Mechanism of action of 5-nitrothiophenes against mycobacterium tuberculosis

Article abstract of DOI:10.1128/AAC.02693-13

On using the streptomycin-starved 18b strain as a model for nonreplicating Mycobacterium tuberculosis, we identified a 5-nitrothiophene compound as highly active but not cytotoxic. Mutants resistant to 5-nitrothiophenes were found be cross-resistant to the nitroimidazole PA-824 and unable to produce the F420 cofactor. Furthermore, 5-nitrothiophenes were shown to be activated by the F420-dependent nitroreductase Ddn and to release nitric oxide, a mechanism of action identical to that described for nitroimidazoles. Copyright

Full text of DOI:10.1128/AAC.02693-13

Mechanism of Action of 5-Nitrothiophenes
against Mycobacterium tuberculosis
Ruben C. Hartkoorn, Olga B. Ryabova, Laurent R. Chiarelli,
Giovanna Riccardi, Vadim Makarov and Stewart T. Cole
Antimicrob. Agents Chemother. 2014, 58(5):2944. DOI:
10.1128/AAC.02693-13.
Published Ahead of Print 18 February 2014.
Updated information and services can be found at:
http://aac.asm.org/content/58/5/2944
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Mechanism of Action of 5-Nitrothiophenes against Mycobacterium
tuberculosis
Ruben C. Hartkoorn,a Olga B. Ryabova,b Laurent R. Chiarelli,c Giovanna Riccardi,c Vadim Makarov,b Stewart T. Colea
Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerlanda; A. N. Bakh Institute of Biochemistry, Russian Academy of Science, Moscow,
Russiab; Università degli Studi di Pavia, Dipartimento di Biologia e Biotecnologie, Pavia, Italyc
On using the streptomycin-starved 18b strain as a model for nonreplicating Mycobacterium tuberculosis, we identified a 5-nitro-
thiophene compound as highly active but not cytotoxic. Mutants resistant to 5-nitrothiophenes were found be cross-resistant to
the nitroimidazole PA-824 and unable to produce the F420 cofactor. Furthermore, 5-nitrothiophenes were shown to be activated
by the F420-dependent nitroreductase Ddn and to release nitric oxide, a mechanism of action identical to that described for
nitroimidazoles.
t is widely considered that compounds that effectively target and sized (see the supplemental material) to confirm activity and to
I
kill persistent or nonreplicating Mycobacterium tuberculosis
determine the role of the nitro group. Data (Table 1) revealed that
activity is associated with the nitro group on C-5 of the thiophene
ring (compounds 1 and 2). Exchange of the nitro by an acetyl and
movement of the nitro group from C-5 to C-3 on the thiophene
ring rendered the compound inactive (compounds 3 and 4). In-
troduction of a second nitro group on C-3 of the ring (compounds
5 and 6) did not improve activity and made the compounds mu-
tagenic, as determined using the SOS chromotest (10). Com-
pound 1 was the most active analogue and was used for further
investigations into the mechanism of action.
should have a major impact on antituberculosis chemotherapy.
Current compounds with this so-called sterilizing activity include
the frontline drugs rifampin and pyrazinamide as well as a number
of compounds in clinical development, bedaquiline (TMC207),
Q203, and the nitroimidazoles PA-824 and delamanid (OPC-
67683) (1–4). While the development of these new drug candi-
dates remains promising, the search for novel chemical entities
effective against nonreplicating bacteria is vital in order to com-
plete and sustain the tuberculosis drug pipeline.
Several in vitro models have been developed that enable myco-
bacteria to remain in nonreplicating states, which are thought to
reproduce some of the characteristics of persistent M. tuberculosis
in vivo (5). Characteristically, such bacteria are phenotypically re-
sistant to drugs targeting the bacterial cell well (such as isoniazid
and ethambutol), while also becoming less susceptible to steriliz-
ing drugs like rifampin. Of the models described, the streptomy-
cin-starved M. tuberculosis strain 18b (ss18b) model is arguably
the most amenable for screening compounds against nonreplicat-
ing bacteria, as it requires minimal manipulation and can be used
both in vitro and in vivo (5–7). Strain 18b is an rrs mutant of M.
tuberculosis that depends on streptomycin for its growth; removal
of streptomycin results in the bacterium being unable to replicate
further, while maintaining viability (6, 7).
We used the resazurin reduction assay (6, 7) to screen for ac-
tivity against ss18b in 519 compounds previously demonstrated to
be effective against the actively growing H37Rv strain of M.
tuberculosis (8, 9). Hits active on ss18b were subsequently tested to
confirm activity against growing H37Rv and for cytotoxicity on
the human liver carcinoma cell line HepG2 and the human lung
epithelial cell line A549. One compound in particular, the nitro-
thiophene 2-(3-methylpiperidin-1-yl)-5-nitrothiophene (Pub-
Chem substance identifier [SID] 24814045), was found to be equi-
potent against replicating H37Rv and nonreplicating ss18b (MIC
of 6.25 ␮g/ml for both). While compounds with such a profile are
frequently cytotoxic, this compound displayed no cytotoxicity
against HepG2 and A549 cells at 20 ␮g/ml (Table 1). 5-Nitrothio-
phenes were therefore deemed interesting for further investiga-
tion to determine their killing mechanisms against both growing
and nonreplicating mycobacteria.
To learn more about the mechanism of action of the 5-nitro-
thiophenes, we isolated H37Rv mutants on solid medium con-
taining compound 1 (20 ␮g/ml). The frequency of isolation of
resistant mutants was high, at 5 ϫ 106, and of the 11 mutants
selected, all displayed a phenotype highly resistant to compound 1
(MIC, Ͼ100 ␮g/ml), with no altered susceptibility to isoniazid,
rifampin, or moxifloxacin.
From a structural point of view, the nitrothiophene resembles
the nitroimidazole part of PA-824 and delamanid. Interestingly,
nitroimidazoles also have potent activity against nonreplicating
bacteria, displaying comparable activity against strains H37Rv
and ss18b. For these reasons we decided to investigate whether
compound 1-resistant mutants were cross-resistant to PA-824.
Data revealed that this was indeed the case as resistant mutants
were fully cross-resistant to PA-824 (Ͼ100 ␮g/ml), suggesting
that these two classes of compounds share a similar mechanism of
activation or action (or both).
In M. tuberculosis, PA-824 has been elegantly shown to be a
prodrug that is enzymatically reduced by the nonessential, deaza-
flavin (F420)-dependent nitroreductase (Ddn or Rv3547) to re-
Received 11 December 2013 Returned for modification 20 January 2014
Accepted 11 February 2014
Published ahead of print 18 February 2014
Address correspondence to Stewart T. Cole, stewart.cole@epfl.ch.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.02693-13.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.02693-13
Six close analogues of the 5-nitrothiophene hit were synthe-
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Antimicrobial Agents and Chemotherapy p. 2944–2947
May 2014 Volume 58 Number 5

Antitubercular Action of 5-Nitrothiophenes
TABLE 1 Summary of activity of 5-nitrothiophenes
a
Activity (MIC [␮g/ml])
Cytotoxicity (TD50
against:
[␮g/ml]) against:
Compound
Structure
Strain H37Rv
6.25
ss18b
HepG2 cells
A549 cells
SOS chromotest
Not done
PubChem SID 24814045
6.25
Ͼ20
Ͼ20
1
2
3.125
6.25
12.5
12.5
110
61
84
21
Not mutagenic
Mutagenic
3
100
Ͼ100
Ͼ100
41
61
29
65
Not mutagenic
Not mutagenic
4
5
Ͼ100
6.25
6.25
6.25
6.25
72
75
4
Mutagenic
Mutagenic
6
8.9
PA-824
0.078
0.001
0.156
0.625
Not done
Rifampin
Ͼ100
Ͼ100
Not mutagenic
a TD50, 50% toxic dose.
lease nitric oxide (NO) that nonspecifically damages and kills fluorescence high-pressure liquid chromatography (HPLC) as de-
tubercle bacilli (4). While resistance to PA-824 through mutations scribed previously (12, 15). This analysis revealed that all the mu-
in Ddn is infrequent, mutations preventing the proper biosynthe- tants were incapable of synthesizing F420-5 and F420-6. Of the 11
sis of its cofactor F420 are much more common (11, 12). The bio- resistant mutants analyzed, six were possible fbiC mutants (being
synthesis of F420 in M. tuberculosis initially involves the formation unable to produce FO), two mutants produced FO but no inter-
of the intermediate, 7,8-didemethyl-8-hydroxy-5-deazariboflavin mediates containing glutamate residues, and three mutants pro-
(FO), for which FbiC and other enzymes have been shown to be duced only short F420 intermediates (F420-2 to F420-4), but no
needed (11, 13). 2-Phospholactate is subsequently attached to FO,
F420-5 or F420-6 (Fig. 1). Taken together, these results indicate that
followed by the addition of predominantly 5 to 6 glutamate resi- compound 1 is most probably converted by an F420-5-dependent
dues to form F420-5 and F420-6, a process involving FbiA and FbiB nitroreductase to release nitric oxide. Unlike results reported for
(11, 12, 14). F420-5 and F420-6 are the preferred cofactors for a PA-824, for which mutations in Ddn and FGD1 (20% of mutants)
number of F420-dependent nitroreductases, and these cofactors were found (11), all 11 mutants we analyzed were defective for F420
are subsequently recycled to their reduced forms by an F420-de- production, although our sample size was smaller than that of the
pendent glucose-6-phosphate dehydrogenase (FGD1) (11).
To determine the reason for the cross-resistance between PA-
PA-824 study.
To determine if compound 1 exhibits its antituberculosis ac-
824 and the 5-nitrothiophenes, the ddn gene was sequenced in all tivity through the release of nitric oxide in a Ddn-dependent man-
11 resistant mutants selected, but it was found to be wild type in all ner, this hypothesis was tested in Mycobacterium smegmatis. In-
cases. Subsequently, the bacterial F420 content was evaluated by trinsically, M. smegmatis is resistant to compound 1, an
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aac.asm.org 2945

Hartkoorn et al.
FIG 2 5-Nitrothiophenes are activated by Ddn to release nitric oxide. Bacte-
rial growth curves (A) in the presence of 32 ␮g/ml of compound 1 show that
wild-type M. smegmatis (solid circles) and M. smegmatis transformed with the
control vector pSODIT (solid squares) grow in the presence of the compound.
M. smegmatis transformed with pSODIT/ddn (open squares) is, however, sen-
sitive to the action of compound 1, and unable to grow. Under the same
conditions the concentration of nitrite (stable end product of NO) in the
supernatant was measured (B). A time-dependent increase in nitrite levels, and
therefore nitric oxide, was observed only in M. smegmatis transformed with
pSODIT/ddn (and not in the controls lacking Ddn). OD600, optical density at
600 nm.
observation also reported for PA-824 (16). The recombinant ex-
pression of ddn (Rv3547) on a pSODIT plasmid in M. smegmatis
was, however, found to make the bacterium susceptible to com-
pound 1 (Fig. 2). Additionally, when measuring nitric oxide re-
lease following exposure of M. smegmatis to compound 1 (32 ␮g/
ml) by the Griess reaction using nitrate reductase (17), we found
that M. smegmatis expressing Ddn showed a time-dependent re-
lease of nitric oxide, while wild-type M. smegmatis and M. smeg-
matis containing the control vector released a basal amount of NO
that did not increase over time (Fig. 2). These data confirm that
compound 1 is a prodrug that gets enzymatically activated by M.
tuberculosis Ddn to release nitric oxide, the active pharmacophore
that nonspecifically kills both growing and nonreplicating bacte-
ria. This does not, however, exclude the possibility that other F420
-
dependent nitroreductases are able to bioactivate 5-nitrothio-
phenes.
Finally, to evaluate if compound 1 is a good scaffold for struc-
ture-based activity efforts, its metabolic stability was determined.
Using both mouse and human liver microsomes, we found that
the intrinsic clearance rates of compound 1 were very high, at 335
and 96 ␮l/min/mg protein, respectively. Similar results were ob-
tained with compound 2. This high intrinsic clearance is a consid-
erable obstacle for the development of 5-nitrothiophenes.
The data provided here demonstrate that 5-nitrothiophenes,
such as compound 1, are active on both replicating and nonrepli-
cating tubercle bacilli. The mechanism of action of these com-
pounds was found to be similar to that of nitroimidazoles such as
PA-824, most likely involving the reduction of the nitrothiophene
by an F420-dependent nitroreductase to release nitric oxide that
nonspecifically kills M. tuberculosis, independently of growth rate.
Indeed, loss of proper F420 production by the bacterium renders
the compounds inactive. The relative simplicity of the nitrothio-
phene compound makes it a potentially interesting scaffold for
further development. However, since the compound has low met-
abolic stability and its activity is relatively weak compared to that
of PA-824, and especially to that of delamanid (3), and resistant
FIG 1 Fluorescence chromatogram of the F420 content of 5-nitrothiophene
resistant H37Rv mutants. (A) H37Rv extract showing the elution of FO and
F420-3 to F420-7. (B) One of six 5-nitrothiophene-resistant mutants that is
unable to produce FO or any fluorescent F420 intermediates. (C) One of two
5-nitrothiophene-resistant mutants that produced only FO. (D) One of three
5-nitrothiophene resistant mutants that produced short-chain F420s but not
F420-5 or F420-6 that are thought to be the active F420 cofactors used in M.
tuberculosis.
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Antimicrobial Agents and Chemotherapy

Antitubercular Action of 5-Nitrothiophenes
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ACKNOWLEDGMENTS
The research leading to these results received funding from the European
Community’s Seventh Framework Programme (grant 260872).
We thank R. C. Goldman and the National Institute of Allergy and
Infectious Diseases (NIAID) for making available the compounds of the
Molecular Libraries Screening Center Network (MLSCN) for the initial
screening performed here.
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