Printer Friendly

Screening and Identification of Microbial Derivatives for Inhibiting Legumain: An In silico Approach.

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 lymphoma (1). LGMN expression has also been reported in Tumor associated macrophages (TAM) also called as M2 macrophages (2). LGMN is sparsely expressed by the normal tissues1. LGMN undergoes series of maturation steps from its pro-enzyme form to become proteolytically active (3). LGMN expression has been correlated with low apoptosis and high invasion and metastasis of cancer cells both in vitro and in vivo (1). 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 cancer (1,2,4).

Microbial derivatives have shown promising results in the development of therapies for cancer (5). 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 Bax (6,7,8). Trichostatin produced from Streptomyces hygroscopicus is a well-known Histone deacetylase (HDAC) inhibitor, a validated target for the development of antitumor therapies (9). Thiocoraline bioactive compound isolated from Micromonospora marina, has shown selective cytotoxicity against lung and colon cancer cell lines as well as melanoma (10). Macrolactin-A a major metabolite of Noctilucascintillans is reported to inhibit B16-F10 murine melanoma cancer cells (11). 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 dock (14) 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.

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.

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 Streptomyces species. Blasticidin S HCl acts as a DNA and protein synthesis inhibitor (17,18).

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.

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.

Interactions of [alpha]-Zearalenol at LGMN catalytic site

[alpha]-Zearalenol is an oestrogenic mycotoxin produced by several species of Fusarium that contaminate cereal crops (19).

[alpha]-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 [alpha]-Zearalenol at the LGMN catalytic site and Fig 4B shows the 3D diagram of interactions of [alpha]-Zearalenol at the 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 activities (20,21)

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.

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-kB22.

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.

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.

http://dx.doi.org/10.22207/JPAM.12.3.69

(Received: 06 July 2018; accepted: 12 August 2018)

ACKNOWLEDGEMENT

The authors gratefully acknowledge the support of Department of Applied Microbiology, Sri Padmavati Mahila Visvavidyalayam (Women's University), Tirupati, Andhra Pradesh, India without which the present study could not have been completed.

REFERENCES

(1.) Liu C, Sun C, Huang H. Overexpression of Legumain in Tumors Is Significant for Invasion / Metastasis and a Candidate Enzymatic Target for Prodrug Therapy. 2003: 2957-2964.

(2.) Luo Y, Zhou H, Krueger J, et al. JCI - Targeting tumor-associated macrophages as a novel strategy against breast cancer. 2006; 116(8). doi:10.1172/JCI27648

(3.) Zhao L, Hua T, Crowley C, et al. Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage. Cell Res. 2014. doi:10.1038/cr.2014.4

(4.) Mai C-W, Chung FF-L, Leong C-O. Targeting Legumain As a Novel Therapeutic Strategy in Cancers. Curr Drug Targets. 2017; 18(11). doi: 10.2174/1389450117666161216125344

(5.) Bernardes N, Seruca R, Chakrabarty AM, Fialho AM. Microbial-based therapy of Cancer Current progress and future prospects. Bioeng Bugs. 2010. doi:10.4161/bbug.1.3.10903

(6.) Gupta DT. Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer. Proc Natl Acad Sci U S A. 2002; 22: 1409814103. doi:10.1073/pnas.222539699

(7.) Yamada T, Hiraoka Y, Ikehata M, et al. Apoptosis or growth arrest: Modulation of tumor suppressor p53's specificity by bacterial redox protein azurin. Proc Natl Acad Sci U S A. 2004. doi:10.1073/pnas.0400899101

(8.) Apiyo D, Wittung-Stafshede P. Unique complex between bacterial azurin and tumor-suppressor protein p53. Biochem Biophys Res Commun. 2005. doi:10.1016/j.bbrc.2005.05.038

(9.) Vigushin DM, Ali S, Pace PE, et al. Trichostatin A is a histone deacetylase inhibitor with potent antitumor activity against breast cancer in vivo. Clin Cancer Res. 2001. doi:10.1016/s00928674(00)80211-1

(10.) Sithranga Boopathy N, Kathiresan K. Anticancer drugs from marine flora: An overview. J Oncol. 2010. doi:10.1155/2010/214186

(11.) Carte BK. Biomedical potential of marine natural products. Bioscience. 1996. doi:10.2307/1312834

(12.) Banker R, Carmeli S. Tenuecyclamides A-D, cyclic hexapeptides from the cyanobacterium Nostoc spongiaeforme var. tenue. J Nat Prod. 1998. doi:10.1021/np980138j

(13.) Davidson BS. New dimensions in natural products research: cultured marine microorganisms. Curr Opin Biotechnol. 1995. doi:10.1016/09581669(95)80049-2

(14.) Diller DJ, Merz KM. High throughput docking for library design and library prioritization. Proteins Struct Funct Genet. 2001. doi:10.1002/10970134(20010501)43:2 <113::AID-PROT 1023> 3.0.C0;2-T

(15.) Dall E, Brandstetter H. Mechanistic and structural studies on legumain explain its zymogenicity, distinct activation pathways, and regulation. Proc Natl Acad Sci U S A. 2013. doi:10.1073/ pnas.1300686110

(16.) Zhou X, Yu S, Su J, Sun L. Computational Study on New Natural Compound Inhibitors of Pyruvate Dehydrogenase Kinases. 2016. doi:10.3390/ijms17030340

(17.) Yamaguchi H, Tanaka N. Inhibition of protein synthesis by blasticidin S: II. studies on the site of action in e. coli polypeptide synthesizing systems. J Biochem. 1966;60(6):632-642. doi:10.1093/oxfordjournals.jbchem.a128489

(18.) S.B. Sullia and D.H. Griffin. Inhibition of DNA synthesis by Cycloheximide and Blasticidin-S is Independent of their effect on protein synthesis. Biochim Biophys Acta, 475.

(19.) Bennett JW, Klich M, Mycotoxins M. Mycotoxins. Clin Microbiol Rev. 2003. doi:10.1128/CMR.16.3.497

(20.) Barbes C, Sanchez J, Yebra MJ, Robert-Gero M, Hardisson C. Effects of sinefungin and S-adenosylhomocysteine on DNA and protein methyltransferases from Streptomyces and other bacteria. FEMS Microbiol Lett. 1990. doi:10.1016/0378-1097(90)90073-Y

(21.) Zheng S, Hausmann S, Liu Q, et al. Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin's antifungal activity. J Biol Chem. 2006. doi:10.1074/jbc.M607292200

(22.) Ishikawa Y, Tachibana M, Matsui C, Obata R, Umezawa K, Nishiyama S. Synthesis and biological evaluation on novel analogs of 9-methylstreptimidone, an inhibitor of NF-kappaB. Bioorg Med Chem Lett. 2009. doi:10.1016/j.bmcl.2009.01.107.

Bandi Deepa Reddy and Ch. M. Kumari Chitturi *

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

* To whom all correspondence should be addressed. Tel.: +9160091739; E-mail: chandi2222002@yahoo.co.in

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

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

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

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

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

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

Caption: Fig. 4A. 2D diagram showing interactions of [alpha]-Zearalenol at LGMN catalytic site.

Caption: Fig. 4B. 3D diagram showing interactions of [alpha]-Zearalenol at LGMN catalytic site.

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

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

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

Caption: Fig. 6B. 3D diagram showing interactions of 9-Methylstreptimidone at LGMN catalytic site.
Table 1. List of 55 successfully docked microbial
derivatives at the active site of LGMN

S.No   Name of the compounds                 Pubchem ID

1      Blasticidin S hydrochloride             356629
2      Bicyclomycin benzoate                  91618023
3      a-Zearalenol                           5284645
4      Sinefungin                              65482
5      9-Methylstreptimidone                  6373950
6      Cerulenin                              5282054
7      Mycophenolic acid                       446541
8      4-Hydroxyalternariol                  118797633
9      LL Z1640-2                             46882176
10     Tetradecanoyl-L-homoserine lactone     58122267
11     Dodecanoyl-L-homoserine lactone        11565426
12     Epitetracycline hydrochloride          54686189
13     Tetracycline                           54675776
14     Tetracycline hydrochloride             54704426
15     Thiamphenicol                           27200
16     Toyocamycin                             11824
17     Toxoflavin                              66541
18     Deacetylanisomycin                     11790817
19     Bestatin                                72172
20     21-Hydroxyoligomycin A                 3016254
21     Corynecin III                         101131598
22     Terrein                                6436830
23     RK-682                                 54678922
24     TAN 1364B                              54690140
25     Sancycline                             54688686
26     Sancycline hydrochloride               54712662
27     Octanoyl-L-homoserine lactone          6914579
28     Chloramphenicol succinate sodium        656833
29     Methacycline                           54675785
30     Methacycline hydrochloride             54685047
31     Avenaciolide                           11747526
32     Anisomycin                              253602
33     LL Z1640-4                             57370130
34     Clavulanate potassium                  23665591
35     Germicidin B                           86169826
36     Florfenicol amine                       156406
37     Corynecin IV                          133562649
38     Brefeldin A                            5287620
39     Germicidin A                          102106080
40     Clindamycin hydrochloride              16051951
41     Dihydroaeruginoic acid                 5381954
42     Tenuazonic acid                        54683011
43     Roquefortine E                         5326324
44     Cycloechinulin                         16088234
45     Butyryl-L-homoserine lactone           10130163
46     Moniliformin                            40452
47     acetyl-L-homoserine lactone            10012012
48     Chloramphenicol acetate                 83940
49     Hexanoyl-L-homoserine lactone          10058590
50     Simvastatin                             54454
51     Aphidicolin                             457964
52     Chloramphenicol                          5959
53     Butyrolactone I                          7302
54     Roquefortine C                         5935070
55     Cellocidin                              10971

S.No   Name of the compounds                 Lib dock score

1      Blasticidin S hydrochloride               117.08
2      Bicyclomycin benzoate                     99.62
3      a-Zearalenol                              89.35
4      Sinefungin                                85.47
5      9-Methylstreptimidone                     85.15
6      Cerulenin                                 83.99
7      Mycophenolic acid                         83.39
8      4-Hydroxyalternariol                      82.72
9      LL Z1640-2                                81.18
10     Tetradecanoyl-L-homoserine lactone        79.71
11     Dodecanoyl-L-homoserine lactone           79.70
12     Epitetracycline hydrochloride             78.96
13     Tetracycline                              78.96
14     Tetracycline hydrochloride                78.96
15     Thiamphenicol                             78.08
16     Toyocamycin                               76.78
17     Toxoflavin                                76.77
18     Deacetylanisomycin                        76.45
19     Bestatin                                  76.20
20     21-Hydroxyoligomycin A                    75.98
21     Corynecin III                             75.95
22     Terrein                                   75.02
23     RK-682                                    74.51
24     TAN 1364B                                 74.51
25     Sancycline                                74.16
26     Sancycline hydrochloride                  74.16
27     Octanoyl-L-homoserine lactone             73.99
28     Chloramphenicol succinate sodium          73.87
29     Methacycline                              73.42
30     Methacycline hydrochloride                73.42
31     Avenaciolide                              72.96
32     Anisomycin                                72.64
33     LL Z1640-4                                72.57
34     Clavulanate potassium                     71.69
35     Germicidin B                              71.30
36     Florfenicol amine                         70.43
37     Corynecin IV                              69.99
38     Brefeldin A                               69.84
39     Germicidin A                              69.39
40     Clindamycin hydrochloride                 68.16
41     Dihydroaeruginoic acid                    67.72
42     Tenuazonic acid                           66.72
43     Roquefortine E                            64.45
44     Cycloechinulin                            64.05
45     Butyryl-L-homoserine lactone              62.80
46     Moniliformin                              62.21
47     acetyl-L-homoserine lactone               61.35
48     Chloramphenicol acetate                   60.58
49     Hexanoyl-L-homoserine lactone             60.45
50     Simvastatin                               59.49
51     Aphidicolin                               58.92
52     Chloramphenicol                           57.79
53     Butyrolactone I                           51.49
54     Roquefortine C                            51.38
55     Cellocidin                                39.88
COPYRIGHT 2018 Oriental Scientific Publishing Company
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2018 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Reddy, Bandi Deepa; Chitturi, Ch. M. Kumari
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Sep 1, 2018
Words:2946
Previous Article:Comparison of Chromogenic Media with the Corn Meal Agar for Speciation of Candida.
Next Article:Extraction of Chitin and Chitosan from Wild Type Pleurotus Spp and its Potential Application Innovative Approach.
Topics:

Terms of use | Privacy policy | Copyright © 2021 Farlex, Inc. | Feedback | For webmasters |