A novel virus was first found infecting human in China on 30 ^th December, 2019. The causative agent for this disease was found to be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus disease 2019 (COVID-19) was declared as a pandemic by the World Health Organization on 11 ^th March, 2020. The virus has been responsible for ’locking down’ much of the human race in most countries of the world for several months. The infectivity of this virus is extremely high and the mortality of this disease is around 5–7 percentage of the total infected cases ¹ . However, the infection fatality rate was reported to be 0.5–3.6% and the case fatality rate 2.3–7.2%, as per other reports since it differs from region to region over time ²

Coronaviruses consist of an enveloped RNA genome, and these viruses are zoonotic in nature. They cause respiratory, hepatic neurological, and enteric diseases in birds and mammals. As per the recent reports, this disease has claimed 962,634 lives worldwide, infecting 31,175,835 people globally till 22 ^nd September, 2020 ³ . SARS-CoV-2 consists of 30 kb nucleotides as its genome ⁴ . Two polyproteins essential for replication and transcription, pp1a and pp1ab, are encoded by the SARS CoV-2 replicase gene. The main protease (3C-like protease) or Mpro (a 33.8 kDa protein) is responsible for releasing the functional viral polypeptides from polyproteins after cleaving pp1a and pp1ab ⁴ . Therefore, Mpro is identified as an important drug target owing to its functional significance in the life cycle of the virus and also due to the absence of its homologues in the human race ⁴ .

A few drugs such as hydroxychloroquine and remdesivir that were found via bioinformatics approach have been reported. Hydroxychloroquine is a drug that is presently used against malaria. But when it was tested on SARS-CoV-2 virus, it showed activity against the virus in vitro. However, it was not found to be effective against the virus in COVID 19 patients during clinical trials ^{5, 6} . Later, this drug was prohibited from use in certain countries due to it side effects. Remdesivir is an antiviral drug that was found to show activity against the virus and it did inhibit the SARS-CoV-2 virus to certain extent. Remdesivir is presently used against COVID-19 in certain countries ⁷ .

In the present work, we carried out high throughput virtual screening (HTVS). It is a rapid way to search for the potential drug against the target. HTVS of Zinc natural database was performed through Python prescription software known as PyRx. This software has in built dock, Vina and Autodock tools along with openbabel. Autodock software utilizes MGLTools that has computer aided drug discovery pipeline. CADD is required for virtual high throughput screening of large databases for probable hits as therapeutic agents. This type of virtual screening helps to dock several ligands on one protein. PyRx is publicly obtainable HTVS software. Analysis and identification of distinct poses visibly and using a scoring algorithm quantitatively forms two basis of docking technique ⁸ .

The free binding energy (ΔG) between the protein and the ligands is calculated in docking. The free binding energy, thus calculated, forms the foundations of complex systems of subjects such as molecular biology and biochemistry ⁸ .

Natural molecules are known derivatives of most of the present drugs in use against many diseases. In this study, we have also found some good leads and most of the 25 selected molecules show medicinal properties. Ramipril benzyl ester (protected form of ramipril), which is an inhibitor of angiotensin-converting enzyme ACE is the host receptor protein where the SARS-CoV-2 spike protein binds. Ramiprilat, which is an active compound of ramipril benzyl ester, is formed on the hydrolysis of the ester group, which later inhibits ACE. This leads to the prevention of the catalytic conversion of angiotensin I to angiotensin II that causes vasodilation ¹⁷ . Stylopine ¹⁸ and quillaic acid exhibits anti-inflammatory activity ¹⁹ . Cinobufagin, CBF, has antitumor activity as it is known to inhibit the growth of PC3 cells both in vitro and in vivo along with inducing apoptosis of tumor cells as per reports ²⁰ . It was found that intake of the plant ligands such as lariciresinol reduced endothelial dysfunction and vascular inflammation in post-menopausal women and middle aged/elderly men ²¹ . Vitisinol C as per earlier reports was found to exhibit an essential activity against amyloid aggregation, which is present in Vitis vinifera grapevine shoots ²² .

Segetalins are known to have vasorelaxant activity ²³ . Scopolamine is known as antimuscarinics that blocks the effects of certain natural substance (acetylcholine) on the central nervous system, it is also used to prevent nausea and vomiting caused by motion sickness or medications used during surgery ²⁴ .

3-oxo-glycyrrhetinic acid has antitumor activity and expectorant (antitussive) properties ²⁵ . Lactimidomycin has antifungal, antiviral, and anti-cancer properties apart from acting as a direct inhibitor of protein translation inribosomes which is a glutarimide antibiotic derived from the bacterium Streptomyces amphibiosporus ²⁶ . Cardinalins dinalins 4 exhibit anti-cancer properties. Conchinone B has an antioxidant activity. Rotenone used to treat head lice on humans, scabies, and parasitic mites on chickens, livestock, and pet animals. Propafenone is used for the treatment of irregular heartbeat like atrial fibrillation, paroxysmal supraventricular and tachycardia in order to restore normal heart rhythm for maintaining a regular, steady heartbeat ²⁷ . These small molecules or potential drugs could be used as therapeutics for COVID-19 disease due to their binding affinity with the viral protein as well as interaction with Mpro. These small molecules were found to interact with the residues involved in enhancing the catalytic activity of Mpro or SARS CoV-2 main protease. Therefore, these could prove to play important role in inhibiting the viral activity. However, there are three small molecules ramipril benzyl ester, propafenone dimer and lariciresinol found from this study that might prove to be useful against this virus especially due to their present medical application, docking score and interaction with Mpro, as illustrated in Figure 16, Figure 17 and Figure 18.

Ramipril benzyl ester, stylopine, quillaic acid, cinobufagin, vitisinol C, segetalin A, scopolamine, 3-oxo glycyrrhetinic acid, lariciresinol, conchinone B, lactimidomycin, cardinalins 4, and propafenone dimer are 13 molecules that are predicted to form good interaction with Mpro. These molecules showed better binding affinity for Mpro than the reference molecule. Ramipril benzyl ester, apart from binding to the main protease of the virus, was also found to inhibit the virus receptor ACE present in humans. ACE2 is the receptor to which virus spike protein is known to bind to the host. The spike protein and Mpro of the SARS-CoV-2 virus are responsible for the virulence of the virus. Ramipril benzyl ester drug is reported to inhibit ACE and also found to inhibit main protease of SARS-CoV-2 virus from this study. Ramipril benzyl ester is presently used against heart stroke. Ramipril benzyl ester and propafenone are two important small molecules found from our study due to their present medicinal use as drugs for other diseases in human along with lariciresinol due to its medical application as stated in the discussion. As a result, these molecules have medicinal properties which could be used for the treatment of COVID-19 disease after further evaluation. This study presents a significant possibility in finding the cure for the COVID-19 disease from the drugs repurposing point of view. The drug targets mentioned in our study could bring relief to all those who are infected with the disease and these may also act as a prophylactic treatment when tested by the frontline healthcare providers. This study could provide relief to everyone if the drugs mentioned in our study proved to inhibit the virus in COVID19 positive patients since they are known natural derivatives with minimal side effects in individuals. Thus, our study could help in flattening the curve with speedy recovery in COVID -19 positive patients.