Aims Cell surface binding immunoglobin protein (csBiP) is predicted to be susceptible to SARS-CoV-2 binding. With a substrate-binding domain (SBD) that binds to polypeptides and a nucleotide-binding domain (NBD) that can initiate extrinsic caspase-dependent apoptosis, csBiP may be a promising therapeutic target for COVID-19. This study aims to identify FDA-approved drugs that can neutralize viral binding and prevent viral replication by targeting the functional domains of csBiP. Methods In silico screening of 1999 FDA-approved drugs against the functional domains of BiP were performed using three molecular docking programs to avoid bias from individual docking programs. Top ligands were selected by averaging the ligand rankings from three programs. Interactions between top ligands and functional domains of BiP were analyzed. Key Findings The top 10 SBD-binding candidates are velpatasvir, irinotecan, netupitant, lapatinib, doramectin, conivaptan, fenoverine, duvelisib, irbesartan, and pazopanib. The top 10 NBD-binding candidates are nilotinib, eltrombopag, grapiprant, topotecan, acetohexamide, vemurafenib, paritaprevir, pixantrone, azosemide, and piperaquine-phosphate. Among them, Velpatasvir and paritaprevir are antiviral agents that target the protease of hepatitis C virus. Netupitant is an anti-inflammatory drug that inhibits neurokinin-1 receptor, which contributes to acute inflammation. Grapiprant is an anti-inflammatory drug that inhibits the prostaglandin E 2 receptor protein subtype 4, which is expressed on immune cells and triggers inflammation. These predicted SBD-binding drugs could disrupt SARS-CoV-2 binding to csBiP, and NBD-binding drugs may falter viral attachment and replication by locking the SBD in closed conformation and triggering apoptosis in infected cells. Significance csBiP appears to be a novel therapeutic target against COVID-19 by preventing viral attachment and replication. These identified drugs could be repurposed to treat COVID-19 patients.
Electronegative clusters (ENCs) made up of acidic residues and/or phosphorylation sites are the most abundant repetitive sequences in RNA‐binding proteins. Previous studies have indicated that ENCs inhibit RNA binding for structured RNA‐binding domains (RBDs). However, this is not the case for the unstructured RBD in histone pre‐mRNA stem‐loop binding protein (SLBP). The SLBP RBD contains 70 amino acids and is followed by a phosphorylatable ENC. ENC phosphorylation increases RNA‐binding affinity of SLBP to the sub‐picomolar range. In this study, we use NMR and molecular dynamics simulations to elucidate the mechanism for this tight binding. Our NMR data demonstrate that the ENC transiently folds apo SLBP into an RNA‐bound resembling state. We find that in the RNA‐bound state, the phosphorylated ENC interacts with the loop region opposite to the RNA‐binding site. This allosteric interaction stabilizes the complex and therefore enhances RNA binding. To evaluate the generality of our findings, we graft an ENC onto endoribonuclease homolog 1's first double‐stranded RNA‐binding motif (DRBM1), an unstructured RBD that shares no homology with SLBP. We find that the engineered ENC increases the folded species of DRBM1 and inhibits RNA binding. On the contrary, introducing basic residues to DRBM1 makes the domain more unfolded, enhances RNA binding, and mitigates the inhibitory effect of the engineered ENC. In summary, our study suggests that ENCs promote folding of unstructured RNA‐binding domains, and their effects on RNA binding depend on the electropositive charges on the RBD surface.
Background: Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) and Apolipoprotein E (ApoE) are two of the strongest genetic risk factors for late-onset Alzheimer's disease (AD), but while their interplay in the microglial response to AD pathology is hotly studied, surprisingly little is known about the mechanism by which their variants impair this role. Method:To define the structural mechanism by which AD-associated variants impair the TREM2-ApoE interaction, we used sequence-based in silico methods to predict the binding site between TREM2 and ApoE, validated the predicted binding site with biolayer interferometry (BLI), then constructed initial models of TREM2-ApoE complex structures using the identified binding site between TREM2 and APOE as restraints for unbiased molecular docking methods. The top four initial models were re-ranked by comparing the effect of key residue mutations in the TREM2-ApoE complex on binding affinities calculated from molecular dynamics-molecular mechanics Poisson-Boltzmann surface area (MD/MM-PBSA) binding free energy analyses with binding affinities measured by BLI. The final validated TREM2-ApoE complex structure model provides for further investigating the molecular and structural effects of AD-associated TREM2 variants on interactions between hydrophobic site of TREM2 and hinge region of ApoE.Result: Our iterative MD/MM-PBSA-BLI procedure generated an all-atom model of the TREM2-ApoE complex that replicates the binding effects of both engineered mutations in TREM2 and AD-associated variants in both TREM2 and ApoE. The validated model shows the interaction centered on the apical hydrophobic site of TREM2 and the hinge region of ApoE. The interaction sites in the TREM2-ApoE complex model are consistent with regions affected by AD-associated variants in simulations of the individual proteins, with biophysical studies using truncated constructs of the two proteins, and with a recently reported minimal active peptide of TREM2 that is sufficient to stimulate its cytoprotective and cytokine regulating roles in primary microglia.
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