A computational perspective on the dynamic behaviour of recurrent drug resistance mutations in the pncA gene from Mycobacterium tuberculosis

Tuberculosis is still one of the top 10 causes of death worldwide, particularly with the emergence of multidrug-resistant tuberculosis. As the most effective first-line anti-tuberculosis drug, pyrazinamide also develops resistance due to the mutation in the pncA gene. Among these mutations, seven mutations at positions F94L, F94S, K96N, K96R, G97C, G97D, and G97S are classified as high-level resistance mutations. However, the resistance mechanism of Mtb to PZA (pyrazinamide) caused by these mutations is still unclear. In this work, we combined molecular dynamics simulation, molecular mechanics/generalized-Born surface area calculation, principal component analysis, and free energy landscape analysis to explore the resistance mechanism of Mtb to PZA due to F94L, F94S, K96N, K96R, G97C, G97D, and G97S mutations, as well as compare interaction changes in wild-type and mutant PZA-bound complexes. The results of molecular mechanics/generalized-Born surface area calculations indicated that the binding free energy of PZA with seven mutants decreased. In mutant systems, the most significant interactions with His137 and Cys138 were lost. Besides, PCA and FEL confirmed significant variations in the protein dynamics during the simulation specifically by altering the Fe²⁺ binding and its destabilization. Furthermore, mutants also flipped the β-sheet 2, which also affects the binding of Fe²⁺ and PZA. In the G97D mutant, reported as most lethal, mutation causes the binding pocket to change considerably, so that the position of PZA has a large movement in the binding pocket. In this study, the resistance mechanism of PZA at the atomic level is proposed. The proposed drug-resistance mechanism will provide valuable guidance for the design of anti-tuberculosis drugs.