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Therapeutic strategy to end Tuberculosis (TB) world: structural and functional characterization of potential weak hotspots of Mycobacterium tuberculosis molecular targets from combinatorial in silico perspective.

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The world has witnessed several decades of Tuberculosis (TB) pandemic and numerous advanced scientific efforts to control the invasiveness of the newly evolving Mycobacterium tuberculosis strains (Mtb) resulting in drug resistance. TB disease has killed hundreds of millions of humans and left millions maimed that need to be rehabilitated; about 10.0 million infections and 1.5 million annually in the last decade. Drug-resistant TB has remained more challenging in the previous 20 years than drug-susceptible TB and is chromosomal mutations-associated in selected genes of the Mtb. Notable mutations identified by biomarkers are related to phenotypic drug resistance, and these include; an 81 bp region in rpoB gene with > 95 % mutations in rifampicin (RIF) clinical isolates and katG gene promoter of the mabA-inhA showed to be associated with INH-resistance. Different Strategies, including the recent WHO End TB approach, have been employed to alleviate or stop TB. The current identification of the critical roles of Mtb demethylmenaquinone methyltransferase (menG) target in the survival, pathogenesis, virulence, and drug resistance created an avenue for the development of efficacious therapeutics that can eradicate TB. MenG is a member of the methyltransferase superfamily. It catalyzes one of the last steps of the menaquinone biosynthesis pathway, requires for maintenance of the Mtb cell envelope. The other two studied targets investigated in this work are N-acetylglucosamine-6-phosphate deacetylase enzyme (NagA), which represents a critical enzymatic step in the production of essential amino sugar required by Mtb for the cell wall biosynthesis and the secreted antigen 85C enzyme (Ag85C) target. The latter target catalyzes the synthesis of trehalose derivatives and attachment of mycolic acids. These targets have gained considerable attention in drug discovery pipelines. However, there is little information about menG, as it lasks structural dynamics due to the lack of crystal structure, active site regions, and amino acids of it Mycobacteria homologs. Similarly, the dynamics of the NagA and Ag85C proteins structure are still unknown. Therefore, justifications led to the modelling of the 3D Structure of menG to understand the structural and functional features that could be investigated at the atomistic level. Homology models were also created for the five (5) mycobacterial homologs. Furthermore, the inevitable need for new drugs has led to the application of in silico techniques including molecular modelling and molecular dynamics simulations, which provide opportunities for the chemists to evaluate and assess numerous compounds that can lead to potential drugs against the mycobacterial disease. Furthermore, these computational techniques justify the present incorporation of several computational tools integrated into this study to provide insights into the conformational changes that illuminate potential inhibitory mechanism, identification of the binding site amino acids, and characterization. Here, we analyze the weak hotspots dynamics specific to each of the Mtb targets, most notably the loop and active residues around or within the ligand-binding sites to obtain useful findings for the design of higher efficacious potential antitubercular drugs. Molecular dynamics simulations were performed to gain molecular standpoints of the conformational binding of the experimental drugs, which were reported to be highly effective against each respective target. Structural dynamics and motions behaviour of menG upon the binding of inhibitor (DG70, biphenyl amide compound) were estimated. Additional in silico thermodynamic analyses were further employed to explore intuitions into the binding mode of each inhibitor mainly for the proposed binding site of menG to identify the residues for binding. Sequence analysis of the homologs of Mycobacterium tuberculosis NagA and Ag85C targets, including those of smegmatis, marinum, leprae, ulcerans, were performed to obtain unique sequence similarities and differences and the structural and functional characterization upon the binding of the ligand. An experimental protocol let to the discovery of a selective covalent inhibitor, β- isomer monocyclic enolphosphorus Cycliphostin, of Ag85C SER-124. Moreover, chapter 4 also unravels the impact of the function of the non-synonymous single nucleotide polymorphisms of NagA target. The desired expectation is that the implementation of the information extricated from this study would provide the structural silhouette for pharmaceutical scientists and molecular biologists to abet in the identification and design of novel antimycobacterial drugs most especially for TB.


Doctoral Degree. University of KwaZulu-Natal, Durban.