SARS-CoV-2 is a single-stranded RNA-enveloped virus whose gene fragments consist of structural and non-structural proteins. Some of the genes (viz., E, M, N, and S) encode structural proteins, whereas some encode important non-structural proteins (viz., papain-like protease- PLpro, 3-chymotrypsin-like/main protease-M^pro, Spike-protein, and RNA-dependent RNA polymerase-RdRp) by the ORF region. The Spike-protein possesses two subunits (S1 and S2), which play important roles in the receptor recognition and membrane fusion process. The S1 subunit encompasses a receptor-binding domain (RBD) which interacts with the host receptor protein [such as angiotensin-converting enzyme 2 [ACE2] or TMPRSS2 protein], whereas the S2 subunit negotiates fusion of the virus to the host cellular membrane. These two events are critical to the entry of the viral particle into the host cells. After entry into the host cells, the viral RNA is released into the cell and the polyproteins are processed by the M^pro protein. The M^pro protease of SARS-CoV-2 is identified as a cysteine protease that possesses a catalytic dyad Cys145-His41 in the active site of the protease, responsible for its activity. M^pro plays an important role in the processing of replicase polyprotein and in the maturation of virus. The RNA-dependent RNA polymerase (RdRp) is essentially required for the viral RNA synthesis, which ultimately enhances the viral virulence. Thus, the SARS-CoV-2 Spike-protein, M^pro, and RdRp proteins are important drugable target to mitigate the viral entry and virulence of the disease. Several preclinical and clinical studies on the chemically synthesized inhibitors of these drugable targets showed significant toxicity and other adverse effects. For instance, antiviral drugs such as lopinavir and remdesivir exert many side-effects in COIVD-19 patients (Barlow et al., 2020; Cao et al., 2020; Liu et al., 2020). Therefore, there is an urgent need to identify potential natural SARAS-CoV-2 drugable target inhibitors. Plant-based small molecules showed efficacy against different diseases/ailment including antiviral disease in in silico and in vitro experiments (Gupta et al., 2020; Kushwaha et al., 2020a; Kushwaha et al., 2020b; Kushwaha et al., 2021a). In the present study we selected two Indian medicinal plants (Azadirachta indica and Aloe vera) based on the rationale reviewed through the literature.

Verma (1974) first time reported antiviral potential (against potato virus X) in Azadirachta indica phytochemical (Verma, 1974). After that several antiviral activities have been reported in extract and/or pure isolated compounds from the plant against Coxsackie B group of viruses, Polio virus, Dengue virus type-2, HIV, Herpes simplex virus type 1, etc. Phytochemicals present in A. indica has potential to modulate the early event of viral replication cycle (Badam et al., 1999). SaiRam et al. (2000) proposed that neem phytochemicals have the ability to neutralize the viral particle before entering the cells (SaiRam et al., 2000). They also indicate the entry point inhibition potential in neem phytochemical against viral entry into the host cell. Parida et al. (2002) in their study also indicated that the neem phytoconstituents could inhibit the viral entry into the host cells (Parida et al., 2002). In a different study, Udeinya et al. (2004) reported that neem extract ameliorates adverse effects of human immunodeficiency virus (HIV) in human subjects possibly by inhibiting the cellular entry of the virus (Udeinya et al., 2004). In an interesting study, Xu et al. (2012) reported that the phytochemicals present in A. indica have the potential to neutralize the viral particle and inhibit the viral entry (Xu et al., 2012). Different studies reported the antiviral efficacy such as disruption of herpes simplex virus type 1 envelope, treatment of genital herpes virus, pigeon paramyxovirus type 1 Replication, anti-influenza activity, porcine epidemic diarrhea anti-viral activity of Aloe vera extracts/phytochemicals in various experimental models and clinical study (Sydiskis et al., 1991; Perfect et al., 2005; Dziewulska et al., 2018; Borges-Argáez et al., 2019). Interestingly, A. vera is an active ingredient of “Bioaron C” syrup, which is a herbal medicine known to prevent upper respiratory tract infections (URTIs). Besides, the plant has been used as medicine for URTIs for many decades (Glatthaar-Saalmüller et al., 2015). Over all the literature revealed the multimechanism-mediated antiviral potential in A. Indica and A. vera phytochemicals.

Our research group identified lead A. Indica phytochemicals having potential to bind human host protein TMPRSS2 involved in Spike-protein entry into the cell (Senapati et al., 2021). Matveeva et al. (2020) studied selected phytochemicals (n=1,911) from 55 plant species to find anti-SARAS-CoV-2 protein inhibitors. This showed about 36 phytochemicals per plant species. Similarly, few studies reported the main protease binding potential of only A. Indica phytochemicals, but they considered less number of phytochemicals (Garg et al., 2020; Adegbola et al., 2021; Umar et al., 2021). Lim et al. (2021) suggested A. indica as a potential herbal source of anti-SARS-CoV-2 agent with a multimodal efficacy of antiviral, anti-inflammatory, and immunomodulatory properties. Recently two different randomized controlled trials of A. indica extract/capsule on COVID-19 positive patients established the significant anti-SARS-CoV-2 potential in A. indica plant (Khan et al., 2020; Nesari et al., 2021). Similarly, few studies reported the Aloe vera phytochemicals as SARS-CoV-2 M^pro and RdRp binding potential (Mpiana et al., 2020; Abouelela et al., 2021; Balkrishna et al., 2021). These studies either considered fewer compounds or concerned only the Aloe vera–specific phytochemicals for the study. In the present study we targeted the three SARS-CoV-2 drugable targets (M^pro, Spike-protein, and RdRp) and considered phytochemicals especially reported in Aloe vera plant. Keeping all the abovementioned facts, we selected the A. indica and A. vera phytochemicals (at larger scale) to revisit the identification of potential antiviral inhibitor of plant origin.

In the present study, a total of 130 phytochemicals present in Azadirachta indica and Aloe vera were identified in literature-based search. The phytochemicals were docked at SARS-CoV-2 Spike, RdRp, and M^pro proteins. Docking score of the Aloe vera and Azadirachta indica lead phytochemicals (≤−6.0 kcal/mole cut-off value) against the test proteins is tabulated in Tables 1 , 2 , respectively. The 2D structures of the top lead compounds against various test proteins are depicted in Figure 1 .

In the present study, Isoquercetin, MS 3, 7-Hydroxyaloin B, and 10-Hydroxyaloin A present in Azadirachta indica and Aloe vera medicinal plants showed potential binding against SARS-CoV-2 drugable targets, viz., Spike-protein, RdRp, and Main protease proteins. The standard inhibitors lopinavir, VE607, and ribinavir were used to compare the docking efficacy of phytochemicals against M^pro, Spike, and RdRp proteins, respectively. The 10-Hydroxyaloin A, 7-Hydroxyaloin B, and 10-Hydroxyaloin B 6’-catate A. vera compounds showed -8.57, -8.37, and -8.35 kcal/mole docking score against M^pro protein. Top three M^pro inhibitors present in A. indica (Isoquercetin, VEPAOL, and Nimbidiol) showed -9.73, -9.43, and -6.8 kcal/mole binding efficacy at the active site of the M^pro protein. The standard inhibitor lopinavir showed -5.33 kcal/mole binding efficacy. The binding pose of the lead M^pro inhibitors (Isoquercetin and 10-Hydroxyaloin A) and standard compound is depicted in Figure 2A . Amino acids involved in the binding with Isoquercetin, 10-Hydroxyaloin A, and standard inhibitor are depicted in Figures 2B–D . Asn142, residue of M^pro, was involved in hydrogen bond formation with lopinavir. Besides, amino acid residues Ser46, Thr45, Cys44, Thr25, Thr24, Leu27, Gly143, Thr26, Met49, Arg188, His41, Asp187, Val186, His164, Met165, Ser144, Phe140, Leu141, Cys145, His163, Glu166, and Gln189 were involved in hydrophobic interaction with protease ( Table 3 ). Isoquercetin formed hydrogen bonds with residues Glu166, Asn146, and His41. Gly143, Leu27, Thr25, Cys44, Val42, Met49, Cys145, Met165, Arg188, Gln189, Thr190, Pro168, Leu167, Leu141, Ser144, His163, and Phe140 amino acid residues of M^pro protein interacted with Isoquercetin by hydrophobic interaction ( Table 3 ). 10-Hydroxyaloin A showed hydrogen bonding with His41, Leu141, Gly143, and Cys145 residues and furthermore interacted with Leu27, Arg188, Thr26, Glu166, Ser144, His164, Asn142, His163, Thr25, Thr45, Cys44, Ser46, Met49, Gln189, Met165 amino acid residues via hydrophobic interaction. The top three lead molecules from Azadirachta indica and Aloe vera (10-Hydroxyaloin A, 7-Hydroxyaloin B, 10-Hydroxyaloin B 6’-catate, Isoquercetin, VEPAOL, and Nimbidiol) showed H-bond interaction with His41, Leu141, Gly143, Cys145, Gln189, Glu166, Thr190, His164, Phe140, Asn146, Thr26, Asn142 residues. Moreover, the active phytochemicals showed hydrophobic interaction at the test protein active site ( Figure 2 and Table 3 ).

MS 3, BB, and Barbaloin phytochemicals in A. vera plant showed -9.57, -8.57, and -8.03 kcal/mole docking score against SARS-CoV-2 Spike-protein. The top three Spike-protein inhibitors present in A. indica (Isoquercetin, VEPAOL, and Kaempferol) showed -8.22, -7.71, and -6.24 kcal/mole binding efficacy at the protein binding site (S1) of the Spike protein. The standard inhibitor VE607 showed -7.81 kcal/mole binding efficacy. The binding pose of the lead Spike protein inhibitors (MS 3 and Isoquercetin) and standard compound is depicted in  Figure 3A . Amino acids involved in the binding with MS 3, Isoquercetin, and standard inhibitor are depicted in Figures 3B–D . Asp422 residue of Spike protein S1 domain was involved in hydrogen bond formation with the VE607. Besides, several amino acid residues Arg346, Thr345, Phe347, Arg509, Ser494, Lys417, Tyr421, Pro491, Leu492, Tyr351, Gln493, Ile418, Val350, Tyr495, Ile402, Ser349, Ala348, Phe497, and Asp442 were involved in hydrophobic interaction with the protein-binding domain ( Table 3 ). MS 3 formed hydrogen with residues Ser494, and Gln493. Leu492, Pro491, Tyr421, Asn422, Lys417, Ile418, Tyr495, Val350, Gly496, and Ser349 amino acid residues of the S1 domain of Spike protein interacted with MS 3 by hydrophobic interaction ( Table 3 ). Isoquercetin showed hydrogen bonding with Phe347, Val350, Asp442, and Phe497. Furthermore, it interacted with Arg509, Arg346, Ala348, Val401, Ser349, Gln493, Tyr495, Tyr351, Ser494, Gln498, Pro499, Ser443 amino acid residues by hydrophobic interaction. The top two lead molecules from Azadirachta indica and top three Aloe vera (MS 3, BB; Barbaloin, Isoquercetin, and VEPAOL) showed H-bond interaction with Ser494, Gln493, Phe497, Asp442, Phe347 Tyr495, Val401, Val350, Val350, Asn422, Pro491, Tyr351 amino acid residues. Moreover, the active phytochemicals showed hydrophobic interaction at the test protein active site ( Figure 3 and Table 3 ).

The 7-Hydroxyaloin B, CHEBI:35671, and UNII-73899319HU A. vera compounds showed -7.07, -6.96, and -6.77 kcal/mole docking score against SARS-CoV-2 RdRp protein. The top RdRp protein inhibitors (above the cut-off value) present in A. indica (Isoquercetin and Isonimocinolide) showed -6.86 and -6.29 kcal/mole binding efficacy at the active site of the protein. The standard inhibitor ribinavir showed -6.13 kcal/mole binding efficacy. The binding pose of the lead RdRp protein inhibitors (7-Hydroxyaloin B and Isoquercetin) and standard compound is depicted in Figure 4A . Amino acids involved in the binding with 7-Hydroxyaloin B, Isoquercetin, and standard inhibitor are depicted in Figures 4B–D . Trp617, Asp618, Lys798, and Glu811 residues of RdRp active site were involved in hydrogen bond formation with ribinavir. Besides, Tyr619, Ser814, Phe812, Asp761, Asp760, Ala762, Gly616, Trp800, and Cys799 amino acid residues showed hydrophobic interaction at the RdRp active site ( Table 3 ). The 7-Hydroxyaloin B formed hydrogen with residues Asp618, Tyr619, Lys621, Asp760, Lys798, and Glu811. Furthermore 7-Hydroxyaloin B interacted with residues Asp761, Asp623, Cys622, Pro620, Lys551, Arg553, and Ser814 hydrophobically ( Table 3 ). Isoquercetin formed hydrogen bonding with Trp617, Asp618, Tyr619, Asp760, and Lys798 amino acid residues. Furthermore, isoquercetin interacted with residues Pro620, Lys621, Cys622, Cys799, Trp800, Gly616, Ala762, Asp761, Glu811, Ser814, and Ser759 hydrophobically. The top two lead molecules from Azadirachta indica and top three from Aloe vera (7-Hydroxyaloin B, CHEBI:35671, and UNII-73899319HU, Isoquercetin, and Isonimocinolide) showed H-bond interaction with Asp618, Tyr619, Lys621, Asp760, Lys798, Glu811, Asp761, Ala762, His810, Arg553, Ser759, Ser814, and Trp617 amino acid residues of the RdRp protein. Moreover, the active phytochemicals showed hydrophobic interaction at the test protein active site ( Figure 4 and Table 3 ).

In the present study we found the potential interaction of phytochemicals with the drugable targets of SARS-CoV-2-mediated infection in human. For this we targeted the three key steps of the viral pathogenesis, viz., viral entry into the host cell (Spike-protein), conversion of viral precursor to functional proteins (M^pro), and viral genome replication (RdRp). Interaction of Spike-protein RBD with the ACE-2 receptor (human host protein) is a critical step in viral entry into the host cell (Senapati et al., 2020). Recently Shang et al. (2020) reported that residues 455, 482–486, 493, 494, and 501 are critical SARS-CoV-2 S1 domain amino acids involved in interaction with human ACE-2 protein (Shang et al., 2020). Our research group reported inhibitory potential of phytochemicals against SARS-CoV-2 drugable proteins. The in silico inhibitory potential of Curcuma longa and Withania somnifera phytochemicals against M^pro protein of SARS-CoV-2 has been reported recently (Gupta et al., 2020; Kushwaha et al., 2021c). The identified lead phytochemicals showed significantly increased binding potential at the M^pro active site in comparison to standard inhibitors in molecular docking and molecular dynamics simulation study (Gupta et al., 2020; Kushwaha et al., 2021c). In the present study, we found that lead A. indica and A. vera phytochemicals interacted with two of the critical amino acids (Gln493 and Ser494) ( Table 3 ). Isoquercetin formed hydrogen bonding, while MS 3 showed hydrophobic interaction with the Gln493 and Ser494 amino acids, which are critical for viral Spike-protein and human host ACE-2 binding receptor. MS 3 showed H-bonding with the Asn422 amino acid residue, which is reported to be involved in ACE-2 binding of Spike-protein (Gordon et al., 2020). Lead phytochemicals (binding efficacy below <−6.0 kcal/mole) also showed hydrophobic and hydrogen bond interaction with the key amino acids involved in Spike-protein RBD domain and ACE-2 protein-protein interaction (Asp442, Ser494, and Gln493). The results indicate that Isoquercetin and MS 3 have potential to disrupt spike glycoprotein-ACE-2 protein-protein interaction by binding at SARS-CoV-2 RBD. Thus, the lead molecules might inhibit viral entry into the cell. It has been shown that RdRp inhibitors (such as remdesivir) potentially block the RNA synthesis and thereby delay the chain termination process in SARS-CoV-2 RNA synthesis (Gordon et al., 2020). SARS-CoV-2 RdRp inhibitors are being studied in clinical trials in various countries of the world. Binding of A. indica and A. vera lead compounds at RdRp active site showed interaction with similar amino acids (Asp618, Tyr619, Asp760, Lys798, Glu811, and Asp761), indicating the SARS-CoV-2 RdRp inhibition potential in the test medicinal plants ( Table 3 ). The SARS-CoV-2 RNA–mediated translation generated polyproteins that later on cleaved at some specific sites by Main protease enzyme to produce functional proteins. This step aids in the virulence of SARS-CoV-2. Literature reports that a Cystine-Histidine dyad is essential for the protease activity of M^pro. Besides, the alanine, glycine, glutamate, serine, and leucine residue also play important roles in the cleavage catalysis process (Gupta et al., 2020). In the present study, the 10-Hydroxyaloin A and Isoquercetin showed hydrogen bond interaction with the Cys and His residues, indicating the Cys-His dyad disruption potential of the lead phytochemicals. The dyad disruption might lead to the decreased M^Pro activity, which in turn inhibits the production of functional protein. Thus, the 10-Hydroxyaloin A and Isoquercetin might be involved in the SARS-CoV-2 virulence mitigation.

RMSD is a parameter that computes the distance between protein atoms. The average distance between the atoms in unbound and ligand/standard inhibitor–bound targeted protein allows us to assess the comparative conformation and stability of the protein (Gupta et al., 2020). The present study indicates that the binding of 10-Hydroxyaloin A and Isoquercetin did not affect the conformational stability of the M^Pro protein ( Figures 5A, B ). The result indicates the stable M^pro-10-Hydroxyaloin A (MHA) and M^pro-Isoquercetin (MNB) complex formation. RMSF is an important parameter to assess the fluctuation of protein atoms across the time duration from a reference position. This allows us to study the comparative fluctuations in the portion of target protein (residue) before and after the ligand binding. In this study, binding of 10-Hydroxyaloin A and Isoquercetin showed the stabilization of the 40–45 and 140–165 amino acid residues during the simulation time ( Figures 5C, D ). The key amino acids required for the catalytic activity of the test protein fall in these areas. Thus, it might be inferred that the lead phytochemicals tightly bind with the key amino acids and stabilize the active site of the protein. In the previous studies in our laboratory, we found that phytochemical binding mediated lesser fluctuations in target protein amino acid residues (Gupta et al., 2020; Kushwaha et al., 2021c). Radius of gyration (Rg) provides valuable information about the folding of regular secondary structure of the targeted protein into the tertiary or functional structure before and after binding of the ligand/inhibitor molecule. In comparison to unbound protein, the 10-Hydroxyaloin A and Isoquercetin binding mediated decreased Rg value during the simulation period, indicating the compact and stabilized folding in the ligand-bound complexes ( Figures 6A, B ). The binding of a ligand at the active site of a protein surrounding a solvent is a solvent-substitution process. Thus, calculations of Solvent accessibility surface area (SASA) of ligand-bound and unbound protein give important information about the potential ligand binding. Isoquercetin binding mediated significantly decreased SASA value of the complex (in comparison to standard inhibitor–bound and non-bound protein), indicating the stabilized protein structure/active site throughout the simulation period ( Figures 6C, D ). Although the various types of interactions among ligand and targeted protein are involved in the complex stabilization process, hydrogen bond formation plays a significant role in this process. The greater the number of hydrogen bond incidence during the ligand-protein complex formation, the greater the stability of the complex. In the present study, the increased hydrogen formation potential (in comparison to standard inhibitor) of the 10-Hydroxyaloin A and Isoquercetin phytochemicals during the simulation period indicates the potential stability of the protein complex ( Figures 7E–G ). The greater number of hydrogen bond formation in the test compound interaction with the M^pro protein corroborates the more negative docking score of the lead compounds in comparison to standard inhibitor. The PCA and free energy landscape results (compact structure and increased centric energy respectively) indicate the compactness of the M^pro protein structure after binding the lead molecules at the active site of the protein ( Figures 8 , 9 ). The analysis of the various secondary structures of the test protein in the presence of 10-Hydroxyaloin A and Isoquercetin indicated the involvement of β-sheet and 5’-helix in the interaction ( Figure 9 ). The DCCM analysis indicated that the 10-Hydroxyaloin A inhibited the amino acid motion in the test protein, but isoquercetin revealed similar pattern of amino acid motions in standard inhibitor–bound protein. Over all, the analysis of the MD simulation trajectory revealed the stable and energetically favorable complex formation in the presence of lead phytochemicals. The MM-PBSA analysis showed the contribution of amino acids in the ligand-protein binding ( Figure 10 ). It should be noted that the His41 and Cys145 amino acid residues involved in catalytic dyad were contributing to the interaction of lead phytochemicals and the M^pro protein. The results substantiate the potential binding and active site inhibition potential in the lead molecules. Over all, the boiled egg diagram, the bioavailability radar, and the physiochemical properties of the A. indica and A. vera lead phytochemicals showed the drug-like potential that could be utilized for anti-SARS-CoV-2 drug discovery ( Figure 11 ).

Plant-derived compounds possess single and/or multitargeted therapeutic potential against various diseases including viral disease. Thus, the computer-based identification of phytochemicals present in the medicinal plants is the need of time to identify potential inhibitors of SARS-CoV-2 drugable targets. The natural compounds possess less toxicity and associated side-effects, which make them a suitable candidate for drug discovery. In the present molecular docking study, we found that the phytochemicals present in A. vera and A. indica medicinal plants possess significant binding potential at the active site/protein-protein interaction sites of the SARS-COV-2 drugable targets (Spike-protein, RdRp, and M^pro proteins). Further, the molecular dynamic (MD) simulation and MMPBSA calculations revealed that 10-Hydroxyaloin A and Isoquercetin phytochemicals present in the A. vera and A. indica, respectively, stabilize the structure and energy of the M^pro-ligand complex. More importantly, the lead phytochemicals showed disruption of His-Cys dyad at the active site of the M^pro protein required for its catalytic activity. In MMPBSA analysis, we found that the His and Cys residues contributed significantly in the binding free energy of the complex. Overall, we conclude that the A. vera and A. indica Indian medicinal plants could be taken as source of lead anti-SARS-CoV-2 agents for future drug discovery.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

SK designed and conceptualized the study. PK, AS, TB, and AY contributed in the data generation. SK wrote the manuscript. The figures and tables were developed by KP, MS, and AS. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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