Novel Multifunctional Cannabidiol-Based Analogues with In Silico, In Vitro, and In Vivo Anti-SARS-CoV-2 Effect

Abstract

Background/Objectives: COVID-19 was responsible for millions of deaths worldwide. This study aimed to identify substances with in vitro and in vivo effects against the SARS-CoV-2 virus. Methods: Compounds PQM-243 and PQM-249, two terpene-N-acyl-aryl-hydrazone analogues, were evaluated in vitro against SARS-CoV-2 to a antiviral activity and inhibitory effect against angiotensin converting enzyme 2 (ACE2). A possible inhibitory effect affecting the interaction between the receptor-binding domain (RBD) protein and/or ACE2 was evaluated using LUMMIT kit. A SARS-CoV-2-induced pulmonary pneumonia model was developed to evaluate the effects of the compounds after 3 days of treatment. Results: Compounds PQM-243 and PQM-249 exhibited IC₅₀ values of 0.0648 ± 0.041 µM and 0.2860 ± 0.057 µM against SARS-CoV-2 with a selective index of >1543.21 and 349.65, respectively, and IC₅₀ values of 12.1 nM and 13.3 nM, respectively, against ACE2. All concentrations used significantly reduced interactions between ACE2 and RBD. Computational studies suggest that these new compounds are potent direct anti-SARS-CoV-2 agents, capable of reducing both virus viability and its invasive ability in the host cells by reducing the interaction between RBD and ACE2. It was also demonstrated that even when administered by the oral route, both compounds reduced SARS-CoV-2-induced lung inflammation. Our data suggests that both compounds can act as potent direct anti-SARS-CoV-2 agents, reducing both viral viability and host cell entry. In addition, they exhibited a significant multi-target-directed pharmacological profile, also reducing SARS-CoV-2-induced lung inflammation when administered orally. Conclusions: Overall, these findings support further investigation of PQM-243 and PQM-249 as promising antiviral and anti-inflammatory multi-target prototypes for the development of innovative drug candidates targeting SARS-CoV-2 and other virus-related respiratory diseases.

Keywords: ACE2 inhibitors; COVID-19; SARS-CoV-2; antiviral effect; multifunctional cannabidiol-based analogues.

Scheme 1

Rational design of two series of CBD-based N-acyl-hydrazones (series 1 and 2) and chemical structures of PQM-243 and PQM-249 with potential antiviral and anti-inflammatory effects against COVID-19.

Figure 1

PQM-243 and PQM-249 did not show cytotoxicity against Vero Cells CCL-81. Four concentrations (0.1 to 100 μM) of PQM-243 and PQM-249 were incubated with the cells for 72 h, then alamarBlue™ reagent was added, and the results were measured by optical density using a spectrophotometer at 577 nm. Data are presented as mean ± standard error of the mean (SEM).Each column color represents one different concentration used.

Figure 2

PQM-243 and -249 exhibited virucidal effects. The graphs display titration results after SARS-CoV-2 particles were incubated at 37 °C with PQM-243 and -249 (at 100 μM) for 1 h. Data are shown as mean ± standard error of the mean (SEM), * p < 0.05 indicates a statistical difference when compared to the viral control by ANOVA. MEM = minimal essential medium (viral control); PFU = plaque-forming units.

Figure 3

Dose–response curve and viral inhibition percentage graph of PQM-243 and PQM-249. Vero cells CCL-81 were infected with SARS-CoV-2. The median and interquartile range of the number of infective SARS-CoV-2 particles were assessed by the viral plaque assay. Trendlines obtained by logistic regression with the fourth logistic parameter are shown in the graphs. IC₅₀ = half-maximal inhibitory concentration. Top viral = indicates the highest value of viral control titer (point with 0 µM of compound), Hollow circle in y axis = average of the viral control titer, used for IC₅₀ calculation, PFU = plaque-forming units.

Figure 4

Docking results for compounds PQM-243 (left, cyan sticks) and PQM-249 (right, yellow sticks) against TMPRSS2 (top) and RdRp (bottom), showing key hydrogen bonds (yellow dashed lines). The reference compounds co-crystallized in the receptor structures (PDB codes 6T7P and 7BV2, respectively, for TMPRSS2 and RdRp) are represented as transparent green sticks.

Figure 5

Detection of RBD:ACE2 interaction inhibitors with Lumit™ SARS-CoV-2 Spike RBD: hACE2 Immunoassay. PQM-243 or PQM-249 (at concentrations between 10 and 300 µM) were incubated with RBD prior to ACE2 addition (upper graph (RBD:ACE2) or PQMs were incubated with ACE2 prior to addition of RBD (lower graph (ACE2:RBD). Luminescence was recorded using a Varioskan equipment (Thermo Scientific Co., Waltham, MA, USA). RLU, relative unit of light. Values represent the mean of 7–13 replicates. Statistical analyses were done by ANOVA followed by Tukey’s post-test. # p < 0.05 indicates a statistical difference when comparing the positive group against the RBD or ACE2 groups. * p < 0.05 indicates a statistical difference when comparing different concentrations of each PQM against the positive group.

Figure 6

PQM-243 and PQM-249 reduce leukocyte infiltration and cytokine production in the bronchoalveolar lavage. Mice were treated with an oral dose of PQM-243 or PQM-249 (at 10 µmol/kg) at days 1, 3, and 5 post-nasal instillations of inactivated SARS-CoV-2 (10⁵ PFU). On the 7th day, mice were euthanized, and bronchoalveolar lavage was collected. Results are expressed as mean ± standard error (n = 5–16). Statistical analyses were done by ANOVA followed by Tukey’s post-test. # p < 0.05 indicates a statistical difference when comparing groups instilled with virus and pre-treated with vehicle to the group instilled with saline and pre-treated with vehicle. * p < 0.05 indicates a statistical difference when comparing groups instilled with virus and pre-treated with PQM-243 or PQM-249 to the group instilled with virus and pre-treated with vehicle. “+”: represent SARS-CoV-2 positive.

Figure 7

PQM-243 and PQM-249 reduced levels of cytokines produced in the lungs. Mice were treated with an oral dose of PQM-243 or PQM-249 (at 10 µmol/kg) at days 1, 3, and 5 post-nasal instillation of inactivated SARS-CoV-2 (10⁵ PFU). On the 7th day, mice were euthanized and lungs collected. The results are expressed as mean ± standard error (n = 6–11). Statistical analysis was performed by ANOVA, followed by Tukey’s post-test. # p < 0.05 indicates a statistical difference between the virus-instilled and infected vehicle-pre-treated groups with to the non-infected saline-treated and vehicle-pre-treated animal groups. * p < 0.05 indicates a statistical difference between the virus-instilled group and the infected group pre-treated with PQM-243 or PQM-249, with the virus-infected groups only treated with vehicle. PFU = plaque-forming units. “+”: represent SARS-CoV-2 positive.