Targeting the Essential Transcription Factor HP1043 of Helicobacter pylori: A Drug Repositioning Study.

Antibiotic-resistant bacterial pathogens are a very challenging problem nowadays. Helicobacter pylori is one of the most widespread and successful human pathogens since it colonizes half of the world population causing chronic and atrophic gastritis, peptic ulcer, mucosa-associated lymphoid tissue-lymphoma, and even gastric adenocarcinoma. Moreover, it displays resistance to numerous antibiotics. One of the H. pylori pivotal transcription factors, HP1043, plays a fundamental role in regulating essential cellular processes. Like other bacterial transcription factors, HP1043 does not display a eukaryote homolog. These characteristics make HP1043 a promising candidate to develop novel antibacterial strategies. Drug repositioning is a relatively recent strategy employed in drug development; testing approved drugs on new targets considerably reduces the time and cost of this process. The combined computational and in vitro approach further reduces the number of compounds to be tested in vivo. Our aim was to identify a subset of known drugs able to prevent HP1043 binding to DNA promoters. This result was reached through evaluation by molecular docking the binding capacity of about 14,350 molecules on the HP1043 dimer in both conformations, bound and unbound to the DNA. Employing an ad hoc pipeline including MMGBSA molecular dynamics, a selection of seven drugs was obtained. These were tested in vitro by electrophoretic mobility shift assay to evaluate the HP1043-DNA interaction. Among these, three returned promising results showing an appreciable reduction of the DNA-binding activity of HP1043. Overall, we applied a computational methodology coupled with experimental validation of the results to screen a large number of known drugs on one of the H. pylori essential transcription factors. This methodology allowed a rapid reduction of the number of drugs to be tested, and the drug repositioning approach considerably reduced the drug design costs. Identified drugs do not belong to the same pharmaceutical category and, by computational analysis, bound different cavities, but all display a reduction of HP1043 binding activity on the DNA.

Definition of the Binding Architecture to a Target Promoter of HP1043, the Essential Master Regulator of Helicobacter pylori.

HP1043 is an essential orphan response regulator of Helicobacter pylori orchestrating multiple crucial cellular processes. Classified as a member of the OmpR/PhoB family of two-component systems, HP1043 exhibits a highly degenerate receiver domain and evolved to function independently of phosphorylation. Here, we investigated the HP1043 binding mode to a target sequence in the hp1227 promoter (Php1227). Scanning mutagenesis of HP1043 DNA-binding domain and consensus sequence led to the identification of residues relevant for the interaction of the protein with a target DNA. These determinants were used as restraints to guide a data-driven protein-DNA docking. Results suggested that, differently from most other response regulators of the same family, HP1043 binds in a head-to-head conformation to the Php1227 target promoter. HP1043 interacts with DNA largely through charged residues and contacts with both major and minor grooves of the DNA are required for a stable binding. Computational alanine scanning on molecular dynamics trajectory was performed to corroborate our findings. Additionally, in vitro transcription assays confirmed that HP1043 positively stimulates the activity of RNA polymerase.

SrnR from Streptomyces griseus is a nickel-binding transcriptional activator.

Nickel ions are crucial components for the catalysis of biological reactions in prokaryotic organisms. As an uncontrolled nickel trafficking is toxic for living organisms, nickel-dependent bacteria have developed tightly regulated strategies to maintain the correct intracellular metal ion quota. These mechanisms require transcriptional regulator proteins that respond to nickel concentration, activating or repressing the expression of specific proteins related to Ni(II) metabolism. In Streptomyces griseus, a Gram-positive bacterium used for antibiotic production, SgSrnR and SgSrnQ regulate the nickel-dependent antagonistic expression of two superoxide dismutase (SOD) enzymes, a Ni-SOD and a FeZn-SOD. According to a previously proposed model, SgSrnR and SgSrnQ form a protein complex in which SgSrnR works as repressor, binding directly to the promoter of the gene coding for FeZn-SOD, while SgSrnQ is the Ni(II)-dependent co-repressor. The present work focuses on the determination of the biophysical and functional properties of SgSrnR. The protein was heterologously expressed and purified from Escherichia coli. The structural and metal-binding analysis, carried out by circular dichroism, light scattering, fluorescence and isothermal titration calorimetry, showed that the protein is a well-structured homodimer, able to bind nickel with moderate affinity. DNase I footprinting and ß-galactosidase gene reporter assays revealed that apo-SgSrnR is able to bind its DNA operator and activates a transcriptional response. The structural and functional properties of this protein are discussed relatively to its role as a Ni(II)-dependent sensor.