IN SILICO FINDING OF THE PUTATIVE DRUG TARGETS FROM HYPOTHETICAL SET OF PROTEINS FOR MYCOBACTERIUM LEPRAE TN

IN SILICO FINDING OF THE PUTATIVE DRUG TARGETS FROM HYPOTHETICAL SET OF PROTEINS FOR MYCOBACTERIUM LEPRAE TN

Utkarsh Raj ¹, Sarvesh Pathak ², Imlimaong Aier ¹, Saurabh Gupta ¹, Pritish Kumar Varadwaj ^{* 1}

Department of Applied Sciences ¹, Indian Institute of Information Technology-Allahabad, Allahabad, Uttar Pradesh India.

Jacob School of Biotechnology and Bio-Engineering ², Sam Higginbottom Institute of Agriculture, Technology and Sciences, Allahabad, Uttar Pradesh, India.

ABSTRACT: Leprosy is a chronic granulomatous disease caused by acid-fast bacilli Mycobacterium leprae. It is most prevalent in tropical countries and poses a threat to over 122 countries across the globe. In 2012, 232857 new cases of leprosy were registered in the world. World Health Organization (WHO), recommended the Multi Drug Therapy (MDT) for treatment of leprosy, but the minimum duration of treatment ranges from 6-12 months. Therefore, the need of a new putative target for treatment of leprosy, which is effective in leprosy and reduces the duration of treatment. The Proteome information available suggests that among the 1603 proteins in M. leprae TN, a staggering 27% or more remains classified as hypothetical uncharacterized set of proteins. In this present work we, assign the probable functions of hypothetical set of proteins present in M. leprae to explore their plausible role as new putative drug targets. Out of 442,177 hypothetical protein sequences had GO term. Of these, 43 sequences had disease ontology (DO) term, 59 sequences had human phenotype (HP) term and out of these 43 sequences, 16 sequences were found to contain only DO term, while 27 sequences had both HP and DO term. Out of 177, 15 sequences were found to be associated with 39 KEGG reference pathway pathways. Out of 39 pathways, inositol phosphate metabolism, fatty acid degradation, ethylbenzene degradation, sulfur relay system and limonene & pinene degradation were common metabolic pathway, which might be used as putative drug target for M. leprae.

Leprosy, Mycobacterium leprae, Hypothetical protein, Inositol metabolic pathway, Putative drug target

INTRODUCTION: Leprosy is a chronic granulomatous disease and is also known as Hansen's disease (HD) ¹. The disease is caused by an acid-fast, rod-shaped bacillus Mycobacterium leprae. The clinical manifestations of leprosy are bacillary infiltration, lesions on skin, peripheral neuropathy and various immunological reactions.² It is an endemic disease and is spread out in tropical counties.

According to the World Health Organization (WHO) more than 115 countries were affected by leprosy, and around 189018 prevalent cases were registered till the first quarter of 2013 ³. Moreover, 232857 new cases of leprosy per 100000 populations were registered during 2012. The regional distribution of the disease is given in Table 1. Overall 25 countries of the African region, 28 Americas region, 20 Eastern Mediterranean regions, 11South-East Asia region and 31 countries of the Western Pacific region were included. However, 134752 new cases were reported in India in the year 2012 ³. The number of cases in 2012 was higher as compared to the cases reported in recent years as shown in Fig. 1.

Leprosy is a Flügge droplet infection, transmission occurs by inhalation of the bacilli contained in nasal secretion, but other possible route of transmissions of leprosy are skin erosions, blood, vertical transmission, breast milk, and insect bites ⁴. The incubation period of leprosy is about 3-10 years, symptom disabilities when, M. leprae invades Schwann cells in the peripheral nervous system leading to damage of peripheral nerve.¹ The main symptom of leprosy is disfiguring skin sores, lumps or bumps with loss of pain and sensation due to damage of peripheral nerve.

In 1982, the WHO classified leprosy, based on the bacterial index (BI). It becomes paucibacillary (PB) when BI goes lower than 2 and multi-bacillary (MB) when BI increases more than or equal to 2⁵. WHO campaign for leprosy with the support of private foundations and pharmaceutical companies was for the recommendation of Multi Drug Therapy (MDT) ⁶. Now-a-days, genome sequencing provides fast and reliable solutions to identify the whole information of the genome. The genome sequencing of M. leprae samples from Tamil Nadu, India was performed by Cole et al. in the year 2001 using a combination of sequenced cosmids from multiplex sequencing and end sequences from the genome shotgun library using dye termination on automated sequencers. M. leprae TN genome belongs under SNP type 1 and subtype A. The molecular weight of the genome is 2.2 x 109 Daltons, containing 3,268,203 base pairs (bp) and G+C (guanine + cytosine) content of 57.8%. The genome contains 49.5% of total genome as protein-coding gene, 27% of pseudo genes and remaining 23.5% of genome does not show any expression ⁷. The total 1603 proteins are available in different protein database out of them, 442 protein sequences are hypothetical and uncharacterized.

Hypothetical proteins are plausible target for drug discovery by computational methods. Literature survey suggested that hypothetical proteins assign the functional information in various organisms by computational methods. Computational analysis of hypothetical proteins for human fetal brain showed that many hypothetical proteins have ligase activity ⁸ No prior comprehensive studies were undertaken on hypothetical proteins for M. leprae TN, therefore functional annotation and detailed investigations of hypothetical proteins, which might be used as putative drug targets for M. leprae TN, has been attempted in the present work.

FIG. 1: REPORTED CASE OF LEPROSY ACROSS THE GLOBE

TABLE 1: WHO REPORT ON THE PREVALENCE OF LEPROSY?

METHODOLOGY:

MATERIALS AND METHODS:

Databases employed: Uncharacterized hypothetical protein sequences for Mycobacterium leprae TN were retrieved from Proteome sets of UniProt-KB database ⁹. Reference proteome set of M. leprae TN was last modified on July 4, 2014. Database of Essential Gene (DEG 10.6) was used for the investigation of essential gene present in M. leprae ¹⁰. KEGG database (Release 69.0) was used to investigate the metabolic pathway information ¹¹.  String Data Base (String DB, version 9.1) was used to identify and analyze the networks for Cluster of Orthologous Groups (COG) between protein families ^{12, 13}.

Tools employed: Hmmscan web server was used to assign the Pfam domain information from the query protein sequences ^{14, 15}. CD batch search was used to assign the functional domain information from the query protein sequences ¹⁶.  dcGO web server was used to predict Genome Ontology (GO), Disease Ontology (DO) and Human Phenotype (HP) from the query protein sequences. ¹⁷ KEGG Automatic Annotation Server (KAAS) was used to predict pathway associations from the query protein sequences ¹⁸. 

Sequence analysis: 442 hypothetical, uncharacterized protein sequences, belonging to M. leprae, were retrieved from UniProt-KB database. Domain information of these sequences was analyzed by hmmscan against Pfam database, with an e valve of 10^-3. We found that out of 422, 318 protein sequences had at least one Pfam domain. Functional annotated conserved domain for 318 protein sequences were identified by NCBI Batch Web CD-Search against CDD--45746PSSMs with an e-value 10­^-3. We obtained 276 sequences, which had at least one functional annotated conserve domain. Further, these 276 protein sequences were analyzed for possible GO, DO and HP term. Out of 268, 177 sequences had GO term. KAAS was used to retrieve the reference metabolic pathway from KEGG for 177 sequences, which had GO term.

RESULTS AND DISCUSSION:

Sequence based drug target identification: The proteome information available suggests that among the 1603 proteins in M. leprae TN, a staggering 442 remains classified as a hypothetical uncharacterized set. The result of Hmmscan suggested that out of 442 hypothetical uncharacterized hypothetical sequences, 311 sequences had at least one Pfam domain associated with the query sequences. After excluding spurious annotations, we predicted the functional annotated conserved domain from CD batch search for 311 sequences with an e value of 10^-3, against conserve domain database (CDD). At last, we found 1685 functional annotated conserved domain which were widely distributed in 268 sequences.

Functional information related to Gene Ontology (GO) was predicted by domain-centric Gene Ontology (dcGO) predictor. The result of dcGO predictor was tabulated in supplementary Table 1. Out of 268 hypothetical/uncharacterized protein sequences, 177 sequences had GO term. Out of 177, 43 sequences had disease ontology (DO) term and 59 sequences had human phenotype (HP) term. Out of these 43 sequences, 16 sequences had only DO term, while 27 sequences had both HP and DO term.

Supplementary:

TABLE 1: FUNCTIONAL ANNOTATION OF 177 SEQUENCES HAVING GO TERM THROUGH DOMAIN-CENTRIC GENE ONTOLOGY (DCGO) PREDICTOR

N/A* - Not available

Further, homology of 43 sequences (which had DO term) with human proteome were analyzed by NCBI BLASTP program. BLASTP program was performed against UniProt-KB database for human proteome with an e value 0.001. Hits were considered as true positive (homologs), when it had an identity of >30% and a coverage of >70%. We found that 13 sequences were homologous to human proteome, while the remaining 30 sequences with DO term did not show homology with Human proteome. These 30 sequences might be used as putative drug target for M. leprae. UniProt-KB Sequence ID for these sequences was tabulated in supplementary Table 1.

Essential genes in prokaryotes have a minimal genome, which play the key roles in metabolism. Database of Essential Gene (DEG) constitute a minimal genome, forming a set of functional modules, which play the important role in metabolism. DEG BLASTp report suggested that out of 177 sequences, 101 sequences had at least one prokaryotic DEG homologous sequences. These 101 sequences were found to contain 494 DEG homologous genes. These sequences might be used as putative drug target for M. leprae (supplementary Table 2).

Supplementary:

TABLE 2: LIST OF 101 SEQUENCES (OUT OF 177 SEQUENCES) CONTAINING 494 DEG HOMOLOGOUS GENES

Total protein-coding genes in your sequence: 177 genes

In our sequence, the no. of genes having homologs with DEG: 101 genes

In DEG, the no. of genes having homologs with our sequence: 494 genes

KASS was used to predict Pathway associations for the 177 sequences that have GO term and had at least one functional annotated conserved domain. KASS was performed using the BBH method (Best bidirectional Hit) from which we found that 50 sequences had KEGG orthology hits. Out of these 50 hits, 15 sequences were found to be associated with 39 KEGG pathways, which are shown in supplementary Table 3. KASS report provides important information about the protein (enzyme) associated in metabolic pathway.

Supplementary:

TABLE 3: LIST OF 50 SEQUENCES HAVING KEGG ORTHOLOGY HITS OBTAINED THROUGH KASS

Homology based metabolic pathway reconstruction: Sequence information for metabolic pathway was retrieved from the KEGG database and MetaCyc ¹⁹.  Enzyme catalyzing each step in the reference metabolic pathway was used as query to search against UniProt-KB database for homologs in M. Leprae TN using NCBI protein blast (BLASTP) at an e value of 10^-3. Hits considered as true positive (homologs), when it had an identity of >30% and a coverage of >70%. Further, homologs founds within M. leprae TN were using COG modules of String database to understand the conservation of gene. Resultant, we found that inositol phosphate metabolism, fatty acid degradation, ethylbenzene degradation, sulfur relay system and limonene and pinene degradation were common metabolic pathways, which might be used as drug target in M. leprae. Here we reconstruct the Inositol phosphate metabolism because it is a common drug target in various Prokaryotes as well as in cancer cell.

Case study: Inositol phosphate metabolism: Inositol phosphate metabolism is common in eukaryotes, but it is also found in some prokaryotes. Inositol derivatives play an important role in Mycobacteria; they are glycosyl-phosphatidylinositol (GPI), phosphatidylinositol mannosides (PIM) and phosphatidylinositol (PI) ²⁰. Myo-inositol-1-phosphate (MIP) synthase is a good target for antipolar drugs ²¹. Mycobacterium tuberculosis, Trypanosomabrucei and Candida albicans able to produce inositol in vitro in order to cause disease or even grow (Trypanosomabrucei) ²². Hence, inositol phosphate metabolism is a possible drug target for Mycobacteria. We constructed the possible inositol phosphate synthetic pathway in M. leprae. Reference pathway for inositol phosphate synthesis is shown in supplementary Fig. 1 and complete list of enzymes that are involved in inositol phosphate synthesis is shown in supplementary Table 4. Homology based possible inositol synthesis pathway in M. leprae was depicted in Fig. 2.

Supplementary:

TABLE 4: LIST OF ENZYMES THAT ARE INVOLVED IN INOSITOL PHOSPHATE SYNTHESIS INOSITOL PHOSPHATE METABOLISM

^**mtb=Mycobacterium tuberculosis 1435

^***mle= Mycobacterium leprae TN

Supplementary: 

FIG. 1: REFERENCE PATHWAY FOR INOSITOL PHOSPHATE SYNTHESIS

FIG. 2: REPRESENTATION OF HOMOLOGY BASED INOSITOL SYNTHESIS PATHWAY IN M. LEPRAE

Interaction of query protein against COG was depicted in Fig. 3A and Phylogenetic analysis of query with other COG proteins were depicted in Fig. 3-B. There are two enzymes, Q57240 (COG1260) andP46813 (COG0483), which catalyzes the reactions in inositol phosphate synthesis and are conserved in M. leprae. Out of these two proteins, one protein (Q57240) is a hypothetical protein.

FIG. 3A: NETWORK ANALYSIS OF QUERY PROTEIN SEARCHED AGAINST COG USING STRING

FIG. 3B: PHYLOGENETIC ASSOCIATION OF QUERY PROTEIN AGAINST COG PROTEINS

CONCLUSION: Sequence based possible drug target for M. leprae has been identified by computational approach ^{23, 24, 25}. 1603 protein sequences for M. leprae were present in UniProt-KB database, out of which, a staggering 27% and more remains classified as a hypothetical uncharacterized set of proteins. Out of these hypothetical uncharacterized set of proteins, 177 set of proteins had GO term. Out of these 177 protein sequences, 43 had DO term while 13 (out of these 43 sequences) were having homology with human proteome. Remaining 30 hypothetical uncharacterized protein sequences were observed which had DO term but were not homologous with human proteome ^{26, 27}. Thus, the 30 uncharacterized proteins may be used as putative drug target for M. leprae.

Out of 177 protein sequences which had GO term, 101 sequences displayed homology with Prokaryotes. These 101 sequences were found to be homologous with 494 DEG genes. KASS was used to predict Pathway associations for these 177 sequences and we found that 50 sequences had KEGG orthology hits. Out of 50, 15 sequences are found to be associated with 39 KEGG pathways ^{28, 29}. These protein sequences regulated the various metabolic pathways ³⁰. However, out of 39 pathways, inositol phosphate metabolism, fatty acid degradation, ethyl-benzene degradation, sulfur relay system and limonene & pinene degradation were common metabolic pathway, which might be used as putative drug target for M. leprae.  Further, we reconstructed the homology based inositol metabolic pathway for M. leprae.

ACKNOWLEDGMENTS: The authors acknowledge the Department of Applied Sciences, Indian Institute of Information Technology, Allahabad for providing computing facility.

CONFLICT OF INTEREST: The authors have no Conflict of Interest.

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    How to cite this article:

    Raj U, Pathak S, Aier I, Gupta S, Varadwaj PK: In silico finding of the putative drug targets from hypothetical set of proteins for Mycobacterium leprae tn. Int J Pharm Sci Res 2017; 8(5): 2100-21.doi: 10.13040/IJPSR.0975-8232.8(5).2100-21.

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