key: cord-0847640-ex4ncggl authors: Li, Changzhi; Zhou, Hongjuan; Guo, Lingling; Xie, Dehuan; He, Huiping; Zhang, Hong; Liu, Yixiu; Peng, Lixia; Zheng, Lisheng; Lu, Wenhua; Mei, Yan; Liu, Zhijie; Huang, Jie; Wang, Mingdian; Shu, Ditian; Ding, Liuyan; Lang, Yanhong; Luo, Feifei; Wang, Jing; Huang, Bijun; Huang, Peng; Gao, Song; Chen, Jindong; Qian, Chao-Nan title: Potential inhibitors for blocking the interaction of the coronavirus SARS-CoV-2 spike protein and its host cell receptor ACE2 date: 2021-12-15 journal: bioRxiv DOI: 10.1101/2021.12.14.472545 sha: 595015f6d5f36cee17683a2ffa85a1964623c6cd doc_id: 847640 cord_uid: ex4ncggl The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social and economic stability. In this study, we established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus S protein and its host receptor ACE2. This platform is a rapid, sensitive, specific, and high throughput system. With this platform, we screened two compound libraries of 2,864 molecules and identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S-protein and the human cell ACE2 receptor. This data suggests that TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug. SIGNIFICANCE The ongoing pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made a serious threat to public health worldwide. Given the urgency of the situation, researchers are attempting to repurpose existing drugs for treating COVID-19. In this present study, we screened two compound libraries of 2,864 molecules and identified a potent inhibitor (TS-984) for blocking the coronavirus S-protein and the human cell ACE2 receptor. TS-984 might have the potential to be developed into an effective anti-coronavirus drug for treating COVID-19. The ongoing pandemic of COVID-19 caused by the severe acute respiratory syndrome 26 coronavirus 2 (SARS-CoV-2) has made a serious threat to public health worldwide. Given the 27 urgency of the situation, researchers are attempting to repurpose existing drugs for treating 28 COVID-19. In this present study, we screened two compound libraries of 2,864 molecules and 29 identified a potent inhibitor (TS-984) for blocking the coronavirus S-protein and the human cell Optimization of HTRF assay for high-throughput screening 57 To obtain the maximal binding effect of the coronavirus S protein and its ACE2 receptor, we 58 first optimized the ratios of S-RBD/ACE2 and S-RBD-His/ACE2-d2 ( Fig. 1A-1D) . We observed 59 that the assay system worked the best with 1.15 μg/ml of ACE2-d2 and 0.88 μg/ml of S-RBD- 60 His. To ensure the HTRF assay was suitable for high throughput screening of the S protein-61 ACE2 inhibitors, natural compound emodin was used as a positive control in this study as it was 62 previously identified to block the binding of the coronavirus S protein to the ACE2 receptor [2] . 63 PBS with 1% BSA was used as a negative control. The average Z factor value of the assay was Nafamostat mesilate inhibits the binding of SARS-CoV-2 S-RBD to ACE2 69 Nafamostat mesilate was reported to inhibit TMPRSS2. To see whether it could also block the 70 interaction of the coronavirus S protein and its ACE2 receptor, we tested its inhibiting potential 71 with our HTRF high throughput screening platform. Our results indicate that nafamostat 72 mesylate inhibits the interaction of the S protein and ACE2, and its inhibiting effect is more 73 powerful compared with the positive control compound. The EC 50 for nafamostat mesilate and 74 positive control emodin were 11.34μM and 126.1μM, respectively (Fig. 1E ). To identify novel S-RBD/ACE2 binding inhibitors with our HTRF-based screening platform, we 78 screened an FDA compound library of 1,280 molecules and a Topscience compound library with 79 1,584 natural products. The compound libraries were initially screened with our high throughput 80 HTRF platform by using a concentration of 100 μM of each compound ( Fig. 2A, 2B ). In the 81 initial screening, we identified 23 candidate compounds that presented an inhibition effect on the 82 interaction between the SARS-CoV-2 S-RBD and ACE2, with an HTRF inhibition signal >50%. 83 Of the 23 compounds, 20 were excluded in the following validation experiments. Finally, only 84 three compounds, tannic acid from the FDA library, TS984 (9-Methoxycanthin-6-one) and 85 TS1276 (anthraquinone) from the Topscience library passed the EC50 (HTRF) determination by 86 the HTRF screening. The EC 50 s (HTRF) for tannic acid, 9-Methoxycanthin-6-one, and 87 anthraquinone was 49.71 μM, 36.21μM, and 55.9 μM, respectively. Negative Positive TS-0961 TS-0962 TS-0963 TS-0964 TS-0965 TS-0966 TS-0967 TS-0968 TS-0969 TS-0970 TS-0971 TS-0972 TS-0973 TS-0974 TS-0975 TS-0976 TS-0977 TS-0978 TS-0979 TS-0980 TS-0981 TS-0982 TS-0983 TS-0984 TS-0985 TS-0986 TS-0987 TS-0988 TS-0989 TS-0990 TS-0991 TS-0992 TS-0993 TS-0994 TS-0995 TS-0996 TS-0997 TS-0998 TS-0999 TS-1000 TS-1001 TS-1002 TS-1003 TS-1004 TS-1005 TS-1006 TS-1007 TS-1008 TS-1009 TS-1010 TS-1011 TS-1012 TS-1013 TS-1014 TS-1015 TS-1016 TS-1017 TS-1018 TS-1019 TS-1020 TS-1021 TS-1022 TS-1023 TS-1024 TS-1025 TS-1026 TS-1027 TS-1028 TS-1029 TS-1030 TS-1031 TS-1032 TS-1033 TS-1034 TS-1035 TS-1036 TS-1037 TS-1038 TS- COVID-19: Epidemiology, Evolution, and Cross-Disciplinary 317 Perspectives Emodin blocks the SARS coronavirus spike protein and 319 angiotensin-converting enzyme 2 interaction COVID-19: Progress in diagnostics, therapy and vaccination Coronaviruses -drug discovery and therapeutic options Remdesivir and chloroquine effectively inhibit the 325 recently emerged novel coronavirus (2019-nCoV) in vitro First Case of 2019 Novel Coronavirus in 327 the United States Compassionate Use of Remdesivir for Patients 329 with Severe Covid-19 Compassionate remdesivir treatment of 331 severe Covid-19 pneumonia in intensive care unit (ICU) and Non-ICU patients: Clinical outcome and 332 differences in post-treatment hospitalisation status Supportive Care in Hospitalized Patients With Severe COVID-19: A Single-Center Experience Remdesivir in adults with severe COVID-19: a 337 randomised, double-blind, placebo-controlled, multicentre trial Protease inhibitors targeting coronavirus 339 and filovirus entry Nafamostat Mesylate Blocks 341 Activation of SARS-CoV-2: New Treatment Option for COVID-19 Reduce Severity of Coronavirus Disease 2019 Sepsis: A First Observation Camostat mesylate against SARS-CoV-2 345 and COVID-19-Rationale, dosing and safety Identification of Nafamostat as a 347 Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion 348 Using the Split-Protein-Based Cell-Cell Fusion Assay Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and 351 Viral Infection In Vitro in a Cell-Type-Dependent Manner Tannic acid suppresses SARS-CoV-2 as a dual 353 inhibitor of the viral main protease and the cellular TMPRSS2 protease Cytotoxic and antimalarial 355 constituents of the roots of Eurycoma longifolia Tongkat Ali (Eurycoma longifolia Jack): a review on its ethnobotany and 357 pharmacological importance Cytotoxic Compounds from Brucea 359 mollis Establishment and validation of a pseudovirus neutralization 361 assay for SARS-CoV-2 Assessing the application of a pseudovirus 363 system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine 364 development AutoDock4 and AutoDockTools4: 366 Automated docking with selective receptor flexibility UCSF Chimera-A visualization 368 system for exploratory research and analysis Structure of the SARS-CoV-2 spike receptor-binding domain 370 bound to the ACE2 receptor Reparametrization of Protein Force Field Nonbonded 372 Interactions Guided by Osmotic Coefficient Measurements from Molecular Dynamics Simulations Acknowledgments: We thank the South China Center for Innovative Pharmaceuticals for the 376 support of some of the work Funding: This study was supported by the grants from National Natural Science Foundation of 378 Writing -original draft Writing -review & editing Anti-novel coronavirus drug based on the binding target 390 of ACE2 and S protein and its application" (China Patent Application No. 202110165852.6) was 391 approved for tannic acid. Patent entitled "An anti-SARS-CoV-2 drug" (China Patent Application 392 No. 2021105827561 and PCT/CN2021/097391) is pending for TS-984 All the other authors declare that they have no 394 competing interest TS-984 is available from TOPSCIENCE under the name 397 of MT4601. All data are available in the main text or the supplementary materials. All data have 398 been uploaded onto the Research Data Deposit (www.researchdata.org.cn) with the approval 399 number RDD2021000XXX the 9-methoxycanthin-6-one and the S protein/ACE2 complex. The yellow sticks 435 represent the ligand 9-methoxycanthin-6-one, the green and blue sticks represent the 436 important residues within 5Å of the ligand, the red dotted line represents the H-bond 437 interaction located in the 9-methoxycanthin-6-one and the S protein/ACE2 complex.