Diarylquinolines Are Bactericidal for Dormant Mycobacteria as a Result of Disturbed ATP Homeostasis

An estimated one-third of the world population is latently infected with Mycobacterium tuberculosis. These nonreplicating, dormant bacilli are tolerant to conventional anti-tuberculosis drugs, such as isoniazid. We recently identified diarylquinoline R207910 (also called TMC207) as an inhibitor of ATP synthase with a remarkable activity against replicating mycobacteria. In the present study, we show that R207910 kills dormant bacilli as effectively as aerobically grown bacilli with the same target specificity. Despite a transcriptional down-regulation of the ATP synthase operon and significantly lower cellular ATP levels, we show that dormant mycobacteria do possess residual ATP synthase enzymatic activity. This activity is blocked by nanomolar concentrations of R207910, thereby further reducing ATP levels and causing a pronounced bactericidal effect. We conclude that this residual ATP synthase activity is indispensable for the survival of dormant mycobacteria, making it a promising drug target to tackle dormant infections. The unique dual bactericidal activity of diarylquinolines on dormant as well as replicating bacterial subpopulations distinguishes them entirely from the current anti-tuberculosis drugs and underlines the potential of R207910 to shorten tuberculosis treatment. An estimated one-third of the world population is latently infected with Mycobacterium tuberculosis. These nonreplicating, dormant bacilli are tolerant to conventional anti-tuberculosis drugs, such as isoniazid. We recently identified diarylquinoline R207910 (also called TMC207) as an inhibitor of ATP synthase with a remarkable activity against replicating mycobacteria. In the present study, we show that R207910 kills dormant bacilli as effectively as aerobically grown bacilli with the same target specificity. Despite a transcriptional down-regulation of the ATP synthase operon and significantly lower cellular ATP levels, we show that dormant mycobacteria do possess residual ATP synthase enzymatic activity. This activity is blocked by nanomolar concentrations of R207910, thereby further reducing ATP levels and causing a pronounced bactericidal effect. We conclude that this residual ATP synthase activity is indispensable for the survival of dormant mycobacteria, making it a promising drug target to tackle dormant infections. The unique dual bactericidal activity of diarylquinolines on dormant as well as replicating bacterial subpopulations distinguishes them entirely from the current anti-tuberculosis drugs and underlines the potential of R207910 to shorten tuberculosis treatment. Mycobacterium tuberculosis infection results in more than 2 million deaths per year and is the leading cause of mortality in people infected with HIV 2The abbreviations used are: HIVhuman immunodeficiency virusDARQdiarylquinolineTBtuberculosisMICminimum inhibitory concentrationMBCminimum bactericidal concentrationWCCWayne cidal concentrationCFUcolony-forming unitDCCDN,N′-dicyclohexylcarbodiimideDETA/NOdiethylenetriamine/nitric oxide. (1Dye C. Scheele S. Dolin P. Pathania V. Raviglione M.C. J. Am. Med. Assoc. 1999; 282: 677-686Crossref PubMed Scopus (2744) Google Scholar). The global epidemic of tuberculosis (TB) is fuelled by co-infection of HIV patients with TB and a rise in multidrug-resistant TB strains (2Corbett E.L. Watt C.J. Walker N. Maher D. Williams B.G. Raviglione M.C. Dye C. Arch. Int. Med. 2003; 163: 1009-1021Crossref PubMed Scopus (2224) Google Scholar). Despite the fact that TB control programs have been in place for decades, approximately one-third of the world population is latently infected with M. tuberculosis. Reactivation of latent TB is a high risk factor for disease development, particularly in immunocompromised individuals, such as HIV-infected patients. For global control of the TB epidemic, there is an urgent medical need for new drugs active against dormant or latent bacilli. These so-called sterilizing drugs would be able to shorten the current 6-month treatment duration for drug-susceptible TB and also offer new treatment opportunities for latent TB. human immunodeficiency virus diarylquinoline tuberculosis minimum inhibitory concentration minimum bactericidal concentration Wayne cidal concentration colony-forming unit N,N′-dicyclohexylcarbodiimide diethylenetriamine/nitric oxide. Tubercle bacilli enter lungs of healthy individuals by inhalation, where they are phagocytosed by the alveolar macrophages that eliminate most of the invading mycobacteria (3Koul A. Herget T. Klebl B. Ullrich A. Nat. Rev. Microbiol. 2004; 2: 189-202Crossref PubMed Scopus (312) Google Scholar). However, a small proportion of bacilli survive and exist in a nonreplicating, hypometabolic state, and these bacilli are tolerant to killing by bactericidal anti-TB drugs, such as isoniazid (4Gomez J.E. McKinney J.D. Tuberculosis. 2004; 84: 29-44Crossref PubMed Scopus (441) Google Scholar). They can linger in these altered physiological environments for an individual's lifetime and maintain the capability of causing active TB after reactivation. The pathophysiological conditions in human lesions, thought to lead to persistence, are reduced oxygen tension, nutrient limitation, and acidic pH (5Flynn J.L. Chan J. Infect. Immun. 2001; 69: 4195-4201Crossref PubMed Scopus (347) Google Scholar, 6Wayne L.G. Eur. J. Clin Microbiol. Infect. Dis. 1994; 13: 908-914Crossref PubMed Scopus (322) Google Scholar). Recently, we identified a new chemical class, diarylquinolines (DARQs) that demonstrate potent anti-mycobacterial activity on replicating bacilli both in vitro and in vivo (7Andries K. Verhasselt P. Guillemont J. Gohlmann H.W. Neefs J.M. Winkler H. Van Gestel J. Timmerman P. Zhu M. Lee E. Williams P. de Chaffoy D. Huitric E. Hoffner S. Cambau E. Truffot-Pernot C. Lounis N. Jarlier V. Science. 2005; 307: 223-227Crossref PubMed Scopus (1813) Google Scholar), and the lead compound, R207910 (or TMC207), is currently in Phase IIb clinical trials for the treatment of patients with multidrug-resistant TB. R207910 acts by specifically targeting the membrane-bound c-subunit of F1F0-ATP synthase, the ATP-synthesizing machinery of the cell (8Koul A. Dendouga N. Vergauwen K. Molenberghs B. Vranckx L. Willebrords R. Ristic Z. Lill H. Dorange I. Guillemont J. Bald D. Andries K. Nat. Chem. Biol. 2007; 3: 323-324Crossref PubMed Scopus (453) Google Scholar). During synthesis of ATP, the energy stored in the electrochemical proton gradient across the membrane is utilized to drive protons from the periplasmic space into the cytoplasm through the F0 subunit and supplying a torque to the F1 unit to convert ADP into ATP (9Junge W. Nelson N. Science. 2005; 308: 642-644Crossref PubMed Scopus (53) Google Scholar, 10Bald D. Noji H. Yoshida M. Hirono-Hara Y. Hisabori T. J. Biol. Chem. 2001; 276: 39505-39507Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Under nonrespiratory conditions, in several bacteria, ATP synthases can function in the reverse direction, hydrolyzing ATP to ADP through ATPase activity and as such pumping protons from the cytoplasm into the periplasmic space (11Deckers-Hebestreit G. Altendorf K. Annu. Rev. Microbiol. 1996; 50: 791-824Crossref PubMed Scopus (175) Google Scholar). In this way, ATP synthase generates the membrane potential required for the uptake of nutrients. In dormant mycobacteria, a considerable remodeling of the respiratory chain has been reported, including down-regulation of cytochrome aa3 type oxidase and up-regulation of the cytochrome bd type menaquinole oxidase (12Shi L. Sohaskey C.D. Kana B.D. Dawes S. North R.J. Mizrahi V. Gennaro M.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15629-15634Crossref PubMed Scopus (254) Google Scholar, 13Schnappinger D. Ehrt S. Voskuil M.I. Liu Y. Mangan J.A. Monahan I.M. Dolganov G. Efron B. Butcher P.D. Nathan C. Schoolnik G.K. J. Exp. Med. 2003; 198: 693-704Crossref PubMed Scopus (1169) Google Scholar). Several studies also suggest that hypometabolic, nonreplicating mycobacteria have decreased requirements for ATP synthase, since the genes encoding the components of the ATP synthase operon are down-regulated, both in vitro and in vivo (12Shi L. Sohaskey C.D. Kana B.D. Dawes S. North R.J. Mizrahi V. Gennaro M.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15629-15634Crossref PubMed Scopus (254) Google Scholar, 13Schnappinger D. Ehrt S. Voskuil M.I. Liu Y. Mangan J.A. Monahan I.M. Dolganov G. Efron B. Butcher P.D. Nathan C. Schoolnik G.K. J. Exp. Med. 2003; 198: 693-704Crossref PubMed Scopus (1169) Google Scholar, 14Karakousis P.C. Yoshimatsu T. Lamichhane G. Woolwine S.C. Nuermberger E.L. Grosset J. Bishai W.R. J. Exp. Med. 2004; 200: 647-657Crossref PubMed Scopus (205) Google Scholar). It is, however, an open question whether ATP synthase plays an essential role in dormant bacilli and whether the enzyme is responsible for ATP synthesis and/or maintenance of the membrane potential. Furthermore, it is not known whether, despite ATP synthase down-regulation, this enzyme can still be utilized as a drug target in dormant bacilli and whether DARQs are still able to effectively kill dormant bacilli. Here we report that, despite a substantial down-regulation of the ATP synthase operon and reduced cellular ATP levels, DARQs effectively kill nonreplicating M. tuberculosis. We find that dormant mycobacteria have active and functional ATP synthase that is capable of synthesizing ATP, a scarce energy resource in a nonreplicating cell. The depletion of ATP in dormant mycobacteria by the R207910-mediated chemical inhibition of ATP synthase leads to potent bactericidal activity. Thus, targeting processes that generate ATP and concurrently disturb the cellular ATP homeostasis is an effective strategy against dormancy. R207910 exhibits no significant effect on the membrane potential in dormant or replicating mycobacteria, suggesting that ATP synthase is not critical for maintaining the membrane potential but is primarily used for the production of ATP. We also demonstrate an increased susceptibility of dormant mycobacteria toward R207910 as compared with actively growing bacteria. Using R207910-resistant mycobacterial strains, we show that the drug specifically targets ATP synthase during dormancy. The unique dual bactericidal action of R207910 on dormant as well as actively replicating bacteria probably contributes to its remarkable sterilizing efficacy in mice, where R207910 as a monotherapy was shown to be as effective as the triple combination of rifampin, isoniazid, and pyrazinamide (7Andries K. Verhasselt P. Guillemont J. Gohlmann H.W. Neefs J.M. Winkler H. Van Gestel J. Timmerman P. Zhu M. Lee E. Williams P. de Chaffoy D. Huitric E. Hoffner S. Cambau E. Truffot-Pernot C. Lounis N. Jarlier V. Science. 2005; 307: 223-227Crossref PubMed Scopus (1813) Google Scholar, 15Mitchison D.A. Nat. Biotechnol. 2005; 23: 187-188Crossref PubMed Scopus (21) Google Scholar). This may also explain the ability of this drug to render lungs of infected mice culture-negative faster than first line anti-TB drugs. In conclusion, despite a number of changes in the energy metabolism during dormancy, the ATP production by ATP synthase remains essential for mycobacterial survival. The current study demonstrates the critical importance of ATP synthase in mycobacterial dormancy and suggests that the ATP synthase is an excellent target to treat latent TB infections. Bacterial Strains and Culture Medium—M. tuberculosis H37Rv, Mycobacterium smegmatis, and Mycobacterium bovis BCG were obtained from the American Type Tissue Culture collection. Mycobacteria were cultured in Middlebrook 7H9 medium (Difco) with 0.05% Tween-80 (Sigma) in log phase for a period of 3–4 days before the start of the experiment. The incubation was done in a 250-ml sterile Duran bottle with stirring conditions 37 °C (500 rpm). Chemical Synthesis of DARQ Analogs—DARQ compounds are synthesized as described in a earlier patent (WO2004/011436) and previous reports (7Andries K. Verhasselt P. Guillemont J. Gohlmann H.W. Neefs J.M. Winkler H. Van Gestel J. Timmerman P. Zhu M. Lee E. Williams P. de Chaffoy D. Huitric E. Hoffner S. Cambau E. Truffot-Pernot C. Lounis N. Jarlier V. Science. 2005; 307: 223-227Crossref PubMed Scopus (1813) Google Scholar). MIC Microdilution Assays and Determination of Minimum Bactericidal Concentration (MBC90) and Wayne Cidal Concentration (WCC90)—Minimum inhibitory concentration (MIC, μg/ml) of M. tuberculosis against several DARQ compounds was determined by using 7H9 liquid broth and 7H10 agar dilution as described in earlier studies (16Lenaerts A.J. Gruppo V. Marietta K.S. Johnson C.M. Driscoll D.K. Tompkins N.M. Rose J.D. Reynolds R.C. Orme I.M. Antimicrob. Agents Chemother. 2005; 49: 2294-2301Crossref PubMed Scopus (289) Google Scholar) with minor modifications using Alamar blue or resazurin for fluorometric measurement (7Andries K. Verhasselt P. Guillemont J. Gohlmann H.W. Neefs J.M. Winkler H. Van Gestel J. Timmerman P. Zhu M. Lee E. Williams P. de Chaffoy D. Huitric E. Hoffner S. Cambau E. Truffot-Pernot C. Lounis N. Jarlier V. Science. 2005; 307: 223-227Crossref PubMed Scopus (1813) Google Scholar). MBC90 (μg/ml) was determined as the concentration where 90% of the aerobic grown bacteria were killed after 5 days of treatment by colony-forming unit (CFU) counting using 7H10 agar dilution according to Clinical and Laboratory Standards Institute guidelines. WCC90 (μg/ml) was determined as the concentration where 90% of the dormant bacteria (grown in the Wayne model) were killed after 5 days of treatment by CFU counting using 7H10 agar dilution. Dormancy Assays—Drastic oxygen depletion (hypoxia model) of mycobacterial cultures was achieved by placing loosely capped tubes containing different drug concentrations inside an anaerobic jar (BBL) along with anaerobic gas generation envelopes (using palladium catalysts), as described previously (17Stover C.K. Warrener P. VanDevanter D.R. Sherman D.R. Arain T.M. Langhorne M.H. Anderson S.W. Towell J.A. Yuan Y. McMurray D.N. Kreiswirth B.N. Barry C.E. Baker W.R. Nature. 2000; 405: 962-966Crossref PubMed Scopus (921) Google Scholar). After 7 days of anaerobiosis, the dormant cultures were harvested by low speed centrifugation, washed twice with 7H9 medium to remove the drugs, and resuspended in drug-free medium. The CFUs of the treated and untreated cultures were determined by plating on 7H10 agar to evaluate the bactericidal activity. In the Wayne model, gradual O2 depletion induces dormancy in mycobacterial cultures, as previously described (18Wayne L.G. Hayes L.G. Infect. Immun. 1996; 64: 2062-2069Crossref PubMed Google Scholar). Briefly, after induction of dormancy for 18 days by gradual O2 depletion, cultures without exposure to oxygen were treated further with various drug concentrations for a period ranging from 4 to 21 days, and CFUs were counted on 7H10 plates. In the NO model, dormancy was obtained by incubating cultures with 150 μm of a NO donor (diethylenetriamine/nitric oxide (DETA/NO)) for 2 h, followed by treatment with several compounds for 10 h as previously described (19Voskuil M.I. Schnappinger D. Visconti K.C. Harrell M.I. Dolganov G.M. Sherman D.R. Schoolnik G.K. J. Exp. Med. 2003; 198: 705-713Crossref PubMed Scopus (783) Google Scholar). RNA Isolation and Real Time Quantitative PCR—Dormant and aerobically grown bacterial pellets were resuspended in Trizol and disrupted with acid-washed glass beads in the Mixer Mill (MM 301). Total RNA was isolated as previously described (20Voskuil M.I. Visconti K.C. Schoolnik G.K. Tuberculosis (Edinb.). 2004; 84: 218-227Crossref PubMed Scopus (353) Google Scholar) and cleaned up with the RNeasy Protect Bacteria minikit (QIAgen). Reverse transcription was carried out from 0.5 μg of total RNA using random hexamer primers and Superscript II RT as described by the manufacturer (Invitrogen). The sequence of the TaqMan FAM (6-carboxyfluorescein) and TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) probes (Eurogentec) and primers for the mycobacterial genes analyzed in this study are available upon request. Real time quantitative PCR was carried out using the ABI PRISM 7900HT sequence detection system (Applied Biosystems) as described by the manufacturer. Amplification of endogenous 16 S ribosomal RNA was performed to standardize the amount of cDNA sample added to each reaction. Total Cellular ATP Measurement Assay—ATP levels of log phase aerobic and dormant cultures of M. tuberculosis were measured using the ATP bioluminescence assay kit (Roche Applied Science), as previously described (8Koul A. Dendouga N. Vergauwen K. Molenberghs B. Vranckx L. Willebrords R. Ristic Z. Lill H. Dorange I. Guillemont J. Bald D. Andries K. Nat. Chem. Biol. 2007; 3: 323-324Crossref PubMed Scopus (453) Google Scholar). ATP levels were followed during dormancy (Wayne model) from day 0 to day 21. Additionally, M. tuberculosis cultivated under Wayne dormancy conditions for 18 days were treated with R207910 (0.006–6 μg/ml), isoniazid (0.3 μg/ml), and dicyclohexylcarbodiimide (DCCD) (100 μg/ml) for 4 days, and ATP levels were measured. Measurement of ATP Synthesis Activity—ATP synthesis of dormant M. smegmatis grown under Wayne conditions for 10 days was measured as described previously (8Koul A. Dendouga N. Vergauwen K. Molenberghs B. Vranckx L. Willebrords R. Ristic Z. Lill H. Dorange I. Guillemont J. Bald D. Andries K. Nat. Chem. Biol. 2007; 3: 323-324Crossref PubMed Scopus (453) Google Scholar). Briefly, inverted membrane vesicles were prepared using a precooled French pressure cell at 20,000 p.s.i. The membrane vesicles were preincubated with R207910 or DCCD under stirring conditions at room temperature for 10 min. The ATP synthesis activity was determined by energizing the membranes with NADH and quantifying the amount of ATP produced using the luciferin/luciferase system (ATP Bioluminescence Assay Kit HS II; Roche Applied Science). Data are presented as averages ± S.E. Measurement of the Membrane Potential—Membrane potential was measured using tetra-[3H]phenylphosphonium bromide (Amersham Biosciences) in aerobic and Wayne dormant (10 days) M. smegmatis, as previously described (21Rao M. Streur T.L. Aldwell F.E. Cook G.M. Microbiology. 2001; 147: 1017-1024Crossref PubMed Scopus (111) Google Scholar). Briefly, cells were energized with 20 mm glucose, tetra-[3H]phenylphosphonium bromide was added, and cells were centrifuged through silicon oil. Supernatant and cell pellet were dissolved in scintillation fluid and counted with a liquid scintillation analyzer (TRI-CARB 2100TR; Packard). Membrane potential (in mV) was calculated according to the Nernst relationship. Values represent averages ± S.E. of four independent experiments. Comparisons were performed using Student's t test (StatXact). p values of <0.05 were considered as statistically significant. Bactericidal Activity of R207910 on Dormant M. tuberculosis—The effect of DARQ lead compound, R207910, on the growth of nonreplicating M. tuberculosis was tested in three different in vitro dormancy models. As a control for target specificity during dormant conditions, we tested the efficacy of R207910 on the R207910-resistant strain of M. tuberculosis (BK12), carrying a point mutation in the α-helix of the c-subunit of the ATP synthase complex (7Andries K. Verhasselt P. Guillemont J. Gohlmann H.W. Neefs J.M. Winkler H. Van Gestel J. Timmerman P. Zhu M. Lee E. Williams P. de Chaffoy D. Huitric E. Hoffner S. Cambau E. Truffot-Pernot C. Lounis N. Jarlier V. Science. 2005; 307: 223-227Crossref PubMed Scopus (1813) Google Scholar). Both wild type and mutant strains were subjected to dormancy using either gradual (Wayne) or drastic (hypoxia) O2 depletion or NO treatment in the presence of 0.1 and 10 μg/ml R207910 (Fig. 1). We treated Wayne dormant M. tuberculosis for 7 days with R207910 to determine its killing efficacy. As can be seen in Fig. 1A, R207910 at 10 μg/ml leads to a 1.8-log10 reduction in CFU counts, whereas no apparent effect was observed at 0.1 μg/ml. The viability of dormant bacilli was not affected by isoniazid treatment, an inhibitor of biosynthesis of cell wall mycolic acids. In contrast, metronidazole at 100 μg/ml led to a 1.9-log10 reduction in CFUs as compared with untreated control. Metronidazole is a prodrug that is effective only under anaerobic conditions. However, it has been shown to be ineffective against latent mycobacteria in a Cornell mouse infection model (22Brooks J.V. Furney S.K. Orme I.M. Antimicrob. Agents Chemother. 1999; 43: 1285-1288Crossref PubMed Google Scholar). DCCD, a nonspecific ATP synthase inhibitor, showed a drastic killing effect at 100 μg/ml (Fig. 1A). In the hypoxia model, M. tuberculosis cultures were subjected to drastic O2 depletion in an anaerobic chamber. R207910 reduced the mycobacterial viability by 2.1-log10 CFUs at 10 μg/ml but had no apparent effect at 0.1 μg/ml (Fig. 1B). Metronidazole tends to be slightly more potent than in the Wayne model, leading to a 2.5-log10 CFU reduction at 100 μg/ml. This can be explained by the extreme hypoxic conditions created in the anaerobic chamber, and as such, the effects of metronidazole, which works best in a severely O2-depleted environment, tend to be enhanced. NO has been shown to inhibit aerobic respiration in several bacterial systems, and it induces a dormancy response in mycobacteria similar to oxygen depletion (19Voskuil M.I. Schnappinger D. Visconti K.C. Harrell M.I. Dolganov G.M. Sherman D.R. Schoolnik G.K. J. Exp. Med. 2003; 198: 705-713Crossref PubMed Scopus (783) Google Scholar). Treatment of midlog aerobically grown cultures of M. tuberculosis with low nontoxic concentrations (150 μm) of DETA/NO induced typical dormancy regulon gene expression (Fig. 3A). In our study, 2 h after treatment with DETA/NO, mycobacteria were exposed for 10 h to R207910 and control drugs. DETA/NO alone had no effect on mycobacterial survival as compared with untreated control (Fig. 1C). Furthermore, isoniazid (10 μg/ml) had no effect on these dormant bacilli, whereas the viability of the bacilli was drastically reduced by DCCD. However, metronidazole (100 μg/ml), in contrast to the O2 depletion dormancy assays, was not bactericidal, because as a prodrug, it is active only in an O2-limiting and not in an NO environment. R207910 (10 μg/ml) led to a 1-log10 drop in CFU counts, whereas no apparent effect was observed at 0.1 μg/ml. It has been previously shown that NO dissipates in the reaction tubes over a period of time, and by 16 h, the bacteria tend to lose their dormant phenotype (19Voskuil M.I. Schnappinger D. Visconti K.C. Harrell M.I. Dolganov G.M. Sherman D.R. Schoolnik G.K. J. Exp. Med. 2003; 198: 705-713Crossref PubMed Scopus (783) Google Scholar). However, the effects of R207910 on NO-treated mycobacteria in this model (10 h of treatment) were reached within the dormant physiological phase. Susceptibility Comparison of Dormant and Aerobically Grown Mycobacteria with R207910—The present first line and second line TB antibiotics exhibit strong bactericidal activity on replicating mycobacteria, but none of them show a comparable efficacy on dormant bacilli. In order to evaluate the bactericidal activity of R207910 on dormant versus actively replicating mycobacteria, we determined the bactericidal concentration of R207910 in aerobic (MBC90) and Wayne dormant (WCC90) M. tuberculosis (Table 1). Rifampicin and isoniazid, included as controls, were both more potent in aerobically grown and significantly less for the dormant bacilli. In contrast, R207910 was slightly more active on dormant bacilli (WCC90 of 1 μg/ml), as compared with aerobically grown bacilli (MBC90 of 2 μg/ml). This suggests that dormant bacteria are slightly more prone (about 2-fold) to R207910 bactericidal activity as compared with actively growing mycobacteria (WCC90/MBC90 ratio of 0.5).TABLE 1Comparative killing activity of TB compounds (rifampicin, isoniazid, and R207910) for actively replicating and dormant M. tuberculosisCompoundMBC90WCC90WCC90/MBC90μg/mlμg/mlRifampicin0.030.517Isoniazid0.25>64>256R207910210.5 Open table in a new tab To further evaluate this result, we measured killing kinetics of R207910 and isoniazid on Wayne dormant and aerobically grown bacilli (Fig. 2). For this purpose, both cultures were diluted to equal A600 values or ∼107 to 108 CFU/ml and treated with R207910 (10 μg/ml). For aerobic cultures, we observed an initial 2.2-log10 drop in CFUs by the end of the first week of treatment with R207910 and an additional 0.8-log10 reduction in CFUs by day 14. In contrast, treatment of dormant mycobacteria resulted in an initial 1.8-log10 drop in CFUs by the end of the first week, but by day 14, killing was more pronounced, with an additional 3.0-log10 drop in CFUs versus untreated control. As expected, isoniazid had no effect on dormant bacterial cultures. However, in aerobic cultures, isoniazid killed about 2.2 log10 during the first week, with no additional killing during the second week. R207910-mediated killing of dormant bacilli at a 10 μg/ml concentration was highly potent, since no bacteria could be counted by day 14 (Fig. 2). In contrast, the killing kinetics of R207910 at 1 μg/ml on Wayne dormant M. tuberculosis suggested a characteristic biphasic killing with a 2.9-log10 drop in CFUs by day 14 and no further reduction by day 21 (Fig. S1). In summary, by measuring the bactericidal concentrations as well as killing kinetics, we demonstrated that R207910 kills dormant M. tuberculosis at least as effectively as actively replicating bacilli. This makes R207910 the only TB drug with equal bactericidal activities on different bacterial subpopulations. Specificity of DARQ Activity in Dormant Mycobacteria—Target specificity of DARQs was assessed by introducing chemical changes in the DARQ scaffold. During dormancy, bacteria might have alternative means of energy or ATP production (e.g. substrate level phosphorylation), and as such, ATP synthase might be redundant. Hence, it is postulated that R207910 might affect other cellular targets as well (23Zhang Y. Post-Martens K. Denkin S. Drug Discov. Today. 2006; 11: 21-27Crossref PubMed Scopus (171) Google Scholar). At first, we studied the correlation between various structural and chemical changes in R207910 and its analogs versus their effect on biological activity. This leads to the determination of a structure-activity relationship. Over 400 DARQ derivatives were prepared and tested on actively replicating and aerobically grown M. tuberculosis cultures in in vitro MIC determination assays (data not shown). Five compounds (Fig. S2A) with MICs between 0.09 and 20 μg/ml were selected for evaluating their bactericidal effect on Wayne dormant M. tuberculosis. R207910 at 1 and 10 μg/ml led to 1.3- and 2.1-log10 CFU reduction, respectively, as compared with untreated dormant control cultures (Table 2). As can be seen in Table 2, an analogue with a methyl substituent on the first aryl moiety of R207910 (named 18040971-AAA) retained its bactericidal activity on dormant bacteria with 1.55-log10 killing at 1 μg/ml. However, replacement of the dimethylamino group by azole groups (25756770-AAA) led to a decrease in the activity on both aerobic (MIC 8.19 μg/ml) and dormant bacilli (0.29-log10 CFU reduction). Interestingly, changing the chain length in R207910 between the hydroxyl and the terminal dimethylamino group (18086601-AAA) also decreased activity on both aerobic (MIC 20.21 μg/ml) as well as dormant bacilli (0.11-log10 CFU reduction). These data suggest a strong correlation between structural variations of the DARQ scaffold and the subsequent effect on their activities against replicating and dormant bacilli (Fig. S2B). The sharp overlap between the bactericidal activities of DARQ analogs on dormant and replicating bacilli implies that their effects are highly target-specific, irrespective of mycobacterial growth conditions and physiological states.TABLE 2Bactericidal action of DARQs in killing aerobic and dormant M. tuberculosisCompound IDMIC90 (aerobic cultures)Log10 reduction (dormant cultures)10 μg/ml1 μg/mlμg/mlR2079100.092.11.3518040971-AAA0.162.131.5517996134-AAA1.741.140.8725756770-AAA8.190.980.2918086601-AAA20.210.230.11 Open table in a new tab In a second approach to characterize the target specificity of R207910 during dormancy, we tested the efficacy of R207910 on the resistant strain of M. tuberculosis (BK12) bearing a mutation in c-subunit of ATP synthase (7Andries K. Verhasselt P. Guillemont J. Gohlmann H.W. Neefs J.M. Winkler H. Van Gestel J. Timmerman P. Zhu M. Lee E. Williams P. de Chaffoy D. Huitric E. Hoffner S. Cambau E. Truffot-Pernot C. Lounis N. Jarlier V. Science. 2005; 307: 223-227Crossref PubMed Scopus (1813) Google Scholar). At the highest concentration of 10 μg/ml, R207910 had no effect on this mutant strain in Wayne (Fig. 1A), hypoxia (Fig. 1B), and NO (Fig. 1C) conditions. Isoniazid had no effect under these conditions, suggesting that bacilli were indeed in a nonreplicating state. The ATP synthase inhibitor DCCD had a drastic killing effect and led to a 5.3-, 2.8-, and 4.3-log10 drop in CFUs in Wayne (Fig. 1A), hypoxia (Fig. 1B), and NO (Fig. 1C), respectively. The lack of cross-resistance between these two compounds suggests that although DCCD binds to the same target as R207910, its binding pocket is different. In summary, our results demonstrate that killing of mycobacteria by DARQs critically depends on the structure of the DARQ compound as well as critical amino acid compositions in the target protein. ATP Synthesis in Nonreplicating Mycobacteria—The dormancy physiological phase involves changes in several metabolic pathways that lead to the transcriptional reprogramming of d