The computational approach in drug discovery helps in predicting the activity and fate of a potential drug candidate, thereby cutting down the cost and time of drug development as well as avoiding unwarranted drug toxicity and reducing the ethical concerns of experimental animals (Malik et al., 2017). With the computer-aided ligand-protein interaction technique (molecular docking), the efficacy of the diverse bioactive compounds present as a composite mixture in a given medicinal plant can be individually evaluated (Johnson et al., 2020 & Johnson et al., 2021) and their respective drug-likeness, pharmacokinetic and pharmacodynamic behaviours can be predicted through the Swiss-ADME server (Ranjith & Ravikumar, 2019 & Ojo et al., 2021) and the toxicity profile through the ProTox-II server (Banerjee et al., 2018 & Ghosh et al., 2019). In this study, 10 compounds were selected from one hundred and fifty bioactive compounds of Neem after the molecular docking evaluation of their inhibitory potential against SARS-CoV-2 3C-like protease. This drug target is a SARS-CoV-2 enzyme belonging to the family of cysteine proteases and it is one of the non-structural proteins (nsps) required for the cleavage of viral RNA-translated polyproteins (Pp1a ad -1ab) into other nsps such as helicases (nsp13), RNA-dependent RNA polymerase (RdRp or nsp12), exonucleases (nsp14) and endonucleases (nsp15); all of which are components of the viral replication-transcription complex (RTC) required for new virion synthesis ( Hao et al., 2017; Asghari, et al., 2020; Ortega et al., 2020 & Wu et al., 2020). Among these 10 selected test compounds, rutin had the highest binding affinity followed by tanninamine, quercitrin, hyperoside and kaempferol, before the standard inhibitor K36. Rutin, which is a glycoside formed by a fusion of flavonol quercetin with disaccharide (Sharma et al., 2013 & Prasad & Prasad, 2019), has been evident to be anticarcinogenic, antidiabetic, antimicrobial, and cardio-, neuro-, haemato-, hepato- and nephroprotective (Ren et al., 2003; Ganeshpurkar & Saluja, 2017; Prasad & Prasad, 2019). Tanninamine, quercitrin, hyperoside and kaempferol have also been reported to display a wide range of biological activities, including antiviral, in different experimental studies (Kim & Park, 2018; Wang et al., 2018; Raza et al., 2017; Chung et al., 1998). Tannin amine which is a tannic acid derivative is a polyphenol used for the treatment of a vast number of diseases, including viral diseases (Sharma et al., 2019). Quercitrin (a rhamnose glycoside) is a glycoside derivative of quercetin, which is a flavonoid with a well-known antiviral potency (Wu et al, 2016). Hyperoside, a quercetin-3-O-galactoside is another flavonoid that has shown antiviral potential against the hepatitis B virus (HBV) (Wu et al, 2007). Kaempferol is a flavonoid aglycone that exists in a glycoside form and has been implicated as an antiviral agent against HIV and coronavirus (Zakaryan et al., 2017). The five compounds interacted with important active site amino acids residues of the enzyme. Rutin, tanninamine, quercitrin, hyperoside and the standard ligand formed one or more hydrogen bonds with Glu-166. Kaempferol was also found to be in association with this residue. Glu-166 is an important residue required by the enzyme for its substrate-induced dimerization, a necessary condition for catalysis (Roe et al., 2021). Mutation of this residue has been found to significantly reduce the substrate-induced dimerization process and subsequently prevent enzyme activation (Cheng et al., 2010 & Roe et al., 2021). Mutation of Glu-166 blocks the substrate-induced dimerization of SARS coronavirus main protease (Hsu et al., 2005 & Roe et al., 2021). The compounds also bind to Asn-142 which is required for blocking the entrance of the substrate-binding subsite in the enzyme monomer, by forming a hydrogen bond with Glu 166 (Cheng et al., 2010a). Also, of high significance is their interaction with His 41 and Cys 145. These residues play very critical roles at the catalytic site of the enzyme (Yoshino et al., 2020). The catalytic site holds a His 41-Cys 145 catalytic dyad in a cleft between two structural domains of the enzyme, where Cys 145 acts as a nucleophile during the first step of the catalytic process and His 41 acts as a base catalyst (Chang, 2010b). Molecular interaction with these important residues is the target of most SARS-CoV-2 3C-like protease inhibitors.
The pharmacophore models of rutin, quercitrin, tanninamine, hyperoside and kaempferol on SARS-CoV-2 3C-like proteinase showed that hydrogen bond donors/acceptors and aromatic rings are the structural features of the compounds responsible for the molecular interactions with the enzyme (Fig. 3). These important features could have contributed to the binding affinity of the compounds. Hydrogen bonds are generally considered to be facilitators of protein-ligand binding (Chen et al., 2016) and their presence is an indication of good docking quality and complex stability (Wu et al., 2012). Aromatic interactions are very significant to molecular recognition and are particularly essential in drug design since about 20% of amino acids are aromatic in nature (Lanzarotti et al., 2020).
Despite the high binding affinity of the compounds for the protein target, a promising drug candidate should fulfil the paramount criteria of drug-likeness. Drug-likeness is achieved when the molecular and structural features of the test compound are under the acceptable range. Such features include water-solubility, lipophilicity, molecular size, flexibility, polarity and saturation of the compound etc, and they determine whether a compound will be orally bioavailable (Duru et al., 2021; Pires et al., 2015). Water solubility is associated with lipophilicity and permeability and this, in turn, determines the bioavailability of molecules at the target site. The compounds (apart from the poorly soluble Nimbaflavone and the moderately soluble Azadirachtannin and Nimbinone), possess high water solubility which is needed for easy passage within the aqueous blood, but in principle could reduce their membrane permeation capacity and bioavailability. However, the compounds possess varying levels of lipophilicity, some of which are good enough for them to penetrate the intestinal linings. The lipophilicity value of most of the selected Neem phytoconstituents falls within the acceptable value of octanol-water partition coefficient (Log P) ≤ 5. Additionally, Kaempferol, Scopuletin, Nimbaflavone and Nimbinone fully obeyed Lipinski and Verber’s rules, and hence, they can be predicted to be orally bioavailable. Myricetin could also be a good oral drug because it violated only one Lipinski rule. The Lipinski rule constitutes the criteria of molecular weight (MW) ≤ 500, octanol/water partition coefficient (C logP) ≤ 5, number of hydrogen bond donors (HBD) ≤ 5 and number of hydrogen bond acceptors (HBA) ≤ 10; and an orally active drug should not violate more than one of these criteria. For Veber's rule, compounds that meet only the two criteria of ≤ 10 rotatable bonds and polar surface area no greater than 140 Å2 are projected to have good oral bioavailability. Apart from passing Lipinski and Veber's rules, these compounds also possess a good bioavailability score of 0.55 as against 0.17 for the remaining compounds. This implies that Kaempferol, Scopuletin, Nimbaflavone, Nimbinone and Myricetin have a 55% probability of at least 10% oral bioavailability in rat or measurable human colon carcinoma (Caco-2) permeability, whereas, the remaining compounds possess only about 17% probability. The oral bioavailability prediction also agrees with the predicted GIA potential (which is high for kaempferol, scopoletin, nimbaflavone and nimbinone) and the blood-brain barrier (BBB) permeability (which is high for scopoletin and nimbinone).
In addition to the drug-likeness properties, the pharmacokinetic properties of the compounds were also considered. The results indicated that kaempferol, quercitrin and hyperoside might escape the efflux pump (P-gp) which is a multidrug resistance protein that offers protection to the organs from oxidative damage by xenobiotics. Another important pharmacokinetic-related factor involves the underlying role played by drug-metabolising enzymes like cytochrome P-450 (van Waterschoot & Schinkel, 2011; Turner & Agatonovic-Kustrin, 2007). Roughly 50–90% known drugs are substrates of the 5 important isoforms CYP 1A2, 2C19, -2C9, -2D6 and − 3A4 (Mishra & Dahima, 2019; Shweta & Rashmi, 2019; Daina et al., 2017; Di, 2014). This implies that Kaempferol, nimbaflavone, nimbinone, myricetin and scopoletin could inhibit the metabolism of drugs that are substrates of one or more of these enzymes, thereby causing some levels of drug-drug interaction (Shweta & Rashmi, 2019; Daina et al., 2017; Kirchmair et al., 2015; Potts & Guy 1992).
Prediction of the toxicity profile of test compounds is an essential part of the early drug discovery process. Roughly 89% of new drug candidates could not accomplish the human clinical trial despite their high efficacy and acceptable pharmacokinetic properties, and 50% of these failures are due to unexpected drug-related toxicity (Nisha et al., 2016 & Kirchmair et al., 2015). From the results obtained in this study, Tannin-amine, Kaempferol and Nimbinone are not likely to induce any toxic effect; but rutin and hyperoside might be immunotoxic, and quercitrin could be both carcinogenic and immunogenic. However, the two most toxic of all the compounds are myricetin and azadirachtannin with LD50 values of 159 and 274 mg/Kg respectively. The remaining compounds, the majority of which belong to the oral toxicity class 5 (narrow probability of being harmful if swallowed) are relatively safe, with LD50 values ranging from 2000 to 5000 mg/Kg.