key: cord-0923331-mr76k9im authors: Esam, Zohreh; Akhavan, Malihe; lotfi, Maryam; Bekhradnia, Ahmadreza title: Molecular docking and dynamics studies of Nicotinamide Riboside as a potential multi-target nutraceutical against SARS-CoV-2 entry, replication, and transcription: A new insight date: 2021-08-30 journal: J Mol Struct DOI: 10.1016/j.molstruc.2021.131394 sha: 9c61cfddbf36402c93108962cf2b615abc848415 doc_id: 923331 cord_uid: mr76k9im The highly contagious Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is a newborn infectious member of the dangerous beta-coronaviruses (β-CoVs) following SARS and MERS‐CoVs, can be regarded as the most significant issue afflicting the whole world shortly after December 2019. Considering CoVs as RNA viruses with a single-stranded RNA genome (+ssRNA), the critical viral enzyme RNA dependent RNA polymerase (RdRp) is a promising therapeutic target for the potentially fatal infection COVID-19. Nicotinamide riboside (NR), which is a naturally occurring analogue of Niacin (vitamin B3), is expected to have therapeutic effects on COVID-19 due to its super close structural similarity to the proven RdRp inhibitors. Thus, at the first phase of the current molecular docking and dynamics simulation studies, we targeted SARS-CoV-2 RdRp. On the next phase, SARS-CoV RdRp, human Angiotensin-converting enzyme 2, Inosine-5’-monophosphate dehydrogenase, and the SARS-CoV-2 Structural Glycoproteins Spike, Nonstructural viral protein 3-Chymotrypsin-like protease, and Papain-like protease were targeted using the docking simulation to find other possible antiviral effects of NR serendipitously. In the current study, the resulted scores from molecular docking and dynamics simulations as the primary determinative factor as well as the observed reliable binding modes have demonstrated that Nicotinamide Riboside and its active metabolite NMN can target human ACE2 and IMPDH, along with the viral S(pro), M(pro), PL(pro), and on top of all, RdRp as a potential competitive inhibitor. The potentially fatal infection coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has begun in December 2019 from Wuhan city of China and affected the whole aspects of life all over the world via becoming a pandemic. The capability of SARS-CoV-2 to attack different parts of the body, particularly vital organs i.e., lungs, heart, brain, and kidneys, makes this highly contagious multi-organ infection a serious global crisis 1, 2 . Although it seems that with the advent of vaccines, the transmission chains in this viral pandemic have been broken, considering the current concerns about their effectiveness against the mutant variants, the reported adverse effects, the probable long-term side effects [3] [4] [5] [6] , and the necessity for saving the infected peoples' lives, we need to find safe, efficient, and preferably affordable, and on top of all, specific drugs instead of the currently prescribed non-specific therapeutic cocktails 7, 8 . In this way, RNA-dependent RNA polymerase (RdRp) is an essential enzyme for viral genome replication and translation; therefore, it can be considered excellent therapeutic target for COVID-19 treatments. RdRp-targeted antivirals structurally fall into two classes: nucleoside inhibitors (NIs) and non-nucleoside inhibitors (NNIs) 9 . NIs, i.e., Ribavirin, Favipiravir, and the related structural analogues 10 , act as purine pseudo bases after phosphorylation and cause irreversible mutations in the viral genome or inhibit the viral RdRp directly, in a concentration-dependent manner 11 . The inhibitory mechanism of NIs is related to bioisosterism and steric similarity with purine nucleoside triphosphates 12 . The NIs binding pocket (enzyme active site) is highly conserved among various RdRp enzymes; therefore, it is not surprising that the above-mentioned broad spectrum NIs, i.e., Favipiravir and Ribavirin, have efficacy against COVID-19 13 . An essential structural element in nucleotide analogue inhibitors of RdRp is the rotating carboxamide moiety attached to the aromatic core (Scheme 1). This structural portion makes the antiviral activity of these compounds possible. The formation of different rotational conformations of the amide bond has been responsible for forming the critical drug-target interactions. Compounds containing a rotatable carboxamide group attached to an aromatic center can mimic the endogenous ligand guanosine triphosphate (GTP) and create base-pair interactions with Cytosine. Such kind functional groups can also create the base-pair interactions with Uracil via mimicking the adenosine triphosphate (ATP) binding properties 14 . Theoretically, the presence of a ribose portion in the chemical structure of NIs, causes incorrect RNA chain elongation, since these compounds are used as alternative substrates for endogenous nucleotides. In this sense, it is worth mentioning that this leads to virus mutagenesis 11 ; but it seems not to be crucial for antiviral activity 15 . 16 . Besides, the other viral DNA polymerase inhibitors Ganciclovir which was successfully administered for the first reported COVID-19 patient with diabetes 17 , and also the clinically approved drug candidate IDX-184 as a direct-acting antiviral drug in numerous theoretical studies 15, 18, 19 , both possess the discussed structural features (Scheme 2), and also the capability to be tightly wrapped to the SARS-CoV-2 RdRp (PDB ID: 6M71), according to the molecular dynamics and docking simulations 20, 21 . In this regard, it can be said that all of these antivirals have the structural pattern of Favipiravir and Ribavirin (Schemes 1,2), albeit with an in-flexible and rigid carboxamide portion which allows these compounds to mimic GTP and base pair with Cytosine 14 . The naturally occurring vitamin Nicotinamide riboside (NR) with a close structural similarity 22 to NIs (Scheme 3) is orally bioavailable and currently sold as a safe supplement 23 25 . The enzymatic and molecular modeling results proved that DMB220 competes with the natural nucleoside triphosphate (NTP) substrates for binding to the RdRp active site, which is different from the binding site of classic NNIs. According to the undeniable principle of Structure-Activity Relationship (SAR), we expect common biological effects from similar chemical structures. Therefore, in this study, the potential inhibitory effects of NR, and NMN against SARS-CoV-2 RdRp were studied using molecular docking and molecular dynamics (MD) simulation as a considerable primary step in the identifying of novel therapeutic agents for COVID-19. This research has been based on the structural similarity of NR, with the proven broad-spectrum inhibitors of viral RdRp which are successfully used for the clinical management of COVID-19 patients, i.e., Ribavirin and Favipiravir. Natural substrates Adenosine triphosphate (ATP), Guanosine-triphosphate (GTP), and the active forms of broad-spectrum RdRp inhibitors Favipiravir and Ribavirin were used as the reference substrates. Considering the obtained molecular docking and MD results, NR can be considered as a Nicotinamide-derived small-molecule that can ultimately be wrapped by the SARS-CoV-2 RdRp active site as the competitive inhibitor DMB220. Along with that, In the next phase of the research, S pro , ACE2 receptor, 3CLpro or the main protease (M pro ), and Papain-like protease (PL pro ) which play essential roles in host cell-virus surface interaction for viral entry into the target cells, and processing of translated polyproteins respectively, was used as the molecular docking targets. Furthermore, IMPDH is an essential enzyme in the biosynthesis of guanine nucleotide, that its inhibitors have revealed proven effects against human coronaviruses 26 , was the last selected target in this represented molecular docking studies since NR shows the same structural pattern with one major group of IMPDH inhibitors including Ribavirin and Mizoribine (as their monophosphate-activated metabolites) which target the binding site of the natural substrate (inosine monophosphate). The obtained results from the present investigation discuss not only NR as a potential small-molecule drug against COVID-19 but also a pharmacophore model for the rational design or screening of the multi-target anti-SARS-CoV-2 agents. The first drug target for the current study is RdRp of SARS-CoV-2 in complex with cofactors (PDB ID: 6M71) 27 . NSP12, which is the RdRp is chain A with 851 amino acids (Supplementary information, Figure S1 (A)). Another drug target is the RdRp structure of SARS-CoV (PDB ID: 6NUR) 28 and identified SARS Coronavirus (SARS-CoV) NSP12 bound to NSP7 and NSP8 cofactors. The NSP12 (chain A) was selected as RdRp, and the cofactors (NSP7 (chain C)) and NSP8 (chains (B) and (D)) were removed from the structure ( Figure S1 (B)). COVID-19 main protease in complex with an inhibitor N3 (PDB ID: 6LU7) 29 (chain A) was chosen as another target in this study ( Figure S1 (C)). Additionally, we carried out the docking using perfusion SARS-CoV-2 Spike glycoprotein (S pro ) with a single receptor-binding domain (chain A) (PDB ID: 6VSB) 30 ( Figure S1 (D)). Moreover, the human type II Inosine Monophosphate Dehydrogenase (PDB ID: 1NF7) 31 is another selected target in this investigation ( Figure S1 (E)). For the molecular docking, we used SARS-CoV-2 Spike receptor-binding domain bound with ACE2 (PDB ID 6M0J) 32 ( Figure S1 (F)). Furthermore, SARS-CoV-2 Papain-like protease (PDB ID: 6WX4) 33 , which is essential for virus maturation and its infectivity 34 , was used as another protease target in this study ( Figure S1 (G)). The three-dimensional structures were selected and obtained from Protein Data Bank (http://www.rscb.org) with proper resolutions. In this study, a total of fourteen compounds was used as docking ligands to screen and identify the potent antiviral agents for COVID-19 (Table 1) . PubChem database (www.pubchem.ncbi.nlm.nih.gov/) was used to retrieve the 3D chemical structures of the selected molecules, which were downloaded in .sdf format. Energy minimization was subjected to all conformers. All the minimizations were performed under an RMSD gradient of 0.01 kcal mol −1 and RMS distance of 0.1 Å with MMFF94X force-field. The ligands were converted to (pdbqt) file format as inputs for docking studies. Molecular docking as a powerful computational modeling tool is a structure-based drug design (SBDD) approach to identify the essential amino acid interactions between the active site of a receptor/enzyme and generated ligands with low energy conformation 35 In order to validate the docking results, 70 ns Molecular Dynamics (MD) simulation was employed for two main drug-able targets of SARS-Cov-2 RdRp and M pro in complex with NR. As a computational approach, MD is used to analyze the dynamic behavior of complexes where atoms and molecules interact as a function of time. The top-predicted docking pose of NR with the highest binding score to proteins was used as a starting point for the MD simulation. GROMACS 2019.1 software was used for all simulations applying the AMBER03 force field. All the MD simulation systems were solvated using the SPC water model. The particle mesh Ewald algorithm (PME) 42 was considered for long-range electrostatic interactions during simulation. To regulate the simulation temperature, the Berendsen thermostat process was used for 1ns at 310K. A cubic cell was generated within 0.9 Å on each side of the system and a periodic boundary condition was preferred during the simulation. During the simulation, the Fourier grid spacing, and Coulomb radius were set at 0.16 and 1.2 nm, respectively, and the van der Waals interactions were limited to 1.2 nm. The MD trajectories were saved at every 10 ps for energy stabilization. Overall, 10,000 frames were obtained from each production simulation. Molecular Mechanics/Poisson-Boltzmann Surface Area method was used for binding free energy calculation 43 . Selected snapshots from the last 20 ns MD simulation were used for two protein-ligand complexes. Binding free energy of protein and drug was calculated using the equation below: Where G complex depicts the total free energy of the protein−ligand complex and G protein and G ligand are the free energies of the protein and ligand in the solvent, respectively. Moreover, the equation below was used to understand further each energy term, such as Van der Waal forces (G vdw ), electrostatics energy(G ele ), polar (G pol ), and non-polar (G npol ) interactions contributing to the total energy (G), while TS refers to the entropic contribution to the free energy in a vacuum where T and S denote the temperature and entropy, respectively. 3 Results and discussion Natural occurring vitamin NR and its active metabolite (NMN) were structurally similar to Favipiravir and Ribavirin. Thus, according to the Structure-Function Relationship, it was expected to have comparable antiviral activity due to a similar mechanism. Therefore, a molecular docking study was used as the first step for a structure-based investigation of this assumption. According to the binding affinities which are shown in The predicted binding site and 2D interaction of the selected ligands for SARS-CoV-2 main protease (M pro ) or 3CL pro are shown in Figure S4 . As presented in Table 4 Carmofur (-6.3 kcal/mol) which is the proved Michael acceptor inhibitor of M pro . In addition, the same as Carmofur, the critical structural features for interaction with essential amino acid residues CYS145 and HIS41 are also present in these three successfully tested ligands [44] [45] [46] . Structural parameters including RMSD, RMSF were used to evaluate the stability of proteindrug complexes. In this study, two docked complexes, including NR against M pro and RdRp 47 . 2D visualization of the interaction between the protein with studied molecules can be seen in Figure S6 . Docking analysis yields high binding affinities, ranging from (-8.6 kcal/mol) to (-8.9 kcal/mol) for all studied ligands ( Table 7) . The vast number of hydrogen interactions with IMPDH in the binding pocket, as shown in Figure S6 , causes the stability of the complexes. The inhibition of IMPDH prevents interleukin-6 production 48 . This effect also can suppress virus replication 49 . Therefore, IMPDH inhibitors deserve more attention as COVID-19 therapeutic agent 26 . In this regard, this docking study hypothesizes that the compound NR as monophosphate metabolite may be effective in COVID-19 patients' management via inhibition of IMPDH. Based on the experimental studies, this docking research proposes that the mechanism of anti-COVID-19 efficiency of Ribavirin 12,19 may occur through the inhibitory effects on IMPDH. Furthermore, Favipiravir showed weaker ligand-protein interactions, and about 0.3 and 0.1 kcal/mol higher free energy of binding than Mizoribine and Ribavirin, respectively, which these docking results are consistent with the results of experimental researches 50 . Table 7 . Molecular docking analysis of studied compounds against IMPDH (1NF7). Tables 8 and 9) show the calculated binding data of the four above-mentioned molecules yielded binding affinities ranging from (-4.8 kcal/mol) to (-6 kcal/mol). Among these studied molecules, NR and Ribavirin yielded higher binding affinities towards these targets. As shown in Table 8, NR and Ribavirin have free energy values of -6 kcal/mol and -5.8 kcal/mol, respectively, against ACE2. According to the reflected data in Table 9 -8.7 SER68, PRO69, ASN303, ARG322, GLY326, SER327, GLY328, ASP364, GLY365, SER388, TYR411 --2 Favipiravir-RMP -8.6 SER68, ASP274, SER275, ASN303, GLY328, MET414, GLY415 ARG322 -3 Mizoribine monophosphate -8.9 SER68, ASN303, GLY326, SER327, GLY328, SER329, ILE330, ASP364, GLY365, GLY366, ILE367, GLY387, SER388, TYR411 --4 Ribavirin monophosphate -8.7 SER68, ASN303, ARG322, GLY324, GLY326, SER327, GLY328, SER329, ASP364, GLY366, SER388, TYR411 --and -4.9 kcal/mol against ACE2) to be a natural consequence of their small sizes. As it can be seen in Figure 4 , the selected ligands bind at the interface region of Spike-RBD and ACE2 and having the potential to inhibit the virus entry inside the host cells. Briefly, in this section of our docking study, we used PDB ID 6M0J (Spike-ACE2 complex) and the junction portion between the S pro -RBD and human ACE2 receptor. In the first phase, we eliminated the S pro as a ligand, then targeted the ACE2 and vice versa at the next phase ( Figures S7 and S8 ). According to the resulted scores, NR can be considered as a primitive ligand for the human ACE2 receptor; therefore, a protective agent against SARS-Cov-2 induced infection via inhibition of the viral entrance. Here the molecular docking studies were carried out for the Nicotinamide, NR, Favipiravir, and Ribavirin against coronavirus S pro to identify the probable molecular interactions serendipitously ( Figure S9 ). According to the resulted scores, all molecules showed binding interactions with SARS-CoV-2 S pro , as can be seen in Table 10 . The highest binding affinity was seen for NR (-5.7 kcal/mol), about 1kcal/mol better than Ribavirin (-5.6 kcal/mol), while Nicotinamide shows the lowest binding affinity (-4.5kcal/mol) towards SARS-CoV-2 S pro . Similarly, the negative free energy calculation indicates the interaction of Favipiravir with SARS-CoV-2 S pro (-5.2 kcal/mole). The number of hydrogen bonds and amino acid residues of the target interacting with each molecule are given in Table 10 . Generally, Considering the docking results of NR and the active metabolite NMN (Table 11) and the obtained binding modes (Figure 4 shown a slightly higher activity than NR, which is following the slightly narrower band energy gap of NMN obtaining from the docking study (Tables 2-4 and 6 ). Considering the approximately identical interactions of NMN and Mizoribine monophosphate against the IMPDH, it also raises the need for further investigation into this possible mechanism (Table 11 , Rows 5a and 5b). Besides, the resulted data from the molecular docking studies of NR against S pro and ACE2 receptors by targeting their interface region has demonstrated the potential effectiveness of the discussed structural pattern in this research (Table 11 , Rows 6-8). The experimental studies support molecular docking studies on a particular target at molecular levels, such as mass spectroscopy of ligand-target complexes to characterize the interactions, and the binding modes 52 , further x-ray crystallography identifications following successful cellular assessments. This strategy can determine the best compounds and their binding modes, which can be used for future in silico investigations and virtual screening approaches. In the current research, among the selected targets of SARS-CoV-2, the main protease has revealed confirmed engagement with specific ligands through covalent and, or other kinds of chemical interactions with the catalytic pair amino acid residues Cys145 as the nucleophilic center and His41 as the general acid/base or a π-π interaction creator group (Table 11 and (Table 4 ). On the other hand, although Favipiravir and Ribavirin both are distinguished via mutagenic and non-mutagenic antiviral activity through viral RdRp inhibition by their three-phosphate metabolites, based on the resulted data from the current research, different assumed mechanisms of action are probable for these drugs 57, 58 . Here, the binding affinity and the docking energy of different non-three phosphate forms of Favipiravir and Ribavirin were also investigated and discussed. These theoretical results raise the possibility that the only active form of these drugs may not be their triphosphate metabolites, and, it creates a new insight about the other possible mechanisms of antiviral activity of Favipiravir and Ribavirin, that seems necessary to be subject for further investigations. Regardless of the extensive researches, in the real battlefield against SARS-CoV-2, a broad spectrum of medicinal agents such as antibiotics, antimalarial drugs, non-specific antivirals, anticoagulants, etc., have been prescribed as therapeutic cocktails to save people's lives. At the primitive phase of a higher level, computational drug design and discovery approaches rationalize our attempts to find COVID-19 treatment via virtually targeting the SARS CoV-2 structural and functional constituents in screening processes. In the current study, the resulted scores from molecular docking and dynamics simulations as the primary determinative factor as well as the observed reliable binding modes have demonstrated that Nicotinamide Riboside and its active metabolite NMN can target human ACE2 and IMPDH, along with the viral S pro , M pro , PL pro , and on top of all, RdRp as a potential competitive inhibitor. Besides, Nicotinamide Riboside is one of the significant nicotinamide adenine dinucleotide (NAD+) precursors, which possess significant health benefits such as anti-inflammatory effects by reducing the expression of inflammatory markers such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 that are responsible for the severity of COVID-19. 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