Molecular investigation against the resistant mechanism of PncA mutated pyrazinamide resistance and insight into the role of pH environment for pyrazinamide activation

: Pyrazinamide (PZA), a crucial component of anti-TB therapy, is a prodrug. PZA interacts with PncA protein to be converted into its functional form i.e. pyrazinoic acid (POA). It has unique feature to kill dormant tubercle bacilli of acidic environment. Although, significance of pH environment in PZA activation has been investigated in several of previous studies, insight into the significant atomistic variations in the interaction pattern of PZA with PncA, at different pH environments, are still required to be explored. On the other hand, continuously emerging PncA mutants, associated with PZA resistance, have also become a serious threat for global TB control program. Therefore, the current study was designed to understand the role of pH environment in the PZA activation and to explore the PZA resistance mechanism in various PncA mutants. The study included various In silico experiments like molecular docking, MD simulation, binding free energy estimation, PCA and FEL. In our study, we have found pH-3 and pH-5 environment as a highly significant environment for PZA activation. It was found that protonation or deprotonation of PZA activation site (PAS) residues, majorly K48, D56, K96 and E107, resulted in rearrangement of the PAS according to the pH conditions. It has also been observed that positioning of PZA binding near to Fe2+ and residues of catalytic triad (i.e. D8, K96 and C138) also play a very crucial role in the activation of PZA. The overall insight from the current study may help to develop new therapeutics against PncA mutated PZA resistance.