Sains Malaysiana 52(12)(2023): 3395-3405

http://doi.org/10.17576/jsm-2023-5212-04

 

Characterisation of Recombinant 3CL Protease from SARS-CoV-2 Produced in E. coli BL21 (DE3) for Screening Anti-Covid Drug Candidates using Rhodamine 110-Synthetic Peptide Conjugate as a Substrate

 (Pencirian Protease 3CL Rekombinan daripada SARS-CoV-2 Dihasilkan dalam E. coli BL21 (DE3) untuk Menyaring Calon Dadah Anti-Covid menggunakan Rhodamine 110-Sintetik Peptida Konjugat sebagai Substrat)

 

I GEDE EKA PERDANA PUTRA1, MARIA ULFAH1, NAUFAL HAFIZH2, ERWAHYUNI ENDANG PRABANDARI3, FIRDAYANI3 & IS HELIANTI1,*

 

1Research Center for Applied Microbiology, National Research and Innovation Agency, Jalan Raya Bogor Km 46, Cibinong, Bogor, Indonesia

2Research Center for Agroindustry, National Research and Innovation Agency, Jalan Raya Bogor Km 46, Cibinong, Bogor, Indonesia

3Research Center for Vaccine and Drug,National Research and Innovation Agency, Jalan Raya Bogor Km 46, Cibinong, Bogor, Indonesia

 

Diserahkan: 5 April 2023/Diterima: 7 November 2023

 

Abstract

The prediction that the pandemic is progressing towards becoming endemic does not change the fact that COVID-19 can still be fatal for individuals with weak immune systems. Therefore, anti-COVID drugs are still needed, even when the disease becomes endemic. With regards to SARS-CoV-2, the roles of 3CL protease are crucial in the formation of new virus particles. Therefore, inhibiting the function of these viral proteases will directly prevent viral replication in the human body. In this study, we report the production of a recombinant 3CL protease from SARS-CoV-2 in E. coli BL21 (DE3), which has not been extensively studied in Indonesia. The purified 3CL protease exhibited high solubility and functional activity. Additionally, the recombinant enzyme was characterised using the Rhodamine 110 fluorogenic peptide substrate. We showed that the recombinant 3CL protease was unstable in the presence of a DMSO concentration above 10%. Using the Rhodamine 110 fluorogenic peptide substrate, we found that the enzyme had a KM of 47.0 µM, Vmax of 0.41 RFU/s, and kcat/KM of 0.0088 RFU/μM2.s while the IC50 of the GC376 was 13.35 nM. We also tested three bioactive compounds (catechin, emodin, and 1,4-naphthoquinone) using this recombinant protease as a protein target, and 1,4-naphthoquinone was the most promising bioactive compound in inhibiting the SARS-CoV-2 virus.

 

Keywords: Characterisation; peptide substrate LGSAVLQ-Rh110; recombinant 3CL protease

 

Abstrak

Ramalan bahawa wabak itu sedang berkembang menjadi endemik tidak mengubah fakta bahawa COVID-19 boleh membawa maut bagi individu yang mempunyai sistem imun yang lemah. Oleh itu, dadah anti-COVID masih diperlukan, walaupun penyakit itu menjadi endemik. Berkenaan dengan SARS-CoV-2, peranan 3CL protease adalah penting dalam pembentukan zarah virus baharu. Oleh itu, merencat fungsi protease virus ini secara langsung akan menghalang replikasi virus dalam tubuh manusia. Dalam kajian ini, kami melaporkan penghasilan 3CL protease rekombinan daripada SARS-CoV-2 dalam E. coli BL21 (DE3), yang belum dikaji secara meluas di Indonesia. 3CL protease yang telah ditulenkan menunjukkan keterlarutan yang tinggi dan aktiviti berfungsi. Tambahan lagi, enzim rekombinan telah dicirikan menggunakan substrat peptida fluorogenik Rhodamine 110. Kami mendapati bahawa 3CL protease rekombinan ini tidak stabil dalam kepekatan DMSO melebihi 10%. Dengan menggunakan substrat peptida fluorogenik Rhodamine 110, kami mendapati bahawa enzim ini mempunyai nilaiKM 47.0 µM, Vmax 0.41 RFU/s dan kcat/KM 0.0088 RFU/μM2.s manakala nilai IC50 GC376 ialah 13.35 nM. Kami juga menguji tiga sebatian bioaktif (katechin, emodin dan 1,4-naftoquinon) menggunakan protease ini sebagai sasaran protein dan 1,4-naftoquinon didapati adalah sebatian bioaktif yang paling berpotensi dalam menghalang virus SARS-CoV-2.

 

Kata kunci: Pencirian; rekombinan 3CL protease; substrat peptida LGSAVLQ-Rh110

 

RUJUKAN

Al Adem, K., Ferreira, J.C., Fadl, S. & Rabeh, W.M. 2023. pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity. Journal of Biological Chemistry 299(2): 102790.

Arya, R., Das, A., Prashar, V. & Kumar, M. 2020. Potential inhibitors against papain-like protease of novel coronavirus (SARS-CoV-2) from FDA approved drugs. ChemRxiv. https://chemrxiv.org/engage/chemrxiv/article-details/60c74880bdbb893898a38fb6

Bollag, D.M., Rozycki, M.D. & Edelstein, S.J. 1996. Protein Methods. 2nd ed. New York : John Wiley-Liss, Inc.

Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72(1): 248-254.

Chang, Y., Tung, Y., Lee, K., Chen, T., Hsiao, Y., Chang, H., Hsieh, T., Su, C., Wang, S., Yu, J., Shih, S., Lin, Y., Lin, Y., Tu, Y.E., Tung, C. & Chen, C. 2020. Potential therapeutic agents for COVID-19 based on the analysis of protease and RNA polymerase docking. Preprints 2020: 2020020242.

Cui, J. & Jia, J. 2021. Discovery of juglone and its derivatives as potent SARS-CoV-2 main proteinase inhibitors. European Journal of Medicinal Chemistry 225: 113789.

De Marco Verissimo, C., López Corrales, J., Dorey, A.L., Cwiklinski, K., Lalor, R., Calvani, N.E.D., Jewhurst, H.L., Flaus, A., Doyle, S. & Dalton, J.P. 2022. Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system. Epidemiology & Infection 150: e128.

Elfahmi, Woerdenbag, H.J. & Kayser, O. 2014. Jamu: Indonesian traditional herbal medicine towards rational phytopharmacological use. Journal of Herbal Medicine 4(2): 51-73.

Gurard-Levin, Z.A., Liu, C., Jekle, A., Jaisinghani, R., Ren, S., Vandyck, K., Jochmans, D., Leyssen, P., Neyts, J., Blatt, L.M., Beigelman, L., Symons, J.A., Raboisson, P., Scholle, M.D. & Deval, J. 2020. Evaluation of SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption ionization mass spectrometry. Antiviral Research 182: 104924.

Haniyya, Ulfah, M., Riswoko, A., Mulyawati, L., Ernawati, T. & Helianti, I. 2022. Production of recombinant SARS-CoV-2 3CL-protease: The key for the development of protease inhibitors screening kit in search of potential herb cure for COVID-19. IOP Conference Series: Earth and Environmental Science 976: 012051.

Jin, Z., Du, X., Xu, Y., Deng, Y., Liu, M., Zhao, Y., Zhang, B., Li, X., Zhang, L., Peng, C., Duan, Y., Yu, J., Wang, L., Yang, K., Liu, F., Jiang, R., Yang, X., You, T., Liu, X., Yang, X., Bai, F., Liu, H., Liu, X., Guddat, L.W., Xu, W., Xiao, G., Qin, C., Shi, Z., Jiang, H., Rao, Z. & Yang, H. 2020. Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature 582(7811): 289-293.

Jin, Z., Wang, H., Duan, Y. & Yang, H. 2021. The main protease and RNA-dependent RNA polymerase are two prime targets for SARS-CoV-2. Biochemical and Biophysical Research Communications 538: 63-71.

Kumar, D., Chandel, V., Raj, S. & Rathi, B. 2020. In silico identification of potent FDA approved drugs against Coronavirus COVID-19 main protease: A drug repurposing approach. Chemical Biology Letters 7(3): 166-175.

Li, Z., Li, X., Huang, Y-Y., Wu, Y., Liu, R., Zhou, L., Lin, Y., Wu, D., Zhang, L., Liu, H., Xu, X., Yu, K., Zhang, Y., Cui, J., Zhan, C-G., Wang, X. & Luo, H-B. 2020. Identify potent SARS-CoV-2 main protease inhibitors via accelerated free energy perturbation-based virtual screening of existing drugs. Proceedings of the National Academy of Sciences 117(44): 27381-27387.

Liu, P., Liu, H., Sun, Q., Liang, H., Li, C., Deng, X., Liu, Y. & Lai, L. 2020. Potent inhibitors of SARS-CoV-2 3C-like protease derived from N-substituted isatin compounds. European Journal of Medicinal Chemistry 206: 112702.

Ma, C., Sacco, M.D., Hurst, B., Townsend, J.A., Hu, Y., Szeto, T., Zhang, X., Tarbet, B., Marty, M.T., Chen, Y. & Wang, J. 2020. Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease. Cell Research 30(8): 678-692.

Narayanan, A., Narwal, M., Majowicz, S.A., Varricchio, C., Toner, S.A., Ballatore, C., Brancale, A., Murakami, K.S. & Jose, J. 2022. Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-protease assay. Communications Biology 5(1): 169.

Nawrot-Hadzik, I., Zmudzinski, M., Matkowski, A., Preissner, R., Kęsik-Brodacka, M., Hadzik, J., Drag, M. & Abel, R. 2021. Reynoutria rhizomes as a natural source of SARS-CoV-2 Mpro inhibitors–molecular docking and in vitro study. Pharmaceuticals (Basel, Switzerland) 14(8): 742.

Nguyen, T.T., Jung, J-H., Kim, M-K., Lim, S., Choi, J-M., Chung, B., Kim, D-W. & Kim, D. 2021. The inhibitory effects of plant derivate polyphenols on the main protease of SARS Coronavirus 2 and their structure–activity relationship. Molecules (Basel, Switzerland) 26(7): 1924.

Pattaro-Júnior, J.R., Araújo, I.G., Moraes, C.B., Barbosa, C.G., Philippsen, G.S., Freitas-Junior, L.H., Guidi, A.C., de Mello, J.C.P., Peralta, R.M., Fernandez, M.A., Teixeira, R.R. & Seixas, F.A.V. 2023. Antiviral activity of Cenostigma pluviosum var. peltophoroides extract and fractions against SARS-CoV-2. Journal of Biomolecular Structure and Dynamics 41(15): 7297-7308.

Srivastava, A.K., Kumar, A., Srivastava, H. & Misra, N. 2022. The role of herbal plants in the inhibition of SARS-CoV-2 main protease: A computational approach. Journal of the Indian Chemical Society 99(9): 100640.

Tahir ul Qamar, M., Alqahtani, S.M., Alamri, M.A. & Chen, L-L. 2020. Structural basis of SARS-CoV-2 3CLpro and anti-COVID-19 drug discovery from medicinal plants. Journal of Pharmaceutical Analysis 10(4): 313-319.

Tomohara, K., Adachi, I., Horino, Y., Kesamaru, H., Abe, H., Suyama, K. & Nose, T. 2019. DMSO-Perturbing assay for identifying promiscuous enzyme inhibitors. ACS Medicinal Chemistry Letters 10(6): 923-928.

Utomo, R.Y., Ikawati, M. & Meiyanto, E. 2020. Revealing the potency of citrus and galangal constituents to halt SARS-CoV-2 infection. Preprints 2020: 2020030214.

Vuong, W., Khan, M.B., Fischer, C., Arutyunova, E., Lamer, T., Shields, J., Saffran, H.A., McKay, R.T., van Belkum, M.J., Joyce, M.A., Young, H.S., Tyrrell, D.L., Vederas, J.C. & Lemieux, M.J. 2020. Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication. Nature Communications 11(1): 4282.

Wang, R., Hu, Q., Wang, H., Zhu, G., Wang, M., Zhang, Q., Zhao, Y., Li, C., Zhang, Y., Ge, G., Chen, H. & Chen, L. 2021. Identification of Vitamin K3 and its analogues as covalent inhibitors of SARS-CoV-2 3CLpro. International Journal of Biological Macromolecules 183: 182-192.

Xiong, M., Su, H., Zhao, W., Xie, H., Shao, Q. & Xu, Y. 2021. What coronavirus 3C-like protease tells us: From structure, substrate selectivity, to inhibitor design. Medicinal Research Reviews 41(4): 1965-1998.

Yan, G., Li, D., Lin, Y., Fu, Z., Qi, H., Liu, X., Zhang, J., Si, S. & Chen, Y. 2021. Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors. Cell & Bioscience 11(1): 199.

Yan, S. & Wu, G. 2021. Potential 3-chymotrypsin-like cysteine protease cleavage sites in the coronavirus polyproteins pp1a and pp1ab and their possible relevance to COVID-19 vaccine and drug development. The FASEB Journal 35(5): e21573.

Zhu, W., Xu, M., Chen, C.Z., Guo, H., Shen, M., Hu, X., Shinn, P., Klumpp-Thomas, C., Michael, S.G. & Zheng, W. 2020. Identification of SARS-CoV-2 3CL protease inhibitors by a quantitative high-throughput screening. ACS Pharmacology & Translational Science 3(5): 1008-1016.

Zuhud, E.A.M. & Siswoyo. 2001. Rencana Strategis Konservasi Tumbuhan Obat Indonesia. Bogor: Kerjasama Pusat Pengendalian Kerusakan Keanekaragaman Hayati BAPEDAL dengan Fakultas Kehutanan IPB.

 

*Pengarang untuk surat-menyurat; email: is.helianti@brin.go.id

 

 

 

 

 

 

 

 

   

sebelumnya