Computational genome analyses of metabolic enzymes in Mycobacterium leprae for drug target identification.

Leprosy is an infectious disease caused by Mycobacterium leprae. M. leprae has undergone a major reductive evolution leaving a minimal set of functional genes for survival. It remains non-cultivable. As M. leprae develops resistance against most of the drugs, novel drug targets are required in order to design new drugs. As most of the essential genes mediate several biosynthetic and metabolic pathways, the pathway predictions can predict essential genes. We used comparative genome analysis of metabolic enzymes in M. leprae and H. sapiens using KEGG pathway database and identified 179 non-homologues enzymes. On further comparison of these 179 non-homologous enzymes to the list of minimal set of 48 essential genes required for cell-wall biosynthesis of M. leprae reveals eight common enzymes. Interestingly, six of these eight common enzymes map to that of peptidoglycan biosynthesis and they all belong to Mur enzymes. The machinery for peptidoglycan biosynthesis is a rich source of crucial targets for antibacterial chemotherapy and thus targeting these enzymes is a step towards facilitating the search for new antibiotics.

present a computational approach to identify the genes essential to M. leprae using comparative pathway analysis followed by mapping of non-homologues genes with list of minimal set of essential genes required for cell-wall biosynthesis of M. leprae. In addition, our approach successfully identified a unique group of common enzymes as promising protein targets for new antibiotic development and further characterization in the laboratory.

Methodology: Collection of metabolic pathway enzymes of M. leprae Kyoto Encyclopedia of Genes and Genomes (KEGG)
[10] is a collection of online databases dealing with genomes, enzymatic pathways, and biological chemicals. KEGG maintains five main databases. They are KEGG Atlas, KEGG Pathway, KEGG Genes, KEGG Ligand and KEGG BRITE. First, we collected all the metabolic pathways of M. leprae and H. sapiens from KEGG pathway database. Each of the pathways of M. leprae was compared with all the available pathways of H. sapiens to identify whether that particular pathway of M. leprae is present in H. sapiens or not. The pathways which were present in both M. leprae and H. sapiens were separated out and were named as shared pathways. The pathways which were present only in M. leprae but were not present in H. sapiens were grouped together and were called as unique pathways. The gene name and the enzyme commission number (EC) of all the enzymes present in both shared and unique pathways were identified and collected from KEGG Genes database.

Retrieval of protein sequences and BLAST
The protein sequence of all enzymes in both shared and unique pathways of m. leprae were retrieved from UNIPROT [11] in FASTA format. Each protein sequence was subjected to BLASTP analysis against the H. sapiens at an E-value cutoff of 10 -4 [12]. BLAST results with no hits with H. sapiens were identified as nonhomologues enzymes of M. lepare.

Identification of essential Enzymes
The minimal set of essential genes required for cell envelope biosynthesis of m. leprae was reported previously using comparative genome sequence method by Vissa and Brennan [13]. The M. leprae enzymes which were non homologous to H. sapiens were mapped with the gene list of Vissa & Brennan and the most common M. leprae genes were identified and further explored.

Prediction of enzymes which were non homologous to human
Removing enzymes from the pathogen that share a similarity with the host protein ensures that the targets have nothing in common with the host proteins and thereby, eliminating undesired host protein-drug interactions. BLASTP similarity search of all these 760 (29 unique + 731 shared) enzymes at an E-value cutoff of 10 -4 resulted 179 non-homologues enzymes of m. leprae of which ten enzymes from the unique pathways and the remaining 169 belong to enzymes from shared pathways. All these 179 enzymes with their corresponding gene-id and EC number were represented in Table 2 (see supplementary material).

Comparison of non-homologues enzymes with essential gene set
The 179 (10 + 169) non-homologues enzymes were further compared to the minimal set of 48 essential genes required for cellwall biosynthesis of M. leprae and reported by Vissa and Brennan [13]. There are eight enzymes common in both data sets ( Table 3 in supplementary material). Among the eight common enzymes only one enzyme was found to be present in unique pathway and the remaining seven enzymes were found to be present in shared pathways. All these eight enzymes were categorized as essential enzymes of m. leprae.

Role of essential enzymes of M. leprae
All the eight essential enzymes were further analyzed for the identification of potential drug targets. One of the eight essential enzymes Alanine racemase (alr) is the enzyme found in D-Alanine metabolism which is a unique pathway of M. leprae. It is also found in Alanine and Aspartate metabolism which is a shared pathway of M. leprae. Another essential enzyme Putative dTDP-4dehydrorhamnose 3, 5-epimerase (rmlC) was found to be essential for Nucleotide sugar metabolism which is a shared pathway of M. leprae. It was also found to be essential for polyketide sugar unit biosynthesis which is a unique pathway of M. leprae. The remaining 6 essential enzymes murC, murD, murE, murF, murG and murY were found to be essential for Peptidoglycan biosynthesis. It is noteworthy that all these 6 enzymes belong to the same family. This particular pathway, peptidoglycan biosynthesis was analyzed for the prediction of drug targets. . This enzyme is peripherally associated with the inner face of the cytoplasmic membrane. Therefore, the peptidoglycan subunit is completely assembled before it traverses the cytoplasmic membrane. Phospho-Nacetylmuramoyl-pentapeptide-transferase (mraY) is an important enzyme in murein synthesis. It is responsible for the formation of the first lipid intermediate of the cell wall peptidoglycan synthesis [15]. As the layer of the bacterial cell wall that confers strength is the peptidoglycan meshwork if we target murC, murD, murE and murF which catalyze the addition of a short polypeptide chain to the UDP-N-acetylmuramic acid (UDPMurNAc), we can easily prevent the synthesis of bacterial cell wall. Thus, these are excellent candidates for further exploration.

Conclusion:
The availability of full genome sequences and computer-aided analysis to identify probable antimicrobial drug targets has become a new trend in pharmacogenomics. The use of a comprehensive set of unique pathways and enzymes present in these pathways of M. leprae to identify new drug targets were documented in this study. We have found peptidoglycan biosynthetic pathway and the six mur enzymes (murC, murD, murE, murF, murG and murY) involved in this pathway to be used as potential drug targets. Protein structure and inhibitors of these important enzymes are not currently available. Further analysis on the structural studies on these mur enzymes is believed to provide valuable insights towards the design of an inhibitor specific to the peptidoglycan biosynthesis of M. leprae for the treatment of leprosy. The availability of the newer anti-leprotic drugs in the future would definitely support our present findings such that there would be a possibility of mur enzymes which were proposed by us for targeting M. leprae.