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Comparative Studies to Uncover Further Mechanisms of Action of N-(1,3,4-oxadiazol-2-yl)benzamide Containing Antibacterial Agents

ABSTRACT: Drug-resistant bacterial pathogens still cause high levels mortality annually despite the availability of many antibiotics. Staphylococcus aureus (MRSA) is especially problematic and the rise in resistance to front line treatments like vancomycin and linezolid calls for new chemical modalities to treat chronic and relapsing S, aureus infections. Halogenated N-(1,3,4-oxadiazol-2-yl)benzamides are interesting class of antimicrobial agents, which have been described by multiple groups to be effective against different bacterial pathogens. The modes of action of a few N-(1,3,4-oxadiazol-2-yl)benzamides have been elucidated. For example, oxadiazoles KKL-35 and MBX-4132, have been described as inhibitors of trans-translation (a ribosome rescue pathway) while HSGN-94 was shown to inhibit lipoteichoic acid. However other similarly halogenated N-(1,3,4-oxadiazol-2-yl)benzamides neither inhibit trans-translation nor LTA but are potent antimicrobial agents. For example, HSGN-220, -218, and -144 are N-(1,3,4-oxadiazol-2-yl)benzamides that are modified with OCF3, SCF3 or SF5, and have remarkable minimum inhibitory concentrations (MICs) ranging from 1 to 0.06 microgram per mL against MRSA clinical isolates and show a low propensity to resistance to MRSA over 30 days. The mechanism of action (MOA) of these highly potent oxadiazoles is however unknown. To provide insights into how these halogenated N-(1,3,4-oxadiazol-2-yl)benzamides inhibit bacterial growth, we performed global proteomics and RNA expression analysis of some essential genes of S. aureus treated with HSGN-220, -218, and -144. These studies revealed that the oxadiazoles HSGN-220, -218, and -144 are multi-action antibiotics that regulate menaquinone biosynthesis and other essential proteins like DnaX, Pol IIIC, BirA, LexA, and DnaC. In addition, these halogenated N-(1,3,4-oxadiazol-2-yl)benzamides were able to depolarize bacterial membranes and regulate siderophore biosynthesis and heme regulation. Iron starvation appears to be part of the MOA that led to bacterial killing. This study demonstrates that N-(1,3,4-oxadiazol-2-yl)benzamides are indeed privileged scaffolds for the development of antibacterial agents and that subtle modifications lead to changes to MOA.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Staphylococcus Aureus Subsp. Aureus (ncbitaxon:46170)

SUBMITTER: Dr. Herman O. Sintim

PROVIDER: MSV000088343 | MassIVE | Wed Nov 10 10:09:00 GMT 2021

REPOSITORIES: MassIVE

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Project description:The rapid emergence of drug resistant Staphylococcus aureus (S. aureus), in particular, methicillin-resistant S. aureus (MRSA) poses a serious threat to public health globally. Reuse of metal-based antimicrobials stands for one of the most promising strategies to resensitize drug resistant bacteria to conventional antibiotics. Silver (Ag) has been used as an antimicrobial agent since antiquity, yet its molecular mechanism of action is elusive largely owing to technical challenges to identify the direct silver-binding protein targets accountable for its action. Herein, using an in-house developed hyphenated technique LC-GE-ICP-MS, we successfully separated and identified 38 authentic Ag+-binding proteins (Ag+-proteome) in S. aureus at the whole-cell scale for the first time. By integration with bioinformatic analysis and systematic biochemical characterizations, we captured the first snapshot on the dynamic action of Ag+ against S. aureus at the molecular level, i.e., Ag+ primarily targets glycolysis via inhibiting multiple enzymes and induces the elevation of ROS through functional disruption of redox homeostasis system at the late stage, leading to an upregulation of oxidative pentose phosphate pathway (oxPPP) to alleviate Ag+ stress. However, the activation of oxPPP is ultimately futile due to key oxPPP enzymes being inhibited by Ag+. We further validated that Ag+ could inhibit 6-phosphogluconate dehydrogenase, a key target from oxPPP through binding to His185 at the active site and morphing the shape of catalytic pocket by X-ray crystallography. Significantly, the multi-target mode of action of silver is accountable for its suppression of antibiotic selection effects towards S. aureus as well as its sustainable antimicrobial activity. Such a unique mode of action of Ag+ (and silver nanoparticles) led to enhanced efficacy of a broad range of antibiotics and resensitization of MRSA to antibiotics. Our study resolves the long-standing question of the molecular targets of silver in S. aureus and offers a novel insight into the sustainable bacterial susceptibility of silver, providing a potential approach for combating antimicrobial resistance.

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Comparative Studies to Uncover Further Mechanisms of Action of N-(1,3,4-oxadiazol-2-yl)benzamide Containing Antibacterial Agents

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