Thymidine kinase inhibitors(Northern Arizona University)

The rise of multidrug-resistant Staphylococcus aureus (S. aureus) has escalated into a public health crisis, underscoring the urgent need for alternative antimicrobial strategies. This study identifies the natural flavonoid Morin hydrate as a potent anti-staphylococcal agent that targets thymidine kinase (TK), an essential bacterial enzyme involved in DNA synthesis. Through a combination of in silico and biophysical approaches, Morin hydrate was shown to bind with high affinity within the ATP-binding pocket of TK, forming stable hydrogen bonds that competitively inhibit ATPase activity. Fluorescence quenching and isothermal titration calorimetry confirmed strong binding, while molecular dynamics simulations indicated minimal conformational changes upon complex formation, suggesting significant structural stabilization. In vitro antimicrobial assays demonstrated a minimum inhibitory concentration (MIC) of 100 μM and a minimum bactericidal concentration of 200 μM against S. aureus ATCC 29213, with time-kill kinetics confirming bactericidal activity at concentrations ≥200 μM. Most notably, in an ex vivo macrophage infection model, Morin hydrate effectively eradicated intracellular S. aureus. A concentration of 250 μM completely cleared bacterial loads within 24 h, matching the efficacy of ciprofloxacin. Lower concentrations also showed significant bacteriostatic and bactericidal effects. These findings establish Morin hydrate as a promising lead compound that not only inhibits TK function but also penetrates eukaryotic cells to eliminate protected bacterial reservoirs. Our findings establish Morin hydrate as a promising lead that could potentially inhibit TK function and penetrate eukaryotic cells to eliminate protected bacterial reservoirs. Its potent, dose-dependent antibacterial activity suggests its potential for future anti-staphylococcal drug development.