Insight into the mode of action of clofazimine and combination therapy with. benzothiazinones against Mycobacterium tuberculosis

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1 AAC Accepted Manuscript Posted Online 18 May 2015 Antimicrob. Agents Chemother. doi: /aac Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 2 Insight into the mode of action of clofazimine and combination therapy with benzothiazinones against Mycobacterium tuberculosis Benoit Lechartier 1,2 and Stewart T. Cole 1* 1 Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland 2 Present address: Department of Medicine, Lausanne University Hospital, CH-1011 Lausanne, Switzerland 9 10 Running title: a new TB drug combination Keywords: clofazimine, menaquinone, combination therapy, tuberculosis, benzothiazinones, PBTZ169, synergy * Corresponding author: Prof. S.T. Cole, Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Station 19, 1015 Lausanne, Switzerland Tel Fax stewart.cole@epfl.ch 23

2 ABSTRACT Clofazimine (CFM) is an anti-leprosy drug that was recently repurposed for treatment of multidrug-resistant tuberculosis. In Mycobacterium tuberculosis, CFM appears to act as a prodrug, which is reduced by NADH dehydrogenase (NDH-2), to release reactive oxygen species upon re-oxidation by O 2. CFM presumably competes with menaquinone (MK-4), a key cofactor in the mycobacterial electron transfer chain, for its reduction by NDH-2. We studied the effect of MK-4 supplementation on the activity of CFM against M. tuberculosis and found direct competition between CFM and MK-4 for the cidal effect of CFM, both against non-replicating and actively growing bacteria, as MK-4 supplementation blocked the drug s activity against nonreplicating bacteria. We demonstrated that CFM, like bedaquiline, is synergistic in vitro with benzothiazinones such as PBTZ169, and this synergy also occurs against non-replicating bacteria. The synergy between CFM and PBTZ169 was lost in a MK-4-rich medium, indicating that MK-4 is the probable link between their activities. The efficacy of the dual combination CFM and PBTZ169 was tested in vivo where a great reduction in bacterial load was obtained in a murine model of chronic tuberculosis. Taken together, these data confirm the potential of CFM, in association with PBTZ169, as the basis for a new regimen against drug-resistant strains of M. tuberculosis

3 INTRODUCTION With approximately 9 million incident cases of tuberculosis (TB) worldwide and around 1.5 million deaths in 2012, Mycobacterium tuberculosis infection is one of the most important causes of death from a single infectious agent (1). The spread of multidrug-resistant TB (MDR-TB), namely resistance to both isoniazid and rifampin, poses additional challenges to treatment with currently available anti-tb drugs. The situation is exacerbated by the increasing emergence of extensively drug-resistant (XDR) strains of M. tuberculosis, which cause diseases essentially untreatable with existing compounds. It is nowadays widely acknowledged that we need to develop new antibiotic combinations for TB and that these new regimens should be tested together at the preclinical stage, rather than testing a series of single drugs separately, in order to fill the TB drug development pipeline more efficiently (2-4). Some of the compounds in advanced clinical trials for TB are molecules that were originally used to treat other infectious diseases and have been repurposed for TB. Among the repurposed molecules, clofazimine (CFM), a riminophenazine originally developed as a drug to treat TB but overlooked for decades, has been used as a standard component of the treatment of leprosy for fifty years. It was recently repurposed for managing MDR-TB cases, notably following the results of the so-called Bangladesh study, which demonstrated that a CFMcontaining regimen could cure such resistant cases in 9 to 12 months (5). Grosset et al. demonstrated substantial benefit of adding CFM to second-line regimens in mice infected with isoniazid-resistant strains of M. tuberculosis (6). Furthermore, CFM was recently demonstrated to reduce the duration of TB in the mouse model of TB (7). However, CFM use is hampered by its common side-effects, in particular skin discoloration, caused by its long half-life and extremely high lipophilicity (8). Through medicinal chemistry, new CFM-analogues have been synthesized and these demonstrated equivalent or better efficacy than CFM in a murine model of 3

4 TB with reduced lipophilicity thus reducing expected side effects (9). Although the exact mechanism of action of CFM is not entirely understood, it was elegantly demonstrated in Mycobacterium smegmatis that CFM is a pro-drug, which is reduced by Type 2 NADH:quinone oxidoreductase (NDH-2) and releases reactive oxygen species (ROS) upon spontaneous reoxidation by O 2 (10). CFM is believed to compete with menaquinone (MK-4, vitamin K 2 ), the sole quinone present in mycobacteria and a key electron acceptor, for its reduction by NDH-2. MK-4, also known as menatetrenone (C 31 H 40 O 2, molecular weight: ), consists of a quinone ring linked to a chain of four isoprenoid groups. Benzothiazinones (BTZ) are an extremely potent class of novel antimycobacterials that act by blocking the synthesis of decaprenyl-phospho-arabinose, the precursor of the arabinans in the mycobacterial cell wall (11). The lead compound BTZ043 was demonstrated to be fully compatible with all the other approved or experimental TB drugs tested (12). Interestingly, both BTZ043 and the 2-piperazino-benzothiazinone PBTZ169, the preclinical drug candidate, were shown to act synergistically in vitro with bedaquiline (BDQ), an ATP synthase inhibitor (13). Compared to BTZ043, PBTZ169 has improved potency, safety and efficacy in zebrafish and mouse models of TB and highly encouraging results were obtained against chronic murine TB when PBTZ169 was administered in combination with BDQ and/or PZA (13). In the present study, we first evaluated the activity of CFM against actively growing and non-replicating bacteria in vitro, as well as the effect of MK-4 supplementation on CFM activity. The mode of action of CFM is related to that of BDQ, since the electron transfer chain is coupled to ATP synthesis to produce energy at the plasma membrane level. We therefore tested the interaction profile between CFM and BTZ in vitro before evaluating the efficacy of a PBTZ169- CFM combination in a murine model of chronic TB. 91 4

5 MATERIALS AND METHODS Bacterial strains and culture conditions. M. tuberculosis strains H37Rv, and 18b were grown at 37 C with shaking in 7H9 broth (Difco) supplemented with Middlebrook albumin-dextrosecatalase (ADC) enrichment, 0.2% glycerol, 0.05% Tween 80, and, in the case of 18b, 50 µg/ml STR; or on solid Middlebrook 7H10 medium (Difco) supplemented with 0.5% glycerol, Middlebrook oleic acid-albumin-dextrose-catalase (OADC), and, in the case of 18b, 50 µg/ml STR. Non-replicating streptomycin-starved 18b cultures (SS18b) were generated as previously described (14). Drugs and chemicals. CFM, isoniazid (INH) and menaquinone (MK-4) were purchased from Sigma-Aldrich. Experimental drugs were provided by K. Andries (BDQ) and V. Makarov (BTZ043, PBTZ169). All the drugs and MK-4 were dissolved in dimethyl sulfoxide, except INH which was dissolved in water. Drugs for the in vivo experiment were prepared as follows: PBTZ in 0.5% carboxymethyl cellulose (CMC) at ph 3.0, as acidification increases PBTZ solubility (13), after grinding in a mortar; CFM in acidified CMC with 0.4 % Tween 80. We used a standardized ph for drug preparations within the same experiment to reduce the risk of variation. Drug solutions were prepared weekly and stored at 4 C. Drug activity measurement by REMA. Compound activity and effect of MK-4 supplementation were evaluated by the resazurin reduction microplate assay (REMA) as previously described in 7H9 medium (12). When applicable, growth medium was supplemented with MK-4 at the concentrations indicated. Briefly, bacterial stocks of M. tuberculosis H37Rv were generated from mid-log cultures and frozen at -80 C to standardize the inoculum. Frozen aliquots of tubercle bacilli were thawed and diluted to an OD 600 of and added to the plates containing drug dilutions to obtain a total volume of 300 µl (48-well plate) or 100 µl (96-well plate). Plates were incubated for 6 days at 37 C before addition of resazurin (0.025% w/v to 1/10 5

6 of well volume). After overnight incubation, fluorescence of the resazurin metabolite resorufin was determined (excitation at 560 nm and emission at 590 nm, measured by using a TECAN infinite M200 microplate reader). The minimal inhibitory concentration (MIC) was defined as the lowest concentration preventing resazurin turnover from blue to pink and confirmed by the level of fluorescence measured by the microplate reader. M. tuberculosis SS18b cultures were prepared as previously described and diluted to an OD 600 of 0.1. The effects of MK-4 supplementation on the REMA dose-response curves were investigated using medium that was supplemented with 0, 10, 100, and 1000 µm MK-4. Evaluation of compounds alone or in combination, against replicating M. tuberculosis H37Rv or non-replicating SS18b using colony forming unit (CFU) assays. As described above, bacteria were incubated in 7H9 liquid medium for 7 days in the presence of combinations of compounds at their respective MICs, or fractions thereof for H37Rv (concentrations used in individual REMA assays), and inhibitory or sub-inhibitory concentrations for SS18b. Dilutions were plated on solid medium (supplemented 7H10, with STR for 18b) and CFU counts determined after 3 to 4 weeks of incubation at 37 C. In vivo evaluation of the PBTZ-CFM combination in a murine model of chronic TB. Female BALB/c mice, aged 5 to 6 weeks, were obtained from Charles River Laboratories (Lyon, France). The in vivo antimicrobial activity of the two drugs alone and in combination was assessed in the chronic model of TB, by gavage 6 days a week for 4 weeks. Infection was established using a low-dose aerosol (~200 CFU) generated by a custom-built aerosol exposure chamber (Mechanical Engineering Shops, University of Wisconsin, Madison). Experiments were approved by the Swiss Cantonal Veterinary Authority (authorization no. 2658). Drug treatment began 4 weeks after infection at the dose of 25 mg/kg for PBTZ169 and 20 mg/kg for CFM. Control and treated mice were sacrificed, the lungs and spleens homogenized, and dilutions 6

7 plated on 7H10 agar enriched with 10% OADC and supplemented with cycloheximide (10 µg/ml), ampicillin (85 µg/ml) and 0.4 % w/v activated charcoal (Sigma) to prevent compound carry-over (15). Statistical analysis. CFU counts were log 10 transformed before analysis as mean log 10 CFU ± s.d. and compared using Student s t-test in Prism version 5.0 (Graphpad). Statistical significance was expressed as follows: * P< 0.05, ** P< 0.005, *** P< RESULTS Effect of menaquinone supplementation on CFM activity against replicating and nonreplicating bacteria. We measured the activity of CFM, BDQ and INH against M. tuberculosis H37Rv, by REMA fluorescence (Figure 1), in the presence or absence of MK-4. CFM, BDQ and INH had an MIC in normal 7H9 medium of ~ 0.5, 0.1 and 0.2 µg/ml, respectively. The effect of MK-4 varied considerably in the three assays. For INH, a negative control, we detected no change in activity at the three MK-4 concentrations tested, but did note a slight increase in absolute levels of fluorescence. CFM and BDQ activities were significantly reduced upon MK-4 addition. The MICs for both drugs increased by around 10-fold in this assay in the medium containing the highest concentration of MK-4 (1000 µm). BDQ activity was not affected by 10 µm MK-4, but its MIC increased at the higher concentrations used. We noticed an inverse correlation between CFM activity and MK-4 levels, at the three concentrations tested (10, 100 and 1000 µm). Assuming that the levels of cofactors like MK-4 are critical to maintain oxidative metabolism in the non-replicating state, we repeated the previous assay with CFM using the SS18b model. Here again, CFM activity on non-replicating bacteria was impacted by MK-4 supplementation, as noted in the REMA assay (Figure 2A), where the effect of 1 µg/ml CFM in 7

8 plain medium was suppressed by a high concentration of MK-4. To confirm this result, we analyzed the effect of MK-4 supplementation using the number of SS18b CFU as a growth readout (Figure 2B). The activity of CFM against non-replicating cells was partially neutralized by MK-4. CFM at 1 µg/ml had essentially no activity against SS18b in an MK-4-rich medium and even at 10 µg/ml its activity was greatly reduced when MK-4 was present. The same trend was observed with actively growing H37Rv cells, as assessed by the CFU assay (Figure 2C), where a difference of 3.6 log units in CFU counts was seen after exposure to CFM at 5 µg/ml, in the absence or presence of 1000 µm MK-4. There were no significant differences between the untreated controls, with or without MK-4. Synergistic interaction between CFM and BTZ. Since CFM and BDQ both interfere with cellular respiration, and their activity is affected by MK-4, we hypothesized that there might also be a synergistic interaction between CFM and BTZ as has previously been found between BTZ and BDQ. To test this possibility we evaluated the interaction between BTZs and CFM against M. tuberculosis H37Rv by CFU determination (Figure 3A & B). We used compounds at their individual MIC in REMA: 1.5, 0.4 and ng/ml for BTZ043, PBTZ169 and CFM, respectively, or fractions thereof. The data in Figure 3A show that the combination of 0.4 ng/ml of BTZ043 (quarter MIC) and 37.5 ng/ml CFM (around quarter MIC), which both have no impact on bacterial growth alone, show clear bacteriostatic activity in combination (mean CFU count equivalent to day 0). The same behavior was noticed between PBTZ169 and CFM, demonstrating that this was a compound class effect. The activity of 31.3 ng/ml of CFM (around quarter MIC) was significantly enhanced when combined with 0.1 ng/ml PBTZ169 (quarter MIC), which had no impact on its own, thus confirming the in vitro synergy. Synergistic interaction between BTZ and BDQ or CFM against non-replicating bacteria. PBTZ169, like other cell wall inhibitors, is poorly active against non-replicating 8

9 bacteria (14). We evaluated by the CFU assay the effect of the two synergistic combinations, PBTZ-BDQ (13) and PBTZ-CFM (Figure 3), against actively growing M. tuberculosis H37Rv and SS18b in vitro (Figure 4). The two PBTZ169 concentrations used, 250 and 62.5 ng/ml, which both had no impact on bacterial activity on their own, significantly improved the effect of BDQ or CFM, both at 250 ng/ml. These synergistic interactions against dormant bacilli were reproducible and results were consistent between duplicates, as well as when BTZ043 was used instead of PBTZ169 (data not shown). Effect of menaquinone supplementation on the synergy between CFM and PBTZ169. We assessed by CFU counts the effect of MK-4 supplementation on the synergy between PBTZ169 and CFM (Figure 5). In a MK-4 rich medium (1000 µm), the cidal effects of PBTZ169 (0.8 ng/ml MIC in MK-4 rich medium, Suppl. Figure 1) or CFM (1000 ng/ml) alone were not significantly improved when the drugs were combined. The synergy between CFM and PBTZ169 is therefore lost when the growth medium is saturated with MK-4. Combination study of PBTZ169 with CFM, in the mouse model of chronic TB. We assessed the synergistic combination discovered in vitro in the murine model of chronic TB after low-dose aerosol infection (Figure 6). PBTZ169 (25 mg/kg) and CFM (20 mg/kg) were tested alone and the two drugs together against M. tuberculosis H37Rv. PBTZ reduced the bacterial burden in the lung and spleen by 1.8 ± 0.2 and 2.4 ± 0.2 log units, respectively, compared to the bacterial load before treatment (D0). CFM activity was even more pronounced, with a log unit reduction of 4.0 ± 0.3 for the lung and 2.7 ± 0.2 for the spleen CFU counts. The dual therapy of PBTZ and CFM in combination displayed promising killing since it reduced the number of bacilli in the lungs and in the spleen by 4.6 ± 0.2 and 4.2 ± 0.2 log units, respectively

10 DISCUSSION Efficacious TB treatment exists, but this requires strict implementation strategies, universal access to drugs, and careful compliance over a prolonged treatment period, particularly for drugresistant cases. We need innovative combination regimens comprising new molecules able to kill drug-susceptible as well as drug-resistant strains of M. tuberculosis, while simultaneously decreasing treatment duration, by targeting both active and persistent tubercle bacilli (16). The mechanism of action of CFM is not entirely clear. It was presumed to compete with menaquinone, the sole quinone cofactor in mycobacteria, for electrons carried by the FAD moiety of reduced NDH-2, although such competition has not yet been demonstrated to our knowledge (10). If the mechanism of action of CFM remains obscure, a new mechanism of resistance was recently identified. CFM and BDQ share cross-resistance due to overexpression of the MmpL5 efflux system, which presumably reduces intracellular concentrations of both drugs (17, 18). MK-4 biosynthesis is essential for mycobacterial growth and selective inhibitors of the menaquinone biosynthetic enzyme, MenA (1,4-dihydroxy-2-naphthoate octaprenyltransferase), kill non-replicating M. tuberculosis and act as indirect inhibitors of ATP synthesis (19). An F dependent mechanism against oxidative stress could also be partly suppressed by menadione addition in M. tuberculosis (20). Isoniazid, moxifloxacin and CFM were shown to elevate oxidative stress and F 420 -deficient mutants were hypersensitive to these molecules. In our work, we show that INH activity, as measured by REMA, is not affected by MK-4 supplementation, unlike CFM (Figure 1). These microbiological findings suggest that the proton gradient and subsequent ATP synthesis may be influenced by MK-4 levels thus validating the notion that MK- 4 can suppress CFM activity in a dose-dependent manner, confirming Yano s hypothesis (10). Bacterial inhibition by CFM could be rescued by increasing MK-4 levels. CFM, and to some extent BDQ, thus behave similarly to Ro , a lipophilic amine inhibitor of MenA, whose 10

11 activity was partly suppressed by 400 µm MK-4 (21). However, the antagonistic effect of MK-4 against CFM activity may not be of clinical relevance as the concentrations used in the present work exceed the physiological levels in humans (22). For an optimal regimen, benzothiazinones should be combined with chemotherapeutic agents able to target latent bacilli, such as BDQ, which inhibits the ATP synthase required to maintain mycobacterial viability during dormancy (23). In addition, the residual metabolism of dormant M. tuberculosis presumably requires some de novo RNA and protein synthesis for survival during this phase or for reactivation (24). Given the sensitivity of non-replicating bacteria to inhibitors of these processes (14, 25), combining PBTZ169 and other drugs active against nonreplicating bacilli, such as BDQ, CFM, oxazolidinones or pyrazinamide, offers an attractive foundation for a new TB regimen. In the present study, the combination of PBTZ169 plus CFM was extremely potent in a murine model of chronic TB and similar results are to be expected with the newer riminophenazines. These highly encouraging results are likely to be reproduced when combining PBTZ169 with other compounds affecting the electron transfer chain, such as the newly identified imidazopyridine amide Q203 (26). The possibility of combining menaquinone synthesis inhibitors with DprE1 inhibitors for putative synergy has already been raised (27). The synergy between BTZ and BDQ was initially explained through weakening of the cell wall by DprE1 inhibition leading to better penetration of the ATP synthase inhibitor and improved access to its target (12, 13). Indeed, in support of this explanation, in vitro synergy was also reported between BDQ and the ethylenediamine SQ109 (28), whose primary target is the transmembrane protein MmpL3 required for trehalose monomycolate transport, loss of which weakens the cell wall (29). This explanation for synergy appeared less likely when another mechanism of action of SQ109 was revealed, namely, inhibition of menaquinone biosynthesis (30). 11

12 Regarding the newly identified synergy between BTZ and CFM, this could also arise through some cell wall damage caused by sub-inhibitory concentrations of DprE1 inhibitors and improved penetration of CFM even in the SS18b latency model where a synergistic interaction between PBTZ and BDQ or CFM was detected. This may indicate that, although non-replicating, dormant cells require some cell wall maintenance and retain limited sensitivity to cell wall inhibitors. For instance, INH monotherapy is known to prevent reactivation in latent TB infection, where M. tuberculosis is thought to persist in a dormant state (31, 32). Another explanation for the in vitro synergy between BTZ and compounds affecting membrane potential stems from the enzymatic activity of DprE1 itself (33). DprE1 catalyzes the first step in the conversion of decaprenylphosphoryl-ribofuranose to decaprenylphosphorylarabinose, the sole precursor of the arabinan of the mycobacterial cell wall (34). This FADdependent process requires an electron acceptor for enzyme turnover and re-oxidation of FADH 2 by several electron acceptors was demonstrated in vitro, in particular by MK-4 (33). Therefore, loss of DprE1 activity, due to binding of BTZ, might result in fewer reducing equivalents from FADH 2 entering the electron transfer chain and thus enhance the effects of CFM and BDQ. Alternatively, by disrupting the proton gradient, CFM or BDQ could indirectly prevent reoxidation of FADH 2 in DprE1 hence improving its enzymatic inhibition by BTZ. Of note, the MIC of BTZ increased 4-fold in MK-4-rich medium (Suppl. Figure 1) where the synergy between CFM and PBTZ169 was also lost (Figure 5). Finally, whatever the explanation, the promising activity of the PBTZ169 and CFM combination is highly encouraging for the design of innovative TB regimens for humans, especially against drug-resistant strains

13 ACKNOWLEDGMENTS We thank K. Andries and V. Makarov for providing drugs, A. Vocat for technical assistance, R. Hartkoorn and J. Neres for helpful discussions. B. Lechartier was the recipient of a grant from the Fondation Jacqueline Beytout. The research leading to these results has received funding from the European Communityʼs Seventh Framework Programme (FP7/ ) under grant agreement n REFERENCES 1. WHO Global Tuberculosis Report , World Health Organization, Geneva Ginsberg A The TB Alliance: overcoming challenges to chart the future course of TB drug development. Future medicinal chemistry 3: Diacon AH, Dawson R, Groote-Bidlingmaier von F, Symons G, Venter A, Donald PR, van Niekerk C, Everitt D, Winter H, Becker P, Mendel CM, Spigelman MK day bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin combinations: a randomised trial. Lancet 380: Zumla A, Nahid P, Cole ST Advances in the development of new tuberculosis drugs and treatment regimens. Nature Reviews Drug Discovery 12: Maug AKJ, Salim MAH, van Deun V, van Deun A Short, Highly Effective, and Inexpensive Standardized Treatment of Multidrug-resistant Tuberculosis. American Journal of Respiratory and Critical Care Medicine 182: Grosset J, Tyagi S, Almeida D, Converse P, Li S, Ammerman N, Bishai W, Enarson D, Trébucq A Assessment of clofazimine activity in a second-line regimen for tuberculosis in mice. American Journal of Respiratory and Critical Care Medicine 188:

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17 Lee J, Brodin P, Cho S, Nam K, Kim J Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nature Medicine 19: Cook GM, Heikal A Bridging the gap between a TB drug and its target. Science Translational Medicine 4:150fs Reddy VM, Einck L, Andries K, Nacy CA In vitro interactions between new antitubercular drug candidates SQ109 and TMC207. Antimicrobial Agents and Chemotherapy 54: Tahlan K, Wilson R, Kastrinsky DB, Arora K, Nair V, Fischer E, Barnes SW, Walker JR, Alland D, 3rd CE, Boshoff HI 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: Li K, Schurig-Briccio LA, Feng X, Upadhyay A, Pujari V, Lechartier B, Fontes FL, Yang H, Rao G, Zhu W, Gulati A, No JH, Cintra G, Bogue S, Liu Y, Molohon K, Orlean P, Mitchell DA, Freitas-Junior L, Ren F, Sun H, Jiang T, Li Y, Guo R, Cole ST, Gennis RB, Crick DC, Oldfield E Multitarget drug discovery for tuberculosis and other infectious diseases. Journal of Medicinal Chemistry 57: Cardona P, Ruiz-Manzano J On the nature of Mycobacterium tuberculosis-latent bacilli. European Respiratory Journal 24: Mack U, Migliori GB, Sester M, Rieder HL, Ehlers S, Goletti D, Bossink A, Magdorf K, Holscher C, Kampmann B, Arend SM, Detjen A, Bothamley G, Zellweger JP, Milburn H, Diel R, Ravn P, Cobelens F, Cardona PJ, Kan B, Solovic I, Duarte R, Cirillo DM LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement. European Respiratory Journal 33:

18 Neres J, Pojer F, Molteni E, Chiarelli LR, Dhar N, Boy-Rottger S, Buroni S, Fullam E, Degiacomi G, Lucarelli AP, Read RJ, Zanoni G, Edmondson DE, de Rossi E, Pasca MR, McKinney JD, Dyson PJ, Riccardi G, Mattevi A, Cole ST, Binda C Structural Basis for Benzothiazinone- Mediated Killing of Mycobacterium tuberculosis. Science Translational Medicine 4:150ra ra Wolucka BA Biosynthesis of D-arabinose in mycobacteria - a novel bacterial pathway with implications for antimycobacterial therapy. FEBS Journal 275: FIGURE LEGENDS FIG 1. Effect of menaquinone supplementation on CFM, BDQ and isoniazid (INH) against M. tuberculosis H37Rv, by REMA. Cells were grown in normal 7H9 medium (no MK-4) or with medium supplemented with increasing concentrations of menaquinone (MK-4 10, 100 and 1000 µm). REMA results are presented as mean ± s.d. values of triplicates. Drug concentrations are in µg/ml FIG 2. Effect of menaquinone supplementation on CFM activity against non-replicating M. tuberculosis 18b cells. (A,B) and actively growing M. tuberculosis H37Rv (C). A: Cell activity assessed by REMA, with increasing concentrations of menaquinone. Results are average values of duplicates. B: 18b cells were plated after 7 days of drug exposure with (black) or without (light gray) MK-4 [1000 µm] for CFU count assessment. C: as in B, but with actively growing M. tuberculosis H37Rv cells. CFM concentrations are in µg/ml. #: CFU count below the limit of detection in this assay (< 200 CFU), ns: no statistical significance, NT: non-treated control

19 FIG 3. Synergistic interaction in vitro between CFM and two benzothiazinones, BTZ043 (A) and PBTZ169 (B). We tested BTZ043, PBTZ169 and CFM effects, alone or in combination (BTZ043 & CFM in A; PBTZ169 & CFM in B), on the viability of M. tuberculosis H37Rv. Following 7 days of incubation, bacteria were plated to determine CFU counts. The non-treated bacteria (NT) were also plated on day 0 and on day FIG 4. In vitro interactions between PBTZ169 and BDQ (A) and between PBTZ169 and CFM (B), against non-replicating M. tuberculosis 18b. Streptomycin-starved 18b cells were exposed to selected dilutions of the drugs for 7 days. The non-treated bacteria (NT) were also plated on day 0 and on day 7. Drug concentrations are in ng/ml FIG 5. In vitro interactions between PBTZ169 and CFM, against M. tuberculosis H37Rv in a menaquinone rich medium. The effect of selected concentrations of PBTZ169 and CFM, alone and in combination on the viability of M. tuberculosis H37Rv was assessed after 7 days exposure in a medium containing 1000 µm of menaquinone. Drug concentrations are in ng/ml. The non-treated bacteria (NT) were also plated on day 0 and on day FIG 6. In vivo efficacy study of PBTZ169 and CFM, alone and in combination, in a mouse model of chronic TB, compared with untreated controls (NT). PBTZ169 and CFM were administered 6/7 days at 25 and 20 mg/kg of body weight per day, respectively. Red and black columns correspond to the bacterial burden in the lungs and spleens, respectively, at day 0 (DO) when treatment initiated, or day 28 (D28) when treatment finished. Bars represent the mean ± s.d. of CFUs from 5 mice per group

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