Characterization and Validation of Arg286 Residue of IL-1RAcP as a Potential Drug Target for Osteoarthritis

Abstract

Osteoarthritis (OA) is the most common form of arthritis and the fastest growing cause of chronic disability in the world. Formation of the ternary IL-1β /IL-1R1/IL-1RAcP protein complex and its downstream signaling has been implicated in osteoarthritis pathology. Current OA therapeutic approaches target either the cytokine IL-1β or the primary receptor IL-1RI but do not exploit the potential of the secondary receptor IL-1RAcP. Our previous work implicated the Arg286 residue of IL-1RAcP as a key mediator of complex formation. Molecular modeling confirmed Arg286 as a high-energy mediator of the ternary IL-1β complex architecture and interaction network. Anti-IL-1RAcP monoclonal antibodies (mAb) targeting the Arg286 residue were created and were shown to effectively reduce the influx of inflammatory cells to damaged joints in a mouse model of osteoarthritis. Inhibitory peptides based on the native sequence of IL-1RAcP were prepared and examined for efficacy at disrupting the complex formation. The most potent peptide inhibitor had an IC50 value of 304 pM in a pull-down model of complex formation, and reduced IL-1β signaling in a cell model by 90% at 2 μM. Overall, therapies that target the Arg286 region surface of IL-1RAcP, and disrupt subsequent interactions with subunits, have the potential to serve as next generation treatments for osteoarthritis.

Protein Painting Mass Spectrometry in the Discovery of Interaction Sites within the Acetylcholine Binding Protein

“…We used Ac-AChBP in a protein painting MS for the study of ligand interactions within soluable macromolecular complexes. − An overview of the protein painting study is shown in Figure . We developed the protein painting strategy through identifying optimal conditions of dye application, peptidase digestion, and detection of enzymatic digestion fragments (EDF).…”

“…Protein painting has been described in previous studies. − Briefly, protein complexes representing the “receptor” Ac-AChBP and the “ligand” were prepared by mixing 2 μg of Ac-AChBP with 10× molar excess of ligand (nicotine, choline, bgtx) or 20× molar excess Aβ42. The receptor and ligand solution were incubated in PBS with gentle rotation for 1 h at room temperature.…”