key: cord-0876778-jiv5rxvk authors: Dasgupta, Subhajit; Bandyopadhyay, Mausumi title: Molecular docking of SARS-COV-2 Spike epitope sequences identifies heterodimeric peptide-protein complex formation with human Zo-1, TLR8 and brain specific glial proteins date: 2021-10-13 journal: Med Hypotheses DOI: 10.1016/j.mehy.2021.110706 sha: 5760ebd744f69d54f0bcacc474c90760fc93b32d doc_id: 876778 cord_uid: jiv5rxvk The SARS-COV-2 infection causes severe respiratory tract illness leading to asphyxia and death. The onset of infection is associated with loss of smell sensation and blurred vision, headache with bronchopulmonary symptoms. The clinical observations of neurological abnormalities lead us to address the question, does the virus enter into brain and what is the underlying mechanism of brain infection? The working hypothesis is, SARS-COV-2 Spike epitopes modify blood brain barrier and infect glial cells to induce brain inflammation in genetically diverse human population. The hypothesis is tested by determining binding or interacting ability of virus Spike epitope peptides M1Lys60 and Ala240Glu300 with human toll-like receptor 8 (TLR 8),brain targeted Vascular Cell adhesion Molecules (VCAM1) proteins, Zonula Occludens (ZO), glial cell specific protein NDRG2 and Apo- S100B. The molecular dynamic experiments are performed, and root mean square deviation (RMSD) values are determined for interactions between the Spike peptides and selected proteins. The observations demonstrate formation of heterodimeric complex between the epitope peptides and selected protein structures. The viral epitopes have ability to bind with HLA-DRB1 15:01, 07:01 or 03.01 alleles thus found immunogenic in nature. The observations altogether suggest entry of these Spike protein epitopes into human brain cause inflammation. The SARS-COV-2 infection causes severe respiratory tract illness leading to asphyxia and death. The onset of infection is associated with loss of smell sensation and blurred vision, headache with bronchopulmonary symptoms. The clinical observations of neurological abnormalities lead us to address the question, does the virus enter into brain and what is the underlying mechanism of brain infection? The working hypothesis is, SARS-COV-2 Spike epitopes modify blood brain barrier and infect glial cells to induce brain inflammation in genetically diverse human population. The hypothesis is tested by determining binding or interacting ability of virus Spike epitope peptides M1Lys60 and Ala240Glu300 with human toll-like receptor 8 (TLR 8),brain targeted Vascular Cell adhesion Molecules (VCAM1) proteins, Zonula Occludens (ZO), glial cell specific protein NDRG2 and Apo-S100B. The molecular dynamic experiments are performed, and root mean square deviation (RMSD) values are determined for interactions between the Spike peptides and selected proteins. The observations demonstrate formation of heterodimeric complex between the epitope peptides and selected protein structures. The viral epitopes have ability to bind with HLA-DRB1 15:01, 07:01 or 03.01 alleles thus found immunogenic in nature. The observations altogether suggest entry of these Spike protein epitopes into human brain cause inflammation. Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-COV-2) causes recent pandemic with heightened mortality rate all over the world. Acute respiratory distress (ARD) with uncontrolled surge of cytokines is found to worsen the disease condition. The viral infection causes lung collapse with pneumonia (1, 2) and cardiovascular failure (3) . The genetic mark up with susceptibility of infection in population was not known until recent genome wide association study (GWAS) which suggests 3p21.31 gene cluster is critical for SARS-COV-2 infection with respiratory failure (4) . Several investigations (5-7) also indicated possible genetic factors for determining hosts' susceptibility to SARS-COV-2. Progressive infection causes kidney malfunction (8) and multi-organ failure (9) . The meta-analysis of COVID-19 patients in pandemic demonstrates close association with neuropsychiatric disorders (10) . The presence of virus particles in cerebrospinal fluid has been demonstrated from infected patients (11, 12) which indicates presence of the virus into central nervous system. It is reported that SARS-COV2 infection activates astrocytes and microglia in patients (13) . Though mechanism is still not clear. We identified unique Spike epitopes which have binding affinity to HLA-DRB1 alleles 15:01; 07:01 and 03:01 with variable immunogenicity to T helper cells. The brain receptor binding efficacy of these viral peptides are tested hereby by demonstration of heterodimer formation with selected human brain targeted proteins. The mapping of viral receptors in human brain and immune therapy approach to block these virus epitopes is currently underway. Our working hypotheses are two folds; we propose (1) SARS-COV-2 spike epitope peptides modify blood brain barrier proteins and enter into brain cause inflammation; and (2) Viral spike epitope peptides induce activated T helper cells recognized by HLA-DRB1 alleles in genetically diverse human population leading to chronic brain inflammation and neurodegeneration. The specific interaction patterns between virus spike epitope sequences and human brain targeted proteins are still not clear. It has been reported that, seven members of human Corona Virus family (hCOV) : SARS-COV, SARS-COV-2, MERS-COV, HCoV-OC43, HCoV-229E, HCoV-HKU1 and HCoV-NL63 are related to each other and possibly infect human central nervous system (CNS) (12) . Virus infection causes alteration in blood brain barrier integrity (14, 15) . The postmortem analysis of COVID-19 patients demonstrates presence of activated astrocytes, microglia and cytotoxic T cells in olfactory bulb, brain stem and cerebellum (13, 16) (17). We selected two Spike peptides with amino acid sequences 1-60 (M1Lys60) and 241-300 (Ala240Glu300) (NH2-terminal sequences) to determine their binding efficacy with brain receptor proteins. The viral peptides are found to bind with HLA-DRB1 alleles 03:01, 07:01 and 15:01 to a variable extent ( Table 1) (IEDB epitope analysis tools). The observations presented in Table1 (20) and rat Apo S100B (PDB: 1B4C) (21) . The md experiments were performed by using protein database (PDB) format of Spike peptides and selected proteins. We aligned the PDB formatted Spike peptides with each of selected brain derived peptides considering outlier rejection in 10 cycles and cut off value 2 Angstrom unit. The md run was set for 2 minutes (120 x 10 9 nsec) , 30 Frames per second with ray tracing parameter and each aligned Spike peptide as mobile selection for each selected host protein (target selection). We determined root mean square deviation (RMSD) value (Angstrom) for each molecular dynamics run. The frame per second versus rendering time plot for each molecular dynamic interaction has been presented with docking results on blood brain barrier tight junction protein Zonula Occluden-1 (ZO-1) (Fig. 1A) , Vascular Cell Adhesion Factor (VCAM) (Fig. 1B) , human Toll-like receptor 8 (TLR8) (Fig. 2) . The molecular docking experiments also demonstrates specificity of Spike peptide epitopes to form complex with human astrocyte specific protein N-myc downstream-regulated gene 2 (NDRG2) (Fig. 3A) and glial specific calcium regulator protein Apo S100B ( Fig. 3B) . The molecular dynamics (md) and docking experiments provide clear demonstrations on mechanism of interaction between Spike epitopes (1-60 and 241-300) and selected blood brain barrier protein Zo, innate immune responder TLR8 and brain derived glial cell proteins NDRG2 and Apo-S100B. The interaction profile indicates of SARS-COV-2 in blood brain barrier endothelium and cause inflammation in brain. The use of md method is a new way to identify interaction between homologous as well as non-homologues proteins on the basis of atom-to-atom alignment profile between two interacting proteins. The computational approach with md has been used to demonstrate mode of action of natural compound as possible drug and therapeutics in cancer (22, 23) . Recently, therapeutic importance of human antimicrobial peptide LL-37 for COVID has been analyzed by md methods (24) . In the aspect, our observations provide new findings on Spike protein action on selected proteins in human brain. The blood brain barrier (BBB) composed of endothelial cells, astrocyte-foot process and axons -all play vital role in maintaining CNS integrity. The junctions between the BBB cells selectively pass nutrients and small molecules from periphery to CNS. Our findings (Fig1A and B) that, viral spike epitopes [1-60: M1Lys60 and 241-300: Ala240Glu300)] form complex with tight junction protein Zo-1 and integrin VCAM-1, suggest a mechanism of Spike peptides on viral entry to CNS through BBB. The Spike peptide interaction pattern with human TLR8 (Fig.2) is another critical observation showing involvement of TLR8. The elevated expression of VCAM1 is demonstrated in microglia in brain lesions of multiple sclerosis patients (25) (26) . The possibility that, chronic inflammation in brain through interaction between Spike epitope and VCAM1 integrin leading to autoimmune onset of multiple sclerosis cannot be ignored in aging population. We found Spike peptides bind with astrocyte activation protein NDRG2 (tumor suppressor protein and stress response gene product). The elevated production of this protein shows activation of astrocyte and presence of abnormal synaptic glutamate during neurodegenerative diseases and neuropathic pain (27, 28) . We pursue these findings to determine specific binding region of SARS-COV-2 Spike peptides for the receptor(s) present in human brain and develop immunotherapy. No conflict of interests. There is no animal model or human subjects involved in the manuscript. The consent statement/ ethical approval is not required. The research is supported by internal funding from NeuroDrug Research LLC. There is no Gov't or other fund associated with the project. Table 1 . SARS-COV2 N-terminal Spike peptide epitopes efficiently bind with HLA-DRB1 alleles and demonstrate CD4 cell immunogenicity COVID-19 lungs in post-mortem computed tomography CT in coronavirus disease 2019 (COVID-19): a systematic review of chest CT findings in 4410 adult patients Cardiovascular complications in COVID-19 Association Study of Severe Covid-19 with Respiratory Failure Chromosome 3 cluster rs11385942 variant links complement activation with severe COVID-19 New insights into genetic susceptibility of COVID-19: an ACE2 and TMPRSS2 polymorphism analysis Human Leukocyte Antigen Complex and Other Immunogenetic and Clinical Factors Influence Susceptibility or Protection to SARS-CoV-2 Infection and Severity of the Disease Course Comorbidities in SARS-CoV-2 Patients: a Systematic Review and Meta-Analysis. mBio Deciphering the COVID-19 cytokine storm: Systematic review and meta-analysis Psychiatric and neuropsychiatric presentations associated with severe coronavirus infections: a systematic review and metaanalysis with comparison to the COVID-19 pandemic Validation of SARS-CoV-2 detection across multiple specimen types Coronaviruses and the central nervous system Neuronophagia and microglial nodules in a SARS-CoV-2 patient with cerebellar hemorrhage SARS-COV2 Alters Blood Brain Barrier Integrity Contributing to Neuro-Inflammation SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human blood-brain barrier Neuropathology of patients with COVID-19 in Germany: a post-mortem case series Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and glial cells: Insights and perspectives The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction Structural reorganization of the Toll-like receptor 8 dimer induced by agonistic ligands Crystal structure of the human N-Myc downstream-regulated gene 2 protein provides insight into its role as a tumor suppressor The use of dipolar couplings for determining the solution structure of rat apo-S100B(betabeta) Critical Regulator of Androgen Receptor Signaling and Prostate Cancer Progression Molecular interaction studies of Deguelin and its derivatives with Cyclin D1 and Cyclin E in cancer cell signaling pathway: The computational approach An in silico scientific basis for LL-37 as a therapeutic for Covid-19 VCAM-1-positive microglia target oligodendrocytes at the border of multiple sclerosis lesions Soluble VCAM-1 impairs human brain endothelial barrier integrity via integrin alpha-4-transduced outside-in signalling NDRG2 as a marker protein for brain astrocytes Nervous NDRGs: the N-myc downstream-regulated gene family in the central and peripheral nervous system (50) value less than 50 nM is considered as high affinity binding of peptide with HLADRB1 allele; IC (50) greater than 50 less than 500 nM is intermediate affinity. IC (50) greater than 500 nM less than 5000 nM is considered as low to poor affinity or no affinity. The core peptides are assessed for their affinity profile with alleles.