BIOPHYSICAL, BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF DRUG TARGETS FROM BACTERIA

Abstract

The rapid emergence of drug resistance and declining rate of development and approval of novel antimicrobials agents has presented a serious challenge to modern medicine. The increment of drug resistance in bacteria has become a public health concern worldwide but only a few new therapies have been developed. Therefore, the efforts toward the discovery and development of novel molecules/new scaffolds with new action of mechanism are essentially required at this crucial stage to combat the problem. The recent technological advancement in the field of structural genomics opens up the doors toward the identification and characterization of novel and potential drug targets in pathogenic bacteria. The exploration of essential biosynthetic pathways present in bacteria has drawn the attention of researchers for the expansion of potent inhibitors by targeting the enzymes of these pathways. Shikimate pathway and lipopolysaccharide (LPS) biosynthesis pathway in microbes are essential and attractive pathways for the development of the inhibitors against the pathogenic bacteria. Since these pathways are absent in animals and humans, the enzymes of the pathways are attractive and potential target for the development and designing of novel drug against the resistant bacteria. This thesis contains two sections, which covers the biochemical, biophysical and structural characterization of a monofunctional 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) enzyme from drug-resistant Gram-negative bacteria Providencia alcalifaciens. Additionally, a bifunctional DAH7PS from Bacillus subtilis has also been characterized and its binding and inhibition aspects with polyphenol compounds (Chlorogenic acid, Prephenate, Shikimate, and Ferulate) have determined. In second section, we have characterized LpxC enzyme from a Gram-negative, drug-resistant pathogenic Moraxella catarrhalis and analyzed the binding features of Lpxc with the most potent inhibitors (LpxC-4 and LpxC-2). In first chapter, we have described the general overview of emerging problem of the antibiotic resistance in bacteria. The molecular mechanisms for the acquisition of drug resistance among microbes have been described. Additionally, the potential approaches and efforts made in the direction to overcome the resistance problem have also been discussed. This chapter describes the shikimate pathway and intracellular localization of enzymes of this pathway in different organisms. We have also described the classification, structural characterization and regulation mechanism of microbial DAH7PS. Furthermore, this chapter encompasses the lipid biosynthesis pathway (LPS) along with the structural components of cell envelop present in Gram-negative bacteria. Additionaly, the activation and stimulation of the host innate immune ii system mediated via Lipid A (Endotoxin) has been elaborated. In last part of the chapter, we have described the structural features and reported inhibitors of LpxC which is a committed enzyme of the LPS pathway. 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), the first committed enzyme of the shikimate pathway, catalyzes the condensation reaction of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) to produce DAHP. It further produces a precursor molecule “chorismate” that leads to production of several essential compounds like aromatic amino acids, folates, phylloquinone (vitamin K), ubiquinone, napthoquinone in bacteria, fungi, apicomplexan and plants. Since this pathway is employed only in plants, fungi, microbes, and apicomplexans, enzymes of this pathway are potential targets for the development of drug molecules and herbicides. In second chapter, the molecular cloning, expression, purification, and characterization of DAH7PS from Providencia alcalifaciens (PaDAH7PS) have been described. DAH7PS is a metalloenzyme, which exhibits vulnerability to the oxidative stress and it undergoes inactivation in multiple ways in the presence of redox metal, H2O2, and superoxide. We have determined the oligomeric state of PaDAH7PS and the effect of redox metal on its stability through the size exclusion chromatography. The FTIR, MALDI-TOF/TOF-MS studies have revealed that methionine residues of PaDAH7PS were modified to methionine sulfoxide in oxidative stress conditions. During oxidation, PaDAH7PS alters into partially folded and unfolded states and determined by CD and Fluorescence studies. A significant loss in enzymatic activity of PaDAH7PS was determined and the formation of amorphous aggregates was visualized using AFM imaging, further confirmed by ThT binding based assay. This is the first report where we have shown a hexameric DAH7PS and the methionine residues of PaDAH7PS get oxidize in the presence of redox metals. The partially folded and unfolded oligomeric states with high β-content of PaDAH7PS might be the critical precursors for aggregation. Furthermore, elaboration of the structural characterization of PaDAH7PS has been described in the third chapter. Here, we have determined 3.3 Å crystal structure of PaDAH7PS in apo form. Crystallization of hexameric PaDAH7PS (non-oxidized most active form) was performed in sitting drop trays by the vapor diffusion method. Crystals were grown in the presence of Bis Tris propane buffer, PEG 3350 as the precipitant, 1mM substrate (PEP), 10 mM DTT as a reducing agent at the 293 K. The crystal belongs to P1211 space group with unit cell parameters a, b, c = 54.55, 165.46, 77.08 and α, β, γ = 90, 106.5, 90. The structural comparison iii of apo PaDAH7PS, with complexed DAH7PS (PEP and metal) of Escherichia coli and apo DAH7PS of Saccahromyces cerevesaeaie reveals that the PaDAH7PS molecule adopted a typical well conserved (β/α)8 TIM-barrel fold and its different type assembly of tetrameric form. In apo PaDAH7PS, loop (L2 and L8) regions associated with the binding of the substrate and catalytic metal ions were found disordered. In future, the optimization and analysis of a high resolution structure of PaDAH7PS could provide more structural insights that will be useful for the rational design of inhibitors. Recently, our group members have reported a crystal structure of truncated N-terminal chorismate mutase (CM) regulatory domain, complexed with chlorogenic acid, from bifunctional DAH7PS of Bacillus subtilis. The structural and biochemical data have evidently shown that chlorogenic acid (a structural analog of chorismic acid) acts as an inhibitor of truncated N-terminal regulatory CM domain (chorismate mutase). We have hypothesized that binding of the substrate, product and their analogs at the active site of CM domain can induce conformational changes in DAH7PS catalytic domain and inhibit its catalytic activity. In this context, chapter fourth describes the cloning, expression, purification and characterization of a full length bifunctional DAH7PS from Bacillus subtilis (BsDAH7PS). We have evaluated the binding and inhibition of full length BsDAH7PS, via CM regulatory domain, with the four phenolic compounds (Chlorogenic acid, Prephenate, Ferulate, and Shikimate) using fluorescence and circular dichroism (CD) spectroscopy as well as the molecular docking studies. The fluorescence data suggested the quenching of intrinsic fluorescence of BsDAH7PS via a static mechanism and Kq values (higher than 1010), indicated the ground state complex formation between phenolic compounds and BsDAH7PS. Isothermal titration calorimetry studies have shown that chlorogenic acid has the strongest binding affinity with the BsDAH7PS (KD = ~10.0 μΜ). The far-UV CD results have shown the increment in the α-helical content of BsDAH7PS upon ligand binding. Molecular docking studies have predicted that all compounds are interacting with the regulatory CM domain and Arg27 (Chain A) and Arg10* (Chain B) are the critical residues which are forming H-bonding. This study may be helpful for the development and further designing of the more potent multi-targeted antibacterial agents. Gram-negative bacteria contain an outer membrane which surrounds the cell wall and protects the bacteria from many antimicrobial agents. The outer membrane is composed of lipopolysaccharide molecule which contains three components: O-antigen, a core iv polysaccharide, and lipid A. Lipid A is mainly responsible for anchoring of the membrane to cell wall and essential for the viability and virulence of the bacteria. The biosynthesis of Lipid A is catalyzed by nine enzymes, and the committed step of the LPS pathway is catalyzed by a zinc metalloenzyme UDP-3-O- [(R)-3hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC). LpxC is conserved and shows no homology to any human enzyme. Thus, LpxC has attracted attention for the new drug development against pathogenic bacteria. In chapter fifth, we have described the cloning, expression, purification and characterization of the zinc metalloenzyme UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) from Moraxella catarrhalis (McLpxC). Several reported potential compounds against LpxC have shown large variation in their potency and efficacy. The differential susceptibility and selective binding features of these inhibitors against LpxC from numerous drug-resistant bacteria are still unexplored. In this chapter, we have investigated its binding features with potent inhibitors LpxC-2 and LpxC-4 using biochemical, biophysical and in silico approaches. The circular dichroism results have revealed the changes in secondary and tertiary structure of McLpxC upon inhibitors binding. The fluorescence quenching mechanism was found to be static with Kq >1010 suggesting a ground state complex formation between the McLpxC and inhibitors. Finally, the overall spectroscopic findings suggest that the interaction of LpxC-4 caused large conformational alterations and significant loss of α-helical content in McLpxC as compared to LpxC-2. In Isothermal titration calorimetry based studies, both inhibitors have shown comparable binding affinities (KD = ~10.0 μΜ) and their interactions were exothermic and driven by enthalpy change. Furthermore, the docking studies have shown that Lys239 and Thr191 are the critical residues for binding of the inhibitors. Thus, this study suggests that further optimization and utilization of molecules based on this scaffold will be helpful in designing the new antimicrobial agents targeting LpxC. Conclusively, the overall findings of our proposed work will enhance the understanding of the potential drug targets (DAH7PS and LpxC) in terms of their structural and inhibition features and could serve as a starting point for the development of new antimicrobial agents against bacteria.