Antiviral potential of phytoligands against chymotrypsin-like protease of COVID‐19 virus using molecular docking studies: An optimistic approach

A recent outbreak of the novel coronavirus, COVID‐19, in the city of China and its ensuing worldwide spread have resulted in lakhs of infections and thousands of deaths. As of now, there are no registered therapies for treating the contagious COVID‐19 infections, henceforth drug repositioning may provide a fast way out. In the present study, a total of thirty-five compounds including commonly used anti-viral drugs were screened against chymotrypsin-like protease (3CLpro) using SwissDock. Interaction between amino acid of targeted protein and ligands was visualized by UCSF Chimera. Docking studies revealed that the phytochemicals such as cordifolin, anisofolin A, apigenin 7-glucoside, luteolin, laballenic acid, quercetin, luteolin-4-glucoside exhibited significant binding energy with the enzyme viz. - 8.77, -8.72, -8.36, -8.35, -8.13, -8.04 and -7.87 Kcal/Mol respectively. Therefore, new lead compounds can be used for drug development against SARS‐CoV‐2 infections.


Introduction
The emergence of the severe acute respiratory syndrome-associated to COVID-19 as pandemic; presents new challenges to global biomedical research. At current scenario, according to Johns Hopkins University & Medicine database, the virus has affected 181 countries. As on April 4, 2020, total confirmed cases suffering from COVID 19 were 1,131,713 and 59,884 people have died 1 . The appearance of COVID-19 has also impacted transplantation worldwide 2 . In December of 2019, reports emerged of pneumonia clusters at Wuhan as they were linked to a wet animal wholesale market in the region. After a lot of epidemiologic investigation with International Committee on Taxonomy of Viruses; WHO officially announced identification of corona virus as COVID-19 or SARS-CoV-2 3,4 .
Original diagnostic findings in family cluster cases at China included fever, dry cough, shortness of breath, rhinitis, fatigue, diarrhea, headache, symptoms of pneumonia and low lymphocyte count.
Chest CT scans of the patients showed bilateral patchy shades in the lungs 5 .
Coronaviruses are enveloped, (+) single stranded RNA viruses with a crown like appearance, belongs to the family Coronaviridae, order Nidovirales which is further divided into four genera (α, β, γ and δ),, subgenera Sarbecovirus, species is SARS-CoV 6 . Four CoVs commonly found among humans: HCoV2-229E, -HKU1, -OC43 and -NL63. Novel CoV-2 is a zoonotic form of the beta-coronavirus which can rapidly mutate and recombine although mutations are natural part of the virus life cycle 7,8 .
COVID-19 usually, has an incubation period of 2-7 days and up to 14 days as longest time from infection to symptoms 9 . Throat swab, nasal swab, sputum, blood samples and stool are tested using viral nucleic acid with different techniques including real-time reverse transcription polymerase chain reaction, whole-genome sequencing, paper-based bio-molecular sensors, nanopore target sequencing, antibody-based immunoassays, and the clustered regularly interspaced short palindromic repeats-Cas system 10 .
Drug development against coronavirus includes inhibition of viral replication through acting on its critical enzymes. As the viral genome (29891nucleotide) encodes more than 20 proteins, among which RNA-dependent RNA polymerase, helicase, spike, two proteases (PLpro and 3CLpro) are vital 11 .
A large segment of population with the highest mortality from CoV-19 were elder people and individuals with weak immune system 12,13 . Therefore, interferons enhancement, monoclonal antibodies administration actively or passively can improve the immune response against the virus.
Zinc is also reported to have antiviral effect by inhibiting CoV RNA polymerase activity and thus decreases viral growth in cell culture set up 14,15 . A receptor-binding domain (RBD) of the spike protein in SARS-CoV, mediates the interaction with host angiotensin-converting enzyme 2 (ACE-2) 16 .
The drug, chloroquine affects the glycosylation process of ACE-2, which is essential for interaction with the host. It increases endosomal pH and thus makes unfavorable environment for the cell/virus fusion 17 .
The treatment with conventional drugs is intricate as multidrug resistance due to accumulating mutations in viruses however natural products provide broad spectrum antivirals agents with minimum side effects 18 . In Ayurveda, several medicinal plants namely; Tinospora cordifolia, Ocimum sanctum, Leucas cephalotes, Allium sativum, Allium cepa, Citrus limon, Piper nigrum, Phyllanthus emblica have been reported for antiviral activities [19][20][21] . They are endowed with a variety of secondary metabolites such as tannins, terpenoids, flavonoids, flavones, glycosides, alkaloids, phytosteroids and thus, herbal agents can be promising candidates for evaluating their effects on pathogenic microbes 22 . Therefore, the present study aims to analyze the docking potential of predominant phytoconstituents in the reported plants as inhibitors of 193CLpro in search of therapeutic potential against COVID 19 infection.

Results
In order to identify the new phytoligands with the top-ranking hit; virtual screening was carried out against 3CLpro. Their minimum binding energy values were compared on the basis of their best fitting with the enzyme, H-bond and LD50 value as depicted in Table 1 and Supplementary Table 1

Discussion
The 3-chymotrypsin-like protease (main protease, M pro ) or non-structure protein (Nsp5) was selected for the study. As it is first automatically cleaved from poly-proteins to produce mature enzymes, and then further cleaves downstream Nsps which is essential in the life cycle of the virus. Most of the lead compound showed hydrophobic interaction with amino acid present at catalytic site of 3CLpro.
Catalytic dyad of the enzyme (His41 and Cys145) is located in the gap between domains I and II, and Cordifolin which is an oxygenated chalcone exhibited inhibition of HIV protease 37 . Anisofolin A exclusively reported in the flowers of Leucas species found to have promising anti-mycobacterium activity against M. tuberculosis H37Ra, antimalarial activity against Plasmodium falciparum (3D7) 38 .
The flowers are given in the form of syrup or with honey as a domestic remedy for bronchial asthma, colds, cough 39 . Luteolin which is a flavonoid exhibited potent antiviral activity against SARS coronavirus 40 , Japanese encephalitis virus 41 . It also inhibited HIV-1 protease in cell-free assays 42 .
Laballenic acid (Octadeca-5, 6-dienoic acid) is an allenic fatty acid showed the anti-inflammatory effect through the suppression of TNF-α production in macrophages 43  In addition, as preventive measures, WHO guidelines mainly focuses on social distancing measures such as quarantines, frequent hand wash, use of mask and avoidance of unwanted contact with wild animals or animal products 51 . Like others CoVs, it is sensitive to heat and UV rays, thus drinking hot water may be useful. Other suggested treatment includes providing oxygen inhalations, nutritional support, psychological calmness, maintaining fluids and electrolyte balances of individual 52     SupplementalTable1.pdf