key: cord-0951164-29wxoac4
authors: Chen, Guan-Yu; Pan, Yi-Cheng; Wu, Tung-Ying; Yao, Tsung-You; Wang, Wei-Jan; Shen, Wan-Jou; Ahmed, Azaj; Chan, Shu-Ting; Tang, Chih-Hsin; Huang, Wei-Chien; Hung, Mien-Chie; Yang, Juan-Cheng; Wu, Yang-Chang
title: Potential natural products that target the SARS-CoV-2 spike protein identified by structure-based virtual screening, isothermal titration calorimetry and lentivirus particles pseudotyped (Vpp) infection assay
date: 2021-09-16
journal: J Tradit Complement Med
DOI: 10.1016/j.jtcme.2021.09.002
sha: 9ba864eed8689ffb8a29804e9cc45e46ab2c1083
doc_id: 951164
cord_uid: 29wxoac4

BACKGROUND AND AIM: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells through the binding of the viral spike protein with human angiotensin-converting enzyme 2 (ACE2), resulting in the development of coronavirus disease 2019 (COVID-19). To date, few antiviral drugs are available that can effectively block viral infection. This study aimed to identify potential natural products from Taiwan Database of Extracts and Compounds (TDEC) that may prevent the binding of viral spike proteins with human ACE2 proteins. METHODS: The structure-based virtual screening was performed using the AutoDock Vina program within PyRX software, the binding affinities of compounds were verified using isothermal titration calorimetry (ITC), the inhibitions of SARS-CoV-2 viral infection efficacy were examined by lentivirus particles pseudotyped (Vpp) infection assay, and the cell viability was tested by 293T cell in MTT assay. RESULTS AND CONCLUSION: We identified 39 natural products targeting the viral receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in silico. In ITC binding assay, dioscin, celastrol, saikosaponin C, epimedin C, torvoside K, and amentoflavone showed dissociation constant (K(d)) = 0.468 μM, 1.712 μM, 6.650 μM, 2.86 μM, 3.761 μM and 4.27 μM, respectively. In Vpp infection assay, the compounds have significantly and consistently inhibition with the 50–90% inhibition of viral infection efficacy. In cell viability, torvoside K, epimedin, amentoflavone, and saikosaponin C showed IC(50) > 100 μM; dioscin and celastrol showed IC(50) = 1.5625 μM and 0.9866 μM, respectively. These natural products may bind to the viral spike protein, preventing SARS-CoV-2 from entering cells. SECTION: 1. Natural Products. TAXONOMY (CLASSIFICATION BY EVISE): SARS-CoV-2, Structure-Based Virtual Screening, Isothermal Titration Calorimetry and Lentivirus Particles Pseudotyped (Vpp) Infection Assay, in silico and in vitro study.

To analyze changes in the SARS-CoV-2 spike protein conformation between the ACE2-free and 170 ACE2-bound forms, structural superimpositions were performed using PyMOL (version 0.99rc6) 171 software 25 . Fig. S3 shows the homology modeling performed for the ACE2-free spike protein, which 172 was obtained from the SWISS-MODEL website, and the ACE2-bound protein (PDB ID: 6M0J), which 173 was obtained from the Protein Data Bank (PDB, https://www.rcsb.org/) 18 ). Subsequently, energy minimization using the steepest descent algorithm was performed with the 181 MMFF94 force field to appropriately add partial charges and polar hydrogens to the atoms in the 182 compounds. Next, those compounds were individually saved as .pdbqt format files by the Open Babel 183 program within PyRx software 31 . The substrates, including ligands, metal ions, and water molecules, 184 that are contained in both the ACE2-free and ACE2-bound spike proteins, as resolved by X-ray 185 crystallography, were removed by DS 2019. Next, the atoms contained in the residues of the protein 186 structures were modified by DS 2019 using the CHARMm force field to appropriately add polar 187 hydrogens and partial charges. For docking calculations, the dimensions of docking search spaces were 188 set to contain the residues of the ACE2 binding site of the spike protein RBD as follows. For the ACE2-189 bound spike protein structure, as resolved by X-ray crystallography, the coordinates of the docking 190 search space were set to x = −38.6172, y = 28.4230, and z = 5.0328, and the size of the dimensions of 191 the x-, y-, and z-axes (in angstroms) were respectively set to x = 30.2373, y = 57.9713, and z = 19. 4614, 192 at the connective interface of complex proteins. Similarly, for the ACE2-free spike protein, the 193 coordinates of the docking search space were set to x = 226.271, y = 195.386, and z = 306.4952, and 194 the sizes of the dimensions of the x-, y-, and z-axes (in angstroms) were respectively set to x = 47. Cell viability was assessed using the MTT assays. Torvoside K, epimedin C, amentoflavone, 217 saikosaponin C, dioscin and celastrol were dissolved in DMSO. 293T cells were seeded on 96-well 218 plates at 4 × 104 cells/well. After incubation for 24 h, cells were treated with 0.044828-100 μM natural 219 compounds for 48 h. The culture medium was removed and cells were washed twice with phosphate-220 buffered saline (PBS). 100 μL MTT/medium solution (2.5 mg/mL) were added to each well and 221 incubated with cells for 1 h. After incubation, the medium was removed and 100 μL aliquots of DMSO 222 were added to each well to solubilize the formazan crystals. Absorbance was measured at 470 nm using surface with the human ACE2 protein 17 . A previous study reported that SARS-CoV could alter the 245 conformation of the C-terminal domain (CTD) of the spike protein to better fit the conformation of the 246 ACE2 protein using various angles 10 . To analyze the conformation of the SARS-Co-V-2 spike protein 247 RBD and to determine whether conformational changes occurred between the ACE2-free state and the 248 ACE2-bound state, structural superimposition was performed using PyMOL software. Fig. 1 shows 249 the superimposition model that was constructed [root-mean-square deviation (RMSD) = 1.735 < 2 Å], 250

showing the ACE2-free spike protein aligned with the ACE2-bound spike protein. We found that the 251 conformation of the viral receptor-binding motif (RBM), which contains many of the residues that 252 contact the human ACE2 protein 27 , differed between the ACE2-free and ACE2-bound spike proteins. 253

The RBM of the ACE2-bound spike protein was more proximal to the RBD core and the ACE2 protein 254 than that of the ACE2-free spike protein (blue ring in Fig. 1 ). Compounds that bind to these regions 255 have a high probability of causing a conformational change in the spike proteins, which would likely 256 reduce the binding affinity between the spike protein and the ACE2 protein. To quickly find and 257 identify potential compounds that target the spike proteins, structured-based virtual screening 258 technology was applied. 259 260

Structure-based virtual screening was performed using the AutoDock Vina program within PyRX 262 software to discover compounds that not only bind to the RBD of the ACE2-free spike protein but also 263 the interface between ACE2 and spike protein. Fig. 2 shows the flowchart followed during the virtual 264 screening calculation performed in this study. The numbers of compounds that were able to bind to the 265 ACE2-free spike protein and the spike-ACE2 complex protein were 53 and 222, respectively (binding 266 energy ≤ −8 kcal/mol). Generally, if compounds with the value of the binding energy were higher than 267 −6 kcal/mol, the binding ability was not expected 23 . The data showed that the 39 potential compounds 268 not only binding to the RBD of the ACE2-free spike protein but also binding to the interface between 269 ACE2 and spike protein in silico (binding energy ≤ −8 kcal/mol, Table S1 ). The structures of the 39 270 compounds were also shown in Fig. S4 . 271

To further estimate the binding strength of the 39 compounds, the natural product glycyrrhizic 272 acid was set as the positive control to do the comparison. Glycyrrhizic acid had been reported that it 273 can bind to SARS-CoV-2 spike protein and has the inhibitory activity against the interaction between 274 viral spike protein and ACE2 protein 33 . The docking results showed that the binding energies of 275 glycyrrhizic acid in ACE2-free spike protein and ACE2-spike complex were -7.8 kcal/mol and -8. Table 1 . Generally, the strength of the binding affinities 292 between the compounds and the protein could be estimated by the Kd value 34 . If the Kd value of the 293 J o u r n a l P r e -p r o o f compound was smaller, the binding affinity was stronger. The data showed that the Kd of dioscin, 294 celastrol, saikosaponin C, epimedin C, torvoside K, candidine, cephalinone D, and amentoflavone were 295 0.468 μM, 1.712 μM, 6.650 μM, 2.86 μM, 3.761 μM, 15.7 μM, 4.371 μM and 4.27 μM, respectively. 296

However, the results of c value estimation reported that candidine and cephalinone D were illegal (c 297 value < 1). Additionally, according to the thermodynamics formulas, the ΔG was primarily contributed 298 by Kd. A negative ΔG indicates a spontaneous reaction process, and smaller ΔG values suggest stronger 299 binding affinities. The results showed that the ΔG values of the 6 legal compounds ranged from −37 to 300 −26 kJ/mol, and the binding between the S1 domain of the spike protein and these compounds occurred 301 as the result of spontaneous reaction processes (Δꓖ < 0 kJ/mol). We discovered that dioscin, celastrol, 302 epimedin C, amentoflavone, torvoside K, and Saikosaponin C might have the ability to bind to the S1 303 domain of the SARS-CoV-2 spike protein. 304 305

To further analyze which amino acids of the ACE2-free and spike-ACE2 complex interacted with 307 these identified compounds, the molecular simulation studies were performed using DS 2019 visualizer 308 software. The results of the molecular simulation are documented in Table 2 . 309 Fig. 10D) . 391

We selected the dioscin, celastrol, epimedin C, amentoflavone, torvoside K, and Saikosaponin C 393 to treat 293T/hACE2 cells to examine their inhibitory activity. We discovered that cells with dioscin, 394 celastrol, epimedin C, amentoflavone, torvoside K, and Saikosaponin C treatments significantly and 395 consistently inhibited the 50-90 % of SARS-CoV-2 viral infection efficacy (Fig. 11) . 396 397

To observe the toxicity of these antiviral potential compounds in normal cells, cytotoxicity 399 assessment of these compounds was carried out by 293T cells in MTT assay. We found that the IC50 400 value of torvoside K, epimedin, amentoflavone, and saikosaponin C were greater than 100 μM; the 401 IC50 value of dioscin and celastrol were 1.5625 μM and 0.9866 μM, respectively (Fig. 12) . 402 403

The predictions of the molecular physicochemical properties and drug-likeness are useful for drug 405 development. The web tools, such as the admetSAR (version 2.0) website, Drug Likeness Tool 406 (DruLiTo) website, and Chemicalize website were useful for the prediction of molecular 407 physicochemical properties and estimation of drug-likeness. In table 3, the data reported that candidine, 408 dioscin, and saikosaponin C had higher possibilities to become successful drugs than others through 409 the estimations of five drug-likeness rules (Lipinski's rule, Ghose Filter, CMC-50 like rule, Veber rule, 410 MDDR-like rule). Table 4 showed the molecular physicochemical properties of the 6 potential natural 411 products. The results predicted that celastrol, saikosaponin C, epimedin C, and amentoflavone had the 412 positive (+) ability in human intestinal absorption; saikosaponin C, epimedin C, and torvoside K had 413 higher water solubility than others; The 6 compounds were safe in carcinogenicity. 414 Table S1 have reasonable binding energies of approximately −8 kcal/mol, which 426 may be considered in future phenotypic assays. These compounds may be able to reduce the binding 427 between the viral spike protein and the human ACE2 protein. Among the 39 identified compounds, we 428 also found that dioscin, actinomycin D, and saikosaponin C have previously been reported to exert 429 antiviral activity against other viruses (Table S2) , which combined with our binding affinity results and 430 druglikeness predictions, suggest that dioscin might be the most promising candidate for further 431 development into a potential drug against SARS-CoV-2 activity. The 6 natural products that we 432 discovered from the database have the potential ability to bind to the viral spike protein to affect the 433 binding affinity between the viral spike protein and the human ACE2 protein in vitro. However, the 434 efficacy of the compounds required additional testing using in vivo assays. 435 J o u r n a l P r e -p r o o f Natural products have been found to be beneficial and have long been used to develop effective 436 drugs against several diseases 41 . Therefore, we examined our endemic TDEC, which is rich and diverse 437 in natural product resources derived from traditional medicine, domestic microbes, and marine 438 organisms. The results reported here describe the performance of a structure-based virtual screening 439 combined with ITC binding assay and Vpp infection assay to identify natural products from among an 440 existing database to identify compounds with the potential to prevent the interaction between the SARS-441

CoV-2 spike protein RBD and the ACE2 receptor of the host cell. Although, the bioactivity of the 6 442 compounds was verified to have good binding affinities with the S1 domain of the spike protein through 443 the ITC binding assay and the ability of inhibitory SARS-CoV-2 virus infection was verified by in vitro 444 lentivirus particles pseudotyped (Vpp) infection assay, the in vivo investigations are remaining 445 necessary to confirm the abilities of those potential compounds for against SARS-CoV-2 infections. 446

Until now, many approaches have been devoted to finding compounds binding to ACE2-free 447 SARS-CoV-2 spike protein against viral infection 42,43 . Besides, several kinds of research using protein-448 protein interaction analysis to find compounds, such as zanamivir, for the inhibition of viral spike 449 protein binding to human ACE2 protein through spike-ACE2 complex analysis 42,44 . For example, 450 hesperidin, a natural product, had been reported its disrupt the binding interface between the spike 451 protein and ACE2 42 . The above approaches are valuable and useful strategies to design antiviral activity 452 compounds for preventing binding between viral spike protein and human ACE2 protein. In this study, 453 the major aim of our approach is to discover potential compounds binding to viral spike protein to 454 prevent viral infection. Our strategy is coupled with the above two strategies that were based on the 455 ACE2-free spike protein and ACE2-complex proteins analysis. The benefit of our virtual screening 456 strategy is not only finding the potential compounds binding to prefusion viral spike protein but also 457 finding compounds binding to the connective interface of the spike-ACE2 complex. Besides, we 458 confirmed the bioactivity of the 6 potential natural products by ITC binding assay and Vpp infection 459 assay. We expected that the 6 compounds not only inhibit the binding between viral spike protein and 460 ACE2 protein but also interfere with the complex process of the viral entry mechanism, such as the 461 coordination between receptor binding and spike protein (S1/S2), at the ACE2-spike complex states 42 . 462 Moreover, the 6 compounds are natural products, we also expected those compounds are safe and 463 beneficial for drug developments. 464 ACE2 plays an important role in Renin-Angiotensin System which regulates blood volume and 465 systemic vascular resistance. SARS-CoV-2 spike protein inhibitors inhibit viral infection by blocking 466 the interaction between spike protein and ACE2 protein, and these inhibitors may interfere with ACE2 467 physiological function to abolish blood pressure regulation. Therefore, the side effect of spike protein 468 inhibitors is necessary to be considered. ACE2 inhibitors have been reported that inhibit spike protein-469 mediated SARS virus infection, and decrease ACE2 enzymatic activity in a dose-dependent manner. In 470 contrast, the ACE-2-interacting Domain of SARS-CoV-2 (AIDS) peptide can decrease SARS-CoV-2 471 infection by blocking ACE2 and SARS-CoV-2 interaction without drug-related side effects. Therefore, 472 the binding site of spike protein inhibitors may modulate Renin-Angiotensin System-associated side 473 effects 45-47 . 474

We found that dioscin, actinomycin D and saikosaponin C had been reported that they had anti-475 virus activity in previous studies (Table S2) . Dioscin can inhibit the initial stage of adenovirus infection 476 in 293 cells 48 ; in our Vpp infection assay, dioscin prevented more than 80% of the virus to infect cells 477 in 8.69mg/L (10μM), which is lower than the solubility of the compound (0.2g/L). Actinomycin D can 478 inhibit measles virus replication and RNA synthesis in Vero cells 49 . Saikosaponin C has anti-HBV 479 replication activity 50 . Among that, dioscin and saikosaponin C had been identified that they can inhibit 480 the SARS-CoV-2 viral infection in vitro. Besides, the estimation of ADME/T and drug-likeness also 481 indicated that dioscin and saikosaponin C had passed the Veber rule and MDDR-like rule estimation 482 (Table 3 and Table 4 ). Dioscin and saikosaponin C may be considered as potential compounds for 483 developing anti-SARS-CoV-2 drugs. 484 test compounds 53 . Our results showed that dioscin, celastrol, saikosaponin C, epimedin C, torvoside K, 510 and amentoflavone display good inhibitory activity decreasing the binding between SARS-CoV-2 spike 511 and ACE2 protein. Therefore, these potential natural products may have the ability against SARS-CoV-512 2 viral infection. 513 514

This study was performed to identify potential drugs for COVID-19 therapy. We virtually 516 screened more than 2,000 drugs against the RBD of the SARS-CoV-2 spike protein. After data mining 517 and filtering out unfitted compounds, we identified 39 compounds with high estimated binding 518 affinities with the targeted spike protein. Among these identified compounds, the 6 natural products, 519 including dioscin, celastrol, saikosaponin C, epimedin C, torvoside K, and amentoflavone, were further 520 analyzed to verify the binding affinity with the target protein using ITC. Besides, we also discovered 521 that cells with dioscin, celastrol, epimedin C, amentoflavone, torvoside K, and Saikosaponin C 522 treatments significantly and consistently inhibited the 50-90 % of SARS-CoV-2 viral infection 523 efficacy. Our results suggested that these 6 natural products binding to the viral spike protein with 524 strong affinities and antiviral efficacy. We believe that these compounds could represent potential 525 drugs for the treatment and prevention of SARS-CoV-2 infection. These identified compounds require 526 further validation in animal-based tests to determine their potential to be developed into anti-SARS-527

CoV-2 therapies. 528 529

Author Shu-Ting Chan was employed by the company TCI CO., Ltd., Taiwan. The remaining 531 authors declare that the research was conducted in the absence of any commercial or financial 532 relationships that could be construed as a potential conflict of interest. Table 2 The residues that interact with the 6 potential natural products within the ACE2-free spike 776 and spike-ACE2 complex proteins. 777 778 Table 3 The physiochemical properties and drug-likeness in 6 potential natural products. 779 780 Table 4 The pharmacokinetics, water solubility, and toxicity prediction in 6 potential natural products. 

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Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection 591 by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a 592 high capacity to mediate membrane fusion

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A highly conserved cryptic epitope in the receptor-binding 597 domains of SARS-CoV-2 and SARS-CoV

Crystal structure of the 2019-nCoV spike receptor-binding domain 599 bound with the ACE2 receptor

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Electrostatic interactions (Å) Hydrophobic interactions (Å) Electrostatic interactions (Å) Hydrophobic interactions (Å) Dioscin Tyr351 (2.25Å), Asn354 (2.45Å)

Electrostatic interactions (Å) Hydrophobic interactions (Å) Electrostatic interactions (Å) Hydrophobic interactions (Å)

35 Å), Asn354 (2.83 Å)

.61 Å)

.08 Å), Phe490 (2.16 Å)

.56 Å)

Asn33 (2.16 Å)

Tyr421

.04 Å), Pro463 (3.02 Å), Leu492

Pro463 (3.94 Å)

.56 Å)

Ala386 (2.25 Å)

51 Å)

Tyr505 (5.94 Å)

The quantification of protein is one of the important processes in carrying out ITC binding assay. 485There are many common and useful methods had been applied to the biochemical analysis of protein, 486 such as Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-487 carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays 51 . Among them, the Bradford assay is 488 a popular, simple, rapid, inexpensive, and sensitive assay. It is based on the direct binding of Coomassie 489 brilliant blue G-250 (CBBG) dye to the proteins 51 . In our study, we employ the Bradford assay to 490 analyze the quantification of SARS-CoV-2 spike protein. It is based on the following reasons. Firstly, 491the Bradford assay has widely been used in biochemical analysis of protein quantitation in ITC binding 492 assay. Secondly, it is faster, cheaper, and easier for us than other protein analyses. Therefore, the 493 Bradford assay is adopted to carry out quantification of protein analysis in this study. Although some 494 of the methods for the quantification of protein is a high accuracy, it is less used to carry out protein 495 analysis for ITC binding assay. For example, amino acid analysis (AAA) is one of the accurate assays 496 for protein quantification 52 . However, it is much less to be adopted to implement quantification of 497 protein by AAA for ITC binding assay. Until now, we do not find the literature that quantification of 498 protein was carried out by AAA for ITC binding assay. Moreover, we lack the necessary types of 499 equipment and skills for carrying out AAA. Therefore, we employ the Bradford assay for protein 500 analysis in this study. It is one of the limitations of this study. 501The SARS-CoV-2 virus enters human cells through viral spike protein binding to human ACE2 502 protein. Considering the SARS-CoV-2 virus is a biosafety-level-3 virus, a simplified assay using 503 pseudotyped biosafety-level-2 viral particles with viral spike protein is necessary 53 . In this study, we 504 employ lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 spike protein infection assay to 505 further verify the antiviral activity of these potential natural products. In this assay system, the 293T 506 cells are transfected with an HIV-based lentiviral system that can produce SARS-CoV-2 Spike-507 pseudotyped lentiviral particles. And then, to make viral particles infect 293T cells expressing human 508 ACE2 53 . Finally, to measure the Luciferase expression to estimate the inhibitory of viral infection of 509 J o u r n a l P r e -p r o o f