Sains Malaysiana 52(11)(2023): 3223-3237

http://doi.org/10.17576/jsm-2023-5211-15

In silico Design and Evaluation of Novel Cell Targeting Melittin-Interleukin-24 Fusion Protein: A Potential Drug Candidate Against Breast Cancer

(Reka Bentuk in silico dan Penilaian Penyasaran Sel Baru Protein Gabungan Melitin-Interleukin-24: Calon Dadah Berpotensi Melawan Kanser Payudara)

HAFIZ MUHAMMAD REHMAN^1,4, HAFIZ MUZZAMMEL REHMAN^5,6, NADEEM AHMED², MUHAMMAD IMRAN AMIRZADA³, SHAKIRA ASLAM¹ & HAMID BASHIR^1,*

       ¹Centre for Applied Molecular Biology (CAMB), 87-West Canal, Bank Road, University of the Punjab, Lahore-53700, Pakistan

           ²Centre of excellence in Molecular Biology (CEMB), 87-West Canal, Bank Road, University of the Punjab, Lahore-53700, Pakistan

            ³Department of Pharmacy COMSATS University, Islamabad Abbottabad, Pakistan

     ⁴University Institute of Medical Laboratory Technology, Faculty of Allied Health Sciences, The University of Lahore -54590 Pakistan

     ⁵School of Biochemistry and Biotechnology University of the Punjab, Lahore-53700, Pakistan

    ⁶Department of Human Genetics and Molecular Biology, University of Health Science, Lahore

Diserahkan: 13 Disember 2022/Diterima: 16 Oktober 2023

Abstract

Fusion proteins are designed to achieve new functionality or improved properties synergistically by incorporating multiple protein domains into one complex. The fusion of two genes to translate a recombinant protein for cancer treatment can enhance the bioactivity of drug and can introduce novel drug candidate with wide range of applications in pharmaceuticals and biotechnology. Interleukin-24 (IL-24) is a novel cancer growth-suppressing and apoptosis inducing cytokine while melittin is a natural honeybee derived cationic polypeptide having anti-tumor activity against breast cancer cells. The current study was aimed to perform in silico design and analyses of a melittin-IL-24 fusion protein against breast cancer. The amino acid sequences of the IL-24 and melittin peptide were used to design the fusion protein via a rigid linker. Using the online softwares we predicted the secondary and tertiary structures along with physicochemical properties of the designed fusion protein. The validation and quality of the fusion protein was confirmed by Rampage and ERRAT2. The top ranked structure from I-TASSER showed 18.1KD molecular weight by ProtParam, quality factor of 94.152 by ERRAT and a valid structure by Ramachandran plot with 88.5% residues in favoured region. The docking and simulation studies were performed using ClusPro and Desmond software. The quality, validity, interaction analysis and stability of the fusion protein depicted a functional molecule. The in silico analysis finding and expression predicted value of 0.86 in E. coli on SOLUPROT tool suggest that the melittin- IL-24 fusion protein can lead to develop a potent therapeutic drug against breast cancer.

Keywords: Breast cancer; fusion protein; interleukin 24; melittin; molecular docking

Abstrak

Protein gabungan telah direka bentuk untuk mencapai fungsi baharu atau sifat yang dipertingkatkan secara sinergi dengan menggabungkan berbilang domain protein ke dalam satu kompleks. Gabungan dua gen untuk menterjemah protein rekombinan untuk rawatan kanser boleh meningkatkan bioaktiviti dadah dan boleh memperkenalkan calon dadah baharu dengan pelbagai pengaplikasian dalam farmaseutikal dan bioteknologi. Interleukin-24 (IL-24) ialah sitokin penekan pertumbuhan kanser baharu dan apoptosis manakala melitin ialah polipeptida kation perolehan lebah madu semula jadi yang mempunyai aktiviti anti-tumor terhadap sel kanser payudara. Kajian semasa ini bertujuan untuk menjalankan reka bentuk in silico dan analisis protein gabungan melitin-IL-24 terhadap kanser payudara. Jujukan asid amino IL-24 dan peptida melitin digunakan untuk mereka bentuk protein gabungan melalui penghubung tegar. Dengan menggunakan perisian atas talian, kami meramalkan struktur sekunder dan tertiari bersama-sama dengan sifat fizikokimia reka bentuk protein gabungan. Pengesahan dan kualiti protein gabungan telah disahkan oleh Rampage dan ERRAT2. Struktur kedudukan teratas daripada I-TASSER menunjukkan berat molekul 18.1KD oleh ProtParam, faktor kualiti 94.152 oleh ERRAT dan struktur yang sahih oleh plot Ramachandran dengan 88.5% residu di kawasan yang digemari. Kajian dok dan simulasi dilakukan menggunakan perisian ClusPro dan Desmond. Kualiti, kesahan, analisis interaksi dan kestabilan protein gabungan menggambarkan molekul fungsian. Penemuan analisis in silico dan pengekspresan nilai ramalan 0.86 dalam E. coli pada perkakas SOLUPROT menunjukkan bahawa protein gabungan melitin-IL-24 boleh membawa kepada pembangunan ubat terapeutik poten terhadap kanser payudara.

Kata kunci: Dok molekul; interleukin 24; kanser payudara; melitin; protein gabungan

RUJUKAN

Balimane, P.V. & Chong, S. 2005. Cell culture-based models for intestinal permeability: A critique. Drug Discovery Today 10(5): 335-343.

Bowie, J.U., Lüthy, R. & Eisenberg, D. 1991. A method to identify protein sequences that fold into a known three-dimensional structure. Science 253(5016): 164-170.

Chabner, B.A. & Roberts, T.G. 2005. Chemotherapy and the war on cancer. Nature Reviews Cancer 5(1): 65-72.

Chakravarty, S., Godbole, S., Zhang, B., Berger, S. & Sanchez, R. 2008. Systematic analysis of the effect of multiple templates on the accuracy of comparative models of protein structure. BMC Structural Biology 8(1): 1-13.

Chen, T., Yang, J., Wang, Y., Zhan, C., Zang, Y. & Qin, J. 2005. Design of recombinant stem cell factor–macrophage colony stimulating factor fusion proteins and their biological activity in vitro. Journal of Computer-Aided Molecular Design 19(5): 319-328.

Chen, Y-Q., Zhu, Z-A., Hao, Y-Q., Dai, K-R. & Zhang, C. 2004. Effect of melittin on apoptosis and necrosis of U2 OS cells. Zhong xi yi jie he xue bao= Journal of Chinese Integrative Medicine 2(3): 208-209.

Cruz, A., Nguyen, B., Sauane, M. & Lopez, G.E. 2016. Structural and functional characterization of interleukin-24 based on atomistic molecular modeling. Chemistry Letters 45(3): 327-329.

DeLano, W.L. 2002. Pymol: An open-source molecular graphics tool. CCP4 Newsl. Protein Crystallogr 40(1): 82-92.

Doytchinova, I.A. & Flower, D.R. 2007. VaxiJen: A server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinformatics 8: 4.

Gajski, G. & Garaj-Vrhovac, V. 2013. Melittin: A lytic peptide with anticancer properties. Environmental Toxicology and Pharmacology 36(2): 697-705.

Gasteiger, E., Hoogland, C., Gattiker, A., Wilkins, M.R., Appel, R.D. & Bairoch, A. 2005. Protein identification and analysis tools on the ExPASy server. The Proteomics Protocols Handbook. pp. 571-607.

Ghavimi, R., Mohammadi, E., Akbari, V., Shafiee, F. & Jahanian-Najafabadi, A. 2020. In silico design of two novel fusion proteins, p28-IL-24 and p28-M4, targeted to breast cancer cells. Research in Pharmaceutical Sciences 15(2): 200-208.

Ghomi, F.A., Kittilä, T. & Welner, D.H. 2020. A benchmark of protein solubility prediction methods on UDP-dependent glycosyltransferases. BioRxiv. https://doi.org/10.1101/2020.02.28.962894

Gul, R., Hanif, M.U., Gul, F., Rehman, H.M., Saleem, M., Ahmad, M.S. & Mirza, M.U. 2022. Molecular cloning, expression, sequence characterization and structural insight of Bubalus bubalis growth hormone-receptor. Molecular Biotechnology 65(7): 1062-1075.

Hait, W.N., Grais, L., Benz, C. & Cadman, E.C. 1985. Inhibition of growth of leukemic cells by inhibitors of calmodulin: Phenothiazines and melittin. Cancer Chemotherapy and Pharmacology 14(3): 202-205.

Hebditch, M., Carballo-Amador, M.A., Charonis, S., Curtis, R. & Warwicker, J. 2017. Protein–Sol: A web tool for predicting protein solubility from sequence. Bioinformatics 33(19): 3098-3100.

Hon, J., Marusiak, M., Martinek, T., Kunka, A., Zendulka, J., Bednar, D. & Damborsky, J. 2021. SoluProt: Prediction of soluble protein expression in Escherichia coli. Bioinformatics 37(1): 23-28.

Hu, H., Chen, D., Li, Y. & Zhang, X. 2006. Effect of polypeptides in bee venom on growth inhibition and apoptosis induction of the human hepatoma cell line SMMC-7721 in-vitro and Balb/c nude mice in-vivo. Journal of Pharmacy and Pharmacology 58(1): 83-89.

Ip, S-W., Liao, S-S., Lin, S-Y., Lin, J-P., Yang, J-S., Lin, M-L., Chen, G-W., Lu, H-F., Lin, M-W. & Han, S-M. 2008. The role of mitochondria in bee venom-induced apoptosis in human breast cancer MCF7 cells. In Vivo 22(2): 237-245.

Jahanian-Najafabadi, A., Ghavimi, R. & Akbari, V. 2020. 10P In vitro and in vivo cytolethal and antitumor effects of a novel fusion protein targeting IL-24 toward breast cancer cells. Annals of Oncology 31(Supplement 1): S2.

Jo, M., Park, M.H., Kollipara, P.S., An, B.J., Song, H.S., Han, S.B., Kim, J.H., Song, M.J. & Hong, J.T. 2012. Anti-cancer effect of bee venom toxin and melittin in ovarian cancer cells through induction of death receptors and inhibition of JAK2/STAT3 pathway. Toxicology and Applied Pharmacology 258(1): 72-81.

Keshtvarz, M., Salimian, J., Amani, J., Douraghi, M. & Rezaie, E. 2021. In silico analysis of STX2a-PE15-P4A8 chimeric protein as a novel immunotoxin for cancer therapy. In silico Pharmacology 9(1): 19.

Khan, M.T., Zeb, M.T., Ahsan, H., Ahmed, A., Ali, A., Akhtar, K., Malik, S.I., Cui, Z., Ali, S. & Khan, A.S. 2021. SARS-CoV-2 nucleocapsid and Nsp3 binding: An in silico study. Archives of Microbiology 203(1): 59-66.

Killion, J.J. & Dunn, J.D. 1986. Differential cytolysis of murine spleen, bone-marrow and leukemia cells by melittin reveals differences in membrane topography. Biochemical and biophysical research communications 139(1): 222-227.

Kisseleva, T., Bhattacharya, S., Braunstein, J. & Schindler, C.W. 2002. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 285(1-2): 1-24.

Ko, J., Park, H., Heo, L. & Seok, C. 2012. GalaxyWEB server for protein structure prediction and refinement. Nucleic Acids Research 40(W1): W294-W297.

Kozakov, D., Hall, D.R., Xia, B., Porter, K.A., Padhorny, D., Yueh, C., Beglov, D. & Vajda, S. 2017. The ClusPro web server for protein–protein docking. Nature Protocols 12(2): 255-278.

Laskowski, R.A. 2009. PDBsum new things. Nucleic Acids Research 37(suppl_1): D355-D359.

Lee, S.Y., Park, H.S., Lee, S.J. & Choi, M-U. 2001. Melittin exerts multiple effects on the release of free fatty acids from L1210 cells: Lack of selective activation of phospholipase A2 by melittin. Archives of Biochemistry and Biophysics 389(1): 57-67.

Li, B., Ling, C.Q., Zhang, C., Gu, W., Li, S.X., Huang, X.Q., Zhang, Y.N. & Yu, C.Q. 2004. The induced apoptosis of recombinant adenovirus carrying melittin gene for hepatocellular carcinoma cell. Zhonghua gan Zang Bing za zhi= Zhonghua Ganzangbing Zazhi= Chinese Journal of Hepatology 12(8): 453-455.

Lim, S.M. & Lee, S-H. 2015. Effectiveness of bee venom acupuncture in alleviating post-stroke shoulder pain: A systematic review and meta-analysis. Journal of Integrative Medicine 13(4): 241-247.

Lischer, K., Sitorus, S.R.A., Guslianto, B.W., Avila, F., Khayrani, A.C. & Sahlan, M. 2021. Anti-breast cancer activity on MCF-7 cells of melittin from Indonesia’s Apis cerana: An in vitro study. Asian Pacific Journal of Cancer Prevention 22(12): 3913-3919.

Liu, X., Chen, D., Xie, L. & Zhang, R. 2002. Effect of honey bee venom on proliferation of K1735M2 mouse melanoma cells in‐vitro and growth of murine B16 melanomas in‐vivo. Journal of Pharmacy and Pharmacology 54(8): 1083-1089.

Long, M. 2000. A new function evolved from gene fusion. Genome Research 10(11): 1655-1657.

Lubkowski, J., Sonmez, C., Smirnov, S.V., Anishkin, A., Kotenko, S.V. & Wlodawer, A. 2018. Crystal structure of the labile complex of IL-24 with the extracellular domains of IL-22R1 and IL-20R2. The Journal of Immunology 201(7): 2082-2093.

Lyu, C., Fang, F. & Li, B. 2019. Anti-tumor effects of melittin and its potential applications in clinic. Current Protein and Peptide Science 20(3): 240-250.

Mandell, J.G., Roberts, V.A., Pique, M.E., Kotlovyi, V., Mitchell, J.C., Nelson, E., Tsigelny, I. & Eyck, L.F.T. 2001. Protein docking using continuum electrostatics and geometric fit. Protein Engineering 14(2): 105-113.

Maurya, N.S., Kushwaha, S. & Mani, A. 2019. Recent advances and computational approaches in peptide drug discovery. Current Pharmaceutical Design 25(31): 3358-3366.

Mir Hassani, Z., Nabiuni, M., Parivar, K., Abdirad, S. & Karimzadeh, L. 2021. Melittin inhibits the expression of key genes involved in tumor microenvironment formation by suppressing HIF-1α signaling in breast cancer cells. Medical Oncology 38(7): 1-8.

Moghadam, Z.M., Halabian, R., Sedighian, H., Behzadi, E., Amani, J. & Fooladi, A.A.I. 2019. Designing and analyzing the structure of DT-STXB fusion protein as an anti-tumor agent: An in silico approach. Iranian Journal of Pathology 14(4): 305-312.

Muhammad Rehman, H., Muzzammel Rehman, H., Naveed, M., Khan, M.T., Shabbir, M.A., Aslam, S. & Bashir, H. 2023. In silico investigation of a chimeric IL24-LK6 fusion protein as a potent candidate against breast cancer. Bioinformatics and Biology Insights 17: 11779322231182560.

Naumov, G.N., Townson, J.L., MacDonald, I.C., Wilson, S.M., Bramwell, V.H.C., Groom, A.C. & Chambers, A.F. 2003. Ineffectiveness of doxorubicin treatment on solitary dormant mammary carcinoma cells or late-developing metastases. Breast Cancer Research and Treatment 82(3): 199-206.

Pourhadi, M., Jamalzade, F., Jahanian-Najafabadi, A. & Shafiee, F. 2019. Expression, purification, and cytotoxic evaluation of IL24-BR2 fusion protein. Research in Pharmaceutical Sciences 14(4): 320-328.

Rehman, H.M., Sajjad, M., Ali, M.A., Gul, R., Irfan, M., Naveed, M., Bhinder, M.A., Ghani, M.U., Hussain, N. & Said, A.S.A. 2023. Identification of NS2B-NS3 protease inhibitors for therapeutic application in ZIKV infection: A pharmacophore-based high-throughput virtual screening and MD simulations approaches. Vaccines 11(1): 131.

Rehman, H.M., Sajjad, M., Ali, M.A., Gul, R., Naveed, M., Aslam, M.S., Shinwari, K., Ahmad Bhinder, M., Ghani, M.U. & Saleem, M. 2023. Identification of RdRp inhibitors against SARS-CoV-2 through E-pharmacophore-based virtual screening, molecular docking and MD simulations approaches. International Journal of Biological Macromolecules 237: 124169.

Roy, A., Kucukural, A. & Zhang, Y. 2010. I-TASSER: A unified platform for automated protein structure and function prediction. Nature Protocols 5(4): 725-738.

Saha, S. & Raghava, G.P.S. 2006. AlgPred: Prediction of allergenic proteins and mapping of IgE epitopes. Nucleic Acids Research 34(suppl_2): W202-W209.

Sahna, K.O., Cakir, B. & Tunali-Akbay, T. 2022. Antiproliferative activity of whey and casein bioactive peptides on breast cancer: An in vitro and in silico study. International Journal of Peptide Research and Therapeutics 28: 128.

Sakurai, Y., Mizuno, T., Hiroaki, H., Gohda, K., Oku, J‐I. & Tanaka, T. 2005. High thermal stability imparted by a designed tandem Arg–Trp stretch in an α‐helical coiled coil. Angewandte Chemie 117(38): 6336-6339.

Sato, A.K., Viswanathan, M., Kent, R.B. & Wood, C.R. 2006. Therapeutic peptides: Technological advances driving peptides into development. Current Opinion in Biotechnology 17(6): 638-642.

Shafique, Q-ul.A., Muzzammel Rehman, H., Zaheer, T., Adnan Tahir, R., Ahmad Bhinder, M., Gul, R. & Saleem, M. 2021. A computational approach to modeling an antagonistic angiogenic VEGFR1-IL2 fusion protein for cancer therapy. Bioinformatics and Biology Insights 15: 11779322211043297.

Smith, L.L., Brown, K., Carthew, P., Lim, C-K., Martin, E.A., Styles, J. & White, I.N.H. 2000. Chemoprevention of breast cancer by tamoxifen: Risks and opportunities. Critical Reviews in Toxicology 30(5): 571-594.

Soleimani, M., Mahnam, K., Mirmohammad-Sadeghi, H., Sadeghi-Aliabadi, H. & Jahanian-Najafabadi, A. 2016. Theoretical design of a new chimeric protein for the treatment of breast cancer. Research in Pharmaceutical Sciences 11(3): 187-199.

Trinidad-Calderón, P.A., Varela-Chinchilla, C.D. & García-Lara, S. 2021. Natural peptides inducing cancer cell death: Mechanisms and properties of specific candidates for cancer therapeutics. Molecules 26(24): 7453.

Vangone, A. & Bonvin, A.M.J.J. 2015. Contacts-based prediction of binding affinity in protein–protein complexes. elife 4: e07454.

Wiederstein, M. & Sippl, M.J. 2007. ProSA-web: Interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Research 35(suppl_2): W407-W410.

Xiao, B., Li, W., Yang, J., Guo, G., Mao, X-H. & Zou, Q-M. 2009. RGD-IL-24, a novel tumor-targeted fusion cytokine: Expression, purification and functional evaluation. Molecular Biotechnology 41(2): 138-144.

Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J. & Zhang, Y. 2015. The I-TASSER Suite: Protein structure and function prediction. Nature Methods 12(1): 7-8.

Yang, J., Zhang, W., Liu, K., Jing, S., Guo, G., Luo, P. & Zou, Q. 2007. Expression, purification, and characterization of recombinant human interleukin 24 in Escherichia coli. Protein Expression and Purification 53(2): 339-345.

Yang, Z.L., Ke, Y.Q., Xu, R.X. & Peng, P. 2007. Melittin inhibits proliferation and induces apoptosis of malignant human glioma cells. Nan Fang yi ke da xue xue bao= Journal of Southern Medical University 27(11): 1775-1777.

Yu, K., Liu, C., Kim, B-G. & Lee, D-Y. 2015. Synthetic fusion protein design and applications. Biotechnology Advances 33(1): 155-164.

Zhang, Y. 2008. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9(1): 1-8.

Zhao, W., Song, L., Guocheng, D. & Jingwen, Z. 2019. An efficient expression tag library based on self-assembling amphipathic peptides. Microbial Cell Factories 18(1): 1-11.

Zheng, W., Zhang, C., Li, Y., Pearce, R., Bell, E.W. & Zhang, Y. 2021. Folding non-homologous proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. Cell Reports Methods 1(3): 100014.

Zhu, H.G., Tayeh, I., Israel, L. & Castagna, M. 1991. Different susceptibility of lung cell lines to inhibitors of tumor promotion and inducers of differentiation. Journal of Biological Regulators and Homeostatic Agents 5(2): 52-58.

*Pengarang untuk surat-menyurat; email: hamid.camb@pu.edu.pk