Merging multi-omics with proteome integral solubility alteration unveils antibiotic mode of action.

Antimicrobial resistance is responsible for an alarming number of deaths, estimated at 5 million per year. To combat priority pathogens, like Helicobacter pylori, the development of novel therapies is of utmost importance. Understanding the molecular alterations induced by medications is critical for the design of multi-targeting treatments capable of eradicating the infection and mitigating its pathogenicity. However, the application of bulk omics approaches for unraveling drug molecular mechanisms of action is limited by their inability to discriminate between target-specific modifications and off-target effects. This study introduces a multi-omics method to overcome the existing limitation. For the first time, the Proteome Integral Solubility Alteration (PISA) assay is utilized in bacteria in the PISA-Express format to link proteome solubility with different and potentially immediate responses to drug treatment, enabling us the resolution to understand target-specific modifications and off-target effects. This study introduces a comprehensive method for understanding drug mechanisms and optimizing the development of multi-targeting antimicrobial therapies.

L-Thyroxine and L-thyroxine-based antimicrobials against Streptococcus pneumoniae and other Gram-positive bacteria.

Objectives: The rise of antibiotic-resistant Streptococcus pneumoniae (Sp) poses a significant global health threat, urging the quest for novel antimicrobial solutions. We have discovered that the human hormone l-thyroxine has antibacterial properties. In order to explore its drugability we perform here the characterization of a series of l-thyroxine analogues and describe the structural determinants influencing their antibacterial efficacy. Method: We performed a high-throughput screening of a library of compounds approved for use in humans, complemented with ITC assays on purified Sp-flavodoxin, to pinpoint molecules binding to this protein. Antimicrobial in vitro susceptibility assays of the hit compound (l-thyroxine) as well as of 13 l-thyroxine analogues were done against a panel of Gram-positive and Gram-negative bacteria. Toxicity of compounds on HepG2 cells was also assessed. A combined structure-activity and computational docking analysis was carried out to uncover functional groups crucial for the antimicrobial potency of these compounds. Results: Human l-thyroxine binds to Sp-flavodoxin, forming a 1:1 complex of low micromolar Kd. While l-thyroxine specifically inhibited Sp growth, some derivatives displayed activity against other Gram-positive bacteria like Staphylococcus aureus and Enterococcus faecalis, while remaining inactive against Gram-negative pathogens. Neither l-thyroxine nor some selected derivatives exhibited toxicity to HepG2 cells. Conclusions: l-thyroxine derivatives targeting bacterial flavodoxins represent a new and promising class of antimicrobials.

Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy, Synergy, Resistance and Mechanistic Studies.

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.

Enhanced photocatalytic activity, transport properties and electronic structure of Mn doped GdFeO3 synthesized using the sol-gel process.

In this work we have synthesized Mn doped GdFeO3 nano-particles using a green and facile sol gel method and studied their photocatalytic, optical, vibrational and electrical properties. The Rietveld refinement of the XRD profiles suggests that all the materials have an orthorhombic Pbnm crystal structure. The transmission electron microscope (TEM) images show the decrease of the average particle size from 140 to 80 nm with the Mn concentration. The high crystallinity of the synthesized particles is confirmed from the HR-TEM images. Raman spectrum is employed to investigate the phonon modes of the materials. The optical band gap of the materials is obtained from the UV-vis reflectance spectroscopy (DRS) using Tauc relation which indicates the reduction of the band gap from 2.18 to 1.72 eV with Mn-doping. The photocatalytic activity of the materials is studied by the photocatalytic degradation of rhodamine B (Rh-B) in aqueous solution under visible light illumination. The substitution of Mn at the Fe site introduces an extra electronic state between the conduction band and the valence band which reduces the electronic band gap and enhances the Rh-B degradation efficiency. A 30% Mn doping at the Fe site (GFMO3) provides an optimum space charge width which assists to attain the maximum rate of degradation of the Rh-B dye. The doping of Mn3+ reduces the photogenerated electron and hole recombination rate and hence more charge carriers take part in the redox reaction which facilitates the photo-catalytic efficiency in GFMO3. The degradation rate enhances by a factor of 2.5 for GFMO3 as compared to pure GdFeO3. The highest photocurrent density of 1.31 µA cm-2 of GFMO3 with respect to other materials promotes the separation and transfer of the photo generated charge carriers. The possible photocatalytic mechanism of the Mn doped GdFeO3 is also critically discussed. Alternating current impedance spectroscopy is used to study the electrical properties of the synthesized materials. The increase in the conductivity with the Mn concentration is explained on the basis of the band gap reduction and this is consistent with the Smit and Wijn theory. Magnetic measurement is performed to measure the magnetization strength which is useful to separate the photocatalyst by simply using a magnet. The temperature dependent magnetization measurement suggests the anti-ferromagnetic (AFM) behaviour of the studied materials with the decrease of Néel temperature (TN) with Mn concentration. The XPS study reveals the presence of multiple oxidation states of Fe(2+/3+) and Mn(4+/3+) in these materials which facilitates the conductivity as well as the oxidation/reduction efficiency at the surface of the catalyst. The band gap reduction and its effect on the enhancement of the photocatalytic degradation efficiency with Mn doping are also discussed from the density of states calculations. Thus, this study describes a promising approach for the organic pollutant degradation by designing an efficient and stable perovskite photocatalyst.

Gold nanoparticle-assisted enhancement in the anti-cancer properties of theaflavin against human ovarian cancer cells.

Redox-active quinones have been reported to show good potential for biological activities, while efforts are directed to explore the usefulness of these materials further in cancer management. Our previous study demonstrated that theaflavin and theaflavin-gallates (tea-extracted polyphenols) selectively induce apoptosis of tumour cells in vitro, but its concentration for showing half-maximal therapeutic response remains a matter of concern. In this report, we demonstrated that if theaflavin is conjugated with gold nanoparticles (AuNPs) to form a nanoconjugate AuNP@TfQ, its apoptotic ability increases significantly in comparison to the bare theaflavin (Tf). The nanoconjugate is prepared by following a one-step green synthesis ̶ a reaction between HAuCl4 and the aflavin at room temperature. AuNP@TfQ is characterized using particle size analysis, FESEM, UV-vis, FTIR, fluorescence, and X-ray photoelectron spectroscopytechniques. We assume that the enhanced anti-cancer effect of AuNP@TfQ appears due to the facile oxidation of the pristine theaflavin to its quinone derivative on the surface of AuNPs. The presence of quinone motif in AuNP@TfQ induces an increased level of ROS generation probably through the depolarization of mitochondria and resulted in the caspase-mediated apoptotic cell death which may hold the potential for a "magic bullet"-mediated ovarian cancer treatment.