Pan-genome analysis and Simulation studies to explore Potential Drug Candidates against MDR pathogens involved in Hospital Acquired Infections

Abstract

The industrial revolution and modern developmental era have put forward with the humongous availability of newer approaches. For over three decades computer aided drug designing emerged as powerful discipline playing critical roles in the design of new drug molecules for bacterial pathogens. The present dissertation focused mainly on the applications of drug designing towards possible drug targets in the genome of multidrug resistance bacterial pathogens especially Burkholderia cepacia, Legionella longbeachae and Enterobacter specie. Additionally, molecular modelling, structure-based drug designing, molecular dynamic simulations, binding energies calculations, axial frequency distribution, Site Identification by Ligand Competitive Saturation (SILCS) based drug designing and biological activities of the potential small drug molecules were taken into consideration for biological systems. This dissertation thus is a blend of both the computational exertion and experimental verification to resolve the prevalent issue of antibiotic resistance. First chapter addresses general perspective about hospital acquired infections and nosocomial pathogens that causes respiratory tract and lungs infections mostly emphasized on Burkholderia cepacia, Legionella longbeachae and Enterobacter specie. Hence, providing a mainstream and inspiration for the researcher to tackle the challenges in current research objectives. This is followed by theoretical details of computational approaches utilized for identification of druggable protein targets/drugs. Chapter third encompasses pan-genome analysis of B. cepacia strains with drug designing against glutamate racemase target. The study also highlights the evolutionary connection between each of the nine B. cepacia strains. Moreover, several other targets have been identified here which follows the core genome of B. cepacia. This includes an efficient drug target from different region of inner/outer membrane, periplasmic space and the most important one a cytoplasm region. These include (Glutamate racemase (GR), (UDP3-O-(3-hydroxymyristoyl) glucosamine N- acyltransferase), (Sigma54-dependent Fis family transcriptional regulator) peptidoglycan associated lipoprotein (Pal). This chapter includes comprehensive comparative analysis of all the strains with mainly focuses on antimicrobial resistant genes. Furthermore, molecular modelling, docking and molecular dynamics (MD) simulations was performed to probe the binding conformation of drug molecules. Dynamics of the complex system was properly demonstrated with binding free x energy calculations. In forthcoming chapter, the role of ring positioning of ligand inside the active cavity of peptidoglycan associated lipoprotein (Pal) is addressed. Dynamic studies with a time scale of 600 ns simulation was performed to investigate the binding conformation. This protein target is responsible in signaling pathways between lipid bilayer and cytoplasm. Comparatively, the target is conserved among all the bacterial pathogens and is a key factor of antibiotic resistance. This target is mostly responsible in bind to epithelial cell of lungs and causes severe lungs infections. Thus, by computational mean a potential drug target have been identified with promising binding capabilities. MD simulations illustrated stable binding of the compound at the docked site with no conformational changes observed in the receptor macromolecule. A unique phenomena of ring positioning is a cherry on top of this research work which depicts the penetrating behavior owing to the stability enhancing the suitability of pyrimidine based fused ring containing compounds. Chapter five illustrates about legionellosis caused by a pathogen Legionella longbeachae. We have performed a proteome wide study that infrared natural inhibitors via dynamic simulation against sugar isomerase (SIS) domain protein. The target is involved in the production of lipopolysaccharides, which catalyzes the isomerization of psedoheptulose 7-phosphate in D-glycero-D-manno-heptose 7 phosphate. Findings have affirmed that abovementioned small molecule binds to its receptor cavity with higher affinity. This further was validated via simulations analysis that showed a stable binding position with time interval of 130 ns. Moreover, Radial Distribution Function (RDF), SER55 and SER83 are the main residues involved in hydrogen bonding and in turn enhances the complex stability. Additionally, findings from an indigenously developed method called Axial Frequency Distribution (AFD) by Saad et al in the Computational Biology Lab showed where the ligand gets closer to the binding pose with the residues SER55 and SER83 respectively. Chapter 6th of the dissertation focuses on novel SILCS based method that determines a protein's functional group affinities using a variety of tiny solutes in aqueous solution. In order to produce functional group free energy maps (FragMaps), this has been done to investigate the binding architecture and dynamics of beta-lactamase CMY-10 with deep binding pockets. This study emphasizes the strategies of combination therapeutics and aims to identify a novel β-lactamase inhibitors that can xi inactivate the biosynthetic pathways and allowing the antibiotics to bind to its penicillin binding protein targets. Followed by the last chapter Thiazole substituted arylamine derivatives were discovered to be FabH enzyme inhibitors using a combination of chemical synthesis, biological assessment, and molecular dynamics research. In this final part of a collaborative work the whole computational and experimental pipeline have been performed in Computational Biology Lab except chemical synthesis. In vitro testing against the compound 3f was discovered to be a potential active substance against bacterial infections. The MD simulations suggested that the anisole ring has a unique placement at the interface of the active pocket, interacting with Arg36 and 2-methylthaizole with the bottom floor. Asn274 from the FabH active pocket was shown to be important in compound stability and stabilization of interaction as the simulation progressed by intermolecular interactions analysis. Hence, the outcomes of this research representing a new avenue for drug discovery