STRUCTURAL STUDIES OF DRUG TARGETS OF PATHOGENS

Abstract

Antibiotic resistance is a serious global public threat to human health and and can undermine the crucial advancements that had been achieved in the past for the treatment of infection. The bacterial world consistently has the molecular machinery to drive resistance. The antibiotic resistance genes are present almost everywhere, including natural, clinical and engineered environments. Antibiotic resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) indicate a big threat to human health globally. The presence of antibiotic resistance genes by ESKAPE pathogens has greatly minimized the treatment alternatives for dreadful infections, increased the disease burden and death rates because of treatment collapse, therefore, the management of antimicrobial resistance demands a cooperative global response. To combat the resistant bacteria, the identification and validation of novel drug targets are urgently required and only potent innovative antibacterial agents with advanced modes of actions that can overcome the current mechanisms of resistance could pave a way to counteract the emergence of antibiotic resistance. The accessibility to the microbial genome represents a large number of novel, potential and therapeutic drug target. Structure-based drug design (SBDD) is considered as one of the foremost techniques to design potent inhibitors by predicting the position of potent inhibitors within a three-dimensional representation of the protein structure and estimate the affinity of ligands to target protein with considerable accuracy and efficiency. Staphylococcus aureus is considered as one of the most widespread bacterial pathogens and continues to be a prevalent cause of mortality and morbidity across the globe. The fatality caused by S. aureus is mainly due to the substantial use of β-lactam antibiotics, which is also responsible for developing its resistant strains. FmtA is a key factor linked with methicillin resistance in S. aureus. FmtA exhibits an esterase activity that removes the D-Ala from teichoic acid. Teichoic acids played a significant role in cell wall synthesis, cell division, colonization, biofilm formation, virulence, antibiotic resistance, and pathogenesis. Consequently, new antibacterial compounds are crucial to combat S. aureus resistance. Moreover, the evolution of methicillin-resistant Staphylococcus aureus (MRSA) strains for developing resistance to vancomycin ensures few treatment strategies that provoke an utmost need to develop novel compounds or antibiotics for treating S. aureus infection. Here, in this thesis we aims to find the potential inhibitors against FmtA of S.aureus and proposed four drug molecules (gemifloxacin, paromomycin, streptomycin, and tobramycin) possessing good binding affinities. The interactions of ligands with the protein was also studied by fluorescence and ITC experiments which showed all the ligands have a dissociation constant (Kd) value in the micromolar range. The screened molecules need to be tested and could be further modified to develop the antimicrobial compounds against S. aureus. A number of bacterial proteins are uncharacterized regardless of the fact that their genome sequences are already studied. The molecular function of more than 30% of proteins is not known; such proteins are called hypothetical proteins (HPs). It serves as an opportunity to characterize such putative proteins with respect to their function and structure. The annotation of HPs aids in identifying novel unexplored targets and respective functions. Klebsiella pneumonia and Acinetobacter baumannii are gram-negative bacteria and known to cause several nosocomial infections in immunocompromised patients. They have developed resistance against a broad range of presently available antibiotics, Therefore, exploration of possible novel drug targets against these opportunistic bacteria needs to be undertaken. In this thesis, we have structurally and functionally annotate the hypothetical proteins of these bacteria and identify unexplored drug target i.e., FmtA like protein in K. pneumonia and LptE in A. baumannii. We also proposed antibacterial molecules against these drug targets and these potent compounds can be further screened via in vitro studies and eventually can be used as antibacterial compounds against K. pneumoniae and A. baumannii. Chapter 1 is dedicated to the introduction and review of literature about the drug resistance in bacteria focussing mainly on three ESKAPE pathogens namely S. aureus, K. pneumonia and A. baumannii. It further illustrated the importance of annotation of hypothetical proteins which aids in identification of unexplored drug targets. Later, the chapter introduces the significance of potent drug targets i.e., FmtA, FmtA like protein and LptE present in S. aureus, K. pneumonia and A. baumannii respectively. Chapter 2 presents the drug-repurposing approach to combat Staphylococcus aureus by targeting FmtA, a key factor linked with methicillin resistance in S. aureus. This chapter presents the virtual screening of a set of compounds against the available crystal structure of FmtA. The findings indicate that gemifloxacin, paromomycin, streptomycin, and tobramycin were the top-ranked potential drug molecules based on the binding affinity.Furthermore, these drug molecules were analyzed with molecular dynamics simulations, which showed that the identified molecules formed highly stable FmtA–inhibitor(s) complexes. Moreover, fluorescence spectroscopy and isothermal calorimetry-based binding studies showed that all the molecules possess dissociation constant values in the micromolar scale, revealing a strong binding affinity with FmtA, leading to stable protein–drug(s) complexes. It presents potential beginning points for the rational development of advanced, safe, and efficacious antibacterial agents targeting FmtA. Chapter 3 deals with in-silico functional and structural annotation of hypothetical protein HP CP995_08280 from K. pneumonia and its characterization as a drug target with the aim to identify potent drug candidates. The functional and structural studies using several bioinformatics tools and databases predicted that HP CP995_08280 is a cytosolic protein that belongs to the β-lactamase family and shares structural similarity with FmtA protein from S. aureus (PDB ID: 5ZH8). The structure of HP CP995_08280 was successfully modelled followed by structure-based virtual screening, docking, and molecular dynamics to identify the potential compounds. The five potent antibacterial molecules, namely BDD 24083171, BDD 24085737, BDE 25098678, BDE 33638819, and BDE 33672484, which exhibited high binding affinity (>−7.5 kcal/mol) and were stabilized by hydrogen bonding and hydrophobic interactions with active site residues (Ser42, Lys45, Tyr126, and Asp128) of protein. Molecular dynamics revealed that HP CP995_08280 – ligand(s) complexes were less dynamic and more stable than native HP CP995_08280. Hence, the present study may serve as a potential lead for developing inhibitors against drug-resistant K. pneumonia. Chapter 4 deals the structural and functional annotation of all the hypothetical proteins (> 18kda) of Acinetobacter baumannii K09-14. The characterization was done based on the sequence analysis, structure homology, family, domain and motif analysis as well as protein-protein interactions. The secondary and tertiary structure of all the proteins were predicted using PSIpred and alpha fold respectively followed by their sequence and structure based functional prediction. Furthermore, the predictions above threshold were further analysed to unravel their function as drug target. The HP QER76570.1 was predicted as lipopolysaccharide assembly protein E (LptE) which is complexed with lipopolysaccharide assembly protein D (LptD) for the assembly of lipopolysaccharide layer at the cell surface and can be considered as a drug target. The structure-based virtual screening was performed against lipid molecule library and binding affinities which favourably bind to protein were considered for further evaluation. Molecular dynamics was performed to evaluate the stability of hypothetical protein-ligand(s) complexes. Chapter 5 gives the brief details of the summary and future perspective for exploiting the studied drug targets i.e., FmtA in S. aureus, FmtA like protein in K. pneumonia and LptE of A. baumannii. Chapter 6 enlists all the references which were used during the course of thesis.