Pseudouridimycin—A Potent Nucleoside Inhibitor of the RNA Polymerase Beta Prime Subunit of Streptococcus pyogenes

[Image: see text] Streptococcus pyogenes (group A streptococcus, GAS), a Gram-positive bacterium, is a major cause of mild to severe life-threatening infections. Antibacterial resistance to penicillin and macrolides poses a major threat in the treatment of GAS and necessitates alternate drugs and newer antibiotics. In this direction, nucleotide-analog inhibitors (NIAs) have emerged as important antiviral, antibacterial, and antifungal agents. Pseudouridimycin (PUM), a nucleoside analogue inhibitor discovered from the soil bacterium Streptomyces sp., has proven to be effective against multidrug-resistant S. pyogenes. However, the mechanism of its activity remains elusive. In this study, subunits of the RNA polymerase of GAS have been identified as targets for PUM inhibition and the binding regions have been mapped to the N-terminal domain of the β′ subunit, using computational methods. The antibacterial activity of PUM against macrolide-resistant GAS was evaluated. PUM showed effective inhibition at 0.1–1 μg/mL concentration, which was higher when compared to earlier reports. The molecular interaction between PUM and the RNA polymerase β′-N terminal subunit was investigated using isothermal titration calorimetry (ITC), circular dichorism (CD), and intrinsic fluorescence spectroscopy. The thermodynamic characterization by ITC showed an affinity constant of 6.175 × 10(5) M(–1) denoting a moderate affinity. Fluorescence studies revealed that the interaction of protein-PUM was spontaneous in nature and follows a static quenching of tyrosine signals from the protein. The near- and far-UV CD spectral analysis concluded that PUM induced local tertiary structural changes in the protein, predominantly contributed by aromatic amino acids rather than notable changes in the secondary structure. Hence PUM could be a promising lead drug target for macrolide-resistant strains of S. pyogenes and enable eradication of pathogen in the host system.