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AbstractPyrazinamide (PZA) is an important component of first-line anti-tuberculosis (anti-TB) drugs. The anti-TB agent is activated into an active form, pyrazinoic acid (POA), by Mycobacterium tuberculosis(MTB) pncAgene encoding pyrazinamidase (PZase). The major cause of PZA-resistance has been associated with mutations in the pncAgene. We have detected several novel mutations including V131F, Q141P, R154T, A170P, and V180F (GeneBank Accession No. MH461111) in the pncAgene of PZA-resistant isolates during PZA drug susceptibility testing followed by pncAgene sequencing. Here, we investigated molecular mechanism of PZA-resistance by comparing the results of experimental and molecular dynamics. The mutants (MTs) and wild type (WT) PZase structures in apo and complex with PZA were subjected to molecular dynamic simulations (MD) at the 40 ns. Multiple factors, including root mean square deviations (RMSD), binding pocket, total energy, dynamic cross correlation, and root mean square fluctuations (RMSF) of MTs and WT were compared. The MTs attained a high deviation and fluctuation compared to WT. Binding pocket volumes of the MTs, were found, lower than the WT, and the docking scores were high than WT while shape complementarity scores were lower than that of the WT. Residual motion in MTs are seemed to be dominant in anti-correlated motion. Mutations at locations, V131F, Q141P, R154T, A170P, and V180F, might be involved in the structural changes, possibly affecting the catalytic property of PZase to convert PZA into POA. Our study provides useful information that will enhance the understanding for better management of TB.AbbreviationsDSTdrug susceptibility testingΔelecelectrostatic energyLJLowenstein–Jensen mediumMGITmycobacterium growth indicator tubesMTsmutantsMDmolecular dynamic simulationsMTBMycobacterium tuberculosisNALC–NaOHN-acetyl-l-cysteine–sodium hydroxideNIHNational Institutes of HealthNPTamount of substance (N), pressure (P) temperature (T)NVTmoles (N), volume (V) temperature (T)PZasepyrazinamidaseΔpspolar solvation energyPTRLProvincial Tuberculosis Reference LaboratoryRMSDroot mean square deviationsRMSFroot mean square fluctuationsΔSASAsolvent accessible surface area energyTBtuberculosisGTotaltotal binding free energyΔvdWVan der Waals energyWTwild typeCommunicated by Ramaswamy H. Sarma

Pyrazinamide (PZA) is an essential first line antitubercular drug, which plays a crucial role in tuberculosis treatment. The PZA, which is considered as a pro-drug needs an enzyme of mycobacterial pyrazinamidase (PZase) for its conversion into an active form pyrazinoic acid. Further, this active form of PZA inhibits the ribosomal proteins S1, which facilitates the transfer-mRNA complex formation throughout the translation. The spontaneous mutations in RpsA have been found to be associated with PZA drug resistance. However, the drug resistance mechanism is still unclear. Furthermore, there is no such information available about the structural dynamics of RpsA protein because of mutations that confer Pyrazinoic acid resistance. Moreover, a total of 18 clinical PZA-resistant isolates were investigated and found to be pncAWT, which allowed exploration of the resistance mechanism of RpsA in the mutated state. Samples were repeated for the drug susceptibility testing followed by RpsA gene sequencing. A total of 11 clinical isolates harbored a total of 15 mutations. Almost half of the total strains (7/15) were observed to be in the conserved region of RpsA and known as Mycobacterium tuberculosisC-terminal domain. In the current study, (2/7) mutation T370P (mutant 1) and W403G (mutant 2) were explored to ensure the RpsA resistance mechanism through essential dynamics simulation. The essential dynamics study results revealed that the distal loop mutations drastically altered the conformation of RpsA both in the absence (−) and presence (+) of pyrazinoic acid drug for two reasons: (1) dramatic alteration or reduction in the binding pattern of pyrazinoic acid with active site residues observed and (2) a clear image of the opening and closing switching mechanism was seen upon the distal site mutation on nearby 310-helixes beside the pyrazinoic acid binding site. This switch was found to consistently remain closed only in wild type systems, while it was open in the mutant systems. We called such distance impact an “allosteric effect.” The overall mechanistic investigations will provide useful information behind drug resistance for better understanding to manage tuberculosis.

In an effort to discover the mechanism of resistance offered by Mycobacterium tuberculosis(Mtb) toward the pyrazinamide (PZA) drug, an extensive molecular dynamics strategy was employed. PZA is a first-line prodrug that effectively cuts therapy time by 33% (from 9 to 6 months). Pyrazinamidase enzyme (PZase), encoded by the pncAgene, is responsible for the activation of prodrug PZA into pyrazinoic acid (POA). POA is toxic and potently inhibits the growth of latent Mtb even at low pH values. PZA resistance is caused by three genes pncA, rpsA, and panD. Among them, the pncAgene contributes 72–99% to the resistance. Hence, the present study focused on the novel mutations N11K, P69T, and D126N in the pncAgene. In the present study, the possible mechanism of these three mutations was studied through molecular dynamics simulation and docking techniques. Our in-depth analysis and results are in strong agreement with our experimental observation. The binding pocket analysis showed that mutations decrease the volume of the active site and hinder the correct orientation of PZA drug in the active site. Moreover, the Patchdock score was found to be low as compared to WT showing the disturbance of shape complementarity between PZase and PZA drug. These mutations were found to disturb the position of the Fe2+ion. Among the mutations, D126N allosterically disturbed the position of the Fe2+ion. MMGBSA analyses showed that these mutations decrease the binding affinity toward the PZA drug. In conclusion, mutations N11K, P69T, and D126N result in weak binding affinity with PZA and also cause significant structural deformations that lead to PZA resistance. This study provides useful information that mutations in other than active parts may also cause protein folding and ligand displacement effects, altering the biological functions.