A review on molecular docking analysis of phytocompounds against SARS-CoV-2 druggable targets

Abstract

The novel β-coronavirus, SARS-CoV-2, responsible for the coronavirus disease 2019 (COVID-19) emerged in China in December 2019. Due to its high transmission and infection rate, it has spread around the world and has transformed into a ravaging global pandemic with enormously unprecedented impacts globally on human, social, and economic health. Just like SARS-CoV and MERS-CoV, there is no specific antiviral drug for its treatment. The only available therapeutics are supportive and symptom-based. Thus, scientists are harnessing various strategies to expedite drug development. One such approach is drug repurposing through computational screening of phytocompounds, which leverages proteins that are essential for the entry, replication, pathogenesis, assembly, and release of SARS-CoV-2. Here, we review the available literature on molecular docking of phytoligands against SARS-CoV-2 integral proteins, in a bid to update our current knowledge and identify the most promising molecules. The overwhelming majority of the promising lead compounds are either phenolics or terpenoids. Furthermore, of the elucidated SARS-CoV-2 targets, the main protease (3CL^pro) appears as one of the most attractive druggable targets. Notably, compounds such as rutin, quercetin, luteolin, neoandrographolide, curcumin, and others with evident anti-inflammatory benefits, in addition to their predicted anti-SARS-CoV-2 properties, deserve further studies to validate their activity.

KEYWORDS:

• SARS-CoV-2
• computational analysis
• COVID-19
• druggable targets
• phytocompounds

Introduction

Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA (26-32 kb) viruses with biological sizes ranging from 65 to 125 nm (Li, Liu et al. 2020; Shereen et al. 2020). They belong to the subfamily Orthocoronavirinae within the family Coronaviridae. The four genera within this subfamily include Alphacoronavirus, Betacoronavirus, Deltacoronavirus, and Gammacoronavirus (Yang and Leibowitz 2015; Banerjee et al. 2019). The α- and β-coronaviruses have great medical and veterinary significance and contain 17 and 12 species, respectively. Members of the β-coronavirus include Human Coronavirus OC43 (HCoV-OC43), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Human coronavirus HKU1 (HCoV-HKU1), Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) (Harapan et al. 2020). In November 2002, the SARS-CoV outbreak, with fatality rate of 11%, that later spread globally, emerged in Guangdong, China (Graham et al. 2013). Exactly a decade later (2012), the MERS outbreak, with an associated mortality rate of 37%, broke out in the Kingdom of Saudi Arabia and later spread to 27 other countries (Zumla et al. 2015).

Recently, a novel β-coronavirus, SARS-CoV-2, responsible for coronavirus disease 2019 (COVID-19) emerged in Wuhan, China, in December 2019. This virus belongs to β-coronavirus as SARS-CoV and MERS-CoV. It is genetically closely related to SARS-CoV with 82% similarity and has up to 50% similarity with MERS-CoV (Chan et al. 2020; Lu et al. 2020). Notably, SARS-CoV-2 has been reported to have high genetic similitude with a bat coronavirus, BatCoV RaTG13 (from the horseshoe bat), with 96.2% sequence homology (Zhou et al. 2020), possibly indicating that its reservoir host is bats. Due to its high infectivity and transmissibility, the virus has constituted a global pandemic and is a major public health menace. As of March 22, 2021, the virus has plagued more than 123 million individuals and resulted in 2,711,071 confirmed deaths in 235 countries (WHO, 2021). Typical symptoms of COVID-19 infection include sore throat, fever, dyspnoea, dry cough, headache, and pneumonia (Zhou et al. 2020). Just like SARS-CoV and MERS-CoV (de Wit et al. 2016; Zumla et al. 2015), there is no specific antiviral drug for the treatment of COVID-19, the only available therapeutic options are symptom-based or based on organ support (Li and Clercq 2020); thus, effective therapeutics are urgently needed.

Generally, four strategies are being employed to elucidate and develop potential anti-coronaviral agents. The first involves repurposing already available antivirals with proven efficacy against other viruses (e.g. remdesivir, an inhibitor of Ebola virus replication), with known dosage and clear metabolic characteristics. Remdesivir and favipiravir were found to exhibit great efficacy (Zhang, Xie et al. 2020). However, some of these antiviral therapeutics exhibit broad-spectrum activity and would not eliminate coronaviruses in a precise, targeted, and specific manner, and might be accompanied by non-negligible side effects (Chan et al. 2013). The second strategy involves the repositioning of already available non-antiviral drugs; for example, the anti-SARS-CoV-2 potential of an antimalarial drug, chloroquine, has been demonstrated (Gautret et al. 2020; Wang, Cao et al. 2020). The third method involves the study and characterisation of genomic, proteomic, and pathological features of coronaviruses in a bid to synthesise potential anti-coronaviral molecules from scratch (Omrani et al. 2014). Although agents discovered through this strategy would be more specific and exhibit better activity against coronaviruses, this procedure is often complex, time-consuming (requiring up to 10 years), and capital-intensive. Lastly, structure–activity relationship approach, involving computational (e.g. molecular docking) and high-throughput screening, is exploited to screen molecules (lead compounds), including phytocompounds, for their anti-coronaviral activity (Wu et al. 2020). This strategy is fast, affordable, and allows swift identification, characterisation, and screening of lead compounds while also limiting the use of animal models.

Plants and phytocompounds are receiving considerable attention as potential anti-coronaviral remedies due to their rich bioactive components and evident pharmacological activities which may guide the development of novel antiviral agents (Fischer et al. 2020; Jamiu et al. 2020). Importantly, many conventional drugs available today were designed using natural compounds as templates. Classic examples include the semisynthetic antimalarial drugs, chloroquine phosphate, and artemisinin obtained from Cinchona tree bark and Artemia annua, respectively. Moreover, emetine, an isoquinoline alkaloid, used as an amoebicidal drug is obtained from Cephaelis ipecacuanha (Weathers et al. 2014; Ganjhu et al. 2015). Other plant-derived semisynthetic drugs include quinidine, aspirin, morphine, digitalis, topotecan, taxol, and colchicine (Weaver 2014; Ganjhu et al. 2015). In addition, the use of Traditional Chinese medicines (TCM) for the management/treatment of SARS-CoV, Ebola virus, and H1N1 has been documented (Luo et al. 2019). In fact, the complementary use of TCM with conventional medicine for the successful treatment of COVID-19 has been exemplified (Wang, Chen et al. 2020).

Coronaviruses encode several proteins, some of which are essential for their life cycle (i.e. viral entry, replication, assembly, and release). Some of the most studied coronavirus proteins include 3-chymotrypsin-like protease (3CL^pro), also referred to as main protease (M^pro), papain-like protease (PL^pro), and spike glycoprotein (Sgp) (Shode et al. 2021). Both 3CL^pro and PL^pro are important for the proteolytic processing of viral polyproteins into functional units, coronaviral replication, and regulation of the immune system (Chen et al. 2020; Yu et al. 2020), while the Sgp is indispensable for viral entry into host cells (Isaacson and Ploegh 2009; Mukherjee et al. 2011). In addition, RNA-dependent RNA polymerase (RdRp) catalyses the replication of viral RNA from RNA templates, and the RdRp of SARS-CoV, MERS-CoV, and SARS-CoV-2 has similar sequences (Lung et al. 2020). Notably, due to the indispensable roles of 3CL^pro, PL^pro, Sgp, and RdRp in viral entry, replication, proliferation, and pathogenesis, they remain attractive targets for antiviral drug development. Unsurprisingly, scientists are already exploiting computational approaches, including molecular docking, to identify natural compounds capable of interacting with and modulating these viable targets. Such natural compounds have been speculated as potential inhibitors of their biological functions in silico; however, this must be well substantiated with appropriate laboratory and clinical tests. Here, we review the available scientific literature on the molecular docking-predicted anti-SARS-CoV-2 activity of phytocompounds, in a bid to update our current knowledge and identify the most promising lead compounds.

Materials and methods

Search strategy

The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) was adopted (Moher et al. 2009). A search through literature databases, including PubMed and ScienceDirect, was made using keywords such as (‘COVID-19’ or ‘SARS-CoV-2’ or ‘coronavirus’) and (In silico or ‘molecular docking’ or ‘computational screening’ or ‘virtual screening’ or ‘drug repurposing’) and (‘Medicinal plants’ or ‘Natural products’ or ‘phytocompounds’).

Study selection

Only studies written in English that fulfilled the following criteria were included: (i) involved virtual screening of plant-derived natural products, (ii) involved natural inhibitors of SARS-CoV-2 from plant origin, and (iii) described the binding energy and binding sites of phytoligands. Articles excluded include (i) articles not written in English, (ii) studies without binding energy of phytoligand–target interaction, (iii) articles that exempted the binding sites of the phytoligand on the protein, (iv) studies that do not include molecular docking of phytocompounds, and (v) articles strictly involving virtual screening of conventional drugs. The authors independently searched, screened, and reviewed potentially pertinent publications. Duplicates were removed, disagreements were resolved when necessary, and this review represents a summary of data extracted from relevant studies. This PRISMA flowchart of this study is illustrated in Figure 1.

Figure 1. PRISMA flowchart showing all literature screened, included, and reviewed for this study.

Results and discussions

A brief overview of coronavirus structure and proteome

The proteome of a typical coronavirus including SARS-CoV-2 consists of at least four structural proteins and several non-structural proteins (nsps). The structural proteins include the nucleocapsid (N), membrane (M), envelope (E), and spike (S) proteins (Figure 2(a, b)), all of which play crucial roles in the regulation of viral structure and functions (Schoeman and Fielding 2019). The N protein has two domains; N-terminal domain, and C-terminal domain, both of which are capable of binding and forming complexes with the viral RNA genome, but with disparate mechanisms. The N protein can also form complex with non-structural protein 3 (nsp3) and performs the packaging of the encapsidated genome into virions (Fehr and Perlman 2015; Chen et al. 2020). In addition, M glycoprotein, the most abundant coronaviral structural protein, has a small size ranging 25–30 kDa (Nal et al. 2005; Neuman et al. 2011). The M protein gives the virion its unique shape and is also involved in viral assembly (Arndt et al. 2010; Kuo et al. 2016). The E protein is very small with a size of ∼8–12 kDa. It is not only important for viral pathogenesis but also plays crucial roles in viral budding, assembly, and release (DeDiego et al. 2007; Nieto-Torres et al. 2014). Lastly, the S glycoprotein (Sgp) is quite big with a size of approximately 150 kDA. This glycoprotein consists of two subunits, S1 and S2 units. It is this surface protein that forms the spikes (petal-shaped surface projections), which are indispensable for viral attachment to host receptor and viral entry (Benaic et al. 2006; Chen et al. 2020). Although the receptor-binding domain (RBD) of SARS-CoV and SARS-CoV- 2 Sgps has about 72% amino acid sequence identity (Hemida and Ba Abduallah, 2020; Sun et al. 2020), SARS-CoV-2 Sgp has been reported to bind more strongly with the host receptor (ACE2) (Ali and Vijayan 2020). In addition, the SARS-CoV-2 Sgp also includes a unique region that can interact with and is activated by another host enzyme called furin (Coutard et al. 2020).

Figure 2. An illustration of the genomic organization and structure of SARS-CoV-2. (A) Genomic organization of SARS-CoV-2. The ORF1a and ORF1b of the viral RNA genome encode 16 non-structural proteins (nsp1–nsp16). Nsp3 and nsp5 encode PL^pro and 3CL^pro, respectively, which are essential for viral replication and pathogenesis. The genomic organisation of S glycoprotein showing its two subunits (S1 and S1) is depicted below the viral genome. Dotted lines within this structure indicate the S1/S2 cleavage site. Other important components of the S glycoprotein such as signal protein (SP), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat (HR1 and HR2), transmembrane domain (TM), and cytoplasm domain (CP) are depicted. (B) Structure of SARS-CoV-2 showing its single-stranded positive-sense RNA genome and essential structural proteins.

Furthermore, the ORFs – ORF1a and ORF1b – of the coronavirus genome encode 16 nsps, nsp1-11 and nsp1-16, that is subsequently cleaved into individual nsp (Ziebuhr et al. 2000; Fehr and Perlman 2015). The papain-like protease (PL^pro) is encoded by nsp3. This protein catalyses the proteolytic processing of polyproteins (pp1a and pp1b) and plays a vital role in viral replication and host infection (Yu et al. 2020). It induces infection via its antagonistic effects on interferons (IFNs) and other immune cells. The nsp5 produces 3-chymotrypsin-like protease (3CL^pro), also known as the main protease (M^pro). This protease is also essential for the cleavage of polyproteins and plays remarkable roles in viral replication and modulation of the immune system (Zhou et al. 2019; Chen et al. 2020). Interestingly, the 3CL^pro of SARS-CoV-2 has a huge sequence resemblance (identity) of up to 96% with that of SARS-CoV (Fischer et al. 2020). In addition, the nsp12 and nsp13 of the coronavirus encode RdRp and helicase, respectively. Whilst RdRp catalyses the replication of viral RNA from the RNA templates, helicase also performs vital roles during viral replication and translation (Lung et al. 2020). Additionally, two other nsps – nsp14 and nsp15 encode exoribonuclease (ExoN) and endoribonuclease (EndoU), respectively (Figure 2a & b). Although ExoN acts as a proof-reader, EndoU cleaves viral polyuridine sequences, and this prevents the activation of host immune signals and viral evasion from immune cells (Graepel et al. 2017; Hackbart et al. 2020). A list of nsps with their functions are illustrated in Table 1.

Table 1. List of 16 coronaviral non-structural proteins with their functions.

nsp	Function	References
nsp1	Degradation of mRNA and inhibition of IFN signals	Tanaka et al. 2012
nsp2	Essential for proofreading viral replication	(Harcourt et al. 2004; Naqvi et al. 2020)
nsp3	PL^pro: proteolytic processing of polyproteins, plays vital role in viral replication and host infection; blocking of host immune response	(Lei et al. 2018; Yu et al. 2020)
nsp4	Formation of DMV; may complex with nsp3 and nsp6	(Gadlage et al. 2010; Beachboard et al. 2015; Chan et al. 2020)
nsp5	3CL^pro: polypeptides cleaving, viral replication, and modulation of immune system – inhibition of IFN	(Zhou et al. 2019; Chen et al. 2020)
nsp6	Formation of DMV; restriction of autophagosome expansion; may complex with nsp3 and nsp4	(Angelini et al. 2013; Cottam et al. 2014; Chan et al. 2020)
nsp7	Complex with nsp8 and nsp12; may act as clamp for RdRp	(Zhai et al. 2005; Fehr and Perlman 2015; Kirchdoerfer and Ward 2019)
nsp8	Forms complex with nsp7 and nsp12; acts as a primase	(Zhai et al. 2005; te Velthius et al. 2012; Kirchdoerfer and Ward 2019)
nsp9	Dimerization, RNA binding, and interaction with nsp8	(Egloff et al. 2004; Sutton et al. 2004; Zeng, Deng et al. 2018)
nsp10	Interaction with ExoN domain (nsp14); scaffold protein for nsp14 and nsp16	(Chen et al. 2011; Decroly et al. 2011; Bouvet et al. 2014; Ma et al. 2015)
nsp11	Unknown	(Fang et al. 2008)
nsp12	RdRp: replication of viral RNA	(Ahn et al. 2012; Kirchdoerfer and Ward 2019; Lung et al. 2020)
nsp13	RNA helicase: unwinding of nucleic acid; 5′ triphosphatase	(Adedeji and Lazarus 2016; Hao et al. 2017; Jia et al. 2019)
nsp14	ExoN acts a proof-reader of viral genome; N7-MTase	(Graepel et al. 2017; Chen et al. 2020)
nsp15	EndoU: cleavage of viral polyuridine sequences, evasion of immune cells, attacks immune system	(Hackbart et al. 2020; Kim et al. 2020)
nsp16	2′-O-MTase: protects viral RNA from MDA5 recognition; negative regulation of host immunity	(Chen et al. 2011; Decroly et al. 2011; Shi et al. 2019)

Note: IFN: interferon, PL^pro: papain-like protease, DMV: double-membrane vesicle, 3CL^pro: chymotrypsin-like protease, RdRp: RNA-dependent RNA polymerase, ExoN: exoribonuclease, EndoU: endoribonuclease, 2′-O-MTase: 2′O-ribose methyltransferase.

Notably, whilst the dipeptidyl peptidase 4 (DPP4) represents the functional receptor of MERS-CoV, angiotensin-converting enzyme 2 (ACE2) is the host receptor that mediates and facilitates the entry of SARS-CoV and SARS-CoV-2 into the host cell, and this is achieved via the attachment and interaction of Sgp with this enzyme (Li et al. 2003; Raj et al. 2013; Li, Geng et al. 2020). Although other body organs such as kidneys, intestines, hearts, and brains contain ACE2, these receptors are found in great abundance in the lungs (alveoli) (Zhang, Xie et al. 2020), and this may partly explain why SARS-CoV-2 infection induces greater damage in the pulmonary system. Moreover, SARS-CoV-2 Sgp reportedly binds ACE2 more strongly than SARS-CoV (Coutard et al. 2020).

Virtual screening of phytocompounds as inhibitors of coronavirus-associated proteins

The traditional process of drug discovery is usually complex, strenuous, and quite inefficient. In addition, this process is often very expensive (more than $ 1 billion) and hugely time-consuming (up to 10 years) (Figure 3) (Kapetanovic 2008; Al Qaraghuli et al. 2017). New approaches such as virtual screening with the use of computer docking models have been introduced. This process involves using computer-based techniques to screen and sieve drug candidates based on their desirability (activity, drug-like, and lead-like) and undesirability (toxicity and inactivity) as drugs, such that only suitable drug candidates proceed to subsequent animal testing and clinical trials (Kapetanovic 2008). This protocol does not only complement, facilitate, and accelerate the traditional drug discovery process, but also allows swift repurposing of already available drugs. In addition to its affordability, this technique can also be used for high-throughput screening of druggable targets and compounds (up to 10,000 compounds per day), to facilitate characterisation of drug side effects, reduce the need for animal models, and predict potential drug resistance (Davies and Davies 2010; Xia 2017). This method may be useful to guide the development of novel therapeutic agents for the present global pandemic. In fact, there is an unprecedent increase in the number of studies involving virtual screening of potential anti-COVID-19 agents ever since the structure of SARS-CoV-2 was characterised and solved in February 2020 (Fischer et al. 2020; Mohamed et al. 2020).

Figure 3. Comparison of traditional and virtual methods of drug development.

In silico modeling includes simulation, molecular docking, target point determination, and chemical stability studies for drug design. More specifically, the computational strategy is guided by the study and characterisation of protein–ligand interaction in a bid to identify potential drug candidates. (Mohammadi and Shaghaghi 2020). The strength of this interaction is elucidated by the calculated binding energy (kcal/mol). In other words, a more negative binding energy is an indicator of a stronger interaction and hence better activity. Notably, the remarkable anti-coronaviral potential of a number of phytocompounds has been highlighted using molecular docking technique (Chen and Du, 2020; Lung et al. 2020; Rane et al. 2020; Sampangi-Ramaiah et al. 2020; ul Qamar et al. 2020; Zhang, Wu et al. 2020). Table 2 summarises molecules with considerable interaction with SARS-CoV-2 non-structural proteins, while Table 3 depicts potential phytochemicals with activity against SARS-CoV-2 structural and host-associated proteins as predicted by computational studies.

Table 2. Compounds with potential against SARS-CoV-2 non-structural proteins evaluated through computational screening.

Compound	Type	Source	Target	Binding energy (kcal/mol)	Binding site(s)	Docking tool	Pharmacological evidence	References
(2S)-Eriodictyol 7-O-(6^″- Ogalloyl)-beta-D-glucopyranoside	Glycoside	Phyllanthus emblica	3CL^pro	−19.47	Thr24, Thr25, Gly143, Met49, Cys145, His41, Thr26, Cys44, Thr45, Glu166, Leu167, Gln189, Thr190, Ala191, Gln192	Software MOE	–	(ul Qamar et al. 2020)
10-Hydroxyusambarensine	Alkaloid	Strychnos usambarensis	3CL^pro	−10.0	Tyr54, Met49, Cys145, His163, Met165, Glu166, Pro168, Gln189	AutoDock vina 4.2	Antimalarial potential	(Gyebi et al. 2020)
22-Hydroxyhopan-3-one	Terpenoid	Cassia siamea	3CL^pro	−8.6	Lys137, Tyr239, Leu275, Leu286, Leu287	AutoDock vina 4.2	Antimicrobial potential	(Gyebi et al.2020)
3,5,7,3′,4′,5′-Hexahydroxy flavanone- 3-O-beta-D-glucopyranoside	Glycoside	Phaseolus vulgaris	3CL^pro	−19.10	Met49, Cys145, His41, Thr24, Thr25, Thr26, Cys44, Ser46, Asn142, His164, Met165, Glu166, Gln189	Software MOE	–	(ul Qamar et al. 2020)
5,7,3′,4′-Tetrahydroxy- 2′-(3,3-dimethylallyl) isoflavone	Phenolic	Psorothamnus arborescens	3CL^pro	−29.57	His41, Cys145, Thr24, Thr25, Thr26, Cys44, Thr45, Ser46, Met49, Asn142, Gly143, His164, Glu166, Gln189	Software MOE	Anti-leishmanial agent	(ul Qamar et al. 2020)
6-Oxoisoiguesterin	Terpenoid	Salacia spp.	3CL^pro	−9.1	His41, Met49, Cys145, Met165, Gln189	AutoDock vina 4.2	Antimicrobial potential	(Gyebi et al. 2020)
Amaranthin	Lectin	Amaranthus tricolour	3CL^pro	−18.14	Thr26, Glu166, Cys145, His41, Thr24, Thr25, Cys44, Cys44, Thr45, Asn142, His163, His164, Met165, Glu166, Leu167, Pro168, Gln189	Software MOE	–	(ul Qamar et al. 2020)
Andrographolide	Terpenoid	Andrographis paniculata	3CL^pro	−3.1	Gly143, Cys145, Glu166	Dock 6	Antiviral, anti-inflammatory, antidiabetic, anticancer activity	(Dai et al. 2018; Enmozhi et al. 2020)
Neoandrographolide	Terpenoid	Andrographis paniculata	3CL^pro, PL^pro, RdRp	−7.4 to −7.1	PLpro: Leu163, Gly164, Asp165, Tyr265, Tyr269, Gln270, Gly273, Tyr274; 3CLpro: Thr26, Ser47, Met50, Phe141, Leu142, Asn143, Gln190; RdRp: Tyr340, Asp337, Arg438, Asp508, Arg509. Ser644	AutoDock vina	Antiviral, anti-inflammatory, anticancer activity	(Murugan et al. 2020)
Anisofolin A	Phenolic	Leucas spp.	3CL^pro	−8.72	Leu4	SwissDock	Anti-mycobacterial, antimalarial activity	(Miyaichi et al. 2006; Mishra et al. 2020)
Apigenin	Phenolic	Allium sativum (garlic), Malus domestica (apple), Thymus vulgaris (thyme), Capsicum annuum (red pepper)	3CL^pro	−7.8	Met49, Ser144, Cys145, Glu166	AutoDock 4.2	Antiviral activity against hepatitis B virus, adenoviruses, African swine fever virus	(Zakaryan et al. 2017; Sampangi- Ramaiah et al. 2020)
Apigenin 7-glucoside	Phenolic	Thymus spp.	3CL^pro	−8.36	Gly71, Asn119	SwissDock	Anti-HIV activity	(Miyaichi et al. 2006; Mishra et al. 2020)
Apiin	Phenolic	Petroselinum crispum	3CL^pro	−8.7	His41, Ser46, Met165, Pro168, Gln189	SwissDock	Anti-inflammatory potential	(Das et al. 2020)
Arjunglucoside-I	Terpenoid	Terminalia chebula	3CL^pro	−7.9	Thr26, Leu27, Thr25, His41, Gly143, Phe140, Leu141, Asn142, Ser144, Cys145, His163, Met165, Glu166	AutoDock 4.2	Antioxidant, anti-inflammatory, antiplasmodial activity	(Umesh et al. 2020)
Baicalin	Phenolic	Scutellaria baicalensis (Chinese Skullcap)	3CL^pro	−8.1	Met49, Ser144, Cys145, Glu166, Pro168	AutoDock vina	Antiallergic, anti-inflammatory activity	(Islam et al. 2020)
Betulinic acid	Saponin	Betula spp.	3CL^pro	−4.23	Asn157, Tyr172, Gln175, Glu204	AutoDock 4	Anti-HIV, anti- inflammatory, anthelmintic, antimalarial property	(Dehelean et al. 2012; Zhang et al. 2016; Saeed et al. 2018; Zhang, Wu et al. 2020)
Calceolarioside B	Phenolic	Fraxinus sieboldiana	3CL^pro	−19.87	His41, Gly143, Cys145, Glu166, Thr24, Thr25, Thr26, Leu27, Ser46, Leu50, Leu141, Asn142, Ser144, His164, Met165, Gln189	Software MOE	Anti-respiratory syncytial viral activity	(Dong et al. 2017; ul Qamar et al. 2020)
Carnosol	Terpenoid	Rosmarinus officinalis (rosemary)	3CL^pro	−8.2	Thr26, Leu27, His41, Cys44, Met49, Cys145, His164, Glu166, Gln189, Met165, Arg188	AutoDock 4.2	Antioxidant, anti- inflammatory, antimicrobial anticancer activity	(Umesh et al. 2020)
Carvacrol	Phenolic	Origanum spp.	3CL^pro	−5.9	Leu27, His41, Met49, Asn142, Gly143, Cys145, Met165	AutoDock vina 4.2	Antiviral (HSV, retrovirus, respiratory syncytial virus), antimicrobial activity	(Marinelli et al. 2019; Kumar, Choudhir et al. 2020)
Cordifolin	Phenolic	Tinospora cordifolia	3CL^pro	−8.77	Leu4, Ser46, Asn142, Gln189	SwissDock	Anti-HIV protease activity	(Mishra et al. 2020)
Coriandrin	Furanocoumarin	Coriandrum sativum (coriander)	3CL^pro	−6.4	His41, Met49, Cys145, Met165, Gln189	AutoDock 4.2	Antiviral, anticancer property	(Sampangi-Ramaiah et al. 2020)
Coumaroyl tyramine	Phenolic	Physalis minima; Allium fistulosum	PL^pro, 3CL^pro	−4.18, −3.22	PLpro: Lys5, Gln127, Lys137, Glu290; 3CLpro: Pro108, Gln110, Thr111, Val202, Asp245, Ile249, Asp295	AutoDock 4	Antioxidant activity, inhibition of acetyl cholinesterase, cell proliferation	(Neelam et al. 2010; Zhang, Wu et al. 2020)
Cryptoquindoline	Alkaloid	Cryptolepis sanguinolenta	3CL^pro	−9.7	Met49, Cys148, Met165	AutoDock vina 4.2	Antimicrobial activity	(Gyebi et al. 2020)
Cryptotanshinone	Terpenoid	Salvia miltiorrhiza	PL^pro,3CL^pro	−5.25, −6.23	PLpro: Asn157, Tyr172; 3CLpro: Gln110, Asn151, Asp153, Asp295	AutoDock 4	Antimicrobial, antiparasitic, anti-inflammatory, anticancer activities	(Zeng, Fan et al. 2018; Zhang, Wu et al. 2020)
Curcumin	Phenolic	Curcuma longa (turmeric)	3CL^pro	−7.0	His41, Met49, Leu141, Gly143, Ser144, Cys145, Met165	AutoDock 4.2	Antioxidant, anti-inflammatory, antimicrobial, antiviral activity against dengue virus, HPV, HIV, Zika, chikungunya viruses	(Zakaryan et al. 2017; Kim et al. 2019; Sampangi-Ramaiah et al. 2020)
			3CL^pro	−8.2	Gly143, Cys145, Pro168, Gln192	SwissDock		(Das et al. 2020)
Desmethoxycurcumin	Phenolic	Curcuma longa (turmeric)	3CL^pro	−7.9	Met49, Cys44, Pro168	SwissDock	Anticancer, anti-inflammatory activity	(Das et al. 2020)
Desmethoxyreserpine	Alkaloid	Rauwolfia canescens	3CL^pro	−3.52	Val104, Arg105, Ile106, Gln110, Asn151, Asp153, Ile249	AutoDock 4	Antihypertensive activity	(Zhang, Wu et al. 2020)
Dihomo-γ-linolenic acid	Fatty acid	Borago officinalis	3CL^pro	−3.88	Gln107, Pro108, Gln110, Thr292, Pro293	AutoDock 4	Attenuation of atherosclerosis, anti-inflammatory property	(Wang et al. 2012; Gallagher et al. 2019; Zhang, Wu et al. 2020)
Epigallocatechin	Phenolic	Camellia sinensis	3CL^pro	−7.3	Thr26, His41, Asn142	SwissDock	Anti-inflammatory, anticancer, antioxidant potential	(Das et al. 2020)
Epigallocatechin gallate	Phenolic	Camellia sinensis	3CL^pro	−7.96	Phe140, Cys145, Glu166, Gln189, Gln192	SwissDock	Antioxidant, anti-inflammatory, anticancer potential	(Das et al. 2020)
Friedelin	Terpenoid	Clerodendrum spp.	3CL^pro, RdRp	−7.9, −7.1	Pro168, Asp623	AutoDock vina	Antimicrobial, anti- obesity, anticancer, anti-inflammatory activity	(Kar et al. 2020)
Glabridin	Phenolic	Glycyrrhiza glabra (liquorice)	3CL^pro	−8.0	Cys145, Pro168	AutoDock 4.2	Antioxidant potential	(Sampangi- Ramaiah et al. 2020)
			3CL^pro	−7.6	His41, Leu27, Cys145	SwissDock		(Das et al. 2020)
			3CL^pro	−8.1	His41, Met49, Met165, Glu166	AutoDock vina		(Islam et al. 2020)
Glucobrassicin	Glucosinolate	Brassica spp. (cabbage, broccoli, mustard)	3CL^pro	−8.1	His41, Leu141, Ser144, Met165, Gln189	AutoDock 4.2	–	(Sampangi- Ramaiah et al. 2020)
Glycycoumarin	Phenolic	Glycyrrhiza glabra (liquorice)	3CL^pro	−7.5	Leu141, Cys145, His163, Met165, Glu166	AutoDock 4.2	Antispasmodic activity	(Kim et al. 2019; Sampangi- Ramaiah et al. 2020)
Glycyrrhizin	Terpenoid	Glycyrrhiza glabra (liquorice)	3CL^pro	−7.8	His41, Met49, Cys145, Met165, Thr190	SwissDock	Antioxidant, anti-inflammatory property	(Das et al. 2020)
			3CL^pro	−7.2	Phe134, Pro184, Pro241	AutoDock 4.2		(Sampangi- Ramaiah et al. 2020)
Hederagenin	Terpenoid	Nigella sativa (black cumin), Luffa cylindrica (sponge gourd), Momordica dioica (spiny gourd)	3CL^pro	−7.6	Met49, Leu141, Ser144, Cys145	AutoDock 4.2	Antitumour potential	(Sampangi- Ramaiah et al. 2020)
Hesperidin	Phenolic	Citrus spp., Valeriana jatamansi	3CL^pro	−9.0	His4, Thr24, Thr25, Thr45, Ser46, Met49, Cys145	SwissDock	Anti-inflammatory, antioxidant, antitumour, antiviral activity	(Das et al. 2020)
Kaempferol	Phenolic	Spinach, kale	3CL^pro	−6.01	Arg105, Ile106, Gln110, Thr111, Asp295	AutoDock 4	Antimicrobial, antitumour, anti-inflammatory, antioxidant, antidiabetic, neuroprotective properties	(Imran et al. 2019; Zhang, Wu et al. 2020)
Leucoefdin	Phenolic	Musa spp. (banana)	3CL^pro	−9.29	Met49, Tyr54, Pro52, Phe140, Leu141, Cys145, His163, His164, Met165, Glu166	Glide	Antimicrobial, hypoglycaemic, antitumour property	(Singh and Mishra 2020)
Licoleafol	Phenolic	Glycyrrhiza uralensis	3CL^pro	−19.64	Cys145, His41, Thr24, Thr25, Thr26, Cys44, Thr45, Met49, Gly143, His163, His163, Met165, Glu166, Gln189	Software MOE	–	(ul Qamar et al. 2020)
Lignan	Phenolic	Grains, nuts, seeds, vegetables	3CL^pro	−4.27	Gln110, Asn151, Thr111, Thr292, Pro293, Asp295, Arg298	AutoDock 4	Antibacterial, antifungal activities	(Thirupathaiah et al. 2015; Zhang, Wu et al. 2020)
Liquiritigenin	Phenolic	Glycyrrhiza glabra (liquorice), Glycyrrhiza uralensis (Chinese liquorice)	3CL^pro	−7.7	Met49, Leu141, Gly143, Ser144, Cys145, His163, Met165, Asp187, Arg188	AutoDock 4.2	–	(Sampangi- Ramaiah et al. 2020)
Luteolin	Phenolic	Lonicera spp. (honeysuckle)	3CL^pro, PL^pro	−7.8 to −5.37	Gln189, Leu4, Asn142, Thr26, Asn180, Arg105, Phe185, Gln965, Ser968, Asn969, Tyr273, Leu270, Val-330	AutoDock vina	Antiviral activity against encephalitis virus, iridovirus	(Liu et al. 2020; Yu et al. 2020)
			3CL^pro	−8.35	Phe219	SwissDock		(Mishra et al. 2020)
Luteolin–4-glucoside		Derivative of luteolin	3CL^pro	−7.87	Glu240	SwissDock	Antioxidant activity	(Mishra et al. 2020)
Methyl rosmarinate	Phenylpropanoid	Hyptis atrorubens Poit	3CL^pro	−20.62	Cys145, His41, Thr24, Thr25, Thr26, Cys44, Thr45, Met49, Leu141, Asn142, Gly143, Ser144, His163, His164, Glu166, Gln189	Software MOE	Antidiabetic activity	(ul Qamar et al. 2020)
Moupinamide	Alkaloid	Piper nigru	PL^pro	−3.05	Asp77, Arg83, Ala154, Asm157, Gln175, Gln204	AutoDock 4	Antioxidant property	(Zhang, Wu et al. 2020)
Myricetin 3-O-beta-D-glucopyranoside	Glycoside	Camellia sinensis (green tea)	3CL^pro	−18.42	Asn142, Glu166, Cys145, His41, Thr24, Thr25, Thr26, Thr45, Ser46, Met49, Leu141, Gly143, Ser144, His163, His164, Met165, Gln189	Software MOE	Anti-inflammatory activity	(ul Qamar et al. 2020)
Myricitrin	Phenolic	Myrica cerifera	3CL^pro	−22.13	Gly143, Cys145, His41, Thr24, Thr25, Thr26, Leu27, Cys44, Ser46, Met49, Leu141, Asn142, Ser144, His163, Glu166, Gln189	Software MOE	Antiallergic, anti-inflammatory, antioxidant, antifibrotic activity	(ul Qamar et al. 2020)
			3CL^pro	−7.9	Thr24, Thr25, Thr26, Asn42, Asn119	SwissDock		(Das et al. 2020)
N-cis-feruloyl tyramine	Phenolic	Capsicum annuum	PL^pro3CL^pro,	−3.11, −4.31	PLpro: Asn157, Tyr172; 3CLpro: Val104, Arg105, Ile106, Gln110, Asn151, Thr292, Asp295	AutoDock 4	Anti-inflammatory potential	(Wishart et al. 2018; Zhang, Wu et al. 2020)
Oleanolic acid	Terpenoid	Thymus vulgaris (thyme), Rosmarinus officinalis (rosemary), Olea europaea (Clove)	3CL^pro	−6.0	Cys145, His163, Gln189	AutoDock vina 4.2	Antioxidant, anticancer, antidiabetic, antimicrobial, antihypertensive, anti-inflammatory property	(Jesus et al. 2015; Kumar, Choudhir et al. 2020)
			3CL^pro	−7.8	His41, Met49, Ser144, Cys145	AutoDock 4.2		(Ayeleso et al. 2017; Sampangi- Ramaiah et al. 2020)
Puerarin	Phenolic	Radix puerariae	3CL^pro	−8.4	His41, Cys44, Gly143, Glu166	SwissDock	Antioxidant, anti-inflammatory, hypoglycaemic, neuroprotective property	(Das et al. 2020)
Quercetin	Phenolic	Raphanus spp., Malus domestica, (apple) Foeniculum vulgare, Allium cepa (onion), Camellia sinensis (green tea)	PL^pro3CL^pro	−4.62, −6.25	PLpro: Tyr155, Ser171, Tyr172, Gln175, His176, Glu204; 3CLpro: Gln110, Thr111, Asn151, Ile249, Thr292, Asp295, Arg298	AutoDock 4	Anti-inflammatory, antithrombotic, antidiabetic, hepatoprotective, antioxidant, antiviral activity	(Zakaryan et al. 2017; Zhang, Wu et al. 2020)
			3CL^pro	−8.04	Arg105	SwissDock		(Mishra et al. 2020)
			3CL^pro	−7.3	Leu141, Gly143, Ser144, Cys145, His163, Met165, Arg188	AutoDock 4.2		(Zakaryan et al. 2017; Sampangi- Ramaiah et al. 2020)
Quercitrin	Phenolic	Allium cepa (onion)	3CL^pro	−8.3	Thr25, Thr45, Gly143, Glu166	SwissDock	Antioxidant, anti-inflammatory	(Das et al. 2020)
Rhoifolin	Phenolic	Rhus succedanea	3CL^pro	−8.4	His41, Cys44, Asn119, His145, Glu166	SwissDock	Antiretroviral potential	(Das et al. 2020)
Rosmanol	Terpenoid	Rosmarinus officinalis (rosemary)	3CL^pro	−8.0	Thr25, His41, Met49, Phe140, Leu141, Asn142, His143, Ser144, Glu166, Met165	AutoDock 4.2	Apoptosis induction	(Umesh et al. 2020)
Rosmarinic acid	Phenolic	Thymus vulgaris (thyme), Ocimum basilicum (basil), Rosmarinus officinalis (rosemary), Salvia officinalis (sage)	3CL^pro	−7.1	Lys102, Val104, Thr111, Asn151, Ser158, Phe294	AutoDock 4.2	Anti-inflammatory, antioxidant potential	(Sampangi- Ramaiah et al. 2020)
Rutin (quercetin-3-O- rutinoside)	Phenolic	Houttuynia cordata, Forsythia suspensa, Morus alba, Prunella vulgaris	3CL^pro	−9.6	His41, Leu141, Asn142, Cys145, Glu166, Thr190, Gln192	SwissDock	Antioxidant, antimicrobial, anti- inflammatory, antitumour activity	(Das et al. 2020)
			3CL^pro	−8.67	Thr26, Phe140, Leu141, Asn142, Ser144, Cys145, His163, Glu166, Gln189			(Hu et al. 2020)
Safficinolide	Terpenoid	Salvia officinalis (sage)	3CL^pro	−6.8	Val104, Gln110, Ser158, Phe294	AutoDock 4.2	Antiviral activity	(Sampangi- Ramaiah et al. 2020)
Sageone	Terpenoid	Salvia officinalis (sage)	3CL^pro	−7.1	His41, Cys145, His164, Met165	AutoDock 4.2	Antiviral property	(Sampangi- Ramaiah et al. 2020)
Scutellarin	Phenolic	Scutellaria baicalensis (Chinese Skullcap)	3CL^pro	−8.3	Thr26, Gly143, Cys145	SwissDock	Antioxidant, anti-inflammatory, vascular relaxation, anti-platelet, anticoagulation	(Das et al. 2020)
Stigmasterol	Terpenoid	Clerodendrum spp.	3CL^pro, RdRp	−7.7, −7.0	Pro168, Lys545, Arg555, Thr687, Ala688	AutoDock vina	Antioxidant potential	(Kar et al. 2020)
Sugiol	Terpenoid	Calocedrus formosana	3CL^pro	−6.04	Gln110, His246, Thr292, Pro293, Phe294, Asp295	AutoDock 4	Antioxidant, antiviral agent, anti-inflammatory antineoplastic property	(Kim et al. 2019; Zhang, Wu et al. 2020)
Tanshinone IIa	Terpenoid	Salvia miltiorrhiza	PL^pro3CL^pro	−5.02, −5.17	PLpro: Asp77, Arg83, Tyr155, An157, Tyr172, His176; 3CLpro: Gln110, Asn151, Asp153, Thr292 Pro293, Phe294	AutoDock 4	Cardioprotective; antioxidant, anti-inflammatory, antithromboticpotential	(Shang et al. 2012; Zhang, Wu et al. 2020)
Taraxerol	Terpenoid	Clerodendrum spp.	3CL^pro, RdRp	−8.4, −7.4	Glu166, Pro168, Ala191, Lys545, Arg555	AutoDock vina	Antimicrobial, anti-inflammatory, anti-obesity, anticancer property	(Kar et al. 2020)
Theaflavin	Phenolic	Camellia sinensis (green tea)	RdRp	−9.11	Asp452, Arg553, Arg624	idock & Blind docking	Broad-spectrum antiviral activity against influenza viruses, hepatitis C virus.	(Yang et al. 2014; Chowdhury et al. 2018; Lung et al. 2020)
Ursolic acid	Terpenoid	Malus domestica (apple), Thymus vulgaris (thyme), Rosmarinus officinalis (rosemary), Mentha piperita (peppermint)	3CL^pro^3	−4.0	Thr24, Thr25, Thr26, Cys44, Thr45, Ser46, Asn142, Gly143, Cys145, Glu166	AutoDock vina 4.2	Antiviral, antioxidant, antimicrobial, anticancer potential	(Jesus et al. 2015; Kumar, Choudhir et al. 2020)
			3CL^pro	−7.6	Val104, Thr111, Phe294	AutoDock 4.2		(Kim et al. 2019; Sampangi- Ramaiah et al. 2020)
Withanone	Lactone	Withania somnifera	3CL^pro	−4.42	Gln19, Thr24, Thr25, Thr26, His41, Cys44, Thr45, Ser46, Glu47, Met49, Pro52, Tyr54, Asn119, Asn142, Gly143, Ser144, Cys145, His164	Glide	Neuroprotective potential	(Kumar, Dhanjal et al. 2020)

Key: Catalytic residues of 3CL^pro are highlighted in bold; MOE: Molecular Operating Environment.

Table 3. Virtually screened phytocompounds with activity against SARS-CoV-2 structural proteins and host-associated proteins.

Compound	Type	Source	Target	Binding energy (kcal/mol) and docking tool	Binding site(s)	Pharmacological evidence	References
1,3,8-Trihydroxy-6-methylanthraquinone (emodin)	Phenolic	Rheum spp.	ACE2	−6.19 (ClusPro)	Ala71, Asp67, Lys74	Anti-inflammatory, anti-proliferative, anticarcinogenic property	(Basu et al. 2020)
24-Methylenecycloartan- 3-one	Terpenoid	Azadirachta indica	M protein	−10 (AutoDock vina)	Ile97, Phe100, Arg101, Ala104, Ser111, Phe112, Thr127, Ile128, Thr130, Arg158	Anticancer potential	(Borkotoky and Banerjee 2020)
24-Methylenecycloartanol	Terpenoid	Azadirachta indica	E protein	−10.8 (AutoDock vina)	Leu18, Leu19, Ala22, Phe23, Val25, Phe26, Val29	Antioxidant, anticancer property	(Kim et al. 2019; Borkotoky and Banerjee 2020)
7-Deacetyl-7- benzoylgedunin	Terpenoid	Azadirachta indica	E protein	−10.9 (AutoDock vina)	Ala15, Leu18, Leu19, Ala22, Val25, Phe26	Anticancer potential	(Kim et al. 2019; Borkotoky and Banerjee 2020)
Anthraquinone derivative	Phenolic	Oroxylum spp.	ACE2	−6.15 (ClusPro)	Ser105, Asn103, Gln102, Leu100, Phe28	Immunomodulatory, antiparasitic, antimicrobial, anticancer	(Basu et al. 2020)
Apigenin	Phenolic	Ocimum basilicum	Sgp	−7.7 (AutoDock vina)	Ser730, His1058, Ala1056, Ile870, Pro863, Leu861	Antiviral activity against hepatitis B virus, adenoviruses, African swine fever virus	(Zakaryan et al. 2017; Rane et al. 2020)
Baicalin	Phenolic	Scutellaria baicalensis (Chinese Skullcap)	ACE2	−8.46 (AutoDock vina)	Asn149, Arg273, His505	Antiallergic, anti-inflammatory activity	(Chen and Du 2020)
Beta-amyrin	Terpenoid	Azadirachta indica	M protein	−9.4 (AutoDock vina)	Ile97, Phe100, Arg101, Ala104, Ser111, Phe112, Ile128, Thr130	Antihyperglycemic, hypolipidemic potential	(Santos et al. 2012; Borkotoky and Banerjee 2020)
Chrysin	Phenolic	Oroxylum spp.	ACE2	−6.87 (ClusPro)	Thr129, Ile126, Thr125	Antioxidant, antispasmodic, anti-inflammatory	(Basu et al. 2020)
Curcumin	Phenolic	Curcuma longa (turmeric)	Sgp	−7.1 (AutoDock vina)	Thr430, Leu517, Ala520, Gln564, Phe565, Asn544, Leu546, Leu390, Val382	Antioxidant, anti- inflammatory, antimicrobial, antiviral activity against dengue virus, HPV, HIV, Zika, chikungunya virus	(Zakaryan et al. 2017; Rane et al. 2020)
Cycloeucalenone	Terpenoid	Azadirachta indica	E protein	−10.5 (AutoDock vina)	Ala15, Leu18, Leu19, Ala22, Phe23, Phe26, Val29	Cardioprotective, anticancer activity	(Kim et al. 2019; Borkotoky and Banerjee 2020)
Dihydrotanshinone	Terpenoid	Salvia miltiorrhiza	Sgp	−5.16 (AutoDock 4)	Gln954, Ala958, Thr961	Atherosclerosis attenuation	(Chaung et al. 2011; Zhang, Wu et al. 2020)
Dithymoquinone	Phenolic	Nigella sativa	S:ACE interface	−8.6 (AutoDock vina)	Phe28, Lys31, Glu35, Gln76, Leu79	Anticancer, antioxidant, anti-inflammatory property	(Ahmad et al. 2020)
Fisetin	Phenolic	Acacia spp.	Sgp	−8.5 (AutoDock vina)	Ile870, Pro880, Ser730, His1058, Thr732, Thr778	Antiviral activity against chikungunya Virus, HIV	Zakaryan et al. 2017; Rane et al. 2020
Friedelin	Terpenoid	Clerodendrum spp.	Sgp	−7.3 (AutoDock vina)	Tyr449, Asn450, Lys444	Antimicrobial, anti-obesity, anticancer, anti-inflammatory activity	(Kar et al. 2020)
Genistein	Phenolic	Genista tinctoria	Sgp	−8.2 (AutoDock vina)	Gly1059. Ser730, His1058, Thr732, Lys733, Pro863, Asn867, Thr870, Phe782, Ala1056	Angiogenesis inhibitor, phytoestrogenic, anticancer activity	(Zakaryan et al. 2017; Rane et al. 2020)
Glyasperin A	Phenolic	Glycyrrhiza glabra	Sgp	−7.9 (AutoDock 4)	Glu298, Phe318, Val320, Gln321, Thr323, Val595, Val620, Ile624	Antioxidant potential	(Sinha et al. 2020)
Glycyrrhizin	Terpenoid	Glycyrrhiza glabra¸ Glycyrrhiza radix	Sgp	−9.2 (AutoDock 4)	His625, Glu298, Arg319, Gln321, Val320, Gln321, Val320, Tyr343, The323, Ile624	Anti-inflammatory, anticancer, immunomodulating, neuroprotection, hepatoprotective property	(Kim et al. 2019; Sinha et al. 2020)
			ACE2	−9 (AutoDock vina)	Arg559, Gln388, Arg393, Asp30		(Chen and Du 2020)
Hesperidin	Phenolic	Citrus spp., Valeriana jatamansi	ACE2	−8.3 (AutoDock vina)	Tyr613, Ser611, Arg482, Glu479	Anti-inflammatory, antioxidant, antitumour, antiviral activity	(Chen and Du 2020)
			ACE2	−8.99 (ClusPro)	Asn63, Ala71, Lys74, Ser44		(Basu et al. 2020)
Isoliquiritin apioside	Phenolic	Glycyrrhiza glabra	Sgp	−7.4 (AutoDock 4)	Tyr612, Glu298, Phe318, Val320, Gln321, Val620, Ile624, His625	Anticancer potential	(Sinha et al. 2020)
Isorhamnetin	Phenolic	Tagetes lucida, pears	Sgp	−8.3 (AutoDock vina)	His1058, Val729, Ser730, Met731, Thr732, Lys733, Val860, Leu861, Pro863, Thr778, Ile870, Phe782, Ala1056	Antioxidant activity, anticancer, antimicrobial, anti- inflammatory	(Zakaryan et al. 2017; Rane et al. 2020)
Isothymol	Phenolic	Ammoides verticillata	ACE2	−5.8 (AutoDock vina 4.2)	Glu406, Lys441, Gln442, Thr445	Antimicrobial property	(Abdelli et al. 2020; Gyebi et al. 2020)
Kaempferol	Phenolic	Ficus benjamina, Rubus idaeus, Phaseolus vulgaris	Sgp	−7.4 (AutoDock vina)	Thr393, Ala522, Leu527, Cys391, Leu390, Asn544, Phe782, Ala1056	Antiviral activity against HCMV, HSV-1, HSV-2, influenza A virus	(Zakaryan et al. 2017; Rane et al. 2020)
Liquiritin	Phenolic	Glycyrrhiza glabra	Sgp	−7.7 (AutoDock 4)	His625, Val620, Gln321, Cys291, Gln321, Ile624, Val320	Antioxidant, antimicrobial, anti-inflammatory activity	(Sinha et al. 2020)
Luteolin	Phenolic	Ocimum basilicum, Petroselinum crispum, Capsicum annuum	Sgp	−8.2 (AutoDock vina)	Ala1056, Gly1059, His1058, Asp867, Ile870, Thr871, Pro863, Leu861, Lys733, Met731	Antiviral activity against HIV, Chikungunya virus, Japanese encephalitis virus, SARS-CoV, Rotavirus, Epstein–Barr virus	(Zakaryan et al. 2017; Rane et al. 2020)
Neoandrographolide	Terpenoid	Andrographis paniculata	S:ACE2 complex	−7.8 (Autodock vina)	Phe23, Ser27, Ser30, Asn34, Thr330, Ala331, Leu342	Antiviral, anti-inflammatory	(Murugan et al. 2020)
Nicotianamine	Metal chelator	Glycine max	ACE2	−5.1 (AutoDock vina)	Arg518, Glu406, Ser409, Gln522, Gln442	Metal translocation	(Chen and Du 2020)
Nimbolin A	Styrene	Azadirachta indica	E & M proteins	−11.2, −9.4 (AutoDock vina)	Asn15, Leu18, Leu19, Ala22, Phe23, Val25, Phe26, Trp31, Ile32, Leu35, Gln36, Phe37, Ala38, Asn41, Arg42, Phe45, Ala81, Ile82, Ala85, Asn203, Tyr204, Leu206, Thr208, Asp209	–	(Borkotoky and Banerjee 2020)
Nimocin	–	Azadirachta indica	E & M proteins	−11, −10.2 (AutoDock vina)	Leu18, Leu19, Ala22, Phe23, Val25, Phe26, Gln36, Ala38, Asn41, Arg42, Phe45, Gly78, Ala81, Ile201, Gly202, Asn203, Tyr204, Asp209	–	(Borkotoky and Banerjee 2020)
Phytosterol	Phenolic	Azadirachta indica	M protein	−9.5 (AutoDock vina)	Ile97, Phe100, Arg101, Ala104, Ser111, Thr127, Ile128, Thr130, Arg158	Hypocholesterolemic, antioxidant activity	(Yoshida and Niki 2003; Borkotoky and Banerjee 2020)
Piceatannol	Phenolic	Rheum palmatum, Euphorbia lagascae	S:ACE complex	−8.2 (AutoDock vina)	Asn33, His34, Glu37, Pro389, Lys403, Ser494, Tyr495, Gly496, Tyr505	Antioxidant, anticancer	(Wahebi et al. 2020)
Pterostilbene	Phenolic	Vaccinium spp., Pterocarpus marsupium	Sgp	−6.7 (AutoDock vina)	Thr393, Ala522, Ala520, His519, Asn544, Gln564, Leu390, Gly545, Phe543, Leu546, Phe565	Antiviral activity against HSV, HPV, influenza virus	(Zakaryan et al. 2017; Rane et al. 2020)
			S:ACE complex	−7.0 (AutoDock vina)	Asn33, His34, Glu37, Phe390, Ser496, Tyr505		(Wahebi et al. 2020)
Quercetin	Phenolic	Raphanus spp., Foeniculum vulgare, Capsicum annuum	Sgp	−8.5 (AutoDock vina)	Ser730, Met730, Met731, Lys733, Val 860, Leu861, Pro863, Ile870, Asp867, Ala1056, Pro1057, Gly1059, His1058	Anti-inflammatory, antithrombotic, antidiabetic, hepatoprotective, antioxidant, antiviral activity	(Zakaryan et al. 2017; Rane et al. 2020)
Resveratrol	Phenolic	Vaccinium spp.	Sgp	−7.9 (AutoDock vina)	Thr732, His1058, Lys733, Leu861, Pro863, Asp867, Ile870, Phe782	Antioxidant, anticancer, Antiviral activity against HIV-1, pseudorabies, MERS-CoV	(Zakaryan et al. 2017; Rane et al. 2020)
			S:ACE complex	−8.0 (AutoDock vina)	Asn33, His34, Glu37, Lys403, Lys353, Pro389, Tyr495, Gly496		(Wahebi et al. 2020)
Scutellarin	Phenolic	Scutellaria baicalensis (Chinese Skullcap)	ACE2	−14.9 (AutoDock vina)	Glu495, Arg482	Antioxidant, anti-inflammatory, vascular relaxation, anti-platelet, anticoagulation	(Chen and Du 2020)
Stigmasterol	Terpenoid	Clerodendrum spp.	Sgp	−7.2 (AutoDock vina)	Tyr449, Phe490	Antioxidant potential	(Kar et al. 2020)
Taraxerol	Terpenoid	Clerodendrum spp.	Sgp	−7.5 (AutoDock vina)	Leu455, Glu484, Tyr489	Antimicrobial, anti-inflammatory, anti-obesity, anticancer property	(Kar et al. 2020)

Note: Sgp: Spike glycoprotein, ACE2: Angiotensin-converting enzyme 2, M: membrane protein, E: envelop protein.

Inhibitors of 3CL^pro

Since the main protease of SARS-CoV-2, 3CL^pro (also referred to as M^pro) is essential for proteolytic processing of polypeptides, viral replication, and immune system modulation. Its inhibition will result in significant inhibition of viral replication and pathogenesis, and subsequent attenuation of infection. Consequently, studies have resorted to structural analyses to identify potential inhibitors of this target in a bid to guide the development of potential drug candidates for COVID-19. Notably, the main protease of SARS-CoV-2 has up to 96% sequence identity with that of SARS-CoV. Moreover, the catalytic dyad residues (His41 and Cys145) of SARS-CoV-2 3CL^pro are identical to that of SARS-CoV 3CL^pro (Yang et al. 2003). This has allowed many studies to swiftly dock molecules with reported anti-SARS-CoV 3CL^pro potential.

Through a series of molecular docking, simulation, and toxicity assessment protocols, huge high-throughput computational screenings of 606 million compounds have highlighted the anti-SARS-CoV -2 3CL^pro activity of 16 compounds including two phytocompounds, (-)-taxifolin and rhamnetin, with docking scores of −16.0 and −16.8 kcal/mol, respectively (Fischer et al. 2020). Notably, the latter is commercially available and can be procured as supplements at pharmacies. Additionally, of the 27 phytoligands docked against 3CL^pro in a study by Sampangi-Ramaiah and co-workers (2020), 12 (i.e. apigenin, glabridin, glycoumarin, glycyrrhizin, hederagenin, liquiritigenin, oleanolic acid, quercetin, rosmarinic acid, sageone, ursolic acid, and glucobrassicin) had considerable docking scores within the cut-off value (<−7 kcal/mol) implemented in the study. In addition, none of the tested compounds had a greater binding affinity than the reference antiviral drug (saquinavir) used in the study. However, most of these molecules interacted considerably with one of the catalytic residues (Cys145) of 3CL^pro. Interestingly, many of these compounds are either phenolic or terpenoid (Sampangi-Ramaiah et al. 2020). Similarly, three terpenoids (taraxerol, friedelin, and stigmasterol) demonstrated better efficacy against SARS-CoV-2 3CL^pro than ritonavir (positive control) (Kar et al. 2020).

Another large screening of 32,297 phytochemical TCM compounds revealed the anti-SARS-CoV-2 3CL^pro activity of nine natural compounds including myricitrin, methyl rosmarinate, licoleafol, and amaranthin with very low binding energy (−29.57 to −18.14 kcal/mol). Interestingly, these phytocompounds exhibited better and stronger interaction with the conserved catalytic dyad residues (His41 and Cys145) of SARS-CoV-2 3CL^pro than the conventional drugs (nelfinavir, colistin, and prulifloxacin) used in the study (ul Qamar et al. 2020). Considerable interaction of other compounds such as ursolic acid, oleanolic acid, andrographolide, neoandrographolide, glycyrrhizin, and 6-oxoisoiguesterin and a number of phenolic compounds, including apiin, baicalin, carvacrol, desmethoxycurcumin, epigallocatechin, glabridin, hesperidin, leucoefdin, puerarin, rhoifolin, myricetin, curcumin, calceolarioside B, and scutellarin with either of the catalytic residues of 3CL^pro have also been predicted (Das et al. 2020; Enmozhi et al. 2020; Gyebi et al. 2020; Islam et al. 2020; Kumar, Choudhir et al. 2020; Murugan et al. 2020; Singh and Mishra 2020). The significance of such prediction is that these molecules will remain in interaction with the SARS-CoV-2 3CL^pro since the conserved sites are hardly susceptible to mutation(s). As such, these compounds may offer a more useful guide in the development of COVID-19 drug candidates with profound efficacy. Additionally, mutations of SARS-CoV-2 3CL^pro are often fatal to the virus (Silvestrini et al. 2021), a property that makes it a very attractive druggable target.

Furthermore, in another study involving docking of 110 plant-derived compounds against SARS-CoV-2 3CL^pro, only 11 complied with the cut-off value (−6 kcal/mol) and most of these phytoligands were phenolic compounds (Hu et al. 2020). Phenolic compounds were predicted to interact better with this protease partly due to their abundant hydroxyl groups with higher affinities for the heteroatoms of the protease amino acids (Hu et al. 2020). In addition, rutin (vitamin P), a widely known antioxidant and an active constituent of many TCM (Houttuynia cordata, Forsythia suspensa, Morus alba, and Prunella vulgaris), had the highest binding energy and displayed considerable interaction with one of the conserved catalytic residues (Cys145) of 3CL^pro. Furthermore, this molecule also interacts with and regulates host toll-like receptors (TLR2, TLR6, and TLR7). Its interaction and regulation of TLRs, in addition to its interaction with SARS-CoV-2 3CL^pro, present this compound as a potential inhibitor of SARS-CoV-2 that deserves further exploration, since TLRs are involved in SARS-CoV-2-induced cytokine storm and inflammation, and blockage of its signals has been indicated to prevent this storm (Conti et al. 2020; Ling et al. 2020). Since the study by Hu and workers (2020) was strictly computer-based, further in vitro and in vivo studies are required to validate the reported promising anti-COVID-19 potential of this phytocompound. In addition, the anti-inflammatory, antioxidant, anti-allergy, and antitumour properties of rutin have been reported by earlier studies, and these previous pharmacological evidences may further strengthen speculations of its anti-SARS-CoV-2 and immunomodulatory potential (Koval’skii et al. 2014).

Although a number of studies have predicted the binding efficiency of small molecules with both the monomeric and dimeric forms of 3CL^pro, it is important to reiterate that, while the monomeric state of this protease also contains the relevant His41-Cys145 catalytic dyads, which makes it appropriate for in silico drug–target interaction studies, it is enzymatically inactive. As such, its dimeric form, the active form, would be more suitable for in vitro drug–target interaction studies (Silvestrini et al. 2021; Tekpinar and Yildirim, 2021).

Furthermore, the anti-coronaviral potential of 13 natural compounds from traditional Chinese herbs was demonstrated. A total of 115 compounds were identified through a literature search of natural compounds with anti-SARS-CoV and anti-MERS-CoV activities. These compounds were subjected to absorption, distribution, metabolism, and excretion (ADME) screening, with 13 compounds showing promise. This was followed by the molecular examination of the interaction of these 13 compounds with SARS-CoV-2 proteases. Of these compounds, six compounds, namely coumaroyl tyramine, cryptotanshinone, kaempferol, N-cis-feruloyltyramine, quercetin, and tanshinone IIa, interacted efficiently with both 3CL^pro and PL^pro. Further, moupinamide interacted exclusively with PL^pro, while betulinic acid, desmethoxyreserpine, dihomo-γ-linolenic acid, lignan, and sugiol interacted with 3CL^pro only (Zhang, Wu et al. 2020). Overall, while 11 (84.6%) of the phytoligands interacted with 3CL^pro, only seven (53.8%) interacted with PL^pro, and this might suggest 3CL^pro as a more attractive druggable target. Moreover, four other studies have highlighted the molecular interaction of quercetin, a phenolic antioxidant found in many fruits, grains, and vegetables with 3CL^pro (Figure 4a) (Das et al. 2020; Hu et al. 2020; Mani et al. 2020; Mishra et al. 2020). It is noteworthy that this compound also demonstrates proven anti-inflammatory, antioxidant, antithrombotic, antidiabetic, antiviral, and hepatoprotective properties (Zakaryan et al. 2017). These available pharmacological evidences present this molecule as an attractive potential anti-SARS-CoV-2 agent that requires further exploration.

Figure 4. Interaction of representatives of phytoligands, quercetin, and tryptanthrin with SARS-CoV-2 3CL^pro and PL^pro, respectively (Mani et al. 2020) (Reuse permission was obtained from Elsevier, Licence number: 5034780990710).

Inhibitors of PL^pro

Similar to 3CL^pro, PL^pro plays important roles in the cleaving of polypeptides and viral replication. Additionally, it plays a role in the induction of viral infection. Inhibition of this essential protease is expected to result in the obstruction of viral replication and pathogenesis. Relative to 3CL^pro, few studies have described the molecular interaction of PL^pro with potential plant-derived inhibitors. The anti-SARS-CoV-2 PL^pro potential of an alkaloid, tryptanthrin, found in Strobilanthes cusia, has been demonstrated (Figure 4) (Mani et al. 2020). This compound has also been found to exhibit anti-HCoV-NL63, antimicrobial, antiparasitic, antioxidant, anticancer, antiallergic, and anti-inflammatory activities (Kaur et al. 2017; Tsai et al. 2020). A similar study has highlighted luteolin (a phenolic) as a potential inhibitor of SARS-CoV-2 PL^pro (Yu et al. 2020).

Furthermore, considerable anti-SARS-CoV-2 PL^pro potential of two terpenoids, platycodin D found in Platycodon grandifloras and sugetriol-3, 9-diacetate found in Cyperus rotundus; an alkaloid, phaitanthrin D found in Isatis indigotica; and seven other phenolic compounds have been reported (Wu et al. 2020).

Inhibitors of rdrp

Since RdRp is important for viral replication, inhibitors of this target are expected to prevent viral RNA synthesis and proliferation. Few reports are available on the molecular interaction of phytocompounds with SARS-CoV-2 RdRp. A study by Lung and co-workers (2020) involving 83 TCMs identified theaflavin, a phenolic compound found in black tea, as a potent inhibitor of SARS-CoV, SARS-CoV-2, and MERS-CoV, with the best binding affinity observed with SARS-CoV-2 RdRp. Prior pharmacological evidence has revealed the broad-spectrum antiviral activity of this molecule against hepatitis and influenza viruses (Yang et al. 2014; Chowdhury et al. 2018). The stronger binding affinity of three terpenoid compounds, taraxerol, friedelin, and stigmasterol with SARS-CoV-2 RdRp, compared to known inhibitors (remdesivir and favipiravir) of viral RdRp, has been observed (Kar et al. 2020; Sanders et al. 2020). Another interesting study highlighted the stronger interaction of luteolin, a phenolic compound found in honeysuckle (a main antiviral component of lianhuaqingwen) with SARS-CoV-2 RdRp, compared to remdesivir and ribavirin (Yu et al. 2020).

Inhibitors of structural proteins

As earlier stated, the structural proteins (N, S, E, M) of coronavirus are essential for its success as a virus and pathogen, as they play vital roles in viral entry, pathogenesis, assembly, budding, and release (Schoeman and Fielding 2019; Asghari et al. 2020; Naqvi et al. 2020). As such, these proteins have also attracted attention as potential druggable targets, although less compared to that received by the nsps. Of these structural proteins, overwhelming attention has been afforded to spike glycoprotein (Sgp), and this could be due to its critical role in viral attachment and subsequent entry into the host (Beniac et al. 2006; Chen et al. 2020). Inhibitors of Sgp will obstruct its attachment and interaction with the host receptor, and ultimately result in inhibition of SARS-CoV-2 entry.

A computational study by Rane and co-workers (2020) highlighted the anti-SARS-CoV Sgp potential of 10 phenolic compounds including curcumin, a widely known constituent of turmeric, and kaempferol, an antioxidant found in many vegetables and fruits. Notably, these compounds exhibited better affinity and stability with the Sgp than hydroxychloroquine (HCQ), suggesting that they may offer a more useful guide in the development of COVID-19 drug candidates. A similar study has demonstrated the Sgp-inhibiting potential of another phenolic compound, luteolin, found in honeysuckle (Yu et al. 2020). Interestingly, an earlier in vitro study has reported the potential activity of this compound against SARS-CoV Sgp (Yi et al. 2004). Moreover, the interaction of dihydrotanshinone, a diterpene found in Salvia miltiorrhiza with SARS-CoV Sgp, has also been reported (Zhang, Wu et al. 2020).

Additionally, a similar study by Kar and co-workers (2020) involving about 29 phytomolecules found in Clerodendrum spp. leaf has demonstrated the inhibitory potential of three terpenoids, taraxerol, friedelin, and stigmasterol against SARS-CoV-2 Sgp with docking scores < −7.0 kcal/mol. Notably, their binding affinities with Sgp were better relative to that of the tested antiviral drug, arbidol. More interestingly, taraxerol interacted with spike’s residues (Leu455, Glu484, and Tyr489) speculatively critical for binding host receptor (ACE2) (Ortega et al. 2020). In addition, 20 bioactive constituents of Glycyrrhiza glabra with prior pharmacological evidence which include antiviral, antimicrobial, anticancer, and immunomodulatory properties were docked against Sgp. Of these compounds, one terpenoid; glycyrrhizin, and three phenolics; liquiritin, glyasperin A, and isoliquiritinapioside were top-ranked inhibitors with binding energies of −9.2, −7.7, −7.9, and −7.4 kcal/mol, respectively (Sinha et al. 2020).

Furthermore, with the use of molecular docking and simulation, another study involving 70 compounds from Indian medicinal plant origin (Azadirachta indica) has identified 7-deacetyl-7- benzoylgedunin, 24-methylenecycloartanol, and cycloeucalenone as inhibitors of SARS-CoV 2 E protein. The same study identified beta-amyrin, phytosterol, and 24-methylenecycloartan-3-one as inhibitors of SARS-CoV 2 M protein. Moreover, nimbolin A and nimocin had remarkable interaction with both proteins (Borkotoky and Banerjee 2020). Notably, most of these compounds are members of the terpenoid class of secondary metabolites. High-affinity interaction of two terpenoids – excoecariatoxin found in Excoecaria agallocha and oleanolic acid found in Swertia kouitchensis – and 10 other phenolic compounds with spike protein has been demonstrated (Wu et al. 2020). However, of these compounds, only hesperidin predictably bound the interface between the spike and ACE2 receptor (Table 3).

Inhibitors of host-associated protein

Unsurprisingly, ACE2 has received considerable attention as a potential druggable target in humans, and this is largely due to its integral role as the functional receptor that facilitates SARS-CoV-2 entry. Inhibitors of ACE2 are expected to obstruct the ACE2–spike protein interaction and ultimately prevent viral entry. Computational studies have predicted the interaction of several phytoligands with ACE2 (Table 3). For example, a docking study by Wu and co-workers (2020) predicted the interaction of some compounds including phyllaemblicin G7 found in Phyllanthus emblica, hesperidin, α-glucosyl hesperidin, and neohesperidin found in Citrus spp., theaflavin 3,3′-di-O-gallate and theliokeep found in Camellia sinensis, platycodin D found in Platycodon grandifloras, excoecariatoxin found in Excoecaria agallocha, andrographolide and 14-deoxy-11,12- didehydroandrographolide found in Andrographis paniculata, rosmarinic acid found in Salvia verticillata, rutin found in Sophora japonica, and kouitchenoid B found in Swertia kouitchensis with ACE2. The remarkable interaction of a metal chelator, nicotianamine; a terpenoid, glycyrrhizin found in Glycyrrhiza radix; and three phenolics, hesperidin, an antitumour and antioxidant agent, baicalin and scutellarin, antioxidant molecules found in Chinese skullcap with ACE2 enzyme have been reported (Chen and Du 2020). Another study by Basu and co-workers (2020) also demonstrated the molecular interaction of four phenolics compounds including hesperidin, emodin, anthraquinone derivative, and chrysin with the ACE2 receptor. Moreover, the interaction of dithymoquinone, an ingredient found in the widely known panacea, Nigella sativa (black seed) with SARS-CoV-2 S:ACE2 interface is noteworthy as it exhibited better binding affinity (<−8.0 kcal/mol) with the interface than HCQ (Ahmad et al. 2020). In addition, the interaction of other phenolic compounds such as piceatannol, pterostilbene, and resveratrol with S:ACE complex has also been predicted (Wahedi et al. 2020).

Conclusion and perspective

The present COVID-19 pandemic caused by SARS-CoV-2 has not only brought the world to its knees economically but has continued to take its toll on social and public health. The virus appears more infectious than SARS-CoV and MERS-CoV, and increasing evidence has shown that it can be transmitted by pre-symptomatic and asymptomatic patients (Xian et al. 2020). Currently, there are no specific antiviral drugs for its treatment. While vaccines have been rolled out, computational screenings are also being adopted to evaluate and validate the ability of phytoligands to interact with druggable targets essential for SARS-CoV-2 replication and pathogenesis. Amongst the predicted SARS-CoV-2 targets, the main protease (3CL^pro) remains a very important druggable target. Fascinatingly, this target is also highly conserved and its mutation would have a fatal effect on the virus, a property that makes it a more significant target (Silvestrini et al. 2021). Several plant-derived compounds have been identified and may offer useful guides in the development of COVID-19 drug candidates with remarkable activity. Many of these compounds are either phenolics or terpenoids. Regarding the mechanisms of action of these phytoligands, a conceptual model (Figure 5) could be proposed for typical inhibitors (such as quercetin, luteolin, and neoandrographolide) of SARS-CoV-2 druggable protein targets. Phenolic compounds; quercetin and luteolin, and a terpenoid; neoandrographolide potentially target and interact with SARS-CoV-2 druggable targets, such as 3CL^pro, PL^pro, and Sgp. Such interaction could result in the disruption of viral replication and pathogenesis. Similarly, neoandrographolide could predictably act against the host receptor of SARS-CoV-2, consequently preventing viral entry into the host cell.

Figure 5. The life cycle of SARS-CoV-2 and potential druggable targets of representative phytocompounds. Quercetin and luteolin predictably interact with 3CL^pro, PL^pro, and S glycoprotein, while neoandrographolide acts against 3CL^pro, PL^pro, host ACE2 receptor, RdRp and may also inhibit RNA replication.

While computational approaches can offer leads for drug repurposing and novel discovery, the available data remain tentative and warrants further computational studies for validation, as well as developmental studies for clinical validation. It is hoped that this review will provide invaluable information that would inspire and guide the development of effective anti-SARS-CoV-2 agents from phytocompounds.

Acknowledgements

We express our sincere gratitude to those in the frontlines, in all realms of life, who are working tirelessly to restore global health and wellbeing. Figures 2, 3, and 5 were created with Biorender.com.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

Data sharing is not applicable to this article as no new data were created or analysed in this study.