Abstract

Background: The pathogen Helicobacter pylori (Hp) is a bacterium that infect near the half of the world´s. In Chile infection with Hp reach overall ~ 80%, depending on the geographic area and socioeconomic factors. Nowadays, it is well established that Hp infection underlie the etiology of several gastrointestinal pathologies like duodenal ulcer, gastritis and gastric cancer. The consensus for treatment of Hp infection considers using two antibiotics and a proton pump inhibitor during 14 days. This schedule often is associated with the report of side-effects, which eventually could lead to drop out eradication therapy and finally to elevate the prevalence of antimicrobial resistance. Due to Hp strains displaying antibiotic resistance phenotypes are dramatically increasing, a plethora of studies has been focused in the search for new natural substances with anti-Hp activity. Regarding this later, our group investigate the effect of natural proanthocyanidins (PAC) upon Hp infection (Fondecyt 11110442). In this study we demonstrate that urease inhibition and Hp-adherence to AGS cells depends on PAC size and flavan-3-ol nature. Recently, Hp carbonic anhydrase (CA) has gained importance because it acts cooperatively with urease and help to neutralize the pH of the surrounding environment. During our previous research a series of PACs-derivatives were prepared via nucleophilic attack using phloroglucinol and orcin. Although these compounds showed poor inhibition upon urease, they display a consistent inhibition of human carbonic anhydrase (hCA I). According to literature, inhibition of CA depends on the "Zinc Binding Properties" of the molecule. In this context, polyphenols have been the most investigated, albeit its activity is by far less potent than classical inhibitors like sulfonamides (e.g., acetazolamide). These observations lead us to propose that these derivatives could be interesting candidates for development of new hpCA inhibitors. In order to improve Hp CA inhibition properties, different synthetic strategies could be applied using various phenolic scaffolds as template. With these antecedents we construct the following hypothesis: Natural polyphenols in conjunction with its semi-synthetic and synthetic derivates exert anti- Hp effects through the concomitant inhibition of carbonic anhydrase and urease activities, limiting the ability of this pathogen to infect AGS cells and colonize mice gastric mucosa. The introduction of specific "Zinc Binding Groups" and the support of in silico tools will guide the refining of such compounds in order to originate more potent, selective and safer molecules valuable for the eradication of this pathogen. Methodology to achieving the specific goals: 1) As source for semi-synthesis of PACs-adducts, extraction and purification of natural polymeric PAC will be performed using adsorption resins 2) fragment-based virtual screening of substructures containing motifs with zinc binding groups (ZBG) will be explored for the designing of new phenolic CA inhibitors 3) The design and synthesis of new CA harboring ZBG will be focused in phenolic skeletons, which will be generated via well-known reactions like aldols condensation, Heck coupling or click chemistry using azide/alkyne partners. This products will be used as molecular frameworks. 4) For in vitro inhibition assays, wild type or recombinant urease and CA from Hp will be prepared and purified using bio-compatible (Centrifugal Partition Chromatography, Bioaffinity). 5) In order to rationalize pharmacological data and predict drug-likeness, CA inhibitory mechanism will be studied in silico by docking experiments and prediction of ADME properties. 6) Finally, those compounds with the most efficient in vitro urease and CA inhibitory properties, will be tested in Hp-infected AGS cells and in a short-term non-invasive model of infection using Mongolian gerbils infected with Hp strains labeled with RFP or Zn(II)-DPA in order to perform in vivo molecular imaging. Expected outcomes: In this proposal, using in silico approaches and in vitro and in vivo experiments it will be possible to obtain a select library of hpCA inhibitors. We expect that the introduction of different "Zinc Binding Groups" in this phenolics compounds originate more specific and potent natural and semisynthetic hpCA inhibitors. Finally, we expect that these inhibitors of CA in conjunction with natural PACs (as urease inhibitors) became an effective dual-targeted strategy to eradicate this pathogen.