Bandi Deepa Reddy and Ch. M. Kumari Chitturi*

Department of Applied Microbiology, Sri Padmavati Mahila Visvavidyalayam
(Women’s University), Tirupati, Andhra Pradesh, India.

Abstract

Legumain an asparginyl endopeptidase expressed by both tumor cells and cells present in tumor microenvironment is an ideal therapeutic target for development of cancer therapies due to its correlation with high metastasis and invasion in various cancers. Microbial derivatives have demonstrated many pharmacological properties such as antioxidant, anti-inflammatory, anti tumor and immunostimulatory activities.In the current study, 541 microbial derivatives were screened for their potential to inhibit legumain using Lib dock .Out of 541 compounds screened we have identified 55 microbial derivatives which showed binding to legumain by docking. Molecular interaction analysis of top five docked derivatives revealed the interaction of derivatives with the catalytic residues of legumain. These compounds need to be further evaluated in vitro and in vivo for Legumain inhibition and ultimately cancer regression.

Keywords: In silico, Legumain, Lib dock, Microbial derivatives.

Introduction

Legumain (LGMN) also known as asparaginyl endopeptidase (AEP) is implicated in various cancer such as prostrate, breast, colon, lung, ovarian, central nervous system (CNS) related cancers, melanoma and lymphoma1.LGMN expression has also been reported in Tumor associated macrophages (TAM) also called as M2 macrophages2. LGMN is sparsely expressed by the normal tissues 1.LGMN undergoes series of maturation steps from its pro-enzyme form to become proteolytically active3. LGMN expression has been correlated with low apoptosis and high invasion and metastasis of cancer cells both in vitro and in vivo1.LGMN is expressed not only in tumor cells but also found in the cells present in tumor microenvironment. Hence it holds the potential of serving as a prognostic factor and as a therapeutic target in cancer1,2,4.

Microbial derivatives have shown promising results in the development of therapies for cancer5.Bacterial Azurin produced from Pseudomonas aeruginosa has demonstrated cytotoxicity towards cancer cell lines such as Melanoma (UISO-Mel-2)6 and breast cancer (MCF-7)7 cell lines in vitro. It has also shown to increase apoptosis mediated by stabilising p53 and increasing the expression of pre-apoptotic protein Bax6,7,8.Trichostatin produced from Streptomyces hygroscopicus is a well-known Histone deacetylase(HDAC)inhibitor , a validated target for the development of antitumor therapies9.Thiocoraline bioactive compound isolated from Micromonospora marina,has shown selective cytotoxicity against lung and colon cancer cell lines as well as melanoma10.Macrolactin-A a major metabolite of  Noctilucascintillans is reported to inhibit B16-F10 murine melanoma cancer cells11.Borophycina boron-containing metabolite, isolated from Nostoclinckia and N. spongiaeforme var. tenue, marine cyanobacterial strains has exhibited cytotoxicity against human epidermoid carcinoma (LoVo) and human colorectal adenocarcinoma (KB) cell lines 12,13.

As evidenced by the literature about the potential of microbial derivatives in the development of antitumor therapies, the current study employs the use of in silico tools for screening and identification of LGMN inhibitors. In silico methods have been efficient and quicker for the virtual screening of compounds with a known target protein. Molecular docking is one of the in silico approaches which plays a major role in computer aided drug designing by predicting the binding of lead compounds in the active sites of target proteins.

In the current study we have screened 541 microbial derivatives for their potential to inhibit LGMN by using Lib dock14 module available in Accelrys Discovery Studio 3.5 (San Diego, CA, USA ).

MATERIALS AND METHODS

Selection of LGMN structure from Protein Data Bank
The Crystal structure of active LGMN in complex with YVAD-CMK at pH 5.0 15 was retrieved from Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB ID: 4AWA) (http://www.rcsb.org/pdb). All bound water molecules, other hetero atoms and ligands were removed manually from the PDB file prior to docking. The protein was prepared using “Prepare Protein”module available in discovery studio 3.5.

Generation of ligand dataset
The structures of 541 microbial derivatives (ligands) were collected from PubChem compound database (https://pubchem.ncbi.nlm.nih.gov/). Prior to docking, the ligands were prepared using the ‘‘prepare ligand’’ module available in Discovery studio 3.5.

Active site analysis of 4AWA structure
Prediction of active site is crucial step in molecular docking studies for identification of potent inhibitors.As per the literature LGMN harbours a catalytic triad consisting of three amino acid residues (Cys189-His148-Asn42)15.A receptor grid was created around the binding cavity (active sites) of protein by specifying the key amino acid residues (Cys 189, His 148 and Asn42 ). Binding site sphere was set and 35.78,24.36 and -7.80 are the dimensions of X,Y and Z respectively.

Molecular Docking using Discovery Studio 3.5
To identify new compounds that could potentially inhibit LGMN through binding to the catalytic triad pocket, a virtual screening is carried out using Lib dock module of Discovery Studio 3.514. Lib dock docks ligand into the active site by calculating hot spots and using polar and a polar probes and these hot spots are further used to align ligands to form interactions 16.The default lib dock protocol available in the module was used for the docking.Details of successful and failed ligands are available in the “docked ligands” and “failed ligands” sections respectively of the result file. Different Poses of protein-ligand complex were obtained after successful docking process with their specific lib dock score displayed on it. The interactions between the ligand and the protein molecules were investigated using “Analyze ligand poses” and “2D diagram” of docked receptor-ligand complexes. This analysis gives better idea of interactions between the key residues of protein and complimentarygroups/atoms of ligands.

RESULTS

The crystal structure of LGMN (PDB ID:4AWA) was retrieved from protein data bank and was prepared using prepare protein module. Active site pocket was created using catalytic residues of LGMN (Cys189-His148-Asn42).

Fig. 1A depicts the 3D structure of LGMN retrieved from PDB. Fig.1B illustrates the prepared structures of the protein after removal of hetero atoms, ligands and water molecules with a sphere around the active site.

Fig 1A. 3D Structure of LGMN (PDB ID: 4AWA)

Fig 1B. 3D Structure of prepared LGMN with active sphere shown (PDB ID: 4AWA)

A total of 541 microbial derivatives were docked at the catalytic site of LGMN using Lib Dock. Among the derivatives docked,55 compounds demonstrated successful docking at the catalytic site of LGMN. All the docked poses were ranked by the Lib dock score. The list of compounds docked successfully with their respective lib dock score has been given in Table 1.

Table 1. List of 55 successfully docked microbial derivatives at the active site of LGMN

S.No Name of the compounds Pubchem ID Lib dock score
1 Blasticidin S hydrochloride 356629 117.08
2 Bicyclomycin benzoate 91618023 99.62
3 α-Zearalenol 5284645 89.35
4 Sinefungin 65482 85.47
5 9-Methylstreptimidone 6373950 85.15
6 Cerulenin 5282054 83.99
7 Mycophenolic acid 446541 83.39
8 4-Hydroxyalternariol 118797633 82.72
9 LL Z1640-2 46882176 81.18
10 Tetradecanoyl-L-homoserine lactone 58122267 79.71
11 Dodecanoyl-L-homoserine lactone 11565426 79.70
12 Epitetracycline hydrochloride 54686189 78.96
13 Tetracycline 54675776 78.96
14 Tetracycline hydrochloride 54704426 78.96
15 Thiamphenicol 27200 78.08
16 Toyocamycin 11824 76.78
17 Toxoflavin 66541 76.77
18 Deacetylanisomycin 11790817 76.45
19 Bestatin 72172 76.20
20 21-Hydroxyoligomycin A 3016254 75.98
21 Corynecin III 101131598 75.95
22 Terrein 6436830 75.02
23 RK-682 54678922 74.51
24 TAN 1364B 54690140 74.51
25 Sancycline 54688686 74.16
26 Sancycline hydrochloride 54712662 74.16
27 Octanoyl-L-homoserine lactone 6914579 73.99
28 Chloramphenicol succinate sodium 656833 73.87
29 Methacycline 54675785 73.42
30 Methacycline hydrochloride 54685047 73.42
31 Avenaciolide 11747526 72.96
32 Anisomycin 253602 72.64
33 LL Z1640-4 57370130 72.57
34 Clavulanate potassium 23665591 71.69
35 Germicidin B 86169826 71.30
36 Florfenicol amine 156406 70.43
37 Corynecin IV 133562649 69.99
38 Brefeldin A 5287620 69.84
39 Germicidin A 102106080 69.39
40 Clindamycin hydrochloride 16051951 68.16
41 Dihydroaeruginoic acid 5381954 67.72
42 Tenuazonic acid 54683011 66.72
43 Roquefortine E 5326324 64.45
44 Cycloechinulin 16088234 64.05
45 Butyryl-L-homoserine lactone 10130163 62.80
46 Moniliformin 40452 62.21
47 acetyl-L-homoserine lactone 10012012 61.35
48 Chloramphenicol acetate 83940 60.58
49 Hexanoyl-L-homoserine lactone 10058590 60.45
50 Simvastatin 54454 59.49
51 Aphidicolin 457964 58.92
52 Chloramphenicol 5959 57.79
53 Butyrolactone I 7302 51.49
54 Roquefortine C 5935070 51.38
55 Cellocidin 10971 39.88

The top 5 derivatives with highest lib dock scores were further used to evaluate the interactions with LGMN.

Interactions of Blasticidin S hydrochloride at LGMN catalytic site
Blastocidin S hydrochoirde is a salt of Blasticidin S a nucleoside antibiotic, produced by Stretomyces species. Blasticidin S HCl acts  as a DNA and protein synthesis inhibitor17,18.

Blasticidin S hydrochloride interacted with all the three amino acids of catalytic residues Cys 189, His 148 and Asn 42 by forming hydrogen bonds. In addition, it has also interacted with Asp 231,Gly 149, Asp 147 with hydrogen bonding. The molecular interaction analysis indicates Blasticidin S hydrochloride as potent inhibitor of LGMN owing to its interaction with the catalytic triad amino acids residues and nine hydrogen bonds at the active site. Fig 2A illustrates 2D diagram of interactions of Blasticidin S hydrochloride at the LGMN catalytic site and

Fig 2B shows the 3D diagram of interactions of Blasticidin S hydrochloride at the LGMN catalytic site

Fig 2A. 2D diagram showing interactions of Blasticidin S hydrochloride at LGMN catalytic site

Fig 2B. 3D diagram showing interactions of Blasticicidn S hydrochroideat LGMN catalytic site

Interactions of Bicyclomycin benzoate at LGMN catalytic site
Bicyclomycin benzoate is an antibiotic produced by Streptomyces sapporonensis and it inhibits gram negative bacteria.

Bicyclomycin benzoate interacts with LGMN at the active site by forming hydrogen bonds with Asn 42 (catalytic aminoacid), Arg 44 and Ala 218.In addition, other interactions such as van der Waals, pi-Alkyl and pi-cation are also observed in the 2D diagram.

Fig 3A illustrates 2D diagram of interactions of Bicyclomycin benzoate at the LGMN catalytic site and Fig 3B shows the 3D diagram of interactions of Bicyclomycin benzoateat the LGMN catalytic site.

Fig 3A. 2D diagram showing interactions of Bicyclomycin benzoate at LGMN catalytic site

Fig 3B. 3D diagram showing interactions of Bicyclomycin benzoate at LGMN catalytic site

Interactions of α-Zearalenol at LGMN catalytic site
α-Zearalenol is an oestrogenic mycotoxin produced by several species of Fusarium that contaminate cereal crops19.

α-Zearalenol interacts with LGMN at the active site by forming two hydrogen bonds with catalytic amino acids Cyst 189 and His 148.In addition,other interactions such as van der Waals, pi-Alkyl and pi-cation are also observed in the 2D diagram.

Fig 4A illustrates 2D diagram of interactions of α-Zearalenol at the LGMN catalytic site and Fig 4B shows the 3D diagram of interactions of α-Zearalenol at the LGMN catalytic site.

Fig 4A. 2D diagram showing interactions of α-Zearalenol at LGMN catalytic site

Fig 4B. 3D diagram showing interactions of α-Zearalenol at LGMN catalytic site

Interactions of Sinefungin at LGMN catalytic site
Sinefungin is an inhibitor of transmethylation reactions associated to DNA, RNA and Proteins. It is a natural nucleoside with antifungal , antiviral and antiprotozoal activities20,21

Sinefungin interacts with LGMN at the active site by forming three hydrogen bonds with Arg 44, Ser 216 and Asp 231. It interacts with the catalytic residues such as Asn 42 with van der Waal and His 148 with Pi-Pi stacked interactions.

Fig 5A illustrates 2D diagram of sinefungin at the LGMN catalytic site and Fig 5B shows the 3D diagram of interactions of sinefungin at the LGMN catalytic site.

Fig 5A. 2D diagram showing interactions of sinefungin at LGMN catalytic site

Fig 5B. 3D diagram showing interactions of sinefungin at LGMN catalytic site

Interactions of 9-Methylstreptimidoneat LGMN catalytic site
9-Methylstreptimidone is isolated from Streptomyces species.
9-Methylstreptimidone exhibits antifungal and antiviral activity. Also known as an inhibitor of the nuclear factor, NF-κB22.

9-Methylstreptimidone interacts with LGMN at the catalytic site by forming three hydrogen bonds with Cys 189(catalytic amino acid), Asp 147 and Gly 149.It interacts with the other catalytic residues such as Asn 42 and His 148 with vander Waal interactions. Other interactions such as carbon hydrogen bond and Pi alkyl stacked interactions are also observed.

Fig 6A illustrates 2D diagram of interactions between 9-Methylstreptimidone at the LGMN catalytic site and Fig 6B shows the 3D diagram of interactions between 9-Methylstreptimidone at the LGMN catalytic site.

Fig 6A. 2D diagram showing interactions of 9-Methylstreptimidone at LGMN catalytic site.

Fig 6B. 3D diagram showing interactions of 9-Methylstreptimidone at LGMN catalytic site.

CONCLUSION

The objective of the current study was to screen and identify microbial derivatives for their potential to inhibit LGMN activity using in silico approaches. Molecular docking of microbial derivatives has identified 55 potential LGMN inhibitors from 541 screened using Lib dock module. The results of this study not only demonstrate the probable binding mode of these derivatives with LGMN, but also encourage further evaluation of these microbial derivatives both in vitro and in vivo for LGMN inhibition and cancer regression.

ACKNOWLEDGEMENT

The authors gratefully acknowledge the support of Department of Applied Microbiology, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati, Andhra Pradesh, Indiawithout which the present study could not have been completed.

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