Phytomolecules having flavone and napthofuran nucleus exhibited better binding G-score against protease and SPIKE protein of novel corona virus COVID-19 CURRENT STATUS: POSTED

The present study aims to screen the different phytoconstituents and drugs for potential treatment of the corona virus COVID-19 and for specificity through virtual screening. The plant molecules selected were based upon traditional knowledge and are prescribed in the Indian system of medicine for infectious/ respiratory conditions. The three target proteins selected for the study are 3CLpro, PLpro, and SPIKE. These proteins have defined pathological roles in disease transmission. The virtual screening was carried out in these proteins using the GLIDE Schrödinger Maestro software version 11.9.011. The efficacy was assessed by the calculated G-score of the ligand interaction with the amino acid side chains of the ligand binding domain. Molecules such as saponarin, mangiferin, and hesperidin exhibited better G-score with 3CLpro and PLpro. Similarly, diphyllin and tuberculatin exhibited better G-score for SPIKE protein. The reference anti malarial drug hydroxychloroquine showed better interactions with 3CLpro and PLpro. Similarly, protease inhibitors and antiviral drugs have shown interaction with 3CLpro specific protease protein. Interestingly, SPIKE protein ligands, diphyllin and tuberculatin from Justicia adhatoda (vasaka), were found to be unique and did not show affinity to protease inhibitor. It can be concluded, that the molecules having flavone scaffolds show better binding affinity with protease proteins 3CLpro and PLpro. SPIKE protein scaffold is different and showed better binding affinity with molecules having naptho-furan ring. The traditionally used plant phytoconstituents did not exhibit good binding affinity; however, we believe that a combination of these herbs might induce human immune system against microbial infection. , Anti-Viral drugs,& Standard WHO approved drugs for COVID-19 were performed using the Schrodinger Maestro v.11.9.011 Ligand docking and the resultant G-scores were obtained for the three COVID-19 target proteins 3CLpro , PL pro &SPIKE.


Introduction
The pandemic of the novel corona virus infection   CoV transmits mainly through droplet infection (respiratory secretions) and close person-to-person contact. Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) infects human by primarily targeting enterocytes, pneumocytes, and thereby establishing a cycle of infection and replication [7].
SARS-CoV-2 attaches to the target cell by interacting with host cell protein (such as angiotensin converting enzyme-2) and releases the viral genome into the cytoplasm of host cells. It acts just like a messenger RNA and directs the host cell to synthesize two long polyproteins, which enables the virus to take command over host ribosomes for their own translation process [8].
There are no approved therapies currently available for COVID-19. Current drugs having indications for other diseases have been tried in these patients. The viral load in these patients has come down with the treatment of anti-malarial drug like hydroxyl chloroquine, anti-viral drugs, and protease drugs. There are also discussions within the healthcare system that for individuals having a good immune system, the infection rate will be less. Consequently, recommendation has also been made to boost the immune system. It is a pandemic and no specific drugs are available, which has made researchers to focus on drug re-purposing and also natural products. The objective is to stop or delay the infection to minimize the future socioeconomic disruption due to this global health-care burden.
Secondary metabolism is a complex defence phenomenon of plants and still serves as a source of countless medicinal compounds in pharmaceutical drug discovery. In comparison to chemical drugs, herbal medicines are less understood mechanistically due to poly-pharmacological interactions by compounds present in the herb. Even now, in the combat of COVID-19 pandemic, the National Health Commission of China recommended traditional Chinese medicine as an early defence treatment option [9].When it comes to infectious diseases, Indian traditional systems of medicine (like Ayurveda and Siddha) emphasizes not only anti-microbials but also on enhancing immunity boosting activities Hence, in the present study, virtual screening of the drugs and phytochemicals were made on three important protein targets of COVID-19 virus SARS-CoV-2 to elucidate the most potentially active molecules. The phytoconstituents selected are the chemical and/ or functional markers of herbs, which are being recommended in the Indian System of medicine for either the management of infectious/ respiratory disorders or strengthening of the immune system [11,12]. This approach will also validate the use of herbal preparations in infectious condition.

Methodology
All the in-silico simulations were performed in Maestro v11.9.011 modelling package provided by Schrödinger, LLC, New York, NY, 2019-1, installed on an Intel Core i7-4770 processor, kernel GNOME™ Linux 2.6 Centos 6.5. 2.5Aº;PDB ID: 6M0J, Resolution: 2.45Aº, respectively) and have been depicted in Fig.1. The force field used during the protein preparation was OPLS3.Proteins were pre-processed to add hydrogen and delete waters beyond 5Å , reviewed , modified, and finally minimized using the Protein Preparation Wizard module in Maestro. Ligands were prepared to desalt, generate stereoisomers & generate possible states at target pH 7±2 using LigPrep module in Maestro. A receptor grid was generated at the binding site using the receptor grid generation tool in Maestro. All the ligands were docked within the grid-generated area. Standard precision (SP), followed by extra precision (XP) mode of docking were performed for selection of top hit ligands.

Results
The screening of phytochemicals and drugs currently used to treat COVID19 with three different proteins having pathological function indicates effective interactions of compounds with the targeted proteins. The G score for all the compounds are given in Table 1 Fig.2).

Ligand Interactions of Compounds with SPIKE protein of SARS-CoV-2(6M0J):
The ligand interaction site for these protein molecules has been developed by our group. Earlier, no reports were available on ligand protein interactions with this target. It has been observed that two Molecules, which have shown interactions with either of these two sets of amino acids showed better G-scores. Hydrophobic interactions at Tyr 505 & Tyr 495 were commonly observed. The standard drugs hydroxychloroquine, oseltamivir, and remedisivir were found to be least active as indicated by their lower G-scores (Table 4; Fig. 4).

Discussion
The objective of the present study is to identify a suitable ligand to interact with the different target  Interestingly, the phytoconstituents from neem, tulsi & andrographis (Nila-Vembu) did not show any binding scores with these protein targets. Likewise, other chemical molecules studied include curcumin, liquitrigenin, iso-liquitrigenin, glabridin, piperine, glycyrrhizic acid, vasicine & vascinone and these showed no or low binding scores. The clinically used protease inhibitors, anti-viral drugs, ivermectin & chloroquine were also docked with these proteins; the results show lower G-scores comparatively with saponarin, mangiferin, hesperidin, diphyllin, and tuberculatin. Hydroxychloroquine and remedesivir showed better G-scores for 3CLpro & PLpro, but these two drugs showed lesser Gscores for SPIKE protein in comparison to diphyllin & tuberculatin. 6M0J can also be targeted, being it a SPIKE protein. But, as the binding site is quite large it is difficult to block it with small molecules and it would be better if it can be blocked with larger molecules with better g-scores and binding affinity, However, this target can be tried for prophylactic purpose. Formulations of traditional system of medicine contain multiple herbs and generally natural products act on either additive or synergistic mechanism to elicit the poly-pharmacological action [23]. The current work was designed based on the hypothesis on antiviral activities of these compounds from selected herbs. However, compounds like curcumin (from Curcurma longa) and azadirachtin (from Azadirachta indica) are well-documented for their immuno-modulatory responses, including its effect on lymphoid cell populations, antigen presentation, humoral and cell-mediated immunity, and cytokine production [24, 25]. Hence, studying the effect of these compounds on immune modulation targets is also needed to elucidate the complete potential of these compounds.
It can be concluded, that the molecules having flavone scaffolds show better binding affinity with protease proteins 3CLpro and PLpro. SPIKE protein scaffold is different and showed better binding affinity with molecules having napthofuran ring. The traditionally used plant phytoconstituents did not exhibit good binding affinity; however, we believe that a combination of these herbs might induce human immune system against microbial infection.

S. No
Pubchem ID Name of the compound 3CLpro (6LU7)  Table 2 The best fit phytomolecules and currently indicated drugs interaction with the amino acids of