Virtual Screening Combined with Phase Transition-FRET for Discovery of Small-Molecule FcRn Antagonists

Neonatal Fc receptor (FcRn) antagonists, which competitively block the binding of immunoglobulin G (IgG) to FcRn, offer a promising therapeutic strategy for treating IgG-mediated autoimmune disorders. However, all currently available FcRn antagonists are macromolecular therapeutic antibodies, which come with certain clinical limitations. The development of alternative small-molecule FcRn antagonists is therefore of considerable importance. To facilitate the discovery of small-molecule FcRn antagonists, we established a novel screening method based on phase transition induced Förster resonance energy transfer (PT-FRET) technique. The PT-FRET technique integrated fluorescence labeled pH-responsive polymers into the FRET system, which could not only amplify the fluorescence intensity but also obviate the necessity for labeling diverse small molecules with varying structures, addressing the labeling challenges in conventional FRET-based methods. Additionally, to further reduce the time and cost as well as improve hit rate, virtual screening was employed prior to PT-FRET, greatly narrowing the scope of small-molecule candidates with potential FcRn binding affinity. After verification and optimization, this novel strategy was successfully applied to the discovery of small-molecule FcRn antagonists. Through virtual screening of a small-molecule compound library containing over 2.0 × 106 compounds, 28 candidate compounds were selected, among which 3 compounds were further identified by PT-FRET. Confirmation results revealed significant antagonistic activity for these 3 identified compounds through a conventional competitive ELISA, demonstrating the reliability and feasibility of the proposed method. Action mechanism was further elucidated via molecular dynamics simulations and binding mode analysis.