SARS-CoV-2 (COVID-19) is a pandemic respiratory infectious disease with serious public health and economic implications [38]. In view of the serious situation, many antiviral drugs and vaccines have been developed against COVID-19 targeting the SARS-CoV-2 spike (S) protein [39, 40]. However, the efficacy of drugs and vaccines is usually limited by multiple spike mutations of the SARS-CoV-2 variants [1, 41, 42]. To address the challenge of treating SARS-CoV-2 variants, identifying highly conserved sequences and potentially druggable pockets for drug development represents a promising strategy [43]. In SARS-CoV-2 infection, heparan sulfate (HS) from host molecules is essential for recognition of the receptor binding domain (RBD) protein in the SARS-CoV-2 spike by ACE2, regardless of the high variability and therefore prevents the virus from easily acquiring drug resistance [44]. On the other hand, specific viral structural proteins with highly conserved protein sequences, such as the spike RBD are expressed during viral infection.
The spike receptor binding domain is located at the bottom of the S1 domain and plays an important role in host ACE2 receptor binding within membrane fusion through HS-assisted [45–47]. In the current study, we observed that RBD is associated with the recognition of ACE2 ability and is also high-conversed in all SARS-CoV-2 variants. The results of sequence alignments showed that the wild type SARS-CoV-2 has highly conserved sequences from Y453 to G476 among other variants such as beta, gamma, delta, kappa, epsilon and omicron. In addition, structural alignments of all SARS-CoV-2 variants revealed that the highly conserved protein sequences were also located in the HS binding region of RBD. We further found that the cellular HS interacted with RBD by forming hydrogen bonds at residues S454, F456, R457, S459 and, E471 and by hydrophobic interactions with residues K458, S469, I472, Y473, Q474 and, P491. It is therefore feasible and reasonable to block this region as a good target and strategy for the design and development of anti-COVID-19. More importantly, the development of antiviral drugs obtained from this approach will be broad-spectrum agents targeting viruses that use the interaction between RBD and HS to facilitate their life cycle, including COVID-19. To avoid the possibility of a high mutation rate in the region where HS binds to RBD, we further selected the active sites including R454, R457, and S459 as essential ‘hot spots’ for the development of a potent antiviral drug.
Natural products showed excellent antiviral activities by targeting structural and non-structural proteins of SARS-CoV-2 [48, 49]. Therefore, the use of natural products, e.g., from traditional Chinese medicine, can be a good option for drug development. Crocin, a terpenoid compound, was studied by Aanouz et al. and showed a promising binding affinity with the major protease of SARS-CoV-2 in the docking study [50]. In addition, broussochalcone A, a flavonoid isolated from Broussonetia papyrifera (L.), has higher affinity and stability in the Mpro of SARS-CoV-2 than lopinavir [51]. Berberine isolated from Hydrastis canadensis L. was shown to have a much lower binding energy to chymotrypsin-like protease (3CLpro). The result of the MD simulation of berberine in a complex with 3CLpro showed high stability and indicated a strong effect against COVID-19 by decreasing the activity of 3CLpro [52].
In the current study, we focused on investigating the active natural compounds in a traditional herbal formula, NRICM101 for the treatment of COVID-19. NRICM101 a traditional Chinese medicine formula developed by the National Research Institute of Chinese Medicine (NRICM) in Taiwan. This formula had successfully shown antiviral activities to prevent SARS-CoV-2 infection [39]. This natural herbal-based formula was proposed by NRICM to target viral respiratory infection and immunomodulation during the SARS-CoV outbreak in 2003 [40, 41]. One of the pharmaceutical functions of NRICM101 was proposed to interfere with host cell invasion and viral replication by binding the viral spike protein [42]. However, little is known about the active compounds in NRICM101 in suppressing SARS-CoV-2. In our study, we performed molecular docking and MD simulations of several natural compounds from NRICM101 with the highly conserved region of RBD spike protein. We found that acetoside, extracted from Scutellaria baicalensis R., exhibited the highest binding affinity. Acetoside, one of the main components in the Scutellaria baicalensis R., was suggested for anti-SARS-CoV-2 effects by inhibiting 3CLpro [53]. In this study, acetoside was found to exhibit the inhibitory potential by forming hydrogen bonded interactions with R454, F456, R457, and E471, as well as hydrophobic interactions with K458, Y473, Q474, and P491 residues within the conserved region of the RBD in the spike protein of SARS-CoV-2. While further in vitro and in vivo testing is essential, our initial studies has uncovered the possibility of using acetoside in the treatment of the sarbecovirus family across different species.
In summary, we used a structure-based computational approach with molecular docking and MD simulation to screen and characterize the potential inhibitors of SARS-CoV2-S-RBD. With the establishment of the ‘hot spot’ model, 1382 natural products from NRICM101 were comprehensively screened, and the compounds acetoside, hyperoside, isoquercitrin, CAG, CGA, and oroxyloside were identified that apparently interfere with the RBD activities. We demonstrated that acetoside blocked the active site of RBD by interacting with residues R454, F456, R457, and E471 via hydrogen bond interactions and hydrophobic contacts with K458, Y473, Q474, and P491, which are key residues for the structure-based lead optimization against RBD protein. The discovery of the RBD inhibitor acetoside from Scutellaria baicalensis R. holds great potential for the development of new and promising therapeutics for the treatment of COVID-19.