s Computational Insight into the Binding Mechanism of Pyrazinoic Acid to RpsA Protein

Background: Resistance to the critical first line anti-tubercular drug, Pyrazinamide, is a significant obstacle to achieving the global end to tuberculosis targets. Approximately 50% of multidrug- resistant tuberculosis and over 90% of extensively drug-resistant tuberculosis strains are also Pyrazinamide resistant. Pyrazinamide is a pro-drug that reduces the duration of tuberculosis therapy time by 9-12 months, while used as an anti-biotic in the 1st- & 2nd-line tuberculosis treatment regimens. Pyrazinamidase is an enzyme encoded by pncA gene, which is responsible for the amide hydrolysis of pyrazinamide into active pyrazinoic acid. Pyrazinoic acid can inhibit trans-translation by binding to ribosomal protein S1 and competing with tmRNA, the natural cofactor of ribosomal protein S1. Although pncA mutations have been commonly associated with pyrazinamide resistance, a small number of resistance cases have been associated with mutations in ribosomal protein S1. Ribosomal protein S1was recently identified as a possible target of pyrazinamide based on its binding activity to pyrazinoic acid and the capacity to inhibit trans-translation. Objective: Despite the critical role played by pyrazinamide, its mechanisms of action are not yet fully understood. Therefore, this study is an effort to explore the resistance mechanism toward pyrazinamide drug in Mycobacterium (M.) tuberculosis. Methods: An extensive molecular dynamics simulation was performed using the AMBER software package. We mutated residues of the binding site (i.e., F307A, F310A, and R357A) in the RpsA S1 domain to address the drug-resistant mechanism of RpsA in complex that might be responsible for pyrazinamide resistance. Moreover, it is challenging to collect the drug mutant to combine the complex of a protein by single- crystal X-ray diffraction. Thus, the total three structures were prepared by inducing mutations in the wild-type protein using PyMol. Results: The dynamic results revealed that a mutation in the binding pocket produced pyrazinamide resistance due to the specificity of these residues in binding pockets, which resulted in the scarcity of hydrophobic and hydrogen bonding interaction with pyrazinoic acid, increasing the CAdistance between the binding pocket residues as compared to wild type RpsA that led to structural instability. Conclusion: The overall dynamic results will provide useful information behind the drug resistance mechanism to manage tuberculosis and also helps in better management for future drug resistance.