LAUSR

The emergence of multiple drug‐resistant “super gonorrhoea” complicates the management and treatment of Neisseria gonorrhoeae infections due to the progressive accumulation of mutations in the biological targets of frontline antimicrobials. Continuous evaluation and reporting of newer molecular targets and their inhibitors are necessary. Here, we present l‐asparaginase of N. gonorrhoeae (NgA) as a new molecular target based on structure‐based high‐throughput screening, molecular dynamics(MD) simulations, and validation by biophysical, biochemical, and cell viability assays. We observed that the NgA is evolutionarily conserved in both the drug‐resistant and susceptible strains of N. gonorrhoeae, indicating its importance in the growth and survival of the pathogen. Three Food and Drug Administration‐approved drugs, pemirolast, thalidomide, and decitabine, were identified as potential inhibitors of NgA using high‐throughput screening. The binding energies of the drugs with NgA were −20.14, −19.67, and −16.47 kcal/mol, respectively, compared to −6.82 ± 1.46 for enzyme–substrate l‐Asn, as obtained through MD simulations. Subsequently, fluorescence quenching and differential scanning calorimetry experiments validated the in silico data. The observance of inhibition of NgA activity at micromolar drug concentrations further strengthened our findings. Conclusive evidence came from the cell viability assays where these drugs were found to impede the growth of N. gonorrhoeae culture effectively. Thus, our study establishes l‐asparaginase as a new molecular target against gonococcal infections. From this study, we propose that targeting of NgA can be explored to control N. gonorrhoeae infections in combination therapy.