Phylogenetic analysis on corona virus main proteases
Main protease (Mpro, also called 3CLpro) is considered as one of the important molecular targets for designing novel drugs against corona viruses[22]. With a view to design drugs/inhibitors specifically targeting main protease of nCoVID’19, in-silico analysis was performed using main protease sequences of SARS-CoV-2, SARS-CoV and MERS-CoV. Multiple sequence alignment identified 12 significant differences between main proteases of SARS CoV and SARS-CoV-2 (Fig. 1). Out of the 12 differences, S45 to A45 was found to reside within in the binding site of SARS-CoV-2 main protease. This may play a crucial role in determining differential binding affinity of the two proteases.
Phylogenetic analysis was performed using main protease sequences sharing >50 percentage similarity against SARS-CoV-2homolog revealed its significant genetic relatedness with main proteases of SARS CoV (96.08% similarity) and bat coronavirus (76.84% similarity) (Fig. 2). Next to this, it shared significant similarity with ORF1ab of Rousettus bat coronavirus. Main protease of nCoVID’19 shared only 50.65% similarity against main proteases of MERS-CoV. Above results clearly indicated the need for a highly specific novel drug specifically inhibiting main proteases of SARS-CoV-2.
Virtual screening of potential herbal ligands against major protein targets of SARS-CoV-2
Virtual screening of 721 ligands belonging to small molecules and active compounds from 37 medicinal herbs against 7 major protein targets of nCOVID’19 identified potential inhibitors. Information regarding the binding site residues predicted using CASTp server is provided in Table 3. Top 10 hits reported with higher binding affinity for each target protein is considered for downstream analysis (Table 4). Seven molecular targets of SARS-CoV-2 include, Main protease, RNA-dependent RNA polymerase (RdRp), NSP3, NSP9, NSP10-NSP16, NSP15 and Spike protein.
Small molecules/herbal compounds exhibiting significant inhibitory activity against Main Protease
Despite of significant structural (RMSD: 0.71 Å) and binding site volume similarity between SARS-CoV and SARS-CoV-2, they showed differential binding affinity against different inhibitors (Table 1).Virtual screening of small molecules against MPro identified agathisflavone as the best inhibitor exhibiting the binding affinity value of -8.2 kcal/mol.
Out of 721 ligands screened against SARS-CoV main protease, a ligand namely rutin abundantly found in Terminalia chebula, Azadirachta indica and Ocimum basilicum exhibited highest binding affinity value of -9.0 kcal/mol. In case of MERS-CoV main protease, amentoflavone predominantly found in Mangifera indica and Garcinia species showed the maximum binding affinity value of -8.6kcal/mol. Interestingly, a cytotoxic biflavonoid agathisflavone found in cashew nut (Anacardium occidentale) was shown to exhibit significant binding affinity with -8.0 kcal/mol against the main protease of SARS-CoV-2. Agathisflavones have been reported for their cytotoxicity against malignant cell lines [23]. Agathisflavone is a biflavanoid derived from plant source and has been found to possess several biological activities[24]. Various studies have found that agathisflavone possesses antioxidant, anti-inflammatory, antiviral, antiparasitic, cytotoxic, neuroprotective, and hepatoprotective activities. It has also been suggested that agathisflavone could be used in the treatment of oxidative stress, inflammatory diseases, microbial infection, hepatic and neurological diseases and cancer[25]. This compound was found to involve in the formation of 3 hydrogen bonds at ASP 187, PRO 52 and ARG 40. This was followed by Rubusic acid (Pedalium murex), solanocapsine (Solanum nigrum), chlorogenin (Solanum torvum), Lupeol (Carica papaya and Azadirachta indica), Cyanin (Zingiber officinale), 3-O-trans-cafeeoyltormentic acid, Luteolin-7-O-(6”-malonylglucoside) (Vitex negundo), Agnuside (Vitex negundo) and Luteolin 7-O-beta-D-glucoside (Vitex negundo) exhibiting significant binding affinity in the decreasing order against MPro.
The phytochemical cyanin found in Zingiber officinale was found to bind effectively against main proteases of all three coronaviruses (Fig. 3a, c and d) and placed in the top 10 hits list. It showed a binding affinity value of -8.3 kcal/mol, -8.2 kcal/moland -7.7 kcal/mol against SARS-CoV-2, SARS COV and MERS CoV main proteases respectively.
RNA-dependent RNA polymerase (RdRp):
RdRp is responsible for replication of COVID19 genome inside the host. Among the ligands tested, ivermectin (Fig. 3b) showed the higher binding affinity value of -9.4 kcal/mol through the hydrogen bonding at ASN 497 residue. Amentoflavone stood at second position with the binding affinity value of -9.3 kcal/mol. This was found to react at three amino acid residues namely ARG 553, THR 556 and ASN 691. Ligands viz., Corilagin (Terminalia bellirica and Terminalia chebula), Agasthisflavone, 3-O-trans-caffeoyltormentic acid, Arjungenin (Terminalia chebula), Crateogolic acid (Syzygium aromaticum), 3,8’-biapigenin, cyanin (Zingiber officinale) were found to exhibit significant binding affinity (< -8.0 kcal/mol).
Spike Protein
X-ray crystal structure of spike glycoprotein (PDB ID: 6M71) was used chosen for performing virtual screening. Virtual screening was performed by choosing ACE interacting region as the binding site (Fig. 4a). 1,8-Dichloro-9,10-diphenylanthracene-9,10-diolfrom Carica papaya was found to exhibit significant binding affinity against spike glycoprotein (-8.2 kcal/mol). GLY 496 residue was found to be involved in the formation of hydrogen bond with the 1, 8-Dichloro-9, 10-diphenylanthracene-9,10-diol. Earlier, leaf extracts of Carica papaya was reported to have significant effect in combating dengue virus infection [26] and its exact role in increasing platelet counts is not clear. 1,8-Dichloro-9,10-diphenylanthracene-9,10-diol was found buried in the binding site of spike glycoprotein exhibiting hydrophobic interactions with residues such as LEU 39, TYR 41, TYR 449, TYR 453, TYR 495, PHE 497 and TYR 505 (Fig. 3e). This was followed by other small molecules viz., agasthisflavone, amentoflavone, ivermectin, agnuside (Vitex negundo), taraxerol (Cissus quadrangularis) and nimbinene (Azadirachta indica) exhibiting significantly higher level of binding affinity towards spike protein of nCOVID’19.
Non-Structural Proteins (NSPs)
Apart from the four major structural proteins (S, E, M and N proteins), non-structural proteins namely NSP3 (cleavage of N-terminal replicase poly protein), NSP9 (ssRNA binding) NSP10-NSP16 (co-factor in activating replicating enzyme) and NSP15 involved in the transcription and replication of nCoVID’19 can also serve as potential targets for containing the virus using inhibitory herbal molecules[27].
Virtual screening of small molecules against NSP3 identified amentoflavone (Mangifera indica) as the top scored ligand with binding affinity of -7.5 kcal/mol. In the decreasing order of binding affinity, luteolin 7-O-(6''-malonylglucoside) (Vitex negundo), rubusic acid(Pedalium murex), acteoside (Clerodendrum serratum), ivermectin, taraxerol acetate (Cissus_quadrangularis), catechin 7-O-gallate, luteolin 7-O-beta-D-glucoside (Vitex negundo), agathisflavone (Anacardium occidentale) and luteolin-7-o-beta-d-glucopyranoside (Vitex negundo)were found to be placed next to amentoflavone. It was observed that three inhibitorsfrom Vitex negundowere reported with the highest binding scores. Vitex negungo belongs to Verbenaceae family known for its effects for ailments like ophthalmia, deafness, indigestion, piles and jaundice [28]. Results of earlier experiments conducted by Wu et al. [29] also revealed similar findings of vitexin from Vitex negundo exhibiting significant binding affinity towards NSP3.
Another small molecule friedelin from Vitex negundo and Acorus calamus was also found to exhibit significant binding affinity of -9.6 kcal/mol against NSP9 (Table 4). Eventhough, many hydrophobic interactions were observed, no hydrogen bond interaction was found in the binding site of NSP9. It is very interesting to observe that five out of the top ten inhibitors are from a single plant source Solanum nigrum. As evidenced from other studies, Solanum nigrum is one of the traditionally known medicinal plant known for its use in treatment ofseizure, pain, ulcer, inflammation, diarrhea, eye infections, jaundice and oxidative stresses [30-32].
Virtual screening against NSP15 identified oleonolic acid and urosolic acid as molecules exhibiting highest binding affinity values of -9.2 kcal/mol. Oleonolic acid and urosoic acid are known for their anticancerous and anti-inflammatory activities [33-35]. Both the phytochemicals are reported to be enriched in Ocimum basilicum and Ocimum tenuiflorum (Table 4).This was followed by crategolic acid (Syzygium aromaticum), arjungenin (Terminalia chebula), hederagenin (Nigella sativa), triterpenoid (Abutilon indicum), beta-amyrin (Cissus quadrangularis), friedelin (Vitexnegundo, Acoruscalamus), catechin 7-O-gallate (Camellia sinensis) and arjunolic acid (Terminalia chebula).
In the case of NSP-NSP16 protein complex, interface of the complex (Fig. 4b) was chosen as the binding site for performing virtual screening. Compounds such as amentoflavone, 10-methoxycamptothecin, 3,8'-biapigenin, taraxerol acetate (Cissus_quadrangularis), corilagin (Terminalia bellirica and Terminalia chebula), lupeol acetate (Pedalium murex), emetine, chlorogenin (Solanum torvum) and spirostan-3-ol (Solanum torvum) were ranked among the top 10 molecules exhibiting highest binding affinity.
Molecules exhibiting inhibitory activity against multiple protein targets of nCOVID’19
Phytochemicals exhibiting inhibitory activity against multiple targets of viruses are expected to confer durable protection to the patients. This will be more beneficial in situations where the virus is developing mutations in one of the targets. Small molecules namely, amentoflavone, agathisflavone, catechin-o-gallate and chlorogenin exhibited significant binding affinity towards multiple targets of nCOVID’19.
Amentoflavone showed inhibitory activity against RdRp (-9.3 kcal/mol), NSP9 (-8.3 kcal/mol), NSP3 (-7.4 kcal/mol), NSP10-NSP16 (-8.5 kcal/mol) and spike glycoprotein (-8.2 kcal/mol). In all the interactions, the target - ligand binding affinity was greater than -8.0 kcal/mol except NSP3. Protein-ligand interactions exhibited by the small molecules amentovlavone and agathisflavone is shown in the Fig. 5a-e and Table S3. Amentoflavone is a naturally occurring biflavanoid reported to be found in more than 120 plants[35]. Many of these plants have been used in traditional medicine for several thousand years in different parts of the world. Several studies have reported that amentoflavone possess anti-inflammatory, anti-oxidative, anti-diabetic, anti-tumor, anti-viral and anti-fungal activities [35]. Evidences have been reported for amentoflavone exhibiting anti-senescence activity in the cardiovascular and central nervous system[36]. Further, Amentoflavone isolated from Torreyanuciferawas demonstrated to possess inhibitory activity against SARS-CoV3CLPro [37].
Similarly, agathisflavone was found to exhibit significant interaction against four different protein targets viz., RNA-dependent RNA polymerase (-8.9kcal/mol), SARS-CoV-2 main protease (-8.2kcal/mol), spike glycoprotein (-8.2 kcal/mol) and NSP 3 (-6.6kcal/mol) (Fig. 5f-I, Table. S3). Catechin-o-gallate was also found to possess significant binding affinity towards spike glycoprotein (-7.3 kcal/mol), NSP3 (-6.6 kcal/mol), RNA-dependent RNA polymerase (-7.9 kcal/mol) and NSP15 (-8.5 kcal/mol). Chlorogenin from Solanum torvumbexhibited binding affinity value of -8.2 kcal/mol (NSP9), -7.7 kcal/mol (MPro) and -7.6 kcalmol (NSP16-NSP10) of SARS-CoV-2.
Fifteen different compounds viz., 3,8'-biapigenin, 3-O-trans-caffeoyltormentic acid, agnuside, arjungenin, corilagin, crategolic acid, cyanin, friedelin, luteolin 7-O-(6''-, malonylglucoside), N-methylsolasodine, rubusic acid, solanocapsine, spirostan-3-ol, taraxerol and taraxerol acetate were found to possess significant interactions with at least two protein targets of SARS-CoV-2 (Table 5).
Effect of FDA approved drugs on SARS-CoV-2 protein targets
Hydroxycholoroquine,chloroquine and ivermectin drugs were selected as positive controls as they were reported to possess anti-viral activity[38, 39]. Hydroxychloroquine was reported to show promising inhibitory activity against nCOVID-19 spike protein [40, 41]. Our results revealed that hydroxycholorquine and chloroquine showed less binding affinity against all the 7 targets of nCOVID-19 compared to ivermectin (Table 6). Ivermectin exhibited significant binding affinity value of -9.4 kcal/mol and -8.2 kcal/mol against RNA - dependent RNA polymerase (RdRp) and spike protein respectively (Fig. 6). Ivermection also exhibited significant binding affinity against NSP9 (-7.5kcal/mol) and spike glycoprotein (-8.2 kcal/mol) (Table S4).
Analysis on top reported plants with best ligand hits
Among the 721 phytochemicals originating from of 37 plant species, 36 (5% approx.) phytochemicals from 22 plants (Fig. 7) were found to be the best hits with higher binding affinities against all the seven targets (Table 5). Among those 27 plants, 6 plants were found to be the ingredients of a traditional siddha herbal formulation namely “Kabasura kudineer” recommended by AYUSH Board of Government of India for boosting immunity. Vitex negundo was reported to possess 34 different phytochemicals included in this study. Out of the 34, five different compounds namely, luteolin 7-O-beta-D-glucoside, luteolin 7-O-(6''-malonylglucoside), agnuside, luteolin-7-o-beta-d-glucopyranoside and friedelin were found to exhibit significant binding affinity against 5 different protein targets of SARS-CoV-2 namely, spike glycoprotein, SARS-CoV-2 main protease, NSP3, NSP9, NSP15 in the SARS-CoV-2.Followed by Vitex negundo, plants such as Solanum nigrum and Pedalium murex were found to possess 5 different antiviral compounds.
Solanocapsine, Spirostan-3-ol, N-methylsolasodine, Diosgenin and Solasodine are the phytochemicals reported in Solanum nigrum which effectively inhibited three SARS-CoV-2 targets (SARS CoV2 MPro, NSP9 and NSP16-NSP10).
Pedalium murex reported Diosgenin (NSP9), Lupeol acetate (NSP16-NSP10), Phytosterol (NSP9), Urosolic acid (NSP15) and Rubusic acid (SARS CoV2 Mpro, NSP3) as SARS CoV2 inhibitors. It is noteworthy that maximum of six targets have been inhibited by the compounds from Pedalium Murex. Inspite of its role as antiulcerogenic, nephroprotective, hypolipidemic, aphrodisiac, antioxidant, antimicrobial and insecticidal activities, Pedalium Murex has been traditionally used in treating ailments like cough and cold as a regular practice[42].
Medicinal plants namely, Azadirachta indica, Terminalia chebula, Cissus quadrangularis, Clerodendrum serratum and Ocimum basilicum were found to contain 4 different inhibitors. These herbal plants may be the potential targets for future research towards developing herbal formulations against SARS-CoV-2. Intensive genomics and proteomics research may lead to identification of novel drugs against this pandemic disease.
Generation of improved knowledge and understanding biochemical and molecular basis of herbals used in traditional Ayurveda and siddha medicine will accelerate development of effective drugs in controlling emerging diseases. In this study, comparative analysis of main proteases of MERS-CoV, SARS-CoV and SARS-CoV-2 revealed significant differences between the three homologs which were confirmed by differential binding affinity exhibited by 721 phytochemicals against the three main proteases. Cyanin from Zingiber officinale exhibited significant inhibitory activity against main proteases of all the three viruses. Table S5 provides details regarding the 2D diagram and binding affinity of cyanin with MPro of three CoVs. Popular fruit trees mango (Mangifera indica) and cashew nut (Anacardium occidentale) rich in amentoflavone and agathisflavone showing significant inhibitory activity against multiple targets of SARS-CoV-2. Vitex negundo, Solanum nigrum, Pedalium Murex, Terminalia chebula, Azadirachta indica, Cissus quadrangularis, Clerodendrum serratum and Ocimum basilicum were also found to contain phytochemicals showing inhibitory activity against SARS-CoV-2 proteins. More interestingly, this study has picked up Carica Papaya exhibiting inhibitory activity against spike glycoprotein and MPro of SARS-CoV-2 which was known for its protective role against dengue virus in humans [26]. Overall, this study has shortlisted potential phytochemicals having inhibitory activity against SARS-CoV-2 which can be taken for further testing and drug formulation studies.