key: cord-0528166-x91860xg
authors: Vardhan, Seshu; Sahoo, Suban K
title: In silico ADMET and molecular docking study on searching potential inhibitors from limonoids and triterpenoids for COVID-19
date: 2020-05-16
journal: nan
DOI: nan
sha: 2051f6d15baa130cf6ac28ed49ed360fb9864c78
doc_id: 528166
cord_uid: x91860xg

The computational strategies like molecular docking, simulation, in silico ADMET and drug-likeness prediction were adopted to search potential compounds that can inhibit the effects of SARS-CoV-2. Considering the published literatures on medicinal importance, total 154 phytochemicals with analogous structure from limonoids and triterpenoids were selected to search potential inhibitors for the five protein targets of SARS-CoV-2, i.e., 3CLpro (main protease), PLpro (papain like protease), SGp-RBD (spike glycoprotein-receptor binding domain), RdRp (RNA dependent RNA polymerase, ACE2 (angiotensin converting enzyme 2). Analyses of the in silico computational results and reported medicinal uses, the phytochemicals such as 7-deacetyl-7-benzoylgedunin, epoxyazadiradione, limonin, maslinic acid, glycyrrhizic acid and ursolic acid were found to be effective against the target proteins of SARS-CoV-2. The protein-ligand interaction study revealed that these phytochemicals bind at the main active site of the target proteins. Therefore, the core structure of these potential hits can be used for further lead optimization to design drugs for SARS-CoV-2. Also, the plants extracts like neem, tulsi, citrus and olives containing these phytochemicals can be used to formulate suitable therapeutics approaches in traditional medicines.

The ongoing research focused globally to formulate suitable therapeutic approaches to control the effects of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to human life that caused the disease COVID-19. The first patient infected with SARS-CoV-2 was detected in December, 2019 at Wuhan, China. Subsequently, the virus spread across 187 countries and territories due to its high human to human contagious nature and infected 4006,257 as of 11 th May 2020 with a total death of 278,892 [1] . The uncontrolled spread pushed the World Health Organization (WHO) to declare the outbreak of SARS-CoV-2 as a public health emergency of international concern (PHEIC) on 30 th Jan 2020, whereas a pandemic on 11 th

March 2020 [2] . Effective steps like identification of infected persons through rapid diagnosis, and quarantined them to stop further spread of the virus are underway to fight against this pandemic. However, the non-availability of medically proven efficacious drugs or vaccines is the main concern of COVID-19 pandemic [3] . Repurpose of some approved drugs like hydroxychloroquine, remdesivir, favipiravir, arbidol, hydroxychloroquine/azithromycin, lopinavir/ritonavir and lopinavir/ritonavir combined with interferon beta etc. are used, but despite some promising results further clinical results are required to examine their mechanisms of inhibition, efficacy and safety in the treatment of COVID-19 [4] [5] [6] [7] [8] . Therefore, both computational and experimental approaches are adopted to search suitable drugs from the library of FDI-approved drugs and also drugs under clinical trial but not yet approved to repurpose against the COVID-19. Simultaneously, the recent reports supporting the use of traditional medicines as adjuvant for treating COVID-19 [9] [10] [11] and therefore, efforts are also going to integrate the use of both western drugs and traditional medicines to formulate suitable therapeutic strategies.

The computational approaches like dynamic simulations, molecular docking, drugs likeness prediction, in silico ADMET study etc. are valuable tools to screen potential drugs/molecules from various databases that contained in lakhs. The computational screening save both experimental cost and time in the field of drugs discovery. Considering the recent results of the use of traditional medicines in managing the epidemic of COVID-19 [9] [10] [11] , our ongoing research is focused on to screen phytochemicals found mainly in the Indian medicinal plants with the important objectives: (i) to search phytochemicals that bind effectively at the active sites of the protein targets of SARC-VoV-2, (ii) to propose important hits that can be further investigated for lead optimization and drugs discovery, and (iii) to provide computational evidences for formulating Indian traditional medicines in controlling the spread and effects of SARC-VoV-2. Our literatures survey revealed that the triterpenoids like 3βfriedelanol from Euphorbia neriifolia, quinone-methide triterpenoids extracted from Tripterygium regelii (Celastraceae) and glycyrrhizin from Glycyrrhiza glabra are experimentally proven to inhibit the effects of SARS-CoV (first identified in Guangdong, China in 2002) [12] [13] [14] [15] . Also, our recent molecular docking studies of phytochemicals against the protein targets of SARS-CoV-2 supported the higher binding affinity towards limonin, a triterpenoid found in citrus [16] . ACE2, PDB ID: 6M17) were retrieved from the PDB database (www.rcsb.org).

The molecular docking studies were carried out by using Autodock Vina1.1.2 [18] , and the binding energies of the limonoids and triterpenoids were estimated towards SARS-CoV-2 therapeutic targets. The proteins 3D structures retrieved from RCSB PDB databases, and some of the missing residues of the proteins were modelled using Swiss model online tools to generate the fine structures. The refined protein structures were analysed using the Ramachandran plot.

Different conformations, hydrophobic sites are identified and analysed using BIOVIA Discovery studio visualization tool.

The protein structure flexibility and dynamics simulations were performed using CABSflex2.0 online simulation tool with the default options [19] . The simulated model generated through trajectory clustering k-medoid method. This tool calculates the protein dynamic simulations at 10 ns, predicts fluctuations and protein aggregation propensity.

After the molecular docking analysis of 154 compounds with the five protein targets of SARS-CoV-2, the absorption, distribution, metabolism, elimination and toxicity (ADMET) of the 47 best dock scored limonoids and triterpenoids were further screened using online tool and antimalarial, and also used routinely in the Indian traditional medicine (ayurveda) to treat various health problems. Therefore, a basic preliminary screening of phytochemicals from limonoids and triterpenoids were carried out based on the published literatures, and total 154 phytochemicals were selected, and then studied against the five protein targets (3CLpro, PLpro, spike glycoprotein, RdRp and ACE2) of SARS-CoV-2 (Scheme 1).

Scheme 1. Flow-chart showing the steps to screen phytochemicals for the COVID-19.

The selected 154 phytochemicals were screened against the five important protein targets,

i.e., Mpro or 3CLpro, PLpro, SGp-RBD, RdRp and ACE2 of SARS-CoV-2 by performing molecular docking analysis using the Autodock Vina computational program. The structural spike glycoprotein (S protein) of SARS-CoV-2 recognizes with the transmembrane protein of host cell receptor ACE2 [22, 23] . This also internalizes the virus into the endosomes where the conformational changes take place in the spike glycoprotein. It acts as a viral fusion peptide that covers up S2 cleavage that occurs during virus endocytosis. Thereafter, RdRp facilitates the viral genome replication [24] . The main protease 3CLpro and PLpro act as proteases in the process of proteolysis of viral polyprotein into functional units [25] . In short, the SpG and ACE2 are collectively involved in disease establishment and 3CLpro, PLpro, RdRp involved in translation and replication lead to virus proliferation in the host cell. Therefore, these five proteins were considered as the therapeutic targets for the molecular docking with the selected 154 phytochemicals.

The dock score of the compounds against each protein are summarized in Table S1 . The table of dock score of 154 phytochemicals against the five target proteins revealed that majority of the phytochemicals showed dock score higher than -6.5 Kcal/mol, and comparably higher dock score than the western drugs hydroxychloroquine and arbidol studied as a control. As the core part of all the compounds are similar, 20 compounds from each protein that showed the maximum dock score were selected for further screening (Table S2) . Accordingly, total 47 compounds were considered for further in silico ADMET and drug-likeness study.

The 47 compounds selected based on their higher dock score were screened further for in silico ADMET and drug-likeness study. Considering the computational prediction and the available experimental evidences on their pharmaceutical properties, the best 6 compounds for each protein target (i.e., total 15 phytochemicals) were selected for protein-ligand interaction study to intendity the potential hits that bind at the catalytic sites of the respective protein ( Table   1) (S1-S6), the active site residues are 140-145 and 163-166 amino acids in the domain II. The best 6 compounds finalised based on the dock score with 3CLpro followed by in silico ADMET and drug-likeness are summarized in Table 2 along with the important molecular interactions.

The main protease complex with the phytochemicals 7-deacetyl-7-benzoylgedunin is

showing binding affinity at the catalytic dyad ( Fig. 1 and Fig. S1 ). Catalytic dyad pocket shows ligand binds to Cys145 forming π-alkyl interaction and His41 forming π-cation interactions [44] . 

PLpro consists of four domains such as thumb, finger, palm and ubiquitin like domain.

The active site located in between the thumb and palm domains [45] . The subunits consist of catalytic triad (Cys112, His273, Asp287), where the active site is located. PLpro NSP3 domain contains S2/S4 inhibitor binding sites. Therefore, the molecular screening of phytochemicals that docked at specific residues of S2/S4 site could inhibit the activity of PLpro [45] . The dock score along with the molecular interactions of the best 6 phytochemicals are summarized in Table 3 .

The protein-ligand interaction study revealed that the epoxyazadiradione binds exactly at Fig. 2 and Fig. S3) . Epoxyazadiradione binding at the pocket of catalytic triad (Cys112, His273, Asp287) residues can hindrance the enzymatic activity of PLpro [46, 47] . Further, the MD simulations generated RMSF plot of PLpro showing available contacts to substrate at chain B (5-10, 170-185, 265-270) residues (Fig. S4) . and maximum binding energy -9.2kcal/mol ( Fig. 3 and Fig. S5) process and those are resembling in the RMSF plot (Fig. S6) . Glycyrrhizic acid is also encapsulated in the receptor cavity with maximum binding energy -9.9kcal/mol and the site located between NiRAN domain and β-hairpin structure that polymerize 3ʹ end [49] . Glycyrrhizic acid could cause interference to β-hairpin polymerize activity due to its binding affinity at the active site residues Tyr129, Ser772, Asn781, Asn138

and closest non-covalent interactions to Gln773, Lys714, Tyr32, Asp711, Ser784, Lys780, Gly774, Ser778, Ala706, Thr710, Asp135, His133, Cys139, Lys47. Other limonoids showing binding affinity to RdRp hydrophobic cavities are 7-deacetyl-7-benzoylgedunin, 7deacetylgedunin, limonin-17beta-D-glucopyranoside and obacunone ( Table 4 ). Lys47, Tyr32. π-π: Ala706

Limonin17bDglucopyranoside -8.9

A :H-His133, Ser784, Lys780, Ala706, Asp711 C: Thr710, Gly774 VDW-Asn138, Phe134, Ser709, Lys714, Ser772, Gln773, Asp135

Obacunone -8.7

A:C:Thr319, Thr252, VDW: Lys267, Tyr265, Arg249, Leu251, Ser255, Val320, Ile266, Phe321, Trp268, Pro323, Pro322

The spike proteins determine the virion-host tropism that includes the entry of the virions into host cells, and their interactions with human ACE2. It constitutes the target prominent that plays a key role in the development of the procatylitics and therapeutics [50] . Two states in receptor binding domains (RBD) namely buried (lying state) or exposed (standing state) were observed in the recent reports coming up with structures of the different CoV's that are inherently flexible with the RBD's [50] . Due to its structural importance, we focused on SGp-RBD inhibition study by screening 157 phytochemicals through molecular docking and binding confirmations at 3 active sites that could inhibit the SGp and hinder SGp-ACE2 complex formation. Spike glycoprotein RDB contains 333-527 residues where the active sites are located [51] . The molecular interactions between the best 6 phytochemicals with the SGp sites are shown in Table 5 .

Maslinic acid binding at the RBD where the initial contact was made towards ACE2.

Maslinic acid with binding energy -9.3kcal/mol, forming a complex at C chain with a possible interactions to site-1: H bond to Arg441, non-covalent (VDW) interactions to Ser456, Leu443, Pro459, ly464, Lys465 and (Alkyl-π-alkyl) interactions to His445, Phe460, Arg444, Pro477, Pro466 ( Fig. 4 and Fig. S7 ). It is also forming a complex at E chain site-2, H bond to Asp480, Tyr440 an unfavourable donor-donor interaction, Alkyl-π-alkyl to Tyr484, tyr491, Lys390 and VDW to Tyr436, Gly482, Thr486, Thr487, Asn479, Tyr481 (Fig. S8) . In site-3, the maslinic acid binding to an active cavity at chain E residues Ser358, Ala331 forming H bond and non-covalent VDW interactions to Phe360, Asn427, Thr332, Asn424, Asn357, Thr359, Ile428, Trp423, Phe329, Tyr356, Thr425, Arg495, Asn330 (Fig. S9) . Maslinic acid collectively forms hydrophobic interactions at site-1, site-2 and site-3 that could hinder the SpG-RBD complex with 

ACE2 plays a key role in cardio renal disease and acts as a human host receptor for the SARS and helps in viral-host interactions [52] . ACE2, a human viral receptor binds to SGp RBD at a specific site that establishes the primary contact for host-pathogen interaction [53] . The active site residues of ACE 2 were studied by using site-directed mutagenesis and it was found that Arg273 plays a critical role in substrate binding, whereas the consequence of its replacement results in the abolishment of the enzyme activity. Also, His 345 plays an important role as a hydrogen bond donor/acceptor to form the tetrahedral peptide intermediate [53] . From the computational approaches, the best 6 phytochemicals showing higher binding affinity at hydrophobic sites along with the molecular interactions are summarized in Table 6 . The interactions of other phytochemicals are summarized in Table 6 . 

In summary, the screening of 154 phytochemicals from limonoids and triterpenoids by molecular docking, in silico ADMET and drug likeness with five protein targets (3CLpro, PLpro, RdRp, SpG, ACE2) resulted 15 effective phytochemicals, from which best six phytochemicals were proposed as potential hits against the SARS-CoV-2. Among the six, the two neem isolates 7-deacetyl-7-benzoylgedunin posing binding energy -9.1kcal/mol to cleave substrate binding site catalytic dyad residues (Cys145 and His41) of 3CLpro, a potential lead to inhibit proteases involved in the process of proteolysis of viral polyprotein into functional units and

Epoxyazadiradione an active lead inhibiting the PLpro S2 subunit with -8.4kcal/mol binding energy to pocket consisting of Catalytic Triad (Cys112, His273, Asp287). A citrus fruit seed isolate limonin inhibiting the RdRp substrate binding site at NSP12-NSP7 residues with the B.E -9.2kcal/mol that inhibits the polymerase activity due to hindrance in NiRAM replication fork.

Neem and Citrus fruits isolates potentially blocks the SARS-CoV-2 translation and replication functions. Maslinic acid, corosolic acid and 2-hydroxyseneganolide are potent inhibitors of SGp-RBD active sites that hinder to dock hACE2 receptor. Glycyrrhizic acid including ursolic acid could potentially interfere with the ACE2-SGp RBD complex with binding energy -10.3kcal/mol. Olives, holy basil and lycorice triterpenoids potentially obstruct SGp and ACE2 receptors disease establishment. Therefore, the computational predictions along with the reported pharmacological properties postulated that the limonoids and triterpenoids are potential against SARS-CoV-2 target proteins. The outcomes will be useful in formulating therapeutic strategies using the traditional medicines as well as the potential hits can be used for lead optimization for drugs discovery for SARS-CoV-2.

Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2

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Traditional Chinese Medicine in the Treatment of Patients Infected with 2019-New Coronavirus (SARS-CoV-2): A Review and Perspective

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SARS-CoV 3CLpro inhibitory effects of quinone-methide triterpenes from Tripterygium regelii

An Overview of Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3CL Protease 24

Inhibitors: Peptidomimetics and Small Molecule Chemotherapy

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Mechanistic Perspectives of Maslinic Acid in Targeting Inflammation

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Oleanane triterpenoids in the prevention and therapy of breast cancer: current evidence and future perspectives

Cytotoxic and Melanogenesis-Inhibitory Activities of Limonoids from the Leaves of Azadirachta indica (Neem)

Cytotoxic and Apoptosis-Inducing Activities of Limonoids from the Seeds ofAzadirachta indica(Neem)

Insights on inhibition of Plasmodium falciparum plasmepsin I by novel epoxyazadiradione derivatives -molecular docking and comparative molecular field analysis

Effect of Limonin and Nomilin on HIV-1 Replication on Infected Human Mononuclear Cells

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Structure of Mpro from COVID-19 virus and discovery of its inhibitors

Catalytic Function and Substrate Specificity of the Papain-Like Protease Domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus

The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds

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