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
|