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Understanding the resistance mechanisms against first line anti-tubercular drugs & finding resistance-defiant novel leads

Student Name: Ms Aditi Singh
Guide: Dr Pallavi Somvanshi
Year of completion: 2019

Abstract:

Tuberculosis (TB) drug resistance has been causing a public health crisis since long and its treatment is getting scarce due to the lesser drug efficacy and increased drug toxicity of the available drugs. Evolution of drug resistant Mycobacterium strains threatens the TB treatment and control programs globally. Rifampicin (RIF), Pyrazinamide (PZA), Isoniazid (INH) and Ethambutol (EMB) are the four essential first-line antitubercular drugs, which play pivotal role in tuberculosis treatment but increasing TB resistance renders these drugs ineffective. Mutations are the key players in the emergence of antibiotic resistance in Mycobacterium, which affect the genes, required for antibiotic action i.e. genes that encode the protein targets of drugs or enzymes that are required for prodrug (INH & PZA) activation.

Resistance to Rifampicin is caused mainly by mutations in its target RNA polymerase beta subunit protein (RpoB). RpoB contains a Rifampicin resistance determining region (RRDR) and has several potent sites for mutations. Pyrazinamide is a prodrug that requires amide hydrolysis by mycobacterial pyrazinamidase enzyme for conversion into pyrazinoic acid (POA). POA is known to target Ribosomal protein S1 (RpsA), Aspartate decarboxylase (PanD) and some other mycobacterial proteins. Spontaneous chromosomal mutations in RpsA have been reported for phenotypic resistance against Pyrazinamide. Similarly, INH also requires activation by mycobacterial catlase peroxidase (KatG) enzyme and mutation in KatG are responsible for poor INH activation. On the contrary, Ethambutol targets a set of protein Arabinosyltransferases (EmbC, EmbA & EmbB) involved in cell wall synthesis. Thus, each drug has its unique mechanism of action via an exclusive target protein, which makes them indispensable for TB treatment regimen. 

Structure guided research approaches provide mechanistic insights into the impact of mutation in drug resistance in infectious diseases. Structural and conformational studies on mutant species furnish valuable information for dynamical aspects of protein drug interactions. This study investigates several clinically significant mutations involved in drug resistance against first-line antituberculars as mutations of RpoB at a single site (H451) to eight different amino acids involved in RIF resistance, Δ438A mutant form of RpsA protein for PZA resistance as well as the KatG double mutant (S315T+R463L) for INH resistance. Long range molecular dynamics studies in combination with per residue binding free energy calculations, free energy landscape analysis and essential dynamics analysis were performed to outline the mechanism underlying the high-level resistance conferred by the most frequently occurring mutants. Residue wise MMPBSA decomposition and protein-drug interaction pattern revealed the difference of energetically favourable binding site in the wild type protein in comparison to the mutant due to the difference in conformational flexibility and collective modes of motions between WT and mutants.

Additionally, high throughput virtual screening of FDA approved and natural compounds library was done to reposition new potent drugs effective to inhibit the function of wild type as well as resistant targets. Virtual screening provided two best leads i.e. Amikacin and Terlipressin with higher binding potential to EmbC, which were further evaluated to elucidate the dynamic binding by several in-silico methods. An understanding of interactions of wild as well as mutant target residues and corresponding drugs will help in modifying the existing drugs and designing better drugs to effectively curb MTB resistance menace. This study provides mechanistic insights into drug resistance mutations, and the results arising out of this study will pave way for design of novel and effective inhibitors targeting the resistant strains of Mycobacterium tuberculosis.

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