Employing bioactive compounds derived from Ipomoea obscura (L.) to evaluate potential inhibitor for SARS‐CoV‐2 main protease and ACE2 protein

Abstract Angiotensin converting enzyme 2 (ACE2) and main protease (MPro) are significant target proteins, mainly involved in the attachment of viral genome to host cells and aid in replication of severe acute respiratory syndrome‐coronaviruses or SARS‐CoV genome. In the present study, we identified 11 potent bioactive compounds from ethanolic leaf extract of Ipomoea obscura (L.) by using GC‐MS analysis. These potential bioactive compounds were considered for molecular docking studies against ACE2 and MPro target proteins to determine the antiviral effects against SARS‐COV. Results exhibits that among 11 compounds from I. obscura (L.), urso‐deoxycholic acid, demeclocycline, tetracycline, chlorotetracycline, and ethyl iso‐allocholate had potential viral inhibitory activity. Hence, the present findings suggested that chemical constitution present in I. obscura (L.) will address inhibition of corona viral replication in host cells.


INTRODUCTION
Convolvulaceae is one of the biggest family of flowering plants and possesses many members with medicinal and economic benefits. It comprises of 59 genera and 1,600 species. This family is widely distributed in both tropical and moderate temperature regions. One of its members Ipomoea obscura (L.) is a little climbing plant with small heart-shaped leaves and sharp-edged apex (Aiping et al., 2020;Saravana Prabha et al., 2017). It is native to parts of Africa, Asia, and certain Pacific Islands. Corolla is comprised of five fully fused petals. Seeds and fruits have been used as a cleansing agent to reduce breathing difficulties, alleviate pain, and improve vision (Shahina, 1994). The phytochemical constituents of I. obscura promote anti-inflammatory activity, antioxidant activity, anticancer activity, and antimicrobial activity against certain microorganisms. Ayurveda has explored numerous medicinal properties of this plant against dysentery, ulcers, hemorrhoids, and swellings (Christophe & Pharm, 2002). This plant grows mainly on fences or low ground cover as substrate in disturbed areas.
Severe acute respiratory syndrome (SARS) is a perilous pulmonary infection triggered by single stranded (+) sense RNA virus. Coronaviruses (CoVs) are largest family of RNA viruses with size ranging from 60 to 140 nm. It is further classified into four genera as α, β, γ, and δ. SARS-CoV-2 falls under the genus of β coronavirus (Markus et al., 2019). The main pathways of COVID-19 infection in humans are through droplets, aerosols, feces, and mouth mucus membranes of infected person and cause adverse symptoms such as typical acute respiratory infection, cough, fever, myalgia, sneezing, acute kidney injury, fatigue, acute liver injury, diarrhea, a sore throat, breathing collapse, as well as vomiting (Anthony & Stanley, 2015). The genome of corona virus composed of nonstructural proteins, accessory proteins, and structural proteins. Structural proteins had four major parts: one is spike surface glycol protein (S), tiny envelope protein (E), matrix protein (M), and nucleo-capsid protein. The corona virus protein is mainly responsible for entry of SARS-CoV-2 in to host cell (Yong & Edward, 2020).
Angiotensin converting enzyme 2 (ACE2) is an integral membrane glycoprotein mainly present in kidney, endothelium, lungs, and heart. The receptor-binding domain (RBD) of spike protein includes important amino acids (L455, F486, Q493, S494, N501, and Y505) that were involved in interaction and communication of single stranded RNA coronavirus genome (Andersen, Rambaut, Lipkin, Holmes, & Garry, 2020;Yifei, 2020). So far, six RBD amino acids have shown best binding activities against ACE2 receptors and for determining the host tropism of SARS-CoV-like virus. The spike envelope glycoprotein interacts with cellular receptors ACE2 to initiate membrane fusion and viral replications after the proteolytic cleavage event. Subsequently, viral genome is released into the cytoplasm and replicated viral particles then ini-tiate the viral replication and cause adverse effect on a host organism. By blocking the ACE2/SARS-CoV-2 interaction, we could cease the viral replication and multiplication. The ACE2 and spike glycol protein may be considered as the attractive drug target for the discovery and development of effective antiviral drug against the viral disease (Bui et al., 2020;Fatima & Florian, 2020;Othman et al., 2020).
One of the best characterized drug targets among CoV is the main protease (M pro ). Besides the papain-like protease, this enzyme is necessarily aimed at the dispensation of polyproteins that endure translated since the viral RNA. Impeding the action of this enzyme would block viral replication. Subsequently, proteases reported from Homo sapiens do not cleave the functional site by hybrid analogues, which is yet to be discovered. (Fatima & Florian, 2020;Mothay & Ramesh, 2020;Macchiagodena, Pagliai, & Procacci, 2020;Nisha Muralidharan, Sakthivel, Velmurugan, & Michael Gromiha, 2020). At present, there are no effective inhibitors against the novel corona virus; therefore, developing protein-based inhibitors for corona virus may emerge as a prerequisite strategy for curbing this virus. Computational methodologies have become an essential tool for drug discovery programs for the identification to lead optimization and formulation. Approaches such as ligand or structural based in silico techniques are commonly used in many discovery efforts. Henceforth, present investigation is carried out to identify bioactive compounds from I. obscura (L) and examine its antiviral activity against COVID-19 by the in silico approach.

Sample leaves collection and hydrodistillation
The healthy mature and fresh leaves of I. obscura (L.) Ker Gawl were collected from Madurai district of Tamil

Chemicals reagents and apparatus
All chemical reagents were purchased from Sigma-Aldrich and applied for analysis with no further purification. The major apparatus included a hydrodistillation apparatus, a polarimeter, a Jasco V630 spectrophotometer, and a gas chromatography-mass spectrometer (GC−MS).

Ethanolic extract preparation
The fresh leaves of I. obscura (L.) were carefully washed in running tap water, shade dried for 1 week, and powdered in an electric mixer grinder. The powdered leaves were subjected to ethanol solvent extraction. In total 300 g of dried plant powder was extracted utilizing Soxhlet extraction with 1.5 L of ethanol in a random shaker for 72 h at room temperature. Solvent extract was then evaporated using a rotary evaporator. The dried extract was collected in airtight bottles and stored at 4 • C for further studies.

Phytochemical analysis
Based on the preliminary phytochemical screening, the ethanolic leaf extract of I. obscura (L.) was subjected to GC-MS analysis on a Perkin Elmer gas chromatograph Clarus 500 Perkin Elmer system comprising an AOC-20i auto sampler and a gas chromatograph interfaced to a mass spectrometer prepared with Elite 5MS (5% diphenyl/95% dimethyl polysiloxane) fused a silica column (30 mm × 0.25 mm × 0.25 μm df) that operated in electron impact mode with an ionization energy at 70 ev. Helium gas (99.999%) was utilized as a carrier gas at a constant flow rate of 0.1 mL/min and volume of 2 L was analyzed. The injector temperature was maintained at constant 250 • C. Compounds present in ethanolic leaf extract were detected and cross-checked by comparing their retention indices and mass spectra fragmentation patterns using stored database in a computer library and with published literature, NIST08s.LIB (Lafferly, 1989) and WILEY8.LIB (Stein, 1990).

Molecular docking simulation
Molecular docking is a computational approach for scrutinizing the interaction between the therapeutic target and a small molecule. In this investigation we performed in silico docking by applying Glide 5.5 (Dik-Lung, Daniel, & Chung, 2011;Glide, 2009), against respiratory therapeutic target ACE2 exhibited in human and M Pro in SARS-CoV-2. The in silico docking exploration involves five steps, which are as follows.

Collection and preparation of therapeutic target proteins
PDB Protein retrieval: The therapeutic targets ACE2 (PDB: 6M0J) and M Pro (PDB: 6LU7) were retrieved from PDB structure, which possessed a resolution value 2.16 and 2.45 Å, respectively (Jin et al., 2020;Lan et al., 2020). Target proteins were preprocessed such as refinement, assigning bond orders, treating metals, and treating disulfides, building missing heavy atoms, formal charges, adjusting bond orders, adding hydrogen atom, and undesirable water molecule using PP-wizard. The protein structure energy was minimized until root mean square deviation (RMSD) cutoff was touched 0.30 Å. The consolidated protein structure was subsequently taken into receptor grid generation panel for receptor lattice generation, as binding pocket

HIGHLIGHTS
• Identified the potential bioactive compounds from Ipomoea obscura (L.) leaf extract.
• Molecular docking studies against ACE2 and M Pro in SARS-CoV genome.
• Molecular interaction study claims that urso-deoxycholic acid competitively binds with hACE2 and M Pro .
information plays an important role in structure-based drug designing. The binding pocket residues were gathered from the literature. The

Preparation of I. obscura (L.) chemical constitution
Eleven bioactive compounds from medicinal plant I. obscura (L.) were identified. The ligands were subjected into Ligprep to optimize lowenergy, three-dimensional (3D) structure with proper chiralities for each molecular arrangement. It generated various structure isolated molecules with ionization states, tautomers, stereo chemistries, and ring conformations. The force field was optimized by OPLS3.

In silico docking into therapeutic
Glide is a popular and reliable tool for docking investigations, and here we employed glide version 5.5 performing for docking studies. The refined target protein structure (PDB ID: 6LU7, 6M0J) was utilized as the receptor, and prepared bioactive compounds from medicinal plant I. obscura (L.) were docked with an active site of target proteins by the Glide XP model . We analyzed the protein-ligand interactions, glide score, and energy by a Glide XP visualizer.

ADME properties assessment
Nowadays a failure rate of drug candidate increases in clinical stages due to undesired pharmacokinetics properties. The Absorption, Distribution, Metabolism, and Excretion (ADME) properties evaluation plays an important role in drug discovery. However, determination of

RESULTS AND DISCUSSION
The GC-MS is extensively used in medical, pharmaceutical, envi-

Docking simulation results of compounds present in I. obscura (L.) with ACE2 protein and M Pro
Interaction exposed that the key residues SER 511, TYR 196,GLN 102,and GLU 208 Table 3 and Figure 2. The 6LU7 is the M pro found in the CoV associated with SARS and emerged as a potential drug target for COVID-19 (Khan, Khan, Khan, Ahmad, & Ansari, 2020 Figure 3. The glide score −7.111 kcal/mol and glide energy −46.632 kcal/mol were calculated (see Table 4).

ADME properties prediction
The bioactive compounds were further assessed for their drug-like behavior of ADME by use of QikProp. For the five bioactive molecules, the aqueous solubility (QPlogS) necessary for absorption and delivery of drug inside the human body range between −5.766 and −2.623, respectively. The percentage of human oral absorption for the bioactive compounds ranged from 23% to 95%. The predicted value of binding to human serum albumin (QPksha) fitted well within the acceptable range (∼ −0.637 to −0.060). The predicted blood/brain barriers were within the acceptable range (∼ −2.242 to −1.357). All the ADME properties are in adequate quality for solubility and permeability of cell membrane (Table 5): S in mol/L (acceptable range −6.5 to 0.5); percentage of human oral absorption (<25% is poor and >80% is high); prediction of binding to human serum albumin (acceptable range −1.0 to 1.5); prediction of brain/blood (acceptable range −3.0 to 1.2); the predicted rotatable bonds fit well with acceptable range 6-8; the predicted hydrophilic surface accessible solvent area (SASA) is under an acceptable range 158.858-314.842; molecular weight (<500 Da); hydrogen bond donor (<5); hydrogen bond acceptor (<10); predicted octanol/water partition coefficient log p (acceptable range −2.0 to 6.5).

Molecular dynamics trajectory analysis
The RMSD and RMSF graphs for ACE2 versus urso-deoxycholic and M pro versus urso-deoxycholic are plotted in Figures 4 and 5. The M pro -urso-deoxycholic showed slight fluctuations at 5 than the entire

ACKNOWLEDGMENTS
The authors thank ICGEB authorities especially to the Director ICGEB, New Delhi for providing the necessary infrastructure and ICGEB core funds for this research work and to analyze and screen the molecules for COVID19 disease from our species of interest. We also thank Director General, ICGEB Trieste, Italy and Department of Biotechnology (DBT) for financially supporting the research activities at ICGEB. We thank the authorities of Karpagam Academy

CONFLICT OF INTEREST
The authors declare no competing financial interest.

AUTHORS CONTRIBUTION
TK, EM, MA, and BB conceived and designed the experiments and wrote the paper. PSP, AM, AJ, and KBH contributed reagents and materials, performed the experiments, analyzed and interpreted the data, and wrote the paper. PS, AVA, KS, and VKG analyzed and interpreted the data and wrote the paper.