ABSTRACT
Neurodegenerative diseases are characterized by increased permeability of the blood-brain barrier (BBB) due to alterations in cellular and structural components of the neurovascular unit, particularly in association with neuroinflammation. A previous screening study of peptide ligands to identify molecular alterations of the BBB in neuroinflammation by phage-display, revealed that phage clone 88 presented specific binding affinity to endothelial cells under inflammatory conditions in vivo and in vitro. Here, we aimed to identify the possible target receptor of the peptide ligand 88 expressed under inflammatory conditions. A cross-link test between phage-peptide-88 with IL-1ß-stimulated human hCMEC cells, followed by mass spectrometry analysis, was used to identify the target of peptide-88. We modeled the epitope-receptor molecular interaction between peptide-88 and its target by using docking simulations. Three proteins were selected as potential target candidates and tested in enzyme-linked immunosorbent assays with peptide-88: fibronectin, laminin subunit α5 and laminin subunit ß-1. Among them, only laminin subunit ß-1 presented measurable interaction with peptide-88. Peptide-88 showed specific interaction with laminin subunit ß-1, highlighting its importance as a potential biomarker of the laminin changes that may occur at the BBB endothelial cells under pathological inflammation conditions.
Subject(s)
Blood-Brain Barrier , Endothelial Cells/metabolism , Inflammation/metabolism , Laminin/metabolism , Animals , Bacteriophage M13 , Biomarkers , Cells, Cultured , Cross-Linking Reagents , Fibronectins/metabolism , Gene Ontology , Humans , Interleukin-1beta/pharmacology , Models, Molecular , Molecular Docking Simulation , Peptide Library , Protein Binding , Protein Conformation , Protein Interaction Mapping , RatsABSTRACT
Dengue and Zika are two mosquito-borne diseases of great impact on public health around the world in tropical and subtropical countries. DENV and ZIKV belong to the Flaviviridae family and the Flavivirus genus. Currently, there are no effective therapeutic agents to treat or prevent these pathologies. The main objective of this work was to evaluate potential inhibitors from active compounds obtained from Marcetia taxifolia by performing inverse molecular docking on ZIKV-NS3-helicase and ZIKV-NS5-RNA polymerase as targets. This computational strategy is based on renormalizing the binding scores of the compounds to these two proteins, allowing a direct comparison of the results across the proteins. The crystallographic structures of the ZIKV-NS3-helicase and ZIKV-NS5-RNA-polymerase proteins share a great similarity with DENV homologous proteins. The P-loop active site of the crystallographic structure of ZIKV-NS3-helicase presents a high percentage of homology with the four dengue serotypes. It was found that most ligands of the active compounds (5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (5DP); 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5HH); myricetin-3-O-rhamnoside (M3OR)) from Marcetia taxifolia had a better affinity for ZIKV-NS3-helicase than for ZIKV-NS5-RNA polymerase, as indicated by the negative multiple active site correction (MASC) score, except for M3RG that showed a higher affinity for ZIKV-NS5-RNA polymerase. On the other hand, the AutoDock Vina scores showed that M3OR had the highest score value (-9.60 kcal/mol) and the highest normalized score (1.13) against ZIKV-NS3-helicase. These results in silico demonstrated that the nonstructural proteins NS3-helicase and NS5-RNA polymerase, which share similar molecular structures between the selected viruses, could become therapeutic targets for some bioactive compounds derived from Marcetia taxifolia.
