In this study, we showed comparative interaction of naproxen to the N terminal RNA- binding domain and C terminal dimerization/oligomerization domain of the nucleocapsid protein of SARS COV–2. We showed several key residues of the N terminal domain coordinate with naproxen that would compete for RNA binding with nucleocapsid. We also stimulated the binding dynamics of naproxen to N and C terminal domains during 100 ns of using molecular dynamics.
Nucleocapsid is one of the structural proteins of SARS COV–2 that is important for many functions including viral ribonucleoprotein (RNP) complex, regulation of replication or transcription. Crystallographic studies of the SARS-CoV N terminal domain revealed that the domain is folded mostly with antiparallel β-sheets core with a positively charged protruding hairpin, similarly, the same folding pattern has been seen for modeled and crystal structure of SARS-CoV–2 nucleocapsid 9. Although the overall structure N terminal domains are the same among many betacoronavirus, the surface charge distribution pattern is markedly different 8 indicating unique RNA binding module for SARS COV–2. The C terminal domain of nucleocapsid also folds into a conserved structural shape resembling the letter C in the monomeric form among coronaviruses. As it has been modeled in this study C terminal domain folds into extended conformation with the dimerization surface at the center of the domain. The amino acids at the dimerization surface have been described for SARS-CoV which are mainly involved in hydrophilic and hydrophobic interactions33. Truncated nucleocapsid protein of SARS-CoV missing amino 285–422 inhibited the dimerization of nucleocapsid and virion assembly 34
The molecular dynamics simulation that was done here, confirmed the data that were obtained by molecular docking and MM-GBSA evaluation and showed naproxen strongly interacted with the RNA binding pocket of the N terminal domain structure. By comparing with empty structure, it is clear that its flexibility did not affect significantly when naproxen bound (data not shown). It also should be noted that an intrinsic property of NP is its flexibility35. Our analysis following the other studies showed the two key arginine residues (R88 and R92) are bonded to naproxen in the whole simulation time. These residues are very important for the native RNA binding process and also present in the nucleocapsid of other viruses such as influenza30,32. In silicomutagenesis of these residues, the affinity of naproxen to the binding site drastically decreased. In addition to the electrostatic interactions, a network of H-bond between the ligand, protein and surrounding water molecules contributes to the stabilizing of the naproxenN terminal domain complex. Figure x11 shows this effect clearly. In this figure you can, the interaction energy completely obeys from the H-bond count between naproxen and the structures (this also was seen for the C terminal domain). For the N terminal domain, these strong interactions mask the RNA binding groove and compete with the viral RNA binding which similar to other studies on the anti-viral effect of this drug36.We also examine the binding potential of naproxen to the C terminal domain because this requires protein dimerization which is essential for its normal function. Fig 10c, d show the way that this domain behaves. It has more flexibility than the N terminal domain which is completely in accordance with other findings that pointed out the flexibility of the C-terminal of NP is reminiscent37,38.Possibly the higher flexibility interferes with the strong binding of naproxen with the key residues at the dimerization interface. By the way, as described in the result, occupying this site by naproxen cannot hinder the dimerization process which was deduced from the protein docking study.
High-throughput virtual screening and repurposing of the existing drugs for targeting SARS COV–2 proteins are the two main strategies for the rapid development of new anti-SARS COV–2 drugs. Using target-based virtual ligand screening, 18 putative proteins encoded by SARS COV- 2 were screened against the ZINC drug database and a local database.the results of this study showed three potential targets including 3-chymotrypsin-like protease (3CLpro), Spike, RNA- dependent RNA polymerase (RdRp), and papain-like protease (PLpro) with several lead compounds. However, such compounds were not identified for the nucleocapsid 39
After the recent global outbreak of SARS COV–2 full-genome sequencing and phylogenic analysis indicated that SARS COV–2 is a betacoronavirus and similar to severe acute respiratory syndrome (SARS) virus. The receptor-binding gene structure of SARS COV–2 is alike with SARS coronavirus, and there is the same receptor for cell entry in both 40. Additionally, the determination of the detailed 3D-structures of key virus proteins, it is very efficient to apply computer-aided drug design techniques to quickly identify promising drug repurposing candidates for virus elimination 41. So, the aim of this insilico study was to determine the antiviral effect of naproxen on SARS COV–2 and the results of the present study showed that naproxen had interaction with two types of arginine, ARG A 49 and ARG A 45, which play a major role in RNA binding in whole transcriptional processes. Naproxen binds to the nucleocapsid site and does not allow the virus to bind to the cells. Previously, Zheng et al show that the NP of influenza B virus (BNP) has a higher binding affinity to naproxen than influenza A virus NP (ANP) and specifically, naproxen targets the NP at residues F209 (BNP) and Y148 (ANP) 27. In another study,Lejal et al by in silico screening showed that naproxen combined antiviral and anti-inflammatory effects by targeting both NP and COX2 in influenza A virus 36.
The result of the present study about COVID–19 and other studies about influenza show that nucleocapsid has a main role in the life cycle of coronaviruses and it is a target for naproxen. Nucleoprotein acts as a single-stranded RNA binding protein that takes on a significant role in ribonucleoprotein particles (RNPs) transporting between the nucleus and cytoplasm and it is a structural protein of RNPs that is required for virus replication 42. Docking studies show that several known drugs such as Carfilzomib, Eravacycline, Valrubicin, Lopinavir, and Elbasvir. Carfilzomib, Streptomycin, and formoterol have an inhibitory effect against SARS COV–2 43,44. These drugs in-comparison with Naproxen are expensive and additionally, Naproxen is a drug in controlling some symptoms of these patients, such as fever and myalgia, so, it seems that is preferred over other drugs. Therefore, due to the antiviral role of naproxen, it is recommended to use naproxen in patients infected with SARS COV–2 without drug contraindication to help control fever and virus clearance time.