Exploring bioactive compounds from Arthrospira platensis related for treatment of Systemic lupus erythematosus using network analysis

Results

Compounds disclosure from A. platensis C1 by multi-retrieving methods

A total of 95 metabolic pathways of A. platensis C1 (Additional file 1) were retrieved from the Spirulina-Proteome Repository (SpirPro) database [14]. The main metabolic pathways of this organism were related to carbohydrate, amino acid, and vitamin metabolism, which consisted of 15, 13, and 11 pathways, respectively. To find the compounds in A. platensis C1, enzymes that produced the metabolic products from substrates were firstly sought. Seventy-nine of these 95 pathways had enzyme-related metabolites, while the other 16 pathways did not. A total of 699 EC numbers (enzyme number in the KEGG database) were retrieved (Additional file 2). The syn00230 pathway associated with purine metabolism had the highest EC numbers of 41 enzymes. Many of these complex pathways contained only a few Spirulina-specific EC numbers. The 415 non-redundant enzymes were collected resulting a total of 968 products produced. After removing duplicated products, the remaining 369 non-redundant products were collected. These products were linked to 369 SIDs in the PubChem database. However, data on the chemical structures were only available for 302 CIDs. Literature review and text mining of published data were further done to find all known and potential chemical compounds in A. platensis C1. A genome-scale metabolic model of A. platensis C1 containing 692 genes, 837 metabolites, and 875 reactions has been reported [17], from which a total of 501 compounds were retrieved with their relevant CIDs. After combining the retrieved data, from SpirPro, Klanchui et al. [17], and literature review (Table 1), and excluding all redundancies, a total of 833 CIDs (Additional file 3) were obtained and used for further analysis.  

Table 1. Compounds of A. platensis C1 retrieved from various sources.

EC; enzyme number in KEGG database, CID; compound identifier in PubChem database, NA; not available

Computational screening of immunosuppressive agents from various databases

Data on both the approved and potential immunosuppressive drugs for SLE treatment and their identity numbers were retrieved from various databases (DrugBank, KEGG, PubChem, TTD, DailyMed, and the ATC/DDD). These drugs and their identify numbers were matched with CID numbers in the PubChem database. Proteins and chemical compounds related to immunologic effects were also searched in PubChem database, using the terms “immunomodulator”, “immunosuppression”, “immunosuppressive”, and “immunosuppressive agents” as recommended by NCBI (Medical Subject Heading; MeSH). The corresponding identity (GI) numbers were also collected. After combined the data and excluding the redundancies, a total of 281 chemical compounds related to immunologic effects were obtained (Additional file 4). These were later matched with the compounds retrieved from A. platensis C1.

Structural similarity matching between compounds retrieved from A. platensis C1 and immunosuppressive agents

To find the algal potential bioactive compounds to be used as SLE drugs or immunosuppressive agents, matching between all potential compounds retrieved from A. platensis C1 and all immunosuppressive agents was done. Details of the 234,073 similarity matchings with their scores are shown in the Additional file 5. The structural similarity matching between the 833 compounds retrieved from this alga and 281 immunosuppressive agents resulting in 1637, 412, 203, 103, 28, and 5 matchings with Tanimoto scores of > 70%, > 80%, > 85%, > 90%, > 95%, and 100%, respectively (Additional file 6). For example (Table 2), there are 5 compounds, including cytidine, desthiobiotin, lomustine, agmatine, and anthranilic acid, matching of 100% with known Immunosuppressive agents. Whereas others, metrotrexate drug had a matching score of 87% with folic acid. Lobenzarit drug had a matching score of 80% with anthranillic acid, 72% with 4- aminobenzoic acid, and 71% with 1-(2-carboxyphenylamino)-1-deoxyribulose 5-phosphate.

Additionally, there were 103 matchings with Tanimoto scores of > 90%. The immunosuppressive agents, namely 8-aminoguanosine, 3868-32-4 (2,8-Diamino-9-[3,4-dihydroxy-5-(hydroxymethyl)-2-oxolanyl]-3H-purin-6-one), fludarabine phosphate, and cladribine had the most frequent matchings of 24 (23.3%), 24 (23.3%), 16 (15.5%), and 11 (10.7%), respectively. The compounds: cytidine, 5'-cytidylic acid, cytidine 5'-diphosphate, desthiobiotin, and cytidine triphosphate had the most frequent matchings of 8 (7.8%), 6 (5.8%), 6 (5.8%), 3 (2.9%), and 3 (2.9%), respectively.

Table 2. Structural similarity scores between the 833 compounds from A. platensis C1 and 281 immunosuppressive agents. 

Bioassay matching between compound bioassay of A. platensis C1 and bioassay associated with SLE

PubChem was searched to find all proteins and bioassays associated with SLE.  A total of 660 bioassays and their relevant assay identifier (AID) numbers were also retrieved (Additional file 7). The five compounds and their related bioassays were matched with the 660 bioassays associated SLE. AIDs and targets (proteins or nucleotides) of these matchings, between bioassays associated with SLE and bioassays associated with immunosuppressive compounds, were as follows (Additional file 8); SLE and cytidine: AID504734 protein target - inhibitor of human Toll-like receptor 9; SLE and lomustine: AID625175 protein target - protease, cathepsin G enzyme inhibition, and AID625176 protein target - peptidase, ELA2 (Neutrophil Elastase 2) enzyme inhibition; SLE and anthranilic acid: AID651758 protein target - interleukin 8 stimulation. Unfortunately, all of the SLE matching bioassays above are non-active bioassays. However, cytidine, a nucleoside molecule, has 7 active bioassays with activities against Plasmodium falciparum, antibabesial activity, and Ewings sarcoma. Whereas, desthiobiotin has only 1 active bioassay for in vivo anticancer drug screening for tumor model Friend Virus Leukemia in mice. Though, cytidine and desthiobiotin were respectively identified in the pyrimidine and biotin metabolic pathways in A. platensis, the latter desthiobiotin has been defined as an immunosuppressive agent in MESH term. Lastly, Lomustine, an alkylating antineoplastic agent, has 39 active bioassays in vivo anticancer drug tests. As most of the approved SLE treatment drugs and the potential drugs that can be used are categorized in the immunosuppressant and antineoplastic groups, so that these 3 compounds which have anticancer activities can be proposed the potential to be used for SLE treatment.

Disease-gene-drug-compound network for SLE

The disease-gene-drug network was created using VisANT 4.0. The network consisted of 471 genes/proteins associated with SLE, 219 antineoplastic drugs, and 54 immunosuppressive drugs (Figure 2). From the matchings between 833 compounds retrieved from A. platensis C1 and 281 immunosuppressive agents with Tanimoto scores of > 90%; two immunosuppressive agents, namely fludarabine phosphate [18] and cladribine [19], were included in this SLE-gene-drug network. Only fludarabine phosphate was found to be associated with the SLE-related genes, as shown in the figure.

The disease-gene-drug-compound network was created by mapping 5 bioactive compounds from A. platensis C1 with the greatest potential, associated-immunosuppressive agents, and 471 genes/proteins associated with SLE onto the interaction network, after which the network was visualized using Cytoscape (Figure 3). 

Integration of these data in the network offered insights into the potential immunologic effect of the compounds and their target genes/proteins. The target genes for cytarabine were MDM2, TP53, and JAK2, and targets for d-dethiobiotin were PPARG and IL1B. All of these 5 genes associated with SLE. Cytidine and desthiobiotin, from A. platensis C1, had a structural similarity score of 100% with cytarabine and d-dethiobiotin, respectively. Lomustine, agmatine, and anthranilic acid, from A. platensis C1, did not have target genes associated with SLE directly. These three compounds are defined as immunosuppressive agents, and their targets are shown in the network (Figure 3). 

Molecular docking and molecular dynamics of the potential bioactive compounds

Cytidine, desthiobiotin, lomustine, agmatine, and anthralinic acid, were identified as the bioactive compounds with potential for SLE treatment from the above analyses. Only the first two bioactive compounds, cytidine and desthiobiotin (Figure 4), were not annotated as the immunosuppressive drugs by themselves. To determine their potential drug effect, molecular docking was used to check the binding affinity between cytarabine drug (ligand), cytidine bioactive compound (ligand) and the following receptors: MDM2, TP53, and JAK2; and the binding affinity between d-dethiobiotin drug (ligand), destiobiotin bioactive compound (ligand) and the following receptors: PPARG and IL1B. Both cytidine and desthiobiotin had good docking energy scores with their corresponding receptor proteins and similar to cytarabine and d-dethibiotin (Table 3). Additionally, MD simulation was performed to confirm the binding affinity of the compound and receptor protein. The RMSD plot for backbone, complex, and ligand of cytidine, desthiobiotin and their corresponding receptor proteins were shown (Figure 5). The RMSD values of all complexes were rather stable and maintained within the fluctuation of only 1 Å. The RMSD values of all complexes showed very small fluctuation and reached the equilibrium within 5 ns. The RMSD of backbone displayed the same fluctuation pattern with the complex. 

Table 3. Binding affinity scores between the ligands including drugs and bioactive compounds, with their corresponding receptors by molecular docking

Discussion

SLE is a chronic autoimmune disease with a wide spectrum of clinical features and unknown etiology [10]. Serious side effects may occur with conventional immunosuppressive treatment. Identification of biological therapeutic agents targeting molecular mediators with limited side effects can be challenging. Spirulina has immunomodulatory activity, and its benefits have been studied in patients with various diseases.

Metabolic pathways and potential bioactive compounds of A. platensis C1 for SLE treatment were investigated in this study. Unlike other strains, A. platensis C1 produces a single colony with non-gliding ability; and can be used for arsenic culture selection. Based on these abilities, this strain is used for genetic studies in the laboratory. Extensive research on both lipid desaturation mechanisms and physiological conditions for cell growth have been performed using this strain. The whole genome sequence of A. platensis C1 at the GenBank database of the Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/genome/?term=Arthrospira+platensis) is 6,089,210 bp long and contains 6,108 protein-coding genes, 45 RNA genes, and no plasmids [20]. The metabolic pathway of A. platensis from the Encyclopedia of Genes and Genomes (KEGG) database belongs to A. platensis NIES39, not C1. Fortunately, an organism-specific database for A. platensis C1 has been created and can be retrieved from the Spirulina-Proteome Repository (SpirPro) database.

Immunosuppressive drugs are the mainstays of treatment for SLE [21]. Some antineoplastic drugs are used to treat autoimmune diseases. Drugs used to treat other autoimmune diseases may also work for SLE treatment because these conditions are characterized by an overactive immune system. In this study, all approved and potential drugs or agents for SLE treatment were searched and used for analysis. Immunosuppressive, immunomodulatory, and antineoplastic agents were collected from various databases, namely DrugBank, KEGG, PubChem, TTD, DailyMed, and ATC/DDD. The DrugBank database is a unique bioinformatics and cheminformatics resource that combines detailed drug data with comprehensive drug target information. It contains 12,147 drug entries including 2,557 approved small molecule drugs, 1,285 approved biotech (protein/peptide) drugs, 130 nutraceuticals, and over 5,865 experimental drugs. Additionally, 5,167 protein (i.e. drug target/enzyme/transporter/carrier) sequences are linked to these drug entries. The DailyMed web contains 108,551 drug listings as submitted to the Food and Drug Administration (FDA). It provides high quality information about marketed drugs. The ATC/DDD is a tool for exchanging and comparing data on drug use at international, national, or local levels. The ATC/DDD has become the gold standard for international drug utilization research. This system is developed and maintained by the WHO Collaborating Centre for Drug Statistics Methodology, http://www.whocc.no. A total of 281 drugs and chemical compounds related to immunologic effect were collected from these databases.

From the structural similarity matchings between the compounds retrieved from A. platensis C1 and immunosuppressive agents with Tanimoto scores of ≥ 90%, fludarabine phosphate was one of the immunosuppressive agents with the most frequent matching. Its chemical structure is almost similar to that of adenosine monophosphate found in A. platensis C1. Fludarabine phosphate was found to be associated with SLE-related genes and may be used to treat this autoimmune disease [18, 22, 23]. Adenosine monophosphate plays an important role in many cellular metabolic processes, and it is also a component in the synthesis of RNA. It is found in many kinds of organism, therefore not a unique and interesting compound in Spirulina.

Cytidine had structural similarity matching with a Tanimoto score of 100% with three immunosuppressive agents: beta-arabinosylcytosine, arabinofuranosylcytosine, and cytarabine. The target genes for cytarabine are MDM2, TP53, and JAK2. All of these genes are associated with SLE. Cytidine also has bioassay activity against Ewings sarcoma. The antineoplastic activity of cytidine may be used for autoimmune disease treatment. Desthiobiotin had structural similarity matching with a Tanimoto score of 100% with d-dethiobiotin, an immunosuppressive agent. The target genes for d-dethiobiotin are PPARG and IL1B; these two genes are associated with SLE. Desthiobiotin has been defined as an immunosuppressive agent in MESH term. Desthiobiotin has one active bioassay for in vivo anticancer drug screening for tumor model Friend Virus Leukemia in mice. Cytidine and desthiobiotin are respectively identified in the pyrimidine and biotin metabolic pathways of A. platensis. Lomustine, agmatine, and anthranilic acid, which are found in A. platensis C1, are classified as immunosuppressive agents in various drug databases. Lomustine also has active bioassays for in vivo antineoplastic drug tests and has been studied in patients with progressive glioblastoma [24].

Our findings identified cytidine and desthiobiotin as bioactive compounds in A. platensis C1 with the highest potential for use as immunosuppressive agents against SLE. The results from disease-gene-drug-compound network for had been confirmed by molecular docking and MD simulation. Both cytidine and desthiobiotin had good docking score with their corresponding receptor proteins and similar to cytarabine and d-dethibiobin, the immunosuppressive agents. The rather stable RMSD of cytidine and desthiobiotin ligands with SLE target proteins indicated the high confidence for disease specific therapy. Lomustine, agmatine, and anthranilic acid may be used for SLE treatment because of their own immunosuppressive activities. All of these bioactive compounds should be clinically studied for their medical use in relation to SLE.

Several studies have shown that Spirulina enhances both the mucosal and systemic immune systems. It has immunosuppressive effects on both humoral and cell-mediated immune responses, and its immunomodulatory activity in patients with allergic rhinitis and in elderly patients are well documented. Bioactive compounds in Spirulina and mechanisms of action in these conditions and other autoimmune diseases also need to be further investigated and established.

Conclusions

This work investigated the targets and mechanisms of the immunosuppressive activity of bioactive compounds in Spirulina (A. platensis C1) against SLE. Cytidine, desthiobiotin, lomustine, agmatine, and anthralinic acid, were identified as the bioactive compounds with enormous potential for SLE treatment, based on structural similarity matching, bioassay matching, disease-gene-drug-compound network analysis, and molecular docking and dynamics.

Abbreviations

SLE: systemic lupus erythematosus; KEGG: Kyoto Encyclopedia of Genes and Genomes; SpirPro: Spirulina-Proteome Repository; CID: compound identifier; SID: substance identifier; AID: assay identifiers; EC: enzyme number

Declarations

Ethics approval and consent to participate: Not applicable

Consent for publication: Not applicable

Availability of data and materials: Sample of the dataset used for this study are included in the additional file 3, 4 and 7 accompanying this article. 

Competing interests: The authors declare that they have no competing interests.

Funding: None

Authors' contributions: AC, MR and TL designed the research. AC performed the research, analyzed the results and prepared the manuscript. HP analyzed molecular docking and MD simulation. MR and TL supervised the research and assisted with results analysis and manuscript preparation. All authors read and approved the final version of this manuscript. 

Acknowledgements

We thank Dr. Weerayuth Kittichotirat and Dr. Yaowaluck Maprang Roshorm, King Mongkut's University of Technology Thonburi, Bangkok Thailand, for their helpful guidance and expert advice. We also thank Mr. Oscar Nnaemeka, School of Bioresource and Technology, King Mongkut's University of Technology Thonburi, Bangkok Thailand, for editing and proofreading the manuscript.

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