WO2018084809A1

WO2018084809A1 - Methods for the treatment or prevention of mycobacterial infections

Info

Publication number: WO2018084809A1
Application number: PCT/SG2017/050553
Authority: WO
Inventors: Kevin Pethe; Gerd Pluschke; Nicole SCHERR; Raphael BIERI
Original assignee: Nanyang Technological University; Schweizerisches Tropen- Und Public Health-Institut; Universität Basel Vizerektorat Forschung
Priority date: 2016-11-02
Filing date: 2017-11-02
Publication date: 2018-05-11

Abstract

The present invention relates to a method of treating or preventing an infection of a mycobacterium deficient for cytochrome bd oxidase (e.g. M. leprae or M. ulcerans) or a disease resulting from said infection, using a compound capable of inhibiting cytochrome bc1 of the respiratory electron transport chain. Also disclosed is a method of treating or preventing an infection of a mycobacterium expressing cytochrome bd oxidase (e.g. M. tuberculosis) or a disease resulting from said infection, using a compound capable of inhibiting cytochrome bc1 of the respiratory electron transport chain in combination with a therapeutic agent capable of inhibiting cytochrome bd oxidase.

Description

METHODS FOR THE TREATMENT OR PREVENTION OF MYCOBACTERIAL INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATION

This application makes reference to and claims the benefit of priority of the Singapore Patent Application No. 102016091 93V filed on 02 November 2016, the content of which is incorporated herein by reference for all purposes, including an incorporation of any element or part of the description, claims or drawings not contained herein and referred to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.

FIELD OF THE INVENTION

The present invention relates generally to compounds and methods for the treatment or prevention of mycobacterial infections, in particular infection of M. tuberculosis, M. leprae, or M. ulcerans.

BACKGROUND OF THE INVENTION

Mycobacterial infections can cause different diseases such as tuberculosis, leprosy, and Buruli ulcer. Additionally, mycobacterial diseases can cause overwhelming, disseminated disease in immunocompromised patients. Despite the tremendous efforts thus far, the eradication of mycobacterial diseases has never been achieved, nor is eradication imminent. Therefore, there remains a considerable need for new technologies for the treatment of mycobacterial infections.

SUMMARY OF THE INVENTION

The present invention satisfies the afore-mentioned need in the art by providing novel compounds and methods of treating mycobacterial infections and diseases resulting therefrom.

In a first aspect, the invention relates to a method of treating or preventing an infection of a mycobacterium deficient for cytochrome bd oxidase or a disease resulting from said infection in a subject, the method comprising administering to the subject an effective amount of a compound capable of inhibiting cytochrome bd of the respiratory electron transport chain in said mycobacterium.

In various embodiments, the subject is a mammal, preferably a human. In various embodiments, the mycobacterium is selected from the group consisting of M. ulcerans, M. leprae, M. lepraemurium, and M. lepromatosis. In preferred embodiments, the mycobacterium is M. leprae and the disease is leprosy.

In preferred embodiments, the mycobacterium is M. ulcerans and the disease is Buruli ulcer.

In various embodiments, the compound is of formula (I) or (II),

(II), wherein each X is independently N, C-R3, or C-FU; with the proviso that no more than two Xs are N;

wherein Ri and R2 are each independently hydrogen, acyl group, alkenyl group, alkoxy group, alkoxycarbonyl group, alkoxycarbonyloxy group, alkoxysulfonyloxy group, alkyl group, alkylamino group, alkylaminocarbonyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkylsulfonyl group, alkylsulfonyloxy group, alkylthio group, alkynyl group, amide group, amidine group, amino group, arylalkoxy group, arylalkyl group, aryl group, arylcarbonyl group, arylcarbonyloxy group, aryloxy group, aryloxycarbonyl group, aryloxycarbonyloxy group, aryloxysulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, azido group, carbamido group, carbamoyl group, carbazoyi group, carbonyl group, carboxylate group, carboxylic acid group, cyanato group, cyano group, cycloalkenyl group, cycloalkyl group, dialkylamino carbonyl group, dialkylamino group, guanidino group, guanyl group, halo group, heteroarylalkoxy group, heteroarylalkyl group, heteroaryl group, heteroarylcarbonyl group, heteroaryloxy group, heterocyclic group, hydroxamino group, hydroxy group, imino group, isocyanato group, isocyano group, mercapto group, nitro group, oxo group, perhaloalkenyl group, perhaloalkoxy group, perhaloalkyl group, perhaloalkynyl group, perhaloarylalkyl group, perhaloaryl group, perhalocycloalkyl group, phosphate group, phosphine group, phospho group, sulfate group, sulfo group, sulfonyl group, oxidized form thereof, substituted form thereof, heteroatom form thereof, or combination thereof;

wherein each Fb is independently hydrogen, "C-group", acyl group, alkenyl group, alkoxy group, alkoxycarbonyl group, alkoxycarbonyloxy group, alkoxysulfonyloxy group, alkyl group, alkylamino group, alkylaminocarbonyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkylsulfonyl group, alkylsulfonyloxy group, alkylthio group, alkynyl group, amide group, amidine group, amino group, arylalkoxy group, arylalkyl group, aryl group, arylcarbonyl group, arylcarbonyloxy group, aryloxy group, aryloxycarbonyl group, aryloxycarbonyloxy group, aryloxysulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, azido group, carbamido group, carbamoyl group, carbazoyi group, carbonyl group, carboxylate group, carboxylic acid group, cyanato group, cyano group, cycloalkenyl group, cycloalkyl group, dialkylamino carbonyl group, dialkylamino group, guanidino group, guanyl group, halo group, heteroarylalkoxy group, heteroarylalkyl group, heteroaryl group, heteroarylcarbonyl group, heteroaryloxy group, heterocyclic group, hydroxamino group, hydroxy group, imino group, isocyanato group, isocyano group, mercapto group, nitro group, oxo group, perhaloalkenyl group, perhaloalkoxy group, perhaloalkyl group, perhaloalkynyl group, perhaloarylalkyl group, perhaloaryl group, perhalocycloalkyl group, phosphate group, phosphine group, phospho group, sulfate group, sulfo group, sulfonyl group, oxidized form thereof, substituted form thereof, heteroatom form thereof, or combination thereof; and

wherein each FU is independently hydrogen, "D-group", acyl group, alkenyl group, alkoxy group, alkoxycarbonyl group, alkoxycarbonyloxy group, alkoxysulfonyloxy group, alkyl group, alkylamino group, alkylaminocarbonyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkylsulfonyl group, alkylsulfonyloxy group, alkylthio group, alkynyl group, amide group, amidine group, amino group, arylalkoxy group, arylalkyl group, aryl group, arylcarbonyl group, arylcarbonyloxy group, aryloxy group, aryloxycarbonyl group, aryloxycarbonyloxy group, aryloxysulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, azido group, carbamido group, carbamoyl group, carbazoyi group, carbonyl group, carboxylate group, carboxylic acid group, cyanato group, cyano group, cycloalkenyl group, cycloalkyl group, dialkylamino carbonyl group, dialkylamino group, guanidino group, guanyl group, halo group, heteroarylalkoxy group, heteroarylalkyl group, heteroaryl group, heteroarylcarbonyl group, heteroaryloxy group, heterocyclic group, hydroxamino group, hydroxy group, imino group, isocyanato group, isocyano group, mercapto group, nitro group, oxo group, perhaloalkenyl group, perhaloalkoxy group, perhaloalkyl group, perhaloalkynyl group, perhaloarylalkyl group, perhaloaryl group, perhalocycloalkyl group, phosphate group, phosphine group, phospho group, sulfate group, sulfo group, sulfonyl group, oxidized form thereof, substituted form thereof, heteroatom form thereof, or combination thereof;

wherein one or more R3 or FU groups may join and form a fused ring with one or more other Fb, FU, or combination of R3 and FU groups; or resonance form thereof, or salt thereof, or salt of resonance form thereof.

In various embodiments, the compound is any one of compounds #1 -9 and 86-87, preferably any one of compounds #1 and 86-87,

In various embodiments, the method kills the mycobacterium.

In a second aspect, the invention relates to a method of treating or preventing an infection of a mycobacterium expressing cytochrome bd oxidase and/or a disease resulting from said infection in a subject, the method comprising administering to the subject an effective amount of a compound capable of inhibiting cytochrome bd in said mycobacterium in combination with an effective amount of an additional therapeutic agent capable of inhibiting cytochrome bd oxidase in said mycobacterium.

In various embodiments, the subject is a mammal, preferably a human. In various embodiments, the mycobacterium is selected from the group consisting of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium "hominissuis",

M. colombiense, M. asiaticum, M. gordonae, M. gastri, M. kansasii, M. hiberniae, M. nonchromogenicum, M. terrae, M. triviale, M. pseudoshottsii, M. shottsii, M. triplex, M. genavense, M. florentinum, M. lentiflavum, M. palustre, M. kubicae, M. parascrofulaceum, M. heidelbergense, M. interjectum, M. simiae, M. branderi, M. cookii, M. celatum, M. bohemicum,

M. haemophilum, M. malmoense, M. szulgai, M. botniense, M. chimaera, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, M. intracellular, M. lacus, M. marinum, M. monacense, M. montefiorense, M. murale, M. nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimoidei, M. tusciae, M. xenopi, M. intermedium, M. abscessus, M. chelonae,

M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum, M. senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M. parafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii, M. wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M. arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense,

M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, M. seoulense, and M. tokaiense.

In preferred embodiments, the mycobacterium is M. tuberculosis and the disease is tuberculosis.

In various embodiments, the compound is as described above.

In various embodiments, the additional therapeutic agent is any one of quinolone compounds, Aurachin, nitric oxide (NO) donors such as PA-824, antibiotics LL-Z1272, Gramicidin S, and derivatives thereof.

In various embodiments, the method kills the mycobacterium.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings. Figure 1 . Oxidative phosphorylation pathway in M. tuberculosis. The molecular targets of Q203 (compound #1 ) and bedaquiline (BDQ) are shown.

Figure 2. Activity of Q203 against M. bovis BCG parental, M. bovis BCG bd oxidase KO (bd ox. KO) and the bd ox. KO complemented strain (bd ox. KO compl.). Q203 was tested in a dose- response in 96-well plates. Optical density at 600nM was recorded 7 days after incubation. Each concentration was tested in triplicate. Bedaquiline and Isoniazid were used as reference drugs. Figure 3. The cytochrome bc1 -aa3 and BD oxidase are jointly required for aerobic respiration. The parental (P), bd ox. KO (M), and complemented (C) strains were incubated in sealed tubes with DMSO (solvent control) or 400nM of Q203 (+Q203) in the presence of methylene blue (oxygen probe). All the strains were able of aerobic respiration with DMSO. In the presence of a high dose of Q203, the Parental and complemented strains were still able of aerobic respiration, whereas the bd ox. KO (M) strain was not (as witnessed by the blue color of the bacterial suspension). These results demonstrate that the cytochrome bd oxidase can act as an efficient terminal electron acceptor upon chemical inhibition of the bc1 -aa3 branch by Q203.

Figure 4. Electron transport chain in M. leprae. Note the absence of the aerobic cytochrome bd oxidase, and of any other terminal electron acceptors.

Figure 5. Sequences of the predicted Q203-binding site in M. tuberculosis, M. leprae and M. ulcerans. Single-letter amino acids code was used. Figure 6. (A) In vitro potency of Q203 and IPA-7 (compound #7) against 4 M. ulcerans strains. The dose-response curves were determined against clinical isolates from Cameroon (black circles and green squares), Togo (blue triangles) and Australia (red triangles) using a resazurin- based assay in 96-well plates. One representative of three independent experiments is shown. (B) Time-to-kill kinetic assays of Q203 and IPA-7 against M. ulcerans. Cameroonian strain S1013 was incubated with different compound concentrations (multiples of the respective minimal inhibitory concentration) for 0, 3, 7, 14, 21 and 28 days. Bacteria were then spread on 7H10 agar without compound and CFUs were counted after 16 weeks of growth at 30 °C

Figure 7. Efficacy of Q203 in a mouse model of Buruli ulcer. Mice were infected with M. ulcerans in the left hind footpad. Five weeks post-infection, mice were treated with rifampin (10mg/kg), Q203 (0.5mg/kg), IPA-7 (inactive IPA derivative; 0.5mg/kg) or vehicle control (control) for four weeks. Drug treatments were administered by oral gavage three times per week. Disease progression was followed by weekly measurement of the food pad thickness (A) or by taking pictures (B) at the end of treatment. (A) Mean values of the foot pad thickness (mm); the error bars represent the S.D. (B) Pictures of representative feet taken at the end of treatment (week 9 post-infection). Figure 8. Efficacy of Q203 and other I PA derivatives against M. ulcerans. Compound #86 was active against three clinical isolates (S1298, S1213, S1013) at a MIC50 ranging from 38.8 nM to 66 nM, whereas #87 was active at a MIC50 ranging from 1.3 nM to 2.3 nM.

Figure 9. Q203 Is a bacteriostatic agent that does not inhibit respiration in M. tuberculosis. (A) Oxygen consumption assay in M. tuberculosis H37Rv using the oxygen sensor Methylene Blue at 0.001%. (8) MiCso of Q203 against M. tuberculosis H37Rv (red circles), bacillus Calmette- Guerin (pink stars), and the clinical isolates N0052 (blue squares), N0072 (purple triangles). N0145 (green inverted triangles), N0157 (red diamonds), and N0155 (orange hexagons) replicating in culture broth medium. Bacterial growth was measured by recording the Optical Density at 600 nm (ODeoo) after 5 d of incubation. (C) Bactericidal activity ol Q203 and BDQ against Af. tuberculosis H37Rv (red circles) and the clinical isolates N0052 (blue squares), N0072 (purple triangles), and N0145 (green triangles). The dotted line represents 90% bacteria! killing compared with the initial inoculum (MBC90). **Siatistical difference (P < 0.001 , Student's t test) between the potency oi BDQ and Q203. All experiments were performed in triplicate and repeated at least once. BDQ was used as a control drug targeting oxidative phosphorylation in ail experiments.

Figure 10. Effect of Q203 on the viability of M. bovis bacillus Caimeite-Guer!n. The dotted line represents 90% bacterial killing compared with the initial inoculum (MBC90). inoc, inoculum size at the start of the experiment. Data are expressed as the mean ± SDs. The experiments were performed in triplicate and repeated once.

Figure 11. The alternate CyX-bd terminal oxidase contributes to cellular respiration under aerobic conditions in M tuberculosis. M. tuberculosis H37Rv (red circles). H37Rv LcydAB (green squares), and LcydABconvp (blue triangles) were Incubated with the oxygen probe MitoXpress In the presence of 1% DMSO (A), Q203 at 400 nM (B), or BDQ at 500 nM (C). Kinetics of oxygen consumption was measured by recording the fluorescence (Exaeo, Emeso) over a 500- min period. Relative fluorescence units were converted into relative units of oxygen consumption (ROC). Insets: Oxygen consumption assay using methylene blue as oxygen sensor. P, H37Rv; M, H37Rv AcydAB; C, H37Rv AcyoCAScomp strains. (D) The inhibitory concentration (IC50) of Q203 and BDQ on oxygen consumption was measured using the MitoXpress oxygen probe. iCso was calculated from measurement of the fluorescence read after 180 rnin of incubation at 37 °C. The experiments were performed in triplicate and repeated at least once. Data are expressed as the mean ± SDs of triplicates for each concentration of a representative experiment.

Figure 12. Q203 is bactericidal and triggers a rapid ATP depletion in M. tuberculosis H37Rv kcydAB strain. ATP ieveis were measured using a iuciferase-based assay in H37Rv (A), H37Rv LcydAB (B), and H37Rv kcydABcomp (C) exposed to a dose-range of Q203 {circies} or BDQ (squares). Relative Light Units (RLU) were recorded after 24 h of Incubation, inset In A depicts the ATP levels in M. tuberculosis H37Rv treated with Q203 at 50 nM or BDQ at 500 nM (BDQ). "Statistical difference (P < 0.01 , Student's i test) in ATP levei between Q203- and BDQ-treated bacteria. (D) Bactericidal potency of Q203 and BDQ against replicating M. tuberculosis H37Rv (red circles), H37Rv AcydAB (green squares), and H37Rv LcydABcomp (blue triangles) strains. The dotted ilne represents 90% bacterial killing compared with the initial inocuium (MBCso). """Statistical difference (P < 0.001 , Student's t test) in CPU number between H37Rv and H37Rv kcydAB treated with Q203. Inoc, inoculum size at the start of the experiment. Data are expressed as the mean + SDs of triplicates for each concentration.

Figure 13. cydAB deletion had no significant impact on growth and ATP homeostasis in M. tuberculosis H37Rv. (A) Growth of M tuberculosis strains H37Rv (red circles), H37Rv LcydAB (green squares), and H37Rv LcydABcomp (blue triangles) was monitored over a 12-d period. (B) ATP levels in replicating M. tuberculosis strains H37Rv (red bars), H37Rv LcydAB (green bars), and H37Rv AcydABcomp (blue bars). Data are expressed as mean ± SDs. The experiments were performed in triplicate.

Figure 14. cydAB deletion had a moderate effect on the M!Cso of Q203 against M. tuberculosis H37Rv (red circles), H37Rv LcydAB (green squares), and H37Rv LcydABcomp (blue triangles) strains replicating in culture broth medium {A and C). MiCso of Q203 against bacillus Calmette- Guirin (red circies), bacillus Ca!mette-Guerin LcydAB (green squares), and bacillus Caimette- Gu6rin AcydABcomp (blue triangles) replicating in culture broth medium (8 and D). Bacterial growth was quantified by recording the ODeoo after 5 d of Incubation.

Figure 15. The alternate Cyt-M terminal oxidase contributes to cellular respiration in bacillus Calmette-Gu0rin. Bacillus Calmette-Guerin (P), bacillus Ca!mette-Guenn LcydAB (M). and bacillus Calmette~Gu6rin AcycMScomp (C) were incubated with the oxygen probe Methylene blue in the presence of 1 % DMSO, 400 nM Q203, or 500 nM BDQ in sealed tubes and incubated under an anaerobic atmosphere to prevent oxygen leak. Pictures were taken after 4 d of incubation at 37 ^«0. Figure 16. The Cyt-ix?«:aas and Cyt-bd contribute to oxygen respiration in mycobacteria! inverted membrane vesicles, inverted membrane vesicles from bacillus Caimette~Gu6rin parental (red circles), LcydAB {green squares), and LcydABcomp (blue triangles) strains were incubated with the oxygen probe MitoXpress in the presence of 1% DMSO (A), Q203 at 10 nM (S), or BDQ at 500 nM (C). Kinetic of oxygen consumption was measured by recording the fluorescence (Ex3so. Eme5o) over a 30-min period. SDs of three rep!icates are shown. The experiment was repeated once.

Figure 17. Q203 is bactericidal and triggers a rapid ATP depletion in bacillus Calmette~Gu0rin LcydAB. ATP leveis were measured using a !uciferase-based assay in bacillus Calmette- Guerin (A), baciiius Calmetie-Guenn LcydAB \B), and bacillus Ca!mette-Guirin LcydABcomp (C) exposed to a dose-range of Q203 (circles) or BDQ (squares). Relative Light Units (RLU) were recorded after 24 h of incubation. Insel in A depicts the ATP levels in baciiius Calmetie- Gu&in treated with Q203 at 25 nM (Q203) or BDQ at 250 nM (BDQ). The dotted line represents 90% bacteria! killing compared with the Initial inoculum (MBC90). *Statist!ca! difference (P < 0.01 , Student's / test) in ATP !eve! between Q203- and BDQ-treated bacteria. (D) Bactericidal potency of Q203 and BDQ against baciiius Calmetle-Guerin (red circles), bacillus Calmelte- Guenn LcydAB (green squares), and bacillus Ca!mette-Guenn LcydABcomp (blue triangles) strains. "Statistical difference (P < 0.001 , Student's t test) in CPU number between H37Rv and H37Rv LcydAB treated with Q203. !noc, inoculum size at the start of the experiment. BDQ was used at 500 nM. Data are expressed as the mean + SD oi triplicates for each concentration.

Figure 18. The Cyt-bci:aa$ and the Cyt-bd terminal oxidases are jointly required for ATP homeostasis and survival in nutrient-starved, phenotypic drug-resistant persisters. ATP levels were quantified in nutrient-starved M. tuberculosis H37Rv (red bars), H37Rv LcydAB (green bars), and H37Rv LcydABcomp (blue bars) treated with a dose-range of Q203 (A) or BDQ (B). (C) Bactericidal potency of Q203, BDQ and isoniazid (INH) was evaluated against the M. tuberculosis strains H37Rv (red circles). H37Rv LcydAB (green squares), and H37Rv LcydABcomp (blue triangles). The dotted line represents 90% bacterial killing compared with the untreated control. **Statisticai difference (P < 0.001 , Student's t test) in CFU number between H37Rv and H37Rv LcydAB treated with Q203. Results are expressed as mean ± SDs. Experiments were performed in triplicate and repeated once.

Figure 19. The Cyt-bct:aa3 and the Cyt-bd are jointly required for ATP homeostasis and survival of nutrient-starved bacillus Caimette~Gu6rin. ATP ieve!s were quantified In nutrient-starved bacillus Ca!mette-Guerin {A), bacillus Calmette-Guerin LcydAB (B), and bacillus Calmette- Gue>in LcydABcomp (C) strains treated with a dose-range of Q203 (black circles) or BDQ (black squares). Inset in A depicts the ATP levels in bacillus Caimette-Guirin treated with Q203 at 62.5 nM (Q203) or BDQ at 1 ,250 nM (BDQ). *Statistical difference (P < 0.01 , Student's t test) in ATP ievei between Q203- and BDQ-treated bacteria. (D) Bactericidai potency of Q203, BDQ, and isoniazid (iNH) was evaiuated against the strains bacillus Calmette-Guerin (red circles), bacillus Ca!mette-Guenn AcydAB (green squares), and bacillus Calmette-Guerin AcydABcomp (blue triangles) after 15 d of incubation. Bacterial counts were determined by CFU determination on nutrient-agar plates. The dotted line represents 90% bacterial killing compared with the untreated control. ""Statistical difference (P < 0.001 , Student's t test) In CFU number between H37Rv and H37Rv AcydAB treated with Q203. Data are expressed as mean ± SDs. The experiments were performed in triplicate and repeated two times. Figure 20. The C t-bci:aa3 and Cyt-M are jointly required for growth in macrophages and for virulence in a mouse model. THP-1 cells were infected with the strains H37Ftv (A), H37Rv AcydAB (B), and H37Rv AcydABcomp (C) and treated with 1% D SO (vehicle control), Q203, or BDQ. Viability of intracellular mycobacteria was determined after 5 d of treatment. Dotted line, initial bacteria! load at 1 h postinfection. *Greater than or equal to 90% reduction in bacterial load compared with the Initial bacteria! load. The means and SDs of three replicates for each experiment are shown. The experiment was repeated once. BALB/c mice were aerosol- Infected with either M. tuberculosis H37Rv (red circles), AcydAB (green squares), or AcydABcomp (blue triangles). Two weeks after infection, treatment was started by oral administration of Q203 at 2 mg kg, BDQ at 10 mg/kg, or vehicle control three times a week. Bac!!lary burden (CFU) in lungs of treated animals was assessed after 2 and A wk treatment with either (D) Q203, (£) vehicle, or (F) BDQ. To compare drug efficacy between different strains, CFU counts were normalized to the time o! treatment start (day 13 after infection). CFU counts are shown in Table 5. Gross pathology (G, /·/, I), and H&E staining (Fig. 21 ) was performed on all lung samples to determine severity of disease and level of inflammation. Error bars represent SDs of at least four replicates. An unpaired Student t test was performed between parental and AcydAB CFU counts. *P < 0.05; **P < 0.01.

Figure 21. Q203 treatment reduced disease severity and level of inflammation in the lungs of mice infected with the M. tuberculosis AcydAB strain. H&E staining was performed In lung sections of animals treated for 4 wk with either Q203 (A, O, and G), vehicle (β, E, and Hi, or BDQ (C, F, and Λ.

Figure 22. A structure-activity relationship study of 85 IPA derivatives against clinically-relevant classical lineage M. ulcerans isolates in vitro.

Figure 23. Absence of tissue necrosis and oedema formation in Q203 treated mice. HE stained histological sections of foot pads from representative control (A), IPA-7 (B), rifampicin (C), or Q203 (D) treated mice as well as of a non-infected control foot pad (E). Scans of whole foot pads (A1 , B1 , C1 , D1 and E1 ) as well as pictures taken at the epidermis (A2, B2, C2, D2 and E2) and at the site of infection (A3, B3, C3, D3 and E3) are shown. Scale bars represent 5 mm (A1 , B1 , C1 , D1 and E1 ), and 80 μιτι (A2, A3, B2, B3, C2, C3, D2, D3, E2 and E3). Figure 24. Treatment with Q203 results in a strong reduction of the bacterial load and induces killing of AFB. (A) Bacterial load during the course of infection as determined by IS2404-specific qPCR. Individual animals are depicted, values are shown as Iog10 (genomes per foot pad) and mean values are linked by connecting lines. (B-E) Tissue sections of foot pads from representative control (B1 and B2), IPA-7 (C1 and C2), rifampicin (D1 and D2) and Q203 treated (E1 and E2) mice stained with ZN for visualization of AFB. Scale bars represent 200 μιτι (B1 , C1 , D1 and E1 ) and 10 μιτι (B2, C2, D2 and E2).

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural, and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes." In case of conflict, the present specification, including explanations of terms, will control.

Provided herein are compounds of formula (I) or (II),

wherein each X is independently N, C-R3, or C-F ; with the proviso that no more than two X's are N;

wherein each R3 is independently hydrogen, "C-group", acyl group, alkenyl group, alkoxy group, alkoxycarbonyl group, alkoxycarbonyloxy group, alkoxysulfonyloxy group, alkyl group, alkylamino group, alkylaminocarbonyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkylsulfonyl group, alkylsulfonyloxy group, alkylthio group, alkynyl group, amide group, amidine group, amino group, arylalkoxy group, arylalkyl group, aryl group, arylcarbonyl group, arylcarbonyloxy group, aryloxy group, aryloxycarbonyl group, aryloxycarbonyloxy group, aryloxysulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, azido group, carbamido group, carbamoyl group, carbazoyi group, carbonyl group, carboxylate group, carboxylic acid group, cyanato group, cyano group, cycloalkenyl group, cycloalkyl group, dialkylamino carbonyl group, dialkylamino group, guanidino group, guanyl group, halo group, heteroarylalkoxy group, heteroarylalkyl group, heteroaryl group, heteroarylcarbonyl group, heteroaryloxy group, heterocyclic group, hydroxamino group, hydroxy group, imino group, isocyanato group, isocyano group, mercapto group, nitro group, oxo group, perhaloalkenyl group, perhaloalkoxy group, perhaloalkyl group, perhaloalkynyl group, perhaloarylalkyl group, perhaloaryl group, perhalocycloalkyl group, phosphate group, phosphine group, phospho group, sulfate group, sulfo group, sulfonyl group, oxidized form thereof, substituted form thereof, heteroatom form thereof, or combination thereof; and

wherein each FU is independently hydrogen, "D-group", acyl group, alkenyl group, alkoxy group, alkoxycarbonyl group, alkoxycarbonyloxy group, alkoxysulfonyloxy group, alkyl group, alkylamino group, alkylaminocarbonyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkylsulfonyl group, alkylsulfonyloxy group, alkylthio group, alkynyl group, amide group, amidine group, amino group, arylalkoxy group, arylalkyl group, aryl group, arylcarbonyl group, arylcarbonyloxy group, aryloxy group, aryloxycarbonyl group, aryloxycarbonyloxy group, aryloxysulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, azido group, carbamido group, carbamoyl group, carbazoyl group, carbonyl group, carboxylate group, carboxylic acid group, cyanato group, cyano group, cycloalkenyl group, cycloalkyl group, dialkylamino carbonyl group, dialkylamino group, guanidino group, guanyl group, halo group, heteroarylalkoxy group, heteroarylalkyl group, heteroaryl group, heteroarylcarbonyl group, heteroaryloxy group, heterocyclic group, hydroxamino group, hydroxy group, imino group, isocyanato group, isocyano group, mercapto group, nitro group, oxo group, perhaloalkenyl group, perhaloalkoxy group, perhaloalkyl group, perhaloalkynyl group, perhaloarylalkyl group, perhaloaryl group, perhalocycloalkyl group, phosphate group, phosphine group, phospho group, sulfate group, sulfo group, sulfonyl group, oxidized form thereof, substituted form thereof, heteroatom form thereof, or combination thereof;

wherein one or more R3 or FU groups may join and form a fused ring with one or more other Fb, FU, or combination of R3 and FU groups;

or resonance form thereof, or salt thereof, or salt of resonance form thereof.

The compounds defined herein are detailed in PCT patent publication No. WO2017049321 , which is hereby incorporated by reference in its entirety together with all the specific compounds disclosed therein. These compounds may be commercially available or may be prepared in accordance with the disclosure of PCT patent publication No. WO2017049321 .

In various embodiments, the compound is of formula (III)

(III),

wherein Rs, Ft6, and R7 are each independently selected from the group consisting of hydrogen, halogen, and substituted or unsubstituted C1 C5 alkyl, and Rs is independently selected from the group consisting of the chemical groups described in Table 1 , wherein the compound is capable of inhibiting the cytochrome bc1 of the respiratory electron transport chain in a mycobacterium.

Table 1 . Re groups

The term "alkyl" as used herein refers to a linear, branched, or cyclic saturated hydrocarbon group. The term "C1 -C5 alkyl" refers to an alkyl group having 1 -5 carbon atoms.

The term "halogen" refers to fluoro, chloro, bromo, and iodo. The term 'inhibit" or "inhibition" indicates a partial or complete reduction in a biological activity compared to a baseline. "Inhibition of cytochrome bc1 " refers to a decrease of, for example, 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 1 00% in cytochrome bd activity in the respiratory electron transport chain as a direct or indirect response to the presence of a compound of the invention relative to the activity of cytochrome bd in the absence of the compound. The decrease in activity may be due to the direct interaction of the compound with cytochrome bd , or due to the interaction of the compound with one or more other factors that in turn affect cytochrome bd activity. For example, the presence of the compound may decrease cytochrome bd activity by directly binding to the cytochrome bd , by causing (directly or indirectly) another factor to decrease cytochrome bd activity, or by (directly or indirectly) decreasing the amount of cytochrome bd present in the cell or organism.

Inhibition of cytochrome bd activity also refers to observable inhibition of cytochrome bd activity in a standard biochemical assay for cytochrome bd activity as known in the art. Preferred inhibitors of cytochrome bd activity have an ICso value less than or equal to 1 0 micromolar, more preferably less than or equal to 1 micromolar, still more preferably less than or equal to 1 00 nanomolar, and most preferably less than or equal to 1 0 nanomolar.

In various embodiments, Rs, R6, and R7 are each independently selected from the group consisting of hydrogen, chloro, bromo, methyl, and ethyl. This means that R5 is hydrogen, chloro, bromo, methyl, or ethyl; R6 is hydrogen, chloro, bromo, methyl, or ethyl ; R7 is hydrogen, chloro, bromo, methyl, or ethyl; and Re is any one of the groups described above. It is to be understood that the various combinations and permutations of said R5, R6, R7 and Re groups, even if such combinations and permutations are not explicitly described herein for the sake of conciseness, are contemplated to be within the scope of the present invention, if not expressly excluded.

In various embodiments, the compound is any one of compounds #1 -54 as described in Figure 22.

In various embodiments, the compound is any one of compounds #1 -9 and 86-87, preferably any one of compounds #1 and 86-87 of Table 2.

Table 2. Non-limiting and preferred compounds

Without wishing to be bound to any particular theory, it is believed that the compounds defined herein are capable of inhibiting cytochrome bd of the respiratory electron transport chain in a mycobacterium.

Further provided are pharmaceutically acceptable compositions comprising the compounds described herein and a pharmaceutically acceptable carrier, which may further comprise an additional therapeutic agent capable of inhibiting the cytochrome bd oxidase of the respiratory electron transport chain in a mycobacterium.

The term "electron transport chain" as used herein refers to a series of redox reactions where ATP is broken down into ADP, producing a net gain of energy in the organism.

Mycobacteria harbor genes for a cytochrome c pathway that consist of a cytochrome bd (related to the mitochondrial complex III, encoded by qcrCAB), and an aa3-type cytochrome c oxidase (complex IV). The cytochrome bd transfers electrons from menaquinol to the cytochrome c oxidase, a process which is linked to proton translocation across the membrane. The cytochrome bd oxidase is a respiratory quinol: 02 oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme i the production of a proton motive force by the vectorial charge transfer of protons.

The additional therapeutic agent of the present invention may be any agent capable of inhibiting any cytochrome bd oxidase respiratory oxygen reductase, such as a chemical compound, a nucleic acid silencing agent (e.g., a CRISPR system, a shRNA, an antisense RNA, a miRNA, or other RNA-based or RNA-like silencing agents), or a protein which inhibits the expression of cytochrome bd oxidase. However more preferably, the additional therapeutic agent of the present invention may be any compound capable of inhibiting mycobacterial cytochrome bd oxidase known in the art. Non-limiting examples of the additional therapeutic agent include quinolone compounds, Aurachin, nitric oxide (NO) donors such as PA-824, antibiotics LL- Z1272, Gramicidin S, derivatives thereof, and others disclosed in the pertinent literature, e.g. Borisov, et al. Biochim Biophys Acta. 201 1 Nov;1807(1 1 ):1398-413; Lu, et al. Sci Rep. 2015 May 27;5:10333; Mogi, et al. Biochim Biophys Acta. 2009 Feb;1787(2):129-33; and Mogi, et al. FEBS Lett. 2008 Jun 25;582(15):2299-302.

The term "pharmaceutically acceptable" is employed herein to refer to those materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject extract from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically- acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol ; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; sterile distilled water; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations. See Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa. : Mack Publishing Co., 1995), which discloses typical carriers and conventional methods of preparing pharmaceutical formulations.

The skilled artisan would also realize that proper formulation is dependent upon the route of administration selected for the specific application, and the proper route and mode of administering the compound described herein to a subject should be determined on a case-by- case basis.

The compound or composition described herein can be administered via any parenteral or non- parenteral (enteral) route that is therapeutically effective for proteinaceous or nucleic acid-based drugs. Parenteral application methods include, for example, intracutaneous, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal or intravenous injection and infusion techniques, e.g. in the form of injection solutions, infusion solutions or tinctures, as well as aerosol installation and inhalation, e.g. In the form of aerosol mixtures, sprays or powders. An overview about pulmonary drug delivery, i.e. either via Inhalation of aerosols (which can also be used in intranasal administration) or intracheal instillation is given by Patton et al. Proc Amer Thoracic Soc 2004; Vol. 1 pages 338-344, for example). Non-parenteral delivery modes are, for instance, orally, e.g. in the form of pills, tablets, capsules, solutions or suspensions, or recta!ly, e.g. in the form of suppositories. Compounds described herein can be administered systemicaily or topically in formulations containing conventional non-toxic pharmaceutically acceptable excipients or carriers, additives and vehicles as desired.

In one embodiment of the present invention the pharmaceutical is administered parenterally to a mammal, and in particular to humans. Corresponding administration methods include, but are not limited to, for example, intracutaneous, subcutaneous, intramuscular, intratracheal or intravenous injection and infusion techniques, e.g. in the form of injection solutions, infusion solutions or tinctures as well as aerosol installation and inhalation, e.g. in the form of aerosol mixtures, sprays or powders. A combination of intravenous and subcutaneous infusion and /or injection might be most convenient in case of compounds with a relatively short serum half life. The pharmaceutical composition may be an aqueous solution, an oil-in water emulsion or a water-in-oil emulsion.

The dosage of the compound described herein applied may vary within wide limits to achieve the desired preventive effect or therapeutic response. It will, for instance, depend on the half-life of the compound in vivo. Further, the optimal dosage will depend on the biodistribution of the compound, the mode of administration, the severity of the disease/disorder being treated as well as the medical condition of the patient. For example, when used in an ointment for topical applications, a high concentration of the compound can be used. However, if wanted, the compound may also be given in a sustained release formulation, for example liposomal dispersions or hydrogel-based polymer microspheres, like PolyActive™ or OctoDEX™ (cf. Bos et a!,, Business Briefing: Pharmatech 2003: 1 -8). Other sustained release formulations available are for example PLGA based polymers (PR pharmaceuticals), PLA-PEG based hydrogels (Medincei!) and PEA based polymers (Medivas).

Accordingly, the compound described herein can be formulated into compositions using pharmaceutically acceptable Ingredients as well as established methods of preparation (Gennaro, A.L. and Gennaro, A.R. (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wiikins, Philadelphia, PA). To prepare the pharmaceutical compositions, pharmaceutically inert Inorganic or organic excipients can be used. To prepare e.g. pills, powders, gelatine capsules or suppositories, for example, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyois, natural and hardened oils can be used. Suitable excipients for the production of solutions, suspensions, emulsions, aerosol mixtures or powders for reconstitution into solutions or aerosol mixtures prior to use include water, alcohols, glycerol, polyois, and suitable mixtures thereof as well as vegetable oils.

The formulations can be sterilized by numerous means, including filtration through a bacteria- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile medium just prior to use.

In a first aspect, the invention relatates to a method of treating or preventing an infection of a mycobacterium or a disease resulting from said infection in a subject, the method comprising administering to the subject an effective amount of a compound capable of inhibiting cytochrome bc1 of the respiratory electron transport chain in said mycobacterium.

The subject may be any human or non-human animal beings, preferably a mammal, more preferably a human. The term "treating" refers to having a therapeutic effect and at least partially alleviating or ameliorating an abnormal condition in the subject. The term "preventing" refers to decreasing the probability that a subject contracts or develops pathogenic infection.

The term "administering" relates to a method of delivering a compound to a cell or tissue of a subject. For cells harbored within a subject, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications. The methods disclosed herein may be used to treat any suitable mycobacterial infection. In various embodiments, the mycobacterium is selected from the group consisting of M. ulcerans, M. leprae, M. lepraemurium, and M. lepromatosis. In preferred embodiments, the mycobacterium is M. leprae and the disease is leprosy.

In various embodiments, the compound may be any compound described above, preferably any one of compounds #1 -54 and 86-87, more preferably any one of compounds #1 -9 and 86-87, most preferably any one of compounds #1 and 86-87, whereby the method kills the mycobacterium. For example, M. leprae or M. ulcerans infection can be cleared by treatment with such a compound alone, whereby leprosy or Buruli ulcer is treated or prevented. In a second aspect, the invention relates to a method of treating or preventing an infection of a mycobacterium expressing cytochrome bd oxidase and/or a disease resulting from said infection in a subject, the method comprising administering to the subject an effective amount of a compound capable of inhibiting cytochrome bd in said mycobacterium in combination with an effective amount of an additional therapeutic agent capable of inhibiting cytochrome bd oxidase in said mycobacterium.

In various embodiments, the subject is a mammal, preferably a human.

In various embodiments, the mycobacterium is selected from the group consisting of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium "hominissuis",

M. haemophilum, M. malmoense, M. szulgai, , M. botniense, M. chimaera, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, M. intracellular, M. lacus, M. marinum, M. monacense, M. montefiorense, M. murale, M. nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimoidei, M. tusciae, M. xenopi, M. intermedium, M. abscessus, M. chelonae, M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum, M. senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M. parafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii, M. wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M. arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, M. seoulense, and M. tokaiense. In preferred embodiments, the mycobacterium is M. tuberculosis and the disease is tuberculosis.

In various embodiments, the compound is as described above, preferably any one of compounds #1 -54 and 86-87, more preferably any one of compounds #1 -9 and 86-87, most preferably any one of compounds #1 and 86-87.

In various embodiments, the additional therapeutic agent capable of inhibiting cytochrome bd oxidase is any one of quinolone compounds, Aurachin, nitric oxide (NO) donors such as PA- 824, antibiotics LL-Z1272, Gramicidin S, derivatives thereof, and others disclosed in the pertinent literature, e.g. Borisov, et al. Biochim Biophys Acta. 201 1 Nov;1807(1 1 ):1398-413; Lu, et al. Sci Rep. 2015 May 27;5:10333; Mogi, et al. Biochim Biophys Acta. 2009 Feb;1787(2):129- 33; and Mogi, et al. FEBS Lett. 2008 Jun 25;582(15):2299-302.

For a mycobacterium expressing cytochrome bd oxidase, by administering a compound inhibiting cytochrome bd and an additional therapeutic agent inhibiting cytochrome bd oxidase either simultaneously, sequentially or separately, the method affords a superior therapeutic effect to that achieved upon administration of only said compound or said additional therapeutic agent alone, and at its conventional dose. The superior therapeutic effect may be measured by, for example, the extent of the response, the response rate, or the time to disease progression or the survival period of the combination therapy, to that achievable on dosing one of said compound and said additional therapeutic agent alone, and at its conventional dose.

Various combinations of the compound and the additional therapeutic agent, although not explicitly disclosed herein, are within the scope of the present application. Determining optimal combination of the two agents is within the knowledge of the person of average skill in the art. Without wishing to be bound to any particular theory, it is believed that the compound capable of inhibiting cytochrome bd can be used to inhibit the proliferation of a mycobacterium expressing functional cytochrome bd oxidase such as M. tuberculosis alone, in which case the drug effect is only bacteriostatic. In preferred embodiments, however, as described herein, the compound capable of inhibiting cytochrome bd is administered in combination (separately, sequentially, or simultaneously) with an additional therapeutic agent capable of inhibiting cytochrome bd oxidase, which combination is able to kill the mycobacterium.

It should be noted that the compounds and the additional therapeutic agents may be formulated into pharmaceutical compositions as described above prior to the administration.

Also encompassed within the scope of the present application are methods of killing a mycobacterium or inhibiting the proliferation of a mycobacterium, said method comprising contacting said mycobacterium with an effective amount of a compound described herein alone or in combination with an effective amount of an additional therapeutic agent capable of inhibiting the cytochrome bd oxidase of the respiratory electron transport chain. Said method may be performed in vitro, ex vivo, or in vivo.

EXAMPLES

Example 1: Use of IP A compounds for the treatment of Buruli ulcer and leprosy

Materials and Methods Bacterial strains, general growth conditions and reagents

M. ulcerans strains S1012, S1013, S1 047 and S1298 (isolated in 2010, 201 1 and 2013 from Cameroonian BU patients; also see (Bratschi MW, et al. PLoS Negl Trap Dis. 2013;7:) were routinely propagated at 30 °C in BacT/Alert culture bottles supplemented with enrichment medium (bioMerieux). For in vitro compound testing, bacteria were grown either in liquid 7H9 medium or on 7H10 agar, supplemented with 1 0 % (vol/vol) OADC. The M. ulcerans strain S1014 (Togo) was received from F. Portaels (ITM, Antwerp) and the Australian strain S1251 from J. Fyfe (VIDRL, Melbourne). Q203, used for initial screenings, was kindly provided by K. Pethe (Pasteur Institute Korea). For subsequent in vitro and in vivo experiments, re-synthesized Q203 was provided by K. Pethe (Nanyang Technological University). The IPA screening panel encompassing 85 IPA compounds was received from BASF SE, Ludwigshafen.

Drug susceptibility assays

Minimal inhibitory concentrations (MIC) were determined as described before (Scherr N, et al. Antimicrob Agents Chemother. 2012;56: 6410-6413) by performing resazurin-based metabolic activity assays in the 96-well plate format. Briefly, two-fold serial dilutions of compounds were set up in duplicates or triplicates in a volume of 100 μΙ. Then, 1 00 μΙ of a diluted M. ulcerans culture (OD=0.04) was added and incubated at 30 °C for 8 days in the presence of the compounds. Upon resazurin (0.125 mg/ml) addition, plates were transferred to 37 °C and incubated o/n. Compound activities were determined by fluorescence measurements (λ=540/588 nm). Unless otherwise stated, the determined MIC values refer to the actual measured minimal concentrations at which growth was still completely inhibited. The MIC values obtained were then classified into different activity categories.

Kinetic survival assays

Serial dilutions of compounds Q203 and IPA-7 (0.25, 1 , 2, 4, 8 and 16 fold the corresponding MIC value) were set up in a volume of 100 μΙ. Then 100 μΙ of a diluted M. ulcerans S1013 (OD600=0.04) was added and bacteria were incubated at 30°C for 0, 3, 7 14, 21 and 28 days in the presence of the compound. At every time point, bacteria were washed and 100 μΙ of undiluted, 1 :100 or 1 :1000 diluted bacterial suspension was plated on 7H1 0 agar. CFUs were assessed after 16 weeks of incubation at 30 °C.

Infection of mice All mice were maintained in specific pathogen-free facilities at the Ecole Polytechnique Federale de Lausanne (EPFL, Switzerland) and the studies were performed under BSL-3 conditions in eight weeks old female BALB/c mice (Harlan). For the infection of mice, the M. ulcerans strain S1013 was used (Bratschi MW, et al. PLoS Negl Trap Dis. 2013;7). For preparing the inoculum, the bacteria were cultivated for 6-8 weeks in Bac/T medium (Biomerieux, 25101 1 ), pelleted by centrifugation and resuspended in sterile PBS to a stock concentration of 125 mg/ml wet weight. The infection was performed by injecting 30 μΙ (about 6 x 103 bacilli) of an appropriate dilution of the stock solution in sterile PBS into the hind left foot pad of the mice. The course of infection was followed by weekly measurements of the foot pad thickness using a caliper. Mice were euthanized at treatment start (week 5), at the end of the treatment (week 9) and 6 weeks after completion of the treatment (week 15). Pictures of the feet were taken using a compact camera (WG-20, RICOH). Foot pads were aseptically removed for the determination of the bacterial load by quantitative RT-PCR or for histopathological analysis.

Treatment of mice

Treatment was started 5 weeks after infection when the first macroscopic signs such as foot pad swelling and reddening of the skin were observed. Treatment was given orally on three days per week during a period of four weeks (18 mice per treatment group). Q203 and IPA-7 were dissolved in 20 % D-a-Tocopherol polyethylene glycol 1000 succinate (TPGS) (Sigma, 57688) / H20 containing 1 % Dimethyl Sulfoxide (Sigma, D2650) and administered at a concentration of 0.5 mpk. Rifampicin (Sigma, R3501 ) was dissolved in H20 and given at a concentration of 10 mpk. As a control, 20 % TPGS / H20 containing 1 % DMSO was administered.

Determination of the bacterial load by quantitative RT-PCR

After euthanasia, mouse feet designated for quantification of M. ulcerans bacteria by qPCR were removed above the ankle, cleaned by 70 % EtOH, cut into 4 pieces and transferred to hard tissue grinding tubes (MK28-R, Precellys, KT03961 -1 -008.2). For the preparation of foot pad lysates, 750 μΙ sterile 7H9 medium was added and homogenization was performed using a Precellys 24-Dual tissue homogenizer (3 x 20 s at 5000 rpm with 30 s break). Afterwards, the lysate was transferred into a new tube and the remaining and still intact tissue was homogenized for a second time after addition of 750 μΙ of sterile 7H9 medium. The lysates were pooled and DNA was isolated from 100 μΙ of a 1 /20 dilution of the pooled lysate as described by Lavender and Fyfe (Lavender CJ, Fyfe JAM. Methods Mol Biol Clifton NJ. 2013;943: 201 -21 6). After DNA isolation, the bacterial load was determined by performing IS2404-specific quantitative RT-PCR analysis as previously described (Lavender CJ, Fyfe JAM. Methods Mol Biol Clifton NJ. 2013;943: 201 -216). Ct values were converted into genome numbers per foot pad by making use of the standard curve established by Fyfe et al. (Fyfe JAM, et al. Appl Environ Microbiol. 2007;73: 4733-4740). Histopathology

Mouse feet used for histopatho logical analysis were removed above the ankle and fixed at room temperature during 48 hours in 10 % neutral-buffered Formalin solution (4 % formaldehyde, Sigma, HT501 128-4L). The feet were decalcified in Formic Acid Bone Decalcifier (ImmunocalTM, StatLab, 1414A) for 6 days at room temperature and subsequently transferred to 70 % ETOH for storage. After dehydration and paraffin embedding, 5 μιτι thin sections were cut, de-paraffinised, rehydrated, and stained according to WHO standard protocols with Haematoxylin/Eosin (HE, Sigma, 51275- 500ML, J.T. Baker, 3874) to stain for connective tissue or Ziehl-Neelsen/Methylene blue (ZN, Sigma, 21820-1 L and 03978-250ML) to stain for mycobacteria. The stained sections were mounted with the Eukitt^® mounting medium (Fluka, 03989) and pictures were taken using a Leica^® DM2500B microscope or an Aperio scanner. Mycobacterium tuberculosis is an obligate aerobe that can survive, but not replicate, under anaerobic conditions. The reasons for the strict dependence on oxygen for growth are poorly understood, but illustrate the prominence of the terminal respiratory oxidases for the biology of the bacteria. Mycobacteria harbor genes for a cytochrome c pathway that consist of a cytochrome bc1 (related to the mitochondrial complex III, encoded by qcrCAB), and a aa3-type cytochrome c oxidase (complex IV).

The cytochrome bc1 transfers electrons from menaquinol to the cytochrome c oxidase, a process which is linked to proton translocation across the membrane. Since the cytochrome c oxidase is also capable of pumping protons, this pathway is the most energetically favorable respiratory branch in mycobacteria.

The cytochrome c oxidase is annotated as essential (1 ), and attempts to delete qcrCAB in M. tuberculosis were unsuccessful (2), suggesting that the cytochrome c pathway is required for the survival of slow-growing mycobacteria.

It is interesting to note that in addition to the bc1 -aa3 branch, M. tuberculosis also possesses a bacterial-specific cytochrome bd oxidase, which acts as an alternate aerobic terminal acceptor (similar function as the bc1 -aa3 branch, Figure 1 ). However, the cytochrome bd oxidase does not appear to be essential in M. tuberculosis since it can be deleted without any obvious growth defect in vitro (3, unpublished observations). In addition, some mycobacteria possess anaerobic terminal electron acceptors (Figure 1 ), but their role remains elusive since mycobacteria are unable to grow in the absence of oxygen.

Several inhibitors of the cytochrome bd known. The archetype is stigmatellin, a natural antibiotic that inhibits most cytochrome bc-1 . Recently, the discovery of small-molecules targeting the cytochrome bd in M. tuberculosis has triggered interest on the respiratory cytochrome c pathway (4-8). A series of imidazopyridine amide (IPA) compounds that interfere with energy metabolism were identified independently by several teams (4-7). The most advanced IPA derivatives is Q203 (7, 9), which is in clinical development (phase I) under a US FDA investigational new drug application. The IPA series is selective to mycobacteria since it has no effect on any other tested microorganisms (4, 7). Genetic evidences suggest that the IPA series inhibits respiration by interfering with the binding of menaquinole at the Qp site of qcrB. Of note, the architecture of the electron transport chain is identical in Mycobacterium tuberculosis and Mycobacterium bovis BCG (attenuated vaccine strain that can be handled in a BSL2 laboratory), and both species are equally susceptible to Q203. Therefore, mechanisms of action studies of the IPA series can be performed in M. bovis BCG.

The cytochrome bd oxidase limits the potency of Q203 in mycobacteria

Even though the cytochrome bd oxidase is not strictly essential in M. tuberculosis and in M. bovis BCG, several observations suggest that this alternate aerobic terminal acceptor restrict the potency of the IPA series by offering an alternate electron flow (Figure 1 ). This was first suggested by the team of Dr Clifton Barry who showed that the upregulation of the bd oxidase in the laboratory- adapted M. tuberculosis H37Rv strain (H37Rv) reduced the potency of IPA series (5). The deletion of the bd oxidase resulted in a dramatic shift in sensitivity to IPA derivatives from a Minimum inhibitory Concentration (MIC) >50 μΜ (apparent inactivity) to a MIC <0.024 μΜ (5) in H37Rv.

Our own data do not fully support these findings, since we have previously shown that the IPA series is active against H37Rv, a strain that was extensively used to develop Q203 (7, 9). In addition, we showed that the deletion of the cytochrome bd oxidase did not affect dramatically the MIC of Q203 in M. tuberculosis H37Rv or in M. bovis BCG (Figure 2 and Table 3). Since the procedure for MIC testing was not detailed in the article published by Clifton Barry and coll. (5), this discrepancy may be the result of a difference in methodology.

Nevertheless, we noticed several interesting observations supporting the notion that the presence of the bd oxidase interfere with the potency of the IPA series. Q203 is highly potent against M. tuberculosis and M. bovis BCG in vitro, but is largely bacteriostatic: the drug candidate inhibits growth, but does not kill the bacteria even at high dose up to 400nM. However, Q203 is bactericidal against a mycobacterial strain deficient for the production of the bd oxidase, having a Minimum Inhibitory Concentration 95% (MBC95) below 20 nM. MBC95 was determined by Colony Forming Unit determination on agar plates. This finding demonstrated that the presence of the cytochrome bd oxidase obliterates the bactericidal potency of Q203 against in mycobacteria (M. bovis BCG and M. tuberculosis). In other words, the electron flow going through the cytochrome bd oxidase branch (Figure 1 ) upon inhibition of the bc1 -aa3 branch by Q203 (or related IPA derivatives) is sufficient to prevent bacterial death, and result in a drug effect that is only bacteriostatic. This notion is reinforced by the finding that Q203-treated mycobacteria still respire oxygen using the alternate cytochrome bd oxidase (Figure 3). In the absence of the cytochrome bd oxidase, the electron flow cannot be diverted anymore upon inhibition of the bc1 -aa3 branch, leading to bacterial death.

Table 3. Minimum Inhibitory Concentration 50% (MICso) of Q203 and reference drugs against M. bovis BCG parental, M. bovis BCG bd oxidase KO (bd ox. KO) and the bd ox. KO complemented strain (bd ox. KO compl.). The values were extracted from the results depicted in Figure 2. MICso values were calculated using GraphPad Prism 7.

The respiratory bd-aa3 branch: an ideal drug target in M. leprae and M. ulcerans

With this knowledge in mind, we explored the genome of non-tuberculosis pathogenic mycobacteria (NTM) to study the structure of their respiratory chain. We focused on Mycobacterium leprae and Mycobacterium ulcerans, which are the pathogenic mycobacteria posing the most serious global health issue after tuberculosis. M. ulcerans is responsible for Buruli ulcer, whereas M. leprae is the etiological agent of leprosy. Both M. leprae and M. ulcerans are under selective pressure to lose genes at a high rate, a phenomenon known as reductive evolution. The genomes of M. ulcerans and M. leprae are much smaller than the genome of M. tuberculosis, and also contain a larger proportion of pseudogenes (inactive genes) compared to M. tuberculosis. The composition of their respiratory chain is particularly interesting in the context of our work. In M. leprae, the only functional terminal electron acceptor is the bc1 -aa3 branch (1 0). Other alternate acceptors (cytochrome bd oxidase fumarate dehydrogenase, nitrate reductase) have been lost during the process of reductive evolution (Figure 4), showing that M. leprae relies exclusively of the bc1 -aa3 branch (target of Q203) to respire. Since the predicted Q203-binding site in qcrB is conserved between M. tuberculosis and M. leprae (Figure 5), it suggested that M. leprae may be exquisitely susceptible to Q203 (and other I PA derivatives) due to the inability of its respiratory chain to reroute the electron flow upon inhibition of the bc1 -aa3 branch. Given the absence of the cytochrome bd oxidase, we also hypothesized that Q203 (and other I PA derivatives) is highly bactericidal against M. leprae.

Very much like M. leprae, M. ulcerans strains belonging to the classical lineage (responsible for >95% of the BU cases worldwide) do not express a functional bd oxidase due to a null mutation in cydA (1 1 ) and retain a conserved Q203-binding site, suggesting a high potency and bactericidal activity of Q203 against M. ulcerans. One of us had sequenced the genome of numerous M. ulcerans clinical isolates and it was confirmed that all African and Australian M. ulcerans isolates sequenced belonging to the classical lineage have a similar null mutation in cydA that result in a non-functional enzyme. The only exceptions that we could find were isolates belonging to the ancestral lineage causing sporadic cases in Japan, China and South America. These strains encode for a functional cytochrome bd oxidase.

Since the predicted Q203-binding site in qcrB is also conserved between M. tuberculosis and M. ulcerans (Figure 5), we also predicted that Q203 would be highly potent and bactericidal against M. ulcerans. We further tested this hypothesis by evaluating the potency of Q203 against M. ulcerans. Our data confirmed that:

1 . Q203 is highly potent against clinically-relevant classical lineage M. ulcerans isolates in vitro (MIC5o<1 nM, Figure 6 and MBC95 of 1 nM). A structure-activity relationship was established by profiling the potency of 85 IPA derivatives (Figure 22).

2. Q203 is extremely potent in a mouse model of Buruli ulcer. At a low dose of 0.5mg/kg given 3 times per week for 4 weeks, Q203 was curative when given alone (Figure 7). The mice treated with Q203 did not experience any relapse up to five months post- treatment (end of the experiment).

The maximum efficacy of Q203 in a mouse model of tuberculosis was achieved at 10mg/kg given 5 times per week for 4 weeks (3 Iog10 reduction in CFU, ref. 7). Since a cure was achieved in a mouse model of Buruli ulcer at 0.5mg/kg given 3 times per week for 4 weeks, Q203 appeared in fact to be even more promising for the treatment of Buruli ulcer than for the treatment of tuberculosis.

In more detail: IPA compounds show potent in vitro inhibitory activity against M. ulcerans

By performing resazurin-based medium-throughput screens as previously described (Scherr N, et al. Antimicrob Agents Chemother. 2012;56: 6410-6413), we tested a pre-selected panel of 85 published and/or commercially available IPA compounds for activity against two M. ulcerans strains isolated from Cameroonian BU patients. Based on the determined minimal inhibitory concentrations we grouped the compounds into activity categories (Figure 22). 20/85 (24 %) compounds were highly active with a MIC <0.1 μg/ml, with 8 of them having a MIC <0.01 μg/ml and a single compound - Q203 - even displaying a MIC <0.001 μg/ml (Figure 22). The SAR study of the IPA against M. ulcerans was found to yield largely similar results as the SAR study against M. tuberculosis (Kang S,et al. J Med Chem. 2014;57: 5293-5305). Small residues like halogen, methyl or ethyl were tolerated on the imidazopyridine core, while the optimum activity of the substitution pattern depended on the amide residue. For the amide residue, pphenylbenzylamides, preferentially substituted with p-CI, showed very high activities in the cell based assay. They were followed by p-(dialkylamino)benzylamides and palkoxybenzylamides. Aliphatic, heterocyclic or branched amides had significantly reduced activities. Recently, pharmacokinetic data for the p-phenylbenzylamides were described, indicating some limitations due to their high lipophilicity and superior pharmacokinetic properties for the Q203 substitution pattern. The IPA compounds Q203 and IPA-7 were selected from the pool of the nine highly active compounds for more detailed profiling. MIC values for Q203 and IPA-7 were determined by performing resazurin-based metabolic activity assays with four M. ulcerans clinical isolates belonging to the classical lineage (Kaser M, et al. BMC Evol Biol. 2007;7: 177). The strains tested included two low passage clinical isolates from Cameroon (S1013 and S1298), and one isolate each from Togo (S1 014) and from Australia (S1251 ). The measured MIC values were in the range of 0.6 ng/ml (1 nM) for Q203 and 10 ng/ml (25 nM) for IPA-7 (Figure 6). The dose- response data showed a sharp threshold for Q203, while the curve progression was less steep for IPA-7. Next, we performed time-kill kinetic assays by cultivating strain S1 013 in liquid broth medium in the presence of different concentrations of Q203 and IPA-7 (equivalent to 0.25x, 1 x, 2x, 4x, 8x and 16x the respective MIC) for different time periods (0, 3, 7, 14, 21 , and 28 days), before plating the bacteria for CFU determination. After incubation of plates for 16 weeks at 30 °C, CFUs were enumerated and in vitro kill-kinetic curves were generated (Figure 6). The results of the kill kinetic assays confirmed the MIC and doseresponse data obtained with the metabolic activity assays (Figure 6). Q203 exerted strong bactericidal effects (4-log10 CFU/ml reduction, 99.99 % killing) when bacteria were exposed for at least 14 days to minimally 1 x the MIC. For IPA-7, a reduction in bacterial numbers could also be observed after two weeks of drug treatment, but killing was less complete when bacteria were exposed to 1 x or 2x the MIC concentration (Figure 6).

Q203 shows potent in vivo activity against M. ulcerans

To monitor the response to antibiotic treatment in vivo, we infected BALB/c mice by foot pad injection with M. ulcerans. Progression of the infection was followed by weekly measurements of the foot pad thickness using a caliper (Figure 7A). Five weeks after infection, when the mice started to show swelling of the infected feet (Figure 7A), the treatment was initiated by orally administering either rifampicin (1 0 mg/kg), Q203 (0.5 mg/kg), IPA-7 (0.5 mg/kg) or a solvent control three times per week for a period of four weeks. A strong increase in foot pad thickness was observed for the controls and the IPA-7 treated mice during the four week treatment course (Figure 7A). Due to the severe progression of the infection, these animals had to be sacrificed after completion of the treatment. In contrast, a complete regression of the foot pad swelling was observed for the Q203 treated mice already 1 .5 weeks after start of treatment (Figure 7A). The foot pad thickness of these mice returned to normal levels and did not increase again for the entire observation period of 10 weeks post treatment (Figure 7A). For rifampicin treated mice, the foot pad thickness reached a plateau after 2 weeks of treatment and started again to slightly increase after completion of treatment (Figure 7A). Responses to the suboptimal treatment with rifampicin (10 mg/kg three times a week instead of the recommended daily administration) varied strongly between animals. The outcome of the different treatments is illustrated in Figure 7B where pictures of representative mice at the end of the treatment are shown. Histopathological analyses of foot pads taken at the end of the treatment revealed for the foot pads of the infected control animals (Figure 23A) and the IPA-7 treated mice (Figure 23B) oedema formation and tissue necrosis, two typical hallmarks of BU histopathology (Guarner J, et al. Emerg Infect Dis. 2003;9: 651 -656; Junghanss T, Johnson RC, Pluschke G. 42 - Mycobacterium ulcerans Disease. Manson's Tropical Infectious Diseases (Twenty-Third Edition). London: W.B. Saunders; 2014. pp. 519-531 .e2). Oedema formation and tissue necrosis were also present in foot pads of mice treated with rifampicin, however, to a much lower extent (Figure 23C). Strikingly, the foot pads of mice treated with Q203 looked almost identical to the noninfected control foot pads (Figure 23, D1 and E1 , respectively) and were completely devoid of oedema (Figure 23, D2) and tissue necrosis (Figure 23, D3). An IS2404- specific qPCR [21 -23] was used to monitor the amounts of M. ulcerans DNA in the infected foot pads. In addition, tissue sections were stained with ZN to assess the integrity of AFBs. The qPCR analysis showed strongly increasing bacterial loads for the control mice and IPA-7 treated mice during the four week treatment period (Figure 6A). These findings are in line with the histopathological analyses, showing large extracellular clusters of solid-stained AFB for these mice (Figure 24, B1 and B2, C1 and C2, respectively). In the mice treated with rifampicin, bacterial multiplication was halted during the four weeks of treatment, but the amount of DNA started again to increase after completion of treatment (Figure 24A). As for the foot pad thickness values, a large in-group variance was observed at week 15 (Figure 24A). Compared to the controls and IPA-7 treated mice the bacteria were present in smaller extracellular clusters (Figure 24, D1 ) and presented as a mix of solid-stained and beaded AFB (Figure 24, D2). In Q203 treated mice the amount of M. ulcerans DNA increased slightly during treatment, but dropped after completion of treatment (Figure 24A). Only small numbers of AFB were found in the tissue sections (Figure 24, E1 ), with the vast majority of these bacilli having a beaded, not solid-stained appearance (Figure 24, E2).

Discussion

Screening of a panel of I PA compounds allowed for the identification of a series of derivatives with high in vitro activities (MIC < 0.01 μg/ml) against M. ulcerans, with Q203 ranking at the top (MIC < 0.001 Mg/ml). Q203 represents the most active derivative of a lead optimization program for M. tuberculosis and it has been shown to have MIC50 values of 2,7 nM (in culture broth medium) and 0.28 nM (inside macrophages) (Pethe K, et al. Nat Med. 2013;19: 1 157-1 160). While the majority of potent anti-tubercular agents are less active against M u!cerans (N. Scherr, personal communication), Q203 displayed an even higher activity (MIC50 = 0.5 nM) against M. ulcerans. This was unexpected since Q203 is more than 5000 fold less effective against M. marinum (MIC50 = 3.5 μΜ) (Pethe et al., 2013), a close relative of M. ulcerans (Doig KD, et al. BMC Genomics. 2012;13: 258). M. ulcerans has diverged from a common ancestor with M. marinum by uptake of a virulence plasmid and by drastic genome reduction (Roltgen K, et al. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis. 2012;12: 522-529). Higher susceptibility to Q203 may thus be related to gene loss or inactivation. In the IPA lead optimization program that resulted in the development of Q203, the activity against M. tuberculosis was mainly improved through the introduction of a long and hydrophobic group at the R3 position (Pethe K, et al. Nat Med. 2013;19: 1 1 57-1 160). This seems to be also relevant for an Increase in potency against M. ulcerans, making Q203 an exceptionally active compound when compared with the in vitro activities of currently used BU drugs such as rifampicin (0.13 Mg/ml), streptomycin (0.25 μς/ιηΙ), ciprofloxacin (1 μς/ιηΙ), clarithromycin (0.25 [ig/m\) and amikacin (1 μ^η\\) (Zhang T, et al. Antimicrob Agents Chemother.201 0;54: 2806-2813; Thangaraj HS, et al. J Antimicrob Chemother. 2000;45: 231 -233; Portaels F, et al. Antimicrob Agents Chemother. 1 998;42: 2070-2073). To our knowledge, MIC values in the low nanomolar range against M. ulcerans have never been observed before. By using the BU mouse foot pad model, we evaluated the in vivo activity of Q203, IPA-7 and rifampicin. As expected, three applications of rifampicin (10 mg/kg) per week for a period of four weeks were not sufficient to achieve complete cure. The high in vitro activity of IPA-7 did not translate into a good in vivo potency at the chosen regimen, which was however highly effective for Q203. Ten weeks after treatment completion, the group of Q203 treated animals was still devoid of relapses. Histopathological analysis showed after treatment with Q203 predominantly beaded and intracellular AFB, reconfirming the efficient killing of the bacteria. To the best of our knowledge, a complete cure of all mice infected with M. ulcerans has never been accomplished before by a one month treatment with a single antibiotic administered only 3 times per week. If clinical development of Q203 against M. tuberculosis will be successful, this also holds great promises for the development of an alternative drug treatment regimen for M. ulcerans. This is the first time that a drug candidate demonstrates curative potency in a preclinical model of Buruli ulcer at such a low dose. Given the long half-life of Q203 (>20 hours), a curative single-dose treatment (or weekly administration) is conceivable. These findings are encouraging for the patient suffering from Buruli ulcer since the current WHO recommended antibiotic treatment is far from being ideal. It relies on the co-administration of streptomycin (injectable) and rifampin for eight weeks (12). The use of streptomycin is complicated due to the mode of administration, and the combination therapy is associated with ototoxicity and other potentially severe side effects.

We also predict that Q203 will be similarly highly potent and curative in a mouse model of leprosy.

Therefore, Q203 has the potential to revolutionize the treatment of Buruli ulcer and leprosy. Interestingly, a drug combination could be developed with bedaquiline (Sirturo^®) since both drugs targets the oxidative phosphorylation pathway, and share similar pharmacokinetic properties (e.g. half-life T1/2 > 20 hours). Furthermore, other IPA derivatives are highly potent against M. ulcerans. #86 was active against three clinical isolates at a MIC50 ranging from 38.8 nM to 66 nM, whereas #87 was active at a MIC50 ranging from 1 .3 nM to 2.3 nM (Figure 8). Therefore, #86 and #87 are promising drugs for the treatment of Buruli ulcer and leprosy.

References

1. Sassetti, C. M., et al. (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Molecular microbiology 48, 77-84

2. Matsoso, L. G., et al. (2005) Function of the cytochrome bc1 -aa3 branch of the respiratory network in mycobacteria and network adaptation occurring in response to its disruption.

Journal of bacteriology 187, 6300-6308

3. Berney M., et al. (2014)A Mycobacterium tuberculosis cytochrome bd oxidase mutant is hypersensitive to bedaquiline. MBio 5(4):e01275-14

4. Abrahams, K. A., et al. (2012) Identification of novel imidazo[1 ,2-a]pyridine inhibitors targeting M. tuberculosis QcrB. PloS one 7, e52951

5. Arora, K., et al. (2014) Respiratory flexibility in response to inhibition of cytochrome C oxidase in Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy 58, 6962-

6965

6. Moraski, G. C, et al. (201 1 ) Advent of lmidazo[1 ,2-a]pyridine-3-carboxamides with Potent Multi- and Extended Drug Resistant Antituberculosis Activity. ACS medicinal chemistry letters 2, 466-470 7. Pethe, K, et al. (2013) Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nature medicine 1 9, 1 157-1 160

8. Rybniker, J., et al. (201 5) Lansoprazole is an antituberculous prodrug targeting cytochrome bc1 . Nature communications 6, 7659

9. Kang, S., et al. (2014) Lead optimization of a novel series of imidazo[1 ,2-a]pyridine amides leading to a clinical candidate (Q203) as a multi- and extensively-drug-resistant antituberculosis agent. Journal of medicinal chemistry 57, 5293-5305

10. Kapopoulou A, Lew JM, Cole ST. (201 1 ) The MycoBrowser portal : a comprehensive and manually annotated resource for mycobacterial genomes. Tuberculosis (Edinb). 91 (1 ):8-13

11. Demangel, C, Stinear, T. P., and Cole, S. T. (2009) Buruli ulcer: reductive evolution enhances pathogenicity of Mycobacterium ulcerans. Nat Rev. Microbiol 7, 50-60

12. World Health Organization. Treatment of mycobacterium ulcerans disease (buruli ulcer).

Geneva: World Health Organization, 2012

Example 2: Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection

Materials and Methods

Strains and Growth Conditions. M. tuberculosis M37Rv, derivative strains, and clinical isolates (25) were maintained in Middlebrook 7H9 broth medium supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% ADS supplement. Hygromycin (75 Mg/mL) or kanamycin (20 Mg/mL) were used when required. Glycerol was omitted to determine drug potency. THP--1 cells were maintained In RPMI medium 1640 supplemented with 10% FBS, 2 mM L-glutamine, I 0 mM sodium pyruvate, and kanamycin (50 ixg/mL). M\Cm and MBC«, Determination, in this study, MIGso was defined as the lowest concentration of compound that inhibited bacterial growth by 50%. MICso was determined by the broth mlcrodilution method using a 96-well flat-bottom plate as described before (31 ), For MBGgo determination, mycobacterial Inoculum adjusted at an QDeoo of 0.005 was incubated in the presence of drugs for 10 d (replicating bacteria) or 15 d (nonreplicating mycobacteria) at 37 °C. Bacterial viability was determined by Colony Forming Units (CPUs) determination on agar plate. The Minimum Bactericidal Concentration leading to 90% reduction in CFU was defined as the MBCso.

Intracellular ATP quantification. The intracellular ATP level was determined with the RacTiter- Gio Microbial Cell Viability Assay (Promega) (10). Nutrient-Starved Culture. Exponentially growing cultures of M. tuberculosis were harvested by centrifugation and washed twice with prewarmed DPBS (Thermo Fisher Scientific) supplemented with Ca²⁺, Mg ²\ and 0.025% Tween 80, Cell density was adjusted to ODeoo of 0, 15 and incubated for 2 wk at 37 ^CC before testing sensitivity to drugs. Gene Knockout and Complementation. Two sets of cydAB (Rv1823c-1622c) deletion strains were constructed independently In the K.P. laboratory and in the M.B. laboratory using similar strategies based on the use of the plasmid pYUB1471 (32). in the K.P, laboratory, the pYUB1471 containing the 5^' and 3' flank of the cydAB locus was UV-irraciiated (33) before electroporatlon into M. tuberculosis, whereas in the M.B. laboratory, specialized transduction was used as described previously (32). Complementation plasmids were created by either Incorporating the cydABDC operon and its native promoter (330 bp upstream of fhe coding region) into the pMV306 vector (34) via Gibson cloning (35) (New England Biolabs), resulting in plasmid pMVSOo-cydABDC,. or by cloning the cydAB genes in the pMV306 plasmid under the control of the hsp60 promoter, resulting in the plasmid pMV308-cyc½S.

THP-1 Infection ModeL THP-1 cells were treated with 200 nM phorbol myristate acetate and were distributed at a density of 3 χ 10⁶ cells per well in 24-well plates. After 24 h of differentiation, the cell monolayers were infected with M. tuberculosis at a multiplicity of infection o; 10 for 80 mln. Prewarmed complete RPMi medium with or without the test drugs was added. Q203 was used at 250 nrn, whereas BDQ was used at 1 ,000 nM. Mycobacterial viability was determined after 5 d o; iniection by CPU determination on agar plates.

Mouse Experiments and Pathology. Mouse studies were performed in accordance with the National institutes of Health guidelines following the recommendations In the Guide for the Care and Use of Laboratory Animals (36). The protocols used in this study were approved by the institutional Animal Care and Use Committee of Albert Einstein College of Medicine (Protocol #201 50208). Female BALB/c mice (The Jackson Laboratory) were infected via aerosol infection at a dose Intended to yield an infection of 10³ CPU per mouse. Drug dosing was initiated 13 d postinfection. Drugs were formulated in 20% D-a-Tocophero! polyethylene glycol 1000 succinate (TPGS) per 1 % DMSO and administered via gavage three times per week. Infection In the lung was determined by CPU determination on agar plates at 13, 27, and 38 d. For pathological analysis and histological staining, lung samples were fixed in 1 0% (vol/Vol) neutral formalin, paraffin embedment, and fhe tissues were sectioned at 5 μηι. Sections were either stained with Hematoxylin & Eosln, or using the Kinyoun method for acid-fast bacilli.

Oxygen Consumption Assays. Oxygen consumption in whole bacteria was measured using methylene blue or the MitoXpress Xtra-Oxygen Consumption Assay (Luxcel Biosciences). Methylene blue-based assay. Mycobacteria culture adjusted to an ODeoo of 0.3 were preincubaied for 4 h in 2-mL screw-cap tubes in the presence of Q203 at 400 nM, BDQ at 500 nM. or 1 % DMSO (vehicle control}. Methylene blue at 0.001 % was added to each tube. The tubes were then tightly sealed, an Incubated in an anaerobic jar to avoid oxygen leak,

MstoXpress-based assay. The assay was performed in black 98-wei! plates (flat, clear bottom). One hundred fifty microliters of mycobacteria culture adjusted to an ODeoo of 0.3 were preincubaied for 6 h in the presence of Q203, BDQ, or 1 % DMSO. Ten microliters of the MitoXpress oxygen probe was added to each well that was covered with a layer of high- sensitivity mineral oil to restrict oxygen back diffusion. Fluorescence (Ex: 380 nm, Em: 650 nm) was recorded on a BioTeK CYTATION 3 multimode reader.

Preparation of inverted Membrane Vesicles, Bacillus Calmette-Guerin was grown at 37 C in 7H9-ADS medium. Bacteria were harvested by centrlfugation when the cultures reached an ODeoo of 0.8. Five grams of cells (wet weight) were suspended in 20 mL of 50 rnM Mops-NaOH (pH 7.5), 2 mM MgCia supplemented with protease Inhibitors (protease inhibitor mixture tablets, Roche). Lysozyme (1 .2 mg/mL), 1 ,500 units of DNase i (Sigma), and 6.7 rnM MgCfe were added, and the cells were incubated under stirring conditions for 45 mln at room temperature. The bacteria were then lysed by five passages using a precooled French pressure cell at 25,000 psi (M-1 10L MlciOfluidlser). The lysate was centrifuged at 4,200 g at 4 °C for 20 rnin to remove unbroken bacteria. The supernatant was ultraeentriiuged at 450,000 g for 1 h at 4 °C. The pellet of inverted Membrane Vesicles (iMVs) was resuspended In an appropriate volume of 50 mM Mops-NaOH (pH 7.5), 2 mM MgClj:, and 15% glycerol. Protein estimation was performed using the BCA Protein assay (Thermo Fisher Scientific).

Oxygen Consumption Assay. The MitoXpress oxygen probe was used to quantify oxygen consumption In iMVs. The iMVs (150 μΙ_ of 300 μρ/Γη!..} were preincubaied for 5 mln with a dose range of Q203 or bedaquiline in a prewarmed 50 mM Mes buffer (pH 6.5} supplemented with 2 mM MgCls. NADH was added at a final concentration of 1 mM as electron donor. Ten micro!iters of the MitoXpress oxygen probe were added to each well and covered with a layer of high- sensitivity mineral oil. Fluorescence (Excitation: 380 nm, Emission: 650 nm) was recorded after 30 rnin of incubation using a BioTeK CYTATION 3 multimode reader.

The emergence and spread of drug resistance in pathogenic mycobacteria poses serious global health concerns. Tuberculosis (TB) continues to cause 1 .4 million deaths in HIV-negative Individuals and 10.4 million new cases in 2015 (1 ). ft was recently evaluated that the number of TB cases in India Is two to three times higher than previously estimated (2), suggesting that the global number of TB cases may be largely underestimated. Despite progress in public health management and the use of fixed-dose combinations, the number of multi- and extensively drug-resistant (M-XDR) TB cases continues to rise {^■). According to the last WHO report, the proportion of multidrug-resistant tuberculosis among newly diagnosed cases Is a staggering 5,6% (^') }. In 2015, 580,000 new patients were eligible for MDR-TB treatment. MDR-TB treatment Is challenging because it requires the administration of second-line drugs for up to 2 y (3), with an estimated global success rate of 52% and an unacceptable mortality rate (3 -, There- Is a pressing clinical need for the development of new drugs able to shorten the treatment of MDR-TB to 6 mo or less. More than new drugs, a rational drug combination made of complementary agents is urgently needed. Despite increasing interest from the scientific community, the global drug pipeline remains thin: only a very few new chemical entitles have entered clinical development in the last 40 y (4). The recent approval of bedaquiline (BDQ, S!riuro) represents a critical milestone In anti-TB drug discovery (5-7). Nevertheless, the successful advance of BDQ Is overshadowed by the emergence of clinical resistance less than 3 y after its Introduction to medical use (8), The rapid emergence of resistance is most likely linked to the absence of potent companion drugs, indeed, BDQ is currently given in combination with weaker second- and third-line drugs, imposing a strong selection pressure for BDQ resistance. This reinforces the notion that a rational drug combination of complementary drugs Is required to shorten the treatment time of MDR-TB.

The discovery of BDQ, a potent inhibitor of the mycobacterial Fi F₀-ATP synthase, validated oxidative phosphorylation (OxPhos) (Fig. 1 } as an attractive drug target in M. tuberculosis. OxPhos is an ubiquitous metabolic pathway, in which the energy contained in nutrients is used to generate an electrochemical gradient, also called the proton motive force {pmf), that drives the synthesis of Adenosine Tri- Phosphate (ATP). The pmf is required for the survival of both replicating and nonrepeating (often referred to as dormant} mycobacteria (9,1 0). Dissipation of the pmf leads to a rapid loss of cell viability and cell death. Therefore, drugs targeting enzymes Involved in pmf generation are predicted to reduce time of therapy by killing phenotypic drug- resistant bacterial subpopulations (1 1 ). in M, tuberculosis, the generation of the pmf is mediated primarily by the proton-pumping components of the electron transport chain (ETC). Under aerobic conditions, the ETC of M tuberculosis branches into two terminal oxidases; the proton-pumping cytochrome bcraa3 supercompiex (Cyt-fcc/iaas) and the less energy efficient, but higher-affinity cytochrome bd oxidase (Cyt- fccf) (12-15). in recent years, the discovery of several small molecules targeting the Cyt-5c_;:aa;i branch (16-21 ) has triggered interest in the heme-copper respiratory oxidase (18, 20). All small-molecule inhibitors discovered to date seem to target the cytochrome b subunit of the bc< complex (16-21 ). The best characterized compounds targeting cytochrome be, are a series of imidazopyriciine amides (ΊΡΑ) (16, 18-20}. The most advanced SPA derivative is Q203, a drug candidate currently In clinical trial phase I under a US FDA investigational New Drug application (22). However, despite the reported susceptibility of the Cyt-bcr.aa.3 to chemical inhibition, the influence of the alternative Cyt-bd terminal oxidase on the potency of Q203 and related drugs remains to be defined. Here, through a combination of chemical biology and genetic approaches, we reveal the existence of a synthetic lethal interaction between the Cyt- bci'.am and the Cyt-fcd terminal oxidases. A synthetic lethal interaction is a well-described phenomenon where the single !nactivation of two genes has little effect on cell viability, whereas the simultaneous inactivation of both genes results in cell death (23), Upon chemical Inhibition of the Cyt-hci:aa3 complex, respiration through Cyt-fccf is sufficient to maintain the viability of replicating and nonreplicating mycobacteria. However, simultaneous Inhibition of both terminal oxidases was sufficient to inhibit respiration, kill phenotypic drug-resistant persisters, and rapidly eradicate M. tuberculosis infection in vivo.

G2Q3 is a Bacteriostatic Agent that Does Not Inhibit Oxygen Respiration

The metabolic consequences of the chemical inhibition of the mycobacterial C^.-bcr.aas have not been studied in detail. A recent study revealed thai Q203 and BDQ treatment triggered an increase in oxygen consumption rate (OCR) up to 1 6 h postf.reat.ment, which Is counterintuitive given the capacity of the drugs to interfere with respiration. Interestingly, increase in OCR was only observed at a very high dose o; drugs {300χ MIC), but not at an Intermediate dose (30 χ MIC) (24). Consequently, we were interested in gaining more mechanistic insight into the ETC adaptations to Q203 and BDQ and their long-term effects on oxygen respiration. Using methylene blue as an oxygen probe, we made the observation that oxygen consumption was significantly inhibited by BDQ treatment over a 96-h period, but was unaffected by Q203 treatment (Fig. 9A). To ensure that these results were not an artifact due io the inability of Inhibitors of the Cyt-bci'.aas io Inhibit growth of laboratory strains of M. tuberculosis (17), we verified the potency of Q203 against five clinical isolates (N0052, N0072, N0145, N0157, N0155) from different M. tuberculosis lineages (25) and M bovis bacillus Calmette-Guerln. We confirmed thai Q203 has excellent growth inhibitory potency against all these strains (Fig. 9B). Q203 had a Minimum Inhibitory Concentration leading to 50% growth inhibition (MiCso) of 1 .5- 2.8 nM, whereas BDQ was active in a MlGso range of 42-133 nM (Fig. 9B and Table 4}. Altogether, these results suggested that chemical inhibition of the CyX-bcf.aae terminal oxidase led to bacterial growth arrest without affecting oxygen consumption. Because BDQ inhibited oxygen respiration over a 96-h period, whereas Q203 did not (Fig. 9A), we were next interested In testing the correlation between inhibition of oxygen consumption and bacterial death. interestingly, we observed that despite the superior potency of Q203 in the growth Inhibition assay, the drug candidate was much less effective at killing M, tuberculosis compared with BDQ. BDQ was bactericidal against four strains of M. tuberculosis at a concentration 5- to 12- fold above its MiCso (Fig, 9C), whereas Q203 was bacteriostatic even at doses exceeding 200- fold its MICso (Fig. 9G). Similar results were observed in Mycobacterium hovis bacillus Ga!mette-Guerin (Fig. 1 0). Because BDQ and Q203 target the same pathway (OxPhos), but have a striking difference on mycobacterial viability, we hypothesized that an alternate branch of the ETC may compensate for the chemical inhibition of the Cyt-6c_f:aa<i terminal oxidase.

Table 4. MICso and MBG90 of Q203 and BDQ against H37Rv, five M. tuberculosis clinical isolates, and M. bovis bacillus Calmette-Guerin

The ratio between the MICse ε tnd the MBCso is shown. 1 ixpenmenis Viiere performed in triplicate and repeated at least once. MICso results are expressed as the mean ± SDs of a representative experiment, n.d., not determined.

Cytochrome bd oxidase-Type Oxidase Compensates for Chemical inhibition of the Cytochrome bci:aas Branch

The Involvement of Gyt-oe? in a possible compensatory mechanism was investigated. The cydAB genes (coding for Cyt-M) were deleted in M. tuberculosis H37Rv and Mycobacterium bovis bacillus Calmette-Guerin (bacillus Caimette-Guerin), leading to strains H37Rv AcydAB, and bacillus Calmette-Guerin AcydAB. Deletion of cydAB did not Impact significantly on bacterial growth and ATP homeostasis (Fig. 13). The synthetic lethal interaction between the Cvi-bcr.aas and the Cyt-M was evaluated by treating the mutant strains with Q203. Deletion of cydAB had a modest effect on the growth inhibitory potency of Q203 (Fig, 14), bur a profound Impact on the capacity of mycobacteria to respire with oxygen over a prolonged period (Fig. 1 1 ). Using methylene blue as an oxygen probe, we observed that treatment of H37Rv AcydAB or bacillus Calmette-Guerin AcydAB with Q203 led to an apparent complete inhibition of oxygen respiration (Fig. 1 1 , insets, and Fig. 15). This phenotype was reversed by expressing the cydAB operon in the mutant strains (AcydABcomp strains} (Fig. 1 1 , Insets, and Fig. 15}. The inability of the Cyt-oc? mutant to utilize oxygen was confirmed by measuring the Relative Oxygen Consumption rate (ROC) using the MitoXpress Oxygen probe in whole ceils over a short period (Fig. 12). Under our experimental conditions, G203 had no significant effect on oxygen respiration in the parental strain, buf triggered a complete inhibition of oxygen consumption in H37Rv AcydAB at an iGso of 3.1 nM (Figure 1 1 Band D). These results were corroborated in Inverted membrane vesicles with NADH as the electron donor (Fig. 18). Furthermore, G203 treatment led to a decrease In ATP levels in the parental H37Rv strain, but to a lesser extent compared with BDQ treatment (Fig. 12A). Q203 treatment was more effective at disrupting ATP homeostasis in H37Rv AcydAB compared with the parental strain (Fig. 12B). Similar results were obtained in bacillus Calmette-Guerin {Fig. 17), Because the effect on oxygen consumption correlated with reduced AT P levels in Q203-treated AcydAB strains, we hypothesized that electron flow diverted to the Cyt-M branch upon chemical Inhibition o; the Cyi-bcf.aas was sufficient to maintain cell viability. Consistent with this hypothesis, Q203 displayed a dose- dependent bactericidal effect against H37Rv AcydAB (Fig. 12D). Under the same conditions, the bactericidal potency of BDQ was unaffected by cydAB deletion (Fig. 12D). Similar results were obtained in bacillus Calmette-Guerin (Fig. 17D). Altogether, these findings established a strong synthetic lethal interaction between Cyt-fcc?:aa.¾ and Cyt-bd and the requirement for at least one terminal oxidase to maintain cell viability in mycobacteria.

Cytochrome bd oxidase Type Oxidase Protects Nonrepeating Mycobacteria from Q203- Induced Bacterial Death

Next, the impact of Q203 treatment on ATP homeostasis and viability of nutrient-starved, phenotypic drug- resistant mycobacteria was evaluated. Q203 treatment in the parental strain resulted in a dose-dependent reduction in ATP levels, but without affecting cell viability (Fig. 18 A and C). Under similar experimental conditions, BDQ was bactericidal (Fig. 18C). It was noted that ATP depletion Induced by Q203 treatment in the parental strain was significantly less compared with BDQ treatment (Fig. 1 8 A and B). As observed under replicating conditions, Q203 treatment triggered a more profound ATP depletion in the nutrient-starved H37Rv AcydAB strain compared with the parental strain (Fig. 18A) and was bactericidal (Fig. 18C). The effect on cell viability was profound because G203 at 100 nM killed more than 99.99% of the nonreplicating H37Rv AcydAB strain (Fig. 1 8C). The phenotype was reversed in the H37Rv AcydABcomp strain (Fig. 18C}. Similar results were obtained in bacillus Calmette-Guerin (Fig. 19). These data further demonstrated that the respiratory terminal oxidases are jointly required for oxidative phosphorylation and that simultaneous inactivation of both has a striking effect on the viability of nonreplicating, phenotypically drug-resistant mycobacteria. Synthetic Lethal interaction Between the Respiratory Terminal Oxidases During Infection To test whether the synthetic lethal interaction between the Cyt-.de_;:aa.3 and the Cyx-bd was relevant during infection, the potency of Q203 was evaluated against the H37Rv, H37Rv AcydAB., and complemented strains replicating in THP-1 cells. Bacterial viability was evaluated after 5 d of treatment with Q203 or BDQ. Results revealed that the multiplication profile of the H37Rv AcydAB strain was comparable to the parental H37Rv strain (Fig, 20 A and B), suggesting that the Cyt-fcd alone does not contribute to growth in macrophages. As reported before, Q203 was active against the parental H37Rv strain replicating in macrophages (18, 20). However, the effect of the drug candidate was bacteriostatic (Fig. 2QA). In line with the in vitro phenotypes, Q203 was bactericidal against the H37Rv AcydAB strain replicating in THP-1 cells (Fig. 20B). BDQ was active against intracellular mycobacteria, regardless of the presence of the Cyt-M (Fig. 20A-C). Phenotypes were reverted In the H37Rv AcydABcomp strain (Fig. 20C). This result showed that oxygen respiration contributes to the virulence of M. tuberculosis in a macrophage model and that at least one of the terminal oxidases was required for respiration and energy production in an ex vivo infection model. This finding prompted us to investigate the joint essentiality oi the terminal oxidases In a mouse model oi tuberculosis. BALB/c mice Infected by the aerosol route with the H37Rv, AcydAB, and AcydABcomp strains were treated with Q203 at 2 mg/kg, BDQ at 10 mg/kg, or with the vehicie control three times per week. The H37Rv AcydAB strain had no obvious attenuation phenotype during the course of the infection, but was dramatically more sensitive to Q203 compared with the parental H37Rv or AcydABcomp strains (Fig. 20D-F). During the first 2 wk of treatment, Q203 reduced the bacterial load in the lungs of animals infected by the mutant strain by more than 99% (Fig. 20D}. During the same time frame, Q203 had no significant efficacy against the parental strain (Fig. 20D). Strikingly, after 4 wk of treatment, the bacterial count in the mice infected by the H37Rv AcydAB strain and treated with Q203 had dropped below the limit of detection in 3 out of 5 mice (Fig. 20 D). Using this suboptimal dosing regimen, Q203 had no significant effect against the parental H37Rv and the AcydABcomp strains (Fig. 20D). Although BDQ was effective against the parental strain, there was still an eightfold increase in sensitivity of the H37Rv AcydAB strain compared with the parental strain after 4 wk of BDQ treatment (Fig. 20F}. It was interesting to note that the potency of Q203 against the H37Rv AcydAB strain was radically superior compared with BDQ (Fig. 20 D and F). Gross lung pathology (Fig. 20 G-l) and FI&E staining (Fig. 21 ) corroborated these results. Lungs infected with H37Rv AcydAB and treated with Q203 showed no signs of typical lesions, nor any inflammation foci after 4 wk of treatment (Fig. 20G and Fig. 21 A). In contrast, multiple lesions and inflamed foci were found in the lungs of the mice Infected with the parental, or the complemented strain, that were treated by Q203 (Fig. 20G and Fig. 21 D and G). These results demonstrate the efficacy of a therapeutic approach that exploits the synthetic lethal interaction between Cyt-fcc,>:a¾ and Cyt-fcd oxidases. Table 5. Mouse infection and ireaimenr with Q203 or BDQ

H37RV .i4>'i¾S SC>47¾SCi7iKp

Tre;3tfS£¾t : ! ::··^> Average SO Average SD Average

(C FU/ίΗΪ. ) (CR;/;sL) (f.Ri/ssi. ) (C PO/i!i!. ) (CRJ/isL)

Veh icle 1 4 EiE÷8i 5 4Ε÷&Ι 7 8£-fg>2 2 . 8C+S2 4. 7E+S2 9. SE-H51

13 6 5Ϊ+84 2 SE-f84 2 4£÷85 4. δ£÷&4 I 5E÷S5 8 , St÷«4

27 4 : 7 ¾£ ÷05 2 '4 7 , iJf.-s-¾?j z si : . 4; _S :.::.·

38 .it -86 1 2E-;-»8 'i / . 1 I < 44 4 4¾ ! 4e

BDQ 1 4 8E-f62 5 ·; · 4; · 7 t43÷82 2 . 8E-f£>2 4 7£-f82 9.8E+«1

17 f= '·.: : 04 9E-;-¾4 2 4Βί-ίί5 I S , 6ii-f¾4

2 ? 4 7E÷<55 2 4 £4i÷i¾ l . SE*g>5 7 ©£+05 5 .

38 2 1E÷8S 9 8E÷94 9 8Ε+84 3 , 2 . 5E+35

02»3 :·. 4 5 7 ' 4 44> 2 . 4 7E÷S2

6 =>· ; «·; 2 2 4E-+S¾ 4 , &ϊ4ί-β4 3 ¾es 4 . 4: _S ;:4

:·^: / i 4 44. ? a; a)': I 5 . ¾^" I •^•4 ! 4e 4 . - 4 > 4\

1 1 / : 44- 3, 3E +S2 v . ^:4 > :/·,· Z 7! ^•- 44 S 4^:>

BALB/c mice were aerosol-infected with either the M, tuberculosis H37Rv, AcydAB, or AcydAScomp strains. Treatment with Q203, BDQ, or with the vehicle control was initiated 14 d postinfection. Baciilary burden (CPU} in lungs of infected animals was assessed after 2 wk (day 27) and 4 wk (day 38) of drug treatment. Bacterial burden was also assessed at day 1 and day ^'13 postinfection to confirm bacterial colonization before drug treatment. The average of four mice per time point and per condition is shown.

Discussion

M. tuberculosis is an obligate aerobe that can survive, but not replicate, under hypoxic conditions. The reasons for the strict dependence on oxygen for growth are poorly understood but illustrate the prominence of aerobic respiration and the terminal respiratory oxidases for the biology of this bacterium (1 1 ). in the past 10 y the discovery of drugs active against enzymes of the mycobacterial oxidative phosphorylation pathway, namely, Inhibitors of ATP-synthase (BDQ) and Cyt-bcr.aas (imidazopyridine amices), have confirmed this vulnerability. Here, we show that rapid killing and bactericidal activity against M. tuberculosis can be achieved by exploiting the synthetic lethal interaction between the two terminal oxidases of the electron transport chain. Synthetic lethal relationships likely arise in biological systems to create functional redundancies that mitigate the impact of loss-of-function mutations or inhibition of a single enzyme. The presence of two terminal respiratory oxidases is a perfect example of such a functional redundancy. In this study, we coniirmed that chemical inhibition of the Cyl-bci'sas branch by Q203 inhibited mycobacterial growth at a very low dose, but revealed that the drug candidate was not bactericidal even at a concentration 200-fold in excess of its MICso. We demonstrated that respiration through the alternate Cyt-bd terminal oxidase alone is sufficient to maintain mycobacterial viability but insufficient to sustain growth. This discrepancy is likely due to a difference in energetic efficiency of the two terminal oxidases as the Cyf-fcC/iaa* complex pumps 6 protons per 2 electrons (H⁺/e- ratio of 3), whereas the ratio is only 1 H⁺/e^" for Cyt-M (1 1 , 28, 27), and this might also explain the failure to isolate deletion mutants of genes that encode Gyt- bciiaas In M. tuberculosis (28, 29). As a logical consequence of the functional redundancy, deletion of cydAB led to hypersusceptlbHity to Q203 with complete Inhibition of oxygen consumption, an enhanced effect on ATP homeostasis, and bactericidal action at low dose against replicating and nonrepeating mycobacteria. Importantly, our results show that oxygen respiration is essential for the survival of nutrient-starved, phenotypic drug-resistant mycobacteria, validating avenues for drug development. Under the in vitro conditions used in this study, the presence of the Cyt-bd did not influence the potency of BDQ. A recent report demonstrated that the early killing rate of BDQ is enhanced In an M. tuberculosis AcydA strain (12). Our results are not necessarily in contradiction because In the present study, the bactericidal potency of BDQ was determined at only one late time point. The observation that the H37Rv AcydAB strain has a slight, yet significant increase in sensitivity to BDQ compared with the parental strain in the mouse model supports the previous observation (12), The most critical finding of this study is the rapid clearance of the H37Rv AcydAB strain by Q203 in a mouse model of tuberculosis. Alter 4 wk of treatment with Q203 at only 2 mg/kg, near- eradication of H37Rv AcydAB was achieved, whereas the same drug treatment had no significant effect against the parental strain. This result illustrates the powerful synthetic lethal Interaction between both terminal oxidases and demonstrates that, at least in the microenvironment of the mouse lung, M. tuberculosis relies primarily on oxygen respiration to multiply and persist.

The synthetic lethal interaction between the Cyt-bci:aas and the Cyt-bd could have consequences for the clinical development of Q203. Because the electron flow through the Cyt- bd is sufficient to maintain respiration and viability of Q203-treated mycobacteria, it is uncertain

If drug candidates targeting the Cyt-bcr.aas will show efficacy in humans. An important characteristic of human tuberculosis disease is the manifestation of a range of lesions with different microenvironmental conditions, including varying oxygen tensions (30). The expression ratio of Gyt-tei :aa3 and Cyt-bd is likely to play an integral role in the adaptation to this heterogeneity, further underlining the Importance of a combination therapy targeting both terminal oxidases. It Is possible that Q203, or other advanced derivatives, will be active against human tuberculosis when used in a combination drug therapy. However, based on the data presented here, it is unlikely that inhibition of the Cyt- ter:aa3 alone would lead to bacterial sterilization under all physiological conditions. To unleash the full potential of drugs targeting the Cyt-i3c_;:a,¾ branch, we advocate for the development of Cyt-bd inhibitors, it was previously suggested that Interference with oxidative phosphorylation at multiple levels is a promising anti- TB strategy (24). Our data indicate that a drug combination targeting simultaneously the Cyt- .dc_;:aa.3, the Gyi-M, and the Fi F_u-ATP synthase may represent the cornerstone of a complementary sterilizing drug combination for the treatment of MDR and XDR tuberculosis.

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36. Anonymous (Committee on Care and Use of Laboratory Animals) (1996) Guide for the Care and Use of Laboratory Animals (Natl Inst Health, Bethesda), DHHS Publ No. 85-23. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. The word "comprise" or variations such as "comprises" or "comprising" will accordingly be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

Claims

CLAIMS What is claimed is:

1 . Method of treating or preventing an infection of a mycobacterium deficient for cytochrome bd oxidase or a disease resulting from said infection in a subject, the method comprising administering to the subject an effective amount of a compound capable of inhibiting cytochrome bd of the respiratory electron transport chain in said mycobacterium.

2. The method of claim 1 , wherein the subject is a mammal, preferably a human.

3. The method of claim 1 or 2, wherein the mycobacterium is selected from the group consisting of M. ulcerans, M. leprae, M. lepraemurium, and M. lepromatosis.

4. The method of any one of claims 1 -3, wherein the mycobacterium is M. leprae and the disease is leprosy.

5. The method of any one of claims 1 -3, wherein the mycobacterium is M. ulcerans and the disease is Buruli ulcer.

6. The method of any one of claims 1 -5, wherein the compound is of formula (I) or (II),

wherein each X is independently N, C-R3, or C-FU; with the proviso that no more than two

X's are N; wherein Ri and R2 are each independently hydrogen, acyl group, alkenyl group, alkoxy group, alkoxycarbonyl group, alkoxycarbonyloxy group, alkoxysulfonyloxy group, alkyl group, alkylamino group, alkylaminocarbonyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkylsulfonyl group, alkylsulfonyloxy group, alkylthio group, alkynyl group, amide group, amidine group, amino group, arylalkoxy group, arylalkyl group, aryl group, arylcarbonyl group, arylcarbonyloxy group, aryloxy group, aryloxycarbonyl group, aryloxycarbonyloxy group, aryloxysulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, azido group, carbamido group, carbamoyl group, carbazoyi group, carbonyl group, carboxylate group, carboxylic acid group, cyanato group, cyano group, cycloalkenyl group, cycloalkyl group, dialkylamino carbonyl group, dialkylamino group, guanidino group, guanyl group, halo group, heteroarylalkoxy group, heteroarylalkyl group, heteroaryl group, heteroarylcarbonyl group, heteroaryloxy group, heterocyclic group, hydroxamino group, hydroxy group, imino group, isocyanato group, isocyano group, mercapto group, nitro group, oxo group, perhaloalkenyl group, perhaloalkoxy group, perhaloalkyl group, perhaloalkynyl group, perhaloarylalkyl group, perhaloaryl group, perhalocycloalkyl group, phosphate group, phosphine group, phospho group, sulfate group, sulfo group, sulfonyl group, oxidized form thereof, substituted form thereof, heteroatom form thereof, or combination thereof;

wherein each R4 is independently hydrogen, "D-group", acyl group, alkenyl group, alkoxy group, alkoxycarbonyl group, alkoxycarbonyloxy group, alkoxysulfonyloxy group, alkyl group, alkylamino group, alkylaminocarbonyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkylsulfonyl group, alkylsulfonyloxy group, alkylthio group, alkynyl group, amide group, amidine group, amino group, arylalkoxy group, arylalkyl group, aryl group, arylcarbonyl group, arylcarbonyloxy group, aryloxy group, aryloxycarbonyl group, aryloxycarbonyloxy group, aryloxysulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, azido group, carbamido group, carbamoyl group, carbazoyl group, carbonyl group, carboxylate group, carboxylic acid group, cyanato group, cyano group, cycloalkenyl group, cycloalkyl group, dialkylamino carbonyl group, dialkylamino group, guanidino group, guanyl group, halo group, heteroarylalkoxy group, heteroarylalkyl group, heteroaryl group, heteroarylcarbonyl group, heteroaryloxy group, heterocyclic group, hydroxamino group, hydroxy group, imino group, isocyanato group, isocyano group, mercapto group, nitro group, oxo group, perhaloalkenyl group, perhaloalkoxy group, perhaloalkyl group, perhaloalkynyl group, perhaloarylalkyl group, perhaloaryl group, perhalocycloalkyl group, phosphate group, phosphine group, phospho group, sulfate group, sulfo group, sulfonyl group, oxidized form thereof, substituted form thereof, heteroatom form thereof, or combination thereof;

wherein one or more R3 or FU groups may join and form a fused ring with one or more other R3, FU, or combination of R3 and FU groups;

or resonance form thereof, or salt thereof, or salt of resonance form thereof.

7. The method of claim 6, wherein the compound is any one of compounds #1 -9 and 86-87, preferably any one of compounds #1 and 86-87,

8. The method of any one of claims 1 -7, wherein the method kills the mycobacterium.

9. Method of treating or preventing an infection of a mycobacterium expressing cytochrome bd oxidase and/or a disease resulting from said infection in a subject, the method comprising administering to the subject an effective amount of a compound capable of inhibiting cytochrome bd in said mycobacterium in combination with an effective amount of an additional therapeutic agent capable of inhibiting cytochrome bd oxidase in said mycobacterium.

10. The method of claim 9, wherein the subject is a mammal, preferably a human.

1 1 . The method of claim 9 or 1 0, wherein the mycobacterium is selected from the group consisting of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium "hominissuis", M. colombiense, M. asiaticum, M. gordonae, M. gastri, M. kansasii, M. hiberniae,

M. nonchromogenicum, M. terrae, M. triviale, M. pseudoshottsii, M. shottsii, M. triplex, M. genavense, M. florentinum, M. lentiflavum, M. palustre, M. kubicae, M. parascrofulaceum, M. heidelbergense, M. interjectum, M. simiae, M. branderi, M. cookii, M. celatum, M. bohemicum,

M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum, M. senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M. parafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii, M. wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M. arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, M. seoulense, and M. tokaiense.

12. The method of any one of claims 9-1 1 , wherein the mycobacterium is M. tuberculosis and the disease is tuberculosis.

13. The method of any one of claims 9-12, wherein the compound is of formula (I) or (II),

wherein each X is independently N, C-R3, or C-FU; with the proviso that no more than two X's are N;

wherein Ri and R2 are each independently hydrogen, acyl group, alkenyl group, alkoxy group, alkoxycarbonyl group, alkoxycarbonyloxy group, alkoxysulfonyloxy group, alkyl group, alkylamino group, alkylaminocarbonyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkylsulfonyl group, alkylsulfonyloxy group, alkylthio group, alkynyl group, amide group, amidine group, amino group, arylalkoxy group, arylalkyl group, aryl group, arylcarbonyl group, arylcarbonyloxy group, aryloxy group, aryloxycarbonyl group, aryloxycarbonyloxy group, aryloxysulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, azido group, carbamido group, carbamoyl group, carbazoyl group, carbonyl group, carboxylate group, carboxylic acid group, cyanato group, cyano group, cycloalkenyl group, cycloalkyl group, dialkylamino carbonyl group, dialkylamino group, guanidino group, guanyl group, halo group, heteroarylalkoxy group, heteroarylalkyl group, heteroaryl group, heteroarylcarbonyl group, heteroaryloxy group, heterocyclic group, hydroxamino group, hydroxy group, imino group, isocyanato group, isocyano group, mercapto group, nitro group, oxo group, perhaloalkenyl group, perhaloalkoxy group, perhaloalkyl group, perhaloalkynyl group, perhaloarylalkyl group, perhaloaryl group, perhalocycloalkyl group, phosphate group, phosphine group, phospho group, sulfate group, sulfo group, sulfonyl group, oxidized form thereof, substituted form thereof, heteroatom form thereof, or combination thereof;

wherein each R3 is independently hydrogen, "C-group", acyl group, alkenyl group, alkoxy group, alkoxycarbonyl group, alkoxycarbonyloxy group, alkoxysulfonyloxy group, alkyl group, alkylamino group, alkylaminocarbonyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkylsulfonyl group, alkylsulfonyloxy group, alkylthio group, alkynyl group, amide group, amidine group, amino group, arylalkoxy group, arylalkyl group, aryl group, arylcarbonyl group, arylcarbonyloxy group, aryloxy group, aryloxycarbonyl group, aryloxycarbonyloxy group, aryloxysulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, azido group, carbamido group, carbamoyl group, carbazoyl group, carbonyl group, carboxylate group, carboxylic acid group, cyanato group, cyano group, cycloalkenyl group, cycloalkyl group, dialkylamino carbonyl group, dialkylamino group, guanidino group, guanyl group, halo group, heteroarylalkoxy group, heteroarylalkyl group, heteroaryl group, heteroarylcarbonyl group, heteroaryloxy group, heterocyclic group, hydroxamino group, hydroxy group, imino group, isocyanato group, isocyano group, mercapto group, nitro group, oxo group, perhaloalkenyl group, perhaloalkoxy group, perhaloalkyl group, perhaloalkynyl group, perhaloarylalkyl group, perhaloaryl group, perhalocycloalkyl group, phosphate group, phosphine group, phospho group, sulfate group, sulfo group, sulfonyl group, oxidized form thereof, substituted form thereof, heteroatom form thereof, or combination thereof; and

wherein each FU is independently hydrogen, "D-group", acyl group, alkenyl group, alkoxy group, alkoxycarbonyl group, alkoxycarbonyloxy group, alkoxysulfonyloxy group, alkyl group, alkylamino group, alkylaminocarbonyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkylsulfonyl group, alkylsulfonyloxy group, alkylthio group, alkynyl group, amide group, amidine group, amino group, arylalkoxy group, arylalkyl group, aryl group, arylcarbonyl group, arylcarbonyloxy group, aryloxy group, aryloxycarbonyl group, aryloxycarbonyloxy group, aryloxysulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, azido group, carbamido group, carbamoyl group, carbazoyl group, carbonyl group, carboxylate group, carboxylic acid group, cyanato group, cyano group, cycloalkenyl group, cycloalkyl group, dialkylamino carbonyl group, dialkylamino group, guanidino group, guanyl group, halo group, heteroarylalkoxy group, heteroarylalkyl group, heteroaryl group, heteroarylcarbonyl group, heteroaryloxy group, heterocyclic group, hydroxamino group, hydroxy group, imino group, isocyanato group, isocyano group, mercapto group, nitro group, oxo group, perhaloalkenyl group, perhaloalkoxy group, perhaloalkyl group, perhaloalkynyl group, perhaloarylalkyl group, perhaloaryl group, perhalocycloalkyl group, phosphate group, phosphine group, phospho group, sulfate group, sulfo group, sulfonyl group, oxidized form thereof, substituted form thereof, heteroatom form thereof, or combination thereof; wherein one or more R3 or R4 groups may join and form a fused ring with one or more other R3, R4, or combination of R3 and R4 groups;

or resonance form thereof, or salt thereof, or salt of resonance form thereof.

14. The method of claim 13, wherein the compound is any one of compounds #1 -9 and 86-87, preferably any one of compounds #1 and 86-87,

15. The method of any one of claims 9-14, wherein the additional therapeutic agent is any one of quinolone compounds, Aurachin, nitric oxide (NO) donors such as PA-824, antibiotics LL- Z1272, Gramicidin S, and derivatives thereof.

16. The method of any one of claims 9-15, wherein the method kills the mycobacterium.