It has long been recognized that viral infections are difficult to treat, and phytochemicals have been used as antiviral agents against several viruses in view of the fact that they can inhibit viruses either at the viral entry point or during the replication stages, as well as through immunomodulation potentials.¹⁹ Some plant constituents have the potential treatment and antiviral properties against SARS-CoV-2.²⁰

COVID-19 infection is reliant on host cell factors, namely, ACE2. The strong attachment of virus spike (S) proteins to cell receptors allows CoVs to enter the host cell, enhancing viral entry and adhesion to the cell surface, which culminates in infection.²¹ The major source of SARS-CoV-2 transmission was shown to be ACE2. As a result, the inhibitory action of effective metabolites on the ACE2 catalytic pocket may alter ACE2 conformation, inhibiting SARS-CoV-2 entrance into host cells.

Many studies have used molecular modeling to investigate the possibilities of countering COVID-19 infection using active ingredients obtained from natural sources. A recent in silico study²² was conducted on certain cannabinoids using the SARS-CoV-2 Mpro (main protease) enzyme as the main target, followed by in vitro antiviral activity against SARS-CoV-2. Δ9-tetrahydrocannabinol and cannabidiol (CBD) showed the most significant antiviral potential, displaying IC50 values of 10.25 and 7.91 μM, respectively, which were comparable to those of lopinavir, chloroquine, and remdesivir, with IC50 values ranging between 8.16 and 13.15 μM.²²

In this research, the cannabinoid and noncannabinoid components of C. sativa were examined in silico inside the binding pocket of ACE2. Our molecular docking analysis revealed that 6-prenylapigenin and CBN-C3 had the highest binding affinity with the lowest energy of −8.3 kcal/mol. This was followed by Δ8-THCA with an energy of −8.0 kcal/mol. Overall, all 3 drug affinity comparisons with the reference molecule MLN-4760, an inhibitor of ACE2, showed promising binding affinities.

A compound’s drug-likeness is assessed qualitatively by the chance for a molecule to become an oral drug concerning bioavailability. It is determined by how similar a compound is to existing drugs in structural and physicochemical properties. The reference and hit compounds do not violate the physicochemical Ghose, Lipinski, and Veber rules, seeing that the values of their HBA, HBD, log P, molecular weight, TPSA, molar refractivity, and the number of rotatable bonds within the accepted range. However, for the pharmacophore point filter, the Mugge rule was not respected for Δ8-THCA and CBN-C3 with 1 violation, and XLOGP3 >5, which predicts the log P value using a known log P value of a reference compound as a starting point. To a large extent, they are all predicted to have good size, polarity, and flexibility, which positively correlate with good bioavailability.

However, the bioavailability radar of 6-prenylapigenin suggests that it is slightly unsaturated, with a fraction of carbons in the sp3 hybridization (Fsp3) valued at 0.15, which is less than 0.25. The bioavailability radar of Δ8-THCA suggests that the compound is poorly soluble and has low lipophilicity with an XLOGP3 higher than 5. Because drugs interact with a variety of molecular targets in the body, the biological activity profiles of potential drug candidates must account for human metabolism, and all hit compounds have strong enzyme inhibitory activity in bioactivity.

The ADMET characteristics of candidate compounds are the major reason for the high drug discovery attrition rates. Aqueous solubility is a key physicochemical attribute required for the characterization of an active pharmaceutical ingredient (API) during drug discovery.²³ Furthermore, with water solubility values less than −4.0 log mol/L, CBN-C3 is the only compound with poor solubility. The most convenient mode of drug delivery for medicines and xenobiotics is oral administration. The Caco-2 cell line is used in most specialized laboratories to screen novel chemical entities by predicting their solubility, bioavailability, and the probability of drug-drug or herb-drug interactions in the gut lumen because they closely resemble the human intestinal epithelium in many ways.²⁴ In our case, CBN-C3 showed the highest and Δ8-THCA the lowest in Caco-2 cell permeability. Along with Caco-2 permeability, all hit compounds have an excellent percentage of human intestinal absorption (HIA), with Δ8-THCA being the highest (98%). Skin permeability is undoubtedly effective, with a value of less than 2.5 log Kp. For all compounds, 6-prenylapigenin has the best dermal permeability value. P-glycoprotein acts as a transmembrane efflux pump, transporting substrates from the inside to the outside of the cell. It is one of the drug transporters responsible for the absorption and efflux of a variety of pharmaceuticals. All compounds are Pgp substrates aside from Δ8-THCA and 6-prenylapigenin, which was predicted to be a Pgp II inhibitor. This may promote the intracellular accumulation of substrates, resulting in potential human toxicity.

For the distribution part, the volume of distribution of steady state (VDSS) is low for all substrates, ranging from −1.33 to 0.615 L/kg, which is needed to maintain uniform distribution to give the same concentration in the plasma. As the fraction unbound in humans to which a drug binds to plasma proteins is low for all hits, in comparison to the reference, which exceeds 0.1, is by far the most available unbound (free) drug capable of interacting with a pharmacological target protein. The BBB is mainly maintained between −1.0 and 0.3. All hit compounds are moderately distributed in the brain, with Δ8-THCA predicted to have the best brain distribution although CBN-C3 has the poorest. Similarly, the reference compound MLN-4760 is unable to permeate the CNS, followed by 6-prenylapigenin.

Metabolism-wise, cytochrome P450 is responsible for the biotransformation of most drugs and is a primary cause of variability in drug pharmacokinetics. Strikingly, 6-prenylapigenin and CBN-C3 are substrates of cytochrome P450 3A4 and inhibitors of CYP P450 1A2, 2C19, 2C9, and 3A4, causing the accumulation of the substrates of these enzymes.

The total clearance of a drug from the bloodstream is the sum of renal clearance plus hepatic clearance plus clearance from all other tissues.²⁵ The renal OCT2 of all hit compounds will not be carried out from plasma into the cells by renal OCT2 and will also have no deleterious interaction when coadministered with renal OCT2 inhibitors.

The toxicity of a molecule is one of the most important aspects of drug and lead propositions; AMES toxicity was detected in the CBN-C3 compound only, which is also an hERG II inhibitor. The maximum tolerated dose in humans is mostly kept at less than 0.477 log (mg/kg/d), which they all attain. We can also observe that Δ8-THCA is the safest in acute toxicity (LD50) and MLN-4760 for chronic toxicity (lowest observed adverse effect level [LOAEL]). Similarly, MLN-4760 is the safest for the Minnow toxicity, and CBN-C3 is the safest for T pyriformis toxicity. In the matter of hepatotoxicity, all lead compounds were found to be nonhepatotoxic and nondermo toxic.

There has not been much study done on the 6-prenylapigenin chemical, but it showed moderate anti-methicillin-resistant Staphylococcus aureus (anti-MRSa) (IC50 = 6.5 µg/mL), weak anticandidal (IC50 = 20.0 µg/mL) and mild antimalarial activity against the Plasmodium falciparum D6 clone and W2 clone with IC50 values of 2.8 and 2.0 µg/mL, respectively.²⁶ It was also shown that it inhibited melanin biosynthesis in B16 melanoma cells and showed antioxidant and antitrichomonal activities.²⁷

Cannabivarin (CBN-C3) was identified in 1969 by Vollner et al²⁸ as a CBD homolog in which the side chain is C3H7 instead of C5H11.²⁹ Δ8-tetrahydrocannabinolic acid-A is an analog of tetrahydrocannabinol (THC) with antiemetic, anxiolytic, appetite-stimulating, analgesic, and neuroprotective properties.³⁰ It is chemically and functionally similar to Δ-9-tetrahydrocannabinol (Δ9-THC), producing similar physiological and toxicological effects.

As previously stated, ACE2 is an important component of the RAS. Angiotensin-converting enzyme II inhibits the activation of the RAS ACE-angiotensin II (Ang II)-angiotensin type 1 receptor (AT1R) axis and protects the lungs from damage. The same is true for 2019-nCoV, which employs ACE2 for cell entrance.³¹ Targeting the spike protein may be a wise choice to contain the virus and may have a positive impact as we do not tamper with human ACE2’s natural function, as shown in a study,³² in which they discovered that cannabigerolic acid and cannabidiolic acid were equally effective against the 2 SARS-CoV-2 variants alpha B.1.1.7 and beta B.1.351. These compounds are orally bioavailable and have a long history of safe human use. However, we chose to target the ACE2 because many studies have shown that ACE inhibitors, as well as angiotensin-receptor blockers (ARBs) used either alone or in combination, can be ideal to boost immune responses while also reducing inflammation and organ damage associated with COVID-19 symptoms.³³

The syndrome of acute respiratory distress (ARDS) is a clinically life-threatening condition induced by the excessive activation of RAS triggered by 2019-nCoV infection, and ACE2 protects against COVID-19-induced ARDS.³¹ Because of ACE2’s ARDS-protective properties, the development of drugs enhancing ACE2 activity may become one of the most promising approaches for the treatment of COVID-19 in the near future. Meanwhile, RAS medications such as ACE inhibitors and angiotensin II receptor blockers of the RAS cascade in the lung may be favorable.

Many other investigations, most notably Wang et al,³⁴ deployed a gene modulation strategy demonstrating ACE2 gene expression modulation using C. sativa extracts. Cannabidiol has been demonstrated to alter gene expression and inflammation, and have anti-inflammatory and anticancer properties. Several C. sativa extracts reduce the gene expression of TMPRSS2, another key protein for SARS-CoV-2 entry into host cells.

Our experiment suggests novel chemical components of C. sativa that could be used as therapeutic molecules against COVID-19. It was designed to test novel cannabis extracts for their ability to prevent spike interaction with ACE2 target, employing ACE2 blockade as an analysis strategy. Finally, our investigation incorporated both cannabinoid and noncannabinoid components of C. sativa. This enables us to highlight the significant impacts of cannabis on ACE2 blockage. Further investigations are being pursued, namely, molecular dynamics and quantitative structure-activity relationship (QSAR) studies, to refine these findings and to correlate the exact activity of the hits with ACE2 and inflammation as well as its outcome.