Naturally occurring phytochemical as inhibitors from Catharanthus roseus: An In-silico approaches for drug development against COVID-19

The current outbreak of the novel 2019 Coronavirus disease (COVID-19) is caused by SARS-CoV-2, has developed a threat to the world's human population. There are no effective therapies or vaccines yet, urging the serious efforts to tackle this pandemic situation.SARS-CoV-2 spike protein, papain like protease protein (PLPRO), main protease (3CLpro) and RNA dependent DNA polymerase are key factors in the virus infectious process and have been identied as potential targets for therapeutic formulation. Most people in India depend on conventional Indian medicine (phytochemical compounds) to treat diseases due to lower cost, easier accessibility and no adverse effects. A lot of studies have recently shown that phytochemicals contain an effective anti-viral activity. This study aims to investigate phytochemicals metabolites from the IMPPAT database (Indian Medicinal Plants Database) in order to identify potential COVID-19 inhibitors using in silico approaches. Certain phytochemical compounds with structure analogs like hydroxychloroquine and chloroquine from the IMPPAT database were taken for interaction with SARS-CoV-2 proteins. The Apparicine, 12-Chlorotabersonine, AC1NSULH and Vindolininol identied from Catharanthus roseus were further checked the ADMET property as well as ‘Lipinski’s rule and resulted in a strong binding anity of (-7.6,-7.5) and (-7.6 -7.5) kcal/mol respectively for spikes and papain like protease protein of SARS-CoV-2. Our results indicate that these specic compounds can be used as effective inhibitors and help to pace up the drug development against SARS-CoV-2. Further investigation and testing of these inhibitors against SARS ‐ CoV ‐ 2 are however required to check their clinical trial candidacy. this work is aimed to identify potential plant-based compounds that can serve as substitutes and can help us in blocking the ‘SARS-CoV2 ‘protein function. In the near future, upon further laboratory experiment these phytochemicals that serve as good contestants for the treatment of diseases caused by eliminating SARSCoV2-associated pathogenesis.


Introduction
The epic coronavirus disease 2019 (COVID- 19), as a result of the extreme acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is within the midst of world panic and global health challenge considering that china, December 2019. As of 12 th May 2020, the World Health Organization (WHO) has stated that 40,88,848 and 2,83,153 death cases had been con rmed global, and it has spread to 197 countries [1].
The COVID-19 spread unexpectedly over the globe, turning into a pandemic inside half a month and apart from China, the information of the carnage started out pouring in from nations like the USA, Italy, Spain, France, Germany and Iran along with India on an everyday basis. After the rst case of the COVID-19 on 30 th January 2020 in India, there turn out to be 83,004 cases and 4,337 deaths as in keeping with legitimate information of the ministry of health and family welfare, India on 27 th May,2020 [1].
Tragically, there has been no perceptible achievement in the administration of this ailment to date and the patient is given a treatment dependent on his recognizable and diagnosable manifestations. Albeit a few endeavours have been made in the research and development of the diagnostics, therapeutics and vaccines for this novel coronavirus [2], there occurs no chemotherapeutic agent up to now which has been shown unequivocally to be compelling in treating human illnesses because of a minute virus. To combat this lethal COVID-19, a number of ordinary drugs [3], [4] like chloroquine, hydroxychloroquine, remdesivir, and so forth, have been tried and located with positive healing effect in vitro. However, the clinical drug response isn't very promising and harmfulness stays an unavoidable issue causing genuine antagonistic effects [5].
Coronaviruses possess the largest genomes (26.4-31.7 kb) among all known RNA viruses, with G + C contents varying from 32 to 43 percent. Variable numbers of small ORFs are present between the various conserved genes (ORF1ab, spike, envelope, membrane and nucleocapsid) and, downstream to the nucleocapsid gene in different coronavirus lineages [6].
Two-thirds of viral RNA, mainly located in the rst ORF (ORF1a/b) translates two polyproteins, pp1a and pp1ab, and encodes 16 non-structural proteins (NSP), while the remaining ORFs encode accessory and structural proteins. The rest part of virus genome encodes four essential structural proteins, including spike (S) glycoprotein, small envelope (E) protein, matrix (M) protein, and nucleocapsid (N) protein [7], and also several accessory proteins, that interfere with the host innate immune response. In SARS coronavirus, a de ned receptor-binding domain (RBD) on S protein mediates attachment of the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2). The most abundant and conserved N protein of SARS-CoV-2 is 90 percent identical to SARS-CoV N protein. Moreover, the SARS-CoV-2 genome showed 80 percent identity with SARS-CoV and 89 percent similarity with Bat SARS-like CoV at nucleotide level [8]. Most SARS-CoV-2 genomic encoded proteins are similar to SARS-CoVs, though some deviations however exist. In order to be able to understand the best phytochemical that could bind to most conserved protein spike ectodomain protein [9], papain-like protease protein, main protease protein [10] and RNA-dependent RNA polymerase and help stop the spread of the virus, many researchers have begun to use molecular docking techniques to identify phytochemical that can be used to inhibit.
Because of the inherent aspect outcomes of the synthetic chemical compounds used in allopathic tablets, a sizeable populace has exchanged over to the traditional system of medicine (natural medication) for his or her number one tness care. Ayurveda, the age-old Indian system of medicine, is an increasing number of becoming a well-known system to nancial institution on. The ayurvedic treatment has become an alternative to conventional medicines due to several reasons including easy availability, less or no side effects and less cost. India has consistently been a rich supply of therapeutic plants in view of a few agro-climatic zones [11]. The act of utilizing therapeutic plants to treat human skin including mending of wounds and consume wounds, antifungal, antiviral, antibacterial applications against skin contaminations has long history and is continuing for essential medicinal healthcare even in the cutting edge [12]- [13].
ZINC is a public database of available commercially for virtual-screening compounds. It contain a library of 727 842 molecules, each with 3D structure, using catalogs of compounds from vendors. Signi cant biological protonation states have been attributed to the molecules and they are annotated with characteristics such as molecular weight, calculated LogP and number of rotatable bonds etc. Each molecule in the library contains vendor and purchasing information and is ready for docking using a number of popular docking programs [14]. IMPPAT is a curated database stands for Indian Medicinal Plants, Phytochemistry and Therapeutics. It is a comprehensive online database on the phytochemistry of Indian medicinal plants will enable computational approaches towards natural product based drug discovery. In this direction, IMPPAT has a manually curated database of 1742 Indian Medicinal Plants, 9596 Phytochemicals, and 1124 therapeutic uses spanning 27074 plant-phytochemical associations and 11514 plant-therapeutic associations. In particular, the cure effort resulted in a non-redundant 9596 phytochemicals in silico library with standard chemical identities and structure properties. Using cheminformatic approaches, scientist has computed the physicochemical, ADMET (absorption, distribution, metabolism, excretion, toxicity) and drug-likeliness properties of the IMPPAT phytochemicals. It also show that the stereochemical complexity and shape complexity of IMPPAT phytochemicals differ from libraries of commercial compounds or diversity-oriented synthesis compounds while being similar to other libraries of natural products [15].
Catharanthus roseus is a one of evergreen consistently sprouting herb which has its inception in focal Madagascar island. Periwinkle is recorded for as back as 50 B.C. in people medication writing of Europe as diuretic, hemorrhagic and wound recuperating. Because of high therapeutic value of many phytochemical residences, globally scientist and researchers perceive dozens of alkaloids: some of them lower blood sugar levels (supplying folk treatments with medical provenance) and blood pressure [16]. The point of present investigation is to check the antiviral capability of C. roseus xings against COVID-19 by methods for computational strategies. The middle intent of this study is to inhibit or weaken the interactions between the receptor and RBD by using phytocompounds that could block or abate viral entry into the host cells [16].
Hence, this work is aimed to identify potential plant-based compounds that can serve as substitutes and can help us in blocking the 'SARS-CoV2 'protein function. In the near future, upon further laboratory experiment these phytochemicals that serve as good contestants for the treatment of diseases caused by eliminating SARSCoV2-associated pathogenesis.

Retrieving of Natural phytochemicals as Ligands:
The smiles format of recently used drugs as ligands (Hydroxychloroquine and chloroquine) [17]; [20] were downloaded from the PubChem database and then potential analog were identi ed in IMPPAT database which include all the photochemical compounds from Indian medicinal plants [15] (Fig. 2) (Table 1).  A drug likeliness property of all potential phytochemical compounds were assessed using the molinspiration server [21].

ADMET analysis:
The SwissADME and PROTOX-II webservers were provided with all potential phytochemical compounds as ligands in smiles format for ADMET analysis. The SwissADME is a tool used to forecast compound ADME and pharmacokinetic properties, toxicity, water solubility, and molar refractivity [22]. PROTOX-II is an oral toxicity rodent server that forecasts the value of LD50 and the level of toxicity of a request compounds in six distinct classes varying from LD5 to 5000 with its implications [23].

In silico docking of phytochemicals with targets Protein of SARS-CoV2:
The active site pocket present in the spike ectodomain protein, papain-like protease protein, main protease protein and RNA-dependent RNA polymerase of SARS-CoV2 were analysed using the CASTp server [24]. Active pocket with the maximum volume as well as surface area were considered for molecular docking studies with all phytochemicals [25] through the AutoDock [26] tool. For further analysis, each potentials phytochemical with the minimum binding energy was considered with proteins of SARS-CoV2 [25]. The results were analyzed using MGL tool 1.5.6 to evaluate the residues of amino acids associated with drug binding [26] and the hydrophilic and hydrophobic interactions were measured using PyMol [27] in docking with the maximum binding energies of Ligands receptors.

Preparation of SARS-CoV2 protein as receptor:
All recently deposited protein structure of SARS-CoV2 protein including spike ectodomain protein (open state) (PDB ID: 6VYB), papain-like protease protein (PDB ID: 6W9C), main protease protein (PDB ID: 6YB7) and RNA-dependent RNA polymerase (PDB ID: 7BTF) retrieved from PDB database in native form after eliminating ligands, carbohydrates etc components attached to it ( Fig.1a and Fig.1b). To remove water molecules and add polar hydrogen charges in MGL tool 1.5.6, all PDB structures were further processed.
CASTp server analyzed all the target protein structures for identi ed amino acid residues present in the active site pocket. Spike ectodomain protein showed smaller surface area versus volume binding pocket with amino acid residues ranges from 24-THR to 192-GLN while papain-like protease protein, main protease protein and RNA-dependent RNA polymerase showed larger surface area versus volume binding pocket with amino acid residues ranges from 9-THR to 302-ASP, 90-MET to 164-ASP, 38-TYR to 1064-HIS respectively ( Fig.3a and Fig.3b; Table 2). Only a few phytochemical compounds were recovered, based on the Tanimoto coe cient. For drugs hydroxychloroquine analog compounds as (-)-Apparicine (CID_5281349), Apparicine (Gomezine) (CID_5320486) and 12-Chlorotabersonine (CID_25109982) while chloroquine drug analog compounds as AC1NSULH ((16R)-Dihydrositsirikine 6519-26-2) (CID_5316739), Vindolininol (CID_101287751) and Velbanamine CID_102399433 were further taken for consideration (Table 1). Majority all the phytochemicals compounds belongs to Catharanthus roseus having wide ranges of alkaloids used for therapeutics.

Discussion
Even the rate of COVID-19 infection in shocking outbreaks worldwide is increasing day by day with a big risk of a pandemic situation. Within such circumstances in the near future, if it will not available then it will be di cult to manage the outbreak without drugs and vaccines. There is an immediate need for a multinational and remarkable effort to quickly identify an effective drug for SARSCoV2 [28]- [29]. For our computational approach we have identi ed four essential viral proteins, i.e. spike protein, papain-like protease protein, main protease protein and RNA-dependent RNA polymerase. Spike protein assists in initial host cell communication with the virus [30]. An important catalytic domain, essential for viral replication, is RNA-dependent polymerase, papain-like protease protein (PLpro) and main protease protein (3CLpro). All are prime focus for the development of coronavirus drugs and vaccines [31]- [32].
In the eld of computer-aided drugs designed especially to recognise a probable compound as ligands in molecular docking is extensively used to explore the different types of binding interaction between the prospective drug and the distinct domains or active sites on the target proteins and the nature of the residues presented at the active site. The information on binding energy (Kcal / mol) facilitates us to study and compare the binding a nity of various ligands with their corresponding target receptor molecule. The most a nated ligand can be assigned as the potential drug for further studies [33].
Rane et al [33] [31]. In this regard, we expect that our approach through phytochemical drugs that obstacle against major novel coronavirus (COVID-19) target proteins can be of great assistance in such a globally initiative.

Conclusions
The framework to modifying drugs would be the quickest and most effective decision to nd therapeutic remedies for the Coronavirus Novel SARSCoV2 (COVID- 19). The methodology to bioinformatics may be a very useful strategy in identifying potential inhibitors against the SARS-CoV-2. In this research, the evaluation of possible natural phytochemical drugs mainly secondary metabolites from the IMPPAT database as apparicine, 12-Chlorotabersonine, AC1NSULH, and Vindolininol identi ed from Catharanthus roseus resulted in a great binding a nity with the spike and papain-like protease-protein . Such phytochemical drugs often resulted in more than one binding to target proteins and at the same time raising the diagnostic accuracy and reducing the likelihood of resistance development to one of the targets which provide justi cation for therapeutic development to combat COVID-19. To the best of our knowledge, we are the rst to identify candidates as phytochemicals from Indian medicinal plants against SARS-CoV-2 viral proteins in the silico approach. In the near future further experiments in vitro and in vivo with our potential phytochemical compounds will nd a way to treat SARS-CoV-2 infections.

Declarations
Compliance with ethical standards Con ict of Interest: The authors declare no potential con icts of interest.
Ethical approval: This research did not involve any experimentation on humans or animals.

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