Characterization of the Putative Active Site of Mycobacterium tuberculosis Pyrazinamidase: An Application of Bioinformatics Softwares for Molecular Modeling, Docking and Testing of Drug Analogues

Abstract

Pyrazinamide (PZA), a first-line pro-drug targeting Mycobacterium tuberculosis (Mtb), is a
cornerstone in tuberculosis combined therapeutic management. It is converted to Pyrazinoic
acid by Pyrazinamidase (PZAse), a 2kD enzyme encoded by pncA gene. Ongoing search for
drug analogues of Pyrazinamide entails costly and labor-intensive in-vitro and in-vivo
studies. This study presented a process to predict and characterize a putative active site of
enzymes using free online softwares and databases. The developed platform was applied to
Mycobacterium tuberculosis Pyrazinamidase to perform in-silico experiments such docking
of its natural substrate and candidate drug analogues. Briefly, a molecular model of PZAse
was constructed through online submission of wild type MTb (H37Rv) PZAse protein
sequence to SwissProt Database. Conserved amino acids were identified through multiple
sequence alignment of Mycobacterial strains 131, ten strains of Mycobacteria and five
organisms expressing closely related nicotinamidase/pyrazinamidase. Conserved residues
were plotted into the model supplemented by crevice and drug volume calculations coupled
with mutation data from existing literature helped identify the putative active site. Drug
docking using HEX software showed that amino acids D8, D49, C138, F13, W68, Y103, H71
and A134 interacted with PZA while residues F94 and Y95 stabilized PZA through non-polar
interactions. Molecular docking of Nicotinamide and Morphazinamide revealed higher
binding affinities to PZAse due to hydrophobic interactions at the binding site. Testing PZA
analogues downloaded from PubChem database suggests Pyrazine-2,6-carboxamide fits
the active site, shared similar proximity with PZA. This platform exhibits potential in exploring
enzyme-substrate interactions that can be extended to other applications, such as exploring
enzyme-substrate or receptor-drug interactions, putative active site identification, and
testing candidate drugs in-silico as initial steps in rational drug design.

Key words: pyrazinamidase, pyrazinamide, docking