The increasing antibiotic resistance evolved by Helicobacter pylori has alarmingly reduced the eradication rates of first-line therapies. To overcome the current circulating resistome, we selected a novel potential therapeutic target in order to identify new candidate drugs for treating H . pylori infection. We screened 1120 FDA-approved drugs for molecules that bind to the essential response regulator HsrA and potentially inhibit its biological function. Seven natural flavonoids were identified as HsrA binders. All of these compounds noticeably inhibited the in vitro DNA binding activity of HsrA, but only four of them, apigenin, chrysin, kaempferol and hesperetin, exhibited high bactericidal activities against H . pylori . Chrysin showed the most potent bactericidal activity and the most synergistic effect in combination with clarithromycin or metronidazole. Flavonoid binding to HsrA occurs preferably at its C-terminal effector domain, interacting with amino acid residues specifically involved in forming the helix-turn-helix DNA binding motif. Our results validate the use of HsrA as a novel and effective therapeutic target in H . pylori infection and provide molecular evidence of a novel antibacterial mechanism of some natural flavonoids against H . pylori . The results further support the valuable potential of natural flavonoids as candidate drugs for novel antibacterial strategies.

Antibiotic resistance is a major cause of the increasing failures in the current eradication therapies against Helicobacter pylori. In this scenario, repurposing drugs could be a valuable strategy to fast-track novel antimicrobial agents. In the present study, we analyzed the inhibitory capability of 1,4-dihydropyridine (DHP) antihypertensive drugs on the essential function of the H. pylori response regulator HsrA and investigated both the in vitro antimicrobial activities and the in vivo efficacy of DHP treatments against H. pylori. Six different commercially available and highly prescribed DHP drugs—namely, Nifedipine, Nicardipine, Nisoldipine, Nimodipine, Nitrendipine, and Lercanidipine—noticeably inhibited the DNA binding activity of HsrA and exhibited potent bactericidal activities against both metronidazole- and clarithromycin-resistant strains of H. pylori, with minimal inhibitory concentration (MIC) values in the range of 4 to 32 mg/L. The dynamics of the decline in the bacterial counts at 2 × MIC appeared to be correlated with the lipophilicity of the drugs, suggesting different translocation efficiencies of DHPs across the bacterial membrane. Oral treatments with 100 mg/kg/day of marketed formulations of Nimodipine or Nitrendipine in combination with omeprazole significantly reduced the H. pylori gastric colonization in mice. The results presented here support a novel therapeutic solution for treatment of antibiotic-resistant H. pylori infections.

Helicobacter pylori (Hp) infection is the main cause of peptic ulcer and gastric cancer. Hp eradication rates have fallen due to increasing bacterial resistance to currently used broadspectrum antimicrobials. We have designed, synthesized and tested redox variants of nitroethylene-and 7-nitrobenzoxadiazole-based inhibitors of the essential Hp protein 2 Page 3 of 53 3 flavodoxin. Derivatives of the 7-nitrobenzoxadiazole lead, carrying reduced forms of the nitro group and/or oxidized forms of a sulphur atom, display high therapeutic indexes against several reference Hp strains. These inhibitors are effective against metronidazole-, clarithromycin-and rifampicin-resistant Hp clinical isolates. Their toxicity for mice after oral administration is low and, when administered individually at single daily doses for 8 days in a mice model of Hp infection, they decrease significantly Hp gastric colonization rates and are able to eradicate the infection in up to 60% of the mice. These flavodoxin inhibitors constitute a novel family of Hp-specific antimicrobials that may help fight the constant increase of Hp antimicrobial-resistant strains.

Flavodoxins are small soluble electron transfer proteins widely present in bacteria and absent in vertebrates. Flavodoxins participate in different metabolic pathways and, in some bacteria, they have been shown to be essential proteins representing promising therapeutic targets to fight bacterial infections. Using purified flavodoxin and chemical libraries, leads can be identified that block flavodoxin function and act as bactericidal molecules, as it has been demonstrated for Helicobacter pylori (Hp), the most prevalent human gastric pathogen. Increasing antimicrobial resistance by this bacterium has led current therapies to lose effectiveness, so alternative treatments are urgently required. Here, we summarize, with a focus on flavodoxin, opportunities for pharmacological intervention offered by the potential protein targets described for this bacterium and provide information on other gastrointestinal pathogens and also on bacteria from the gut microbiota that contain flavodoxin. The process of discovery and development of novel antimicrobials specific for Hp flavodoxin that is being carried out in our group is explained, as it can be extrapolated to the discovery of inhibitors specific for other gastric pathogens. The high specificity for Hp of the antimicrobials developed may be of help to reduce damage to the gut microbiota and to slow down the development of resistant Hp mutants.