All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity
Abstract
1. Introduction
2. Materials and Methods
2.1. Cells, Virus and Compounds
2.2. Wheat Germ Cell-Free Protein Synthesis and Protein Purification
2.3. SARS-CoV-2 3CLpro Activity Assay
2.4. Docking Simulation
2.5. Infectivity Assay
2.6. Cell Viability Assay
2.7. RNA Extraction and Real-Time Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR)
2.8. Immunofluorescence Microscopy
2.9. Time of Addition Assay
3. Results
3.1. Development of the SARS-CoV-2 3CLpro Enzyme Assay Using AlphaScreen and Screening of Compounds That Inhibit Enzyme Activity
3.2. ATRA as a Potent SARS-CoV-2 3CLpro Inhibitor
3.3. ATRA Inhibits SARS-CoV-2 Replication
3.4. ATRA Inhibits SARS-CoV-2 Replication Independent of RIG-I Expression
3.5. ATRA Is Effective against the SARS-CoV-2 Variants of Concern in Human Lung Cell Line
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
Zhu, N.; Zhang, D.; Wang, W.; Li, X.; Yang, B.; Song, J.; Zhao, X.; Huang, B.; Shi, W.; Lu, R.; et al. A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. 2020 , 382 , 727–733. [ Google Scholar ] [ CrossRef ] Wang, P.; Nair, M.S.; Liu, L.; Iketani, S.; Luo, Y.; Guo, Y.; Wang, M.; Yu, J.; Zhang, B.; Kwong, P.D.; et al. Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 2021 , 593 , 130–135. [ Google Scholar ] [ CrossRef ] [ PubMed ] Sanders, J.M.; Monogue, M.L.; Jodlowski, T.Z.; Cutrell, J.B. Pharmacologic treatments for coronavirus disease 2019 (COVID-19): A review. JAMA J. Am. Med. Assoc. 2020 , 323 , 1824–1836. [ Google Scholar ] [ CrossRef ] Zhang, L.; Lin, D.; Sun, X.; Curth, U.; Drosten, C.; Sauerhering, L.; Becker, S.; Rox, K.; Hilgenfeld, R. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved a-ketoamide inhibitors. Science 2020 , 368 , 409–412. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ] Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J.R.; Hilgenfeld, R. Coronavirus Main Proteinase (3CL Pro) Structure: Basis for Design of Anti-SARS Drugs. Science 2003 , 300 , 1763–1767. [ Google Scholar ] [ CrossRef ] [ Green Version ] Pillaiyar, T.; Manickam, M.; Namasivayam, V.; Hayashi, Y.; Jung, S.H. An overview of severe acute respiratory syndrome-coronavirus (SARS-CoV) 3CL protease inhibitors: Peptidomimetics and small molecule chemotherapy. J. Med. Chem. 2016 , 59 , 6595–6628. [ Google Scholar ] [ CrossRef ] [ PubMed ] Jin, Z.; Du, X.; Xu, Y.; Deng, Y.; Liu, M.; Zhao, Y.; Zhang, B.; Li, X.; Zhang, L.; Peng, C.; et al. Structure of M pro from SARS-CoV-2 and discovery of its inhibitors. Nature 2020 , 582 , 289–293. [ Google Scholar ] [ CrossRef ] [ Green Version ] 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.; et al. Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication. Nat. Commun. 2020 , 11 , 4282. [ Google Scholar ] [ CrossRef ] [ PubMed ] Chelbi-Alix, M.K.; Pelicano, L. Retinoic Acid and Interferon Signaling cross Talk in Normal and RA-Resistant APL Cells. Leukemia 1999 , 13 , 1167–1174. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ] Hamamoto, S.; Fukuda, R.; Ishimura, N.; Rumi, M.A.K.; Kazumori, H.; Uchida, Y.; Kadowaki, Y.; Ishihara, S.; Kinoshita, Y. 9-cis retinoic acid enhances the antiviral effect of interferon on hepatitis C virus replication through increased expression of type I interferon receptor. J. Lab. Clin. Med. 2003 , 141 , 58–66. [ Google Scholar ] [ CrossRef ] Soye, K.J.; Trottier, C.; Richardson, C.D.; Ward, B.J.; Miller, W.H. RIG-I is required for the inhibition of measles virus by retinoids. PLoS ONE 2011 , 6 , e22323. [ Google Scholar ] [ CrossRef ] [ Green Version ] Soye, K.J.; Trottier, C.; Di Lenardo, T.Z.; Restori, K.H.; Reichman, L.; Miller, W.H.; Ward, B.J. In vitro inhibition of mumps virus by retinoids. Virol. J. 2013 , 10 , 337. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ] Maeda, Y.; Yamaguchi, T.; Hijikata, Y.; Morita, Y.; Tanaka, M.; Hirase, C.; Takai, S.; Tatsumi, Y.; Kanamaru, A. All-trans retinoic acid attacks reverse transcriptase resulting in inhibition of HIV-1 replication. Hematology 2007 , 12 , 263–266. [ Google Scholar ] [ CrossRef ] [ PubMed ] Yamaguchi, T.; Maeda, Y.; Ueda, S.; Hijikata, Y.; Morita, Y.; Miyatake, J.I.; Matsuda, M.; Kanamaru, A. Dichotomy of all-trans retinoic acid inducing signals for adult T-cell leukemia. Leukemia 2005 , 19 , 1010–1017. [ Google Scholar ] [ CrossRef ] [ PubMed ] Glickman, J.F.; Wu, X.; Mercuri, R.; Illy, C.; Bowen, B.R.; He, Y.; Sills, M. A Comparison of ALPHAScreen, TR-FRET, and TRF as Assay Methods for FXR Nuclear Receptors. J. Biomol. Screen 2002 , 7 , 3–10. [ Google Scholar ] [ CrossRef ] [ Green Version ] Matsuyama, S.; Nao, N.; Shirato, K.; Kawase, M.; Saito, S.; Takayama, I.; Nagata, N.; Sekizuka, T.; Katoh, H.; Kato, F.; et al. Enhanced isolation of SARS-CoV-2 by TMPRSS2- expressing cells. Proc. Natl. Acad. Sci. USA 2020 , 117 , 7001–7003. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ] Matsunaga, S.; Masaoka, T.; Sawasaki, T.; Morishita, R.; Iwatani, Y.; Tatsumi, M.; Endo, Y.; Yamamoto, N.; Sugiura, W.; Ryo, A. A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors. Front. Microbiol. 2015 , 6 . [ Google Scholar ] [ CrossRef ] [ Green Version ] Yamaoka, Y.; Matsunaga, S.; Jeremiah, S.S.; Nishi, M.; Miyakawa, K.; Morita, T.; Khatun, H.; Shimizu, H.; Okabe, N.; Kimura, H.; et al. Zika virus protease induces caspase-independent pyroptotic cell death by directly cleaving gasdermin D. Biochem. Biophys. Res. Commun. 2021 , 534 , 666–671. [ Google Scholar ] [ CrossRef ] Yamaoka, Y.; Matsuyama, S.; Fukushi, S.; Matsunaga, S.; Matsushima, Y.; Kuroyama, H.; Kimura, H.; Takeda, M.; Chimuro, T.; Ryo, A. Development of monoclonal antibody and diagnostic test for Middle East respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen. Front. Microbiol. 2016 , 7 , 509. [ Google Scholar ] [ CrossRef ] [ Green Version ] Trott, O.; Olson, A.J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2009 , 31 , 455–461. [ Google Scholar ] [ CrossRef ] [ Green Version ] Du, R.; Cooper, L.; Chen, Z.; Lee, H.; Rong, L.; Cui, Q. Discovery of Chebulagic Acid and Punicalagin as Novel Allosteric Inhibitors of SARS-CoV-2 3CLpro. Available online: https://reader.elsevier.com/reader/sd/pii/S0166354221000656?token=067A519D50C243D8DB69226FA5814A6B5F535267D684F0327D013091EF027785765816A014815C1A69403D353EB45C3F&originRegion=us -east-1&originCreation=20210729065020 (accessed on 29 July 2021). Zhu, W.; Xu, M.; Chen, C.Z.; Guo, H.; Shen, M.; Hu, X.; Shinn, P.; Klumpp-Thomas, C.; Michael, S.G.; Zheng, W. Identification of SARS-CoV-2 3CL protease inhibitors by a quantitative high-throughput screening. ACS Pharmacol. Transl. Sci. 2020 , 3 , 1008–1016. [ Google Scholar ] [ CrossRef ] Luo, X.M.; Ross, A.C. Retinoic acid exerts dual regulatory actions on the expression and nuclear localization of interferon regulatory factor-1. Exp. Biol. Med. 2006 , 231 , 619–631. [ Google Scholar ] [ CrossRef ] [ Green Version ] Naoki, O.; Arihiro, K.; Toshiyuki, Y.; Noriko, H.; Fumio, K.; Suyoshi, S.; Makoto, K.; Kentaro, H.; Hattori, M. The genome landscape of the African Green Monkey kidney-derived vero cell line. DNA Res. 2014 , 21 , 673–683. [ Google Scholar ] [ CrossRef ] [ Green Version ] Wang, M.; Cao, R.; Zhang, L.; Yang, X.; Liu, J.; Xu, M.; Shi, Z.; Hu, Z.; Zhong, W.; Xiao, G. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020 , 30 , 269–271. [ Google Scholar ] [ CrossRef ] Hoffmann, M.; Kleine-Weber, H.; Schroeder, S.; Krüger, N.; Herrler, T.; Erichsen, S.; Schiergens, T.S.; Herrler, G.; Wu, N.H.; Nitsche, A.; et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Available online: https://reader.elsevier.com/reader/sd/pii/S0092867420302294?token=9EBCF58606E2B75994845A858932AF32E87A4870844A01D4C9A8810584C7FC69E3D6BBAC9349F8716F2A547B5A3E5055&originRegion=us -east-1&originCreation=20210518035149 (accessed on 18 May 2021). Yamada, T.; Sato, S.; Sotoyama, Y.; Orba, Y.; Sawa, H.; Yamauchi, H.; Sasaki, M.; Takaoka, A. RIG-I triggers a signaling-abortive anti-SARS-CoV-2 defense in human lung cells. Nat. Immunol. 2021 . [ Google Scholar ] [ CrossRef ] [ PubMed ] Emerging Variants of SARS-CoV-2 and Novel Therapeutics against Coronavirus (COVID-19)-StatPearls-NCBI Bookshelf. Available online: https://www.ncbi.nlm.nih.gov/books/NBK570580/ (accessed on 29 June 2021). Huang, M.E.; Ye, Y.C.; Chen, S.R.; Chai, J.R.; Lu, J.X.; Zhao, L.; Gu, L.J.; Wang, Z.Y. Use of All-Trans Retinoic Acid in the Treatment of Acute Promyelocytic Leukemia ; Springer: Berlin/Heidelberg, Germany, 1989; Volume 32. [ Google Scholar ] El-Baba, T.J.; Lutomski, C.A.; Kantsadi, A.L.; Malla, T.R.; John, T.; Mikhailov, V.; Bolla, J.R.; Schofield, C.J.; Zitzmann, N.; Vakonakis, I.; et al. Allosteric inhibition of the SARS-CoV-2 main protease: Insights from mass spectrometry based assays. Angew. Chemie-Int. Ed. 2020 , 59 , 23544–23548. [ Google Scholar ] [ CrossRef ] Ghosh, A.K.; Chapsal, B.D.; Weber, I.T.; Mitsuya, H. Design of HIV protease inhibitors targeting protein backbone: An effective strategy for combating drug resistance. Acc. Chem. Res. 2008 , 41 , 78–86. [ Google Scholar ] [ CrossRef ] [ PubMed ] Dollé, P. Developmental expression of retinoic acid receptors (RARs). Nucl. Recept. Signal. 2009 , 7 , 6. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ] Gudas, L.J. Retinoids and vertebrate development. J. Biol. Chem. 1994 , 269 , 15399–15402. [ Google Scholar ] [ CrossRef ] Mark, M.; Ghyselinck, N.B.; Chambon, P. Function of retinoic acid receptors during embryonic development. Nucl. Recept. Signal. 2009 , 7 , e002. [ Google Scholar ] [ CrossRef ] [ Green Version ] Mucida, D.; Park, Y.; Kim, G.; Turovskaya, O.; Scott, I.; Kronenberg, M.; Cheroutre, H. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 2007 , 317 , 256–260. [ Google Scholar ] [ CrossRef ] [ Green Version ] Caly, L.; Druce, J.D.; Catton, M.G.; Jans, D.A.; Wagstaff, K.M. The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antivir. Res. 2020 , 178 , 3–6. [ Google Scholar ] [ CrossRef ] [ PubMed ] Adamson, P.C. All-trans-retinoic acid pharmacology and its impact on the treatment of acute promyelocytic leukemia. Oncologist 1996 , 1 , 305–314. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ] Horby, P.W.; Mafham, M.; Bell, J.L.; Linsell, L.; Staplin, N.; Emberson, J.; Palfreeman, A.; Raw, J.; Elmahi, E.; Prudon, B.; et al. Lopinavir–ritonavir in patients admitted to hospital with COVID-19 (RECOVERY): A randomised, controlled, open-label, platform trial. Lancet 2020 , 396 , 1345–1352. [ Google Scholar ] [ CrossRef ]