Eugenol Inhibits the Binding of SARS-CoV-2 Spike S1 with ACE2
The interaction of SARS-CoV-2 with its receptor ACE2 is critical for the infection of host cells and the pathogenesis of COVID-19. Therefore, we screened different molecules based on the inhibition of interaction between ACE2 and the receptor-binding domain (RBD) of SARS-CoV-2 spike S1 (Du et al. 2009). Since supplementation of leaves or leaf extracts of holy basil or Tulsi (Ocimum tenuiflorum) induces antiviral immunity (Mondal et al. 2011; Sahoo et al. 2016), we decided to screen major components (ursolic acid, oleanolic acid, eugenol, and β-caryophylline) of Tulsi leaf. As described recently (Paidi et al. 2021), we employed a chemiluminescence-based ACE2:SARS-CoV-2 spike S1 binding assay (catalog# 79,936; BPS Bioscience). We noticed that SARS-CoV-2 spike S1 binding to immobilized ACE2 was inhibited by different doses of eugenol (Fig. 1A). However, ursolic acid, oleanolic acid and β-caryophylline remained unable to inhibit the association between SARS-CoV-2 spike S1 and ACE2 (Fig. 1A), indicating the specificity of the effect.
In an effort to understand whether eugenol binds to SARS-CoV-2 spike S1 or ACE2, we performed an in silico analysis. We applied a rigid-body protein-protein interaction tool to model the interaction between ACE2 and receptor-binding domain (RBD) of spike S1 in the absence or presence of eugenol. As expected, in the absence of eugenol, various residues (Lys417, Tyr449, Gly496, Asn501, and Tyr505) of spike S1 interacted with Asp30, Asp38, Gln42, and Lys353 residues of ACE2 (Fig. 1B). However, eugenol showed association with spike S1, not ACE2 (Fig. 1C). Interestingly, eugenol interacted with Lys417 residue of spike S1 and due to this binding, the ionic bond (salt bridge) that bound with Asp30 of ACE2 was broken and Lys417 exhibited a different rotameric pose (Fig. 1C).
To confirm the binding of eugenol with spike S1, not ACE2, we employed thermal shift assay, which is an important tool for analyzing ligand-to-receptor binding. The melting profile of full-length spike S1 and human ACE2 was monitored with the aid of a SYBR Green reaction at 27-94 °C. Typical sigmoidal melting curves clearly indicated that recombinant spike S1 (Fig. 1D) and ACE2 (Fig. 1E) were conformationally stable. Interestingly, the melting assay revealed that 10 µM of eugenol was capable of strongly shifting the melting curve of spike S1 by 6 °C from 72.71 °C to 78.71 °C (Fig. 1D). In contrast, eugenol exhibited a thermal shift of only 0.7 °C with human ACE2 protein from 61.5 to 62.2 °C (Fig. 1E). In order to understand whether the complex of spike S1 and ACE2 was conformationally stable and whether eugenol was capable of influencing the preexisting complex, we examined the melting profile of the combination of spike S1 and ACE2 in the absence and presence of eugenol. As evident from sigmoidal melting curve, the complex of spike S1 and ACE2 was conformationally stable (Fig. 1F). However, the presence of eugenol led to a shift of the melting curve of the spike S1:ACE2 complex by 3.27 °C from 59.66 °C to 62.93 °C (Fig. 1F). Together, these results indicate that eugenol binds to SARS-CoV-2 spike S1, but not ACE2, and that eugenol is also capable of associating with the established spike S1:ACE2 complex.
Does Eugenol Inhibit Viral Entry?
Since eugenol suppressed the binding of SARS-CoV-2 spike S1 with ACE2, next, we investigated whether eugenol inhibited viral entry. Pseudoviruses are suitable for virus entry assays, as they permit viral entry to be distinguished from other virus life-cycle stages. Therefore, we used lentiviral particles pseudotyped with the SARS-CoV-2 Spike S1 protein. Since human embryonic kidney 293 (HEK293) cells do not have any detectable ACE2 receptors, we used HEK293 cells expressing human ACE2 for entry assay. In pseudovirus luciferase assay, viral entry into cells correlates to the levels of luciferase signals in the cells. While lenti-naked infection did not increase luciferase signals in hACE2-expressing HEK293 cells, marked increase in luciferase activity was seen in pseudo-SARS-CoV-2-infected cells (Fig. 2A), indicating the entry of pseudo-SARS-CoV-2 into hACE2-HEK293 cells. However, eugenol at 5, 10 and 20 µM concentrations strongly inhibited pseudo-SARS-CoV-2-induced luciferase activity (Fig. 2A), suggesting that eugenol inhibits the entry of pseudo-SARS-CoV-2 into hACE2-HEK293 cells. Similar to pseudo-SARS-CoV-2, infection with pseudo-VSV also led to marked increase in luciferase activity in hACE2-HEK293 cells (Fig. 2B). However, in contrast to that seen with pseudo-SARS-CoV-2, eugenol did not inhibit pseudo-VSV-induced luciferase activity in hACE2-HEK293 cells (Fig. 2B).
To further confirm eugenol-mediated inhibition of viral entry, we checked GFP fluorescence as a measure of infection. Marked GFP expression was found in cells infected with both SARS-CoV-2 pseudovirus (Fig. 2C) and VSV pseudovirus (Fig. 2D) while we did not observe GFP expression in the cells infected with lenti-naked viral particles (Fig. 2C, D). However, eugenol strongly inhibited GFP expression induced by pseudo-SARS-CoV-2 (Fig. 2C, E). On the other hand, eugenol had no effect on VSV pseudovirus-induced GFP expression in hACE2-HEK293 cells (Fig. 2D, F). Together, these results suggest that eugenol is capable of inhibiting the entry of pseudo-SARS-CoV-2, but not pseudo-VSV, into hACE2-HEK293 cells.
Eugenol Inhibits the Activation of NF-κB and the Expression of Proinflammatory Molecules in SARS-CoV-2 Spike S1-stimulated A549 Lung Cell Line
Some COVID-19 patients present a severe symptom of acute respiratory distress syndrome (ARDS) with high mortality. This high severity is dependent on pulmonary inflammation induced by a cytokine storm (Pia 2020), which is most likely mediated by interleukin-6 (IL-6) and other proinflammatory cytokines. NF-κB is a proinflammatory transcription factor (Vallabhapurapu and Karin 2009) and recently we have demonstrated that recombinant SARS-CoV-2 spike S1 induces the activation of NF-κB and the expression of IL-6 in human A549 lung cells (Paidi et al. 2021). While heat-denatured spike S1 does not induce the expression of proinflammatory molecules in A549 cells, anti-spike S1 neutralizing antibody (BioVision; Cat# A3000-50) abrogates the proinflammatory function of recombinant SARS-CoV-2 spike S1 (Paidi et al. 2021). Therefore, we examined if eugenol was capable of suppressing inflammation in human A549 lung cells induced by recombinant SARS-CoV-2 spike S1. As evident by EMSA, recombinant spike S1 induced the DNA-binding activity of NF-κB in A549 cells (Fig. 3A). However, eugenol inhibited spike S1-induced activation of NF-κB (Fig. 3A). To confirm these results, we monitored the expression of TNFα, IL-1β and IL-6, proinflammatory cytokines that are driven by activated NF-κB. Spike S1 increased the expression of TNFα (Fig. 3B), IL-1β (Fig. 3C) and IL-6 (Fig. 3D) in A549 cells. However, eugenol dose-dependently inhibited SARS-CoV-2 spike S1-induced mRNA expression of TNFα (Fig. 3B), IL-1β (Fig. 3C) and IL-6 (Fig. 3D) in A549 cells.
Oral Administration of Eugenol Inhibits Lung Inflammation and Reduces Fever in SARS-CoV-2 Spike S1-intoxicated Mice
Although SARS-CoV-2 does not easily bind to ACE2 and infect normal mice, we have observed that intranasal intoxication of SARS-CoV-2 spike S1 induces fever and important cardiac and respiratory symptoms of COVID-19 in normal C57/BL6 mice (Paidi et al. 2021). Therefore, we studied whether eugenol could reduce these symptoms in mice. Since COVID-19 patients are and/or will be treated with drugs after diagnosis of the disease, we examined whether eugenol administered 5 d after initiation of the disease (Fig. 4A) was still capable of defending mice from COVID-19 related complications. We selected the 5-day window as all SARS-CoV-2 spike S1-intoxicated mice exhibited a body temperature of around 1000 F on 5 d of intoxication (Fig. 4B). Parallel to that observed in human lung cells, intranasal exposure with recombinant SARS-CoV-2 spike S1 (Fig. 4A) led to the expression of TNFα (Fig. 4C), IL-1β (Fig. 4D) and IL-6 (Fig. 4E) in vivo in the lung of C57/BL6 mice. However, oral administration of eugenol strongly inhibited the mRNA expression of TNFα (Fig. 4C), IL-1β (Fig. 4D) and IL-6 (Fig. 4E) in the lungs of SARS-CoV-2 spike S1-insulted mice. Similarly, eugenol treatment also suppressed the level of TNFα in serum of SARS-CoV-2 spike S1-insulted mice (Fig. 4F). Fever is probably one of the prominent symptoms of COVID-19 (Machhi et al. 2020; Pahan and Pahan 2020) and oral administration of eugenol also led to the normalization of body temperature of SARS-CoV-2 spike S1-intoxicated mice (Fig. 4G).
Oral Eugenol Improves Heart Functions in SARS-CoV-2 Spike S1-intoxicated Mice
Since many cardiac related problems of COVID-19 are modeled in SARS-CoV-2 spike S1-intoxicated mice (Paidi et al. 2021), we examined if oral eugenol was capable of improving heart functions in these mice. Non-invasive ECG demonstrated cardiac arrhythmias in SARS-CoV-2 spike S1-intoxicated mice as compared to control untreated mice (Fig. 5A, B). However, eugenol treatment normalized electrical activity of the heart as evident from ECG (Fig. 5A−C). Similarly, eugenol also steadied heart rate (Fig. 5D), heart rate variability (Fig. 5E), JT interval (Fig. 5F), ORS interval (Fig. 5G), QT interval (Fig. 5H), and RR interval (Fig. 5I) in SARS-CoV-2 spike S1-intoxicated mice. As expected, the level of LDH was also markedly higher in serum of SARS-CoV-2 spike S1-intoxicated mice than normal mice (Fig. 5J). However, eugenol treatment normalized serum LDH in spike S1-intoxicated mice (Fig. 5J).
Oral Administration of Eugenol Increases Locomotor Performance in SARS-CoV-2 Spike S1-intoxicated Mice
Recently, we have demonstrated that SARS-CoV-2 spike S1 insult causes functional discrepancies in C57/BL6 mice (Paidi et al. 2021). Similarly, we found a decrease in overall locomotor activities in SARS-CoV-2 spike S1-intoxicated mice (Fig. 6A–G). Therefore, we investigated whether oral eugenol could improve such behavioral deficits. Interestingly, eugenol treatment increased overall locomotor activities as evident by heat map (Fig. 6A), distance travelled (Fig. 6B), velocity (Fig. 6C), cumulative duration (Fig. 6D), center zone frequency (Fig. 6E), center zone cumulative duration (Fig. 6F), and rotorod performance (Fig. 6G). We did not notice any drug-related side effect (e.g. hair loss, appetite loss, weight loss, untoward infection, irritation, etc.) in any mouse upon treatment with oral eugenol at a dose of 25 mg/kg body weight/d.