Systematic identification and repurposing of FDA-approved drugs as antibacterial agents against Streptococcus
Judith Gracia1, Damodharan Perumal2, Prabu Dhandapani2
1Centre for Advanced Studies in Crystallography and Biophysics, University of Madras, Guindy, India.
Abstract:
Streptococcus pyogenes (Group A Streptococcus - GAS) is a human pathogen causing wide range of infections and toxin-mediated diseases in human beings of all age groups with fatality of 10-30 %. The limited success of antibiotics and the non-availability of vaccines makes GAS a global burden. The multi-subunit RNA polymerase (RNAP) is a validated bacterial therapeutic target as it is involved in transcription and can arrest growth. Of the five subunits of this enzyme complex, the β-subunit (RpoC) has attracted specific attention as a drug target, particularly in the switch region. Here we attempt to repurpose non-antimicrobial drugs to act as RpoC inhibitors against S. pyogenes. In this study, 1826 FDA approved drugs have been identified through high-throughput virtual screening. Free Energy Perturbation (FEP) based binding free energy calculations have been performed at the final step of the virtual screening funnel to ensure high accuracy in silico results. Three compounds identified have been tested for susceptibility of S. pyogenes MTCC 442 strain and two antibiotic-resistant clinical isolates of S. pyogenes using microdilution assay. Among the three, two drugs Amlodipine Besylate (Amd) and Ranitidine hydrochloride (Rnt) have shown inhibition against all the tested strains and its mechanism of interaction with RpoC has been studied. The docked complexes were analyzed to understand the binding mode of the drugs to the target. Classical Molecular Dynamics studies for RpoC-Rnt complex and the two stable conformations of RpoC-Amd complex was carried out. Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration (RoG) and Solvent Accessible Surface Area (SASA) of the complexes were plotted and studied. The thermodynamic parameters of protein-drug were experimentally determined using Isothermal Titration Calorimetry (ITC). Infrared spectroscopic studies and Fluorescence quenching studies provided insights into the secondary structural changes in RpoC on binding to the drugs.
Insights
Repurposing non-antibiotic drugs like Amlodipine Besylate and Ranitidine hydrochloride shows promise in inhibiting Streptococcus pyogenes by targeting its RNA polymerase RpoC subunit. This offers a potential new strategy against this dangerous pathogen.
Area of Science:
- Microbiology and Infectious Diseases
- Drug Discovery and Development
- Computational Chemistry
Background:
- Streptococcus pyogenes (Group A Streptococcus - GAS) causes severe infections with high fatality rates (10-30%) and limited treatment options due to antibiotic resistance and lack of vaccines.
- The bacterial RNA polymerase (RNAP) is a crucial target for antimicrobial therapies, with the β-subunit (RpoC) being a specific focus for drug development.
- Repurposing existing non-antimicrobial drugs presents a viable strategy to identify novel therapeutic agents against GAS.
Purpose of the Study:
- To identify and validate non-antimicrobial drugs that can inhibit the RpoC subunit of Streptococcus pyogenes RNA polymerase.
- To investigate the binding mechanisms and interactions of potential drug candidates with the RpoC target.
- To assess the efficacy of identified drugs against GAS strains, including antibiotic-resistant isolates.
Main Methods:
- High-throughput virtual screening of 1826 FDA-approved drugs to identify potential RpoC inhibitors.
- Free Energy Perturbation (FEP) calculations for accurate in silico binding free energy estimation.
- In vitro susceptibility testing using microdilution assays against GAS strains.
- Molecular Dynamics (MD) simulations, Isothermal Titration Calorimetry (ITC), Infrared spectroscopy, and Fluorescence quenching studies to elucidate drug-target interactions and structural changes.
Main Results:
- Two FDA-approved drugs, Amlodipine Besylate (Amd) and Ranitidine hydrochloride (Rnt), demonstrated significant inhibition against all tested S. pyogenes strains, including antibiotic-resistant isolates.
- Detailed analysis of docked complexes, MD simulations, and thermodynamic studies elucidated the binding modes and interactions of Amd and Rnt with the RpoC subunit.
- Spectroscopic studies confirmed drug-induced secondary structural changes in RpoC upon binding.
Conclusions:
- Amlodipine Besylate and Ranitidine hydrochloride are identified as promising non-antimicrobial drug candidates for repurposing against Streptococcus pyogenes infections.
- Targeting the RpoC subunit of bacterial RNA polymerase with these repurposed drugs offers a novel therapeutic avenue.
- Further investigation into these compounds could lead to new treatment strategies for GAS infections, addressing the challenge of antibiotic resistance.
