key: cord-0882966-yybunc6z
authors: Sinha, Saurabh K.; Shakya, Anshul; Prasad, Satyendra K.; Singh, Shashikant; Gurav, Nilambari S.; Prasad, Rupali S.; Gurav, Shailendra S.
title: An in-silico evaluation of different Saikosaponins for their potency against SARS-CoV-2 using NSP15 and fusion spike glycoprotein as targets
date: 2020-05-13
journal: J Biomol Struct Dyn
DOI: 10.1080/07391102.2020.1762741
sha: cfbe98d29de39a1880ef61867f33c3fdddaf77ac
doc_id: 882966
cord_uid: yybunc6z

The Public Health Emergency of International Concern declared the widespread outbreak of SARS-CoV-2 as a global pandemic emergency, which has resulted in 1,773,086 confirmed cases including 111,652 human deaths, as on 13 April 2020, as reported to World Health Organization. As of now, there are no vaccines or antiviral drugs declared to be officially useful against the infection. Saikosaponin is a group of oleanane derivatives reported in Chinese medicinal plants and are described for their anti-viral, anti-tumor, anti-inflammatory, anticonvulsant, antinephritis and hepatoprotective activities. They have also been known to have anti-coronaviral property by interfering the early stage of viral replication including absorption and penetration of the virus. Thus, the present study was undertaken to screen and evaluate the potency of different Saikosaponins against different sets of SARS-CoV-2 binding protein via computational molecular docking simulations. Docking was carried out on a Glide module of Schrodinger Maestro 2018-1 MM Share Version on NSP15 (PDB ID: 6W01) and Prefusion 2019-nCoV spike glycoprotein (PDB ID: 6VSB) from SARS-CoV-2. From the binding energy and interaction studies, the Saikosaponins U and V showed the best affinity towards both the proteins suggesting them to be future research molecule as they mark the desire interaction with NSP15, which is responsible for replication of RNA and also with 2019-nCoV spike glycoprotein which manage the connection with ACE2. [Image: see text] Communicated by Ramaswamy H. Sarma

In the last week of December 2019, Wuhan city, Hubei Province, China emerged with an outbreak of an acute resolved COVID-19 pneumonia, which has subsequently affected around 206 countries around the globe with a 6.1% fatality rate . The genome sequencing has revealed novel coronavirus SARS-CoV-2, formally known as 2019-nCoV as etiological agent for COVID-19 . The genome of CoVs (enveloped, positive single-stranded RNA) is the largest genome of known RNA viruses with varying size (26 kb-32kb in length). Coronaviridae family can be divided in four genera comprising of aand b-CoV, known to infect mammals, whereas dand c-CoVs infect birds. Severe acute respiratory syndrome coronavirus i.e. SARS (2002) and the Middle East respiratory syndrome coronavirus i.e. MERS (2012) were viral pneumonia outbreaks due to b-CoVs and resulted in approximately 800 deaths (Song et al., 2019; Abdo & Eman, 2020) . Whereas, this 'Public Health Emergency of International Concern' (PHEIC) declared due to widespread outbreak of a SARS-CoV-2 resulted in 1,773,086 confirmed cases of COVID-19, including 111,652 deaths, as of 05:00 pm CEST, 13 April 2020, reported to World Health Organization (WHO) (https://who. sprinklr.com) (World Health Organization. 2020).

The SARS-CoV-2 genome (30 kb in size) encodes a large non-structural polyprotein, open reading frame (ORF) 1a/b, which is further proteolytically cleaved to generate 15/16 proteins, five accessory proteins (ORF3a, ORF6, ORF7, ORF8 and ORF9) and four structural proteins (S, M, E and N) deliberated to be essential for SARS-CoV2 assembly and infection Ortega et al., 2020; Boopathi et al., 2020) .

Protrusion of the CoV spike (S) proteins from the virion envelope to cellular receptors and host cell proteases priming plays a vital role in regulation of entry of coronaviruses into cell. Key atomic-level interactions of the SARS-CoV spike protein receptor-binding domain (RBD) with its host receptor angiotensin-converting enzyme-2 (ACE-2) evidenced the regulation of human-to-human and cross-species transmissions of SARS-CoV (Chen & Du 2020) . The recent report on the threedimensional structure of the crystal structure of the 2019-nCoV main protease (PDB: 6LU7) revealed, that the protein is encoded within the viral genome , in complex with an inhibitor. Thus, both main protease and spike protein complexed with human ACE-2 receptor might be important targets for researchers to discover and develop vaccines and other therapeutic agents to control this new CoV.

In the absence of any validated approved therapeutic interventions this pandemic is spreading drastically all over the globe. Indeed, in current emmergent situation, the concept of drug repositioning might be a cost-effective and time-efficient option for meticulous development of possible therapeutic and/or prophylatic lead condidate(s) from wellknown traditional and/or approved drugs (Li et al., 2019) . Recent studies comprising reverse vaccinology and via integrated computational approaches have highlighted repurposing of FDA approved drugs and known drugs from various databases against various corona virus enzymes either individually or in combination to combat the virulence in COVID 19 (Muralidharan et al., 2020; Elmezayen et al., 2020; Gupta et al., 2020; Sarma et al., 2020; Pant et al., 2020; Enayatkhani et al. 2020; . The interaction of ACE-2 and with spike protein with furin cleavage site resulting in the invasion of SARS-CoV-2 is also reported (Hasan et al. 2020) . Additionally, several phytochemicals were found effective in inhibiting the 3CL pro of SARS-CoV and blocking the entry of SARS-CoV into host cells viz. phenolic compounds of Isatis indigotica root (Lin et al., 2005) , baicalin (Chen et al., 2004) , glycyrrhizin (Cinatl et al., 2003) , quercetin (Chen et al., 2006) and Saikosaponin (Cheng et al., 2006) . Moreover, 'leads' from natural products has also been explored for their efficacy against SARS-CoV-2 (Aanouz et al., 2020) , and hesperetin, a natural citrus fruit flavonoid is found promising in suppressing the cleavage activity of the 3 C-like protease (3CL pro ) of SARS-CoV in cell-free and cell-based assays .

Saikosaponins represent a group of oleanane derivatives, usually glucosides and are major bioactive compounds found in plants of Traditional Chinese Medicine (TCM) such as Bupleurum spp., Heteromorpha spp. and Scrophularia scorodonia (Li et al. 2018a; Li et al., 2018b) . Saikosaponins have been reported for various biological activities like anti-viral, antitumor, anti-inflammatory, anticonvulsant, antinephritis and hepatoprotective activity both in-vivo and in-vitro (Chen et al., 2015; Yang et al., 2017; Xiao et al., 2018) . Saikosaponins exhibit immunomodulatory effects with enhanced production of immunosuppressive mediators i.e. T helper (Th) 1/Th2 cells, IL-4 and IL-10 and suppression of pro-immune mediators (IFN-c and TNF-a in splenocytes) (Huang et al., 2017) . Inhibitory potential of Saikosaponins for HBV-DNA replication and HBsAg secretion, without inhibition of cell proliferation has been found to be superior to lamivudine (well-known antiviral drug) (Lin et al., 2015) . Moreover, Saikosaponins (A, B 2 , C and D) have also been reported for their anti-coronaviral efficacy by interfering the early stage of viral replication including absorption and penetration of the virus (Cheng et al., 2006) . A recent study, performed by Goswami and Bagehi (2020) reported the potential of Saikosaponins against SARS-CoV-2, showing favorable affinity towards RBD region of the spike glycoprotein using docking simulation study (Goswami & Bagchi 2020) . However, it will be interesting to investigate the potency of different class of Saikosaponins towards the other protein such as NSP15 and fusion spike (S1 and S2) protein, which will give a clear indication about the selectivity of the Saikosaponins towards SARS-CoV-2. Therefore, in the current investigation attempt has been made to screen and evaluate the potency of different Saikosaponins against different sets of SARS-CoV-2 binding protein via computational molecular docking simulations.

The docking simulation study of total 23 Saikosaponins (Supplementary file) was performed on the 1.9 A crystal structure of NSP15 Endoribonuclease from SARS CoV-2 in the complex with a citrate (PDB ID: 6W01) and prefusion 2019-nCoV spike glycoprotein with a single receptor-binding domain up (PDB ID: 6VSB) which was retrieved from protein data bank (https://www.rcsb.org). The protein preparation wizard of Schr€ odinger maestro 2018-1 MM share version was used to prepare the structure by adding hydrogen, treating metal, deleting water molecules and assigning partial charges by using the OPLS-2005 force field, then assigning protonation states and determining restrained and further partial energy was minimized with 0.3 Å RMSD limit. The binding sites were defined after removing the ligand then grid was generated using a grid box volume of 10_10_10 Å.

The 2 D structure of ligands were downloaded from pubchem compound database (https://pubchem.ncbi.nlm.nih. gov) in SDF format and uploaded to the work space. Then ligprep module of maestro was used to generate the 3 D structure and OPLS-2005 force field was used to generate the different conformation of each ligand. The stable conformer of each Saikosaponin with minimum potential energy was further processed.

Glide module of Schr€ odinger suite was used to dock the each ligand into the identified binding site of grid, and the lowest binding pose of each docking run was retained. The results of simulations were analyzed using glide XP visualizer, and the important active site interactions were analyzed along with the scoring functions.

To check the potency of different Saikosaponins, the first docking simulation study was done on the crystal structure of NSP15 Endoribonuclease from SARS CoV-2 (PDB Code: Table 1 whereas details of binding interactions of top five Saikosaponins against both protein targets comprising details of protein residues involved in hydrogen bonding and hydrophobic interactions are summarized in Tables 2 and 3 (Figure 10 ).

The corona viruses are said to be enveloped RNA viruses having the largest genome among all RNA viruses and containing a replicase gene covered with nonstructural proteins (NSP). Among these NSPs, the NSP15 is a nidoviral RNA uridylate-specific Endoribonuclease (NendoU) which carries a catalytic C-terminal domain, which is specific for uridine (U). A recent study has reported that the NSP15 NendoU activity is mainly responsible for the interference of protein replication and innate immune response (Deng et al., 2017) and therefore, NSP15 is said to be very essential for biological progression of coronavirus.

The studies conducted on the structures of SARS-and MERS-CoV NSP15 revealed that the virus protein constitutes of hexamers i.e. dimers of trimers. Further, each monomeric unit is comprised of $345 amino acids which are folded into three domains viz. N-terminal, middle and NendoU C-terminal catalytic domain, where the NendoU C-terminal catalytic domain comprises two b-sheets which are antiparallel. Study have reported that the NSP15 is responsible for cutting the double-stranded (ds) RNA substrates with specificity via the Mn þ 2-dependent Endoribonuclease activity. The main active sites, conserved among SARS-CoV-2, SARS-CoV and MERS-CoV proteins are located in a shallow groove between the two b-sheets that contain six key amino acids viz. His235, His250, Lys290, Thr341, Tyr343, and Ser294.Among them, His235, His250, Lys290 constitute the catalytic triad, His235 serves as general acid, His250 acts a base, while Ser294 together withTyr343 have been found to govern U specificity.

Our observations revealed that, the Saikosaponin V having two oxane rings substituted with 3 hydroxyl group and the side chain contains 4 hydroxyl and an ester linkage showed very high binding with active site having a furrow between the two b-sheets, carries amino acids Lys290, Thr341, Tyr343 and Ser29. Among these Lys290 have been proposed to comprise the catalytic triad and Ser294 with Tyr343 are believed to govern U specificity. Four hydroxyl group of side chain provides a proper grip between hydrophobic pockets. Saikosaponin U having 2 hydroxyl and hydroxymethyl group of oxane ring and other 2 hydroxyl group and an ester linkage in side chain showed very high binding with active site carries key residues Lys290, Thr341, Tyr343, and Ser294. An ester linkage of side chain provides a proper grip between hydrophobic pockets. Saikosaponin C having two oxane ring substituted with 4 hydroxyl groups and the other chain containing furan ring, showed very high binding with active site carries residues Lys290 Thr341, Tyr343, and Ser294. Hydroxyl group of oxane ring provides a proper grip between hydrophobic pockets. Saikosaponin K is having 3 hydroxyl and hydroxymethyl group of oxane ring and other hydroxymethyl group in other chain showed very high binding with active site carrier key residues Lys290, Thr341, Tyr343, and Ser294. Hydroxyl and hydroxymethyl group of oxane ring provides a proper grip between hydrophobic pockets. Saikosaponin 1 b having two oxane ring substituted with 3 hydroxyl and hydroxymethyl group and the other chain contains hydroxyl and hydroxymethyl group showed very high binding with active site carries residues Lys290 Thr341and Tyr343. Hydroxymethyl group of other ring provides a proper grip between hydrophobic pocket.

It has been reported that, 2019-nCoV uses a densely glycosylated, homotrimeric class I fusion spike protein to make entry into the host cells. This S protein occurs in a metastable prefusion conformation which undergoes structural rearrangement so that the viral membrane gets fuses with the host cell membrane (Li, 2016; Bosch et al., 2003) . This fusion process is accelerated by binding of S1 subunit to the host-cell receptor and by transition of the S2 subunit to a highly stable postfusion conformation (Walls et al., 2017) . The receptor-binding domain (RBD) of S1 subunit undergoes hinge-like conformational movement to engage the host-cell receptor, which transiently hide/expose the determinants of receptor binding. Further, the S1/S2 subunits have been reported to possess arginine-arginine-alanine-arginine (RRAR) a protease cleavage site. Recent studies have also reported that, 2019-nCoV S and SARS-CoV S shares the same functional host cell receptor i.e. angiotensin-converting enzyme 2 (ACE2). In addition, the 2019-nCoV RBD-SD1 106 fragment (S residues from 319-591) have reported to have ACE2 binding site. The affinity with which ACE2 bounds to 2019-nCoV S ectodomain is 10-to 20-fold higher than ACE2 binding to SARS-CoV S. Due to the higher binding affinity of 2019-nCoV S for human ACE2, the spread of 2019-nCoV became apparently much prevalent from human-to-human (Chan et al., 2020) .

Simulation study on crystal structure of 2019-nCoV spike glycoprotein revealed that all the Saikosaponin buried properly into the RBD of S1 subunit and also positioned into the N-terminal domain. Due to the help of RBD this virus makes connection with ACE 2 receptor and gets entry into the host cell. Based on the binding energy, Saikosaponin U and V were the top ranked ligand. The Saikosaponin U has octadecahydropicene ring in which one side is substituted with 3 oxane ring and other side with one oxane ring. The Saikosaponin V having the same ring substituted with 2 oxane ring at one side whereas other side with one oxane rings. The extra oxane ring in Saikosaponin U provides better grip into the binding and hydrophobic pocket. The Saikosaponin U showed good interaction with triple arginine, which is responsible for recognition and cleavage of furin, this step facilitates the 2019-nCoV spike with ACE 2 receptor. Saikosaponin R also having two oxane ring substituted with 4 hydroxyl and the side chain contains one hydroxymethyl group showed very high binding affinity and buried into the domain of binding site, out of which one hydroxyl group of oxane ring which provides a proper grip between hydrophobic pocket.

In a nutshell, from all the simulation studies on both proteins, it can be concluded that Saikosaponin U and V have octadecahydropicene ring with substituted oxane ring but one more extra oxane ring of Saikosaponin U provide better grip into the wide spread binding pocket (residues from 319-519) of spike glycoprotein. The smaller structure of Saikosaponin V makes it best fit ligand into the narrow binding pocket of NSP 15. Thus we can say that Saikosaponin U and V would be the future research interest ligand as they mark the desire interaction with NSP15, which is responsible for replication of RNA and also with 2019-nCov spike glycoprotein which manage the connection with ACE2.

Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission

Moroccan Medicinal plants as inhibitors of COVID-19: Computational investigations

Novel Guanosine Derivatives against MERS CoV polymerase: An in silico perspective

Novel 2019 Coronavirus Structure, Mechanism of Action, Antiviral drug promises and rule out against its treatment

The coronavirus spike protein is a class I virus fusion protein: Structural and functional characterization of the fusion core complex

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: A study of a family cluster

In-vitro susceptibility of 10 clinical isolates of SARS coronavirus to selected antiviral compounds

Potential natural compounds for preventing 2019-nCoV infection

Saikosaponin A inhibits influenza A virus replication and lung immunopathology

Binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with SARS-CoV 3CL(pro): Structure-activity relationship studies reveal salient pharmacophore features

Structure analysis of the receptor binding of 2019-nCoV

Antiviral effects of saikosaponins on human coronavirus 229E in vitro

Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus

Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages

Drug repurposing for coronavirus (COVID-19): Insilico screening of known drugs against coronavirus 3CL hydrolase and protease enzymes

Reverse vaccinology approach to design a novel multi-epitope vaccine candidate against COVID-19: An in silico study

Molecular docking study of receptor binding domain of SARS-CoV-2 spike glycoprotein with saikosaponin, a triterpenoid natural product

In-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel

A review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme-2 and furin

Stimulation of osteogenic differentiation by saikosaponin-A in bone marrow stromal cells Via WNT/beta-catenin pathway

Targeting SARS-CoV-2: A systematic drug repurposing approach to identify promising inhibitors against 3C-like proteinase and 2 0 -O-ribose methyltransferase

Identification of chymotrypsin-like protease inhibitors of SARS-CoV-2 via integrated computational approach

Repurposing host-based therapeutics to control coronavirus and influenza virus

Structure, function, and evolution of coronavirus spike proteins

Early transmission dynamics in Wuhan, China, of novel Coronavirus-infected pneumonia

Saikosaponins: A review of pharmacological effects

A comprehensive review and perspectives on pharmacology and toxicology of saikosaponins

Anti-SARS coronavirus 3C-like protease effects of Isatis indigotica root and plant-derived phenolic compounds

Saikosaponin b2 is a naturally occurring terpenoid that efficiently inhibits hepatitis C virus entry

The crystal structure of COVID-19 main protease in complex with an inhibitor N3

Computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with SARS-CoV-2 Protease against COVID-19

Unrevealing sequence and structural features of novel coronavirus using in silico approaches: The main protease as molecular target

Peptide-like and small-molecule inhibitors against Covid-19

In-silico homology assisted identification of inhibitor of RNA binding against 2019-nCoV N-protein (N terminal domain)

From SARS to MERS, thrusting coronaviruses into the spotlight

Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion

Coronavirus Disease (COVID-2019

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Antiviral and immunoregulatory role against PCV2 in-vivo of Chinese herbal medicinal ingredients

All authors are thankful to Dr. Prajwal Nandekar, Senior Scientist, Schrodinger for helping in computational studies in present investigation.

No potential conflict of interest was reported by the author(s).

http://orcid.org/0000-0002-8232-4476 Nilambari S. Gurav http://orcid.org/0000-0001-6369-9961 Shailendra S. Gurav http://orcid.org/0000-0001-5564-2121