All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity
Takeshi Morita, Kei Miyakawa, Sundararaj Stanleyraj Jeremiah, Yutaro Yamaoka, Mitsuru Sada, Tomoko Kuniyoshi, Jinwei Yang, Hirokazu Kimura, Akihide Ryo
Viruses, doi:10.3390/v13081669
The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC 50 ) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC 50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/v13081669/s1, Figure S1 : The comparison of FRET and AlphaScreen, Figure S2 : Graphical determination of the type pf inhibition, Figure S3 : Cytotoxicity of ATRA, Figure S4 : Protein expression in ATRA treated Calu-3 cells during SARS-CoV-2 infection, Figure S5 : Amino acid sequence alignment of 3CLpro, Table S1 : The list of compounds screened by the enzyme assay.
Conflicts of Interest: The authors declare no competing financial interest. Y.Y. is a current employee of Kanto Chemical Co., Inc. T.K. and J.Y. are a current employee of TOKIWA Phytochemical Co., Ltd.
References
Anand, Ziebuhr, Wadhwani, Mesters, Hilgenfeld, Coronavirus Main Proteinase (3CL Pro) Structure: Basis for Design of Anti-SARS Drugs, Science,
doi:10.1126/science.1085658Chelbi-Alix, Pelicano, Retinoic Acid and Interferon Signaling cross Talk in Normal and RA-Resistant APL Cells, Leukemia,
doi:10.1038/sj.leu.2401469Dollé, Developmental expression of retinoic acid receptors (RARs), Nucl. Recept. Signal,
doi:10.1621/nrs.07006Du, Cooper, Chen, Lee, Rong et al., Discovery of Chebulagic Acid and Punicalagin as Novel Allosteric Inhibitors of SARS-CoV-2 3CLpro
El-Baba, Lutomski, Kantsadi, Malla, John et al., Allosteric inhibition of the SARS-CoV-2 main protease: Insights from mass spectrometry based assays, Angew. Chemie-Int. Ed,
doi:10.1002/anie.202010316Ghosh, Chapsal, Weber, Mitsuya, Design of HIV protease inhibitors targeting protein backbone: An effective strategy for combating drug resistance, Acc. Chem. Res,
doi:10.1021/ar7001232Glickman, Wu, Mercuri, Illy, Bowen et al., A Comparison of ALPHAScreen, TR-FRET, and TRF as Assay Methods for FXR Nuclear Receptors, J. Biomol. Screen,
doi:10.1177/108705710200700102Hamamoto, Fukuda, Ishimura, Rumi, Kazumori et al., 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,
doi:10.1067/mlc.2003.8Hoffmann, Kleine-Weber, Schroeder, Krüger, Herrler et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor
Horby, Mafham, Bell, Linsell, Staplin et al., Lopinavir-ritonavir in patients admitted to hospital with COVID-19 (RECOVERY): A randomised, controlled, open-label, platform trial, Lancet,
doi:10.1016/S0140-6736(20)32013-4Huang, Ye, Chen, Chai, Lu et al., Use of All-Trans Retinoic Acid in the Treatment of Acute Promyelocytic Leukemia
Luo, Ross, Retinoic acid exerts dual regulatory actions on the expression and nuclear localization of interferon regulatory factor-1, Exp. Biol. Med,
doi:10.1177/153537020623100517Maeda, Yamaguchi, Hijikata, Morita, Tanaka et al., All-trans retinoic acid attacks reverse transcriptase resulting in inhibition of HIV-1 replication, Hematology,
doi:10.1080/10245330701255130Mark, Ghyselinck, Chambon, Function of retinoic acid receptors during embryonic development, Nucl. Recept. Signal,
doi:10.1621/nrs.07002Matsunaga, Masaoka, Sawasaki, Morishita, Iwatani et al., A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors, Front. Microbiol
Matsuyama, Nao, Shirato, Kawase, Saito et al., Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells,
doi:10.1073/pnas.2002589117Mucida, Park, Kim, Turovskaya, Scott et al., Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid, Science,
doi:10.1126/science.1145697Naoki, Arihiro, Toshiyuki, Noriko, Fumio et al., The genome landscape of the African Green Monkey kidney-derived vero cell line, DNA Res,
doi:10.1093/dnares/dsu029Pillaiyar, Manickam, Namasivayam, Hayashi, Jung, An overview of severe acute respiratory syndromecoronavirus (SARS-CoV) 3CL protease inhibitors: Peptidomimetics and small molecule chemotherapy, J. Med. Chem,
doi:10.1021/acs.jmedchem.5b01461Sanders, Monogue, Jodlowski, Cutrell, Pharmacologic treatments for coronavirus disease 2019 (COVID-19): A review, JAMA J. Am. Med. Assoc,
doi:10.1001/jama.2020.6019Soye, Trottier, Di Lenardo, Restori, Reichman et al., In vitro inhibition of mumps virus by retinoids, Virol. J,
doi:10.1186/1743-422X-10-337Trott, Olson, Vina, Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, J. Comput. Chem,
doi:10.1002/jcc.21334Vuong, Khan, Fischer, Arutyunova, Lamer et al., Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication, Nat. Commun,
doi:10.1038/s41467-020-18096-2Wang, Cao, Zhang, Yang, Liu et al., Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro, Cell Res,
doi:10.1038/s41422-020-0282-0Yamada, Sato, Sotoyama, Orba, Sawa et al., RIG-I triggers a signaling-abortive anti-SARS-CoV-2 defense in human lung cells, Nat. Immunol,
doi:10.1038/s41590-021-00942-0Yamaguchi, Maeda, Ueda, Hijikata, Morita et al., Dichotomy of all-trans retinoic acid inducing signals for adult T-cell leukemia, Leukemia,
doi:10.1038/sj.leu.2403760Yamaoka, Matsunaga, Jeremiah, Nishi, Miyakawa et al., Zika virus protease induces caspase-independent pyroptotic cell death by directly cleaving gasdermin D, Biochem. Biophys. Res. Commun,
doi:10.1016/j.bbrc.2020.11.023Yamaoka, Matsuyama, Fukushi, Matsunaga, Matsushima et al., Development of monoclonal antibody and diagnostic test for Middle East respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen, Front. Microbiol,
doi:10.3389/fmicb.2016.00509Zhang, Lin, Sun, Curth, Drosten et al., Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved a-ketoamide inhibitors, Science,
doi:10.1126/science.abb3405Zhu, Xu, Chen, Guo, Shen et al., Identification of SARS-CoV-2 3CL protease inhibitors by a quantitative high-throughput screening, ACS Pharmacol. Transl. Sci
Zhu, Zhang, Wang, Li, Yang et al., A novel coronavirus from patients with pneumonia in China, 2019, N. Engl. J. Med,
doi:10.1056/NEJMoa2001017
