Activity of THC, CBD, and CBN on Human ACE2 and SARS-CoV1/2 Main Protease to Understand Antiviral Defense Mechanism

• Abstract
• Introduction
• Results
• Discussion
• Material and Methods
• Chemicals
• Inhibitory activity of SARS-CoV1 and SARS-CoV2 Main Proteases
• Inhibitory activity against hACE2
• Statistical analysis
• In silico study by molecular docking
• 3D cannabinoids chemical structures preparation
• Preparation of SARS-CoV2 main protease, SARS-CoV2 receptor-binding domain (SARS-CoV2 RBD), hACE2 proteins and their native ligands
• Molecular docking experiment
• Post-docking analysis
• Contributorsʼ Statement
• References

Abstract

THC, CBD, and CBN were reported as promising candidates against SARS-CoV2 infection, but the mechanism of action of these three cannabinoids is not understood. This study aims to determine the mechanism of action of THC, CBD, and CBN by selecting two essential targets that directly affect the coronavirus infections as viral main proteases and human angiotensin-converting enzyme2. Tested THC and CBD presented a dual-action action against both selected targets. Only CBD acted as a potent viral main protease inhibitor at the IC₅₀ value of 1.86 ± 0.04 µM and exhibited only moderate activity against human angiotensin-converting enzyme2 at the IC₅₀ value of 14.65 ± 0.47 µM. THC acted as a moderate inhibitor against both viral main protease and human angiotensin-converting enzymes2 at the IC₅₀ value of 16.23 ± 1.71 µM and 11.47 ± 3.60 µM, respectively. Here, we discuss cannabinoid-associated antiviral activity mechanisms based on in silico docking studies and in vitro receptor binding studies.

Introduction

Plant secondary metabolites have been intensively studied for novel antiviral substances long before the severe acute respiratory syndrome coronavirus2 or SARS-CoV2 pandemic occurred [1], [2], [3], [4], but today they gain more attention than before [5], [6], [7]. The principle of antiviral drug discovery from plants is that plants developed defensive metabolites against pathogenic microorganisms such as bacteria, fungi, and viruses to survive [8], [9]. A success story of an approved plant metabolite against the human viral infection is podophyllotoxin, a lignan from Podophyllum peltatum L. or Podophyllum emodi ROYLE, approved to treat human papillomavirus infection topically [9], [10]. However, there are much more plant metabolites that are currently under investigation [11]. Three examples will be given here. First is homoharringtonine, a plant alkaloid, from Cephalotaxus harringtonii (Knight ex J.Forbes) K.Koch [12], [13] showing a wide range of antiviral activities against hepatitis, herpes simplex1, varicella-zoster, foot and mouse disease, Ebola, and SARS-CoV2 viruses [11], [14], [15], [16], [17]. Another example is emetine (a tetrahydroisoquinoline alkaloid) from the plant Psychotria ipecacuanha (Brot.) Stoves [18], [19]. Emetine also exhibits a wide range of antiviral activities against hepatitis, Zika, Ebola, Middle-East respiratory syndrome coronavirus or MERS-CoV, SARS-CoV1, and SARS-CoV2 viruses [11], [17], [18], [20], [21]. Ribaudo et al. (2021) reported that artemisinin (a known antimalarial drug) blocked the entry of SARS-CoV2 into human cells by inhibiting human angiotensin-converting enzyme2 (hACE2) [22].

Since the uprise of this pandemic, the interest in cannabinoids for the SARS-CoV2 treatment has been increased remarkably, but the direct antiviral activity of cannabinoids is still low [23]. Most of the studies were review article focusing on the supportive therapy for SARS-CoV2 infection such as lowering the inflammation by reducing the cytokine storm, especially interleukin-6 and the agonistic effect of the cannabinoids at PPARγ receptor at lung tissue [24], [25], [26], [27]. Only a few studies have reported the activity of cannabinoids against SARS-CoV2 infection experimentally. One study from Wang et al. (2020) showed the downregulation of hACE2 from CBD extract high content [28]. Another study is the first and only study from Raj et al. (2021) that reported the direct and remarkably activity of THC, CBD, and CBN against SARS-CoV2 infection with the same potency as the standard drugs such as chloroquine, remdesivir, and lopinavir in the cell-based assay [29]. However, the mechanism of action of these three cannabinoids against SARS-CoV2 infection is not yet known for certain. Therefore, this study aims to reveal the antiviral mechanism of THC, CBD, and CBN.

Based on the coronavirus pathogenesis (both SARS-CoV1 and SARS-CoV2), there are two main strategies to stop these coronaviruses infection. The first strategy is to prevent viruses from entering the cell. The receptor protein hACE2, is located on the membrane of immune cells, is considered essential for cell invasion, and the viral spike protein, a transmembrane protease, serine 2 (or TRPRSS2), is critical for entering the cell. The second strategy is to stop replicating the virus in the host cell by inhibiting the viral proteolysis (stopping the viral main protease activity). Therefore, we selected hACE2 and viral main proteases from both SARS-CoV1 and SARS-CoV2 as the target proteins to determine the mechanism of action and evaluate the potency of THC, CBD, and CBN against coronavirus infections, as presented in [Fig. 1].

Results

The antiviral activity of THC, CBD, and CBN against SARS-CoV2 infection was recently reported from a cell-based assay, but still the mechanism of action of mentioned cannabinoids has not yet been elucidated [29]. Currently, only the predicted mechanism was proposed by a computational experiment [29], [30]. Our main objective was to evaluate and confirm the antiviral action of three cannabinoids against coronavirus main proteases by tracking the fluorogenic substrate to monitor the enzyme activity. As shown in [Fig. 2], only THC and CBD have strong inhibitory activity against SARS-CoV1 and SARS-CoV2 main proteases at the screening concentration of 100 µg/mL with a minor difference from the positive standard GC376 (Fig. S7, Supporting Information). [Fig. 2] demonstrates high inhibitory activity (IC₅₀) of THC and CBD at 104.43 ± 6.27% and 100.78 ± 1.10% for SARS-CoV1 main proteases, 100.00 ± 6.15% and 102.12 ± 1.66% for SARS-CoV2 main protease, respectively. Remarkably, inhibitions were superior to the activity of GC376 as reference (88.15 ± 19.41%) against SARS-CoV1 main protease and were equal to SARS-CoV2 main protease at 100.62 ± 4.80%, respectively. CBN showed only a minor effect against SARS-CoV1 and SARS-CoV2 main proteases (39.59 ± 10.79% and 31.46 ± 18.49%, respectively). Data support the proposed mechanism from an earlier report suggesting that all three cannabinoids (THC, CBD, and CBN) inhibit coronavirus main protease activity [29]. Since both THC and CBD were characterized as positive candidates (% inhibition higher than 80%), both cannabinoids were subjected to further investigation to determine individual IC₅₀ values.

To examine the efficacy of THC and CBD, both candidates from our first screening study were retested. IC₅₀ value of each cannabinoid was evaluated by equation (2) and compared to the IC₅₀ value of GC376 ([Fig. 3 a] to [c]). CBD showed a strong effect against SARS-CoV2 main protease. In contrast, THC anticipated as an active cannabinoid in the first screening exhibited a moderate potency compared to CBD in this test.

In [Fig. 3 c], the graph demonstrated the minor activity of THC against SARS-CoV2 main protease activity with an IC₅₀ value of 16.23 ± 1.71 µM (R² = 0.99). In contrast, CBD exhibited intense activity against SARS-CoV2 main protease with the IC₅₀ value of 1.86 ± 0.04 µM (R² = 0.99). This IC₅₀ value of CBD was only four times higher than the IC₅₀ value of GC376 at 0.42 ± 0.06 µM (R² = 0.98) as presented in [Fig. 3 a]. CBD test standard deviation (SD) values were more extensive than those from the THC test due to poor solubility. Higher SD values in a biological test are more frequently found for poor soluble compounds as reported by Schulz et al. (2014) and Aleo et al. (2006) [31], [32]. After combining the anti-SARS-CoV2 main protease activities and THC, CBD, and CBN chemical structures, we give a suggestion to explain the structure-activity relationship among these three (see discussion part).

Despite close structural similarity between THC, CBD, and CBN, the inhibitory activity against SARS-CoV1 and SARS-CoV2 main proteases of all three cannabinoids was significantly different. The basic core structure of the cannabinoids consists of two cyclic rings, a resorcinyl ring A and a cyclohexene ring B in all, but a dihydropyran ring C in THC and CBN only ([Fig. 4]). Molecular modelling supported that the cyclohexene substitution was most likely to have better activity than the aromatic benzene ring. Moreover, if there was a free rotation of rings A and B, the inhibitory activity seemed to increase ([Fig. 4]). This might explain why CBD showed the strongest activity among tested cannabinoids against coronavirus main proteases activity.

After correlating the inhibitory activity of tested cannabinoids and chemical structures, we identified CBN as weakest against SARS-CoV1 and SARS-CoV2 main proteases by approximately 30% to 40%. In comparison, THC and CBD strongly inhibited both proteases completely (100%) ([Fig. 4]). Interestingly, the only difference between THC and CBN is a cyclohexene for THC and aromatic ring for CBN. We assumed that the aromatic ring B was not favorable for the inhibitory activity against SARS-CoV1 and SARS-CoV2 main proteases. We also located the essential variation between THC and CBD in the active center. Free rotation of ring B in CBD seemed to be in favor for strong activity (IC₅₀ = 1.86 ± 0.04 µM) compared to the rigid structure in THC (IC₅₀ = 16.23 ± 1.71 µM). This structural feature maximized the antiviral protease potency up to 8.7 times based on our experiments. We did not evaluate the IC₅₀ value of THC and CBD against SARS-CoV1 main protease because both SARS-CoV1 and SARS-CoV2 proteases are highly identical (95%) [33].

As discussed earlier, we already knew that the aromatic ring at ring B in CBN was most likely to negatively affect the inhibitory activity ([Fig. 4], red circle). We performed the molecular docking study to understand better the possible molecular interaction between these cannabinoids and SARS-CoV2 main protease. The molecular docking hinted at the possible reason why CBN did not fit into the binding pocket of the SARS-CoV2 main protease perfectly because of the interaction between the aromatic ring and the catalytic residue, Cys145, as presented in [Fig. 5], red circle. Moreover, molecular docking supported our observation earlier in the structure-activity relationship in CBD. Docking showed that free rotation of CBD allowed the ortho-hydroxy group in ring A to establish a hydrogen bonding with His164, the amino acid in the active site, as shown in [Fig. 5], green circle. In addition, this was not present in THC. Results were in line with our in vitro experiments earlier and extended the existing knowledge regarding the molecular interaction between these cannabinoids (THC, CBD, and CBN) and SARS-CoV2 main protease.

Our validation showed the RMSD of 1 Å, which passed the acceptance criterion of 1.5 to 2 Å [34], [35]. With these limitations, our docking protocol provided acceptable and significant calculations to allow good predictions regarding stable confirmation and remarkable binding.

As shown in [Fig. 5 a], the binding pocket of SARS-CoV2 main protease were distinguished in six sub-pockets (S1, S2, S3, S4, S1′, and S2′). GC376 as the standard inhibitor was fitted into S1, S2, and S3 sub-pockets. The important amino acids playing a pivotal role were Cys145, the catalytic amino acid, Glu166 from the S1 sub-pocket, and Arg40, His41, Met49, Tyr54, Asp187 from the S2 sub-pocket [36]. The molecular docking results showed that THC, CBD, and CBN docked in the same position as GC376, as mentioned earlier ([Fig. 5 b] to [h]). Remarkably, CBN docking revealed that the aromatic ring interacted with Cys145, the catalytic amino acid ([Fig. 5 h], red circle). Even though the molecular docking reported that this interaction was favorable as a pi-donor hydrogen bond, we knew that this interaction must be unfavorable based on the previous in vitro experiment presented in [Fig. 5]. Having a closer look at position and poses of CBN may not fully explain less inhibitory activity ([Fig. 5 f – h]). Our docking model failed to explain reduced inhibition despite the fact that molecular interaction is quite similar to THC and CBD. Raj et al. (2021) determined stability conformation estimations of cannabinoids through density functional theory (DFT) [29]. The DFT approach was applied to determine energy gaps between HOMO and LUMO molecular orbitals. Molecules with a small frontier energy orbital gap are chemically reactive and have low kinetic stability. A higher HOMO to LUMO gap between frontier orbitals confers better stability. The authors showed that THC and CBD have slightly higher HOMO to LUMO energy gaps (ΔE 5.63, 5.80, respectively) than CBN (ΔE 4.85), which indicated that THC and CBD have a better stability profile [29], [37]. Chemical hardness (η) defined as the measure of resistance to distortion of the electron cloud represents compound stability in the binding pocket. The authors determined chemical hardness for THC and CBD (η 2.92, 2.90, respectively) being slightly higher than CBN (η 2.42) supporting again better stability for THC and CBD in the active site. Without x-ray data of co-incubated cannabinoids with the viral main protease, no final explanation can be delivered, but published data give good indications how this inhibition can be explained.

Moreover, our molecular docking supported our hypothesis why CBD had a higher activity than THC. As shown in [Fig. 5 c] and [d], the docked chemical positions between CBD and THC in the binding pocket of SARS-CoV2 main protease were different. For the CBD pose, the cyclohexene ring was pushed toward Cys145. Free rotation allowed the aromatic ring A to form a pi-donor hydrogen interaction with Gln189 from the S3 sub-pocket, as presented in a blue dash line from [Fig. 5 f]. This interaction led to a more stable hydrogen binding between the free hydroxy group in the para position of ring A and His164, the amino acid from the S1 sub-pocket presented in [Fig. 5 f], green circle. It was not confirmed for THC due to the rigid structure from ring C ([Fig. 5 g]). Moreover, this inflexible structure of THC caused an unfavorable hydrogen binding between the available hydroxyl group in ring A position and Glu166, the amino acid from the S4 sub-pocket as presented in a red dash line from [Fig. 5 g]. Finally, a summary of molecular interactions between amino acids in the active site of SARS-CoV2 main protease and compounds of interest (CBD, THC, CBN, and GC376) is provided in [Table 1], and the conserved amino acids among the compounds are given in bold.

After the coronaviruses attached at the hACE2 receptor, viruses are uptaken into the human host cells. Cellular replication in the human body will trigger chemokines overproduction, and proinflammatory cytokines are released we know as a cytokine storm. This storm can be downregulated by drug interaction with CB2 receptors. Eventually, this will cause organ damage, especially in the lung, as coronaviruses main target [38], [39]. Inhibition of hACE2 can slow down the viral spread in the human host or prevent viral infection [33], [40], [41]. Therefore, two active cannabinoids from earlier experiments, such as THC and CBD, were selected to evaluate the efficacy to interact with hACE2. As presented in [Fig. 6 a] to [c], we report the moderate inhibitory activity of THC and CBD against hACE2 compared to MLN-4760 as reference.

The IC₅₀ value of both THC and CBD was calculated by equation (2). The concentration-response curve ([Fig. 6]) showed the same potency for anti-hACE2 activity between THC and CBD as demonstrated by IC₅₀ values of 11.47 ± 3.60 µM (R² = 0.93) and 14.65 ± 0.47 µM (R² = 0.9986) respectively. Calculated IC₅₀ values are considered as moderate based on the previous reports [42], [43]. However, THC and CBD demonstrated a weaker activity than the reference compound MLN-4760, presented by the IC₅₀ value of 0.03 ± 0.1 µM (R² = 0.9899). A similar result on larger SD values was obtained from CBD again, especially at higher concentrations [44], [45]. Wang et al. (2020) reported the down-regulation effect of CBD on the hACE2 receptor on the cell surface only [28], our study has shown the effect of CBD on hACE2 as an inhibitor in combination with THC.

If hACE2 forms a complex with an inhibitor, the specific region of hACE2, namely subdomain I, will move by 13 Å from its original position, as presented in [Fig. 7 a]. This shift can be explained by the induced-fit model [46]. It is important to select the right hACE2 domain to perform the molecular docking of THC and CBD. Furthermore, the hACE2 subdomain I plays an essential role in the interaction between hACE2 and its counterpart of the viral spike protein, namely the SARS-CoV2 receptor-binding domain (SARS-CoV2 RBD) [47]. Based on this concept, it is likely that the hACE2 inhibitor can be used against SARS-CoV2 infection by interfering between hACE2 and SARS-CoV2 spike protein to prevent the virus from entering the cells [47]. Our earlier in vitro study has demonstrated moderate activity for both THC and CBD against hACE2. Therefore, molecular docking was performed to evaluate the possible binding mode of THC and CBD at the active site of hACE2. MLN-4760 as a competitive reference inhibitor was used to navigate the active site, as shown in [Fig. 7 b]. As a result, THC and CBD shared the same binding pocket with MLN-4760 at the active site of hACE2 ([Fig. 7 c]).

As we anticipated, our docking protocol provided an accurate outcome with a small RMSD value of about 1.2 Å, similar to the previous docking experiment on SARS-CoV2 main protease [34], [35]. Our protocol passed the acceptance criterion as mentioned earlier (see supplementary material). The same selection criteria for the selected CBD and THC poses used earlier in a previous docking experiment were also applied. The binding pocket of hACE2 can be divided into two sub-pockets S1 and S1′, as shown in [Fig. 7 b]. At least sixteen amino acids were involved in the inhibitory activity of MLN-4760, the reference, against hACE2. Five amino acids were catalytic residuals such as Arg273, His345, Pro346, Thr371, and His505. Another eight amino acids, like Glu145, Asn149, Phe274, Cys344, Met360, Cys361, Lys363, and Asp368 were from the S1′ sub-pocket. Finally, the last three amino acids from the S1 sub-pocket were Thr510, Arg514, and Try515 ([Fig. 7 b]) [46].

Our docking result suggested that THC and CBD docked at the same binding site as MLN-4760, as presented in [Fig. 7 c]. Only slight differences between CBD and THC poses were observed for ring A and its side-chain of CBD in the S1′ sub-pocket ([Fig. 7 d]). However, ring B of both cannabinoids was in similar orientation as shown in [Fig. 7 d] and [e]. Unlike the finding from SARS-CoV2 main protease, all hydroxy groups from both noncyclic (from THC) and cyclic positions (ring C of CBD) did not impact blocking hACE2 activity. The main reason is free rotation and unhindered interaction to form a hydrogen bonding with Thr371 of the catalytic residual (blue dash lines in [Fig. 7 f] and [g]). The menthenyl ring (ring B) in CBD and THC is identical, why similar molecular interactions were observed for Phe274 close to the catalytic residual of Arg273. In conclusion, there was no significant difference in terms of the molecular interaction between THC and CBD. Docking result provided supportive information suggesting why THC and CBD had the same potency in the anti-hACE2 activity. Finally, a summary of molecular interactions between amino acids in the active site of hACE2 and compounds of interest (CBD, THC, and MLN-4760 as a reference) is provided in [Table 2].

Discussion

Based on recent reports, our data provided more insight information regarding the molecular interaction how THC and CBD inhibit the SARS-CoV2 infection in a cell-based assay [29]. In a previous study, Raj et al. (2021) reported strong antiviral activity against SARS-CoV2 infection from THC, CBD, and CBN at the same potency as remdesivir and lopinavir (clinical references) in a cell-based assay [29]. Unlike Remdesivir (a viral RNA-dependence RNA polymerase inhibitor) [48], [49] and lopinavir (a viral main protease inhibitor) [17], [50], the mechanism of THC, CBD, and CBN against SARS-CoV2 infection are still not fully understood. Only the predicted mechanism as an inhibitor against viral protease was proposed from computational experiments [29], [30]. Our experiments showed that THC and CBD might inhibit SARS-CoV1 and SARS-CoV2 main proteases but not CBN. Therefore, we partially agree with the predicted mechanism from previous studies and propose the hypothesis that CBN may have a different mechanism of action against SARS-CoV2 infection. Notably, we evaluated the structure-activity relationship among THC, CBD, and CBN against SARS-CoV2 main protease. Two major chemical features that are likely to play an essential role in this inhibitory concept are the cyclohexene ring B and the free rotation between ring A and B. Remarkably, THC and CBD are not just only inhibiting the viral proteases like lopinavir, but also impact hACE2 interaction.

We have not discussed any activity of THC, CBD and CBN as potent CB2 agonists know as important target to reduce the proinflammatory effect of SARS-CoV2. THC is known as a partial agonist, as well as CBN being agonist and inverse agonist. If we also consider the pharmacological level of CB receptor-mediated effects of the endocannabinoid system, we see that cannabinoids can probably take a major role in the therapy of SARS-COv2 infections. It is exciting to see that a systems pharmacological approach gives us the confidence that we donʼt have to deal with just THC, CBD and CBN. We know that more than 130 cannabinoids are known in the cannabis plant and the function and pharmacological action of the minor cannabinoids like CBC, CBL, CBE gives needs to be explored. We hope that prenylated terpenophenolics being the typical scaffold of cannabinoids become an interesting class of future antiviral agents.

Material and Methods

Chemicals

THC and CBD (both purity ≥ 98%) were purchased from Cayman Chemical Company, but CBN was synthesized in our laboratory. The synthesis routine of CBN (purity ≥ 98%) is explained below. The work is carried out with permission of the Federal German Opium Agency (4 586 416).

Reaction: to a solution of CBD (314 mg, 1.0 mmol, 1.0 eq) in toluene (30 mL) iodine was added (634.5 mg, 2.5 mmol, 2.5 eq) under nitrogen. The mixture was refluxed for 2 h. After then, the reaction was quenched by cooling to room temperature and sequentially washed with 20 mL of sodium thiosulfate 10% (Na₂S₂O₃). The organic layers were washed by brine and water. After drying through magnesium sulfate, the organic phase was evaporated, and the residue was purified with flash column chromatography (ethyl acetate: petroleum ether = 1 : 99 to 3 : 97) to afford CBN as a pale red oil (189.1 mg, 61%).

TLC: EtOAc : petroleum ether = 9 : 1; R_f-CBD = 0.45, R_f-CBN = 0.35.

The nuclear magnetic resonance or NMR: ¹H NMR (400 MHz; CDCl₃): δ = 0.89 (t, 3H, J = 7.2 Hz), 1.28 – 1.33 (m, 4H), 1.60 (s, 6H), 1.61 – 1.64 (m, 2H), 2.31 (s, 3H) 2.40 (t, 2H, J = 7.3 Hz), 5.30 (br, 1H, -OH), 6.26 (s,1H), 6.41 (s, 1H,), 7.02 (d, 1H, J = 7.4 Hz), 7.11 (d, 1H, J = 7.8 Hz), 8.18 (s, 1H). ¹³C NMR (100 MHz; CDCl₃): δ = 14.3, 21.4, 22.7, 27.3, 30.6, 31.6, 35.8, 87.4, 108.6,110.0, 110.2, 111.1, 122.4, 126.2, 127.3, 127.6, 137.3, 144.2, 153.3, 154.9. ¹H/¹³C-NMR spectrogram of CBN to be found in the supplementary file.

Inhibitory activity of SARS-CoV1 and SARS-CoV2 Main Proteases

The assays were performed by using the SARS-CoV1 and SARS-CoV2 protease assay kits from BPS Bioscience. The authors performed these assays by following the manufactory guideline strictly. The used protocols were summarized here. Firstly, the proper concentration of SARS-CoV1 and SARS-CoV2 protease was adjusted to 5 µg/mL and 4 µg/mL, respectively as recommended by adding 1 mM DTT assay buffer. Secondly, 30 µl of the protease solution was mixed with 10 µl of 0.5 mg/mL sample solution or 500 µM GC376 solution for the positive control or 5% DMSO for the negative control. Thirdly, these mixtures were incubated at room temperature for 30 minutes before adding 10 µl of 10 µM fluorogenic substrate solution. Finally, the reaction was read by a FLUOStar Omega microplate reader at λ_excitation/_emission of 360 nm/460 nm after 4 hours and overnight incubation times for SARS-CoV1 and SARS-CoV2 protease assays consequently. Each cannabinoid was studied in triplicate. The percent inhibition was calculated after the background color elimination. The equation that was used to calculate the percent inhibition of each sample was presented below as equation (1).

% Inhibition = ((FI_control – FI_{test sample})/FI_control) × 100(1)

When FI stands for a fluorescence intensity that was read by the microplate reader and the percent inhibition of each sample was presented in a standard form of mean ± SD values.

All cannabinoids that showed a percent inhibition higher than 80% were subjected to determine a half inhibitory activity concentration or IC₅₀ to evaluate their potencies. Five concentrations of each active cannabinoid in a range of 318 µM to 4 µM were prepared by half fold. Finally, the IC₅₀ value was calculated by the second equation or equation (2), which was described below.

% Inhibition = 100/(1+(IC₅₀/Concentration)^Hill)(2)

When concentration referred to the concentration providing the percent inhibition and Hill value referred to the Hill coefficient and the obtained IC₅₀ values of the active cannabinoids were compared to the IC₅₀ value of GC376, the standard control provided by BPS Bioscience.

Inhibitory activity against hACE2

The anti-hACE2 activity was performed only with the active cannabinoids from the earlier experiment using the ACE2 inhibitor screening assay kit from BPS Bioscience. The assay protocol from the manufacturing was applied here strictly and would be explained shortly. In the beginning, the concentration of hACE2 was diluted by the assay buffer until reaching the desire concentration of 0.5 µg/mL. After that, 20 µl of the prepared hACE2 solution as mention earlier was mixed with 5 µl of 10 mg/mL sample solution or 10% DMSO solution as a control. Then, incubation of 15 minutes was applied at room temperature. Finally, 25 µl of hACE2 fluorogenic substrate solution was added to the wells. The reaction was read after 1 hour incubation time by the same microplate reader as mentioned in the previous section λ_excitation/_emission of 555 nm/585 nm. Each sample was tested three times. The IC₅₀ value of the cannabinoids was also calculated by equation 2 as presented earlier. Once again, the IC₅₀ value of MLN-4760 as a positive control was provided by BPS Bioscience.

Statistical analysis

The descriptive statistics from Microsoft Excel 365 such as mean, and SD were used to calculate the percent inhibition from equation (1) and the R² value from equation (2) was also calculated by the correlation function from MS Excel. Moreover, all graphs in this study were generated by Microsoft Excel as well [51], [52]. First, the solver Add-in package from Microsoft Excel was applied to calculate an initial IC₅₀ value from equation (2) [53], [54]. Finally, the nonlinear least squares (nls) function from RStudio (version 1.4.1717) was used to fit the model and determine all parameters (IC₅₀, standard error (SE), 95% confident interval (95%CI), Hill coefficient, and R²) [55], [56], [57], [58].

In silico study by molecular docking

3D cannabinoids chemical structures preparation

All cannabinoid chemical structures were downloaded from the PubChem database (https://pubchem-ncbi-nlm-nih-gov.accesdistant.sorbonne-universite.fr/), such as THC (PubChem CID: 16 078), CBD (PubChem CID: 644 019), and CBN (PubChem CID: 2543). The geometric optimization and MMFF94 energetic minimization of all cannabinoids were performed by Avogadro version 1.2.0 [59]. All optimized cannabinoids were subjected to AutoDockTools version 1.5.6 for the final preparation before performing the molecular docking experiment [60].

Preparation of SARS-CoV2 main protease, SARS-CoV2 receptor-binding domain (SARS-CoV2 RBD), hACE2 proteins and their native ligands

All of the crystal structures as mention above were obtained from the RSCB protein databank (https://www.rcsb.org/) such as SARS-CoV2 main protease with GC376 (PDB ID: 7C6U) [36], SARS-CoV2 RBD in complex with hACE2 (PDB ID: 6LZG) [47], and hACE2 with MLN-4760 (PDB ID: 1R4L) [46]. USFC Chimera version 1.11.2 was used to remove water molecules from the crystal structures and extract the native ligand. The inhibitor came with the crystal structure was used to validate the established docking protocol later [61]. Once again, Autodock Tools from the earlier section was applied here to prepare these protein structures and their extracted native ligand before the docking experiment [60].

Molecular docking experiment

AutoDock Vina version 1.1.2 was selected to perform the docking experiment and the binding pocket (active site) of each protein was guided by the native ligand as mention above. Each protein had a specific coordinate for its binding pocket as also known as grid box, which was demonstrated in terms of the x, y, and z coordinate system [60]. The grid box of SARS-CoV2 main protease (PDB ID: 7C6U) was set as x = − 21.1, y = − 26.5 and z = 2.3, while the grid box of hACE2 was set as x = 40.2, y = 6.0 and z = 29.0. Even though these coordinates were different, but the size of these grid boxes was set the same as 18 Å × 18 Å × 18 Å. The other docking parameters were set as default [60]. Before applying these established docking protocols to examine the binding mode of the cannabinoids as described above, these protocols had to be validated by redocking the extracted native ligand back into its original position in the protein. Only the protocol that imitated the original pose of the native ligand with root-mean-square deviation (RMSD) less than 2 Å compared before and after redocking was used in this study [34], [62], [63], [64]. The docking pose was selected using the combination of four main criteria and defined as the best pose. First, native ligands such as GC376 and MLN-4760 were used as a reference. Second, docking poses with no hydrogen bonding were excluded. Third, only a pose that exhibited the lowest binding energy was selected. Finally, the docking pose of each cannabinoid was selected independently [65], [66].

Post-docking analysis

The ViewDock package visualized the docking results of each cannabinoid from the Chimera program as mentioned earlier [61]. For the interaction diagram, Discovery Studio Visualizer free version was applied [67].

Contributorsʼ Statement

Conceptualization: T. Pitakbut and O. Kayser; investigation both in vitro and docking experiments: T. Pitakbut; chemical synthesis and structure elucidation: GN. Nguyen; writing, original draft preparation: T. Pitakbut; writing, review and editing: O. Kayser; supervision: O. Kayser. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

We acknowledge financial support by the German Academic Exchange Service (DAAD), grant number 57 299 294.

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Correspondence

Publication History

Received: 01 May 2021

Accepted after revision: 03 August 2021

Article published online:
12 October 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

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