key: cord-0868189-s8t1emgf authors: Khan, Faez Iqbal; Hassan, Fakhrul; Lai, Dakun title: In Silico Studies on Psilocybin Drug Derivatives Against SARS-CoV-2 and Cytokine Storm of Human Interleukin-6 Receptor date: 2022-01-14 journal: Front Immunol DOI: 10.3389/fimmu.2021.794780 sha: d3a7368ffb99b1607f5da0e6fd2def718e68a190 doc_id: 868189 cord_uid: s8t1emgf Various metabolites identified with therapeutic mushrooms have been found from different sources and are known to have antibacterial, antiviral, and anticancer properties. Over thousands soil growth-based mushroom metabolites have been discovered, and utilized worldwide to combat malignancy. In this study, psilocybin-mushroom that contains the psychedelic compounds such as psilacetin, psilocin, and psilocybine were screened and found to be inhibitors of SARS-CoV-2 Mprotease. It has been found that psilacetin, psilocin, and psilocybine bind to Mprotease with −6.0, −5.4, and −5.8 kcal/mol, respectively. Additionally, the psilacetin was found to inhibit human interleukin-6 receptors to reduce cytokine storm. The binding of psilacetin to Mprotease of SARS-CoV-2 and human interleukin-6 receptors changes the structural dynamics and Gibbs free energy patterns of proteins. These results suggested that psilocybin-mushroom could be utilized as viable potential chemotherapeutic agents for SARS-CoV-2. Coronaviruses (CoVs) represent the family Coronaviridae and sub-family Orthocoronavirinae. The sub family Orthocoronavirinae is further divided into four genera namely, a-coronavirus, b-coronavirus, g-coronavirus, and d coronavirus. The host of aand b-coronaviruses are mammals, while the host of gand d-coronaviruses are birds (1, 2) . Some of the b-coronavirus like Severe Acute Respiratory Syndrome CoV 1 & 2 (SARS-CoV1 and SARS-CoV2), and Middle-East Respiratory Syndrome CoV (MERS-CoV) are the leading cause of pneumonia in this century. The outbreaks of SARS-CoV1, MERS-CoV and SARS-CoV2 were caused by zoonotic viruses in 2003, 2012 and 2019-20 with fatality ratio of 10%, 35% and 5%, respectively (3) (4) (5) (6) (7) (8) . The International Virus Classification Commission (ICTV), termed this 2019 novel CoV as SARS-CoV-2 (9, 10) . SARS-CoV-2 possesses the positive-sense single-stranded~30 kb RNA genome and consists of 12 open reading frames. The genes located on the 3′-terminus of the virus genome code for four structural proteins such as membrane glycoprotein (M), envelope (E), nucleocapsid (N), spike (S) and eight accessory such as 3a, 3b, p6, 7a, 7b, 8b, 9b, and ORF14 proteins (11, 12) . ORFIa and ORF1ab genes encode the non-structural proteins that form the replication/ transcription complex, respectively. These genes such as ORFIa and ORF1ab encode two polypeptides called pp1a and pp1ab, respectively. The polypeptides are directly translated and catalytically processed by main protease (Mprotease). Mprotease is accountable for the cleavage of polyprotein downstream of non-structural protein 4 to form distinctive nonstructural proteins that show important roles in the life cycle of the virus (13, 14) . The phenomenal spread of the current COVID-19 pandemic to every part of the sphere has lifted the key concerns for the healthcare system. To combat this pandemic, the researchers are using all possible approaches and practices to inhibit the synthesis of crucial non-structural viral proteins, inhibition of the viral replicase enzyme, inhibition of formation of viral-RNA, prevention of selfassembly of viruses or to boost the human immune response against the virus (15, 16) . Main protease Mprotease or 3CLpro has a significant part in SARS-CoV-2 maturation and replication. Therefore, Mprotease is a striking and potential target for the anti-coronavirus drug development to tackle this current COVID-19 pandemic (17, 18) . In several studies, psilocybin drugs have been tested against COVID-19 for mental health problems caused by the pandemic, and the results of the clinical trials indicate that psychedelics are physiologically and psychologically safe (19) . Psilocybin has been reported to be effective in treating depression recently (20) . A study showed that psilocybin therapy has the potential to play an important therapeutic role in treating a variety of psychological disorders after COVID-19 (21) . The COVID-19 poses challenges to mental health, but it also offers the opportunity to develop promising new therapeutic approaches. Recently, researchers have designed a peptide inhibitor against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. It was found to bind at the RBD of the SARS-CoV-2 spike at the Angiotensin-converting enzyme 2 (ACE2) binding site with high affinity (22) . It has been suggested that the blockage of SARS-CoV-2 spike protein binding to ACE2 in the presence of this peptide may prevent virus entry into host cells (23) . Here, we applied molecular docking and molecular dynamics simulations to study the interaction of different metabolites of psilocybin mushrooms such as psilocybine, psilocin, and psilacetin with Mprotease of SARS-CoV-2. Our findings revealed that psilacetin might inhibit the Mprotease of SARS-CoV-2 and reduce the cytokine storm by decreasing the expression of human interleukin-6 receptors (HIK6). Several clinical trials are underway regarding the effectiveness of these mushroom compounds. The compounds such as psilacetin, psilocin, and psilocybine were evaluated by Lipinski filter to check drug-likeness (24) . In silico pharmacokinetic parameters were estimated to analyse the absorption, distribution, metabolism, and excretion (ADME) properties and toxicity of psilacetin, psilocin, and psilocybine. The ADME, carcinogenicity and toxicity were predicted by SwissADME (25) , CarcinoPred-EL (26) , and PreADMET servers, respectively. The molecular interactions of psilacetin, psilocin, and psilocybine with SARS-CoV-2 Mprotease (PDB: 6LU7) were achieved by means of AutoDock and AutoDock vina (27) . The ligands were docked by defining a grid box with spacing of 1 Å and size of 12 × 22 × 14 pointing in x, y and z directions around the protein active site of coordinates −9.9454 × 11.9130 × 68.3144, respectively. The grid size and coordinates are limited to only important active residues of interest. The Lamarckian Genetic Algorithm was used as the search algorithm with default parameter values. The best-docked postures of Mproteasepsilacetin, Mprotease-psilocin, and Mprotease-psilocybine were chosen based on binding energy and proper orientations of ligands in the active pocket of protein. PyMol (28) and Discovery Studio visualizer (BIOVIA) were used for the visualization of the docked complex. The detailed methodology has been reported previously (29, 30) . MD simulation is executed effectively to understand structurefunction relationships in a protein (31) (32) (33) . MD simulations were executed on Mprotease, Mprotease-psilacetin, HIK6, and HIK6psilacetin at 298 K at the molecular mechanics level implemented in GROMACS 2018.2 (34) using the GROMOS96 43a1 forcefield. PRODRG server generated the topology files of psilacetin, psilocin, and psilocybine molecules. The Mprotease, Mproteasepsilacetin, HIK6, and HIK6-psilacetin were soaked in a cubic box of water molecules using the gmx editconf module for creating boundary conditions. The gmx solvate module was used for the solvation. The spc216 water model was used to solvate the system. The charges on the Mprotease, Mprotease-psilacetin, HIK6, and HIK6psilacetin were neutralized by addition of Na + and Clions using gmx genion module to preserve physiological concentration (0.15 M). A total number of 84/81, 84/82, 195/198, and 195/199 Na + /Clwere added in case of Mprotease, Mprotease-psilacetin, HIK6, and HIK6-psilacetin, respectively. The system was further minimized using 1500 steps of steepest descent. Equilibration took place in two phases using NVT and NPT ensembles. The particle-mesh Ewald method was applied after the equilibration phase (35) was applied. The production phases consisted of 100 ns and were conducted at a temperature of 298 K. The detailed methodology has been published in various publications (36) (37) (38) (39) (40) . PCA or ED was performed for Mprotease, Mprotease-psilacetin, HIK6, and HIK6-psilacetin by diagonalizing the covariance matrix C: The r i denotes the Cartesian coordinate, i th Ca atom, N signifies the number of Ca atoms, and the indicates time average over all the configurations. GFE landscape can suggest structural and conformational changes in Mprotease, Mprotease-psilacetin, HIK6, and HIK6psilacetin molecules. For 2D and 3D conformational depiction, the GFE landscape was projected on PC1 and PC2 for Mprotease, Mprotease-psilacetin, HIK6, and HIK6-psilacetin, respectively. The k B , T and P (PC1, PC2) denote Boltzmann constant, temperature, and normalized joint probability distribution for Mprotease, Mprotease-psilacetin, HIK6, and HIK6psilacetin respectively. Clinical trials of drugs fail for a variety of reasons, including toxicity and poor pharmacokinetic properties. Pharmacokinetic profiles for psilacetin, psilocin, and psilocybine were calculated to determine their bioavailability. Lipinski's rule of five assists in distinguishing between drug-like and non-drug-like molecules. A good drug should have these parameters: mass (< 500 Dalton), lipophilicity (LogP < 5), hydrogen bond donors (< 5), hydrogen bond acceptors (< 10) and molar refractivity . The Lipinski filter and ADMET properties of psilacetin, psilocin, and psilocybine are listed in Table 1 . These drugs passed Lipinski filters and were non-carcinogenic. The binding orientations of psilacetin, psilocin, and psilocybine with SARS-CoV-2 Mprotease (PDB: 6LU7) were analyzed by superimposing the inhibitor N3 present in the pocket of Mprotease. The co-crystallized inhibitor N3 showed electrostatic (ES) interactions with residues Phe140, Gly143, His163, His164, His172, Glu166, Gln189, Thr190, and Ala191 of Mprotease. Further, it shows van der Waals (vdW) contacts with residues Thr24, Thr26, Thr25, Met49, His41, Asn142, Leu141, Cys145, Ser144, Pro168, Met165, Asp187, and Gln192 of SARS-CoV-2 Mprotease. The N3 inhibitor was covalently linked with Cys145 of Mprotease ( Figures 1A, B) . The binding energy of psilacetin with Mprotease was -6.0 kcal/mol. The psilacetin showed ES interactions with residues His41, Tyr54, Glu166, Asp187 and Thr190 of SARS-CoV-2 Mprotease. Further, it showed vdW interactions with residues Met49, His164, Met165, Ley167, Pro168, Arg188, Gln198, Gln192 of SARS-CoV-2 Mprotease ( Figures 1C, D) . The binding energy of psilocin with Mprotease was found to be -5.4 kcal/mol. The psilocin displayed ES interactions with residues His41, Leu141, Gly143, Ser144, Cys145, His163, and Gln189 of SARS-CoV-2 Mprotease. Further, it shows vdW interactions with Thr25, Thr26, Leu27, Phe140, Asn142, Met165, Glu166, and His172 of SARS-CoV-2 Mprotease (Figures 1E, F) . The binding energy of psilocybine with Mprotease was -5.8 kcal/mol. The psilocybine showed ES contacts with His41, Asn142, Phe140, Leu141, Gly143, Cys145, Ser144, His164, His163, Met165, and Gln189 of Mprotease. Further, it shows vdW interactions with residues Thr25, Thr26, Leu27, Met49, Glu166, and His172 of Mprotease ( Figures 1G, H) . The detailed interacting residues are mentioned in Table 2 . It has been found that psilacetin strongly binds with Mprotease. The binding of these compounds was further analyzed using MD simulations. The binding orientations of the co-crystallized ligand with human interleukin-6 receptor (PDB: 1N26) were analyzed. The cocrystallized ligand showed electrostatic interactions with residues Asn110, Glu144, Gln147, Gln158, and Asn226 of HIK-6. Further, it shows van der Waals interactions with residues Ser109, Val112, Pro145, and Ser227 of HIK-6. Additionally, it forms one covalent bond with residue Asn226 (Figures 2A, B) . The binding orientations of psilacetin with HIK-6 were analyzed by superimposing with the co-crystallized ligand present in the active pocket of HIK-6. The binding energy of psilacetin with HIK-6 was found to be -4.1 kcal/ mol. The psilacetin showed electrostatic interactions with residues Ser106, Asn110, Gln158, Ser224, and Asn226 of HIK-6. Further, it shows van der Waals interactions with residues Phe103, Lys105, Ser109, and Val112 of HIK-6 ( Figure 2C, D) . A severe inflammatory response and cytokine storm are mediated by IL-6. It is reported that IL-6 is elevated in COVID-19, especially in severely ill patients (41) . In another study, increased levels of IL-6 in the blood was associated with a fatal outcome in COVID-19 infections. (42) . The early detection of cytokine storms and fast treatment can help to defeat COVID-19 symptoms. This study shows that molecular interactions of psilacetin with the active pocket of HIK-6, and thereby blocking IL-6 could be possibly favorable for patients with severe lung inflammation caused by a cytokine storm. psilacetin binds to a membrane-bound IL-6 receptor, hence stopping IL-6 signaling and inflammatory response. In contrast, the S glycoprotein on the envelope of the CoV binds to the ACE2 receptor on the membrane of host cells, which allows the virus to go into the cells. After binding, the coronavirus is activated, which results in the discharge of viral-RNA into the cytoplasm that leads to infection. Intact ACE2 is integrated with the virus in coronavirus infection. Several researches indicated that SARS-CoV infection can downregulate the expression of ACE2 resulting in a physiological imbalance between angiotensin and ACE2 that could cause multiple organ injuries. It is reported that a few proteins and proteinases are perhaps engaged in the binding and membrane fusion activity (43) . The ability of SARS-CoV2 to replicate in host cells depends on its Mprotease. The Mprotease of SARS-CoV-2 is an important enzyme that is crucial in conciliating viral transcription and replication by processing the polyproteins (44) . The SARS-CoV-2 Mprotease cleaves the overlaying pp1a and pp1ab polyproteins to functional proteins that is a crucial level in viral replication. An attractive drug that targets to block Mprotease of SARS-CoV-2 could aid in inhibiting the processing of polyproteins. The present study shows the molecular interactions of psilacetin, psilocin, and psilocybine with the active pocket of SARS-CoV-2 Mprotease and could be resulted in stopping the RNA replication and thus lessen the symptoms of coronavirus. The structural dynamics of Mprotease, Mprotease-psilacetin, HIK6, and HIK6-psilacetin were explored using the RMSD, RMSF, and the R g . The mean RMSD of Mprotease and Mprotease-psilacetin were 0.32 nm and 0.26 nm, respectively. The mean RMSD of HIK6, and HIK6-psilacetin were 0.69 nm and 0.65 nm, respectively. It has been discovered that RMSD values and residual fluctuations decrease upon binding of psilacetin to Mprotease and HIK6 ( Figure 3 ). The mean R g for Mprotease and Mprotese-psilacetin were 2.07 nm and 2.11 nm, respectively. The mean R g for HIK6, and HIK6-psilacetin were 2.80 nm and 2.85 nm, respectively. It was discovered that Mprotease and HIK6 have less Rg and less compact packing due to attachment of psilacetin. The structural dynamics results indicate that attachment of psilacetin with Mprotease and HIK6 alter the structure dynamics and properties of these proteins. The mean SASA for Mprotease and Mprotease-psilacetin were found to be 129.55 nm 2 and 134.01 nm 2 , respectively. The mean SASA for HIK6 and HIK6-psilacetin were 160.23 nm 2 and 159.05 nm 2 , respectively ( Figure 4) . The SASA scheme indicated that core amino acids in Mprotease are uncovered to solvent molecules after psilacetin attaches to it. There SASA are not affected much for HIK6. Further, the solvation energy of Mprotease, Mproteasepsilacetin, HIK6, and HIK6-psilacetin were found to be 207. 35, 218.54, 237 .03, and 233.75 kJ/mol/nm 2 , respectively. The solvation energy of Mprotease-psilacetin was more than Mprotease, while HIK6-psilacetin has less solvation energy than HIK6. Further, the secondary structure allocations in Mprotease, Mprotease-psilacetin, HIK6, and HIK6-psilacetin were highlighted at each period. The mean amino acid residues involved in structure development of Mprotease, Mproteasepsilacetin, HIK6, and HIK6-psilacetin were noted ( Table 3 ). It was found that the mean amino acid contributed in structure formation for Mprotease, Mprotease-psilacetin, HIK6, and HIK6-psilacetin were 60%, 59%, 52%, and 52%, respectively ( Figures 3C, D) . It has been found that binding of psilacetin inhibits and unfolds b-sheet and a-helix of Mprotease. In the case of HIK6, there were no such changes in the secondary structure reported. The volume of Mprotease, Mprotease- psilacetin, HIK6, and HIK6-psilacetin were 54.98 nm 3 , 55.23 nm 3 , 57.30 nm 3 , and 57.27 nm 3 , respectively. The average density of Mprotease, Mprotease-psilacetin, HIK6, and HIK6-psilacetin were found to be 1020.79 g/l, 1016.11 g/l, 971.59 g/l, and 972.14 g/l, respectively. There were slight changes reported when psilacetin binds to Mprotease and HIK6. The PCA shows a general expansion of Mprotease, Mproteasepsilacetin, HIK6, and HIK6-psilacetin during the course of simulation. It classifies mean fluctuations and atomic mobility of Mprotease, Mprotease-psilacetin, HIK6, and HIK6-psilacetin. The mean of eigenvalues is an amount of the total motion in the system. It also estimates the flexibility of a molecule. 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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.