Rapid advances in whole genome sequencing and computational methods have facilitated the discovery of novel antimicrobial agents and could enable us to combat antibiotic resistance in bacteria. In addition, they have provided important insights into the molecular mechanisms underlying diseases. The development of new bioinformatics approaches and modulation and optimization of existing strategies can increase the accuracy of drug target identification (Read et al., 2001).
The current study has demonstrated how bioinformatics and in silico approaches can be used in drug discovery processes. Since biological systems are complex networks of many metabolic and non-metabolic pathways, the explanation of such systems may be obtained by considering large-scale studies. Focusing on a single drug target at a time in conventional drug discovery methods, may not offer the acceptable answers. Considering biological pathways and whole cell system could provide wider insights about the fitness of a potential drug target. Some proteins in organism that may be predicted as good drug targets, when viewed in the context of biological system, may not actually be vital. Analyzing drug targets in the context of biological system can help in evaluating criticality of the individual targets in the cell pathways.
Of 4358 analyzed proteins, 18 proteins (Redlist) were selected as final putative drug targets in Legionellaceae family (Tabla 2). Putative targets were filtered to exclude non-viable candidates based mostly on importance survival, lack of homology to the human host, well- known the biological function and conserved between Legionellaceae species. The Redlist consists of proteins involved in metabolism (amino acid, energy and lipid metabolisms), cellular transport, cell division and cell motility. These essential genes that play a great role to survive the cell, encode the proteins to maintain a central metabolism, replicate DNA, translate genes into proteins, maintain a basic cellular structure, and mediate transport processes with in or out of the cell (Zhang & Lin, 2009). The essential proteins are involved in many biological pathways that represent molecular interaction networks between pathogen and host. Essentiality search reveals some vital proteins which are required by the pathogen to perform important roles for their survival, growth and replication. Therefore, essentiality search is a critical stage to predict potential drug target in the pathogens. Proteins involved in metabolism and cellular transport provide a major contribution to the Redlist of final targets (14 of 18 proteins). Of 18 Redlist protein targets, 8 targets (lpp0252, lpp1542, lpp1671, lpp1673, lpp2256, lpp2454, lpp2758 and lpp2973) are involved in more than one metabolic pathway (Table 2). Restricted targeting of some specific pathways may cause development of multi-drug resistance among pathogenic bacteria (Shanmugham & Pan, 2013). In general, approaches that consider all vital pathways of organisms can be more successful to identification of efficient drug targets. Targets involving in multiple metabolic pathways are thought to be more efficient drug target and preventing the activity of such targets could increase lethal effects by blocking the activity of several metabolic pathways of the microorganism. Targeting of metabolic enzymes for cancer therapy is a hot topic for drug discovery (Vander Heiden, 2011). Membrane transporter proteins are crucial co-players in cellular processes and are known molecular components of many disease processes. The membrane transporter proteins are targeted by several presently used drugs, and have a large potential as targets for new drug development, a large number (60–70%) of the presently known drug targets are proteins set in a cellular membrane, and membrane proteins are among the most interesting macromolecules to study by structural biology techniques. High-resolution structural knowledge about proteins set in a cellular membrane is of pivotal importance for developing new drugs with therapeutic potential but is also important for the understanding of the molecular mechanisms of cellular communication and function (Ravna et al., 2008). The lpp2758 target is involved in the bacterial two-component system. By means of two-component system, the pathogens perceive the changes in the environment and response to it. Moreover, involvement in pathogenicity of the organisms and the absence of these systems in human makes them attractive drug targets (Capra & Laub, 2012). Therefore, inhibition of these proteins could reduce growth and virulence of the pathogen. Targeting of proteins involved in DNA replication, DNA repair, DNA recombination and cell division (lpp2664 and lpp1690) may disrupt the pathways essential for pathogen survival, growth and reproduction. Seven broad spectrum targets are involved in the crucial processes such as cellular transport, environmental information processing and metabolism (Table 2 and 3). Drug targets involved in the vital metabolic process, seem to be broader spectrum than the other targets such as virulence factors. Because of high specificity, virulence factors are not often broad spectrum drug targets. Targeting of broad spectrum proteins by drug molecules, may facilitate the destruction of wide range of pathogenic bacteria. Eleven Legionellaceae specific targets are suitable for development of narrow-spectrum antibiotic. Such specific target proteins may decrease the threat of development of antimicrobial resistance in wide range of pathogenic bacteria. Based on the localization analysis most of targeting proteins were located on membranes (Table 4). Inhibition of target proteins located on membrane and extracellular proteins is important because of their crucial role as virulence factors aiding pathogens to spread and proliferate within the host. Knowledge of protein localization is valuable for learning their function as well as the interaction of different proteins. When other information is not available, the subcellular localization will also be effective in the annotation for new proteins. In the medical microbiology, subcellular location data can help understand therapeutic intervention points rapidly during the drug discovery process. For example, as of their localization, secreted proteins and membrane proteins are easily available by drug molecules (Peng & Gao, 2014).The Gram-negative bacteria are usually bounded by two membranous structures. The inner one (IM), called the plasma membrane, is a trilamellar structure that bounds the bacterial protoplasm and is composed of a phospholipids bilayer. Many of the membrane proteins that function in energy production, lipid biosynthesis, protein secretion, and transport are conserved in bacteria, but their cellular location is different. In bacteria, these proteins are located in the IM (Chatterjee & Chaudhuri, 2012; Silhavy et al., 2010). The outer membrane (OM) also presents a trilamellar structure (with couple electron dense leaflets, outer and inner) in the electron micrograph and consists of proteins, containing porins, receptors, and an asymmetric distribution of lipids (Chatterjee & Chaudhuri, 2012). The outer membrane of Gram-negative bacteria provides a difficult barrier that must be overcome. There are essentially two pathways that antibiotics can take within the outer membrane: a lipid-mediated pathway for hydrophobic antibiotics, and general dissemination porins for hydrophilic antibiotics, this is due to antibiotic resistance (Delcour, 2009). Molecular structures of membrane transporter proteins is important for drug discovery. The three-dimensional (3D) molecular structure of a protein contains information about the active site and possible ligand binding, and about evolutionary relationships within the protein family (Ravna et al., 2008). Druggability refers to ability of a target molecule to bind with high affinity to the drug molecules. Druggability is one of the most important characteristics of a target molecule. Four druggable targets (lpp0252, lpp1671, lpp1673 and lpp2454) are involved in vital process such as oxidant-antioxidant system, metabolism and biosynthesis of macromolecule. Either of these proteins is a target for conventional antibiotics. Fourteen novel drug targets are suitable for development of new antimicrobials and should be further evaluated experimentally. Currently, several computational methods such as comparative genomics, data mining, structure and sequence to function and metabolic pathways are used for identification of potential drug targets. These approaches consider specificity or essentiality as the main criteria for potential drug candidates. Beside these properties, a suitable drug target must be specific to the pathogen for avoiding harmful side effect and should be a vital protein for survival of the pathogen. Suppression of such drug targets can result in effective control of the pathogen without any harmful effects on the host. Our homology based method considers the essentiality, specificity, druggability, subcellular localization, function and broad spectrum condition of drug targets. Although sequence similarity of protein does not ensure the same structures or binding properties, using such homology based methods could ease the optimization and production of new drugs and vaccines.
Conclusion
In the present study, we applied the homology based method (Butt et al., 2012; Raman et al., 2008; Shanmugham & Pan, 2013) to identify novel putative drug targets in Legionellaceae family. Results of this study identified several proteins in the genomes of Enterobacteriaceae pathogens that can be targeted for effective drug design and development. As regards many of these putative drug targets involve in several vital metabolic pathways such as energy metabolism, amino acid metabolism and lipid metabolism, designing drug molecules against these targets could be very effective for the treatment of Legionella infections. Development of new drug against such targets will be specific to the pathogen and substantially decrease the harmful side effects to the host. The efficiency of already available antimicrobial drugs can be test by this method. Targeting of proteins involved in several crucial metabolic pathways, may facilitate the efficient treatment of infections. The findings of such studies facilitate designing and development of novel antimicrobial drugs against Legionellaceae and other pathogens.