Sandra Salillas

Miriam Alías

Valérie Michel

Alejandro Mahía

Ainhoa Lucía

Liliana Rodrigues

Jessica Bueno

Juan José Galano-Frutos

Hilde De Reuse

Adrián Velázquez-Campoy

José Alberto Carrodeguas

Carlos Sostres

Javier Castillo

José Antonio Aínsa

Dolores Díaz-De-Villegas

Ángel Lanas

Eliette Touati

Javier Sancho

Design, Synthesis and Efficacy testing of nitroethylene-and 7-nitrobenzoxadiazol-based flavodoxin inhibitors against Helicobacter pylori drug-resistant clinical strains and in Helicobacter pylori-infected mice

Keywords: Novel antimicrobials, antimicrobial resistance, Helicobacter pylori, drug discovery, flavodoxin

INTRODUCTION

Helicobacter pylori (Hp) is a Gram-negative bacterium that establishes life-long infections in humans by colonising their gastric mucosa, usually during childhood. [START_REF] Marshall | Gastric Spirochaetes: 100 Years of Discovery before and after Kobayashi[END_REF][START_REF] Polk | Helicobacter Pylori: Gastric Cancer and Beyond[END_REF] Hp infection is the most common bacterial infection all over the world [START_REF] Polk | Helicobacter Pylori: Gastric Cancer and Beyond[END_REF][3][START_REF] Touati | Chronic Helicobacter Pylori Infections Induce Gastric Mutations in Mice[END_REF] with a prevalence that varies from 10 to 94 % in different countries. [START_REF]IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Schistosomes, Liver Flukes and Helicobacter Pylori[END_REF][START_REF] Eusebi | Epidemiology of Helicobacter Pylori Infection[END_REF][START_REF] Roberts | Review Article: The Prevalence of Helicobacter Pylori and the Incidence of Gastric Cancer across Europe[END_REF][START_REF] Mentis | Epidemiology and Diagnosis of Helicobacter Pylori Infection[END_REF] Hp transmission follows person-to-person, oraloral, faecal-oral, waterborne or iatrogenic routes. [START_REF]IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Schistosomes, Liver Flukes and Helicobacter Pylori[END_REF][START_REF]IARC Working Group on the Evaluation of Carcinogenic Risks to Humans[END_REF] In most cases, Hp infection is asymptomatic, but it can progressively damage the gastric mucosa 10 by inducing chronic gastritis [START_REF] Marshall | Gastric Spirochaetes: 100 Years of Discovery before and after Kobayashi[END_REF] and then diseases from peptic ulcer to gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric adenocarcinoma. [START_REF] Polk | Helicobacter Pylori: Gastric Cancer and Beyond[END_REF]3,[START_REF] Sugano | Kyoto Global Consensus Report on Helicobacter Pylori Gastritis[END_REF] Hp is the main cause of gastric cancer all over the world. [START_REF] Sugano | Kyoto Global Consensus Report on Helicobacter Pylori Gastritis[END_REF][START_REF] Malfertheiner | The European Helicobacter Study Group. Management of Helicobacter Pylori Infection-the Maastricht IV/ Florence Consensus Report[END_REF] Up to now, it is the only bacterium classified as a group I carcinogen. [START_REF] Touati | Chronic Helicobacter Pylori Infections Induce Gastric Mutations in Mice[END_REF][START_REF]IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Schistosomes, Liver Flukes and Helicobacter Pylori[END_REF][START_REF]IARC Working Group on the Evaluation of Carcinogenic Risks to Humans[END_REF][START_REF] Malfertheiner | The European Helicobacter Study Group. Management of Helicobacter Pylori Infection-the Maastricht IV/ Florence Consensus Report[END_REF] Eradication of Hp infection is the gold standard in peptic ulcer disease treatment and might prevent gastric cancer, [START_REF] Malfertheiner | The European Helicobacter Study Group. Management of Helicobacter Pylori Infection-the Maastricht IV/ Florence Consensus Report[END_REF][START_REF]Helicobacter Pylori Eradication as a Strategy for Preventing Gastric Cancer[END_REF][START_REF] Leung | Effects of Helicobacter Pylori Treatment on Incidence of Gastric Cancer in Older Individuals[END_REF] the third most common cause of cancer death. [START_REF] O'connor | Population Screening and Treatment of Helicobacter Pylori Infection[END_REF] Effective and affordable treatments are needed to reduce the impact of Hp infection in those diseases. Moreover, Hp infection has been suggested to be involved in other extragastrointestinal disorders [START_REF] Malfertheiner | The European Helicobacter Study Group. Management of Helicobacter Pylori Infection-the Maastricht IV/ Florence Consensus Report[END_REF][START_REF] Franceschi | Role of Helicobacter Pylori Infection on Nutrition and Metabolism[END_REF][START_REF] Franceschi | Extragastric Diseases and Helicobacter Pylori[END_REF][START_REF] Malfertheiner | European Helicobacter and Microbiota Study Group and Consensus panel. Management of Helicobacter Pylori Infection-the Maastricht V/Florence Consensus Report[END_REF] and may modify the bioavailability and absorption of essential nutrients and the plasma levels of metabolic hormones. [START_REF] Franceschi | Role of Helicobacter Pylori Infection on Nutrition and Metabolism[END_REF] Conventional therapy against Hp consists of empirical triple or, more often now, quadruple treatments based on a proton pump inhibitor plus a combination of two or three broad spectrum antimicrobials such as clarithromycin (Cla), amoxicillin, metronidazole (Mnz), tetracycline and bismuth salts. [START_REF] Malfertheiner | The European Helicobacter Study Group. Management of Helicobacter Pylori Infection-the Maastricht IV/ Florence Consensus Report[END_REF][START_REF] Malfertheiner | European Helicobacter and Microbiota Study Group and Consensus panel. Management of Helicobacter Pylori Infection-the Maastricht V/Florence Consensus Report[END_REF][START_REF] Smith | Antimicrobial Susceptibility Testing for Helicobacter Pylori in Times of Increasing Antibiotic Resistance[END_REF][START_REF] Safavi | Treatment of Helicobacter Pylori Infection: Current and Future Insights[END_REF] Increasing Hp resistance to these common antimicrobial drugs, especially to Cla and Mnz, is growing worldwide, reducing the effectiveness of available therapies. [START_REF] Malfertheiner | The European Helicobacter Study Group. Management of Helicobacter Pylori Infection-the Maastricht IV/ Florence Consensus Report[END_REF][START_REF] Smith | Antimicrobial Susceptibility Testing for Helicobacter Pylori in Times of Increasing Antibiotic Resistance[END_REF][START_REF] Safavi | Treatment of Helicobacter Pylori Infection: Current and Future Insights[END_REF][START_REF] Gerrits | Helicobacter Pylori and Antimicrobial Resistance: Molecular Mechanisms and Clinical Implications[END_REF][START_REF] Tacconelli | Research, and Development of New Antibiotics: The WHO Priority List of Antibiotic-Resistant Bacteria and Tuberculosis[END_REF] Therefore, new specific treatments are required, [START_REF] Debraekeleer | Future Perspective for Potential Helicobacter Pylori Eradication Therapies[END_REF] and eradication strategies need to be adapted geographically. [START_REF] Malfertheiner | The European Helicobacter Study Group. Management of Helicobacter Pylori Infection-the Maastricht IV/ Florence Consensus Report[END_REF][START_REF] Smith | Antimicrobial Susceptibility Testing for Helicobacter Pylori in Times of Increasing Antibiotic Resistance[END_REF][START_REF] Savoldi | Prevalence of Antibiotic Resistance in Helicobacter Pylori: A Systematic Review and Meta-Analysis in World Health Organization Regions[END_REF] Ongoing research on vaccines against Hp has not been successful until now, probably due to Hp genetic variability and to the complex host immune response against the bacterium. 9, [START_REF] Del Giudice | Development of Vaccines against Helicobacter Pylori[END_REF][START_REF] Abadi | Vaccine against Helicobacter Pylori: Inevitable Approach[END_REF][START_REF] Blosse | Helicobacter: Inflammation, Immunology, and Vaccines[END_REF] Current alternatives proposed include the application of therapeutic regimes based on local patterns of antimicrobial susceptibility and the use of personalized treatments consisting of the pre-identification of Hp susceptibility/resistance to conventional antimicrobials by molecular or by cultured-guided methods. [START_REF]Helicobacter Pylori Eradication as a Strategy for Preventing Gastric Cancer[END_REF][START_REF] Smith | Antimicrobial Susceptibility Testing for Helicobacter Pylori in Times of Increasing Antibiotic Resistance[END_REF][START_REF] Gerrits | Helicobacter Pylori and Antimicrobial Resistance: Molecular Mechanisms and Clinical Implications[END_REF][START_REF] Papastergiou | Treatment of Helicobacter Pylori Infection: Meeting the Challenge of Antimicrobial Resistance[END_REF] The use of antimicrobial peptides and the development of novel compounds acting on specific Hp targets have also been proposed.

Following the latter approach, specific Hp targets, essential for the bacterium and absent in humans, have been identified. [START_REF] Follmer | Ureases as a Target for the Treatment of Gastric and Urinary Infections[END_REF][START_REF] Neelapu | Identification of Potential Drug Targets in Helicobacter Pylori Strain HPAG1 by in Silico Genome Analysis[END_REF][START_REF] Freigang | Crystal Structure of Oxidized Flavodoxin, an Essential Protein in Helicobacter Pylori[END_REF] One such target is Hp flavodoxin (Hp-Fld), a small acidic redox protein that contains one molecule of tightly bound flavin mononucleotide cofactor. [START_REF] Freigang | Crystal Structure of Oxidized Flavodoxin, an Essential Protein in Helicobacter Pylori[END_REF][START_REF] Martıínez-Júlvez | Common Conformational Changes in Flavodoxins Induced by FMN and Anion Binding: The Structure of Helicobacter Pylori Apoflavodoxin[END_REF][START_REF] Cremades | The Flavodoxin from Helicobacter Pylori: Structural Determinants of Thermostability and FMN Cofactor Binding[END_REF][START_REF] Ayuso-Tejedor | Structural Analysis of an Equilibrium Folding Intermediate in the Apoflavodoxin Native Ensemble by Small-Angle X-Ray Scattering[END_REF] Flavodoxins [START_REF] Sancho | Flavodoxins: Sequence, Folding, Binding, Function and Beyond[END_REF][START_REF] Rodríguez-Cárdenas | Streptococcus Pneumoniae TIGR4 Flavodoxin: Structural and Biophysical Characterization of a Novel Drug Target[END_REF] are bacterial proteins that take part in a variety of electron transfer reactions. In particular, Hp-Fld accepts electrons from the pyruvate ferredoxin oxidoreductase complex (PFOR), which catalyses the oxidative decarboxylation of pyruvate, and transfers them to flavodoxin quinone reductase. [START_REF] Freigang | Crystal Structure of Oxidized Flavodoxin, an Essential Protein in Helicobacter Pylori[END_REF][START_REF] Hughes | Identification of Carboxylation Enzymes and Characterization of a Novel Four-Subunit Pyruvate:flavodoxin Oxidoreductase from Helicobacter Pylori[END_REF][START_REF] St | Quinone Reductase (FqrB): A Redox Partner of Pyruvate:Ferredoxin Oxidoreductase That Reversibly Couples Pyruvate Oxidation to NADPH Production in Helicobacter Pylori and Campylobacter Jejuni[END_REF] Both Hp-Fld and Hp-PFOR are essential for the bacterium survival [START_REF] Freigang | Crystal Structure of Oxidized Flavodoxin, an Essential Protein in Helicobacter Pylori[END_REF][START_REF] Hughes | Helicobacter Pylori porCDAB and oorDABC Genes Encode Distinct Pyruvate:Flavodoxin and 2-Oxoglutarate:Acceptor Oxidoreductases Which Mediate Electron Transport to NADP[END_REF] and compounds interfering with this pathway might be suited for Hp eradication therapies (Figure 1). [START_REF] Freigang | Crystal Structure of Oxidized Flavodoxin, an Essential Protein in Helicobacter Pylori[END_REF]39 Unlike most other flavodoxins, Hp-Fld contains a pocket near the active site where small molecules could bind [START_REF] Freigang | Crystal Structure of Oxidized Flavodoxin, an Essential Protein in Helicobacter Pylori[END_REF][START_REF] Martıínez-Júlvez | Common Conformational Changes in Flavodoxins Induced by FMN and Anion Binding: The Structure of Helicobacter Pylori Apoflavodoxin[END_REF] interfering with binding or electron transfer to Hp-Fld partner proteins. 39 Because flavodoxins as such are not present in humans, and the Fld-like domain found in human cytochrome P450 reductase lacks an equivalent pocket near its active site, no side effects are anticipated for a therapy targeting Hp-Fld. 39

6

In earlier work we have characterized the biophysical properties of Hp-Fld [START_REF] Martıínez-Júlvez | Common Conformational Changes in Flavodoxins Induced by FMN and Anion Binding: The Structure of Helicobacter Pylori Apoflavodoxin[END_REF][START_REF] Cremades | The Flavodoxin from Helicobacter Pylori: Structural Determinants of Thermostability and FMN Cofactor Binding[END_REF][START_REF] Ayuso-Tejedor | Structural Analysis of an Equilibrium Folding Intermediate in the Apoflavodoxin Native Ensemble by Small-Angle X-Ray Scattering[END_REF]39,[START_REF] Cremades | Conformational Stability of Helicobacter Pylori Flavodoxin Fit to Function at pH 5[END_REF] and have identified through experimental screening several small interacting molecules that bind to it. Three of them (named compounds I, II and IV) inhibit Hp-Fld-mediated pyruvate decarboxylation and act as specific bactericidal agents for Hp. [START_REF] Cremades | Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter Pylori Infection[END_REF] Based on those three hits, 123 new derivative molecules were developed, some of which showed better bactericidal properties than the three original ones. [START_REF] Galano | Improved Flavodoxin Inhibitors with Potential Therapeutic Effects against Helicobacter Pylori Infection[END_REF] Because the hits and many of the improved derivatives contain a nitro group and Hp is known to activate nitro groups in certain prodrugs through redox dependent reactions, [START_REF] Gerrits | Helicobacter Pylori and Antimicrobial Resistance: Molecular Mechanisms and Clinical Implications[END_REF][START_REF] Edwards | Nitroimidazole Drugs -Action and Resistance Mechanisms I. Mechanisms of Action[END_REF][START_REF] Olekhnovich | Characterization of the NAD(P)H Oxidase and Metronidazole Reductase Activities of the RdxA Nitroreductase of Helicobacter Pylori[END_REF][START_REF] Martínez-Júlvez | Structure of RdxA: An Oxygen Insensitive Nitroreductase Essential for Metronidazole Activation in Helicobacter Pylori[END_REF][START_REF] Ang | Molecular Fireworks That Combat a Broad Spectrum of Infectious Diseases[END_REF] we have synthesized new anti-Hp compounds that contain partially or fully reduced forms of the nitro groups present in the parent molecules, and additional redox variants modifying sulphur and vinyl groups of the inhibitors. The efficacy of these compounds has been tested in reference Hp strains, Hp clinical isolates, and in Hp-infected mice.

RESULTS AND DISCUSSION

Synthetic

HOAc

O

The amino-sulfone IV-e was synthesized by oxidation of compound IV-a using metachloroperbenzoic acid in ethyl acetate as the oxidant. 51 Compound IV-e was obtained as a mixture with compoundIV-d, from which it was separated by flash chromatography (Scheme 7). Commercially available metronidazole was transformed to Mnz-a by catalytic reduction using palladium-on-carbon as the catalyst (Scheme 8). [START_REF] Iaroshenko | Facile Synthesis of Fluorinated 1-Desazapurines[END_REF] The resulting amino-imidazole proved to be sensitive to oxygen, heat and acid and therefore of limited utility; nevertheless, its toxicity and activity were determined. In each group the upper line provides the formula of the lead and derivatives preliminary tested in vitro in previous work. [START_REF] Cremades | Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter Pylori Infection[END_REF][START_REF] Galano | Improved Flavodoxin Inhibitors with Potential Therapeutic Effects against Helicobacter Pylori Infection[END_REF] The middle line gives the formula of newly synthesized derivatives (see Schemes 1-8 and Synthesis details below). The lower line for II-related compounds provides the formula of commercial compounds not previously tested as antimicrobials against Hp. All of them are less cytotoxic toward human HeLa cells than their corresponding parent compounds (Table 1). In fact, most derivatives are not cytotoxic at concentrations as high as 1 mM (Figure S2). The in vitro anti-Hp activities of these derivatives have been evaluated using three Hp reference strains (Table 1). d Therapeutic Indexes (TI) have been calculated as MCC 50 /MIC Compounds I and II share the presence of a nitroethylene moiety and their derivatives will be jointly discussed. In this family of compounds, reduction of the nitro groups to oxime lowers similarly cytotoxicity and antimicrobial activity, barely changing the TIs for the three Hp strains tested (Table 1). On the other hand, reduction of the ethylene group leads to a drastic decrease of toxicity with a notable reduction of antimicrobial activity, which results in a small increase of TI. Since reduction of the ethylene group lowers so much toxicity, differences in TIs among the ethylene-reduced variants cannot be properly evaluated. In Mnz, the reduction of the nitro group to amine clearly lowers the antimicrobial activity, while possible changes in cytotoxicity cannot be evaluated as neither

14
Mnz nor the amine variant are toxic at the higher concentration tested. Globally considered, reduction of the nitro or the ethylene group in the leads carrying a nitroethylene moiety (I, II and Mnz), decreases to a similar extent toxicity and antimicrobial activity. Only compounds II-a (previously tested by Galano et al., 2013 [START_REF] Galano | Improved Flavodoxin Inhibitors with Potential Therapeutic Effects against Helicobacter Pylori Infection[END_REF] and here re-evaluated), II-b, IIc and II-f slightly improve (about 2-fold) the TI of their parent compound.

Compound IV also contains a nitro group, which, in this case, is attached to a benzoxadiazole ring. In this lead, reduction of the nitro group to amine lowers toxicity so much (>100-fold) that the concomitant moderate decrease in activity observed (around 10fold) combines to a 10-fold higher TI in compound IV-a. The toxicity of compound IV is also greatly reduced (25-fold) by oxidation of the sulphur atom to sulfoxide, which causes a less pronounced decrease in activity and therefore also raises the TI. Oxidation of the sulphur atom to sulfoxide in IV-a, compound IV-d, similarly causes a smaller decrease in activity, while a possible further decrease in toxicity cannot be determined because IV-a is already non-toxic at the higher concentration tested (1 mM). Further oxidation of the sulphur atom in IV-a to sulfone, compound IV-e, lowers the activity. However, chlorination of IV-a increases the antimicrobial activity 2-4 fold and does not introduce toxicity, leading to the high TIs of compound IV-c (from >85 to >170, depending on the Hp reference strain tested). We have also tested an additional IV-related compound: IV-b, that was considered promising in previous assays [START_REF] Galano | Improved Flavodoxin Inhibitors with Potential Therapeutic Effects against Helicobacter Pylori Infection[END_REF] . Unlike all other IV variants, IV-b lacks the benzoxadiazol ring and, therefore, the nitro group. Its toxicity is very low (>1000 µM)

and its anti-Hp activity is similar to that of compound IV. Therefore, the TI of IV-b is quite high (from 164 to 333 depending on the Hp strain). Our analysis of the effect of the flavodoxin inhibitors on these clinical isolates (Table 3 and Table S2) confirms the activity trends previously observed when evaluated against reference strains (Table 1). While compounds II-b, II-d, II-e, IV-d, IV-e display low TIs,

16
Efficacy of compounds against antimicrobial-resistant Hp-strains. Most compounds in Table 1 display higher TI than Mnz for the Mnz-resistant strain NCTC 11637. In addition, several nitro-reduced derivatives of compound IV (i.e IV-a, IV-c, IV-d) and compound IV-b display, for the two Mnz-sensitive strains tested (strains SS1 and 26695), TIs close to that of Mnz, or even higher. To explore the potential usefulness of these flavodoxin inhibitors towards other Hp drug-resistant strains, we have determined the activity of 6

representative commonly used antimicrobials on six Hp strains obtained from patients who have relapsed or are refractory to, at least, two conventional therapies [START_REF] Gisbert | IV Spanish Consensus Conference on Helicobacter Pylori Infection Treatment[END_REF] . Three of these strains (isolates 1-3) are Mnz-resistant, two are both Mnz-and Cla-resistant (isolates 4 and 6) and one is rifampicin-resistant (isolate 5) (Table 2). as reported in Table 1. In bold, TI values indicative of effectivity, according to EUCAST criteria.

Efficacy of compounds against Hp gastric colonization in mice.

The anti-Hp activity of these novel compounds has also been investigated in the mouse model of Hp infection. [START_REF] Lee | A Standardized Mouse Model of Helicobacter Pylori Infection: Introducing the Sydney Strain[END_REF][START_REF] Correia | Docosahexaenoic Acid Inhibits Helicobacter Pylori Growth In Vitro and Mice Gastric Mucosa Colonization[END_REF] First, the toxicity of three representative inhibitors (II-a, IV and IV-a) at doses of 10, 20

17
with the exception of II-e on the isolate 4 (a Cla-and Mnz-resistant strain), compounds IV-a and IV-c show high TIs for most clinical strains. Indeed, according to EUCAST criteria, both IV-c and IV-a are effective against two Mnz-resistant strains (isolates 1 and

2) and against the two Cla-and Mnz-resistant strains (isolates 4 and 6). In addition, IV-c is also effective against the rifampicin-resistant strain (isolate 5). Only one Mnz-resistant strain (isolate 3) displayed low TIs for both IV-c and IV-a (Table 3). and 40 mg/100 g bw has been evaluated. Daily administration of compound IV-a for 8 days had no deleterious effects in stomach, liver, heart, lung and kidney at any dose, as determined by histopathological examination (Figure S3) and analysis of their biochemical parameters (data not shown). Compounds IV and II-a did not induce significant alterations at any dose in liver, heart, lung or kidney either, but they did provoke histological alterations in stomach when administered at the highest dose of 40 mg/100 g (compound As reported in Figure 3A, parent compound I was able to eradicate the Hp infection in some mice (1/5 and 1/7) at 0.1 mg and 1 mg/100 g bw daily doses, respectively, but the overall reduction of the gastric colonization rate observed was not statistically significant.

IV
The oxime variant I-a decreased significantly the colonization rate at 10 mg/100 g bw 22 (p=0,016), but did not eradicate Hp infection from any mouse. The performance of the IIrelated derivatives was also limited. Parent compound II had no inhibitory effect on the gastric colonization; however, the ethylene-reduced II-b eradicated Hp infection from some mice (1/6) at a 20 mg/100 g bw dose; the ethylene-reduced and nitro reduced II-c amine derivative also eradicated Hp infection from some mice (1/4) and significantly reduced the colonization rate (p=0.024) at 20 mg/100 g dose. In addition, compound II-a reduced the colonization (p=0.0009) at a 1 mg/100 g bw dose.

The best performance comes from IV-related compounds, in agreement with in vitro testing. Both the parent compound IV, the amine-derivatives IV-a and IV-d, and the nonbenzoxadiazole containing compound IV-b significantly lowered the colonization rate at most of the doses tested (Figure 4A) and eradicated the infection in some mice (Figure 4B). Finally, compound IV-d, the sulfoxide version of compound IV-a, decreased colonization at 1, 10 and 20 mg/100 g bw doses (p=0.005, 0.0002 and 0.0122, respectively) and eradicate the infection in 60 % (3/5) of animals at the higher dose and in 5 % (1/20) of mice at 10 mg/100 g bw.

23

we discovered flavodoxin inhibitors I, II and IV [START_REF] Cremades | Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter Pylori Infection[END_REF][START_REF] Galano | Improved Flavodoxin Inhibitors with Potential Therapeutic Effects against Helicobacter Pylori Infection[END_REF] and explored the feasibility of improving their antibacterial activity by replacing some of their substituents. [START_REF] Galano | Improved Flavodoxin Inhibitors with Potential Therapeutic Effects against Helicobacter Pylori Infection[END_REF] To try to further improve their therapeutic indexes, we have synthesized new variants exhibiting different redox forms of the nitro, ethylene or sulphur groups present in the original inhibitors. Their potential for Hp eradication has been tested in the mice model of infection, and their activity against drug-resistant clinical Hp isolates have been determined. In all variants tested, partial or full reduction of the nitro group, reduction of the ethylene group, or partial or full oxidation of the sulphur atom largely reduces their cytotoxicity towards both HeLa cells and mice. While in the I and II series concomitant decreases in antibacterial activity are similarly large and the TIs are not improved, within the IV series, the activity decreases are only moderate and the resulting derivatives exhibit much larger TIs for Hp reference strains than the parent compound (Table 1). In fact, some of these novel flavodoxin inhibitors display TIs comparable to those of Mnz and are effective against a variety of Hp clinical isolates resistant to common antimicrobials such as Cla, Mnz or rifampicin. Importantly, these inhibitors, used as sole agents against Hp infection in the mice model, can significantly lower the gastric Hp load (Figures 3A and4A) and, in some mice, they have eradicated the infection (Figures 3B and4B). Thus, compound IV and its derivatives constitute a promising new family of antimicrobial chemicals with potential against the increase of Hp drug-resistant strains.

While their usefulness in novel combination therapies may be already worth investigating to provide alternatives for the existing drugs used against Hp infection, it is also clear that these inhibitors need to be improved before they can be used as stand-alone eradication drugs. Two research lines are being followed toward this end. On one hand, the mechanism of action of these inhibitors needs to be clarified. As they were discovered using a simple binding assay to the target protein, [START_REF] Cremades | Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter Pylori Infection[END_REF][START_REF] Velazquez-Campoy | Biophysical Screening for Identifying Pharmacological Chaperones and Inhibitors Against Conformational and Infectious Diseases[END_REF] a straightforward inhibitory mechanism was proposed [START_REF] Cremades | Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter Pylori Infection[END_REF] consisting on the inhibitors sterically blocking the flavodoxin interaction with physiological partner proteins. [START_REF] Freigang | Crystal Structure of Oxidized Flavodoxin, an Essential Protein in Helicobacter Pylori[END_REF][START_REF] Hughes | Identification of Carboxylation Enzymes and Characterization of a Novel Four-Subunit Pyruvate:flavodoxin Oxidoreductase from Helicobacter Pylori[END_REF][START_REF] St | Quinone Reductase (FqrB): A Redox Partner of Pyruvate:Ferredoxin Oxidoreductase That Reversibly Couples Pyruvate Oxidation to NADPH Production in Helicobacter Pylori and Campylobacter Jejuni[END_REF] The fact that, besides the original inhibitors, [START_REF] Cremades | Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter Pylori Infection[END_REF] all the new derivatives herein tested bind to Hp flavodoxin (as determined by isothermal titration calorimetry and thermal shift assay, not shown) lends support to this mechanism and provides direct experimental evidence for in vitro target engagement. Work is in progress to obtain structural information on flavodoxin/inhibitor complexes as this could facilitate their rational redesign and improvement. In addition, the possibility that flavodoxin activates the compounds needs to be considered as the inhibitors contain nitro groups whose reduction could generate radical cytotoxic products ultimately responsible for the observed antibacterial activity. In this respect, the antibacterial activity of Mnz towards Hp and other microaerophilic pathogens is thought to be mediated by one such redox activation mechanism. [START_REF] Edwards | Nitroimidazole Drugs -Action and Resistance Mechanisms I. Mechanisms of Action[END_REF][START_REF] Ang | Molecular Fireworks That Combat a Broad Spectrum of Infectious Diseases[END_REF][START_REF] Müller | Mode of Action of Metronidazole on Anaerobic Bacteria and Protozoa[END_REF][START_REF] Edwards | Mechanisms of Selective Toxicity of Metronidazole and Other Nitroimidazole Drugs[END_REF][START_REF] Edwards | Nitroimidazole Drugs -Action and Resistance Mechanisms II. Mechanisms of Resistance[END_REF] This alternative activating mechanism cannot be invoked, though, in the case of compound IV-b, the analogue of IV lacking a nitro group. Interestingly, the fact that inhibitors such as IV-a retain antibacterial activity when carrying an amine instead of the reducible nitro group of lead compound IV indicates that, if radical species played a role in the bactericidal activity, they would have to be generated in a reverse manner from cellular oxidation of the amine. On the other hand, a second line for improvement is suggested by the poor aqueous solubility of the inhibitors, reflected in their logS values (Table S1 and Figure S1), and possibly related to the toxicity observed in some of the mice stomachs at the highest dose of the parent compound IV, which we interpret as a chemical direct toxicity since no other alterations are observed. The solubility of the inhibitors needs to be increased by a judicious incorporation of polar groups in order to increase their 25 bioavailability and ensure a systemic effect. Work is in progress to synthesise and test more soluble variants of compound IV-derived inhibitors. Finally, unlike broad-spectrum antimicrobials targeting very fundamental cellular components such as DNA, ribosomes or the cell wall, flavodoxin inhibitors target a protein that is not essential for all bacteria and whose sequence conservation is not particularly high. Our preliminary analysis of the inhibitors' specificity revealed that compound IV was not effective against H. pylori distant bacteria such as E. coli or S. aureus. [START_REF] Cremades | Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter Pylori Infection[END_REF] We have now confirmed the same is true for the novel non-toxic inhibitors described here (not shown). These flavodoxin inhibitors thus appear to be selective and may be less damaging to the commensal microbiota than broadspectrum antimicrobials. 60 analyzer, and spectra were recorded from methanolic solutions using the positive electrospray ionization mode (ESI+). Reactions were magnetically stirred and whenever possible monitored by TLC. TLC was performed on precoated silica gel polyester plates and products were visualized using ultraviolet light (254 nm), ninhydrin, potassium permanganate and phosphomolybdic acid solution followed by heating. Column chromatography was performed using silica gel (Kiesegel 60, 230-400 mesh).

CONCLUSION

1-(2-nitroethyl)-3,5-bis(trifluoromethyl)benzene (II-b).

To a vigorously stirred mixture of compound II (522 mg, 1.83 mmol), silica gel (3.7 g, column chromatography grade, -63.00 ppm. [START_REF] Leung | Effects of Helicobacter Pylori Treatment on Incidence of Gastric Cancer in Older Individuals[END_REF] C NMR (100 MHz, CDCl 3 ): δ 138.4, 132.5 (q, J = 33.5 Hz), 129.0 (q, J = Page 29 of 53

3.6 Hz), 123.2 (q, J = 272.7 Hz), 121.8 (m, J = 3.8 Hz), 75.2, 32.8 ppm. HRMS (ESI + ) m/z

[M+H] + calc for C 10 H 8 F 6 NO 2 288.0454, found 288.0460.

2-(3,5-bis(trifluoromethyl)phenyl)ethan-1-amine (II-c).

A solution of II-b (57.4 mg, 0.2 mmol) in ethanol (10 mL) was hydrogenated under hydrogen atmosphere (1 atm) at room temperature in the presence of 10 % palladium/carbon (10 mg). The reaction was stirred for 5 h or until complete as judged by TLC. Filtration of the catalyst and evaporation of the solvent afforded 51.4 mg (≈ 100 %) of pure desired product. [START_REF] Marshall | Gastric Spirochaetes: 100 Years of Discovery before and after Kobayashi[END_REF] H NMR (400 MHz, CDCl 3 ): δ 7.74 (s, 1H), 7.66 (s, 2H), 3.05 (t, J = 6.8 Hz, 2H), 2.90 (t, J = 6.8 Hz, 2H), 1.96 (bs, 1H) ppm. [START_REF] Safavi | Treatment of Helicobacter Pylori Infection: Current and Future Insights[END_REF] F NMR (376 MHz, CDCl 3 ): δ -62.83 ppm. [START_REF] Leung | Effects of Helicobacter Pylori Treatment on Incidence of Gastric Cancer in Older Individuals[END_REF] C NMR (100 MHz, CDCl 3 ): δ 142.4, 131.8 (q, J = 33.2 Hz), 129.1 (q, J = 2.6 Hz), 123.5 (q, J = 272.2 Hz), 120.5 (m, J = 3.9 Hz), 43.0, 39.5 ppm. HRMS (ESI + ) m/z [M+H] + calc for C 10 H 9 F 6 N 258.0712, found 258.0711.

2-(3,5-bis(trifluoromethyl)phenyl)acetaldehyde oxime (II-d).

A round-bottom flask was charged with Pd(OAc) 2 (10 mg, 0.04mmol), the nitroarene II (214 mg, 0.75 mmol) and freshly distilled THF (4.5 mL). The flask was sealed and purged with Ar. Degassed H 2 O 29 Sigma-Aldrich), 2-propanol (5.5 mL) and chloroform (30 mL), NaBH 4 (284 mg, 7.5 mmol)

was added in about 40 mg portions over a period of 15 min at 25 ºC. The mixture was stirred for additional 2 hours or until completion of the reaction. Excess NaBH 4 was decomposed with diluted hydrochloric acid and the mixture was filtered. The filter was extracted with dichloromethane (3×50 mL) and the combined filtrates were washed with brine (50 mL), dried over anhydrous magnesium sulphate, filtered and evaporated in vacuo to dryness to give 504 mg (96 % yield) of the desired product which was used in the next step without further purification. [START_REF] Marshall | Gastric Spirochaetes: 100 Years of Discovery before and after Kobayashi[END_REF] H NMR (400 MHz, CDCl 3 ): δ 7.81 (s, 1H), 7.70 (s, 2H), 4.69 (t, J = 6.9 Hz, 2H), 3.46 (t, J = 6.9 Hz, 2H) ppm. [START_REF] Safavi | Treatment of Helicobacter Pylori Infection: Current and Future Insights[END_REF] F NMR (376 MHz, CDCl 3 ): δ

(1.75 mL) and i Pr 3 SiH (1.1 g, 7.0 mmol) were sequentially, slowly added dropwise with a syringe. The reaction was stirred for 2 h or until completion as judged by TLC. At that time, the reaction flask was opened to the atmosphere and the reaction mixture was diluted with diethyl ether (25-50 mL). The layers were separated and the aqueous layer was backextracted with diethyl ether (3×20 mL). The combined organic layers were dried over anhydrous magnesium sulphate, filtered and concentrated in vacuo. Purification of the residue by flash chromatography (eluent: Hex/EtOAc: 8/2) gave 61 mg (30 % yield) of the desired product as a 1:1mixture of Z/E isomers. [START_REF] Marshall | Gastric Spirochaetes: 100 Years of Discovery before and after Kobayashi[END_REF] H NMR (400 MHz, CDCl 3 ): δ 7.78 (s, 1H) and 7.78 (s, 1H), 7.70 (s, 2H) and 7,68 (s, 2H), 7.58 (t, J = 6 Hz, 1H) and 6.92 (t, J = 5.6

Hz, 1H), 3.86 (d, J = 5.6 Hz, 2H) and 3.67 (d, J = 6 Hz, 2H) ppm. [START_REF] Safavi | Treatment of Helicobacter Pylori Infection: Current and Future Insights[END_REF] F NMR (376 MHz, CDCl 3 ): δ -62.87 and -62.88 ppm. [START_REF] Leung | Effects of Helicobacter Pylori Treatment on Incidence of Gastric Cancer in Older Individuals[END_REF] C NMR (100 MHz, CDCl 3 ): δ 148.8 and 148.5, 139.0 (q, J = 33.4 Hz), 132.2 (q, J = 33.4 Hz) and 132.2 (q, J = 33.3 Hz), 129. After extracting with ethyl acetate (3 x 3 mL), the combined organic layers were dried over anhydrous magnesium sulphate, filtered and the solvent was evaporated under reduced pressure. TLC monitoring revealed that a mixture of IV-d and IV-e had been obtained. The crude was purified by flash chromatography (eluent: Hex/EtOAc: 3/7) to provide 27 mg (85 % yield) of pure IV-e. hydrogen peroxide (242 µL) and the reaction was stirred at 25 ºC for 24 h. The obtained orange-brown oily mixture was extracted with dichloromethane (3×10 mL). The combined organic layers were dried over anhydrous magnesium sulphate, filtered and the solvent was evaporated under reduced pressure. Flash chromatography of the mixture was done in order to purify the desired product. In this way 84.3 mg (88 % yield) of pure IV-f, which was stored in a sealed flask under an argon atmosphere at 4 °C until testing to avoid spontaneous decomposition because of its sensitivity to oxygen, were obtained. [START_REF] Tomb | The Complete Genome Sequence of the Gastric Pathogen Helicobacter Pylori[END_REF] and SS1 (Sydney Strain 1) [START_REF] Lee | A Standardized Mouse Model of Helicobacter Pylori Infection: Introducing the Sydney Strain[END_REF] (Biosafety Level 2 pathogen) were grown in Brain-Heart Infusion (BHI) broth supplemented with 4 % fetal bovine serum at 37 °C under microaerophilic conditions and then diluted to an optical density at 600 nm of 0.01. Samples of 100 µL of the diluted bacterial suspension were dispensed in a 96-well plate, except for the first well of each row in which 200 µL of the bacterial suspension along with 2 µL of the compound (from a stock solution in dimethyl sulfoxide (DMSO) at 6.4 µg/µL) were added. Two-fold serial dilutions were made as described, [START_REF] Cremades | Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter Pylori Infection[END_REF][START_REF] Galano | Improved Flavodoxin Inhibitors with Potential Therapeutic Effects against Helicobacter Pylori Infection[END_REF] which allowed to test the compounds in a concentration range from 64 to 0.031 µg/mL. Positive and negative controls consisted of brain-heart infusion broth supplemented with 4 % fetal bovine serum and inoculated or not with the diluted Hp bacterial suspension, respectively. We ensured that DMSO concentration was kept at 1 % v/v or below, so that no toxic effect was found for Hp cells. The untreated control consisted in 30,000 cells per well in 100 µL of complete medium (with 0.1 % DMSO). Cell viability was calculated from the wells' absorbance at 450 and 650 nm, as described. [START_REF] Cremades | Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter Pylori Infection[END_REF][START_REF] Galano | Improved Flavodoxin Inhibitors with Potential Therapeutic Effects against Helicobacter Pylori Infection[END_REF] Cytotoxicity curves (i.e percentage of cell viability versus compound concentration) were used to calculate the compounds concentration producing 50 % cell lysis (MCC 50 ), as described. [START_REF] Cremades | Discovery of Specific Flavodoxin Inhibitors as Potential Therapeutic Agents against Helicobacter Pylori Infection[END_REF][START_REF] Galano | Improved Flavodoxin Inhibitors with Potential Therapeutic Effects against Helicobacter Pylori Infection[END_REF] All experiments were performed twice in triplicate. Therapeutic Indexes (TI) were calculated as MCC 50 /MIC.

Minimal Cytotoxic Concentrations (MCC 50

Toxicity assays in mice. The in vivo toxicity of compounds was tested in 48 twenty-week old specific-pathogen-free female C57BL/6J mice obtained from Charles River Laboratories España (Barcelona, Spain), fed with a standard commercial rodent diet and water ad libitum. Females were selected because of their less aggressiveness. All

ASSOCIATED CONTENT

Supporting information

The supporting information is available free of charge on the ACS Publication website.

Figure 1 .

 1 Figure 1. Graphical summary of the rational followed for the development of new

Scheme 1 .Scheme 2 .Scheme 3 . 50 Scheme 5 .Scheme 6 . 9 Scheme 4 .

 123505694 Scheme 1. Reduction of nitro-compound I to oxime I-a

Scheme 7 .

 7 Scheme 7. Oxidation of sulphur IV-a to a mixture of sulfoxide IV-d and sulfone IV-e

Scheme 8 .

 8 Scheme 8. Reduction of metronidazole to amino-imidazole Mnz-a

Figure 2 .

 2 Figure 2. Chemical structure of 19 flavodoxin inhibitors grouped by similarity to lead

a

  HeLa cells have been used to determine the Minimal Cytotoxic Concentration (MCC 50 ),

19 Figure 3 .

 193 Figure 3. Inhibition of Hp SS1 gastric colonization of mice by treatment with I and II-

Figure 4 .

 4 Figure 4. Inhibition of Hp SS1 gastric colonization in mice treated with IV-related

Compound

  IV lowered colonization at 0.1, 1, 5 and 10 mg/100 g bw doses (p=0.044; 0.041; 0.048 and 0.0002, respectively) and eradicated infection in 43 % (3/7) and 17 % (3/18) of mice treated with 0.1 and 10 mg/100 g bw, respectively. Compound IV-a also inhibited the gastric colonization at 1, 5 and 10 mg/100 g bw doses (p=0.0001; 0.0018 and 0.0004, respectively) and eradicated the infection in 13 % (4/31) of treated mice at 1 mg/100 g bw. Compound IV-b, the derivative lacking the nitrobenzoxadiazole moiety, inhibited gastric colonization (p=0.0277 and 0.0042) at 10 and 20 mg/100 g bw, respectively, and eradicated the infection in 29 % (2/7) of mice at a 20 mg/100 g bw dose.

  Several lead compounds previously shown to inhibit the flavodoxin from Hp have been redesigned to improve their antimicrobial activity. By synthesizing new variants exhibiting different redox forms of the nitro, ethylene or sulphur groups, a family of nitrobenzoxadiazol-based flavodoxin inhibitors with low toxicity for HeLa cells and for mice has been developed that exhibits high therapeutic indexes against reference Hp strains. Importantly, some of these new inhibitors are effective against drug-resistant clinical Hp isolates (including Cla-, Mnz-and rifampicin-resistant strains) and are able to significantly reduce Hp gastric colonization in the mouse model of infection and to eradicate the infection in some mice. After improvement of their pharmacokinetic properties, these compounds could constitute a new, Hp-specific family of antimicrobials that could be less damaging for the gut microbiota than currently used broad-spectrum antibiotics and might be used on their own or in new combinations with existing antibiotics to fight Hp resistant strains.EXPERIMENTAL SECTIONGeneral synthetic and analytic procedures. All reagents were of analytical grade and were used as obtained from commercial sources. Compounds II-e, II-f and II-g are commercially available and were acquired from FCH group, Acros Organics and UkrOrgSyntez Ltd. (UORSY), respectively. Reactions were carried out using anhydrous solvents.[START_REF] Marshall | Gastric Spirochaetes: 100 Years of Discovery before and after Kobayashi[END_REF] H NMR,[START_REF] Leung | Effects of Helicobacter Pylori Treatment on Incidence of Gastric Cancer in Older Individuals[END_REF] C NMR and[START_REF] Safavi | Treatment of Helicobacter Pylori Infection: Current and Future Insights[END_REF] F NMR spectra were acquired at room temperature at 400, 100 and 376 MHz, respectively, using a 5 mm probe. Chemical shifts (δ) are reported in parts per million from tetramethylsilane with the solvent resonance as the internal standard. Coupling constants (J) are quoted in hertz. The splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), bs (broad singlet). Attached proton test spectra were acquired to determine the types of carbon signals, and Heteronuclear Multiple Bond Correlation spectra were acquired to confirm the position of heteroatoms. Special precautions such as degassing of the sample were not taken. All compounds used for biological assays are ≥ 95% purity based on NMR and HPLC chromatography. These last experiments have been performed using a Waters HPLC system equipped with a 600-E pump, a 2996 Photodiode Array detector, and a 2707 autosampler. The LC system was fitted with a C18 reversed phase column (VYDAC 238TP C18 5 μm, 4.6 mm × 250 mm) and operated, unless otherwise stated, using a linear gradient of buffer B (100% in 40 min) from 100% buffer A (buffer A consisting of 0.1% TFA in H2O, buffer B consisting of 0.085% TFA in CH3CN/H2O, 95:5 v/v) at a flow rate of 1 mL/min. High-resolution mass measurements were made using a microTOF (time-of-flight)

6, 8 -

 8 dichloro-2-(4-fluorophenyl)chroman-3-one oxime (I-a). Compound I (615 mg, 1.81 mmol) was dissolved in a mixture of dichloromethane (65 mL), conc. hydrochloric acid (4 mL) and methanol (29 mL). After the addition of iron powder (688 mg, 12.32 mmol), the mixture was sonicated (JP Selecta ultrasonic bath, 40 kHz) at room temperature for 10-40 minutes. TLC monitoring was done till completion of the reaction. The reaction mixture was filtered to remove excess iron, and the solvent was evaporated under reduced pressure to dryness. The residue was dissolved in water (35 mL) and extracted with ethyl acetate (3×50 mL). The organic layers were dried over anhydrous magnesium sulphate, filtered and concentrated in vacuo. The crude product was then purified by flash chromatography (eluent: Hex/EtOAc: 8/2) to give 313 mg (53 % yield) of the desired product.[START_REF] Marshall | Gastric Spirochaetes: 100 Years of Discovery before and after Kobayashi[END_REF] H NMR (400 MHz, DMSO-d 6 ): δ 11.54 (s, 1H), 7.45 (d, J = 2.5 Hz, 1H), 7.37-7.31 (m, 2H), 7.30 (d, J = 2.5 Hz, 1H), 7.23-7.18 (m, 2H), 5.93 (s, 1H), 3.94 (d, J = 22.2 Hz, 1H), 3.68 (d, J = 22.2 Hz, 1H) ppm. 19 F NMR (376 MHz, DMSO-d 6 ): δ -113.87 ppm. 13 C NMR (100 MHz, DMSO-d 6 ): δ 161.9 (d, J = 244.6 Hz), 150.9, 148.0, 133.2, 128.6 (d, J = 8.5 Hz), 128.2, 127.3, 125.5, 124.6, 122.4, 115.4 (d, J = 21.7 Hz), 77.2, 24.4 ppm. HRMS (ESI + ) m/z [M+H] + calc for C 15 H 11 Cl 2 FNO 2 326.0146, found 326.0125.

J = 272. 2

 2 Hz) and 123.3 (q, J = 272.6 Hz), 121.2 (m, 3.8 Hz) and 121.0 (m, 3.8 Hz),35.6 and 29.9 ppm. HRMS (ESI + ) m/z [M] + calc for C 10 H 7 F 6 NO 271.0427, found 271.0433.5-chloro-7-((4-methoxybenzyl)thio)benzo[c][1,2,5]oxadiazol-4-amine (IV-c).A mixture of dichloromethane (2.83 mL), conc. hydrochloric acid (170 µL) and methanol (1.29 mL) was set in a flask containing compound IV (25 mg, 0.08 mmol). After adding iron powder (30.42 mg, 0.54 mmol), the reaction was stirred at room temperature until disappearance of the nitro-compound, as judged by TLC. The mixture was poured into water (3 mL) and extracted with dichloromethane (4×3 mL). The organic layers were combined, dried over anhydrous magnesium sulphate, filtered and then evaporated in vacuo. TLC monitoring revealed that a mixture of two products had been obtained, one of them being a compound previously synthesised, namely IV-a, and the other one being IV-c. The crude was purified by column chromatography over silica gel (eluent: Hex/EtOAc: 6/4

7-(( 4 -

 4 methoxybenzyl)sulfonyl)benzo[c][1,2,5]oxadiazol-4-amine (IV-e).To a stirred solution of compound IV-a (29 mg, 0.1 mmol) in ethyl acetate (1.43 mL) at room temperature, 3-chloroperbenzoic acid (34.5 mg, 0.2 mmol)) was added. TLC monitoring was done till the completion of the reaction (1.5 h). Traces of 3-chloroperbenzoic acid and 3-chlorobenzoic acid were neutralized with saturated aqueous NaHCO 3 solution (2×2 mL).

  procedures followed for toxicity analyses in mice were approved by the Ethics Committee for Animal Experiments of the University of Zaragoza, and all the experiments were performed according to the approved ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for the care and manipulation of laboratory animals. Toxicity of compounds IV, II-a, and IV-a was tested at three different doses: 10, 20 and 40 mg/100 g bw. Assuming the administered compounds are uniformly distributed throughout the animal body, these doses range from 12 to 1600 times the MICs of the compounds. Ten groups of 4-5 animals were used. To nine of them, and for each compound, the corresponding daily dose was administered in volumes of 100 µl by oral gavage for 7 days.To the control group, vehicle alone (olive oil, 5 % DMSO) was administered. After treatment, mice were sacrificed by CO 2 asphyxiation and blood was collected by cardiac puncture. An automatic analyser (VetScan, Abaxis) was used to determine biochemical alterations. Histopathology evaluation was done on heart, stomach, liver, lung and kidney samples by haematoxylin-eosin staining of tissue sections.Antibacterial assays in mice.In vivo analysis of the anti-Hp activity of the compounds was carried out in 6-week old (SPF) NMRI (Naval Medical Research Institute) mice (Charles River laboratories, France). The level of Hp gastric colonization in the mice model is similar in male and female, so assays were carried out in females because they are less aggressive than males under the hosting conditions. Experiments were performed according to the European Directives (2010/63/UE), approved by the Committee of Central Animal Facility board of the Institute Pasteur and following the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. The project was authorized by the Comité d'Éthique en Expérimentation Animale, Institut Pasteur (Ref 00317.02). Four different experiments were performed to test compounds I, II,IV, I-a, II-a, II-b, II-c, II-f, IV-a, IV-b, IV-cand IV-d at some of the following doses of 0.1, 1, 5, 10 and 20 mg/100 g bw. In all experiments, 7 mice were analysed for each studied condition. Mice were orogastrically infected at days 1 and 3 during the first week of the experiment with 100 µL of a suspension of Hp strain SS1 (5•10 8 CFU/ml) known to colonize efficiently the mouse gastric mucosa.[START_REF] Lee | A Standardized Mouse Model of Helicobacter Pylori Infection: Introducing the Sydney Strain[END_REF] According to a previously established model of antimicrobial treatment of Hp infection,[START_REF] Correia | Docosahexaenoic Acid Inhibits Helicobacter Pylori Growth In Vitro and Mice Gastric Mucosa Colonization[END_REF] administration of the compounds started at day 8. One-hundred µL of each compound solubilised in 5-10 % DMSO (Euromedex)/95-90 % olive oil (Sigma-Aldrich) as vehicle at the defined doses were daily administered by oral gavage for 8 consecutive days. Non-infected control groups received orally 100 µL of peptone trypsin broth alone. In addition, infected and non-treated control groups were administered 100 µL of vehicle. No toxicity signs associated to the vehicle or the compounds treatment were noticed: i.e. no weight variations (FigureS1) and no appetite alterations. At 8 days post-treatment (day 24), mice were sacrificed by CO 2 inhalation, stomachs were isolated and Hp gastric colonization determined as described.[START_REF] Ferrero | Immune Responses of Specific-Pathogen-Free Mice to Chronic Helicobacter Pylori (Strain SS1) Infection[END_REF] Plates were incubated for 5 days at 37 °C under a microaerophilic atmosphere created by the Anoxomat AN2CTS system (MART Microbiology B.V.). The antibacterial activity of the compounds was evaluated by counting the number of viable bacterial colonies expressed as CFU per gram of gastric tissue.[START_REF] Ferrero | Immune Responses of Specific-Pathogen-Free Mice to Chronic Helicobacter Pylori (Strain SS1) Infection[END_REF] Statistical analysis. Statistical analyses of efficacy assays in mice were performed with GraphPad Prism 7 Software (GraphPad Software, Inc. CA, USA). Mann-Whitney U and Unpaired t tests were used to assess differences in mouse gastric colonization between Hpinfected vehicle-treated and compound-treated groups. Differences were considered statistically significant for p≤0.05.

  Compound characterization data (by NMR, mass spectrometry, HPLC and theoretical determination of logS values), additional information about in vitro and in vivo efficacy experiments (a table where compound doses for mice experiment are detailed, the MIC values for the developed compounds against clinical Hp isolates and the mice weight 37 variation during efficacy experiments), in vitro and in vivo toxicity data (viability of HeLa cells in the presence of the compounds, histological study of in vivo toxicity of some compounds, in vitro acid stability evaluation of compounds), and SMILES format representations of compounds (Molecular Formula Strings).

Table 1 . In vitro activity of flavodoxin inhibitors I, II and IV and related compounds against H. pylori reference strains a MCC 50 b

 1

						MIC c			TI d
	Compound	Structure	(µM)		(µM)			(MCC 50 /MIC)
				HeLa	SS1	NCTC	26695 SS1	NCTC	26695
						11637			11637
	I	Cl	NO2	5	0.37	0.37	0.37	14	14	14
		O
		Cl	F
	I-a	Cl	NOH	117	12	6.1	6.1	10	19	19
		O
		Cl	F
		F3C	NO2
	II			15	1.8	3.5	1.8	8.3	4.3	8.3
		CF3
		F
	II-a		NO2	36	2.3	2.3	2.3	16	16	16
		Cl	F

Table 2

 2

	Compound	Isolate 1	Isolate 2	Isolate 3	Isolate 4	Isolate 5	Isolate 6
	Amoxicillin	S	S	S	S	S	S
	Clarithromycin	S	S	S	R	S	R
	Tetracycline	S	S	S	S	S	S
	Levofloxacin	S	S	S	S	S	S
	Metronidazole	R	R	R	R	S	R
	Rifampicin	S	S	S	S	R	S

. Antimicrobial resistance profiles of H. pylori drug-resistant clinical isolates following the EUCAST criteria. (S = sensitive; R = resistant)

Table 3 . TI values (MCC 50 /MIC) of some developed compounds against H. pylori drug-resistant clinical isolates a

 3

	Compound	Isolate 1 Isolate 2	Isolate 3	Isolate 4	Isolate 5	Isolate 6
	II-b	9.0	9.0	9.0	9.0	9.0	9.0
	II-d	4.2	4.2	4.2	34	4.2	4.2
	II-e	4.7	4.7	4.7	605	4.7	4.7

  The reaction was stirred for 24 h at room temperature and, after checking for complexion, the reaction mixture was filtered through Celite ® and washed a few times with anhydrous THF (3×3 mL). The solvent was removed under reduced pressure to afford the 26,5 mg (94 %) of crude product as an oil. The thus formed amino-imidazole is sensitive to O 2 and heat, and therefore it was stored without further purification in a sealed flask under an argon atmosphere at -20 °C until its MCC 50 and MIC determinations. Just before MIC determinations, the crude was purified by flash chromatography (First eluent:

	dichloromethane /methanol: 1/1, second eluent: methanol, third eluent: methanol containing
	0.1% triethylamine) and used immediately. 1 H NMR (400 MHz, DMSO-d 6 ): δ 5.91 (s, 1H),
	4.16 (bs, 2H), 3.75 (t, J = 5.6 Hz, 2H), 3.54 (t, J = 5.6 Hz, 2H), 2.16 (s, 3H) ppm.
	1 H-NMR
	(400 MHz, CDCl 3 ): δ 8.47 (d, J=7.2 Hz, 1H), 7.75 (d, J=7.2 Hz, 1H), 6.89-6.87 (m, 2H),
	6.77-6.74 (m, 2H), 4.61 (d, J=13.6 Hz, 1H), 4.32 (d, J=13.6 Hz, 1H), 3.77 (s, 3H) ppm. 13 C-
	NMR (100 MHz, CDCl 3 ): δ 160.2, 146.1, 142.6, 142.3, 131.3, 129.1, 128.7, 119.6, 114.1,
	59.5, 55.3 ppm. HRMS (ESI + ) m/z [M+Na] + calc for C 14 H 11 N 3 NaO 5 S 356.0312, found
	356.0308.
	2-(5-amino-2-methyl-1H-imidazol-1-yl)ethan-1-ol (Mnz-a). Commercially available

metronidazole (34.2 mg, 0.2 mmol) and 25 % Pd/C (9 mg) were placed in a 50 mL twonecked round-bottomed flask under argon atmosphere. Anhydrous, degassed THF (10 mL) was added.

Minimal Inhibitory Concentrations (MIC) on reference

Hp strains (NCTC 11637, 26695 and SS1). For microdilution MIC testing, Hp reference strains NCTC 11637 (ATCC 43504, Mnz-resistant strain), 26695 (ATCC 700392)

  ). HeLa cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a 5% CO 2 atmosphere. The toxicity of the

	compounds towards HeLa cells was determined by the XTT (2,3-Bis-(2-methoxy-4-nitro-5-
	sulphenyl)-2H-tetrazolium-5-carboxanilide sodium salt) method using the Cell
	Proliferation Kit II (Roche) as described. 41,42 Briefly, 0.1-µL volumes of compound
	dissolved in DMSO were added to give final compound concentrations of 25, 50, 75, 100,
	250, 500, 750, and 1000 µM, in 96-well plates (with 30,000 cells in 100 µL in each well).

ACKNOWLEDGMENTS

Plates were incubated at 37 ºC for 48 hours under a microaerophilic atmosphere. MICs correspond to the lowest concentrations of compounds leading to a complete inhibition of Hp growth. The MIC values were determined by recording the colour change observed after addition of 30 µL of resazurin (0.1 mg/ml; Sigma-Aldrich) to each well in the 96-well dish. The MICs were additionally confirmed by spotting 10 µL of two consecutive wells (one with the highest concentration of compound that allowed bacterial growth, and the adjacent well with the lowest concentration of compound that prevented bacterial growth) onto Columbia Agar with Sheep Blood plates (Oxoid) followed by incubation at 37 °C for 48 h under microaerophilic conditions. Growth or absence of Hp growth allowed to determine the Minimal Bactericidal Concentration of the compounds. Each experiment was performed twice in triplicate. Both assays confirmed the MIC values obtained by the microdilution method.

MIC on drug-resistant Hp strains. Six Hp drug-resistant strains obtained at the University Hospital Lozano Blesa (Spain) from patients with gastric pathologies (dyspepsia and/or peptic ulcer disease) who had failed to at least two conventional therapies recommended in national guidelines [START_REF] Gisbert | IV Spanish Consensus Conference on Helicobacter Pylori Infection Treatment[END_REF] were selected. These strains were isolated from gastric biopsies obtained during routine upper gastrointestinal endoscopic procedures and cultured. According to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, [START_REF]The European Committee on Antimicrobial Susceptibility Testing[END_REF] three of these Hp strains were Mnz-resistant, two were Mnz/Cla resistant, and one was rifampicin-resistant. Compounds II-b, II-d, II-e, IV-a, IV-c, IV-d and IV-e were tested on all of these strains. The determination of their MIC was performed by microdilution and followed by the colorimetric method using resazurin, as explained above. Written informed consent was obtained from patients and the procedures were approved by the Regional Ethical Committee of Aragón.

AUTHOR INFORMATION Corresponding Author

* Javier Sancho, Biocomputation and Complex Systems Physics Institute (BIFI), Edificio I+D. Mariano Esquillor s/n. 50018-Zaragoza, Spain. E-mail: jsancho@unizar.es. Phone: (34)976761286. Fax: (34)976762123. ORCID: 0000-0002-2879-9200.

Author Contributions

Study concept and design: JS. Acquisition of data: SS, MA, VM, AM, AL, LR, JB, CS.

Notes

The authors declare that a patent has been filled concerning derivatives of compound IV.

ETHICS APPROVAL

Ethics Committee for Animal Experiments of the University of Zaragoza (Spain) and Committee of Central Animal Facility board of the Institute Pasteur (Paris, France).


H Engels

D Bourguet
email: bourguet@supagro.inra.fr

L ' Caga ´n

B Manachini

I Schuphan

T J Stodola

A Micoud

C Brazier

C Mottet

D A Andow

Evaluating Resistance to Bt Toxin Cry1Ab by F 2 Screen in European Populations of Ostrinia nubilalis (Lepidoptera: Crambidae)

Keywords: European corn borer, Bt maize, Mon810, resistance management, HDR strategy

The large-scale cultivation of transgenic crops producing Bacillus thuringiensis (Bt) toxins have already lead to the evolution of Bt resistance in some pest populations targeted by these crops. We used the F 2 screening method for further estimating the frequency of resistance alleles of the European corn borer, Ostrinia nubilalis (Hu ¨bner) (Lepidoptera: Crambidae), to Bt maize, Zea mays L., producing the Cry1Ab toxin. In France, Germany, and Italy, 784, 455, and 80 lines of European corn borer were screened for resistance to Mon810 maize, respectively. In Slovakia, 26 lines were screened for resistance to the Cry1Ab toxin. The cost of F 2 screen performed in the four countries varied from US$300 to $1,300 per line screened. The major difference in cost was mostly due to a severe loss of univoltine lines during the screen in Germany and Slovakia. In none of the screened lines did we detect alleles conferring resistance to Mon810 maize or to the Cry1Ab toxin. The frequency of resistance alleles were Ͻ1.0 ϫ 10 Ð3 , Ͻ1.6 ϫ 10 Ð3 , Ͻ9.2 ϫ 10 Ð3 , and Ͻ2.6 ϫ 10 Ð2 in France, Germany, Italy, and Slovakia, with 95% probability, respectively. The average detection probability over all lines was Ϸ90%. Making the assumption that European corn borer populations in these countries belong to the same genetic entity, the frequency of alleles conferring resistance to the Cry1Ab produced by the Mon810 maize in western and central Europe was 1.0 ϫ 10 Ϫ4 , with a 95% conÞdence interval of 0 Ð3.0 ϫ 10 Ϫ4 .

Transgenic crops producing Bacillus thuringiensis (Bt) toxinsÑreferred to as Bt cropsÑare widely planted in the United States, India, Argentina, Brazil, Canada, China, and Spain [START_REF] James | Global status of commercialized biotech/ GM[END_REF]. For example, maize (Zea mays L.), which provides control of lepidopteran and coleopteran maize pests, has been planted on Ͼ200 million ha during the last decade, corresponding to 30.5% of the global biotech area [START_REF] James | Global status of commercialized biotech/ GM[END_REF]. Large-scale cultivation of these Bt crops is exerting a tremendous selection pressure on target pest species, which prove to evolve Þeld resistance [START_REF] Matten | How governmental regulation can help or hinder the integration of Bt crops within IPM programs[END_REF][START_REF] Kruger | Perspective on the development of stem borer resistance to Bt maize and refuge compliance at the Vaalharts irrigation scheme in South Africa[END_REF][START_REF] Tabashnik | Insect resistance to Bt crops: evidence versus theory[END_REF][START_REF] Tabashnik | Field-evolved insect resistance to Bt crops: deÞnition, theory, and data[END_REF], diminishing the beneÞts of these crops.

The risk of target pests becoming resistant to Bt crops has led to the development of several strategies to manage transgenic crops [START_REF] Gould | Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology[END_REF][START_REF] Roush | Two-toxin strategies for management of insecticidal transgenic crops: can pyramiding succeed where pesticide mixtures have not? Phil[END_REF], Shelton et al. 2002[START_REF] Vacher | Modelling the spatial conÞguration of refuges for a sustainable control of pests: a case study of Bt cotton[END_REF][START_REF] Zhao | Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants[END_REF][START_REF] Tuytuynov | Landscape refuges delay resistance of the European corn borer to Bt-maize: a demo-genetic dynamic model[END_REF]). Among these the most widely accepted and implemented strategy is the "high-dose/ refuge" (HDR) strategy [START_REF] Alstad | Managing the evolution of insect resistance to transgenic plants[END_REF]. This strategy has two components. First, the Bt crops need to produce Bt toxin at high concentration, with the expectation that this concentration would be suf-Þcient to kill most if not all heterozygous individuals. The other part of this strategy is refugesÑthe occurrence of non-Bt plants, crops, or weeds, preserving a pool of susceptible individuals. The HDR is expected to work best when high dose of toxin is expressed in plant tissue, resistance alleles are rare (initial frequencies Ͻ10 Ϫ3 ), and functionally recessive and refuges are close to Bt plants, so that nearly all resistant survivors from the Bt plants will mate with susceptible individuals from refuge plants to produce heterozygous offspring that cannot survive on Bt plants the following generation [START_REF] Alstad | Managing the evolution of insect resistance to transgenic plants[END_REF].

Several techniques have been used to estimate the frequency of resistance alleles in natural populations, including in-Þeld screening [START_REF] Venette | An in-Þeld screen for early detection and monitoring of insect resistance to Bacillus thuringiensis in transgenic crops[END_REF] and screening of Þeld-collected egg masses or larvae [START_REF] Siegfried | Ten years of monitoring for Bt resistance in the European corn borer: what we know, what we donÕt know and what we can do better[END_REF]. When resistance alleles have not been identiÞed and are believed to be rare and recessive, Andow andAlstad (1998, 1999) and [START_REF] Andow | Monitoring and adaptive resistance management[END_REF] suggest that the most efÞcient method is an F 2 screen. This technique preserves genetic variation among isofemale lines and concentrates resistance alleles into homozygous genotypes in the F 2 generation. F 2 screens have been used for the European corn borer, Ostrinia nubilalis (Hu ¨bner) (Lepidoptera: Crambidae) (Andow et al. 1998[START_REF] Andow | Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in an Iowa population of European corn borer (Lepidoptera: Crambidae)[END_REF][START_REF] Bourguet | Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of Ostrinia nubilalis[END_REF][START_REF] Stodola | Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in southern U.S. Corn Belt population of European corn borer (Lepidoptera: Crambidae)[END_REF]. Unlike F2 screens performed on other pest species [START_REF] He | Using F 2 genetic method of isofemale lines to detect the frequency of resistance alleles to Bacillus thuringiensis toxin from transgenic Bt cotton in cotton bollworm (Lepidoptera: Noctuidae)[END_REF][START_REF] Ge ´nissel | Initial frequency of alleles conferring resistance to Bacillus thuringiensis poplar in a Þeld population of Chrysomela tremulae[END_REF][START_REF] Wenes | Frequency and Þtness cost of resistance to Bacillus thuringiensis in Chrysomela tremulae (Coleoptera: Chrysomelidae)[END_REF][START_REF] Downes | Monitoring and adaptive resistance management in Australia for Btcotton: current status and future challenges[END_REF][START_REF] Huang | Sugarcane borer (Lepidoptera: Crambidae) resistance to transgenic Bacillus thuringiensis maize[END_REF][START_REF] Xu | Using an F 2 screen to monitor frequency of resistance alleles to Bt cotton in Þeld populations of Helicoverpa armigera (Hu ¨bner) (Lepidoptera: Noctuidae)[END_REF]) no major Bt resistance alleles, i.e., alleles conferring a level of resistance sufÞciently high to allow survival and development of homozygous individuals on Bt crops, have been detected in the European corn borer. Accordingly, all laboratory selections [START_REF] Bolin | Long-term selection for resistance to Bacillus thuringien-sis CryIA(c) endotoxin in a Minnesota population of European corn borer (Lepidoptera: Crambidae)[END_REF][START_REF] Chaufaux | Chronic exposure of the European corn borer (Lepidoptera: Crambidae) to Cry1Ab Bacillus thuringiensis toxin[END_REF] or Þeld monitoring using discriminating doses of toxin [START_REF] Farinos | Resistance monitoring of Þeld populations of the corn borers Sesamia nonagrioides and Ostrinia nubilalis after 5 years of Bt maize cultivation in Spain[END_REF][START_REF] Siegfried | Ten years of monitoring for Bt resistance in the European corn borer: what we know, what we donÕt know and what we can do better[END_REF]) performed on this pest failed to reveal such major Bt resistance alleles. Still, the F 2 screen allowed an estimation of the frequency of resistance alleles in natural populations, which has been valuable for resistance management planning.

The goal of this study is to continue the investigation of Bt resistance alleles in European European corn borer populations by using the F 2 screening method. Andow et al. (1998[START_REF] Andow | Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in an Iowa population of European corn borer (Lepidoptera: Crambidae)[END_REF], [START_REF] Stodola | Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in southern U.S. Corn Belt population of European corn borer (Lepidoptera: Crambidae)[END_REF][START_REF] Bourguet | Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of Ostrinia nubilalis[END_REF] performed F 2 screens on U.S. Corn Belt and French populations of this pest. Here, we extend the investigation in western and central Europe to determine whether 1) the highdose refuge strategy could be implemented with success for managing the cultivation of Bt maize expressing the Cry1Ab toxin such as the Mon810 and 2) the cost of the F 2 screen makes it a sustainable method to monitor the evolution of Bt resistance in European populations of the European corn borer.

Materials and Methods

F 2 Screen

The F 2 screen protocol for O. nubilalis was adapted from Andow and Alstad (1998). Mated females are considered to be the preferred stage for initiating an F 2 screen, but many variant methods have been proposed [START_REF] Bentur | Frequency of alleles conferring resistance to a Bacillus thuringiensis toxin in a Philippine population of Scirpophaga incertulas (Lepidoptera: Pyralidae)[END_REF], Zhao et al. 2002[START_REF] Stodola | F 2 screen variations and associated statistics[END_REF][START_REF] Stodola | Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in southern U.S. Corn Belt population of European corn borer (Lepidoptera: Crambidae)[END_REF]). For our F 2 screens either mated females, pair-mated adults originating from Þeld-collected late instars, pair-mated adults originating from egg masses collected in the Þeld or large Þeld-collected egg masses were used. For the pair-mated adults from egg masses, only one adult was taken from an egg mass for pair-mating. For the large Þeld-collected egg masses, only large (Ͼ60 eggs) egg masses were used to set F 1 families from an isofemale line, bypassing the need to establish the P 1 generation. In France, lines were exclusively started from paired adults of Þeld-collected diapausing larvae. In Italy and Slovakia, wild-mated females were used to establish isofemale lines. From each isofemale line, the F 1 offspring were reared and sib-mated and the F 2 neonates were screened either on Bt maize (hybrids expressing the Mon810 transformation event and therefore producing a truncated Cry1Ab toxin) leaves or diet containing the Cry1Ab toxin. Starting from the two P 1 adults, each isofemale line allowed us to characterize at least four haplotypes. By sib-mating the F 1 generation, 1/16 of the F 2 individuals were expected to be homozygous for the resistance allele if one of the P 1 adults carried such an allele.

Insect Collection, Rearing, and Screening

France. Insect Collection. Pest insects were collected from 1999 to 2004 in four different regions in France: Aquitaine, Centre, Midi-Pyre ´ne ´es, and Rho ˆnes-Alpes. By comparison, the F 2 reported by [START_REF] Bourguet | Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of Ostrinia nubilalis[END_REF] was performed on populations collected in Midi-Pyre ´ne ´es only. All European corn borers were collected as diapausing larvae on maize stalks during the autumn or early winter. Only one larva was taken per stalk to minimize the probability that siblings were collected. Each isofemale line was started by pairing a virgin male and a virgin female obtained after breaking diapause (for details, see [START_REF] Bourguet | Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of Ostrinia nubilalis[END_REF]. Each pair of female and male was placed at 25ЊC, 70% RH, and a photoperiod of 16:8 (L:D) h in a small plastic container equipped with papersheets on top for oviposition. All pairs were held until enough F 1 egg masses were collected or females died.

F 1 Rearing and F 2 Screening. F 1 egg masses hatched on average after 6 d. For each line, F 1 larvae were reared at 25ЊC, 70% RH, and a photoperiod of 16:8 (L:D) h in a large plastic box (13 by 11 cm) Þlled with the artiÞcial maize diet described by [START_REF] Gahukar | Nouvelles techniques adopte ´es pour lÕe ´levage dÕOstrinia nubilalis Hu ¨bner sur milieu artiÞciel[END_REF]. Diet was added or changed twice a week. The larval development before pupation took on average 30 d. F 1 adults were transferred to a new large plastic box equipped with papersheets on top for oviposition [START_REF] Bourguet | Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of Ostrinia nubilalis[END_REF]. The F 2 generation of each isofemale line was screened for resistance by inoculating neonates on leaves of at least two different Bt maize plants (Elgina, event Mon810). These leaves were evaluated for surviving larvae at 5 d after inoculation. All lines with survivors were scored as potential positives.

Germany. Insect Collection. European corn borer sampling took place in 2003Ð2005 at four different German locations. During the summer, adults were collected using light trap cages. Moths were brought to the laboratory and transferred in pairs to small cages covered with waxed paper for oviposition. Caging females together with a male was to ensure the mating of females that might not have mated in the Þeld. Late instars of O. nubilalis were collected during fall 2003 and 2004 from Bonn and Heilbronn. Only one larva was taken from a single maize plant to avoid the collection of brothers and sisters originated from the same egg mass. Larvae were kept in groups of 15 individuals in petri dishes and provided with an arti-Þcial European corn borer diet based on [START_REF] Wyniger | Insektenzucht. Methoden der Zucht und Haltung von Insekten und Milben im Laboratorium[END_REF] and [START_REF] Farinos | Resistance monitoring of Þeld populations of the corn borers Sesamia nonagrioides and Ostrinia nubilalis after 5 years of Bt maize cultivation in Spain[END_REF] and held at 25ЊC, 70% RH, and continuous day light to try to prevent diapause. Pupation time varied strongly between indi-viduals and many larvae did not pupate at all. Once a week the dishes were checked for pupae, which were removed and males and females were kept separately in plastic boxes until the adults emerged. After emergence, males and females were paired. Moths were provided with a solution of 10% honey in water and held at 25ЊC, 70% RH, and a photoperiod of 16:8 (L:D) h. Rearing procedures of F 1 larvae, F 1 adults, and F 2 screening on Bt maize has been performed using identical protocol decribed below, regardless of the initiation method of the line.

F 1 Rearing and F 2 Screening. After mating the P 1 adults in the cages, egg masses were collected daily, placed in petri dishes with European corn borer diet and held at 25ЊC, 70% RH, and a photoperiod of 16:8 (L:D) h until the blackened head capsules were visible within the eggs. To synchronize the offspring of a female, egg masses were held at 10ЊC until the female had Þnished oviposition. The egg masses from a single female were combined and larvae were reared at 25ЊC, 70% RH, and a photoperiod of 16:8 (L:D) h. Larvae were transferred weekly to new dishes containing fresh diet, and after the second larval instar the density was limited to 15 individuals per dish. F 1 pupae from different families were kept separately, and emerged adults were transferred daily to F 1 family cages for sib-mating. Moths were provided with a solution of 10% honey in water to maximize the oviposition rate.

Neonates hatching from F 2 egg masses were screened on excised Bt maize (Novelis, event Mon810) leaves. F 2 screens were performed in plastic boxes (10 by 10 cm). Five to 10 European corn borer egg masses were transferred into each box together with six to eight leaves of Bt maize tissue over a moistened Þlter paper. Every three days, new Bt leaves were added and the Þlter paper was remoistened. The boxes were held in the same environmental conditions as the F 1 generation. All dishes were checked for surviving larvae after 7Ð10 d. Resistance was only assumed when larvae survived beyond the second larval instar with additional evidence of feeding on Bt maize leaves.

Italy. Insect Collection. Adult European corn borer females were collected in Italy from 2003 to 2004 in three different regions (Lombardia, Piemonte, and Veneto). Wild-mated females had a life span of Ϸ6 d. They were individually held in cylindrical mesh cages with paper on top for oviposition. To synchronize the emergence of Þrst instars, egg masses were collected daily and were stored at 13ЊC and 85% RH until eggs were blackheaded. Larvae and moths were reared at 25ЊC, high relative humidity, and a photoperiod of 16:8 (L:D) h.

F 1 Rearing and F 2 Screening. Larvae hatched after 3Ð 4 d. They were reared in plastic dishes with artiÞcial diet [START_REF] Lozzia | Susceptibility of Ostrinia nubilalis Hu ¨bner (Lepidoptera: Crambidae) to Bacillus thuringiensis var. kurstaki[END_REF], added when necessary. After Ϸ30 d, larvae pupated and pupae were stored in white folded paper. If necessary pupae were synchronized and held at 13ЊC with wet cotton until 30 pupae were collected. F 1 adults were transferred to family cages for sib-mating and were provided with a waterÐ honey solution. Egg masses were collected from paper placed on top of the cages. Hatching F 2 larvae were screened on excised Bt maize (Elgina, event Mon810) leaves or Cry1Ab toxin.

Slovakia. Insect Collection. Wild-mated European corn borer females were collected by sweep netting during 2004 and 2005 at Komjatice and Nitra. Those females had a lifespan from 2 to 4 d and were held in small mesh cages with paper on top for oviposition. Eggs were collected daily, stored in petri dishes at 85% RH and 10ЊC until the last egg laying of each female. The eggs were stored at 25ЊC for the next few days until the occurrence of black heads of future larvae. Black headed egg masses were placed on pieces of aluminum foil and added on the diet (Ϸ150 ml) in canning jars. In 2004, the artiÞcial diet described by [START_REF] Wyniger | Insektenzucht. Methoden der Zucht und Haltung von Insekten und Milben im Laboratorium[END_REF] was used in the experiment. A sim-pliÞed artiÞcial diet described by [START_REF] Nagy | Rearing of the European corn borer (Ostrinia nubilalis Hbn.) on a simpliÞed artiÞcial diet[END_REF] was used in 2005. Larvae and moths were reared at 25ЊC and 70% RH.

F 1 Rearing and F 2 Screening. F 1 egg masses were transferred to petri dishes. Larvae hatched after 3Ð 4 d with an estimated proportion of Ϸ50%. One hundred larvae per dish were reared in 250-ml canning jars with diet. Pupae were collected daily from dishes and were placed in F 1 family cages, where they emerged after Ϸ7 d. Egg masses were collected from paper hanging inside the cages. Neonates hatching from F 2 egg masses were screened on diet with Cry1Ab toxin. We performed the F2 screen using the Cry1Ab toxin (provided by Johannes A. Jehle, Agricultural Service Centre for Rhineland-Palatinate, Germany) laid on the top of the diet at a concentration of 2 g/cm 2 .

Statistical Analysis

Results from each F 2 screen were analyzed for the expected resistance allele frequency (E[p R ]) with its 95% credibility interval and the probability of a false negative (P No ). Bayesian analysis was used to estimate E[p R ] and credibility intervals, which allow conclusions about the populations sampled (Andow andAlstad 1998, 1999;[START_REF] Schneider | ConÞdence interval for Bayesian estimates of resistance allele frequencies[END_REF]. A custom program compiled in C ϩϩ was used to compute P No . It depended on the number of F 1 males and F 1 females, the number of F 2 offspring screened per isofemale line, and the nonscreen mortality of F 2 larvae. Analytical methods for calculating P No are described in [START_REF] Stodola | F 2 screen variations and associated statistics[END_REF].

Cost of F 2 Screens

We estimated the Þxed and variable costs for the F 2 screens performed in each country. We took into account the salaries for scientiÞc and nonscientiÞc staff, personnel for insect collection and rearing, travel costs for Þeld sampling, and all expenses for consumables and associated infrastructure, including cage construction. The overall cost was divided by the number of isofemale lines tested, providing an average cost per screened line.

Results

Multiple methods for starting F 2 screen lines have been used from 1999 to 2005 in the four countries involved in this study. Despite the methodological variation, all methods provided comparable results.

In France, several lines were lost before they were screened because of unmated P 1 females and hatching problems of the F 1 egg masses. The proportion of hatched F 1 eggs varied widely from 20 to 80%. In Germany, the losses were due to several factors. P 1 moths died before oviposition or did not mate, F 1 egg masses did not hatch or collected larvae did not pupate. Losses of collected larvae and adults were high and varied from 43 to 75%. The highest losses of adults were due to their transport to the laboratory. The lowest losses were from starting the lines with large egg masses, which circumvented the need to establish a P 1 generation. In Italy, the main losses during the F 2 screen were due to unmated P 1 and F 1 females. The proportion of hatched F 1 eggs was only 56%. Finally, in Slovakia, the massive loss of lines was due to both unmated females and larvae that remained in dia-pause. The proportion of hatched F 1 eggs was not exactly recorded, but it was estimated to be Ϸ50%.

In total, 784, 455, 80, and 26 isofemale lines were screened in France, Germany, Italy, and Slovakia, respectively (Table 1). For all these lines, there were no surviving F 2 larvae. Thus, major resistance alleles to Cry1Ab were detected in none of the European populations tested in this study (Table 2). Hence, E[p R ] ϭ 3.0 ϫ 10 Ϫ4 , 4.0 ϫ 10 Ϫ4 , 3.1 ϫ 10 Ϫ3 and 9 ϫ 10 Ϫ3 in France, Germany, Italy, and Slovakia, respectively (Table 3). The detection probability associated with these estimates varied from 84 to 98% (Table 3).

The cost for the F 2 screen was ϷUS$300, $700, $400, and $1,300 per screened line in France, Germany, Italy, and Slovakia, respectively.

Discussion

Frequency of Alleles Conferring Cry1Ab Resistance. The F 2 generations of the lines tested so far in U.S. and French populations of the European corn borer were all susceptible to the concentration of Cry1Ab toxin produced by Mon810 Bt maize. Similarly, none of 1,345 lines screened in the current study contained alleles allowing European corn borer larvae to actively feed on Mon810 maize. Hence, the frequency of such alleles are Ͻ1.0 ϫ 10 Ð3 , Ͻ1.6 ϫ 10 Ð3 , Ͻ9.2 ϫ 10 Ð3 , and Ͻ2.6 ϫ 10 Ð2 in France, Germany, Italy, and Slovakia, with 95% probability, respectively.

In Europe, the population genetic structure is not fully understood yet. Populations of Ostrinia feeding on mugwort and hop in France belong to O. scapulalis, a sibling species of the European corn borer [START_REF] Frolov | Reconsidering the taxonomy of several Ostrinia species in the light of reproductive isolation: a tale for E. Mayr[END_REF]). Conversely, no or low genetic differentiation has been detected among European corn borer populations collected on maize throughout France [START_REF] Bourguet | Gene ßow in the European corn borer Ostrinia nubilalis: implications for the sustainability of transgenic insecticidal maize[END_REF][START_REF] Malausa | Genetic structure and gene ßow in French populations of two Ostrinia taxa: host races or sibling species?[END_REF] or among several European corn borer populationsÑalso collected on maizeÑfrom France, Germany, Italy, Slovakia, Spain, and Greece [START_REF] Gaspers | nubilalis, Hbn.), its susceptibility to the Bt-toxin Cry1F, its pheromone races and its gene ßow in Europe in view of an insect resistance management[END_REF]) and among Italian European corn borer population feeding on rice, Oryza sativa L., and on maize [START_REF] Manachini | Using AFLP to investigate the genetic similarity of Ostrinia nubilalis (Lepidoptera: Crambidae) infesting rice and corn[END_REF]. Similarly, there is no clear genetic differentiation between European corn borer populations feeding on maize in western and central Europe [START_REF] Gaspers | nubilalis, Hbn.), its susceptibility to the Bt-toxin Cry1F, its pheromone races and its gene ßow in Europe in view of an insect resistance management[END_REF]. Consequently, in western and central Europe, O. nubilalis probably constitute more or less a single a A full positive result required many larvae feeding and growing to at least the third instar on the screening media. A partial positive required a rating Ն 2 on the Bolin scale (when tested on Bt maize), evidence of feeding and tunneling (when tested on diet treated with Bt), or larvae surviving to the second instar on either Bt maize or diet treated with Bt.

b Could not interpret results due to problems with toxin.

panmictic population so that resistance management and monitoring could be considered at this large scale.

If we considered populations of France, Germany, Italy, and Slovakia as part of the same population, then we can pool the results of the various F 2 screens reported here as well as those published previously by [START_REF] Bourguet | Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of Ostrinia nubilalis[END_REF]. Over these four countries, a total of 2,156 lines of European corn borer have been screened from 1999 to 2004 (Table 3) with no evidence of resistance to Bt maize in any of those lines. The expected frequency of resistance over these 2,156 lines is 1.0 ϫ 10 Ϫ4 with a 95% CI of 0 Ð3.0 ϫ 10 Ϫ4 and an experiment-wise detection probability of Ϸ90%. This provides the most extensive monitoring data on resistance in any target insect pest species, including all transgenic insecticidal crops and all insecticides. It indicates that resistance is rare enough for a successful deployment of the HDR strategy throughout western Europe and possibly also in central Europe, although additional F 2 screens and further investigation of the genetic structure from this area must be performed before any Þrm conclusion.

Cost of F 2 Screen. The F 2 screen is a complex, timeand labor-consuming method that requires skilled staff experienced in insect rearing and susceptibility testing. Its cost strongly depends on the number of isofemales tested in a row and, more importantly, on the proportion of lines that are ultimately screened for susceptibility. Hence, the cost varied from ϷUS$300 (for the screens performed in France) to ϷUS$1,300 per line (for the screens performed in Slovakia). The cost of US$700 per line for the screens performed in Germany is similar to the F 2 screen expenses of [START_REF] Andreadis | Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in Greek and Spanish populations of Sesamia nonagrioides (Lepidoptera: Noctuidae)[END_REF] on S. nonagrioides. However, this latter pest is considerably more difÞcult to rear than O. nubilalis.

The highest costs found in Slovakia (US$1,300) and in Germany (US$700) are mostly due to the presence, in these two countries, of univoltine populations (Caga ´n ˇ1998). These univoltine individuals are genetically constrained [START_REF] Stengel | Etude comparative de la vitesse de croissance et de la sensibilite ´a `la photope ´riode de deux races de pyrales du maõ ¨s (Ostrinia nubilalis Hu ¨bner, Lepidoptera, Pyralidae) et de leurs hybrides[END_REF], and a large fraction of the larvae do not pupate in the laboratory, strongly reducing the number of lines that can be tested. Univoltine populations also occur in France, but they are mostly located in north-eastern France. Localities where sampling took place in France were typically not in the northeast, and probably had a low percentage of univoltine larvae.

In Italy and in southern France, populations are mainly bivoltine. This offers two advantages. First, all larvae reared at 25ЊC with a photoperiod of 16:8 (L:D) h pupate Ϸ10 d after molting to the Þfth instar. Hence, most P 1 lines producing offspring are likely to be tested for their susceptibility to Bt maize. The second advantage is the occurrence of two European corn borer ßights during the summer. This increases the window of time during which wild-mated females can be collected and screened. Indeed, compared with diapausing larvae, which can be stored at 4ЊC for several months, wild moths must be handled immediately during the days after their capture. Because all lines must be managed more or less at the same time, this limits the number of lines that can be started. The extended window of time offered by the bivoltine populations could be very interesting when a limited fraction of wild females produce fertile egg masses likeÑa situation encountered in Slovakia but not during the previous F 2 screens performed in France [START_REF] Bourguet | Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of Ostrinia nubilalis[END_REF].

Finally, in Germany, the relatively high cost per line is partly due to the lowÑsometimes very lowÑ density of European corn borer in maize Þeld. Indeed, the low levels of European corn borer infestation increased the costs of sampling. Conversely, these costs were lower in Italy, notably in northern Italy and southern France where maize Þelds are often heavily infested by O. nubilalis. Noteworthy, European countries and regions suffering strong bivoltine infestations are those where Bt maize is more likely to be planted and where European corn borer resistance to those maize are more likely to be selected.

In conclusion, the F 2 screen is a valuable technique for screening the susceptibility of European corn borer populations to Bt toxins and Bt maize. Isofemale lines can be started using either wild-moths captured within or around maize Þelds during the summer or adults emerging from diapausing larvae collected during the fallÐwinter. In studies performed so far on European corn borer (Andow et al. 1998[START_REF] Andow | Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in an Iowa population of European corn borer (Lepidoptera: Crambidae)[END_REF][START_REF] Bourguet | Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of Ostrinia nubilalis[END_REF][START_REF] Stodola | Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in southern U.S. Corn Belt population of European corn borer (Lepidoptera: Crambidae)[END_REF]this study), the experiment-wise detection probability was often higher than 90% and even reached 99%. However, the current study reveals some limitations of the F 2 screen. The screen must occur during the second laboratory generation. Hence, it requires the complete development of at least one generation in the

Table 1 . Number of lines progressing through the F 2 screen experiment

 1

	Yr Location	P 1 lines started	P 1 mated a	F 1 larvae produced	F 1 adults produced	Screened
		F 2 screen lines started from wild-mated female
	2003 Germany	37	24	24	15	9
	Italy	69		47	46	22
	2004 Germany	118	61	61	60	60
	Italy	189		147	112	58
	Slovakia	426	131	79	53	16
	2005 Slovakia	753	99	51	50	10
			Lines started from paired adults of
				diapausing larvae
	1999 France	152	78	74	62	52
	2000 France	347	229	229	127	104
	2001 France	339	226	143	116	95
	2002 France	461	295	190	180	169
	2003 France	750	541	311	310	186
	2004 France	329	223	193	190	178
	Germany Ϸ150	100	100	100	93
	2005 Germany	144	144	144	144	140
	2005 Germany	Lines started from single large F 1 egg mass 200 b 167 167 153

a Line was considered mated if a female laid fertile eggs. b Number of F 1 egg masses collected for this purpose.

Table 2 . Results from F 2 screen and subsequent rescreening of positive lines a

 2

	Country	Yr	Screen generation	Screening method	Partial positives	Full positives
	France	1999Ð2004	F 2	Bt maize	0	0
	Germany Italy	2003Ð2005 2003	F 2 F 2	Bt maize Diet, incorporated	0 Ñ b	0 Ñ b
		2004	F 2	Bt maize	0	0
	Slovakia	2004	F 2	Diet, surface	3	0
		2005	F 2	Diet, surface	0	0
		2005	F 3	Bt maize, incorporation to diet	0	0

Table 3 . Expected frequencies of major recessive resistance alleles in various European populations of O. nubilalis, with experi- mentwise probability of detecting a resistance allele if one were present in the line

 3 laboratory. In the German and Slovakian screens, most larvae originated from a univoltine population and did not pupate in the laboratory. As a result, many lines were lost, greatly increasing the cost per line screened. A cost ϾUS$500 per univoltine line is certainly too high for any kind of monitoring program. Hence, although F 2 screen is probably the best option for resistance monitoring in European regions where maize Þelds are infested by bivoltine or multivoltine populations of O. nubilalis, this question remains open in other regions where maize Þelds host univoltine populations of this pest. Additional research on breaking diapause in univoltine populations is needed before F 2 screens can be used effectively in these populations.

	Location	Yr	Lines screened	E͓p R ͔ (95% Cl)	Experimentwise dectection prob.
	France	1999Ð2004	784	0.0003 (0Ð0.0010)	0.950
		1999Ð2004 a	1,595	0.0002 (0Ð0.0005)	0.872
	Germany	2003Ð2005	455	0.0004 (0Ð0.0016)	0.975
	Italy	2003Ð2004	80	0.0031 (0Ð0.0092)	0.914
	Slovakia	2004Ð2005	26	0.0090 (0Ð0.026)	0.837 b

a Includes data previously published in[START_REF] Bourguet | Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of Ostrinia nubilalis[END_REF]. b The screen results from 10 of these lines could not be interpreted due to problems with the toxin. They are not included in further calculations of E͓p R ͔ and P No .

Acknowledgments

We thank Claudia Gaspers, Katarina Gottwaldova, Simona Landi, Katharina Pietsch, Kai Priesnitz, Stefan Rauschen, Christiane Saeglitz, Ulrike Schuller, Arti Sinha, and Arnica Zanardo for helping to collect European corn borer adults and larvae and for assistance during the laboratory work performed in Italy, Germany, and Slovakia. We thank two anonymous reviewers for clarifying several points of this article. This work was funded by grants from the EU, Proposal ProBenBt (Protecting the BeneÞts of Bt-toxin from Insect Resistance Development by Monitoring and Management), for the studies performed in Germany, Italy, and Slovakia and by the French Ministry of Agriculture for the study perform in France.