Identification of potent COVID-19 main protease (Mpro) inhibitors from natural polyphenols: An in silico strategy unveils a hope against CORONA

COVID-19, a rapidly spreading new strain of coronavirus, has affected more than 150 countries and received worldwide attention. The lack of efficacious drugs or vaccines against SARS-CoV-2 has further worsened the situation. Thus, there is an urgent need to boost up research for the development of effective therapeutics and affordable diagnostic against COVID-19. The crystallized form of SARS-CoV-2 main protease (Mpro) was demonstrated by a Chinese researcher Liu et al. (2020) which is a novel therapeutic drug target. This study was conducted to evaluate the efficacy of medicinal plant-based bioactive compounds against COVID-19 Mpro by molecular docking study. Molecular docking investigations were performed by using Molegro Virtual Docker 7 to analyze the inhibition probability of these compounds against COVID-19. COVID-19 Mpro was docked with 80 flavonoid compounds and the binding energies were obtained from the docking of (PDB ID: 6LU7: Resolution 2.16 Å) with the native ligand. According to obtained results, hesperidin, rutin, diosmin, apiin, diacetylcurcumin, (E)-1-(2-Hydroxy-4-methoxyphenyl)-3-[3-[(E)-3-(2-hydroxy-4methoxyphenyl)-3-oxoprop-1-enyl]phenyl]prop-2-en-1-one, and beta,beta'-(4-Methoxy-1,3phenylene)bis(2'-hydroxy-4',6'-dimethoxyacrylophenone have been found as more effective on COVID-19 than nelfinavir. So, this study will pave a way for doing advanced experimental research to evaluate the real medicinal potential of these compounds to cure COVID-19.

The SARS-CoV-2 has been identified as β-coronavirus, a non-segmented enveloped positivesense RNA virus, with 29.9 kb genome (5, 3). SARS-CoV-2 causes severe respiratory tract infection in humans and utilize angiotensin-converting enzyme 2 (ACE2) receptors to infect humans (6). Chinese scientists isolated SARS-CoV-2 and sequenced the genome SARS-CoV-2 on January 7, 2020 (7). The crystallized form of COVID-19 main protease (Mpro) was demonstrated by a Chinese researcher Liu et al. (2020) that it is a potential drug target protein for the inhibition of SARS-CoV-2 replication. The Mpro is a key protein required for the proteolytic maturation of the virus (8). Thus, targeting Mpro has the potential to provide effective treatment against SARS-CoV-2 by inhibition of the viral polypeptide cleavage.
In-silico based screening has proven to be a very useful tool to meet the challenges of antiviral drug discovery. Screening of natural or synthetic virtual compound libraries by computational screening methods as docking saves resources in terms of money as well as time (9). Natural compounds have served humans as cheaper and safer drug candidates against several diseases (10,11). Thus, we have screened a small library of natural compounds against Mpro by in silico based screening and in this study, we report the identification of natural compounds (Hesperidin, diosmin, rutin, and apiin) as potent inhibitors of Mpro by using molecular docking approach.

Methods
Computational or theoretical chemistry is a sub-branch of chemistry. It explains the formation possibility of chemical bonding between atoms, molecular activation in terms of molecular dynamics, chemical reaction formation possibilities in the scope of thermodynamic and molecular orbital theory takin into consideration valence orbital interaction and potential energy molecular orbitals (12). The theoretical chemistry bases on two strong physicochemical phenomena; these are quantum mechanics (QM) and molecular dynamics (MD). The subatomic particle (protons, electrons, and neutrons) interactions form the drawback of quantum chemistry that also describes the molecular properties depending on subatomic interactions (13,12). The molecular dynamic theory is based on the spatial conformation of molecule interaction from their active sites by intermolecular interactions like weak Van der Waals interactions or hydrogen bonding. Therefore, the frontier molecular orbital theory (FMOT) has been used to get information about the stability and reactivity of the examined compounds against target bioactive molecules like enzyme, protein, etc.

Results
The binding energies obtained from the docking of 6LU7 were presented in Table 1.
Hesperidin exhibited the highest binding energy at the active site of Covid 19 ( Figure 1). It  Table 2).

Discussion
Coronaviruses have a long history of infecting humans and animals and causing respiratory, digestive, liver and central nervous system diseases in them (16). A novel newly emerged SARS-CoV-2 is presenting major threats to human health nowadays (3). Currently, no specific clinical therapeutics are available for the treatment of SARS-CoV-2-mediated infections (17). Thus, the need of the hour is to identify and characterize novel drug candidate to overcome the health loses caused by SARS-CoV-2. In this context, natural products have gained importance as potent anti-viral agents during recent years (18,19). Considering the immediate need of therapeutics against COVID-19 and services of natural products in drug discovery, we have screened flavonoids against novel drug target, Mpro, of SARS-CoV-2 for the identification of Mpro inhibitors to provide natural scaffolds for drug development.
Antiviral effects of flavonoids have been the subject matter of several reports (20)(21)(22). It has been previously reported that flavonoids exert their antiviral effects via blockage of cellular receptors, inhibiting viral antigenic determinants, loss of enzymatic functions, and/or inhibition of particle biosynthesis which is consistent with our findings (23)(24)(25). Furthermore, the antiviral activity of specific flavonoid subclass groups, such as catechins, flavanones, flavonols has been reported previously against various viral strains (26,27). Song et al. (2005) reported reduced viral infectivity by catechins (26). Antiviral natural product-based medicines have also been used for two previous coronavirus outbreaks of SARS-CoV and MERS-CoV which suggests that nature has tremendous potential to provide treatment for the ongoing epidemic of COVID-19 (28)(29)(30).
Previous reports also suggest anti-influenza virus potential of hesperidin and apiin (31,32), the anti-DENV activity of rutin (33) while the anti-rotavirus potential of diosmin (34) which further affirm the potential of these compounds against COVID-19. Hesperidin and diosmin are flavanone glycoside which is richly found in the citrus including lemons, grapefruits and sweet oranges (35,36). Rutin is a vital nutritional component and abundant flavonol found in tea, and apples (37). Parsely has been reported to be enriched with apiin (38). Interestingly, all the compounds possessing binding energies more than Nelfinavir are nutraceuticals and important nutritional components of fruits and vegetables, thus, we anticipate that consumption of citrus fruits, cherries and apples has the potential to boost immunity to fight against COVID-19 infections.

Conclusions
The rapidly spreading outbreak of COVID-19 has challenged the healthcare sector of the world in the last few months. To contribute to this fight against COVID-19, virtual screeningbased molecular docking was performed to identify novel compounds having the potential to bind M pro of COVID-19. Our results propose that flavonoids such as hesperidin and rutin have a better binding affinity to M pro of COVID-19 than Nelfinavir. According to moldock binding score, the potent flavonoids can be ranked as following by affinity hesperidin > rutin > diosmin > apiin > diacetylcurcumin ( Figure 5). All the compounds bearing good binding potency are components of dietary foods that suggest the biologically safe profile of these compounds further supporting the potential of these compounds as starting points for therapeutics against COVID-19. However, further studies should be conducted for the validation of these compounds using in vitro and in vivo models to pave a way for these compounds in drug discovery.