Corona viruses are a class of single-stranded ribonucleic acid (RNA) viruses and have been classified into four types name as α-corona virus (α-COV), β-corona virus (β-COV), δ-corona virus (δ-COV) and γ-corona virus (γ-COV) [1,2]. It is well-known that these viruses affect a range of living species including humans where two of them, namely Severe Acute Respiratory Syndrome Corona virus (SARS-CoV) and Middle East Respiratory Syndrome Corona virus (MERS-CoV) infect human, which have been responsible for unprecedented pandemic situation, caused a large number of deaths worldwide [2,3].

An intense work carried out by structural biologists has revealed the potential drug targets for the structural and non-structural proteins of SARS-CoV-2. They are 3-chymotrypsin-like protease (3CLpro), papain like protease (PLpro), RNA-dependent RNA polymerase, spike (S) proteins and human angiotensin converting enzyme (ACE) [[4], [5], [6], [7], [8]]. Currently, SARS-CoV-2s have evolved to many different variants as a result of alteration in the spike protein. So, it is difficult to gain effective vaccines (https://www.gisaid.org/, accessed on August 23, 2024) for these viruses. Therefore, enzymes remain alternative antiviral agent targets since their active sites are rarely mutated as they are essential in sustaining viral pathogenicity. The homodimers of WT 3CLpro are 67.60 kDA and each domain comprises of three domains. The residues 8–101, 102–184 and 201–303 are referred to the Domains I, II, and III, respectively. Among these domains the Domain II includes active site. The active site of 3CLpro includes the His41 and Cys145 catalytic dyad and Glu166 residue which serves in dimerization process. The catalytic site of 3CLpro consists of several subsites, such as S1, S1′, S2, S3 and S4 sites [9,10]. 3CLpro has a crucial role in the viral replication. Translation of the viral RNA by host ribosome results in production of two large polyproteins (pp1a and pp1ab) which are needed to be processed by virus's proteases 3CLpro and PLpro. The proteolytic processing of these polyproteins is vital for virus replication [[11], [12], [13], [14], [15], [16]]. Having 11 cleavage sites is the reason that 3CLpro referred to as main protease as well. 3CLpro recognizes peptide substrate residues as numbered with -P4-P3-P2-P1↓P1′-P2′-P3′- from N terminus to the C terminus. The cleavage occurs between P1 and P1′ residues of substrates, where a Gln is at P1, P2 is the one of Ser, Val, Asn or Ala residues. Since this cleavage property is not represented by human proteases, it makes 3CLpro an important drug target [9,[17], [18], [19], [20]].

The available well-defined crystal structures of the target proteins complexed with a chemical structure are also quite valuable for the drug development processes. There are fortunately several X-ray crystal structures of 3CLpro of SARS-CoV-2 complexed with inhibitors [19,21,22]. Hence, the current study considers 3CLpro as a good COVID-19 target because of its essential activity for viral processing. Although there are few compounds (saquinavir, remdesivir, and darunavir and two natural drugs, flavone and coumarin derivatives) used to inhibit 3CLpro [23], there is still a need to develop new more effective inhibitors having low toxicity. In addition, new SARS-CoV-2 variants has been reported from different areas of the world [24] which may decrease the efficacy of the drugs used in treatment of COVID-19.

Classical drug development and improvement techniques are expensive and time-consuming methods comparing to in silico methods. It was reported that computer-aided drug design (CADD) methods can reduce the cost of drug discovery up to 50 %. [25,26]. Nowadays computer-aided drug design (CADD) methods have extensively been used in drug design, discovery and development processes [24,[27], [28], [29]] as well in ligand library designs [30,31]. In fact, CADD methods could be investigated in two categories as its nature, where if a biological target molecule exist generally structure based drug design (SBDD) method followed and in the absence of the knowledge of biological target ligand-based drug design (LBDD) method should be chosen which focuses on the known drug molecules. Molecular docking is one of the essential tools of SBDD method. Although the method was originally developed to define protein-protein interactions [32], nevertheless the method has been adapted to use in docking of lipid-protein, lipid-lipid, enzyme-substrate, drug-enzyme, drug-nucleic acid, protein-nucleic acid, nucleic acid-nucleic acid, protein-drug, and protein-protein systems [[33], [34], [35]]. The technique repose upon investigation of the interactions observed between two molecules at atomic levels [36,37]. The results obtained by molecular docking methods generally followed by molecular dynamics (MD) simulations, where the method dates back to 1950's [38]. The application of the method to the protein systems has been developed in the late 1970's [39]. In its most general definition, the MD simulations are prediction of the motion of every atom included in a system over time [40]. Medicinal chemistry [41], materials science [42], biophysics [43], or biochemistry [40] are some fields that method could be applied [44]. The trajectories of protein-ligand systems obtained from MD simulations generally have been forwarded to the binding free energy calculations in drug design and development processes to calculate binding affinity of a small molecule to a macromolecule which is a more accurate method than docking methods [45]. Molecular Mechanics Poisson-Boltzmann (Generalized Born) Surface Area (MM/PB(GB)SA) is one of the endpoint methods [46,47] that accelerate low-cost binding free energy calculations [47].

The work involves the virtual screening of 756958 compounds against 3CLpro as a target for the treatment of COVID-19. The study contains molecular dynamics and binding free energy calculations of the best nine compounds according to docking results and co-crystalized ligand N3, as well. The results obtained in this study may be useful to those who consider these drugs as potential anti-COVID-19 agents.