Cannabinoids Block Cellular Entry of SARS-CoV-2 and the Emerging Variants
- Richard B. van Breemen*, Ruth N. Muchiri, Timothy A. Bates, Jules B. Weinstein, Hans C. Leier, Scotland Farley, and Fikadu G. Tafesse Cite this: J. Nat. Prod. 2022, XXXX, XXX, XXX-XXX Publication Date:January 10, 2022
Abstract

As a complement to vaccines, small-molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, which cause COVID-19. Affinity selection–mass spectrometry was used for the discovery of botanical ligands to the SARS-CoV-2 spike protein. Cannabinoid acids from hemp (Cannabis sativa) were found to be allosteric as well as orthosteric ligands with micromolar affinity for the spike protein. In follow-up virus neutralization assays, cannabigerolic acid and cannabidiolic acid prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein and prevented entry of live SARS-CoV-2 into cells. Importantly, cannabigerolic acid and cannabidiolic acid were equally effective against the SARS-CoV-2 alpha variant B.1.1.7 and the beta variant B.1.351. Orally bioavailable and with a long history of safe human use, these cannabinoids, isolated or in hemp extracts, have the potential to prevent as well as treat infection by SARS-CoV-2.
Figure 1

Figure 1. Affinity selection–mass spectrometric (AS-MS) discovery of natural ligands to the SARS-CoV-2 spike protein. (A) The spike protein of SARS-CoV-2 consists of trimers of a protein containing an S1 subunit, an S2 subunit, and a transmembrane domain. The S1 subunit binds to human ACE2 to initiate cell entry. Recombinant S1 containing a His-tag was immobilized on magnetic microbeads for affinity selection of ligands. (B) AS-MS was used to isolate and identify natural ligands to the spike protein S1 subunit. A magnetic probe retained the microbeads containing the S1 subunit and bound ligands, while unbound compounds were washed away. Ligands were released using organic solvent and then analyzed using UHPLC-MS. (C) During AS-MS, the SBP-1 peptide bound to immobilized S1 (equivalent to 0.17 μM) (positive control) but not to immobilized denatured S1 (negative control). (D) MagMASS was used for the affinity selection and identification of cannabinoid acids (0.10 μM each in this confirmatory chromatogram) as ligands from hemp extracts. Negative controls using denatured S1 showed no significant binding of cannabinoid acids.
Results and Discussion
Discovery of Hemp Ligands against SARS-CoV-2
cannabinoidb | UHPLC retention time (min) | fold peak area enrichmentc |
---|---|---|
cannabigerolic acid (CBGA) | 3.8 | 20.5 ± 0.51 |
tetrahydrocannabinolic acid (THCA-A) | 8.2 | 16.7 ± 2.2 |
cannabidiolic acid (CBDA) | 3.7 | 12.2 ± 0.52 |
cannabinolic acid (CBNA) | 6.5 | 5.6 ± 1.4 |
cannabigerol (CBG) | 4.1 | 3.4 ± 0.82 |
cannabinol (CBN) | 5.7 | 3.4 ± 0.78 |
Δ8-tetrahydrocannabinol (Δ8-THC) | 6.8 | 3.1 ± 0.81 |
Δ9-tetrahydrocannabinol (Δ9-THC) | 6.8 | 3.0 ± 0.77 |
cannabidiol (CBD) | 4.2 | 2.9 ± 0.72 |
cannabichromene (CBC) | 8.1 | 2.9 ± 0.75 |
Cannabidivarin (CBDV) | 3.0 | 1.6 ± 0.17 |
Mean ± SE (n = 3).
Equimolar cannabinoid mixture (0.10 μM) incubated with the S1 subunit of spike protein (0.17 μM).
Fold peak area enrichment = (UHPLC-MS/MS peak area experiment)/(UHPLC-MS/MS peak area negative control using denatured spike protein S1 subunit).
Figure 2

Figure 2. Computational based modeling of the binding of cannabinoid acids to the SARS-CoV-2 spike protein S1 C-terminal domain using AutoDock Vina. The active site residues of the S1 subunit are shown in yellow. (A) CBGA (pink) is predicted to bind to the anallosteric site (−6.6 kcal/mol free energy of binding). (B) Although less favorable (−6.2 kcal/mol), CBGA (magenta) can also bind to the orthosteric site on the S1 C-terminal domain. (C) THCA-A (cyan) and (D) CBDA (teal) are predicted to bind at the orthosteric site with free energies of binding of −6.5 kcal/mol and −6.3 kcal/mol, respectively.
Figure 3

Figure 3. CBD compounds block viral entry of SARS-CoV-2 through spike binding. Neutralization of spike protein pseudotyped lentivirus and multiple variants of live SARS-CoV-2 virus by cannabinoids CBDA and CBGA. (A) Representative images of high-resolution microscopy of SARS-CoV-2 (WA1/2020)-infected Vero E6 cells treated with 25 μg/mL CBDA, CBGA, or vehicle (control). Cells were stained with anti-ds-RNA (red) antibody to visualize replication sites formed during infection. DAPI (blue) was used to stain nuclei. (B) Infection of ACE2 293T cells with SARS-CoV-2 spike pseudotyped lentivirus in the presence of CBDA or CBGA. Percent neutralization was determined by quantification of total GFP signal resulting from successful pseudovirus infection, normalized to vehicle control (n = 3). (C) Table of IC50 values for pseudovirus experiments. (D and E) Live-virus infection of Vero E6 cells with SARS-CoV-2 variants (WA1/2020, B.1.1.7, and B.1.351) in the presence of CBDA (D) or CBGA (E). Percent neutralization was normalized to vehicle control wells (n = 3). (F) Table of IC50 values for live-virus experiments shown in D and E. IC50 values were determined by fitting data to a three-parameter model for pseudotype infection (C) and live-infection (F) experiments.
Figure 4

Figure 4. Orthosteric site residues of the spike S1 receptor binding domain. The residues in magenta are mutated in the B.1.351 variant (K417N, E484 K, N501Y). The B.1.1.7 variant mutation occurs at N501Y.
Dissociation Constants and Ligand Docking
Inhibition of SARS-CoV-2 Cell Entry
Experimental Section
General Experimental Procedures
Plant Material
Affinity Selection–Mass Spectrometry
Equilibrium Dissociation Constants
Ligand Docking
Pseudotyped Lentivirus Production
SARS-CoV-2 Virus Propagation
Pseudovirus Neutralization Assay
Focus Forming Assay for Live SARS-CoV-2
Immunofluorescence
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.1c00946.
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Tandem mass spectra of affinity selected CBDA, CBGA, and THCA-A and the corresponding standards; cytotoxicity of CBDA in mammalian cell lines (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
The authors thank Shimadzu Scientific Instruments for mass spectrometry support, the Global Hemp Innovation Center for supplying hemp extracts, and the EmerTher company for providing the Ni-NTA magnetic microbeads used in this investigation
References
This article references 49 other publications.
-
1https://www.worldometers.info/coronavirus/, accessed 15 Dec 2021.
-
2Centers for Disease Control and Prevention. https://www.cdc.gov/coronavirus/2019-ncov/science/science-briefs/scientific-brief-emerging-variants.html, accessed 15 Dec 2021.
-
3Walensky, R. P.; Walke, H. T.; Fauci, A. S. JAMA 2021, 325, 1037– 1038, DOI: 10.1001/jama.2021.2294
-
4Tahir ul Qamar, M.; Alqahtani, S. M.; Alamri, M. A.; Chen, L. L. J. Pharm. Anal. 2020, 10, 313– 319, DOI: 10.1016/j.jpha.2020.03.009
-
5Huang, C.; Wang, Y.; Li, X.; Ren, L.; Zhao, J.; Hu, Y.; Zhang, L.; Fan, G.; Xu, J.; Gu, X.; Cheng, Z.; Yu, T.; Xia, J.; Wei, Y.; Wu, W.; Xie, X.; Yin, W.; Li, H.; Liu, M.; Xiao, Y.; Gao, H.; Guo, L.; Xie, J.; Wang, G.; Jiang, R.; Gao, Z.; Jin, Q.; Wang, J.; Cao, B. Lancet 2020, 395, 497– 506, DOI: 10.1016/S0140-6736(20)30183-5
-
6Wu, F.; Zhao, S.; Yu, B.; Chen, Y.-M.; Wang, W.; Song, Z.-G.; Hu, Y.; Tao, Z.-W.; Tian, J.-H.; Pei, Y.-Y.; Yuan, M.-L.; Zhang, Y.-L.; Dai, F.-H.; Liu, Y.; Wang, Q.-M.; Zheng, J.-J.; Xu, L.; Holmes, E. C.; Zhang, Y.-Z. Nature 2020, 579, 265– 269, DOI: 10.1038/s41586-020-2008-3
-
7Jiang, S.; Hillyer, C.; Du, L. Trends Immunol. 2020, 41, 355– 359, DOI: 10.1016/j.it.2020.03.007
-
8Rabi, F. A.; Al Zoubi, M. S.; Kasasbeh, G. A.; Salameh, D. M.; Al-Nasser, A. D. Pathogens 2020, 9, 231, DOI: 10.3390/pathogens9030231
-
9Tai, W.; He, L.; Zhang, X.; Pu, J.; Voronin, D.; Jiang, S.; Zhou, Y.; Du, L. Cell. Mol. Immunol. 2020, 17, 613– 620, DOI: 10.1038/s41423-020-0400-4
-
10Turner, A. J. In Protective Arm of the Renin Angiotensin System (RAS); Unger, T.; Steckelings, U. M.; dos Santos, R. A. S., Eds.; Elsevier: New York, 2015; pp 185– 189. DOI: 10.1016/B978-0-12-801364-9.00025-0
-
11Hoffmann, M.; Kleine-Weber, H.; Schroeder, S.; Krüger, N.; Herrler, T.; Erichsen, S.; Schiergens, T. S.; Herrler, G.; Wu, N.-H.; Nitsche, A.; Müller, M. A.; Drosten, C.; Pöhlmann, S. Cell. 2020, 181, 271– 280, DOI: 10.1016/j.cell.2020.02.052
-
12Guo, Y.-R.; Cao, Q.-D.; Hong, Z.-S.; Tan, Y.-Y.; Chen, S.-D.; Jin, H.-J.; Tan, K.-S.; Wang, D.-Y.; Yan, Y. Military Med. Res. 2020, 7, 11, DOI: 10.1186/s40779-020-00240-0
-
13Du, L.; He, Y.; Zhou, Y.; Liu, S.; Zheng, B.-J.; Jiang, S. Nat. Rev. Microbiol. 2009, 7, 226– 236, DOI: 10.1038/nrmicro2090
-
14Sainz, B., Jr; Mossel, E. C.; Gallaher, W. R.; Wimley, W. C.; Peters, C. J.; Wilson, R. B.; Garry, R. F. Virus Res. 2006, 120, 146– 155, DOI: 10.1016/j.virusres.2006.03.001
-
15Yuan, K.; Yi, L.; Chen, J.; Qu, X.; Qing, T.; Rao, X.; Jiang, P.; Hu, J.; Xiong, Z.; Nie, Y.; Shi, X.; Wang, W.; Ling, C.; Yin, X.; Fan, K.; Lai, L.; Ding, M.; Deng, H. Biochem. Biophys. Res. Commun. 2004, 319, 746– 752, DOI: 10.1016/j.bbrc.2004.05.046
-
16De Clercq, E. J. Clin. Virol. 2001, 22, 73– 89, DOI: 10.1016/S1386-6532(01)00167-6
-
17VanCompernolle, S. E.; Wiznycia, A. V.; Rush, J. R.; Dhanasekaran, M.; Baures, P. W.; Todd, S. C. Virology 2003, 314, 371– 380, DOI: 10.1016/S0042-6822(03)00406-9
-
18Cragg, G. M.; Newman, D. J. Pure Appl. Chem. 2005, 77, 7– 24, DOI: 10.1351/pac200577010007
-
19Dias, D. A.; Urban, S.; Roessner, U. Metabolites 2012, 2, 303– 336, DOI: 10.3390/metabo2020303
-
20von Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.; Habich, D. Angew.Chem. Int. Ed. 2006, 45, 5072– 5129, DOI: 10.1002/anie.200600350
-
21Mishra, B. B.; Tiwari, V. K. Eur. J. Med. Chem. 2011, 46, 4769– 4807, DOI: 10.1016/j.ejmech.2011.07.057
-
22Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629– 661, DOI: 10.1021/acs.jnatprod.5b01055
-
23Kanjanasirirat, P.; Suksatu, A.; Manopwisedjaroen, S.; Munyoo, B.; Tuchinda, P.; Jearawuttanakul, K.; Seemakhan, S.; Charoensutthivarakul, S.; Wongtrakoongate, P.; Rangkasenee, N.; Pitiporn, S.; Waranuch, N.; Chabang, N.; Khemawoot, P.; Sa-Ngiamsuntorn, K.; Pewkliang, Y.; Thongsri, P.; Chutipongtanate, S.; Hongeng, S.; Borwornpinyo, S.; Thitithanyanont, A. Sci. Rep. 2020, 10, 19963, DOI: 10.1038/s41598-020-77003-3
-
24Muchiri, R. N.; van Breemen, R. B. J. Mass Spectrom. 2021, 56, e4647, DOI: 10.1002/jms.4647
-
25van Breemen, R. B.; Huang, C. R.; Nikolic, D.; Woodbury, C. P.; Zhao, Y. Z.; Venton, D. L. Anal. Chem. 1997, 69, 2159– 2164, DOI: 10.1021/ac970132j
-
26Kaur, S.; McGuire, L.; Tang, D.; Dollinger, G.; Huebner, V. J. Protein Chem. 1997, 16, 505– 511, DOI: 10.1023/A:1026369729393
-
27Choi, Y.; van Breemen, R. B. Combin. Chem. High Throughput Screen. 2008, 11, 1– 6, DOI: 10.2174/138620708783398340
-
28Rush, M. D.; Walker, E. M.; Prehna, G.; Burton, T.; van Breemen, R. B. J. Am. Soc. Mass Spectrom. 2017, 28, 479– 448, DOI: 10.1007/s13361-016-1564-0
-
29van Breemen, R. B. Curr. Trends Mass Spectrom. 2020, 18, 18– 25
-
30Citti, C.; Linciano, P.; Panseri, S.; Vezzalini, F.; Forni, F.; Vandelli, M. A.; Cannazza, G. Front. Plant Sci. 2019, 10, 120, DOI: 10.3389/fpls.2019.00120
-
31Hazekamp, A.; Fischedick, J. T.; Díez, M. L.; Lubbe, A.; Ruhaak, R. L. In Comprehensive Natural Products II; Mander, L.; Lui, H.-W.; Eds.; Elsevier: Oxford, UK, 2010; pp 1033– 1084.
-
32Pellesi, L.; Licata, M.; Verri, P.; Vandelli, D.; Palazzoli, F.; Marchesi, F.; Cainazzo, M. M.; Pini, L. A.; Guerzoni, S. Eur. J. Clin. Pharmacol. 2018, 74, 1427– 1436, DOI: 10.1007/s00228-018-2516-3
-
34Sun, Y.; Gu, C.; Liu, X.; Liang, W.; Yao, P.; Bolton, J. L.; van Breemen, R. B. J. Am. Soc. Mass Spectrom. 2005, 16, 271– 279, DOI: 10.1016/j.jasms.2004.11.002
-
35Zhao, Y. Z.; van Breemen, R. B.; Nikolic, D.; Huang, C. R.; Woodbury, C. P.; Schilling, A.; Venton, D. L. J. Med. Chem. 1997, 40, 4006– 4012, DOI: 10.1021/jm960729b
-
36Liu, J.; Burdette, J. E.; Xu, H.; Gu, C.; van Breemen, R. B.; Bhat, K. P.; Booth, N.; Constantinou, A. I.; Pezzuto, J. M.; Fong, H. H.; Farnsworth, N. R.; Bolton, J. L. J. Agric. Food Chem. 2001, 49, 2472– 2479, DOI: 10.1021/jf0014157
-
37Rush, M. D.; Walker, E. M.; Burton, T.; van Breemen, R. B. J. Nat. Prod. 2016, 79, 2898– 2902, DOI: 10.1021/acs.jnatprod.6b00693
-
38Wang, Q.; Zhang, Y.; Wu, L.; Niu, S.; Song, C.; Zhang, Z.; Lu, G.; Qiao, C.; Hu, Y.; Yuen, K. Y.; Wang, Q.; Zhou, H.; Yan, J.; Qi, J. Cell 2020, 181, 894– 904, DOI: 10.1016/j.cell.2020.03.045
-
39Yi, C.; Sun, X.; Ye, J.; Ding, L.; Liu, M.; Yang, Z.; Lu, X.; Zhang, Y.; Ma, L.; Gu, W.; Qu, A.; Xu, J.; Shi, Z.; Ling, Z.; Sun, B. Cell Mol. Immunol. 2020, 17, 621– 630, DOI: 10.1038/s41423-020-0458-z
-
40Tegally, H.; Wilkinson, E.; Giovanetti, M.; Iranzadeh, A.; Fonseca, V.; Giandhari, J.; Doolabh, D.; Pillay, S.; San, E. J.; Msomi, N.; Mlisana, K.; von Gottberg, A.; Walaza, S.; Allam, M.; Ismail, A.; Mohale, T.; Glass, A. J.; Engelbrecht, S.; Van Zyl, G.; Preiser, W.; Petruccione, F.; Sigal, A.; Hardie, D.; Marais, G.; Hsiao, N. Y.; Korsman, S.; Davies, M. A.; Tyers, L.; Mudau, I.; York, D.; Maslo, C.; Goedhals, D.; Abrahams, S.; Laguda-Akingba, O.; Alisoltani-Dehkordi, A.; Godzik, A.; Wibmer, C. K.; Sewell, B. T.; Lourenço, J.; Alcantara, L. C. J.; Kosakovsky Pond, S. L.; Weaver, S.; Martin, D.; Lessells, R. J.; Bhiman, J. N.; Williamson, C.; de Oliveira, T. Nature 2021, 592, 438– 443, DOI: 10.1038/s41586-021-03402-9
-
41Wakshlag, J. J.; Schwark, W. S.; Deabold, K. A.; Talsma, B. N.; Cital, S.; Lyubimov, A.; Iqbal, A.; Zakharov, A. Front. Vet. Sci. 2020, 7, 505, DOI: 10.3389/fvets.2020.00505
-
42Nguyen, L. C.; Yang, D.; Nicolaescu, V.; Best, T. J.; Ohtsuki, T.; Chen, S.-N.; Friesen, J. B.; Drayman, N.; Mohamed, A.; Dann, C.; Silva, D.; Gula, H.; Jones, K. A.; Millis, J. M.; Dickinson, B. C.; Tay, S.; Oakes, S. A.; Pauli, G. F.; Meltzer, D. O.; Randall, G.; Rosner, M. R. bioRxiv 2021, 2021.03.10.432967.
-
43Tautenhahn, R.; Patti, G. J.; Rinehart, D.; Siuzdak, G. Anal. Chem. 2012, 84, 5035– 5039, DOI: 10.1021/ac300698c
-
44Trott, O.; Olson, A. J. J. Comput. Chem. 2009, 31, 455– 461, DOI: 10.1002/jcc.21334
-
45Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235– 242, DOI: 10.1093/nar/28.1.235
-
46Bates, T. A.; Weinstein, J. B.; Farley, S.; Leier, H. C.; Messer, W. B.; Tafesse, F. G. Cell Reports 2021, 34 (7), 108737, DOI: 10.1016/j.celrep.2021.108737
-
47Crawford, K. H. D.; Eguia, R.; Dingens, A. S.; Loes, A. N.; Malone, K. D.; Wolf, C. R.; Chu, H. Y.; Tortorici, M. A.; Veesler, D.; Murphy, M.; Pettie, D.; King, N. P.; Balazs, A. B.; Bloom, J. D. Viruses 2020, 12, 513, DOI: 10.3390/v12050513
-
48Case, J. B.; Bailey, A. L.; Kim, A. S.; Chen, R. E.; Diamond, M. S. Virology 2020, 548, 39– 48, DOI: 10.1016/j.virol.2020.05.015
-
49Katzelnick, L. C.; Coello Escoto, A.; McElvany, B. D.; Chávez, C.; Salje, H.; Luo, W.; Rodriguez-Barraquer, I.; Jarman, R.; Durbin, A. P.; Diehl, S. A.; Smith, D. J.; Whitehead, S. S.; Cummings, D. A. T. PLOS Neglect. Trop. Dis. 2018, 12, e0006862, DOI: 10.1371/journal.pntd.0006862
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