Identification of SARS-CoV-2 Main Protease Inhibitors from a Library of Minor Cannabinoids by Biochemical Inhibition Assay and Surface Plasmon Resonance Characterized Binding Affinity


“The replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its main protease (Mpro), which is a plausible therapeutic target for coronavirus disease 2019 (COVID-19). Although numerous in silico studies reported the potential inhibitory effects of natural products including cannabis and cannabinoids on SARS-CoV-2 Mpro, their anti-Mpro activities are not well validated by biological experimental data. Herein, a library of minor cannabinoids belonging to several chemotypes including tetrahydrocannabinols, cannabidiols, cannabigerols, cannabichromenes, cannabinodiols, cannabicyclols, cannabinols, and cannabitriols was evaluated for their anti-Mpro activity using a biochemical assay. Additionally, the binding affinities and molecular interactions between the active cannabinoids and the Mpro protein were studied by a biophysical technique (surface plasmon resonance; SPR) and molecular docking, respectively. Cannabinoids tetrahydrocannabutol and cannabigerolic acid were the most active Mpro inhibitors (IC50 = 3.62 and 14.40 μM, respectively) and cannabigerolic acid had a binding affinity KD=2.16×10-4 M). A preliminary structure and activity relationship study revealed that the anti-Mpro effects of cannabinoids were influenced by the decarboxylation of cannabinoids and the length of cannabinoids’ alkyl side chain. Findings from the biochemical, biophysical, and computational assays support the growing evidence of cannabinoids’ inhibitory effects on SARS-CoV-2 Mpro.”

“In summary, the inhibitory effects of a collection of cannabinoids on SARS-CoV-2 3CL Mpro were screened by a biochemical assay. Several minor cannabinoids (e.g., THCB and CBGA) showed promising anti-Mpro activity. In addition, we observed that decarboxylated cannabinoids, such as CBG and CBD, showed undermined inhibition capacity, as compared to the precursing cannabinoid acids (i.e., CBGA and CBDA, respectively). This SAR was supported by the binding affinities between these cannabinoids and the Mpro protein obtained from the SPR assays. Furthermore, the impact of the length of the alkyl side chain of cannabinoids on their anti-Mpro activity was explored. Our study is the first to evaluate the anti-Mpro activity of minor cannabinoids and their mechanisms of action, which contribute to a better understanding of cannabinoids’ potential roles in the management of COVID-19.”

Cannabinoids receptors in Covid-19: Perpetrators and victims

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“COVID-19 is caused by SARS-CoV-2 and leads to acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and extrapulmonary manifestations in severely affected cases. However, most of the affected cases are mild or asymptomatic.

Cannabinoids (CBs) such as tetrahydrocannabinol (THC) and cannabidiol (CBD), which act on G-protein-coupled receptors called CB1 and CB2, have anti-inflammatory effects. Many published studies show that CBs are effective in various inflammatory disorders, viral infections, and attenuation of ALI and ARDS.

Therefore, the aim of the present narrative review was to summarize the possible immunological role of CBs in COVID-19. The effects of CBs are controversial, although they have beneficial effects via CB2 receptors and adverse effects via CB1 receptors against ALI, ARDS, and hyperinflammation, which are hallmarks of COVID-19.

The present narrative review has shown that CBs effectively manage ALI and ARDS by suppressing pro-inflammatory cytokines, which are common in COVID-19. Therefore, CBs may be used to manage COVID-19 because of their potent anti-inflammatory effects with suppression of pro-inflammatory cytokines and inhibition of inflammatory signaling pathways.”

Affinity selection-mass spectrometry in the discovery of anti-SARS-CoV-2 compounds

Mass Spectrometry Reviews

“Small molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which is the cause of the COVID-19 pandemic.

To expedite the discovery of lead compounds for development, assays have been developed based on affinity selection-mass spectrometry (AS-MS), which enables the rapid screening of mixtures such as combinatorial libraries and extracts of botanicals or other sources of natural products. AS-MS assays have been used to find ligands to the SARS-CoV-2 spike protein for inhibition of cell entry as well as to the 3-chymotrypsin-like cysteine protease (3CLpro) and the RNA-dependent RNA polymerase complex constituent Nsp9, which are targets for inhibition of viral replication.

The AS-MS approach of magnetic microbead affinity selection screening has been used to discover high-affinity peptide ligands to the spike protein as well as the hemp cannabinoids cannabidiolic acid and cannabigerolic acid, which can prevent cell infection by SARS-CoV-2.

Another AS-MS method, native mass spectrometry, has been used to discover that the flavonoids baicalein, scutellarein, and ganhuangenin, can inhibit the SARS-CoV-2 protease 3CLpro. Native mass spectrometry has also been used to find an ent-kaurane natural product, oridonin, that can bind to the viral protein Nsp9 and interfere with RNA replication.

These natural lead compounds are under investigation for the development of therapeutic agents to prevent or treat SARS-CoV-2 infection.”

Cannabis consumption is associated with lower COVID-19 severity among hospitalized patients: a retrospective cohort analysis

ISRCTN - Publish with BMC

“Background: While cannabis is known to have immunomodulatory properties, the clinical consequences of its use on outcomes in COVID-19 have not been extensively evaluated. We aimed to assess whether cannabis users hospitalized for COVID-19 had improved outcomes compared to non-users.

Methods: We conducted a retrospective analysis of 1831 patients admitted to two medical centers in Southern California with a diagnosis of COVID-19. We evaluated outcomes including NIH COVID-19 Severity Score, need for supplemental oxygen, ICU (intensive care unit) admission, mechanical ventilation, length of hospitalization, and in-hospital death for cannabis users and non-users. Cannabis use was reported in the patient’s social history. Propensity matching was used to account for differences in age, body-mass index, sex, race, tobacco smoking history, and comorbidities known to be risk factors for COVID-19 mortality between cannabis users and non-users.

Results: Of 1831 patients admitted with COVID-19, 69 patients reported active cannabis use (4% of the cohort). Active users were younger (44 years vs. 62 years, p < 0.001), less often diabetic (23.2% vs 37.2%, p < 0.021), and more frequently active tobacco smokers (20.3% vs. 4.1%, p < 0.001) compared to non-users. Notably, active users had lower levels of inflammatory markers upon admission than non-users-CRP (C-reactive protein) (3.7 mg/L vs 7.6 mg/L, p < 0.001), ferritin (282 μg/L vs 622 μg/L, p < 0.001), D-dimer (468 ng/mL vs 1140 ng/mL, p = 0.017), and procalcitonin (0.10 ng/mL vs 0.15 ng/mL, p = 0.001). Based on univariate analysis, cannabis users had significantly better outcomes compared to non-users as reflected in lower NIH scores (5.1 vs 6.0, p < 0.001), shorter hospitalization (4 days vs 6 days, p < 0.001), lower ICU admission rates (12% vs 31%, p < 0.001), and less need for mechanical ventilation (6% vs 17%, p = 0.027). Using propensity matching, differences in overall survival were not statistically significant between cannabis users and non-users, nevertheless ICU admission was 12 percentage points lower (p = 0.018) and intubation rates were 6 percentage points lower (p = 0.017) in cannabis users.

Conclusions: This retrospective cohort study suggests that active cannabis users hospitalized with COVID-19 had better clinical outcomes compared with non-users, including decreased need for ICU admission or mechanical ventilation. However, our results need to be interpreted with caution given the limitations of a retrospective analysis. Prospective and observational studies will better elucidate the effects cannabis use in COVID-19 patients.”

“In this retrospective review of 1831 COVID-19 patients requiring hospital admission, current cannabis use was associated with decreased disease severity. This was demonstrated in lower NIH severity scores as well as less need for oxygen supplementation, ICU admission and mechanical ventilation. While there was a trend toward improved survival in cannabis users”

Antiviral activity of medicinal plant-derived products against SARS-CoV-2

SAGE Journals

“This review presents information from several studies that have demonstrated the antiviral activity of extracts (Andrographis paniculataArtemisia annuaArtemisia afraCannabis sativaCurcuma longaEchinacea purpureaOlea europaeaPiper nigrum, and Punica granatum) and phytocompounds derived from medicinal plants (artemisinins, glycyrrhizin, and phenolic compounds) against SARS-CoV-2.

A brief background of the plant products studied, the methodology used to evaluate the antiviral activity, the main findings from the research, and the possible mechanisms of action are presented.

These plant products have been shown to impede the adsorption of SARS-CoV-2 to the host cell, and prevent multiplication of the virus post its entry into the host cell. In addition to antiviral activity, the plant products have also been demonstrated to exert an immunomodulatory effect by controlling the excessive release of cytokines, which is commonly associated with SARS-CoV-2 infections.”

“Antimicrobial and Antiviral (SARS-CoV-2) Potential of Cannabinoids and Cannabis sativa: A Comprehensive Review”

“Anti-Inflammatory and Antiviral Effects of Cannabinoids in Inhibiting and Preventing SARS-CoV-2 Infection”

“Cannabinoids Block Cellular Entry of SARS-CoV-2 and the Emerging Variants”

Cross-Talk between the (Endo)Cannabinoid and Renin-Angiotensin Systems: Basic Evidence and Potential Therapeutic Significance


“This review is dedicated to the cross-talk between the (endo)cannabinoid and renin angiotensin systems (RAS). Activation of AT1 receptors (AT1Rs) by angiotensin II (Ang II) can release endocannabinoids that, by acting at cannabinoid CB1 receptors (CB1Rs), modify the response to AT1R stimulation.

CB1R blockade may enhance AT1R-mediated responses (mainly vasoconstrictor effects) or reduce them (mainly central nervous system-mediated effects). The final effects depend on whether stimulation of CB1Rs and AT1Rs induces opposite or the same effects. Second, CB1R blockade may diminish AT1R levels. Third, phytocannabinoids modulate angiotensin-converting enzyme-2. Additional studies are required to clarify (1) the existence of a cross-talk between the protective axis of the RAS (Ang II-AT2 receptor system or angiotensin 1-7-Mas receptor system) with components of the endocannabinoid system, (2) the influence of Ang II on constituents of the endocannabinoid system and (3) the (patho)physiological significance of AT1R-CB1R heteromerization.

As a therapeutic consequence, CB1R antagonists may influence effects elicited by the activation or blockade of the RAS; phytocannabinoids may be useful as adjuvant therapy against COVID-19; single drugs acting on the (endo)cannabinoid system (cannabidiol) and the RAS (telmisartan) may show pharmacokinetic interactions since they are substrates of the same metabolizing enzyme of the transport mechanism.”

High-CBD Extract (CBD-X) Downregulates Cytokine Storm Systemically and Locally in Inflamed Lungs

New scientific publication in Frontiers in Immunology - Antineo

“Cytokine storm refers to the dysregulated production of inflammatory mediators leading to hyperinflammation. They are often detrimental, and worsen the severity of COVID-19 and other infectious or inflammatory diseases. Cannabinoids are known to have anti-inflammatory effects but their possible therapeutic value on cytokine storms has not been fully elucidated. In vivo and ex vivo studies were carried out to investigate the effects of high-THC and high-CBD extracts on cytokine production in immune cells. Significant differences between the extracts were observed. Subsequent experiments focusing on a specific high CBD extract (CBD-X) showed significant reductions in pro-inflammatory cytokines in human-derived PBMCs, neutrophils and T cells. In vivo mouse studies, using a systemically inflamed mouse model, showed reductions in pro-inflammatory cytokines TNFα and IL-1β and a concurrent increase in the anti-inflammatory cytokine IL-10 in response to CBD-X extract treatment. Lung inflammation, as in severe COVID-19 disease, is characterized by increased T-cell homing to the lungs. Our investigation revealed that CBD-X extract impaired T-cell migration induced by the chemoattractant SDF1. In addition, the phosphorylation levels of T cell receptor (TCR) signaling proteins Lck and Zap70 were significantly reduced, demonstrating an inhibitory effect on the early events downstream to TCR activation. In a lung inflamed mouse model, we observed a reduction in leukocytes including neutrophil migration to the lungs and decreased levels of IL-1β, MCP-1, IL-6 and TNFα, in response to the administration of the high-CBD extract. The results presented in this work offer that certain high-CBD extract has a high potential in the management of pathological conditions, in which the secretion of cytokines is dysregulated, as it is in severe COVID-19 disease or other infectious or inflammatory diseases.”

Comparative study of CNR1 and CNR2 cannabinoid receptors expression levels in COVID-19 patients with and without diabetes mellitus: Recommendations for future research targets

Diabetes & Metabolic Syndrome: Clinical Research & Reviews

“Background and aims: The COVID-19 pandemic has prompted researchers to look for effective therapeutic targets. The effect of endocannabinoid system against infectious diseases is investigated for several years. In this study, we evaluated the expression level of CNR1 and CNR2 genes in patients with COVID-19 with and without diabetes to provide new insights regarding these receptors and their potential effect in COVID-19 disease.

Methods: In this study, peripheral blood monocytes cells (PBMCs) were isolated from eight different groups including COVID-19 patients, diabetic patients, and healthy individuals. RNA were extracted to evaluate the expression level of CNR1 and CNR2 genes using real-time PCR. The correlation between the expression levels of these genes in different groups were assessed.

Results: A total of 80 samples were divided into 8 groups, with each group consisting of ten samples. When comparing severe and moderate COVID-19 groups to healthy control group, the expression levels of the CNR1 and CNR2 genes were significantly higher in the severe and moderate COVID-19 groups. There were no significant differences between the mild COVID-19 group and the healthy control group. It was found that the expression levels of these genes in patients with diabetes who were infected with SARS-COV-2 did not differ across COVID-19 groups with varying severity, but they were significantly higher when compared to healthy controls.

Conclusion: Our study suggests the possible role of endocannabinoid system during SARS-COV-2 pathogenicity as the expression of CNR1 and CNR2 were elevated during the disease.”

“In conclusion, the outcomes of this research supports the possible role of endocannabinoid system during SARS-COV-2 pathogenicity as the expression of CNR1 and CNR2 were elevated during the disease. Moreover, despite their limitations due to psychiatric side effects, the regulated use of cannabinoids should be examined by researchers to identify their potential effectiveness as a therapeutic target in COVID-19 disease.”

Effect of cannabidiol on apoptosis and cellular interferon and interferon-stimulated gene responses to the SARS-CoV-2 genes ORF8, ORF10 and M protein

Life Sciences

“Aims: To study effects on cellular innate immune responses to ORF8, ORF10, and Membrane protein (M protein) from the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, in combination with cannabidiol (CBD).

Main methods: HEK293 cells transfected with plasmids expressing control vector, ORF8, ORF10, or M protein were assayed for cell number and markers of apoptosis at 24 h, and interferon and interferon-stimulated gene expression at 14 h, with or without CBD. Cells transfected with polyinosinic:polycytidylic acid (Poly (I:C)) were also studied as a general model of RNA-type viral infection.

Key findings: Reduced cell number and increased early and late apoptosis were found when expression of viral genes was combined with 1-2 μM CBD treatment, but not in control-transfected cells treated with CBD, or in cells expressing viral genes but treated only with vehicle. In cells expressing viral genes, CBD augmented expression of IFNγ, IFNλ1 and IFNλ2/3, as well as the 2′-5′-oligoadenylate synthetase (OAS) family members OAS1, OAS2, OAS3, and OASL. CBD also augmented expression of these genes in control cells not expressing viral genes, but without enhancing apoptosis. CBD similarly enhanced the cellular anti-viral response to Poly (I:C).

Significance: Our results demonstrate a poor ability of HEK293 cells to respond to SARS-CoV-2 genes alone, but an augmented innate anti-viral response to these genes in the presence of CBD. Thus, CBD may prime components of the innate immune system, increasing readiness to respond to RNA-type viral infection without activating apoptosis, and could be studied for potential in prophylaxis.”

The Role of Cannabis sativa L. as a Source of Cannabinoids against Coronavirus 2 (SARS-CoV-2): An In Silico Study to Evaluate Their Activities and ADMET Properties


“Cannabis sativa L. is an annual herbaceous plant that belongs to the family Cannabinaceae. In this study, the potential use of forty-five cannabinoids, previously identified from Cannabis sativa to alleviate COVID-19 infection via prohibition of crucial SARS-CoV-2 proteins using molecular docking, was examined. In silico studies were performed on three vital enzymes that serve as principle therapeutic targets to prevent SARS-CoV-2 replication. These enzymes are the main protease SARS-CoV-2 MPro, papain-like protease SARS-CoV-2 PLpro and angiotensin-converting enzyme 2 (ACE2). Regarding SARS-CoV-2 MPro, cannabichromanon (32) showed the best fitting within its active centers, followed by cannabinolic acid (22) and cannabinol (21), displaying ∆G of -33.63, -23.24, and -21.60 kcal/mol, respectively. Concerning SARS-CoV-2 PLpro, cannabichromanon (32) followed by cannabinolic acid (22) and cannabicyclolic acid (41) revealed the best binding within its active pockets owing to multiple bond formation with ∆G values of -28.36, -22.81, and -19.89 kcal/mol. Furthermore, cannabichromanon (32), cannabinolic acid (22), and cannabinol (21) showed considerable fitting within the active sites of angiotensin-converting enzyme 2 (ACE2) evidenced by their significant ∆G values that were estimated as -41.77, -31.34, and -30.36 kcal/mol, respectively. ADME/TOPKAT (absorption, distribution, metabolism, excretion, and toxicity) evaluation was performed on the tested cannabinoids to further explore their pharmacokinetics, pharmacodynamics, and toxicity properties. The results indicated the considerable pharmacokinetic, pharmacodynamic, and toxicity properties of cannabinol (21), cannabinolic acid (22), cannabichromanon (32), and cannabicyclolic acid (41) that showed best fitting scores within the active sites of the tested enzymes. Multivariate data analysis revealed that cannabichromanon and cannabinolic acid showed a discriminant nature and hence can be incorporated in pharmaceutical dosage forms to alleviate COVID-19 infection.”