Benzothiazinones: Prodrugs that covalently modify the decaprenylphosphoryl- β-D-ribose 2′-epimerase DprE1 of mycobacterium tuberculosis

Article abstract of DOI:10.1021/ja106357w

Benzothiazinones (BTZs) form a new class of potent antimycobacterial agents. Although the target of BTZs has been identified as decaprenylphosphoryl- β-d-ribose 2′-epimerase (DprE1), their detailed mechanism of action remains obscure. Here we demonstrate that BTZs are activated in the bacterium by reduction of an essential nitro group to a nitroso derivative, which then specifically reacts with a cysteine residue in the active site of DprE1.

Full text of DOI:10.1021/ja106357w

Published on Web 09/09/2010
Benzothiazinones: Prodrugs That Covalently Modify the
Decaprenylphosphoryl-ꢀ-D-ribose 2′-epimerase DprE1 of Mycobacterium
tuberculosis
Claudia Trefzer,†,‡ Monica Rengifo-Gonzalez,†,‡ Marlon J. Hinner,‡ Patricia Schneider,†,§
Vadim Makarov,†,| Stewart T. Cole,†,§ and Kai Johnsson*,†,‡
New Medicines for Tuberculosis (NM4TB) Consortium, Institute of Chemical Sciences and Engineering and Global
´
Health Institute, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland, and Bakh
Institute of Biochemistry, Russian Academy of Science, 119071 Moscow, Russia
Received July 18, 2010; E-mail: kai.johnsson@epfl.ch
Abstract: Benzothiazinones (BTZs) form a new class of potent
antimycobacterial agents. Although the target of BTZs has been
identified as decaprenylphosphoryl-ꢀ-D-ribose 2′-epimerase
(DprE1), their detailed mechanism of action remains obscure.
Here we demonstrate that BTZs are activated in the bacterium
by reduction of an essential nitro group to a nitroso derivative,
which then specifically reacts with a cysteine residue in the active
site of DprE1.
1,3-Benzothiazin-4-ones (benzothiazinones, BTZs; Figure 1a) are
tuberculosis (TB) drug candidates with high activity against
Mycobacterium tuberculosis in Vitro and in ViVo.1 A minimal
inhibitory concentration (MIC) of 1 ng/mL against M. tuberculosis
H37Rv was measured for the most promising BTZ, BTZ043; this
MIC is a factor of 20 below that of the frontline TB drug, isoniazid.
Figure 1. BTZs and DprE1. (a) BTZ043 and other compounds used in
Genetic and biochemical analysis revealed that the target of BTZs
this study. (b) Epimerization of decaprenylphosphoryl-ꢀ-D-ribose (DPR)
is the enzyme decaprenylphosphoryl-ꢀ-D-ribose 2′-epimerase (DprE1,
via decaprenylphosphoryl-2-keto-D-erythro-pentofuranose (DPX) to deca-
Rv3790).1 DprE1, together with DprE2 (Rv3791), catalyzes the
prenylphosphoryl-ꢀ-D-arabinose (DPA).2 Both DprE1 and DprE2 are
epimerization of decaprenylphosphoryl-ꢀ-D-ribose (DPR) to deca-
prenylphosphoryl-ꢀ-D-arabinose (DPA; Figure 1b), which is a
central precursor for the synthesis of cell-wall arabinans.2,3 Due to
difficulties in isolating active DprE1, details of the mechanism of
action of BTZs are scarce. It is known that a single point mutation
in DprE1 (Cys387Ser or Cys387Gly) results in a more than 250-
fold increased MIC (Table 1).1
required for the epimerization of DPR.
Table 1. MIC Values of Compounds Used in This Study against
Different Mycobacterial Strains and M. smegmatis mc2155
Overexpressing Different DprE1 Mutants
MIC (µg/mL)
strain
residue 387 in DprE1
BTZ043
MTX
CT319
M. tuberculosis H37Rv
M. boVis BCG
Cys
Cys
Ser
Cys
Gly
Ser
Cys
Cys
Gly
0.001
0.002
16
0.004
4
0.125 0.31
Furthermore, the nitro group of BTZs is essential for activity;
its reduction to either the amine or the hydroxylamine increased
the MIC at least 500-fold.1 Following the discovery of BTZs,
another class of compounds that target DprE1 was identified:4
dinitrobenzamide DNB1 and its derivatives (Figure 1a). As observed
for BTZs, point mutations of Cys387 of DprE1 resulted in resistance
to DNB1, and reduction of one of the nitro groups to the amine or
the hydroxylamine abolished activity.4 These data suggest that BTZs
and dinitrobenzamides operate by the same mechanism of action.
n.d.
n.d.
0.125 0.062
0.15
>100
M. boVis BCG BN2a
M. smegmatis mc2155
M. smegmatis MN47a
M. smegmatis MN84a
His-DprE1b
>100
>100
n.d.
n.d.
n.d.
>100
>100
7.81
7.81
n.d.
16
0.05
0.05
12.5
His-G129DprE1b
His-G129,G387DprE1b
a Strains described in ref 1. b Protein coexpressed with DprE2 in M.
smegmatis mc2155. n.d., not determined.
Both BTZs and the dinitrobenzamides are nitroaromatic com-
pounds that contain an electron-withdrawing substituent in the
position meta to the nitro group. Given the prevalence of such
structures in organic chemistry, we searched the literature for related
compounds with reported antimycobacterial activity and identified
the xanthone derivative 1-methyl-2,4,7-trinitroxanthone (MTX;
Figure 1a)5 and another class of dinitrobenzene derivatives6 (here
abbreviated as DNDs; Figure 1a).
We first tested the activity of MTX against BTZ-sensitive and
BTZ-resistant mycobacteria and found that mutations in DprE1 also
confer resistance to MTX (Table 1). This confirms our hypothesis
that MTX and BTZ share the same mechanism of action. Comparing
the structures of the reported DNDs with those of BTZs and
dinitrobenzamides, we predicted that, in the DNDs, only one of
the nitro groups is essential for antimycobacterial activity. We
† NM4TB Consortium (www.nm4tb.org).
‡ Institute of Chemical Sciences and Engineering, EPFL.
§ Global Health Institute, EPFL.
| Russian Academy of Science.
9
10.1021/ja106357w  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 13663–13665 13663

C O M M U N I C A T I O N S
therefore synthesized CT319, a DND derivative in which one of
the nitro groups is replaced by a trifluoromethyl group (Figure 1a;
see also Supporting Information), and measured its activity against
different mycobacteria (Table 1). As predicted, CT319 showed high
activity against M. smegmatis and M. tuberculosis H37Rv but no
significant activity against BTZ-resistant strains with the mutations
Cys387Ser or Cys387Gly in DprE1. Furthermore, reduction of the
nitro group to the amine abolished all antimycobacterial activity
(compound CT318; Supporting Information). These data reveal that
CT319, MTX, BTZs, and DNBs, despite their structural diversity,
share a common mechanism of action and thereby constitute a new
family of antimycobacterial agents.
Electron-deficient nitroaromatic compounds are known to be readily
reduced in biological systems, yielding first the corresponding ni-
trosoarene, which is subsequently further reduced to the corresponding
hydroxylamine and then to the amine.7 Accordingly, it has been
observed that BTZs are reduced to the corresponding amines in bacteria
and mice.1 Nitrosoarenes are electrophiles that readily react with thiols
to form semimercaptals (Figure 2a)8-10 and also can covalently modify
cysteine residues in proteins.11,12 In the absence of an excess of thiols,
the semimercaptals generated from electron-deficient nitrosoarenes are
relatively stable and only slowly rearrange to the corresponding
sulfinamide.9 As the mutation of Cys387 in DprE1 leads to BTZ
resistance, we speculated that the nitroso derivatives of BTZs or related
compounds form a semimercaptal with Cys387 (Figure 2b). To test
this hypothesis, we first attempted to study the reaction of a nitroso
derivative of BTZs with thiols. While the preparation of the nitroso
derivative of BTZ043 proved to be difficult, we succeeded in preparing
the nitroso derivative of CT319, CT325, and analyzed its reaction with
glutathione (Figure S1 and discussion in the Supporting Information).
Mixing equimolar solutions of glutathione and CT325 resulted in the
immediate and almost quantitative formation of the corresponding
semimercaptal (Figure S1 and discussion in the Supporting Informa-
tion). Neither CT319 nor the amine CT318 reacted with glutathione
under these conditions.
purified the protein under denaturing conditions. In addition to Cys387,
His-DprE1 possesses another cysteine, Cys129. The relatively low
stability of the proposed semimercaptal formed between BTZs and
DprE1 required its isolation in the absence of thiols (Vide infra), and
the mutation of Cys129 to glycine facilitated experiments under such
conditions (Supporting Information). Coexpression of either His-
G129DprE1 or His-DprE1 with DprE2 in M. smegmatis decreased the
sensitivity toward BTZ043 relative to wild-type M. smegmatis to the
same extent (Table 1), indicating that His-G129DprE1 from M. tuber-
culosis is functional in M. smegmatis. M. smegmatis strains coex-
pressing His-G129DprE1 and DprE2 were incubated for 4 h with low
concentrations of BTZ043 (0.5 µg/mL) and subsequently lysed in the
presence of 8 M urea and purified by affinity chromatography. His-
DprE1 was isolated, together with GroEL1 as a major contaminant
(Figure 3a). Subsequent analysis by mass spectrometry permitted the
detection of a protein with a mass of 51 786 Da (Figure 3b), which
corresponds to a semimercaptal formed from the nitroso derivative of
BTZ043 and His-G129DprE1 (calculated mass of 51 786.6 Da). Reduc-
ing the sample prior to mass analysis using DTT permitted only the
detection of unmodified His-G129DprE1 (Figure 3b). The sensitivity of
the enzyme-drug adduct toward thiols is in agreement with the known
reactivity of semimercaptals.9 When the experiment was repeated
without incubation of the mycobacteria with BTZ043 and without
reduction of the sample prior to analysis by mass spectrometry, His-
G129DprE1 could not be detected (Figure 3b). We assume that oxidation
of unmodified Cys387 of His-G129DprE1 during lysis and purification
prevents the detection of His-G129DprE1 by mass spectrometry. The
significance of this experiment is that the failure to detect unmodified
His-G129DprE1 in the presence of BTZ043-modified His-G129DprE1
therefore cannot be interpreted as evidence for a complete labeling of
His-G129DprE1. We furthermore incubated M. smegmatis coexpressing
His-G129DprE1 and DprE2 with four BTZ derivatives with masses
different than BTZ043, and in each case a protein with the mass of
the adduct between His-G129DprE1 and the corresponding nitroso
derivative was detected (Table S4 and Figures S3-S6). In comparison,
the copurified GroEL1, which does not possess a cysteine residue,
was not modified by any of the BTZ derivatives and was detected in
the presence and in the absence of thiols (Figure 3b). To demonstrate
that the observed labeling depends on Cys387, we mutated Cys387 in
His-G129DprE1 to glycine, yielding His-G129,G387DprE1. Coexpression
of His-G129,G387DprE1 with DprE2 in M. smegmatis results in high-
level resistance to BTZ043 (Table 1), indicating that also His-
G129,G387DprE1 from M. tuberculosis is functional. Analysis of His-
G129,G387DprE1 by mass spectrometry after incubation of M. smegmatis
with BTZ043 as described above allowed only the detection of
unmodified His-G129,G387DprE1 (Figure 3b). Together, these experiments
support our hypothesis of the formation of a semimercaptal between
Cys387 and a BTZ nitroso derivative. Furthermore, they reveal a
remarkable efficiency of this reaction inside the bacterium.
Figure 2. Nitrosoarenes and thiols. (a) Reaction of nitrosoarenes (Ar-NO)
with thiols (R-SH). The semimercaptal can react with another thiol to form
the hydroxylamine or rearrange to the sulfinamide. (b) Proposed mechanism
of action of BTZs.
To independently confirm the observed covalent modification
of DprE1 by BTZs, we synthesized a 14C-labeled BTZ derivative
(BTZ003; Figure 3c and Supporting Information). M. smegmatis
strains coexpressing His-G129DprE1 and DprE2 were incubated with
radioactive BTZ003, and His-G129DprE1 was purified as described
above. The degree of labeling of His-G129DprE1 with BTZ003 after
purification was determined to be 25% (Figure 3c and Supporting
Information). When these experiments were repeated with His-
G129,G387DprE1, the degree of labeling of His-G129,G387DprE1 was 20-
fold lower than that of His-G129DprE1 (Figure 3c).
To investigate if the nitroso derivative of BTZ043 specifically reacts
with Cys387 of DprE1 in cells, we envisioned incubating M. smegmatis
coexpressing DprE1 and DprE2 from M. tuberculosis with BTZ043
and isolating the protein for further analysis. Overexpression of DprE1
and DprE2 from M. tuberculosis in M. smegmatis mc2155 results in a
decreased sensitivity relative to wild-type M. smegmatis mc2155.1 The
decreased sensitivity is due to an increased concentration of the drug
target, represented by overexpressed DprE1 and DprE2 from M.
tuberculosis and the natively present orthologous DprE1 and DprE2
from M. smegmatis.1 As our attempts to purify native DprE1 failed,
we expressed DprE1 with an N-terminal His-tag (His-DprE1) and
DprE1 belongs to the family of vanillyl alcohol oxidases (VAO),
a family of flavin adenine dinucleotide (FAD)-dependent oxi-
doreductases consisting of an FAD-binding domain and a substrate-
binding domain.13
9
13664 J. AM. CHEM. SOC. VOL. 132, NO. 39, 2010

C O M M U N I C A T I O N S
Figure 3. Covalent binding of BTZs to DprE1. (a) SDS-PAGE of isolated His-G129DprE1. M. smegmatis strains coexpressing His-G129DprE1 and DprE2
were incubated for 4 h with BTZ043 (0.5 µg/mL), lysed in buffer containing 8 M urea, and His-G129DprE1 was purified by Ni-NTA affinity chromatography.
The copurification of GroEL1 is due to a string of histidine residues naturally occurring at its C terminus. Proteins were stained with Coomassie blue. (b)
Mass spectrometric analysis of different DprE1 samples: (i) sample as described in (a); (ii) sample as described in (a) but analyzed after incubation with 10
mM dithiothreitol (DTT); (iii) sample as described in (a) but without BTZ043 incubation; (iv) sample as described in (a) but using M. smegmatis expressing
His-G129,G387DprE1. Samples were analyzed on a Q-TOF Ultima spectrometer (Waters). Calculated masses: His-G129DprE1, 51 371.4 Da; His-G129DprE1-
BTZ043 semimercaptal, 51 786.6 Da; His-G129,G387DprE1, 51 325.4; GroEL1, 56 021.3 Da. (c) Quantification of radioactive labeling of His-G129DprE1 and
His-G129,G387DprE1 after incubation of M. smegmatis expressing either His-G129DprE1 or His-G129,G387DprE1 with 14C-labeled BTZ003 and purification of the
protein as described in (a).
Among the enzymes of this family with known structure, alditol
oxidase possesses the highest sequence identity to DprE1 (18%).14
To gain further insight into the interaction of BTZs with DprE1,
we built a structural model of DprE1 by homology modeling
(Supporting Information). In our model, Cys387 points into the
substrate binding pocket of DprE1 (Figure S2). Thus, modification
of Cys387 should block the substrate binding site of the enzyme.
The mechanism of action of BTZs proposed also raises the question
of the activation of the prodrug inside the mycobacterium. One
possibility would be that the nitroso derivative is generated through
reduction of BTZ by an unknown enzyme and then reacts with
DprE1. Alternatively, DprE1 itself might reduce BTZ to the nitroso
derivative, which then directly undergoes a reaction with Cys387.
This hypothesis is supported by the observation that bacterial
oxygen-insensitive nitroreductases are also flavin-dependent en-
zymes.15 More experiments are needed to characterize the activation
and to explain the remarkable specificity of BTZs, but it is
noteworthy that another exciting TB drug candidate, the bicyclic
nitroimidazole PA-824,16,17 is also a prodrug that is activated by
reduction of an essential nitro group. In the case of PA-824, the
mechanism of action is based on the resulting intracellular release
of nitric oxide,17 and DprE1 is not a target (our unpublished data).
In summary, BTZs and other electron-deficient nitroaromatic
compounds form a new family of antimycobacterial prodrugs
defined by a unique mechanism of action. The mechanistic insights
presented here represent an important step in the further develop-
ment of BTZs as TB drug candidates.
sharing materials. The NM4TB Consortium is funded by the
European Commission (LHSP-CT-2005-018923).
Supporting Information Available: Detailed description of experi-
ments, additional analytical data, and complete refs 1 and 4. This
material is available free of charge via the Internet at http://pubs.acs.org.
References
(1) Makarov, V.; et al. Science 2009, 324, 801.
(2) Mikusova, K.; Huang, H.; Yagi, T.; Holsters, M.; Vereecke, D.; D’Haeze,
W.; Scherman, M. S.; Brennan, P. J.; McNeil, M. R.; Crick, D. C. J.
Bacteriol. 2005, 187, 8020.
(3) Wolucka, B. A. FEBS J. 2008, 275, 2691.
(4) Christophe, T.; et al. PLoS Pathog. 2009, 5, e1000645.
(5) Schaper, K.-J.; Pickert, M.; Frahm, A. W. Arch. Pharm. (Weinheim, Ger.)
1999, 332, 91.
(6) Kappes, B.; Neumann, T.; Unger, E.; Dahse, H.-M.; Mollmann, U.; Schlegel,
B. Patent DE-10158057, 2003.
(7) Spain, J. C. Annu. ReV. Microbiol. 1995, 49, 523.
(8) Eyer, P. Chem.-Biol. Interact. 1979, 24, 227.
(9) Kazanis, S.; Mcclelland, R. A. J. Am. Chem. Soc. 1992, 114, 3052.
(10) Ellis, M. K.; Hill, S.; Foster, P. M. D. Chem.-Biol. Interact. 1992, 82, 151.
(11) Reeve, I. T.; Voss, J. C.; Miller, M. G. Chem. Res. Toxicol. 2002, 15, 361.
(12) Callan, H. E.; Jenkins, R. E.; Maggs, J. L.; Lavergne, S. N.; Clarke, S. E.;
Naisbitt, D. J.; Park, B. K. Chem. Res. Toxicol. 2009, 22, 937.
(13) Leferink, N. G.; Heuts, D. P.; Fraaije, M. W.; van Berkel, W. J. Arch.
Biochem. Biophys. 2008, 474, 292.
(14) Forneris, F.; Heuts, D. P.; Delvecchio, M.; Rovida, S.; Fraaije, M. W.;
Mattevi, A. Biochemistry 2008, 47, 978.
(15) Roldan, M. D.; Perez-Reinado, E.; Castillo, F.; Moreno-Vivian, C. FEMS
Microbiol. ReV. 2008, 32, 474.
(16) Stover, C. K.; Warrener, P.; VanDevanter, D. R.; Sherman, D. R.; Arain,
T. M.; Langhorne, M. H.; Anderson, S. W.; Towell, J. A.; Yuan, Y.;
McMurray, D. N.; Kreiswirth, B. N.; Barry, C. E.; Baker, W. R. Nature
2000, 405, 962.
(17) Singh, R.; Manjunatha, U.; Boshoff, H. I.; Ha, Y. H.; Niyomrattanakit, P.;
Ledwidge, R.; Dowd, C. S.; Lee, I. Y.; Kim, P.; Zhang, L.; Kang, S.; Keller,
T. H.; Jiricek, J.; Barry, C. E. Science 2008, 322, 1392.
Acknowledgment. The authors thank Laure Menin, Katarina
Mikusova, Giovanna Riccardi, August Wilhelm Frahm, Sandeep
Kumar Sharma, and Simone Schmitt for technical assistance and
JA106357W
9
J. AM. CHEM. SOC. VOL. 132, NO. 39, 2010 13665