screening on non culturable mtb.pdf

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New 2-thiopyridines as potential candidates for killing both actively growing and1

dormantMycobacterium tuberculosis2

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Elena Salina#, Olga Ryabova, Arseny Kaprelyants and Vadim Makarov4

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Institution of theRussian Academy of Sciences A.N. Bach Institute of Biochemistry6

RAS, Moscow, Russia7

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Running title: New 2-thiopyridines9

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# Address for correspondence to Elena Salina, [email protected]

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AAC Accepts, published online ahead of print on 14 October 2013

Antimicrob. Agents Chemother. doi:10.1128/AAC.01308-13

Copyright 2013, American Society for Microbiology. All Rights Reserved.


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Abstract27

From in vivo observations, majority of M. tuberculosis cells in latently infected28

individuals are in a dormant and probably non-culturable state, display little metabolic29

activity and are phenotypically resistant to antibiotics. Despite many attempts no specific30

antimicrobials effective for latent tuberculosis have yet been found partially because of lack of31

reliable and adequate in vitromodels for screening for drug candidates. We propose here a32

novel in vitro model of M. tuberculosis dormancy which meets the important criteria of33

latency, namely non-culturability of cells, considerable reduction of metabolic activity and34

significant phenotypic resistance to the first-line antibiotics rifampicin and isoniazid. Using35

this model we found a new group of 2-thiopyridine derivatives which had potent antibacterial36

activity against both actively growing and dormant M. tuberculosis cells. By means of the37

model of M. tuberculosis non-culturability several new 2-thiopyridine derivatives were38

found to have potent antitubercular activity. The compounds are effective against both active39

and dormant M. tuberculosis cells. The bactericidal effect of compounds for dormant M.40

tuberculosiswas confirmed by using three different in vitromodels of tuberculosis dormancy.41

The model of non-culturability could be used as a reliable tool for screening drug candidates42

and 2-thiopyridine derivatives may be regarded as prominent compounds for further43

development of new drugs for curingM. tuberculosislatent infection.44

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Introduction53

Curing latent tuberculosis (TB) is a big challenge for modern chemotherapy (1). One54

third of the entire world's population carries latent TB infection with a lifelong risk of disease55

reactivation, which increases several times in immunocompromised patients (1, 2).56

Mycobacterium tuberculosis cells associated with latent tuberculosis in the human host are57

believed to be in a special non-replicating (dormant) state characterized by the development of58

considerable antibiotic tolerance (2). This tolerance, described as phenotypic drug resistance,59

is due to changes in the physiological state of the bacteria characterized by the low metabolic60

status of cells (2).61

Since specific and highly effective TB drugs for latency are still absent, traditional62

antibiotics such as isoniazid (INH) and rifampicin (RIF) are used in current chemotherapy of63

latent TB. There are some therapeutic regimens based on long-term cure of latently infected64

patients by INH or RIF (3, 4) although INH is weakly effective for latent TB infection because65

it targets the processes of bacterial cell wall biosynthesis (1). Probably the role of INH may be66

to kill emerging bacilli that grow actively as a result of dormant cells reactivation or67

conversion of slowly growing persisters to actively growing bacilli susceptible to INH68

treatment (5). Significant tolerance of dormant cells to RIF was shown in the in vivoCornell69

model of TB persistence (6) thus questioning the efficacy of RIF to cure latent TB. Moreover,70

it was postulated that dormant M. tuberculosis cells are phenotypically resistant to the71

sterilizing activity of RIF and this feature is one of the hallmarks of dormant TB (7).72

Interestingly, in accordance with the Cornell model and some clinical studies, dormant73

cells in latently infected individuals are characterized by non-culturability i.e. a74

phenomenon of transient inability to divide and grow on non-selective solid media (8-10). A75

resuscitation procedure is required for such cells to enter into an active growth state (8, 11).76

Consequently, there is a need for new drugs against latent TB infection. Since genome-77

derived, target-based approaches have had little success in the antibacterial therapeutic area in78


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general (1),whole bacterial cell screening is believed to be a preferable approach for finding79

new drugs for TB as it allows to test drug candidates in a relevant physiological state of the80

pathogen (1, 12).81

Several in vitromodels ofM. tuberculosisdormancy that were developed recently are82

currently being used to searching for anti-latency drugs (7, 13-15), however, dormant cells83

obtained in the vast majority of these in vitromodels are fully culturable and metabolically84

active and are characterized by limited antibiotic resistance that may compromise their use in85

the search for drugs for latent TB.86

We propose here a new in vitromodel that mimics the phenomenon of TB latency. In87

this model we managed to eliminate many of the limitations of the in vitromodels mentioned88

above. In particular, dormant cells in this model are non-culturable (NC), of low metabolic89

activity and characterized by phenotypic resistance both to INH and RIF. Therefore, this in90

vitromodel meets the key criteria of true dormancy and may be applied as a relevant tool for91

latent TB drug discovery. Using this new model we found a new group of 2-thiopyridine92

derivatives which had potent antibacterial activity against both actively growing and dormant93

M. tuberculosiscells.94

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Materials and methods96

Chemistry97

All reagents and solvents were purchased from commercial suppliers and used without98

further purification. Melting points were determined on Electothermal 9001 and are99

uncorrected.1

H NMR was measured in DMSO-d6at 400 MHz on a Varian Unity +400 (USA).100

Shifts for NMR are reported in ppm downfield from TMS (). A Waters Micromass ZQ101

detector (USA) was used in ESI MS for identification of various products. Elemental analyses102

were carried out on a Carlo-Erba 5500 elemental analyzer for C, H, N, and the results are103


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within 0.3% of the theoretical values. Merck silica gel 60 F254plates were used for analytical104

TLC; column chromatography was performed on Merck silica gel 60 (70-230 mesh).105

Compounds 11026101 and 10026127 were obtained by a series of successive106

transformations of 2-hydroxynicotinamide which was first reacted with fuming nitric acid in107

concentrated sulfuric acid to give the corresponding 5-nitroderivative. The reflux of the latter108

with excess thionyl chloride results in 2-chloro-5-nitronicotinic acid chloroanhydride which109

was immediately used in a reaction with 12% ammonia solution in water and the substituted110

nicotinamide was isolated with high yield. Subsequently, boiling with thionyl chloride111

resulted in the key intermediate 2-chloro-3-cyano-5-nitropyridine. A mixture of 2-chloro-3-112

cyano-5-nitropyridine and 1.15 mol of potassium thiocyanate was refluxed for 4 hours and113

dissolved in water. Light yellow crystal of 3-cyano-5-nitro-2-thiocyanopyridine 11026101114

was formatted and filtered off. Yield 67% (ethanol). MS m/z+.

206.1H NMR (dmso-d6) 9.68115

(1H, s, C(6)H), 8.66 (H, s, C(4)H) ppm. Anal. C7H2N4O2S, C,H,N. The same synthesis116

procedure was used to obtain 3-cyano-2-diethyldithiocarbamoyl-5-nitronopyridine 10026127117

using sodium diethyldithiocarbamate as nucleophylic agent in the last step. Yield 82%118

(ethanol). MS m/z+.

296.1H NMR (dmso-d6) 9.73 (1H, s, C(6)H), 8.75 (H, s, C(4)H), 4,22119

and 3.93 (4H, two q, N(CH2CH3)2), 1.32 (6H, t N(CH2CH3)2) ppm. Anal. C11H12N4O2S2,120

C,H,N.121

A series of 1-oxidopyridines 11026103, 11026115 and 11026114 was synthesized122

from the corresponding 5-R-2-chloropyridines, which were oxidized by complex123

thiourea/H2O2 and trifluoroacetic acid anhydride in methylene chloride. These124

chloroderivatives were treated with potassium thiocyanate for synthesis of compounds125

11026103 and 11026115, and sodium thioacetic acid for synthesis of compound 11026114.126

All reactions were done in ethanol as solvent. 1-Oxido-5-(trifluoromethyl)pyridin-2-yl127

thiocyanate (11026103). The yield was 53% (etanol), MS m/z+.

220.1H NMR (dmso-d6) 128

8.39 (1H, s, C(6)H), 7.79 (H, d, C(3)H), 7.44 (H, d, C(4)H) ppm. Anal. C 7H3F3N2OS, C,H,N.129


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Ethyl 6-thiocyanatonicotinate 1-oxide (11026115). The yiels was 42 % (ethyl acetate). MS130

m/z+.

224.1H NMR (dmso-d6) 8.51 (1H, s, C(6)H), 8.02 (H, d, C(3)H), 7.88 (H, d, C(4)H),131

4.38 (2H, q, COOCH2CH3), 1.41 (3H, t, COOCH2CH3) ppm. Anal. C9H8N2O3S, C,H,N. Ethyl132

6-(acetylthio)nicotinate 1-oxide (11026114). The yiels was 36 % (ethanol). MS m/z+.

241.1H133

NMR (dmso-d6) 8.53 (1H, s, C(6)H), 8.07 (H, d, C(4)H), 7.52 (H, d, C(3)H), 4.38 (2H, q,134

COOCH2CH3), 2.35 (3H, s, SC(O)CH3), 1.41 (3H, t, COOCH2CH3) ppm. Anal. C10H11NO4S,135

C,H,N.136

Bacterium and media.137

M. tuberculosisstrain H37Rv was grown from frozen stocks for 1012 days in Sauton138

medium, containing: KH2PO4, 0.5 g; MgSO47H2O, 1.4 g; L-asparagine, 4 g; glycerol, 60 ml;139

ferric ammonium citrate, 0.05 g; sodium citrate, 2 g; 1% ZnSO47H2O, 0.1 ml; H2O, to l L; pH140

7.0 (adjusted with 1 M NaOH) (16) and supplemented with ADC (albumin, glucose and NaCl)141

and 0.05% of Tween-80 (37 C, 200 rpm).142

Dormant cells preparation.143

To obtain non-culturable (dormant) cells the culture grown in Sauton medium to144

OD600=5 served as an inoculum that was added to potassium-deficient Sauton medium145

supplemented with ADC and 0.05% of Tween-80 (37 C, 200 rpm) at a concentration 5x105146

cells per ml. Potassium-deficient Sauton medium contains: Na2HPO412H2O, 8.9 g;147

MgSO47H2O, 1.4 g; L-asparagine, 4 g; glycerol, 60 ml; ferric ammonium citrate, 0.05 g;148

sodium citrate, 2 g; 1% ZnSO47H2O, 0.1 ml; H2O, to l L; pH 7.0 (adjusted with 1 M NaOH)149

To obtain dormant M. tuberculosis cells in the Wayne hypoxia model and Betts150

starvation model the appropriate conditions were applied (13, 14). Briefly, for Wayne151

dormancy model M. tuberculosiswas grown in Dubos media at 37 C in sealed tubes. Cells152

ceased replicating after 7-8 days when oxygen concentrations decrease to the microaerobic153

level (1% oxygen saturation) and entered a non-replicating persistence state. With continued154

incubation the oxygen tension decrease to anaerobic level (0.06% oxygen saturation) and155


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anaerobic culture was harvested at 14 days. For Betts starvation model cultures grown for 7156

days in nutrient-rich media were pelleted, washed twice with PBS and then resuspended in157

PBS and left standing at 37 C in sealed bottles for a 6 week period.158

Resuscitation of non-culturable cells.159

Resuscitation procedure was performed in liquid Sauton diluted medium (1:1, v/v, the160

final concentration of glycerol 0.6%) supplemented with ADC. Non-culturable cells161

obtained in potassium-deficient Sauton medium were harvested, washed twice with fresh162

media, resuspended in ADC-supplemented Sauton diluted medium and left standing at 37 C.163

The number of resuscitated cells was estimated by Most Probable Number (MPN) assay (17).164

Viability estimation.165

For CFU-counting ten-fold dilutions of M. tuberculosis suspensions were plated in166

triplicate on agar-solidified ADC-supplemented Sauton medium (limit of detection is 5 CFU167

per ml). Plates were incubated at 37 C for 21-25 days. For MPN assay (17) ten-fold bacterial168

dilutions were resuspended in ADC-supplemented liquid Sauton diluted medium in 48-well169

Corning microplates. Microplates were incubated at 37 C for 30 days without agitation.170

Wells with visible bacterial growth were counted as positive, and MPN values were calculated171

using standard statistical methods (17).172

Incorporation of radioactive uracil.173

Culture samples (1 ml) were placed in 2-ml screw cup tubes with 5,6-3H uracil (1 Ci)174

and incubated at 37 C with agitation for 20 h. Afterwards, 0.2 ml of the culture was placed in175

a 15-ml Falcon tube with 3 ml of 10% ice-cold Cl3and incubated in ice for 15 min.176

The mixture was then filtered through a Whatman glass microfiber filter (GFC) and washed177

with 3 ml 7% Cl3 and 6 ml 96% ethanol. Filters were then placed in 10 ml of178

scintillation mixture, and counts determined using a LS analyzer (Beckman Instruments, Inc).179

MICs determination.180


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The determination of the MIC (Minimal Inhibitory Concentration) for M. tuberculosis181

H37Rv was determined according to the NCCLS guidelines(18) using a broth microdilution182

method in Middlebrook 7H9 media supplemented with ADC with a final inoculum of 5x105183

cell/mL. The compounds were dissolved in DMSO (1 mg/ml) and used as a stock solution.184

Concentrations ranging from 31.25 to 4000 ng/ml (at these concentrations the compounds185

were soluble in the medium) were used to assess the effectiveness of compounds. Microtitre186

plates were incubated at 37oC for 72 h, the MIC value represents the lowest dilution of the187

compound in which no bacterial growth was detected.188

Estimation of bactericidal effect.189

Both log-phase and dormant cells were exposed to different concentrations of RIF,190

INH and 2-thiopyridines for 7 days (37 C, 200 rpm). Both treated and untreated ten-fold191

diluted suspensions were employed in triplicate for MPN assays in ADC-supplemented liquid192

Sauton diluted medium in 48-well Corning microplates at 37 C for 30 days without agitation.193

MPN values were calculated using standard statistical methods (17).194

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Results and discussion196

Themodel of M. tuberculosis non-culturability197

As previously demonstrated, potassium-deficiency led to the development of non-198

culturability in Mycobacterium smegmatis cells (19). The application of a similar approach199

induced non-culturability inM. tuberculosisbacilli: the cultivation of cells under potassium-200

deficient conditions led to their transition to a reversible NC state after a 30-day incubation201

period (Fig. 1). More than 99% of M. tuberculosis cells were unable to form colonies on202

standard solid medium after a 30-day incubation period in potassium-limiting conditions but203

the NC cells remained viable and could be resuscitated. Resuscitation of cells starved for 15-204

30 days by incubation in liquid Sauton diluted medium demonstrated the presence of 108

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potentially viable cells per ml in the starved culture according to MPN assay. However, about206


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106cells/ml (or less than 1% of the population) in such a 30-day-starved population remained207

fully culturable.208

The main problem of all in vitro dormancy models is the presence of cells with209

different physiological status in the population (11). Therefore, elimination of210

replicating/active cells is a crucial point for establishing a model with a high proportion of211

truly dormant cells. In particular, penicillin treatment was used for enrichment ofMicrococcus212

luteus dormant cells(20) and Hu and colleagues have suggested applying RIF to kill viable213

cells in the population of long-stationary-phase M. tuberculosis cells (21). As a result, they214

obtained a population of persistent M. tuberculosis cells, however, their concentration was215

very low (ca 102cells /ml).216

In order to obtain a population enriched with dormant NC cells and to remove217

metabolically active cells a 15-day-starvedM. tuberculosisculture was additionally incubated218

in the presence of a moderate concentration of RIF (5 g/ml), which should not affect viability219

of dormant cells (7). After such additional treatment of a 15-day-starved culture by RIF for220

10-12 days, a zero-CFU population of NC cells was obtained. These cells were unable to221

produce colonies on solid medium but were still characterized by a high recovery potential222

estimated by MPN assay when cultivated in Sauton diluted medium, 1x108 cells/ml (close to223

MPN for starved cells without rifampicin) were able to resuscitate and transit into a fully224

culturable state (Fig. 1). Such a high recovery potential of the zero-CFU population was225

maintained for at least the following 10-14 days afterM. tuberculosiscells entered in a zero-226

CFU state.227

Despite the ability to recover from the NC state, the metabolic activity of cells in the228

zero-CFU population was very low according to the rate of radioactive uracil incorporation229

correlating with transcriptional activity of cells. While viable log-phase M. tuberculosiscells230

demonstrated a rate of radioactivity incorporation at approx. 40 000 cpm estimated for 1x107

231

cells, this parameter was at least 100-fold less for the same amount of NC cells. This was in232


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contrast to the metabolic activity of persisters obtained after exposure to a high concentration233

of RIF (100 g/ml) which was just 4 times less that parameter in log-phase cells (21), which234

means that Hus persisters did not demonstrate a dramatic metabolic reduction. This235

discrepancy could be explained by the different physiological state of cells in these two236

models.237

Apart from reduced metabolic activity, NC cells were morphologically distinct from238

actively growing cells (they were sphere-shape) and characterized by considerable resistance239

to anti-TB drugs. Namely, the treatment of NC cells in the zero-CFU population by two240

first-line anti-TB drugs RIF and INH (both at 5 g/ml) resulted in no effect on their ability to241

recover from the NC state in diluted liquid Sauton medium (Fig. 2). Poor killing effect was242

demonstrated even after treating the cells with ten-fold higher doses (Fig. 2). The sensitivity243

of cells obtained after the resuscitation to RIF and INH and the suppression of resuscitation244

from the NC state in the presence of 5 g/ml RIF and INH (not shown) showed that the245

antibiotic resistance that developed in NC cells is phenotypic.246

We compared the sensitivity to antibiotics of cells obtained in different in vitro247

dormancy models with the cells produced in the model described in this study (Table 1).248

Given that the population of dormant cells possibly contains a fraction of NC cells and that it249

is impossible to reveal susceptibility of NC cells to drugs by plating, we applied cell counting250

in a liquid medium (MPN assay) along with a standard CFU counting to estimate the killing251

effect of antibiotics. Cells in all dormancy models were expected to be characterized by their252

considerable resistance to INH (7, 13-15) because the latter impacts the biosynthesis of cell253

wall mycolic acids, a process which is evidently inactive in dormant and even in aged cells.254

However, dormant cells in all previously published M. tuberculosis dormancy models255

demonstrated significant susceptibility to RIF, which, in turn, may indicate some256

transcriptional activity in such cells. Resistant dormant NC cells in this study are evidently257

characterized by more deep dormancy (11) in comparison to previously published models.258


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Similarly, deep persistence was reported to be characterised by very low metabolic activity259

and ability to survive under longer antibiotic exposure and their high concentrations (22)260

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2-thiopyridine derivatives are effective against both M. tuberculosis active and262

dormant cells.263

Earlier, some 2-thiopyridine derivatives were found to have antileprosy activity (23).264

We therefore proposed that this group of chemical compounds may also possess anti-TB265

activity. In the present study we removed the potentially toxic nitro group and introduced a266

thiocyanato group as a result of different chemical modifications of the basic structures of 2-267

thiopyridine together with SAR studies. During SAR research we also suggested that one of268

the key factors of the antimicrobial activity of this group in the compounds is the level of269

positive charge on the second carbon atom of the pyridine ring. To confirm this hypothesis we270

synthesized several compounds where the pyridine nitrogen atom was oxidized. Then, the271

anti-TB activity of five 2-thiopyridine derivatives was studied (Fig. 3). Two compounds from272

this series, namely #11026103 and #11026115 demonstrated MICs of 0.25 g/ml (Table 2).273

The effectiveness of these five compounds was checked in the model of non-274

culturability of M. tuberculosiscells. As these NC cells are unable to produce colonies on275

non-selective agar-solidified media, we applied the MPN assay to check the effect of these276

compounds on the viability of dormant NC cells. In contrast to CFU counting this approach is277

able to uncover a sub-population of cells with decreased culturability which may arise as a278

result of antibiotic treatment.279

It was found that incubation of NC M. tuberculosis cells with some of the above280

mentioned compounds did affect their viability. In particular, incubation of NC cells with 10281

g/ml of both #11026114 and #11026115 for 7 days led to a more than 2-log killing effect282

(Fig. 4). At the same time NC cells are highly resistant to RIF and INH even at a high283

concentration (50 g/ml) (Fig. 2). The susceptibility of NC cells to the 2-thiopyridine284


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derivatives may indicate the existence of some residual metabolic activity in the latent state of285

the pathogen, which may be potentially targeted by novel antimicrobials. Whilst the target(s)286

of studied compounds in M. tuberculosisis not clear, some thiopyridines were identified as287

inhibitors of M. tuberculosis alanine racemase which plays an essential role in cell wall288

synthesis (24), or enoyl-acyl carrier protein reductase which catalyzes an essential step in fatty289

acid biosynthesis (25). However, MIC reported forM. tuberculosisfor the first inhibitor is 50290

times higher then for the compounds described in present study, and the second inhibitor was291

not tested on mycobacteria. Thus, the mechanism of action of the compounds discovered in292

the present work remains to be elucidated.293

For further studying the effectiveness of 2-thiopyridine derivatives on dormant M.294

tuberculosiscells we selected the compound #11026115 and performed two other well-known295

in vitrodormancy models: the Wayne hypoxia model (13) and the Betts starvation model (14).296

Dormant cells obtained in different models were treated with 10 g/ml of #11026115 for 7297

days. As the population of dormant cells in these models may contain a fraction of NC cells298

(arising before or after treatment) we applied the MPN assay for estimation of the killing299

effect of #11026115 instead of a standard CFU counting. According to the MPN assay,300

dormant cells in these models are characterized by slightly higher resistance to both RIF and301

INH than was reported previously (7, 13-15).Probably, antibiotic treatment may induce the302

partial transition of cells to a NC state instead of cell death, which was not noticed earlier303

because of applying standard CFU counting. We also observed a significant killing effect of304

compound #11026115 on these dormantM. tuberculosiscells obtained in the Wayne hypoxia305

model and the Betts starvation modelin vitro (Fig. 5). Despite the fact that dormant cells in306

both models are rather susceptible to RIF, treatment with compound #11026115 demonstrated307

an even stronger effect in comparison to RIF in all three of M. tuberculosisin vitrodormancy308

models and their effectiveness on actively growing cells was comparable to that of both RIF309


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and INH. So, we may conclude that several 2-thiopyridinas are able to kill dormant M.310

tuberculosisbacteria.311

Despite the recent success with drug candidates such as diarylquinolines (TMC207),312

which target ATP synthesis (26, 27), and benzothiazinones (BTZ043), which target essential313

cell-wall arabinan synthesis (28), the efficacy of both candidates at killing dormant bacilli was314

found to be low. For example, BTZ043 failed to kill cells with low metabolic status (15, 28,315

29)and the specific TMC207 affected the viability of dormant cells in the Wayne model but316

not the viability of nutrient-starved organisms (30).317

By a combination of two approaches - potassium deficiency and RIF treatment - we318

obtained a population containing dormant NC cells in a high concentration. The in vitromodel319

proposed in this study produces dormant NC cells which are characterized by remarkable320

metabolic cessation and considerable phenotypic resistance to both RIF and INH. Therefore,321

this model meets the key criteria of latent tuberculosis and may be applied as a relevant tool322

for latent TB drug discovery. Here, we demonstrated for the first time that 2-thiopyridines are323

able to kill dormantM.tbcells obtained under three different types of stress: hypoxia, nutrient324

starvation and non-culturability under potassium limitation. The most active compound was325

#11026115, which kills dormantM. tuberculosisbacteria obtained in all three in vitromodels326

of dormancy. We suggest 2-thiopyridinederivatives as a prominent group of compounds for327

further development of drugs for curing latent TB.328

Funding329

This work was supported by the Program of the Presidium of the Russian Academy of330

Sciences Molecular and Cellular Biology, FP7 project More medicines for tuberculosis331

(Grant 260872) and Russian Foundation of Basic Research grant number 12-04-01760-.332

Acknowledgements333

We are grateful to Prof. Stewart Cole (Global Health Institute, EPFL) for his critical334

reading of the manuscript and valuable comments.335


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References336

1.Koul A, Arnoult E, Lounis N, Guillemont J, Andries K.2011. The challenge of337

new drug discovery for tuberculosis. Nature. 469:483-90.338

2.Zhang Y.2004. Persistent and dormant tubercle bacilli and latent tuberculosis. Front339

Biosci. 9:1136-56.340

3.Lobue P, Menzies D.2010. Treatment of latent tuberculosis infection: An update.341

Respirology 15:603-22.342

4.Menzies D, Al Jahdali H, Al Otaibi B.2011. Recent developments in treatment of343

latent tuberculosis infection. Indian J. Med. Res. 133:257-66.344

5.Zhang Y, Yew WW, Barer MR.2012. Targeting persisters for tuberculosis control.345

Antimicrob Agents Chemother. 56:2223-30346

6.Dhillon J, Dickinson JM, Sole K, Mitchison DA.1996. Preventive chemotherapy of347

tuberculosis in Cornell model mice with combinations of rifampin, isoniazid, and348

pyrazinamide. Antimicrob. Agents Chemother. 40:552-5.349

7. Deb C, Lee CM, Dubey VS, Daniel J, Abomoelak B, Sirakova TD, Pawar S,350

Rogers L, Kolattukudy PE. 2009. A novel in vitro multiple-stress dormancy model for351

Mycobacterium tuberculosisgenerates a lipid-loaded, drug-tolerant, dormant pathogen. PLoS352

One. 4:e6077.353

8.Chao MC, Rubin EJ.2010. Letting sleeping dos lie: does dormancy play a role in354

tuberculosis? Annu Rev. Microbiol.64:293-311.355

9.Dhillon J, Lowrie DB, Mitchison DA. 2004. Mycobacterium tuberculosis from356

chronic murine infections that grows in liquid but not on solid medium. BMC Infect. Dis.357

4:51.358

10.Khomenko AG, Golyshevskaya VI. 1984. Filtrable forms of Mycobacteria359tuberculosis. Z. Erkr. Atmungsorgane. 162:147-54.360


15/26

15

11.Young M, Mukamolova GV, Kaprelyants AS.2005. Mycobacterial dormancy and361its relation to persistence. In: Parish T (ed). Mycobacterium: Molecular Microbiology.362

London: Horizon bioscience. 265320.363

12.Payne DJ, Gwynn MN, Holmes DJ Pompliano DL. 2007. Drugs for bad bugs:364confronting the challenges of antibacterial discovery. Nature Rev. Drug Discov. 6:2940.365

13.Wayne LG, Hayes LG.1996. An in vitromodel for sequential study of shiftdown366of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect.367

Immun. 64:2062-2069.368

14.Betts JC, Lukey PT, Robb LC, McAdam RA, Duncan K.2002. Evaluation of a369nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein370

expression profiling. Mol. Microbiol. 43:717-731.371

15.Sala C, Dhar N, Hartkoorn RC, Zhang M, Ha YH, Schneider P, Cole ST. 2010.372Simple model for testing drugs against nonreplicating Mycobacterium tuberculosis.373

Antimicrob. Agents Chemother. 54:4150-4158.374

16.Connell ND. 1994. Mycobacterium: isolation, maintenance, transformation, and375mutant selection. Methods Cell Biol. 45:107-25.376

17.de Man JC.1975. The probability of most probable numbers. J. Appl. Microbiol. 3771:67-78.378

18.National Committee for Clinical Laboratory Standards. 2007. Methods for379Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Fourth380

Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.381

19.Shleeva MO, Mukamolova GV, Young M, Williams HD, Kaprelyants AS.2004.382Formation of 'non-culturable' cells of Mycobacterium smegmatis in stationary phase in383

response to growth under suboptimal conditions and their Rpf-mediated resuscitation.384

Microbiology. 150:1687-1697.385


16/26

16

20.Kaprelyants AS, Kell DB. 1993. Dormancy in stationary-phase cultures of386Micrococcus luteus: flow cytometric analysis of starvation and resuscitation. Appl.387

Environment. Microbiol.59:3187-3196.388

21.Hu Y, Mangan JA, Dhillon J, Sole KM, Mitchison DA, Butcher PD, Coates AR.3892000. Detection of mRNA transcripts and active transcription in persistent Mycobacterium390

tuberculosisinduced by exposure to rifampin or pyrazinamide. J. Bacteriol.182:6358-6365.391

22.Ma C, Sim S, Shi W, Du L, Xing D, Zhang Y. 2010. Energy production genes392sucB and ubiF are involved in persister survival and tolerance to multiple antibiotics and393

stresses inEscherichia coli. FEMS Microbiol Lett. 303:33-40394

23.Makarov V, Riabova OB, Yuschenko A, Urlyapova N, Daudova A, Zipfel PF,395Mllmann U. 2006. Synthesis and antileprosy activity of some dialkyldithiocarbamates. J.396

Antimicrob. Chemother. 57:11341138.397

24.Anthony KG, Strych U, Yeung KR, Shoen CS, Perez O, Krause KL, Cynamon398MH, Aristoff PA, Koski RA.2011. New classes of alanine racemase inhibitors identified by399

high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis.400

PLoS One. 6:e20374401

25.Ling LL, Xian J, Ali S, Geng B, Fan J, Mills DM, Arvanites AC, Orgueira H,402Ashwell MA, Carmel G, Xiang Y, Moir DT. 2004. Identification and characterization of403

inhibitors of bacterial enoyl-acyl carrier protein reductase. Antimicrob Agents Chemother.404

48:1541-7.405

26.Andries K, Verhasselt P, Guillemont J, Ghlmann HW, Neefs JM, Winkler H,406Van Gestel J, Timmerman P, Zhu M, Lee E, Williams P, de Chaffoy D, Huitric E,407

Hoffner S, Cambau E, Truffot-Pernot C, Lounis N, Jarlier V. 2005. A diarylquinoline408

drug active on the ATP synthase ofMycobacterium tuberculosis. Science. 307:2237.409


17/26

17

27.Koul A, Dendouga N, Vergauwen K, Molenberghs B, Vranckx L, Willebrords R,410Ristic Z, Lill H, Dorange I, Guillemont J, Bald D, Andries K. 2007. Diarylquinolines target411

subunit c of mycobacterial ATP synthase. Nature Chem. Biol. 3:323324.412

28.Makarov V, Manina G, Mikusova K, Mllmann U, Ryabova O, Saint-Joanis B,413Dhar N, Pasca MR, Buroni S, Lucarelli AP, Milano A, De Rossi E, Belanova M,414

Bobovska A, Dianiskova P, Kordulakova J, Sala C, Fullam E, Schneider P, McKinney415

JD, Brodin P, Christophe T, Waddell S, Butcher P, Albrethsen J, Rosenkrands I, Brosch416

R, Nandi V, Bharath S, Gaonkar S, Shandil RK, Balasubramanian V, Balganesh T,417

Tyagi S, Grosset J, Riccardi G, Cole ST. 2009. Benzothiazinones kill Mycobacterium418

tuberculosisby blocking arabinan synthesis. Science. 324:801814.419

29.Zhang M, Sala C, Hartkoorn RC, Dhar N, Mendoza-Losana A, Cole ST. 2012.420Streptomycin-starved Mycobacterium tuberculosis 18b, a drug discovery tool for latent421

tuberculosis. Antimicrob. Agents Chemother. 56:5782-5789.422

30.Gengenbacher M, Rao SP, Pethe K, Dick T. 2010. Nutrient-starved, non-423replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate424

lyase for maintenance of ATP homeostasis and viability. Microbiology. 156:81-87.425

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Figure legends.436

Figure 1. Formation of NC M. tuberculosis cells under potassium-limiting437

conditions. Filled squares - gradual CFU decreasing in prolonged stationary phase; filled438

circles formation of a zero-CFU population after an additional treatment a 15-day-starved439

culture with a moderate concentration of RIF (5 g/ml). A potential viability of NC cells was440

estimated after resuscitation in liquid Sauton diluted medium by MPN assay. The arrow shows441

MPN value for 25-days starved with the additional treatment with RIF. Similar MPN values442

were also found for 15-30 days starved culture without RIF treatment. This experiment was443

repeated five times with similar results. Typical experiment is shown. Standard deviation for444

CFU did not exceed 10-20% for CFU mean and 20-30% for MPN mean.445

446

Figure 2.Resistance of NC M. tuberculosiscells to INH and RIF treatment. NC447

cells were washed and treated by different concentrations of antibiotics for 7 days. Viability of448

both treated and untreated NC cells was tested by the concentration of cells, which were able449

to recover from NC state (by MPN assay). The no-drug data represent an untreated control.450

Results are displayed as means and standard deviations of three independent experiments.451

452

Figure 3. Chemical structures of 11026101, 11026103, 11026114, 11026115 and453

10026127.454

455

Figure 4. Bactericidal effect of 2-thiopyridines on NC (dormant) M. tuberculosis456

cells.NC cells were treated by the compounds (10 g/ml) for 7 days at 37 C. Viability of both457

treated and untreated NC cells was tested by MPN assay. Bars represent 95% confidence458

limits.459

460


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Figure 5. The bactericidal effect of RIF, INH and compound #11026115 for log-461

phase and dormantM. tuberculosiscells obtained in different in vitromodels.Dark bars 462

the model of non-culturability in potassium limiting conditions; grey bars Wayne hypoxia463

model; light-grey bars Betts starvation model; white bars actively growing cells. Cells464

were treated by the 10 g/mlantimicrobials for 7 days. Viability of both treated and untreated465

dormant cells was estimated by the MPN assay. Bars represent 95% confidence limits.466

467

468

469

470

471

472

473

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477

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Table 1. Resistance ofM. tuberculosisdormant cells obtained in different models486

of TB dormancy in vitro to RIF and INH (percentage of resistant cells after a 7-day-487

treatment at 37 C). Ten-fold dilutions of M. tuberculosis suspensions were plated in488

triplicate onto agar-solidified Sauton medium (CFU counting) or in liquid Sauton ADC-489

supplemented medium containing Tween-80 0.05% (v/v) (MPN counting) in 48-well Corning490

microplates, wells with visible bacterial growth were scored as positive. Figures obtained in491

this study are in bold and calculated as means of three independent experiments.492

Models

Percentage of resistant

cells calculated by CFU

counting

Percentage of resistant

cells calculated by MPN

counting

RIF

(5-10

g/ml)

INH

(0.5-1

g/ml)

RIF

(5 g/ml)

INH

(1 g/ml)

Wayne model (Wayne and Heys,

1996)

< 0.10 96 0.8 100

Nutrient starvation (Betts et al.,

2002)

< 0.01 701.0 100

Multiple stress (Deb et al., 2009)

12.5 84.4 ND ND

Non-replicating state (Sala et al.,

2010)

< 0.01 30 ND ND

Non-culturability in

potassium-limiting conditions

(zero-CFU population)

* * 100 100

* CFU counting could not be performed due to non-culturability of cells493

494


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Table 2. Minimal Inhibitory Concentration of 2-thiopyridines forM. tuberculosis495

H37Rv cells. The MIC was determined according to the NCCLS guidelines using a broth496

microdilution method in Middlebrook 7H9 media supplemented with ADC with a final497

inoculum of 5x105cell/ml. The results of five independent experiments are shown.498

499

Compound MIC, g/ml

11026101 4.0

11026103 0.250

11026114 0.750

11026115 0.250

10026127 4.0

500

501

502

503

504

505

506

507

508

509

510

511


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