Innovations in molecular biology and proteomics adapted to in vivo peptide phage display define active protein domains as biomarkers in multiple sclerosis neuroinflammation

Abstract

With the double goal to streamline biomarker discovery of Multiple Sclerosis neuroinflammatory pathology entangled by non-affected Central Nervous System tissue and to better characterize the complex molecular interactions at the altered blood brain barrier (BBB) that becomes permissive to extravasation of blood derived immune cells and compounds, we choose to identify peptide ligands reacting with inflammatory CNS lesions. With no “a-priori” for targets by applying in vivo the phage displayed 12aa peptide screening in CNS neuroinflammation of the experimental autoimmune encephalomyelitis (EAE) rat model of MS and healthy controls, we generated two complex phage displayed peptide repertoires. By developing molecular DNA subtraction of both phage repertoires we generated a subtraction phage repertoire of EAE specific peptides. In a bioinformatics approach aligning the 270,612 EAE peptides to the human proteome the formed peptide clusters allowed the ranking of proteins being potential “mimicked” indicators of functional protein interactions. Experimentally, among the most high score mimicked proteins, derived synthesized short peptides (22aa) of mimicked domains were tested for labeling of BBB alterations in CNS sections of MS and other chronic neurodegenerative diseases. In vitro studies using the BBB model hCMEC/D3 confirmed their binding to human endothelial cells under proinflammatory stimulation. Finally, with a derived peptide covalently linked to a nanocarrier contrast agent, we could target in vivo and monitor by MRI, experimentally induced inflammatory CNS lesions. Our strategy of combining the developed molecular biology and bioinformatics approaches adapted to phage display is applicable to many disease models to identify biomarkers and functional proteins/domains.