Targeting the G-quadruplex as a novel strategy for developing antibiotics against hypervirulent drug-resistant Staphylococcus aureus

Background: The rapid emergence of multiple drug-resistant (MDR) Bacterial pathogens and the lack of a novel Antibiotic pipeline pose a serious threat to global healthcare. The limited number of established targets further restricts the identification of novel Antibiotics to treat life-threatening MDR infections caused by Staphylococcus aureus strains. Therefore, novel targets for developing Antibiotics are urgently required. In this study, we hypothesized that the G-quadruplex (G4)-binding ligands can be used as novel Antibiotics as their binding can possibly downregulate/block the expression of vital genes.

Methods: To test this, first we screened the Antibiotic properties of representative G4-binding ligands against hypervirulent and MDR S. aureus USA300 and determined the in vitro and in vivo Antibacterial activity; and proposed the mechanism of action by applying various microbiological, Infection, microscopic, and biophysicochemical techniques.

Results: Herein, among screened G4-binding ligands, N-methyl mesoporphyrin IX (NMM) showed the highest Antibacterial activity against S. aureus USA300. NMM exhibited a minimum inhibitory concentration (MIC) of 5 μM against S. aureus USA300, impacting cell division and the cell wall by repressing the expressions of genes in the division cell wall (dcw) gene cluster. Genome-wide bioinformatics analysis of G4 motifs and their mapping on S. aureus genome, identified the presence of G4-motif in the promoter of mraZ, a conserved master regulator of the dcw cluster regulating the coordinated cell division and cell wall synthesis. Physicochemical assessments using UV-visible, circular dichroism, and nuclear magnetic resonance spectroscopy confirmed that the G4-motif present in the mraZ promoter formed an intramolecular parallel G4 structure, interacting with NMM. In vivo reporter followed by coupled in vitro transcription/translation (IVT) assays confirmed the role of mraZ G4 as a target interacting NMM to impose extreme Antibacterial activity against both the gram-positive and -negative bacteria. In-cell and in vivo validation of NMM using RAW264.7 cells and Galleria mellonella; respectively, demonstrated that NMM exhibited superior Antibiotic activity compared to well-established Antibiotics, with no observed cytotoxicity.

Conclusions: In summary, the current study identified NMM as a broad-spectrum potent Antibacterial agent and elucidated its plausible mechanism of action primarily by targeting G4-motif in the mraZ promoter of the dcw gene cluster.

S. aureus USA300; dcw cluster; mraZ; Division cell wall; G-quadruplex; G4 ligands; N-methyl mesoporphyrin IX (NMM).