involved in PA-824 resistance in Mycobacterium
tuberculosis. Proceedings of the National Academy of Sciences of the United
States of America 103:431-436.
14.
Matsumoto M, Hashizume H, Tomishige T, Kawasaki M, Tsubouchi H, Sasaki
H, Shimokawa Y, Komatsu M. 2006. OPC-67683,
a
nitro-dihydro-
27

646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
imidazooxazole derivative with promising action against tuberculosis in vitro
and in mice. PLoS medicine 3:e466.
Singh R, Manjunatha U, Boshoff HI, Ha YH, Niyomrattanakit P, Ledwidge R,
Dowd CS, Lee IY, Kim P, Zhang L, Kang S, Keller TH, Jiricek J, Barry CE, 3rd.
2008. PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular
NO release. Science 322:1392-1395.
Chiarelli LR, Mori G, Esposito M, Orena BS, Pasca MR. 2016. New and Old Hot
Drug Targets in Tuberculosis. Current medicinal chemistry 23:3813-3846.
Mikusova K, Ekins S. 2016. Learning from the past for TB drug discovery in the
future. Drug discovery today.
Abrahams KA, Chung CW, Ghidelli-Disse S, Rullas J, Rebollo-Lopez MJ,
Gurcha SS, Cox JA, Mendoza A, Jimenez-Navarro E, Martinez-Martinez MS,
Neu M, Shillings A, Homes P, Argyrou A, Casanueva R, Loman NJ, Moynihan
PJ, Lelievre J, Selenski C, Axtman M, Kremer L, Bantscheff M, Angulo-
Barturen I, Izquierdo MC, Cammack NC, Drewes G, Ballell L, Barros D, Besra
GS, Bates RH. 2016. Identification of KasA as the cellular target of an anti-
tubercular scaffold. Nature communications 7:12581.
Manjunatha UH, SP SR, Kondreddi RR, Noble CG, Camacho LR, Tan BH, Ng
SH, Ng PS, Ma NL, Lakshminarayana SB, Herve M, Barnes SW, Yu W, Kuhen
K, Blasco F, Beer D, Walker JR, Tonge PJ, Glynne R, Smith PW, Diagana TT.
2015. Direct inhibitors of InhA are active against Mycobacterium tuberculosis.
Science translational medicine 7:269ra263.
Lee M, Cho SN, Barry CE, 3rd, Song T, Kim Y, Jeong I. 2015. Linezolid for XDR-
TB--Final Study Outcomes. The New England journal of medicine 373:290-291.
Gopal M, Padayatchi N, Metcalfe JZ, O'Donnell MR. 2013. Systematic review of
clofazimine for the treatment of drug-resistant tuberculosis. The international
journal of tuberculosis and lung disease : the official journal of the International
Union against Tuberculosis and Lung Disease 17:1001-1007.
Sotgiu G, D'Ambrosio L, Centis R, Tiberi S, Esposito S, Dore S, Spanevello A,
Migliori GB. 2016. Carbapenems to Treat Multidrug and Extensively Drug-
Resistant Tuberculosis: A Systematic Review. International journal of molecular
sciences 17:373.
Dawson R, Narunsky K, Carman D, Gupte N, Whitelaw A, Efron A, Barnes GL,
Hoffman J, Chaisson RE, McIlleron H, Dorman SE. 2015. Two-stage activity-
safety study of daily rifapentine during intensive phase treatment of pulmonary
tuberculosis. The international journal of tuberculosis and lung disease : the
official journal of the International Union against Tuberculosis and Lung Disease
19:780-786.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Sterling TR, Scott NA, Miro JM, Calvet G, La Rosa A, Infante R, Chen MP,
Benator DA, Gordin F, Benson CA, Chaisson RE, Villarino ME, Tuberculosis
Trials Consortium tACTGftPTBTTiotTBTC, the Aids Clinical Trials Group for
the Prevent Tb Trial are listed in the Supplement i. 2016. Three months of
weekly rifapentine and isoniazid for treatment of Mycobacterium tuberculosis
infection in HIV-coinfected persons. Aids 30:1607-1615.
Wilson R, Kumar P, Parashar V, Vilcheze C, Veyron-Churlet R, Freundlich JS,
Barnes SW, Walker JR, Szymonifka MJ, Marchiano E, Shenai S, Colangeli R,
Jacobs WR, Jr., Neiditch MB, Kremer L, Alland D. 2013. Antituberculosis
thiophenes define a requirement for Pks13 in mycolic acid biosynthesis. Nature
chemical biology 9:499-506.
28

695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
26.
27.
Jones WJ, Nagle DP, Jr., Whitman WB. 1987. Methanogens and the diversity of
archaebacteria. Microbiological reviews 51:135-177.
Bleicher K, Winter J. 1991. Purification and properties of F420- and NADP(+)-
dependent alcohol dehydrogenases of Methanogenium liminatans and
Methanobacterium palustre, specific for secondary alcohols. European journal of
biochemistry / FEBS 200:43-51.
Purwantini E, Daniels L. 1996. Purification of a novel coenzyme F420-
dependent glucose-6-phosphate dehydrogenase from Mycobacterium
smegmatis. Journal of bacteriology 178:2861-2866.
Hartkoorn RC, Ryabova OB, Chiarelli LR, Riccardi G, Makarov V, Cole ST.
2014. Mechanism of action of 5-nitrothiophenes against Mycobacterium
tuberculosis. Antimicrobial agents and chemotherapy 58:2944-2947.
Katzianer DS, Yano T, Rubin H, Zhu J. 2014. A high-throughput small-molecule
screen to identify a novel chemical inhibitor of Clostridium difficile. International
journal of antimicrobial agents 44:69-73.
Viganor L, Galdino AC, Nunes AP, Santos KR, Branquinha MH, Devereux M,
Kellett A, McCann M, Santos AL. 2016. Anti-Pseudomonas aeruginosa activity
of 1,10-phenanthroline-based drugs against both planktonic- and biofilm-
growing cells. The Journal of antimicrobial chemotherapy 71:128-134.
Tay CX, Quah SY, Lui JN, Yu VS, Tan KS. 2015. Matrix Metalloproteinase
Inhibitor as an Antimicrobial Agent to Eradicate Enterococcus faecalis Biofilm.
Journal of endodontics 41:858-863.
Granato MQ, Massapust Pde A, Rozental S, Alviano CS, dos Santos AL,
Kneipp LF. 2015. 1,10-phenanthroline inhibits the metallopeptidase secreted by
Phialophora verrucosa and modulates its growth, morphology and
differentiation. Mycopathologia 179:231-242.
Tahlan K, Wilson R, Kastrinsky DB, Arora K, Nair V, Fischer E, Barnes SW,
Walker JR, Alland D, Barry CE, 3rd, Boshoff HI. 2012. SQ109 targets MmpL3, a
membrane transporter of trehalose monomycolate involved in mycolic acid
donation to the cell wall core of Mycobacterium tuberculosis. Antimicrobial
agents and chemotherapy 56:1797-1809.
Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V. 2004.
Autophagy is a defense mechanism inhibiting BCG and Mycobacterium
tuberculosis survival in infected macrophages. Cell 119:753-766.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
Levine B, Deretic V. 2007. Unveiling the roles of autophagy in innate and
adaptive immunity. Nature reviews. Immunology 7:767-777.
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T,
Kominami E, Ohsumi Y, Yoshimori T. 2000. LC3, a mammalian homologue of
yeast Apg8p, is localized in autophagosome membranes after processing. The
EMBO journal 19:5720-5728.
Khare G, Kumar P, Tyagi AK. 2013. Whole-cell screening-based identification of
inhibitors against the intraphagosomal survival of Mycobacterium tuberculosis.
Antimicrobial agents and chemotherapy 57:6372-6377.
Khorasani-Motlagh M, Noroozifar M, Moodi A, Niroomand S. 2013.
Biochemical investigation of yttrium(III) complex containing 1,10-
phenanthroline: DNA binding and antibacterial activity. Journal of
photochemistry and photobiology. B, Biology 120:148-155.
38.
39.
29

742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
40.
41.
Liu X, Li X, Zhang Z, Dong Y, Liu P, Zhang C. 2013. Studies on antibacterial
mechanisms of copper complexes with 1,10-phenanthroline and amino acid on
Escherichia coli. Biological trace element research 154:150-155.
Zhu X, Li T, Gu X, Zhang S, Liu Y, Wang Y, Tan X. 2013. Structural and
functional investigation into acetyl-coenzyme A synthase and methyltransferase
from human pathogen Clostridium difficile. Metallomics : integrated biometal
science 5:551-558.
Kumar D, Nath L, Kamal MA, Varshney A, Jain A, Singh S, Rao KV. 2010.
Genome-wide analysis of the host intracellular network that regulates survival of
Mycobacterium tuberculosis. Cell 140:731-743.
Intemann CD, Thye T, Niemann S, Browne EN, Amanua Chinbuah M, Enimil
A, Gyapong J, Osei I, Owusu-Dabo E, Helm S, Rusch-Gerdes S, Horstmann RD,
Meyer CG. 2009. Autophagy gene variant IRGM -261T contributes to protection
from tuberculosis caused by Mycobacterium tuberculosis but not by M.
africanum strains. PLoS pathogens 5:e1000577.
Che N, Li S, Gao T, Zhang Z, Han Y, Zhang X, Sun Y, Liu Y, Sun Z, Zhang J, Ren
W, Tian M, Li Y, Li W, Cheng J, Li C. 2010. Identification of a novel IRGM
promoter single nucleotide polymorphism associated with tuberculosis. Clinica
chimica acta; international journal of clinical chemistry 411:1645-1649.
King KY, Lew JD, Ha NP, Lin JS, Ma X, Graviss EA, Goodell MA. 2011.
Polymorphic allele of human IRGM1 is associated with susceptibility to
tuberculosis in African Americans. PloS one 6:e16317.
Songane M, Kleinnijenhuis J, Alisjahbana B, Sahiratmadja E, Parwati I,
Oosting M, Plantinga TS, Joosten LA, Netea MG, Ottenhoff TH, van de Vosse
E, van Crevel R. 2012. Polymorphisms in autophagy genes and susceptibility to
tuberculosis. PloS one 7:e41618.
Songane M, Kleinnijenhuis J, Netea MG, van Crevel R. 2012. The role of
autophagy in host defence against Mycobacterium tuberculosis infection.
Tuberculosis 92:388-396.
Yu Q, Zhu X, Di W, Jiang Y. 2016. High Beclin-1 Expression in Human Alveolar
Macrophage Significantly Correlate with the Bacteriologic Sterilization in
Pulmonary Tuberculosis Patients. Clinical laboratory 62:1003-1007.
Floto RA, Sarkar S, Perlstein EO, Kampmann B, Schreiber SL, Rubinsztein
DC. 2007. Small molecule enhancers of rapamycin-induced TOR inhibition
promote autophagy, reduce toxicity in Huntington's disease models and enhance
killing of mycobacteria by macrophages. Autophagy 3:620-622.
42.
43.
44.
45.
46.
47.
48.
49.
50.
Shoji-Kawata S, Sumpter R, Leveno M, Campbell GR, Zou Z, Kinch L, Wilkins
AD, Sun Q, Pallauf K, MacDuff D, Huerta C, Virgin HW, Helms JB, Eerland R,
Tooze SA, Xavier R, Lenschow DJ, Yamamoto A, King D, Lichtarge O, Grishin
NV, Spector SA, Kaloyanova DV, Levine B. 2013. Identification of a candidate
therapeutic autophagy-inducing peptide. Nature 494:201-206.
Sundaramurthy V, Barsacchi R, Samusik N, Marsico G, Gilleron J, Kalaidzidis
I, Meyenhofer F, Bickle M, Kalaidzidis Y, Zerial M. 2013. Integration of
chemical and RNAi multiparametric profiles identifies triggers of intracellular
mycobacterial killing. Cell host & microbe 13:129-142.
51.
52.
Zumla A, Maeurer M. 2016. Host-directed therapies for multidrug resistant
tuberculosis. International journal of mycobacteriology 5 Suppl 1:S21-S22.
30