Plant Products as Inhibitors of Coronavirus 3CL Protease

Background: The ongoing COVID-19 pandemic has created an alarming situation due to extensive loss of human lives and economy, posing enormous threat to global health security. Till date, no antiviral drug or vaccine against SARS-CoV-2 has reached the market, although a number of clinical trials are under way. The viral 3-chymotrypsin-like cysteine protease (3CLpro), playing pivotal roles in coronavirus replication and polyprotein processing, is essential for its life cycle. In fact, 3CLpro is already a proven drug discovery target for SARS- and MERS-CoVs. This underlines the importance of 3CL protease in the design of potent drugs against COVID-19. Methods: We have collected one hundred twenty-seven relevant literatures to prepare the review article. PubMed, Google Scholar and other scientific search engines were used to collect the literature based on keywords, like “SARS-CoVs-3CL protease,” “medicinal plant and anti-SARS-CoVs-3CL protease” published during 2003–2020. However, earlier publications related to this topic are also cited for necessary illustration and discussion. Repetitive articles and non-English studies were excluded. Results: From the literature search, we have enlisted medicinal plants reported to inhibit coronavirus 3CL protease. Some of the plants like Isatis tinctoria L. (syn. Isatis indigotica Fort.), Torreya nucifera (L.) Siebold and Zucc., Psoralea corylifolia L., and Rheum palmatum L. have exhibited strong anti-3CLpro activity. We have also discussed about the phytochemicals with encouraging antiviral activity, such as, bavachinin, psoralidin, betulinic acid, curcumin and hinokinin, isolated from traditional medicinal plants. Conclusion: Currently, searching for a plant-derived novel drug with better therapeutic index is highly desirable due to lack of specific treatment for SARS-CoV-2. It is expected that in-depth evaluation of medicinally important plants would reveal new molecules with significant potential to inhibit coronavirus 3CL protease for development into approved antiviral drug against COVID-19 in future.


INTRODUCTION
Recently, a novel coronavirus was discovered in patients suffering from respiratory ailment accompanied by fever, dry cough and tiredness. Other symptoms, like sore throat, nasal congestion, headache, conjunctivitis, diarrhea, loss of taste or smell, were usually mild, and appeared gradually. This was coronavirus disease 2019 , unknown to the world before its outbreak in December 2019 in Wuhan, China (Burki, 2020). The highly infectious virus started to spread rapidly among the population in many countries all over the world, and created a pandemic situation within a couple of months (Bedford et al., 2020). Reports have indicated that even asymptomatic people can transmit the virus, mainly through respiratory droplets that can cause human-to-human transmission. The extremely contagious novel coronavirus 2019  was responsible for about 1.76 million deaths and 79.67 million confirmed cases globally till date (WHO, 2020).
The major phenotype of COVID-19 is severe acute respiratory distress syndrome (ARDS), similar to that caused by SARS-CoV and MERS-CoV (Hirano and Murakami, 2020). At present, there is no specific treatment for COVID-19, while vaccines will take some more time to come to the market. Hence, clinical management of COVID-19 is currently limited to preventive and supportive treatments, and mostly designed to alleviate further complications and organ damage (Rodríguez-Morales et al., 2020). Clinical studies have shown promising results in patients using the protease inhibitor drug lopinavir in combination with ritonavir, commonly used to treat HIV (Lu, 2020). Also, hydroxychloroquine, an antimalarial drug, and remdesivir, a nucleoside analogue of SARS-CoVs, were used to treat COVID-19 patients (Lee and Hsueh, 2020;Wu et al., 2020).
SARS-CoV-2 encodes two proteases, a papain like protease (PL Pro ), and a 3-chymotrypsin-like cysteine protease (3CL Pro ) also known as viral main protease (M pro ), for proteolytic processing during viral maturation. The PL Pro of coronavirus cleaves at no less than two sites on the pp1a polyprotein, whereas 3CL Pro has at least eleven inter-domain sites on the pp1a and pp1ab polyproteins (Krichel et al., 2020). The functional importance of this proteolytic enzyme in the viral life cycle makes 3CL pro a promising target for drug development against SARS-CoV-2 and other coronaviruses. Zhang et al. have successfully crystallized the 3CL protease from SARS-CoV-2 (Zhang et al., 2020a). This protease contains several highly conserved substrate-binding sites within the active site of the enzyme. Therefore, this is an opportunity for designing a wide variety of inhibitors against coronavirus 3CL pro . It is also exciting that the structures of 3CL pro in SARS-CoV-2 and SARS-CoV differ by only twelve amino acids with comparable ligand binding efficiency (Macchiagodena et al., 2020). This demonstrated the possibility that inhibitors of SARS-CoV-3CL pro will be active against SARS-CoV-2-3CL pro , too. In fact, protease inhibitors have drastically reduced the mortality against HCV/HIV, and maximized the therapeutic benefit (Kurt Yilmaz et al., 2016). Altogether, it is speculated that 3CL protease represents a potential target for the inhibition of CoV replication and this will definitely escalate the ongoing search for a new drug against SARS-CoV-2.
Natural products and their derivatives are used for treatment of various ailments since pre-historic times. A large number of herbal products and their constituents have shown promising inhibitory activity related to viral infections in humans (Ganta et al., 2017;Panda et al., 2017;Shen et al., 2019;Bahramsoltani and Rahimi, 2020). In 2002, during SARS-CoV outbreak in China, several clinical research projects were initiated on administration of Traditional Chinese Medicine (TCM) in combination with Western medicine for treatment of SARS-CoV infection (WHO, 2004). On verification of the clinical data, WHO declared that judicious use of TCM against SARS-CoV would help to reduce the mortality rate as compared to Western medicine alone. Furthermore, the combination therapy could lower the overall cost, and highlighted the importance of introducing plant products for the treatment of SARS-CoV (WHO, 2004).
In this background, it would be relevant to search for plant products with potential efficacy to inhibit coronavirus 3CL protease ( Figure 1). In fact, SARS-CoV-3CL Pro was reportedly inhibited by phytochemicals abundant in green tea extracts . The therapeutic importance of green tea is evident in customary Indian and Chinese medicinal systems for prevention of various diseases (Haqqi et al., 1999;Kavanagh et al., 2001;Sueoka et al., 2001), and also for its, neuroprotective (Weinreb et al., 2004), anti-bacterial (Roccaro et al., 2004), and antiviral (Weber et al., 2003) properties. Flavanols and flavonols, including (-)-epigallocatechin-3-gallate (EGCG), are mainly responsible for health promoting effects of green tea (Higdon and Frei, 2003;Moyers and Kumar, 2004). Another traditional plant, Angelica keiskei (Miq.) Koidz (Umbelliferae), commonly used as a mild cathartic and diuretic tonic (Kimura and Baba, 2003), showed inhibitory activity against SARS-CoV-3CLpro with IC50 values of 11.4 µM (Park et al., 2016). This plant extract was also reported for its anti-bacterial, hepato-protective and other activities (Kil et al., 2017). Cullen corylifolium (L.) Medik. (syn. Psoralea corylifolia L.) is used in Chinese medicine and traditional Ayurveda against different types of skin diseases, such as leprosy, leukoderma and psoriasis (Sah et al., 2006). This plant is also known for its anti-inflammatory and antimicrobial properties (Khushboo et al., 2010). Later on, six aromatic compounds isolated from seeds of Cullen corylifolium (L)., Medik. were noted for their inhibitory activity against PL pro (Kim et al., 2014); the isolated phytochemicals inhibited the enzyme in a dose-dependent manner with IC 50 ranging from 4.2 to 38.4 µM. Similarly, many natural products have shown antiviral activity at nanomolar concentration against SARS-CoV (e.g., lycorine, homoharringtonine, silvestrol, ouabain, tylophorine and 7-methoxycryptopleurine), and might contribute to future drug discovery pipeline (Islam et al., 2020). In fact, clinical trials of a few herbal compounds against SARS-CoV-2-3CL Pro aroused hope for plant-derived anti-SARS-CoV-2 drugs. Very recently, 3CL protease inhibitor NLC-001, a plant product administered orally as dietary supplement, got US FDA approval. Presently, clinical studies of NLC-001 to treat COVID-19 is going on in Israel while the pharmaceutical company Todos Medical Ltd. is evaluating its commercialization options worldwide (Golodetz, 2020). In another study, a natural product, baicalein, derived from TCM was identified as inhibitor of SARS-CoV-2-3CL pro (Figure 1). Clinical trial showed that baicalein was well tolerated in treatment of acute, or chronic hepatitis in China (clinical trials registration number CTR20132944) (Li et al., 2014b).
In this review, we provided a compilation of well-documented plant-derived compounds and their derivatives acting against coronavirus 3CL protease, and their current stage of investigation. A detailed insight into the active compounds with regard to their mode of inhibition have been presented along with predictive studies in silico. Finally, a discussion is focused on several plants with ethno-pharmacological reputation. Further, we have proposed a few selected phytochemicals for evaluation against this protease enzyme with a view to providing clues toward coronavirus drug discovery in future.

CORONAVIRUS LIFE CYCLE
SARS-CoV-2 is an enveloped, non-segmented, positive-sense RNA virus with genome size of ∼30 kb, belonging to the Nidovirales order and Coronaviridae family (Khailany et al., 2020). The viral genome contains at least six open reading frames (ORFs). The first ORF (ORF1a/b) is about two-thirds of the whole genome length and encodes 16 nonstructural proteins (nsp1-16). The virus contains four major structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N), all located within the 3′ end of the viral genome. Spike protein is responsible for binding with cellular receptors as well as fusion of viral and cellular membrane. M protein plays a central role in the assembly of viruses, making cellular membranes as workshops where viruses and host factors come together to create new virus particles. E protein is necessary for viral pathogenesis and its smaller size (9-12 kDa) makes it easier for the virus to assemble before release (Bianchi et al., 2020). N protein is the only protein in nucleocapsid that binds to the viral genome in a conformation of the "beads-ona-string" type ( Figure 3A) (Gordon et al., 2020). SARS-CoV-2 was found with ∼80% nucleotide identity to SARS coronavirus .
The attachment of SARS-CoV-2 to the host cell is initiated by interaction between S protein and its host cell receptor known as angiotensin-converting enzyme (ACE2) ( Figure 3B) (Choudhary et al., 2020). This interaction is followed by a series of events leading to the delivery of viral genome into the cytoplasm. The 5′ end of the RNA genome, ORFs 1a and 1b, are translated into pp1a and pp1ab; upon entry into the cell pp1ab is translated through a frameshift mechanism. The non-structural proteins (nsps) 1-11 and 1-16 are encoded by polyproteins pp1a and pp1ab, respectively. These polyproteins are then cleaved into individual non-structural proteins by two viral proteases, namely, PLP and 3CL pro /M pro . The PLP encoded in nsp3, cleaves between nsp1/2, nsp2/3 and nsp3/4, while 3CL pro /M pro encoded by nsp5 is responsible for the remaining 11 cleavage events (Krichel et al., 2020). Most of the non-structural proteins in the replicase-transcriptase complex (RTC) contribute to establish an environment favorable for RNA synthesis .
The synthesis of viral RNA produces genomic as well as subgenomic RNAs. After viral RNA translation, the structural proteins S, E, and M are moved through secretory pathway into the endoplasmic reticulum-golgi intermediate compartment. The viral genomes, encapsulated in the compartment membrane, interact with the structural proteins to form mature virions and get transported to the cell surface to be released by exocytosis (Shereen et al., 2020).

STRUCTURE OF SARS-COV-3CL PRO
SARS-CoVs are single-stranded RNA viruses with the largest known viral RNA genomes to date. The genome is a polyadenylated RNA of ∼30 kb, and 41 percent of the residues are G or C rich (Rota et al., 2003). Functional polypeptides are released by extensive proteolytic processing from each polyprotein, primarily by 33.8-kDa 3C-like protease (Yang et al., 2003). The crystal structure of 3CL pro (2.54 Å) reveals that the molecule is composed of three domains. Domains I, II and III contain amino acid residues 8 to 99, 100 to 183, and 184 to 303, respectively. Domains I and II are six-stranded antiparallel β barrels, and together mimic the architecture of chymotrypsin and 3C proteinases of picornavirus. Domain III is the substratum binding site found in a cleft between the two. A domain III loop connects domain II with C-terminal end of domain III (Anand et al., 2003). Domain III (residues 198-303), a globular cluster of five helices, regulates the dimerization of 3CL pro through interaction of the salt-bridge between Glu290 of chain-A and Arg4 of chain-B (Shi and Song, 2006). The 3CL pro active site contains a catalytic dyad in which a cysteine residue (Cys145) acts as a nucleophile, and histidine residue (His41) acts as a base ( Figure 4) (Anand et al., 2003). SARS-CoV-2-3CL pro has 96% structural similarity with SARS-CoV-3CL protease forming a dimeric structure for catalytic activity (Zhang et al., 2020a).

PLANTS WITH INHIBITORY ACTIVITY AGAINST 3CL PROTEASE
Natural products have always played a crucial role in drug development against various diseases. Therefore, traditional herbs from diverse geographical locations and various habitats could be considered as potential sources of new drugs for treatment of viral infections, including those caused by SARS-CoVs and its emergent mutants. For centuries, the medicinal plant Isatis tinctoria L. (Brassicaceae) have been esteemed in Europe, Central Asia, and in TCM for therapeutic and cosmetic application, and indigo-blue dyeing character (Speranza et al., 2020). All parts of this plant are utilized in complementary and alternative medicinal preparations against eruptive epidemic diseases, pharyngitis, laryngitis, hepatitis, various kinds of fevers, influenza and viral skin diseases. Currently, I. tinctoria root is recognized in European phytotherapy for medicinal properties, mostly due to its antiviral activities (Hamburger, 2002). During the outbreaks of SARS-CoV in China, Hong Kong, and Taiwan, Isatis tinctoria L. and several phenolic herbs were commonly used against the viral disease. I. tinctoria L. root contains phytochemicals such as indigo, indirubin, indican, β-sitosterol, sinigrin and γ-sitosterol. Seven other compounds from various sources, namely aloe-emodin, hesperetin, quercetin, naringenin, daidzein, emodin and chrysophanol were also tested for their SARS-CoV-3CL pro inhibitory effect (vide Table 1; Figure 5). Among these, aloe-emodin, sinigrin and hesperetin reportedly inhibited cleavage activity of the 3CL pro in a cell-based assay in dose-dependent manner .
Torreya nucifera (L.) Siebold and Zucc. is historically used as a medicinal herb in Asia. Ethanol extract of T. nucifera leaves showed promising inhibitory activity against SARS-CoV-3CL pro (62% viral inhibition at 100 μg/ml). Eight diterpenoids and four biflavonoids were isolated and evaluated for SARS-CoV-3CL pro inhibition using fluorescence resonance energy transfer analysis following bioactivity-guided fractionation. Out of these compounds, the most potent inhibitory effect on 3CL pro was shown by a biflavone, namely, amentoflavone (IC 50 8.3 µM). Three more biflavones, viz. apigenin, luteolin and quercetin inhibited 3CL pro activity with IC 50 values of 280.8, 20.2, and 23.8 µM, respectively (Bae et al., 2010).
Ethanolic extract of Cullen corylifolium (L.) Medik (syn. Psoralea corylifolia L.) seeds showed high activity against the SARS-CoV-PL pro with an IC 50 value of 15 μg/ml. Subsequently, bioactivity-guided fractionation of the ethanol extract resulted in six aromatic compounds known as bavachinin, neobavaisoflavone, isobavachalcone, 4′-O-methylbavachalcone, psoralidin and corylifol A. All the isolated flavonoids inhibited PL pro to exhibit their inhibitory potency in a dose-dependent manner (Kim et al., 2014).
Another study with twenty phytocompounds, including abietane and labdane-type diterpenes, lupane-type triterpenes, lignoids and curcumin, were tested for their anti-SARS-CoVs activity. The tested phytocompounds exhibited substantial levels of anti-SARS-CoV activity at 10 μM. Betulinic acid, betulonic acid, hinokinin, curcumin, niclosamide and savinin were among the twenty phytocompounds showing substantial inhibition of 3CL protease. It was claimed as the first report to demonstrate that natural lupane-type triterpenes and lignan could block 3CL protease activity by competitive inhibition (Wen et al., 2007).
Various types of tea extracts including black tea, green tea, oolong tea, and pu'er tea were investigated for 3CL Pro inhibitory activity. Results suggested that extracts from pu'er and black tea were more potent than those from green or oolong tea. Finally, water extracts from different types of tea were prepared and tested for their inhibitory activities against 3CL Pro . The authors found that 3CL Pro inhibitors were tannic acid, theaflavin-3′-gallate and theaflavin-3, 3′-digallate, as revealed by the fluorogenic substrate assay. The inhibitory effect of sixty-four purified natural compounds on the function of SARS-CoV helicase, nsP13, and HCV helicase was determined by double-strand DNA unwinding assay, or fluorescence resonance energy transfer, or colorimetry-based ATP hydrolysis assay. The study showed that a few selected natural flavonoids, including myricetin and scutellarein, may serve as potent inhibitors of SARS-CoV (Yu et al., 2012). Recently, traditional Chinese medicinal plant Scutellaria baicalensis Georgi, widely used as broad spectrum antiviral agent, demonstrated anti-SARS-CoV-2-3CL pro activity in vitro with EC 50 value 0.74 μg/ml . The active molecule baicalein strongly inhibited the enzymatic activity with IC 50 value 0.39 µM. Again, the authors identified four baicalein analogues, namely, scutellarein, dihydromyricetin, quercetagetin, and myricetin could inhibit SARS-CoV-2-3CL pro activity at micromolar concentration.
The alcoholic extract of Chinese medicinal plant Rheum palmatum L. roots and rhizomes were studied against the protease enzyme; the semi-purified fractions such as RH11, RH121, and RH125 significantly inhibited 3CL protease activity of SARS coronavirus (Luo et al., 2009).

FUTURE PROSPECT Promising Plants and Related Natural Products Waiting to be Evaluated against SARS-CoV-2-3CL pro
Considering the extensive global research focusing on antiviral plant products, the prospect of discovering anti-SARS-CoV-2 drugs remains hopeful. In fact, several bioactive constituents derived from traditional medicinal plants have now gained much attention as prophylactic immunity-boosters and/or adjuvant therapy for management of SARS-CoV-2. In the earlier sections, we have presented a number of phytochemicals and plant products inhibiting coronavirus 3CL protease. Now we shall enlist some of the prospective medicinal plants waiting for evaluation against coronavirus 3CL pro , and present them in Tables 2-6. In future, it would be more meaningful if advanced technology is used for cross-screening of these plants, as shown in Figure 6, for their potential activity against SARS-CoV-2.
It has been reported that HIV protease inhibitors could be used against coronavirus by targeting SARS-CoV-2-3CL pro . To deal with the COVID-19 pandemic, HIV protease inhibitors (lopinavir/ritonavir) are currently being used with some success. Both the drugs are active against SARS-CoV-2-3CL pro . Hence, we searched for the plants which have already demonstrated inhibitory activity against HIV protease, for example, Justicia adhatoda L (Acanthaceae) leaf extract and Andrographis paniculata (Burm. f.) Nees listed in Table 2. Therefore, these plants should be specifically investigated for the probability of anti-SARS-CoV-2-3CL pro activity as well. Again, mangiferin, isolated from Mangifera indica L., demonstrated efficacy against mutant strains of HIV-1 protease. Hence, mangiferin should be tasted against resistance strains of SARS-CoV-2-3CL pro . Flavones/menthalactone isolated from Mentha villosa Huds. inhibited HIV-protease at posttranslational level, therefore, may also inhibit polyprotein processing activity of SARS-CoV-2-3CL pro . Similarly, natural products like camelliatanin H and maslinic acid could be tested for application against coronavirus.
Structural similarity of HCV NS3/4A protease and SARS-CoV-2-3CL pro suggested a promising approach for finding useful plant products for COVID-19 therapy (Bafna et al., 2020). Both the proteases have a striking 3D-structural similarity, particularly in the key active site residues. Hence, plants which are already known to possess inhibitory activity against HCV NS3/4A protease need to be tested against coronavirus also (Table 3) Following the outbreak of SARS-CoV in 2002, research efforts to find new antiviral agents were rationally focused on traditional medicinal plants reputed in different parts of the world for treatment of pulmonary disorder, asthma and fevers related to cough and cold problems and bronchial infections. The plants enlisted in Table 4 have been commonly used as anti-allergic/ anti-histamine/bronchodilator/muscle relaxant in folk cultures, and well-known for immunomodulatory, anti-inflammatory and antioxidant properties. All of these features are useful for the treatment of respiratory disorders (Savithramma et al., 2007). Therefore, these medicinal plants should be evaluated for anti-SARS-CoV-2 activity, since respiratory disorder is an expected outcome of COVID-19 infection. For example, Broussonetia papyrifera L., and Artemisia scoparia Waldst. and Kit. are traditionally used for common cough, lung inflammation, bronchitis and asthma. Medicinal plants such as Salvia officinalis Linn. and Carum carvi L. pharmacologically act as bronchodilators, while Ficus religiosa L, Mimosa pudica Linn. and Hyoscyamus niger Linn. are used for anti-asthmatic purposes. Some such plants, enlisted in Table 5, are waiting to be explored for unknown antiviral constituents, as well as for their prospective anti-SARS-CoV-2-3CL pro activity.
Lycorine isolated from Lycoris radiata (L'Hér.) Herb. is an effective antiviral compound against coronavirus. Again, resveratrol (terpenoid) and luteolin (flavonoid) extracted from Veronica linariifolia Pall. ex Link strikingly inhibited coronavirus entry by interfering with the binding between ACE2 receptor and spike protein of SARS-CoV. These compounds have antiviral potential against coronavirus and need experimental validation in order to confirm their anti-SARS-CoV-3CL pro activity.

Selection of Phytochemicals Suitable for Future Development Against SARS-CoV-3CL pro
Following the outbreak of SARS-CoV in 2002, research efforts were focused on the antiviral prospect of selected constituents from medicinal plants which are traditionally used for community health care in different parts of the world. Some of them are presented below with relevance to their development against COVID-19, in future.

Betulin Analogues
Betulin and betulinic acid (pentacyclic lupane-type triterpenes) are ubiquitous secondary metabolites found in a variety of medicinal plants, mainly of Betulaceae family, possessing a wide range of pharmacological activities. The bark extracts of several species of  birch trees (Betula spp.) have been recommended in traditional medicinal systems for the treatment of rheumatism and arthritis, hepatitis, skin rash, intestinal worms and scurvy (Rastogi et al., 2015). Plenty of investigations have been undertaken to clarify the ethnopharmacological aspects, which showed that betulinic acid and its analogues could be useful for the treatment of cancer, inflammatory diseases, metabolic disorders, cardiovascular conditions, and neurological ailments (Ebeling et al., 2014;Amiri et al., 2020). Moreover, betulin and betulinic acid were shown to be non-toxic, with favourable therapeutic index at doses up to 500 mg/kg body weight in mice (Jäger et al., 2008). The promising antiviral activity of betulin and betulinic acid was reported by several workers (Fujioka et al., 1994;Pavlova et al., 2003). Further studies indicated that betulinic acid acts in the early phase of HIV infection by preventing cleavage of Gag protein, thereby hindering maturation of the virus (Bildziukevich et al., 2019). Again, Navid et al., 2014 demonstrated effective inhibition of herpes virus by analogues of betulin against acyclovir-resistant clinical isolates of HSV-1; also, hepatitis B virus has been found to be susceptible to betulin analogues (Yao et al., 2009;Navid et al., 2014). In a study by Wen et al. (2007), phyto-compounds were evaluated for antiviral activity against SARS-CoV (Wen et al., 2007). Out of 221 compounds, betulinic acid was found to be the most effective inhibitor of the coronavirus. Then, on the basis of molecular modeling analysis, it was observed that the competitive inhibitory activity of betulinic acid on 3CL pro activity was consistent with the formation of multiple hydrogen bond interactions between the compound and specific amino acid residues located at the active site of the pocket of the protease enzyme (Pillaiyar et al., 2016). Taken together, this indicated practical applicability of betulinic acid and its derivatives to provide a precise direction for development of novel anti-SARS-CoVs-3CL pro drugs from natural sources.

Griffithsin
Griffithsin, an algal protein, was isolated from an aqueous extract of Griffithsia sp (Ceramiaceae genus) of marine red alga (Rhodophyta). Traditionally, red algae have a long history of use as functional foods for their richness in protein, fatty acids, minerals and vitamins (Li et al., 2014a). The potent anti-HIV activity of griffithsin was found with EC 50 at a pico-molar range, and much lesser toxicity to the host cells (Mori et al., 2005). Later on, griffithsin was considered for topical application as a preexposure prophylactic against HIV (O'Keefe et al., 2009). In fact, it has been found that the lectin could inactivate other enveloped viruses also, especially those with highly glycosylated proteins on their surface (Kouokam et al., 2016). The protein showed inhibitory activity against a broad spectrum of animal and human coronaviruses, as it could bind specifically to the SARS-CoV spike glycoprotein and inhibited entry of the virus (O'Keefe et al., 2010). The outstanding efficacy of griffithsin in SARS-CoV-infected mice suggested that it merits further investigation for prophylaxis or treatment of respiratory infection caused by emerging viruses of the Coronaviridae family. Thus, by virtue of its broad antiviral spectrum, griffithsin holds great promise for development into universal Frontiers in Pharmacology | www.frontiersin.org March 2021 | Volume 12 | Article 583387 antiviral therapeutics. Therefore, this molecule needs further exploration on its putative anti-SARS-CoV-3CL pro activity.

Glycyrrhizin
Glycyrrhizin (or glycyrrhizic acid) is a major constituent of liquorice root obtained from Glycyrrhiza glabra L., and Glycyrrhiza uralensis Fisch. ex DC. from Fabaceae family of plants. Liquorice extract is a well-known phytomedicine in use since prehistoric times to alleviate common ailments like bronchitis, gastritis and jaundice. Currently, it is added as edible emulsifier and gel-forming agent in modern foodstuffs, and investigated for its anti-inflammatory, anti-allergenic, antimicrobial and antiviral properties in order to validate its traditional medicinal applications (Li et al., 2014a). Oriental medicinal systems recommended it as antitussive treatment of viral respiratory tract infections, such as dry cough or hoarse voice, and also for chronic fevers (Shibata, 2000). Actually, randomized controlled trials conducted with glycyrrhizin and its derivatives reduced hepatocellular damage in patients with HIV-1, and chronic hepatitis B and C virus infection. Additionally, animal studies on glycyrrhizin demonstrated a reduction of mortality in HSV, and influenza A virus (Fiore et al., 2008). In vitro studies revealed antiviral activity against HIV-1, SARS-CoV, respiratory syncytial virus, arboviruses, vaccinia virus and vesicular stomatitis virus (Baltina et al., 2015). Further studies on clinical isolates of coronavirus showed efficacy of glycyrrhizin to inhibit viral replication, as well as the adsorption and penetration of virus into host cell at non-cytotoxic concentration (Cinatl et al., 2003). Further, chemically modified glycyrrhizin derivatives exhibited enhanced inhibition of SARS-CoV replication in vitro (Hoever et al., 2005). Recent studies based on integrated computational approach and pharmacological aspects lend further support to glycyrrhizin playing an auxiliary role in COVID-19 treatment (Luo et al., 2020;Muhseen et al., 2020). In a timely review, Bailly and Vergoten (2020) have critically analyzed the prospective development of glycyrrhizin analogues not only as antiviral drugs, but also as adjuvant therapy for their protective effects on the vulnerable organs in patients suffering from SARS-CoV-2 infection (Bailly and Vergoten., 2020). Taking all this into consideration it is a high time to explore this time-tested safe phytochemical against SARS-CoV-2-3CL pro to an approved drug in future.

Lycorine
Lycorine is a bioactive constituent of Amaryllidaceae family of plants, like Hymenocallis littoralis (Jacq.) Salisb., Lycoris radiata (L'Hér.) Herb., and Narcissus pseudonarcissus L. cv. Dutch Master, which are traditionally used in many countries for wound healing, and treatment of cancer and infectious diseases (Nair and Van Staden, 2014). In China, the bulbs of Lycoris radiata have been traditionally used in the treatment of laryngeal complications, wounds and carbuncles (He et al., 2013). Lycorine potently inhibited flaviviruses in cell culture mainly through suppression of viral RNA replication (Zou et al., 2009). In fact, this alkaloid has been found to exhibit a wide range of antiviral activities against ZIKA, HIV-1, HCV, and SARS-CoV (Liu et al., 2011). A high throughput screening study on two hundred herbal extracts demonstrated Lycoris radiata (L'Hér.) Herb. to be the most potent antiviral plant against SARS-CoV with EC 50 2.4 ± 0.2 μg/ml. Further, meticulous fractionation and analysis of L. radiata extract led to the identification of lycorine as the active principle, with EC 50 15.7 ± 1.2 nM (Li et al., 2005). In a recent study, the mechanism behind the anti-SARS-CoV-2 activity of lycorine has been attributed to modulating the host factors (Zhang et al., 2020b). Nevertheless, it would be interesting to explore this molecule as a candidate for anti-3CL pro activity in future.

Tanshinone
Tanshinones are a class of phenanthrene-quinone diterpenoids that are the major lipophilic constituents of the root of Salvia miltiorrhiza Bunge, a well-known traditional Chinese medicinal herb (Danshen), primarily used for treating cardiovascular and cerebrovascular diseases. More than 40 types of tanshinone molecules have been characterized exhibiting a variety of biological activities in traditional clinical applications (Li et al., 2013), and pharmacological properties such as anti-cancer, antibacterial and antiviral effects (Jiang et al., 2019). Investigation on S. miltiorrhiza showed that its lipophilic fraction possesses marked inhibitory activity against both the proteases (3CL Pro and PL Pro ) of SARS-CoV, and also found the inhibitory effects of dihydrotanshinone against viral entry in MERS-CoV (Park et al., 2012). In fact, dihydrotanshinone may play a dual role by blocking the entry of coronavirus as well as its post-attachment replication inside the host cell (Kim et al., 2018). Obviously, further validation and clinical trials are needed to establish its antiviral efficacy against SARS-CoV-3CL Pro .

CONCLUSION
Presently, the world has witnessed the extraordinary outbreak of COVID-19 caused by SARS-CoV-2, raising widespread health concerns. Earlier, in 2003, soon after the emergence of SARS-CoV, the X-ray crystallographic structure of SARS-CoV-3CL pro dimer with a covalently bound inhibitor was elucidated. Since then, numerous inhibitors of 3CL pro enzyme, some of these originating from plants, have been identified, although none of them has reached the clinic till this day. In this perspective, we have discussed several traditional medicinal plant-products screened against 3CL pro activity through in vitro and in silico studies (Figure 7). In addition, we have proposed promising plant-products which have not yet been explored for inhibition of 3CL pro . Needless to say that all the prospective molecules would require in vivo antiviral assessment and pharmacokinetic evaluation before going for clinical trial. The structural similarity of HIV-and HCV-protease with SARS-CoV-3CL pro is a promising approach to find useful plant products for COVID-19 therapy, as elaborated in this article. Also, the plants known for treatment of respiratory disorders have been suggested in this regard. Thus, it is a challenge to repurpose and develop these natural products into potent, low molecular weight inhibitors of SARS-CoV-3CL pro , with minimum toxicity, to combat the onslaught of emerging coronavirus diseases. We hope that this review will be useful for phytochemists and virologists targeting 3CL pro to identify novel therapeutics against SARS-CoVs.

AUTHOR CONTRIBUTIONS
AM and BH designed the study and collected the information. AM and AJ wrote the article. AM revised the text under supervision of BH. All authors contributed to the article and approved the submitted version.