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The current therapies against gastric pathogen Helicobacter pylori are ineffective in over 20% of patients. Enzymes belonging to the purine salvage pathway are considered as novel drug targets in this pathogen. Therefore, the main aim of the current study was to determine the antibacterial activity of pyridoxal 5'-phosphate (PLP), an active form of vitamin B6, against reference and clinical strains of H. pylori . Using a broad set of microbiological, physicochemical (UV absorption, LC-MS, X-ray analysis) and in silico experiments, we were able to prove that PLP inhibits adenylosuccinate synthetase (AdSS) from H. pylori by the competition with GTP (IC 50 eq ∼30 nM). This behaviour was attributed to formation of a Schiff base with a lysine residue (a covalent bond with Lys322 in the GTP binding site of AdSS) and was potentiated by the presence of vitamin C. This antibacterial activity of PLP gives hope for its future use against H. pylori .

Urease catalyzes the hydrolysis of urea, leading to an increase in stomach pH and supporting Helicobacter pylori survival, which is linked to several gastrointestinal disorders. In this study, thiazine-based Schiff bases were explored as promising urease inhibitors. Various spectroscopic techniques characterized the synthetic library of thiazine Schiff bases 2-36 and also evaluated for their inhibitory activities against urease. The derivatives demonstrated significant inhibitory potential with IC 50 values ranging from 0.14 ± 0.08 to 3.66 ± 0.21 μM, outperforming the standard inhibitor thiourea (IC 50  = 19.43 ± 0.18 μM). Structure-activity relationship (SAR) studies revealed that specific substitutions (type and positions) on the aryl ring significantly affect the inhibition potential. The most potent derivative, compound 7, possessed 2-methoxy-5-trifluoromethyl substitutions and exhibited an IC 50 of 0.14 ± 0.08 μM. Enzyme kinetics studies revealed that the most potent derivatives function as competitive inhibitors. Additionally, molecular docking studies provided insights into the binding interactions between the molecule and the urease active site, highlighting key residues involved in inhibitor binding. These findings highlight the therapeutic potential of thiazine-based Schiff bases as urease inhibitors and provide insights for the development of new anti-ulcer agents., Competing Interests: Declaration of competing interest The authors declare no conflict of interest, financial or otherwise. No writing assistance was utilized in the production of this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The eradication of Helicobacter pylori, the etiologic agent of gastric ulcer and adenocarcinoma, is a big concern in clinics due to the increasing drug resistance phenomena and the limited number of efficacious treatment options. The exploitation of the H. pylori carbonic anhydrases (HpCAs) as promising pharmacological targets has been validated by the antibacterial activity of previously reported CA inhibitors due to the role of these enzymes in the bacterium survival in the gastric mucosa. The development of new HpCA inhibitors seems to be on the way to filling the existing antibiotics gap. Due to the recent evidence on the ability of the coumarin scaffold to inhibit microbial α-CAs, a large library of derivatives has been developed by means of a pH-regulated cyclization reaction of coumarin-bearing acyl thiosemicarbazide intermediates. The obtained 1,3,4-thiadiazoles (10-18a,b) and 1,2,4-triazole-3-thiones (19-26a,b) were found to strongly and selectively inhibit HpαCA and computational studies were fundamental to gaining an understanding of the interaction networks governing the enzyme-inhibitor complex. Antibacterial evaluations on H. pylori ATCC 43504 highlighted some compounds that maintained potency on a resistant clinical isolate. Also, their combinations with metronidazole decreased both the minimal inhibitory concentration and minimal bactericidal concentration values of the antibiotic, with no synergistic effect., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Background: Qurom quenching enzyme have an impact on treatment efficacy and prevent the recurrence of Helicobacter pylori biofilm-related infections, although it has not been thoroughly investigated in vitro and in silico. The current study aims to characterize the N-acyl homoserine lactonase, the quorum quenching AiiA protein of Bacillus licheniformis against H. pylori biofilm., Methods and Results: In this study, AiiA protein were screened for their anti-biofilm activity, was found to effectively control biofilm formation of H. pylori with concentrations ranging from 2 to 10 µg/mL. According to CLSM and COMSTAT analysis, the untreated substratum had the robust biofilm biomass of 25-18 µM and biovolume of 3-4 mm 3 /mm 2 . The total biofilm biovolume and average biofilm thickness were considerably reduced by 40% with a single application of 10 µg/mL of AiiA protein. The biofilm treated with AiiA exhibited a lower urease and polysaccharides than to the untreated biofilm. Further, in silico analysis, exhibited a greater interaction of AiiA against the outer membrane proteins of H. pylori compared to virulence factors. The conserved domains in the binding pockets of AiiA proteins showed a highest binding affinity proving the catalytic activity of the protein., Conclusion: In this study, the H. pylori biofilm architecture, exopolysaccharide and urease were significantly controlled by our purified N-acyl homoserine lactonase from B. licheniformis. Furthermore, the molecular docking showed the significant interaction between AiiA and key biofilm forming and virulence proteins proved an excellent antibiofilm activity controlling the infections of H. pylori human pathogen., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The human gastric pathogen Helicobacter pylori chronically affects the gastric mucosal layer of approximately half of world's population. The emergence of resistant strains urges the need for identification of novel and selective drug against new molecular targets. A ubiquitous enzyme, Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), is considered as first line of defense against uracil mis-incorporation into DNA, and essential for genome integrity. Lack of dUTPase triggers an elevated recombination frequency, DNA breaks and ultimately cell death. Hence, dUTPase can be considered as a promising target for development of novel lead inhibitor compounds in H. pylori treatment. Herein, we report the generation of three-dimensional model of the target protein using comparative modelling and its validation. To identify dUTPase inhibitors, a high throughput virtual screening approach utilizing Knowledge-based inhibitors and DrugBank database was implemented. Top ranked compounds were scrutinized based on investigations of the protein-ligand interaction fingerprints, molecular interaction maps and binding affinities and the drug potentiality. The best ligands were studied further for complex stability and intermolecular interaction profiling with respect to time under 100 ns classical molecular dynamic stimulation, establishing significant stability in dynamic states as observed from RMSD and RMSF parameters and interactions with the catalytic site residues. The binding free energy calculation computed using MM-GBSA method from the MD simulation trajectories demonstrated that our molecules possess strong binding affinity towards the Helicobacter pylori dUTPase protein. We conclude that our proposed molecules may be potential lead molecules for effective inhibition against the H. pylori dUTPase protein subject to experimental validation.Communicated by Ramaswamy H. Sarma.

The Asp-tRNA Asn /Glu-tRNA Gln amidotransferase (GatCAB) has been proposed as a novel antibacterial drug target due to its indispensability in prominent human pathogens. While several inhibitors with in vitro activity have been identified, none have been demonstrated to have potent activity against live bacteria. In this work, seven non-hydrolyzable transition state mimics of GatCAB were synthesized and tested as the transamidase inhibitors against GatCAB from the human pathogen Helicobacter pylori. Notably, the methyl sulfone analog of glutamyl-adenosine significantly reduced GatCAB's transamination rate. Additionally, four lipid-conjugates of these mimics displayed antibacterial activity against Bacillus subtilis, likely due to enhanced cell permeability. Inhibitory activity against GatCAB in live bacteria was confirmed using a sensitive gain-of-function dual luciferase reporter in Mycobacterium bovis-BCG. Only the lipid-conjugated methyl sulfone analog exhibited a significant increase in mistranslation rate, highlighting its cell permeability and inhibitory potential. This study provides insights for developing urgently needed novel antibacterial agents amidst emerging antimicrobial drug resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Heme trafficking is essential for cellular function, yet mechanisms of transport and/or heme interaction are not well defined. The System I and System II bacterial cytochrome c biogenesis pathways are developing into model systems for heme trafficking due to their functions in heme transport, heme stereospecific positioning, and mediation of heme attachment to apocytochrome c. Here we focus on the System II pathway, CcsBA, that is proposed to be a bi-functional heme transporter and holocytochrome c synthase. An extensive structure-function analysis of recombinantly expressed Helicobacter pylori and Campylobacter jejuni CcsBAs revealed key residues required for heme interaction and holocytochrome c synthase activity. Homologous residues were previously identified to be required for heme interaction in Helicobacter hepaticus CcsBA. This study provides direct, biochemical evidence that mechanisms of heme interaction are conserved, leading to the proposal that the CcsBA WWD heme-handling domain represents a novel target for therapeutics., (© 2024. The Author(s).)

Background: Theoretically, a rapid urease test (RUT) using a swab of the gastric wall (Swab-RUT) for Helicobacter pylori (H. pylori) is safe. However, the validity and utility of Swab-RUT remain unclear. Therefore, we assessed the validity and utility of Swab-RUT compared to RUT using mucosal forceps of the gastric wall (Forceps-RUT) and 13 C-urea breath test (UBT)., Methods: This study was a multicenter prospective observational study. When the examinees were suspected of H. pylori infection during esophagogastroduodenoscopy, we performed Swab-RUT and Forceps-RUT continuously. When the examinees were not suspected of H. pylori infection, we performed Swab-RUT alone. We validated the status of H. pylori infection using UBT., Results: Ninety-four examinees were enrolled from four institutions between May 2016 and December 2020 (median age [range], 56.5 [26-88] years). In this study, the sensitivity, specificity, and accuracy of Swab-RUT to UBT were 0.933 (95% confidence interval: 0.779-0.992), 0.922 (0.827-0.974), and 0.926 (0.853-0.970), respectively. The Kappa coefficient of Swab-RUT to UBT was 0.833, and that of Swab-RUT to forceps-RUT was 0.936. No complications were observed in this study., Conclusions: Swab-RUT is a valid examination for the status of H. pylori infection compared to the conventional Forceps-RUT., (© 2024. The Author(s).)

Helicobacter pylori is the main causative agent of gastric cancer, especially non-cardiac gastric cancers. This bacterium relies on urease producing much ammonia to colonize the host. Herein, the study provides valuable insights into structural patterns driving urease inhibition for high-activity molecules designed via exploring known inhibitors. Firstly, an ensemble model was devised to predict the inhibitory activity of novel compounds in an automated workflow (R 2  = 0.761) that combines four machine learning approaches. The dataset was characterized in terms of chemical space, including molecular scaffolds, clustering analysis, distribution for physicochemical properties, and activity cliffs. Through these analyses, the hydroxamic acid group and the benzene ring responsible for distinct activity were highlighted. Activity cliff pairs uncovered substituents of the benzene ring on hydroxamic acid derivatives are key structures for substantial activity enhancement. Moreover, 11 hydroxamic acid derivatives were designed, named mol1-11. Results of molecular dynamic simulations showed that the mol9 exhibited stabilization of the active site flap's closed conformation and are expected to be promising drug candidates for Helicobacter pylori infection and further in vitro, in vivo, and clinical trials to demonstrate in future., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

We have characterized the catalytic cycle of the Helicobacter pylori KatA catalase (HPC). H. pylori is a human and animal pathogen responsible for gastrointestinal infections. Multifrequency (9-285 GHz) EPR spectroscopy was applied to identify the high-valent intermediates (5 ≤ pH ≤ 8.5). The broad (2000 G) 9-GHz EPR spectrum consistent with the [Fe(IV) = O Por •+ ] intermediate was detected, and showed a clear pH dependence on the exchange-coupling of the radical (delocalized over the porphyrin moiety) due to the magnetic interaction with the ferryl iron. In addition, Trp • (for pH ≤ 6) and Tyr • (for 5 ≤ pH ≤ 8.5) species were distinguished by the advantageous resolution of their g-values in the 285-GHz EPR spectrum. The unequivocal identification of the high-valent intermediates in HPC by their distinct EPR spectra allowed us to address their reactivity towards substrates. The stabilization of an [Fe(IV) = O Trp • ] species in HPC, unprecedented in monofunctional catalases and possibly involved in the oxidation of formate to the formyloxyl radical at pH ≤ 6, is reminiscent of intermediates previously identified in the catalytic cycle of bifunctional catalase-peroxidases. The 2e - oxidation of formate by the [Fe(IV) = O Por •+ ] species, both at basic and acidic pH conditions, involving a 1H + /2e - oxidation in a cytochrome P450 peroxygenase-like reaction is proposed. Our findings demonstrate that moonlighting by the H. pylori catalase includes formate oxidation, an enzymatic reaction possibly related to the unique strategy of the neutrophile bacterium for gastric colonization, that is the release of CO 2 to regulate the pH in the acidic environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His 6 -tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His 6 -tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked.

The prevalence of Helicobacter pylori infection has been increasing rapidly due to the genetic heterogeneity and antibacterial resistance shown by the bacteria, affecting over 50% of the world population and over 80% of the Indian population, in particular. In this regard, novel drug targets are currently being explored, one of which is the crucial metabolic enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) involved in the de novo nucleotide biosynthesis pathway, in order to combat the infection and devise efficient therapeutic strategies. The present study reports the development of methylpyrazole-substituted benzimidazoles as small molecule inhibitors of H. pylori IMPDH with a nanomolar range of enzyme inhibition. A set of 19 small molecules have been designed, synthesized, and further evaluated for their inhibitory potential against H. pylori IMPDH using in silico , in vitro , biochemical, and biophysical techniques. Compound 7j was found to inhibit H. pylori IMPDH with an IC 50 value of 0.095 ± 0.023 μM, which is close to 1.5-fold increase in the inhibitory activity, in comparison to the previously reported benzimidazole-based hit C91 . Moreover, kinetic characterization has provided significant insights into the uncompetitive inhibition shown by these small molecules on H. pylori IMPDH, thus providing details about the enzyme inhibition mechanism. In conclusion, methylpyrazole-based small molecules indicate a promising path to develop cheap and bioavailable drugs to efficiently treat H. pylori infection in the coming years, in comparison to the currently available therapy.

A series of sulfonate and sulfamate derivatives bearing benzofuran or benzothiophene scaffold exhibited potent inhibitory effect on urease enzyme. Most of the derivatives exhibited significantly higher potency than thiourea, the standard inhibitor. Compound 1s was identified as the most potent urease inhibitor with an IC 50 value of 0.42 ± 0.08 μM, which is 53-fold more potent than thiourea, positive control (IC 50  = 22.3 ± 0.031 μM). The docking results further revealed the binding interactions towards the urease active site. Phenotypic screening revealed that compounds 1c, 1d, 1e, 1f, 1j, 1n, and 1t exhibit high potency against H. pylori with MIC values ranging from 0.00625 to 0.05 mM and IC 50 values ranging from 0.0031 to 0.0095 mM, much more potent than the positive control, acetohydroxamic acid (MIC and IC 50 values were 12.5 and 7.38 mM, respectively). Additional studies were performed to investigate the toxicity of these compounds against the gastric epithelial cell line (AGS) and their selectivity profile against E. coli, and five Lactobacillus species representative of the gut microflora. Permeability characteristics of the most promising derivatives were investigated in Caco-2 cell line. The results indicate that the compounds could be targeted in the GIT only without systemic side effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori . The secretion system is encoded by the  cag  pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect., Competing Interests: The authors declare there are no competing interests.

Lacto- N -difucohexaose II (LNDFH II) is a typical fucosylated human milk oligosaccharide and can be enzymatically produced from lacto- N -tetraose (LNT) by a specific α1,3/4-fucosyltransferase from Helicobacter pylori DMS 6709, referred to as FucT14. Previously, we constructed an engineered Escherichia coli BL21(DE3) with a single plasmid for highly efficient biosynthesis of LNT. In this study, two additional plasmids harboring the de novo GDP-L-fucose pathway module and FucT14, respectively, were further introduced to construct the strain for successful biosynthesis of LNDFH II. FucT14 was actively expressed, and the engineered strain produced LNDFH II as the major product, lacto- N -fucopentaose (LNFP) V as the minor product, and a trace amount of LNFP II and 3-fucosyllactose as very minor products. Additional expression of the α1,3-fucosyltransferase FutM1 from a Bacteroidaceae bacterium from the gut metagenome could obviously enhance the LNDFH II biosynthesis. After optimization of induction conditions, the maximum titer reached 3.011 g/L by shake-flask cultivation. During the fed-batch cultivation, LNDFH II was highly efficiently produced with the highest titer of 18.062 g/L and the productivity yield of 0.301 g/L·h.

In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway., (Copyright © 2024 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Urease, a pivotal enzyme in nitrogen metabolism, plays a crucial role in various microorganisms, including the pathogenic Helicobacter pylori . Inhibiting urease activity offers a promising approach to combating infections and associated ailments, such as chronic kidney diseases and gastric cancer. However, identifying potent urease inhibitors remains challenging due to resistance issues that hinder traditional approaches. Recently, machine learning (ML)-based models have demonstrated the ability to predict the bioactivity of molecules rapidly and effectively. In this study, we present ML models designed to predict urease inhibitors by leveraging essential physicochemical properties. The methodological approach involved constructing a dataset of urease inhibitors through an extensive literature search. Subsequently, these inhibitors were characterized based on physicochemical properties calculations. An exploratory data analysis was then conducted to identify and analyze critical features. Ultimately, 252 classification models were trained, utilizing a combination of seven ML algorithms, three attribute selection methods, and six different strategies for categorizing inhibitory activity. The investigation unveiled discernible trends distinguishing urease inhibitors from non-inhibitors. This differentiation enabled the identification of essential features that are crucial for precise classification. Through a comprehensive comparison of ML algorithms, tree-based methods like random forest, decision tree, and XGBoost exhibited superior performance. Additionally, incorporating the "chemical family type" attribute significantly enhanced model accuracy. Strategies involving a gray-zone categorization demonstrated marked improvements in predictive precision. This research underscores the transformative potential of ML in predicting urease inhibitors. The meticulous methodology outlined herein offers actionable insights for developing robust predictive models within biochemical systems.

Infections from Helicobacter pylori (Hp) are endangering Public Health safety worldwide, due to the associated high risk of developing severe diseases, such as peptic ulcer, gastric cancer, diabetes, and cardiovascular diseases. Current therapies are becoming less effective due to the rise of (multi)drug-resistant phenotypes and an urgent need for new antibacterial agents with innovative mechanisms of action is pressing. Among the most promising pharmacological targets, Carbonic Anhydrases (EC: 4.2.1.1) from Hp, namely HpαCA and HpβCA, emerged for their high druggability and crucial role in the survival of the pathogen in the host. Thereby, in the last decades, the two isoenzymes were isolated and characterized offering the opportunity to profile their kinetics and test different series of inhibitors., (Copyright © 2024. Published by Elsevier Inc.)

Helicobacter pylori is a gram negative spiral shaped bacteria that causes peptic ulcer and gastric cancer. It Is the sixth most prevalent cancer in the world and the third leading cause of cancer death. The increase in reported cases of H. pylori resistance to the drugs and antibiotics shows the need for the development of new and efficient drugs against the pathogen. In the present study, D-glycero-D-manno-heptose-1,7-bisphosphate 7-phosphatase (GmhB), an enzyme involved in the biosynthesis of lipopolysaccharides that encourages bacterial adherence, self-aggregation and identifying the host cells was modelled and the active sites were predicted through POCASA which is an automated ligand binding site prediction server. Natural product activity and species source (NPASS) is a database of 96,481 natural compounds that were subjected to virtual screening workflow that includes Qikprop, Lipinski rule, filtering out reactive functional groups followed by high throughput virtual screening and the top 10 compounds were selected for further induced fit docking along with the substrate D-glycero-β-D-manno-heptose 1,7-bisphosphate. The compound NPC170742 (Alpha, Beta, 3,4,5,2',4',6'-Octahydroxy dihydrochalcone) showed higher affinity than the substrate, and both the substrate D-glycero-β-D-manno-heptose 1,7-bisphosphate and the compound NPC170742 were subjected to molecular dynamics simulation. The results exposed the compound NPC170742 could be a potential lead compound against the enzyme D-glycero-D-manno-heptose-1,7-bisphosphate 7-phosphatase of H. pylori .Communicated by Ramaswamy H. Sarma.

Background: Helicobacter pylori (HP) infection could lead to various gastrointestinal diseases. Urease is the most important virulence factor of HP. It protects the bacterium against gastric acid., Objective: Therefore, we aimed to design urease inhibitors as drugs against HP infection., Methods: The DrugBank-approved library was assigned with 3D conformations and the structure of the urease was prepared. Using a re-docking strategy, the proper settings were determined for docking by PyRx and GOLD software. Virtual screening was performed to select the best inhibitory drugs based on binding affinity, FitnessScore, and binding orientation to critical amino acids of the active site. The best inhibitory drug was then evaluated by IC 50 and the diameter of the zone of inhibition for bacterial growth., Results: The structures of prepared drugs were screened against urease structure using the determined settings. Clodronic acid was determined to be the best-identified drug, due to higher PyRx binding energy, better GOLD FitnessScore, and interaction with critical amino acids of urease. In vitro results were also in line with the computational data. IC 50 values of Clodronic acid and Acetohydroxamic Acid (AHA) were 29.78 ± 1.13 and 47.29 ± 2.06 μg/ml, respectively. Diameters of the zones of inhibition were 18 and 15 mm for Clodronic acid and AHA, respectively., Conclusion: Clodronic acid has better HP urease inhibition potential than AHA. Given its approved status, the development of a repurposed drug based on Clodronic acid would require less time and cost. Further, in vivo studies would unveil the efficacy of Clodronic acid as a urease inhibitor., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The development of new natural drugs for Helicobacter pylori (H. pylori) management has recently received significant attention. Iris confusa (I. confusa) was long used for the treatment of bacterial infections and gastritis. This study aimed at evaluating its effect on management of H. pylori infection and exploring its bioactive metabolites. The inhibitory potential of the polar (PF), non-polar (NPF) fractions and the isolated compounds against H. pylori using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in addition to their cyclooxygenases (COX-1 and COX-2), and nitric oxide (NO) inhibitory activities were assessed. The most biologically active compound was tested for its selective H. pylori inosine-5'-monophosphate dehydrogenase (HpIMPDH) inhibitory potential. Chromatographic purification of PF and NPF allowed isolation of tectoridin, orientin, irigenin, tectorigenin, isoarborinol and stigmasterol. The PF exhibited significant anti-H. pylori (MIC 62.50 µg/mL), COX-1, COX-2 (IC 50 of 112.08 ± 0.60 and 47.90 ± 1.50 µg/mL respectively, selectivity index SI of 2.34), and NO (IC 50 47.80 ± 0.89 µg/mL) inhibitory activities, while irigenin was the most potent isolated compound. Irigenin was found to have a promising activity against HpIMPDH enzyme (IC 50 of 2.07 ± 1.90 μM) with low activity against human hIMPDH2 (IC 50  > 10 μM) than clarithromycin, assuring its selectivity. Overall, I. confusa and its isolated compounds may serve as a potential source of plant-based drugs for H. pylori control. This study scientifically validated the claimed anti-bacterial activity of I. confusa and revealed irigenin potential as a novel lead exhibiting anti H. pylori activity in a first record., (© 2022. The Author(s).)

Helicobacter pylori urease remains a validated drug target for the eradication of pervasive chronic stomach infection that leads to severe human health diseases such as gastritis and stomach cancer. The increased failure of current treatment protocols because of resistance to broadband antibiotics, severe side effects and low compliance underscore the need for a targeted eradication therapy. Therefore, in the present research, we have developed a new series of acetylphenol-based acyl thioureas that can potentially provide a new template for drug candidates to inhibit urease enzyme. Newly synthesized compounds 7a-j were evaluated for urease inhibitory strength using thiourea as a positive control. In vitro inhibitory results revealed that all the tested compounds were significantly potent than the standard drug. The most active lead 7f competitively inhibited the enzyme and displayed an IC 50 value of 0.054 ± 0.002 μM, a ~413-fold strong inhibitory potential than thiourea (IC 50  = 22.3 ± 0.031 μM). Various insightful structure-activity relationships were developed showing the key structural requirements for potent inhibitory effects. Molecular docking analysis of 7f inside the active pocket of urease suggested several important interactions with amino acid residues such as ILE411, MET637, ARG439, GLN635, ALA636 and ALA440. Finally, pharmacokinetic properties suggested that the tested derivatives are safe to develop as low-molecular-weight drugs to treat ureolytic bacterial infections., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The discovery of life-changing medicines continues to be the driving force for the rapid exploration and expansion of chemical space, enabling access to innovative small molecules of medicinal importance. These small molecules remain the backbone for modern drug discovery. In this context, the treatment of ureolytic bacterial infections inspires the identification of potent and effective inhibitors of urease, a promising and highly needed target for H. pylori eradication. The present study explores the evaluation of sulfamate derivatives for the inhibition of urease enzyme. The tested compounds showed remarkable inhibitory effect and high level of potency. Compound 1q emerged as the lead inhibitor with an IC 50 value of 0.062 ± 0.001 µM, ∼360-fold more potent than thiourea (IC 50  = 22.31 ± 0.031 µM). The assessment of various contributing factors towards the inhibition profile allowed for the establishment of diverse structure-activity relationships. Kinetics studies revealed the competitive mode of inhibition of compound 1q while molecular modeling analysis identified various crucial binding interactions with ARG609, ARG439, HIS519, HIS492, HIS593, ALA440, and ALA636 in the active pocket of the enzyme. Finally, the calculated pharmacokinetic properties suggest a promising profile of our potent sulfamate-based urease inhibitors., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The urease enzyme has been an important target for the discovery of effective pharmacological and agricultural products. Thirteen regio-selectively alkylated benzimidazole-2-thione derivatives have been designed to carry the essential features of urease inhibitors. The urease enzyme was isolated from Helicobacter pylori as a recombinant urease utilizing the His-tag method. The isolated enzyme was purified and characterized using chromatographic and FPLC techniques showing a maximal activity of 200 mg/mL. Additionally, the commercial Jack bean urease was purchased and included in this study for comparative and mechanistic investigations. The designed compounds were synthesized and screened for their inhibitory activity against the two ureases. Compound 2 inhibited H. pylori and Jack bean ureases with IC 50 values of 0.11; and 0.26 mM; respectively. While compound 5 showed IC 50 values of 0.01; and 0.29 mM; respectively. Compounds 2 and 5 were docked against Helicobacter pylori urease (PDB ID: 1E9Y; resolution: 3.00 Å) and exhibited correct binding modes with free energy (ΔG) values of -9.74 and -13.82 kcal mol -1 ; respectively. Further; the in silico ADMET and toxicity properties of 2 and 5 indicated their general safeties and likeness to be used as drugs. Finally, the compounds' safety was authenticated by an in vitro cytotoxicity assay against fibroblast cells.

Thirty-eight disulfides containing N-arylacetamide were designed and synthesized in an effort to develop novel urease inhibitors. Biological evaluation revealed that some of the synthetic compounds exhibited strong inhibitory potency against both cell-free urease and urease in intact cell with low cytotoxicity to mammalian cells even at concentration up to 250 μM. Of note, 2,2'-dithiobis(N-(2-fluorophenyl)acetamide) (d7), 2,2'-dithiobis(N-(3,5-difluorophenyl)acetamide) (d24), and 2,2'-dithiobis(N-(3-fluorophenyl)acetamide) (d8) were here identified as the most active inhibitors with IC 50 of 0.074, 0.44, and 0.81 μM, showing 32- to 355-fold higher potency than the positive control acetohydroxamic acid. These disulfides were confirmed to bind urease without covalent modification of the cysteine residue and to inhibit urease reversibly with a mixed inhibition mechanism. They also showed very good anti-Helicobacter pylori activities with d8 showing a comparable potency to the clinical used drug amoxicillin. The impressive in vitro biological profile indicated their immense potential as therapeutic agents to tackle H. pylori caused infections., (© 2021 Wiley-VCH GmbH.)

The paralogues RrpA and RrpB, which are members of the MarR family of DNA binding proteins, are important for the survival of the global bacterial foodborne pathogen Campylobacter jejuni under redox stress. We report that RrpA is a positive regulator of mdaB , encoding a flavin-dependent quinone reductase that contributes to the protection from redox stress mediated by structurally diverse quinones, while RrpB negatively regulates the expression of cj1555c (renamed nfrA for NADPH-flavin reductase A), encoding a flavin reductase. NfrA reduces riboflavin at a greater rate than its derivatives, suggesting that exogenous free flavins are the natural substrate. MdaB and NfrA both prefer NADPH as an electron donor. Cysteine substitution and posttranslational modification analyses indicated that RrpA and RrpB employ a cysteine-based redox switch. Complete genome sequence analyses revealed that mdaB is frequently found in Campylobacter and related Helicobacter spp., while nfrA is predominant in C. jejuni strains. Quinones and flavins are redox cycling agents secreted by a wide range of cell types that can form damaging superoxide by one-electron reactions. We propose a model for stress adaptation where MdaB and NfrA facilitate a two-electron reduction mechanism to the less toxic hydroquinones, thus aiding survival and persistence of this major pathogen. IMPORTANCE Changes in cellular redox potential result in alteration in the oxidation state of intracellular metabolites and enzymes; consequently, cells make adjustments that favor growth and survival. The work we present here answers some of the many questions that have remained elusive over the years of investigation into the enigmatic microaerophile bacterium Campylobacter jejuni. We employed molecular approaches to understand the regulation mechanisms and functional analyses to reveal the roles of two novel quinone and flavin reductases; both serve as major pools of cellular redox-active molecules. This work extends our knowledge on bacterial redox sensing mechanisms and the significance of hemostasis.

Accessing large numbers of structurally diverse glycans and derivatives is essential to functional glycomics. We showed a general tolerance of galactosyltransferases toward uridine-diphosphate-galactosamine (UDP-GalN), which is not a commonly used sugar nucleotide donor. The property was harnessed to develop a two-step chemoenzymatic strategy for facile synthesis of novel and divergent N-acetylgalactosamine (GalNAc)-glycosides and derivatives in preparative scales. The discovery and the application of the new property of existing glycosyltransferases expand their catalytic capabilities in generating novel carbohydrate linkages, thus prompting the synthesis of diverse glycans and glycoconjugates for biological studies., (© 2021 Wiley-VCH GmbH.)

Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori . Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D- glycero -D- manno -heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L- glycero -D- manno -heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D- glycero -D- manno -heptose-1,7-bisphosphate into D- glycero -D- manno -heptose-1-phosphate with a preference for the β-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.

Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter pylori, a bacterium etiologically related to 95% of gastric cancers. Here, we present the crystal structures of FabX alone and in complexes with an octanoyl-acyl carrier protein (ACP) substrate or with holo-ACP. FabX belongs to the nitronate monooxygenase (NMO) flavoprotein family but contains an atypical [4Fe-4S] cluster absent in all other family members characterized to date. FabX binds ACP via its positively charged α7 helix that interacts with the negatively charged α2 and α3 helices of ACP. We demonstrate that the [4Fe-4S] cluster potentiates FMN oxidation during dehydrogenase catalysis, generating superoxide from an oxygen molecule that is locked in an oxyanion hole between the FMN and the active site residue His182. Both the [4Fe-4S] and FMN cofactors are essential for UFA synthesis, and the superoxide is subsequently excreted by H. pylori as a major resource of peroxide which may contribute to its pathogenic function in the corrosion of gastric mucosa., (© 2021. The Author(s).)

This article describes the design and synthesis of a series of novel amantadine-thiourea conjugates ( 3a - j ) as Jack bean urease inhibitors. The synthesized hybrids were assayed for their in vitro urease inhibition. Accordingly, N -(adamantan-1-ylcarbamothioyl)octanamide ( 3j ) possessing a 7-carbon alkyl chain showed excellent activity with IC 50 value 0.0085 ± 0.0011 µM indicating that the long alkyl chain plays a vital role in enzyme inhibition. Whilst N -(adamantan-1-ylcarbamothioyl)-2-chlorobenzamide ( 3g ) possessing a 2-chlorophenyl substitution was the next most efficient compound belonging to the aryl series with IC 50 value of 0.0087 ± 0.001 µM. The kinetic mechanism analyzed by Lineweaver-Burk plots revealed the non-competitive mode of inhibition for compound 3j . Moreover, in silico molecular docking against target protein (PDBID 4H9M) indicated that most of the synthesized compounds exhibit good binding affinity with protein. The compound 3j forms two hydrogen bonds with amino acid residue VAL391 having a binding distance of 1.858 Å and 2.240 Å. The interaction of 3j with amino acid residue located outside the catalytic site showed its non-competitive mode of inhibition. Based upon these results, it is anticipated that compound 3j may serve as a lead structure for the design of more potent urease inhibitors.

Gastrointestinal tract infection caused by Helicobacter pylori is a common virulent disease found worldwide, and the infection rate is much higher in developing countries than in developed ones. In the pathogenesis of H. pylori in the gastrointestinal tract, the secretion of the urease enzyme plays a major role. Therefore, inhibition of urease is a better approach against H. pylori infection. In the present study, a series of syn and anti isomers of N -substituted indole-3-carbaldehyde oxime derivatives was synthesized via Schiff base reaction of appropriate carbaldehyde derivatives with hydroxylamine hydrochloride. The in vitro urease inhibitory activities of those derivatives were evaluated against that of Macrotyloma uniflorum urease using the modified Berthelot reaction. Out of the tested compounds, compound 8 (IC 50 = 0.0516 ± 0.0035 mM) and compound 9 (IC 50 = 0.0345 ± 0.0008 mM) were identified as the derivatives with potent urease inhibitory activity with compared to thiourea (IC 50 = 0.2387 ± 0.0048 mM). Additionally, in silico studies for all oxime compounds were performed to investigate the binding interactions with the active site of the urease enzyme compared to thiourea. Furthermore, the drug-likeness of the synthesized oxime compounds was also predicted.

To date, little attempt has been made to develop new treatments for Helicobacter pylori (H. pylori), although the community is aware of the shortage of treatments for H. pylori. In this study, we developed a 192-tandem-microwell-based high-throughput assay for ammonia that is a known virulence factor of H. pylori and a product of urease. We could identify few drugs, that is, panobinostat, dacinostat, ebselen, captan, and disulfiram, to potently inhibit the activity of ureases from bacterial or plant species. These inhibitors suppress the activity of urease via substrate-competitive or covalent-allosteric mechanism, but all except captan prevent the antibiotic-resistant H. pylori strain from killing human gastric cells, with a more pronounced effect than acetohydroxamic acid, a well-known urease inhibitor and clinically used drug for the treatment of bacterial infection. This study offers several bases for the development of new treatments for urease-containing pathogens and to study the mechanism responsible for the regulation of urease activity., (© 2021 Federation of American Societies for Experimental Biology.)

It is now well established that the Gram-negative bacterium, Helicobacter pylori, causes gastritis in humans. In recent years, it has become apparent that the so-called non-pylori Helicobacters, normally infecting pigs, cats, and dogs, may also be involved in human pathology via zoonotic transmission. Indeed, more than 30 species of non-pylori Helicobacters have been identified thus far. One such organism is Helicobacter canadensis, an emerging pathogen whose genome sequence was published in 2009. Given our long-standing interest in the biosynthesis of N-formylated sugars found in the O-antigens of some Gram-negative bacteria, we were curious as to whether H. canadensis produces such unusual carbohydrates. Here, we demonstrate using both biochemical and structural techniques that the proteins encoded by the HCAN_0198, HCAN_0204, and HCAN_0200 genes in H. canadensis, correspond to a 3,4-ketoisomerase, a pyridoxal 5'-phosphate aminotransferase, and an N-formyltransferase, respectively. For this investigation, five high-resolution X-ray structures were determined and the kinetic parameters for the isomerase and the N-formyltransferase were measured. Based on these data, we suggest that the unusual sugar, 3-formamido-3,6-dideoxy-d-glucose, will most likely be found in the O-antigen of H. canadensis. Whether N-formylated sugars found in the O-antigen contribute to virulence is presently unclear, but it is intriguing that they have been observed in such pathogens as Francisella tularensis, Mycobacterium tuberculosis, and Brucella melitensis., (© 2021 The Protein Society.)

Due to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium's recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori - two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori. KEY POINTS: • A method for assessing H. pylori cells penetration by drug candidates is described. • Three alternative detection assays that complement each other can be used. • The method may be adapted for other bacteria as well., (© 2021. The Author(s).)

Methionine aminopeptidases (MetAPs) are an important class of enzymes that work co-translationally for the removal of initiator methionine. Chemical inhibition or gene knockdown is lethal to the microbes suggesting that they can be used as antibiotic targets. However, sequence and structural similarity between the microbial and host MetAPs has been a challenge in the identification of selective inhibitors. In this study, we have analyzed several thousands of MetAP sequences and established a pattern of variation in the S1 pocket of the enzyme. Based on this knowledge, we have designed a library of 17 azaindole based hydroxamic acid derivatives which selectively inhibited the MetAP from H. pylori compared to the human counterpart. Structural studies provided the molecular basis for the selectivity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31 P, 1 H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19 F and 27 Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers., (© 2021. The Author(s).)

Two novel series of Dihydropyrimidine-hydroxamic acid hybrids (4a-4l and 5a-5l) were designed, synthesized and evaluated for in vitro Helicobacter pylori urease inhibition. In vitro enzyme inhibition screening led to the discovery of three potent urease inhibitors 2-[[4-(4-hydroxy phenyl)-6-oxo-1,6-dihydropyrimidine-2-yl]-amino]-N-hydroxy acetamide (4g), 2-[[4-(4-chloro phenyl)-6-oxo-1,6-dihydropyrimidine-2-yl]-amino]-N-hydroxy acetamide (4b) and 3-[[4-(3-methoxy phenyl)-6-oxo-1,6-dihydropyrimidine-2-yl]-amino]-N-hydroxy propanamide (5l). Compound 4g showed excellent urease inhibition with IC 50 value of 14 ± 1 nM, indicated by its strong interactions with both metallic Ni ++ ions, Gly279, His221, Ala365, Asp362, Asn168, Arg338 and His322 residues of the active site of urease. Further, compounds 4b and 5l displayed very good activity with IC 50 value of 0.082 ± 0.004 µM and 0.14 ± 0.013 µM respectively compared to standard Acetohydroxamic acid (IC 50 - 27.4 ± 1.2 µM). Kinetic studies revealed that a mixed inhibition with both competitive and non-competitive aspects is involved in the urease inhibition mechanism. The in vitro urease inhibition results were supported by molecular docking studies. Collectively, this study indicates that 4g could be considered as promising lead molecule that can be further developed as a potent drug molecule for the treatment of Helicobacter pylori caused gastritis for further studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: the neoplastic B cells of the Helicobacter pylori -related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma proliferate in response to H. pylori , however, the nature of the H. pylori antigen responsible for proliferation is still unknown. The purpose of the study was to dissect whether CagY might be the H. pylori antigen able to drive B cell proliferation., Methods: the B cells and the clonal progeny of T cells from the gastric mucosa of five patients with MALT lymphoma were compared with those of T cell clones obtained from five H. pylori -infected patients with chronic gastritis. The T cell clones were assessed for their specificity to H. pylori CagY, cytokine profile and helper function for B cell proliferation., Results: 22 of 158 CD4 + (13.9%) gastric clones from MALT lymphoma and three of 179 CD4 + (1.7%) clones from chronic gastritis recognized CagY. CagY predominantly drives Interferon-gamma (IFN-γ) and Interleukin-17 (IL-17) secretion by gastric CD4 + T cells from H. pylori -infected patients with low-grade gastric MALT lymphoma. All MALT lymphoma-derived clones dose dependently increased their B cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1., Conclusion: the results obtained indicate that CagY drives both B cell proliferation and T cell activation in gastric MALT lymphomas.

Helicobacter pylori ( H. pylori ) is a pathogen that can remain in the stomach of an infected person for their entire life. As a result, this leads to the development of severe gastric diseases such as gastric cancer. In addition, current therapies have several problems including antibiotics resistance. Therefore, new practical options to eliminate this bacterium, and its induced affections, are required to avoid morbidity and mortality worldwide. One strategy in the search for new drugs is to detect compounds that inhibit a limiting step in a central metabolic pathway of the pathogen of interest. In this work, we tested 55 compounds to gain insights into their possible use as new inhibitory drugs of H. pylori glucose-6-phosphate dehydrogenase (HpG6PD) activity. The compounds YGC-1 ; MGD-1, MGD-2; TDA-1 ; and JMM-3 with their respective scaffold 1,3-thiazolidine-2,4-dione; 1H -benzimidazole; 1,3-benzoxazole, morpholine, and biphenylcarbonitrile showed the best inhibitory activity (IC 50 = 310, 465, 340, 204 and 304 μM, respectively). We then modeled the HpG6PD protein by homology modeling to conduct an in silico study of the chemical compounds and discovers its possible interactions with the HpG6PD enzyme. We found that compounds can be internalized at the NADP + catalytic binding site. Hence, they probably exert a competitive inhibitory effect with NADP + and a non-competitive or uncompetitive effect with G6P, that of the compounds binding far from the enzyme's active site. Based on these findings, the tested compounds inhibiting HpG6PD represent promising novel drug candidates against H. pylori .

Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase ( Hp MTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase ( Hp AFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S -adenosylhomocysteine. The k cat / K m value for AFL is 6.8 × 10 4 M -1 s -1 , 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for Hp AFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo- Hp AFLDA and with MTCF at the catalytic sites show a catalytic site Zn 2+ or Fe 2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp 3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that Hp AFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with Hp MTAN producing adenine and depurinated futalosine and Hp AFLDA producing futalosine. Inhibition of cellular Hp MTAN or Hp AFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes a crucial step in the biosynthesis of DNA and RNA, and it has been exploited as a promising target for antimicrobial therapy. The present study discusses the development and synthesis of a series of sulfonyl-α-l-amino acids coupled with the anisamide scaffold and evaluates their activities as anti-Helicobacter pylori and IMPDH inhibitors. Twenty derivatives were synthesized and their structures were established by high-resolution mass spectrometry and  1 H and 13 C nuclear magnetic resonance measurements. Four compounds (6, 10, 11, and 21) were found to be the most potent and selective molecules in the series with minimum inhibitory concentration (MIC) values <17 µM, which were selected to test their inhibitory activities against HpIMPDH and human (h)IMPDH2 enzymes. In all tests, amoxicillin and clarithromycin were used as reference drugs. Compounds 6 and 10 were found to have a promising activity against the HpIMPDH enzyme, with IC 50  = 2.42 and 2.56 µM, respectively. Moreover, the four compounds were found to be less active and safer against hIMPDH2 than the reference drugs, with IC 50  > 17.17 µM, which makes sure that their selectivity is toward HpIMPDH and reverse to that of amoxicillin and clarithromycin. Also, the synergistic antibacterial activity of compounds 6, 10, amoxicillin, and clarithromycin was investigated in vitro. The combination of amoxicillin/compound 6 (2:1 by weight) exhibited a significant antibacterial activity against H. pylori, with MIC = 0.12 µg/ml. The molecular docking study and ADMET analysis of the most active compounds were used to elucidate the mode-of-action mechanism., (© 2021 Deutsche Pharmazeutische Gesellschaft.)

The prevalence of peptic ulcer diseases has decreased over the past decades. The contribution of Helicobacter pylori to these changes has not been clearly delineated. Two cohorts of patients receiving esophagogastroduodenoscopy examination together with urease test were enrolled, 1 from year 2001 (n = 1030), the other from year 2019 (n = 600). The prevalence changes of peptic ulcer diseases as well as the associated clinical factors were analyzed. An independent cohort of gastric biopsy samples (n = 151) positive for H. pylori were retrieved for ureC gene genotype analysis. Comparison between the patients recruited from 2001 and 2019 revealed significant decrease in H. pylori infection (P < 0.001), duodenal ulcer prevalence (P < 0.001) and gastric ulcer prevalence (P < 0.001). Multivariate analysis showed that the decreases of these factors were independent (adjusted P < 0.001 for all). Intriguingly, in H. pylori positive patients, the prevalence of duodenal ulcer still decreased with year (P < 0.001), which was not found in gastric ulcer (P = 0.345). Genetic analysis of H. pylori urease gene showed that MboI-restriction fragment length polymorphism-defined genotype 3 UreC was significantly more prevalent in gastric ulcer patients than in others (P = 0.022). Independent decreases of H. pylori infection, gastric ulcer and duodenal ulcer over decades were found. In H. pylori positive patients, duodenal ulcer prevalence decreased overtime while gastric ulcer prevalence remained unchanged. Gastric ulcer/cancer had a higher prevalence of MboI-defined genotype 3 UreC gene., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3'-5' exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The 'prototypical' Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, which include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with K i values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 μM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[( N -3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.

The Helicobacter pylori HtrA protein (HtrA Hp ) is an important virulence factor involved in the infection process by proteolysis of components of the tight (claudin-8 and occludin) and adherens junctions (E-cadherin) between epithelial cells. As a protease and chaperone, HtrA Hp is involved in protein quality control, which is particularly important under stress conditions. HtrA Hp contains a protease domain and two C-terminal PDZ domains (PDZ1 and PDZ2). In the HtrA protein family, the PDZ domains are proposed to play important roles, including regulation of proteolytic activity. We therefore mutated the PDZ1 and PDZ2 domains in HtrA Hp and studied the maintenance of proteolytic activity, assembly and rearrangement of the corresponding oligomeric forms. Our in vitro experiments demonstrated that at least PDZ1 is important for efficient substrate cleavage , while both PDZ domains are dispensable for the chaperone-like activity. However, in living H. pylori cells, only the mutant containing at least PDZ1, but not PDZ2, ensured bacterial growth under stressful conditions. Moreover, we can demonstrate that PDZ1 is crucial for HtrA Hp oligomerization. Interestingly, all truncated proteolytically active HtrA Hp variants were functional in the in vitro infection assay and caused damage to the E-cadherin-based adherens junctions. These findings provide valuable new insights into the function of HtrA Hp in an important pathogen of humans., (© 2020 John Wiley & Sons Ltd.)

The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (-153ESEEKAWQKLCSL165-) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122-Trp159-Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57-Glu155-Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Aims: Helicobacter pylori (Hp) infection plays an important role in the development of gastric cancer. Hp can secrete gamma-glutamyltransferase (GGT), however, the impact of GGT of Hp on the human gastric cells is not clear. Although it has been demonstrated that ten-eleven translocation 1 (TET1) is overexpressed in gastric cancer, the relationship between the GGT of Hp and TET1 has not been studied. The aim of this study was to explore the relationship between GGT and TET1, and the role of TET1 in the development of gastric cancer induced by Hp was also explored., Materials and Methods: The correlation between TET1 and prognosis of gastric adenoma cancer was analyzed by bioinformatics. The GGT gene of Hp26695 was knocked out by electroporation with plasmid to construct the GGT knockout strain of Hp (Hp-KS-1). The shTET1 lentivirus transfected GES-1, MGC-803 and SGC-7901 cell lines were constructed. The biological characteristics of the three kind of cells were detected., Key Findings: TET1 was overexpressed in gastric tissues of Hp infected patients and mice. Bioinformatics analysis showed that in patients with gastric cancer, higher TET1 expression would result in poorer prognosis. The GGT gene of Hp can lead to overexpression of TET1 in GES-1, MGC-803 and SGC-7901 cells, along with the activation of Wnt/β-catenin signaling pathway, and then promoting tumorigenesis. After silencing TET1, the Wnt/β-catenin signaling pathway which was activated by GGT of Hp was inhibited., Significance: GGT of Helicobacter pylori can promote gastric carcinogenesis by activating Wnt/β-catenin signaling pathway trough up-regulating TET1., (Copyright © 2020. Published by Elsevier Inc.)

The main post-translational reversible modulation of proteins is phosphorylation and dephosphorylation, catalyzed by protein kinases (PKs) and protein phosphatases (PPs) which is crucial for homeostasis. Imbalance in this crosstalk can be related to diseases, including cancer. Plenty of evidence indicates that protein tyrosine phosphatases (PTPs) can act as tumor suppressors and tumor promoters. In gastric cancer (GC), there is a lack of understanding of the molecular aspects behind the tumoral onset and progression. Here we describe several members of the PTP family related to gastric carcinogenesis. We discuss the associated molecular mechanisms which support the down or up modulation of different PTPs. We emphasize the Helicobacter pylori ( H. pylori ) virulence which is in part associated with the activation of PTP receptors. We also explore the involvement of intracellular redox state in response to H. pylori infection. In addition, some PTP members are under influence by genetic mutations, epigenetics mechanisms, and miRNA modulation. The understanding of multiple aspects of PTPs in GC may provide new targets and perspectives on drug development., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Helicobacter pylori is the most infectious human pathogen that causes gastritis, peptic ulcers and stomach cancer. H. pylori DNA polymerase I (HpPol I) is found to be essential for the viability of H. pylori, but its intrinsic property and attribution to the H. pylori DNA replication remain unclear. HpPol I contains a 5'→3' exonuclease (5'-Exo) and DNA polymerase (Pol) domain, respectively, but lacks a 3'→5' exonuclease, or error proofreading activity. In this study, we characterized the 5'-Exo and Pol functions of HpPol I and found that HpPol I is a multifunctional protein displaying DNA nick translation, strand-displacement synthesis, RNase H-like, structure-specific endonuclease and exonuclease activities. In the in vitro DNA replication assay, we further demonstrated that the 5'-Exo and Pol domains of HpPol I can cooperate to fill in the DNA gap, remove the unwanted RNA primer from a RNA/DNA hybrid and create a ligatable nick for the DNA ligase A of H. pylori to restore the normal duplex DNA. Altogether, our study suggests that the two catalytic domains of HpPol I may synergistically play an important role in the maturation of Okazaki fragments during the lagging-strand DNA synthesis in H. pylori. Like the functions of DNA polymerase I in Escherichia coli, HpPol I may involve in both DNA replication and repair in H. pylori., (© 2020 Federation of European Biochemical Societies.)