Exploring combined verses single mode of inhibition of Mycobacterium Tuberculosis RNA polymerase as a therapeutic intervention to overcome drug resistance challenges: atomistic perspectives.

Abstract

The impact of Rifampin resistance on the overall global epidemic of antimicrobial resistance has become very prominent in recent years and is eventually stifling current efforts being made to control tuberculosis drug resistance. Rifampin resistance has significantly contributed to making TB the leading cause of morbidity from an infectious disease globally. The RNA polymerase of Mycobacterium tuberculosis has been extensively explored as a therapeutic target for Rifampin resistance with recent studies exploring synergistic inhibition as an effective approach, by combining Rifampin and other drugs in the TB drug resistance. Apart from the paucity of data elucidating the structural mechanism of action of the synergistic interaction between Rifampin and DAAPI, previous studies did not also utilize the X-ray crystal structure of Mtb RNAP due its unavailability. This thesis used advanced computational tools to unravel molecular insights into the suppression of the emergence of resistance to Rifampin by a novel Nα-aroyl-N-aryl-phenylalaninamides (AAPI) prototype inhibitor, DAAPI, co-bound to Mtb RNAP with Rifampin. Our studies revealed co-binding induced a stable Mtb RNAP protein structure, increased the degree of compactness of binding site residues around Rifampin and subsequently improved the binding affinity of Rifampin. Studies in this thesis further provide an atomistic mechanism behind Rifampin resistance when the recently resolved crystal structure of Mycobacterium tuberculosis RNA polymerase is subjected to a single active site mutation. We also identified and rationalized the structural interplay of this single active site mutation upon co-binding of Rifampin with the novel inhibitor, DAAPI. Our findings report that the mutation distorted the overall conformational landscape of Mycobacterium tuberculosis RNA polymerase, resulting in a reduction of binding affinity of Rifampin and an overall shift in the residue interaction network of Mycobacterium tuberculosis RNA polymerase and upon single binding. Interestingly, co-binding with DAAPI, though impacted by the mutation exhibited improved Rifampin binding interactions amidst a distorted residue interaction network. Findings establish a structural mechanism by which the novel inhibitor DAAPI stabilizes Mycobacterium tuberculosis RNA polymerase upon co-binding with Rifampin, thus suppressing Rifampin resistance. We also provide vital conformational dynamics and structural mechanisms of mutant enzyme-single ligand and mutant enzyme-dual ligand interactions which could potentially shift the current therapeutic protocol of TB infections, thus aiding in the design of novel Mycobacterium tuberculosis RNA polymerase inhibitors with improved therapeutic features against the mutant proteins.