key: cord-345999-iiw4cs8p
authors: Khare, Prashant; Sahu, Utkarsha; Pandey, Satish Chandra; Samant, Mukesh
title: Current approaches for target-specific drug discovery using natural compounds against SARS-CoV-2 infection
date: 2020-09-24
journal: Virus Res
DOI: 10.1016/j.virusres.2020.198169
sha: 
doc_id: 345999
cord_uid: iiw4cs8p

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) recently caused a pandemic outbreak called coronavirus disease 2019 (COVID-19). This disease has initially been reported in China and also now it is expeditiously spreading around the globe directly among individuals through coughing and sneezing. Since it is a newly emerging viral disease and obviously there is a lack of anti-SARS-CoV-2 therapeutic agents, it is urgently required to develop an effective anti-SARS-CoV-2-agent.Through recent advancements in computational biology and biological assays, several natural compounds and their derivatives have been reported to confirm their target specific antiviral potential against Middle East respiratory syndrome coronavirus (MERS-CoV) or Severe Acute Respiratory Syndrome(SARS-CoV).These targets including an important host cell receptor, i.e., angiotensin-converting enzyme ACE2 and several viral proteins e.g. spike glycoprotein (S) containing S1 and S2 domains, SARS CoV Chymotrypsin-like cysteine protease (3CL(pro)), papain-like cysteine protease (PL(pro)), helicases and RNA-dependent RNA polymerase (RdRp). Due to physical, chemical, and some genetic similarities of SARS CoV-2 with SARS−COV and MERS−COV, repurposing various anti-SARS−COV or anti-MERS−COV natural therapeutic agents could be helpful for the development of anti−COVID-19 herbal medicine. Here we have summarized various drug targets in SARS−COV and MERS−COV using several natural products and their derivatives, which could guide researchers to design and develop a safe and cost-effective anti-SARS−COV-2 drugs.

The outbreaks of coronavirus (CoV) infection that have already threatened the world by SARS and MERS in the first decade of 21 st century have recently come up with a novel strain of lethal coronavirus named as 2019 novel coronavirus (SARS-CoV-2). In December 2019, the disease was originally started in the local seafood market of Wuhan of China (Hui et al., 2020; Perlman et al., 2020; Zhu et al., 2020) . Since then this new coronavirus strain has spread across the globe very rapidly with the catastrophic effects. Coronaviruses are the non-segmented, enveloped viruses with positive-sense RNA as their genetic material belonging to the family Coronaviridae. They are pleomorphic and club-shaped spikes are present on their cell surface.

The disease is characterized as respiratory disorders with flu-like symptoms such as a sore throat, fever, cold, cough and severe pneumonia is also reported in more critical cases. SARS-CoV-2 can be transmitted through coughing and sneezing droplets of infected individuals; these virions containing droplets retained on the hard surfaces for a longer time and can spread to a fresh individual by direct inhalation or by touching the infected surfaces. As of 31 st August 2020, the complete number of affirmed COVID-19 cases reported globally is more than 25 million and the mortality has crossed more than 850,600.

Recently many efforts have been made to develop the therapeutic agents to control COVID-19, but so far no medicine is significantly effective against SARS-CoV-2 (Tu et al., 2020) , and further supportive care is also needed to the individual for proper breathing. While the development of a vaccine may also take 12-18 months (Pandey et al., 2020) , repurposing of the drugs (from Ebola to malaria to arthritis) is the only feasible option for treating the patients in this current situation (Simsek Yavuz and Unal, 2020) . Progress in drug discovery and development largely depends on the identification of potential drug targets. For the management J o u r n a l P r e -p r o o f of COVID-19 infection, various molecular targets playing important role in the SARS-CoV-2 life cycle including host cell receptor-Angiotensin-converting enzyme ACE2 (PDB ID 3D0G) and viral proteins such as S protein (containing S1 and S2 domains) (PDB ID 6XM0); various cysteine proteases such as papain-like cysteine protease (PL pro ) (PDB ID 6WX4) or Chymotrypsin like nprotease (3CL pro ) (PDB ID 1P9U), helicases and RNA-dependent RNA polymerase (RdRp) (PDB ID 6M71) could be evaluated .

Nature has provided us with an immense supply of natural products. Interestingly, the nutraceuticals market hugely depends on the success of natural drugs for the treatment of infectious diseases (Williamson et al., 2020) . So these natural products and their derivatives could offer new scope for the control and prevention of various ailments including viral infections (Figure-1A-D and Table- 1 A-C) (Chen and Du, 2020; Ganjhu et al., 2015; Islam et al., 2020; Jo et al., 2020; Lin et al., 2014; Wang et al., 2014) . This article gathers information on the use of herbal-based drugs and/or their derivatives for target-specific drug discovery against SARS CoV2 infection (Figure-2).

Initially, CoV was known to cause mild disease, but the recent outbreaks (SARS-CoV outbreak of China and MERS-CoV outbreak of Saudi Arabia and now COVID-19 originated from Wuhan, Hubei, China) signifies the importance of understanding the structure, metabolism, and pathophysiology of CoV-associated diseases to identify major drug targets (J Alsaadi et al.,

The viral RNA codes for some conserved genes: ORF1a, ORF1b, OEF3S, E, M, and N gene. The ORF1a/b genes code for viral replicase polyproteins (PPs) PP1A and PP1ab. These J o u r n a l P r e -p r o o f PPs are further processed to form sixteen mature non-structural proteins (NSPs), which play a crucial role in the formation of the replicase transcriptase complex. Other structural proteins viz. membrane (M), envelope (E), spike (S), nucleocapsid as well as other accessory proteins are encoded by rest of the genome (McBride et al., 2014) and the beta-CoVs also have hemagglutinin esterase (HE) glycoprotein (Hilgenfeld, 2014) . All these proteins play a significant role in virulence and for viral multiplication. Hence these viral proteins could be the potential targets for the treatment of SARS CoV2 infection.

Spike proteins are glycoprotein which facilitates the attachment of coronavirus to the target cells via a specific receptor present on the cell surface of host i.e. Angiotensin-converting enzyme ACE2 receptor in SARS-CoV(Li et al., 2003; Zhou et al., 2020) and dipeptidyl peptidase-4 in MERS-CoV(Mubarak et al., 2019) . The coronavirus relies on the association of viral envelope protein with host cell membrane for delivering their nucleocapsid. The spike proteins (S) are responsible for viral entry inside the host cell and are accountable for disease progression in a specific types of host cells. During the fusion of S protein with a specific receptor on the host cell membrane, a crucial conformational change occurs in S glycoprotein (Belouzard et al., 2012) . So the S-glycoprotein could be evaluated as a potential drug target. So far various natural compounds and their derivatives have been tested for anti-SARS-CoV activity against this protein (Ho et al., 2007) . Several extracts/derivatives from the herbs belonging to family polygonaceae have been reported to inhibit the SARS-CoV S protein interaction with Angiotensin-converting enzyme ACE2 receptor. Anthraquinone compound namely emodin (1), a plant extract isolated from genus Polygonum, and Rheum has efficiently impeded the interaction of S protein and Angiotensin-converting enzyme ACE2 receptor. Moreover, it also J o u r n a l P r e -p r o o f hampered S protein-pseudo typed retrovirus infectivity to Vero E6 cells. These observations indicated the potential role of emodin as a drug candidate against S protein (Ho et al., 2007; Yi et al., 2004) . Two naturally occurring compounds tetra-O-galloyl-β-d-glucose (TGG) (2) and luteolin (3) derived from Galla chinensis were reported to possess anti-SARS-CoV activities.

TGG and luteolin have a high affinity for S2 domain of spike protein. This indicates the anti-SARS activity of TGG and luteolin is due to inhibition of virus and host cell fusion however the exact mechanism remains unknown (Yi et al., 2004) . These observations indicate that TGG and luteolin could be used for drug development against COVID-19 targeting S2 domain

Helicase also known as NTPase is involved in the replication of viral genomic RNA as well as in transcription and translation (Frick and Lam, 2006) . SARS-CoV helicase is an enzyme of the SF1 family, which hydrolyzes all NTPs and utilizes ATP, dATP, and dCTP as substrates (Karpe and Lole, 2010). CoV helicase nsP13 has been reported to retain dsRNA unwinding activity with translocation along the nucleic acid by ATP hydrolysis (Adedeji et al., 2012) . Various natural compounds have also been reported to inhibit helicases of SARS-CoV-2. The activity of two naturally occurring flavonoids namely myricetin (4) and scutellarein (5) have been shown to inhibit potential against SARS CoV helicase nsP13. These compounds have been reported to inhibit helicase protein by affecting the ATPase activity (Yu et al., 2012) .Therefore, helicases could be a potential drug target for anti-COVID-19 therapy.

J o u r n a l P r e -p r o o f

Angiotensin-converting enzyme ACE2 receptor is a human receptor to the SARS and SARS-CoV-2 . Angiotensin-converting enzyme ACE2 receptor is mostly present as cell surface receptors and rarely circulates in soluble form. These receptors facilitate entry of three CoV strains (e.g. NL63, SARS-CoV, and SARS-CoV-2), which are present most abundantly in the lungs (predominantly in type 2 pneumocytes and macrophages), testis, brain, heart, blood vessels, and kidney (Verdecchia et al., 2020) . The overexpression of ACE2 receptor from human, pig, civet in HeLa cells permitted replication of SARS-CoV-2, thus proving it to be the principal receptor for CoV entry (Zhou et al., 2020) . Drugs targeting the ACE2 receptor could be efficient for anti CoV drugs. Various natural compounds such as baicalin, (6) scutellarin (7), nicotianamine (8) (docking score -5.1) and glycyrrhizin (9) (docking score -9) (supplementary Table 1 ) have been reported to have potential anti-2019-CoV effects by preventing the attachment and entry of virus (Chen and Du, 2020) , Particularly baicalin, extracted from plant Scutellaria baicalensis Georgi demonstrated an excellent antiviral and anti-SARS activity . Another such compound scutellarin, is reported to reduce ACE2 activity in brain tissues (Wang et al., 2016) and therefore this compound can also be evaluated as an ACE2 receptor inhibitor to block the entry of SARSCoV2. Stilbenoids belonging to other phenolic natural compounds were reported to possess inhibitory activity against ACE2 receptor (Wahedi et al., 2020) . Furthermore, natural extracts isolated from garlic were also observed to have inhibitory effects against ACE2 receptor (Thuy et al., 2020) . (11) (Docking Score --9.565), and pectolinarin (12) demonstrated anti-SARS-CoV 3CL pro activity (Jo et al., 2020). 3CL pro has 3 domains at substrate binding site -S1, S2, and S3. S1 represents the polar site of 3CL pro , S2 represents the hydrophobic site, while S3 has no strong tendency.

Molecular docking showed the binding affinity of three flavonoids with 3 domains of 3CL pro (Jo et al., 2020). Another flavonoid amentoflavone (13) (Docking Score −11.42) is the most effective flavonoid inhibiting SARS-CoV 3CL pro (Ryu et al., 2010) (supplementary Table 1 (19), isotheaflavin-3-gallate [(TF2B) (20)] and theaflavin-3, 3'-digallate [(TF3) (21)] belonging to polyphenols of tea were reported to exhibit antiviral properties by their inhibitory potential against 3CL Pro . Triterpenes [betulinic acid (22) and savinin (23)] were reported to possess anti 3CLpro activity (Wen et al., 2007) . Recently, a sum of 28 natural compounds was identified from the Shuanghuanglian preparations. Out of which two major bioactive compounds baicalin (6) and baicalein, (24) were found to possess significant inhibitory activity against SARS-CoV 3CL pro by inhibiting the proliferation in Vero E6 cells (Su et al., 2020) 

The papain-like cysteine protease (PL pro ) plays an important role in SARS-CoV viral genomic RNA replication. It cleaves the N terminal site of polyproteins (PPs) to generate three nonstructural proteins (NSPs-1, 2, and 3) (Hilgenfeld, 2014; Lindner et al., 2005) . PL pro also contains a catalytic core domain and a consensus sequence LXGG which is required for cleaving replicase substrate (Barretto et al., 2005) . Thus PL pro could be used as a crucial drug target for anti-SARS drug development (Park et al., 2017) . Recently 13 chalcones that includes isobavachalcone (25) (Dockind Score -8.82) , 4-hydroxyderricin (26) 

It is a big challenge to develop an effective antiviral therapeutic agent. Various inverse agonists are currently being explored against COVID-19. The nucleoside inhibitor (Gilead's Nuc inhibitor) which has shown disappointment in the treatment of Ebola is effective in the treatment of a 2019-CoV patient in the USA, but the higher rate of mutation in this virus have restricted the use of this drug for treating the n-Cov patients (Nguyen et al., 2020) . Moreover, remdesivir another drug recommended for the treatment of Ebola and other RNA viruses have also been found useful in some of the patients (Gordon et al., 2020; Hillaker et al., 2020; Shannon et al., 2020) . Recently anti-influenza drug favipiravir or avigan was considered as an efficient treatment regimen for COVID-19 patients as compared to other antiviral agents (Chibber et al., 2020; Rosa and Santos, 2020; Zhu et al., 2020) . Likewise, chloroquine and hydroxychloroquine which is effective against malaria, lupus, and rheumatoid arthritis (Garcia-Cremades et al., 2020; Rosa and Santos, 2020; Zhu et al., 2020) have also been found effective in coronavirus infection . Due to the involvement of in silico approaches in pharmaceutical research, now it is quite possible to identify the specific drug targets and understanding the mechanism of action of various natural products and their derivatives (Supplementary information). In this review, we have summarized various drug targets for natural drugs and their synthetic compounds, which were used to treat SARS CoV and MERS CoV. We have discussed the importance of various herbal-based compounds that can inhibit viral infectivity by blocking the ACE2 receptor of host or interrupt the activity of various viral proteins/enzymes such as spike glycoproteins (S protein), 3CL protease, PL pro , helicase, and RNA dependent RNA polymerase. We have documented the mechanism of action of various herbal-based drugs so; these natural compounds could be important substitutes of synthetic drugs for the treatment of viral infections due to their low cost and safety efficacy.

In summary, we have identified and discussed the target-specific antiviral potential of several natural compounds against various strains of CoV, which might directly impede the COVID-19

pandemics. Further pharmaceutical companies should also give more emphasis on natural product research for the development of novel therapeutic agents against various viral infections to achieve sustainable development goals on health.

J o u r n a l P r e -p r o o f (Yi, 2004) 3. Luteolin 10.6μM Spike Protein (S) (Yi, 2004) 4. Myricetin 2.5-3.0 μM Helicase (Yu, 2012) 5. Scutellarein 0.4-1.24 μM Helicase (Yu, 2012) 6. Baicalin 2.24mM Angiotensin-converting enzyme 2 (ACE2) receptor (Deng et al., 2012) 7. Scutellarin 44-52 μM ACE2 receptor (Wang et al., 2016) 8. Nicotianami ne 84nM ACE2 receptor 9. Glycyrrhizin NA ACE2 receptor J o u r n a l P r e -p r o o f Chymotrypsin like protease (3CL pro ) (Wen, 2007) 23. Savinin 25 μM Chymotrypsin like protease (3CL pro ) (Wen, 2007) 6. Baicalin 6.41 ± 0.95 μM Chymotrypsin like protease (3CL pro ) (Su et al., 2020) 24. Baicalein 0.94 ± 0.20 μM Chymotrypsin like protease (3CL pro ) (Su et al., 2020) J o u r n a l P r e -p r o o f 54. Saikosaponin B2 1.7 ± 0.1 μmol/L 55. Saikosaponin C 19.9 ± 0.1 μmol/L 56. Saikosaponin D 0.02 ± 0.001μmol/L 57. R-Halitunal NA (Koehn et al., 1991b) Diterpenes 58. ferruginol 0.40 µg/ml (Wen et al., 2007) 59. dehydroabieta-7one 4.00 μM (Wen et al., 2007) 60. Sugiol NA (Wen et al., 2007) 61. cryptojaponol >3.3 µg/ml (Wen et al., 2007) 62. 8β-hydroxyabieta-9(11), 13-dien-12-0.44 µg/ml (Wen et al., 2007) one 63. 7βhydroxydeoxycrypt ojaponol 1.15 μM (Wen et al., 2007) 64. 6,7dehydroroyleanone 5.55 μM (Wen et al., 2007) 65. 3β, 12diacetoxyabieta-6, 8,11,13-tetraene 0.48 µg/ml (Wen et al., 2007) 66. pinusolidic acid 4.71 μM (Wen et al., 2007) 67. forskolin 3.1 µg/ml (Wen et al., 2007) Sesquiterpenes 68. cedrane-3β,12-diol >2.3 µg/ml (Wen et al., 2007) 69. Cadinol 1.04µg/ml (Wen et al., 2007) Triterpenes 22. betulinic acid >4.5 µg/ml (Wen et al., 2007) 70. betulonic acid 0.29 µg/ml (Wen et al., 2007) Lignins:

71. hinokinin >10μM (Wen et al., 2007) 23. savinin 0.40 µg/ml (Wen et al., 2007) 72. 4,4′-Obenzoylisolariciresinol NA (Wen et al., 2007) 73. Honokiol 6.5μM (Wen et al., 2007) 74. Magnolol 3.80μM (Wen et al., 2007) 75. Curcumin >10μM (Wen et al., 2007) 76. Niclosamide <0.1μM (Wen et al., 2007) 77. Valinomycin 1.82 µg/ml (Wen et al., 2007) 78 

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