Discovery of novel small-molecule binders targeting IL-6 receptor conformational ensembles to regulate IL-6 signaling.

Abstract

INTRODUCTION: Interleukin-6 (IL-6) signaling via its receptor (IL-6R) is a central driver of inflammation and immune-related diseases. Although monoclonal antibodies targeting this pathway are clinically effective, their limitations, including high costs and parenteral administration, create an urgent need for orally available small-molecule inhibitors. However, targeting the extensive protein‒protein interaction interface between IL-6 and IL-6R remains a significant challenge. OBJECTIVES: This study aims to discover novel small-molecule inhibitors that directly bind to IL-6R and disrupt IL-6/IL-6R complex formation through an integrated computational and experimental strategy while also delineating the druggable binding site on IL-6R to guide rational drug design. METHODS: Ensemble docking was performed against thirteen distinct IL-6R conformations to screen five diverse compound libraries totaling 98,992 molecules. Iterative rounds of molecular dynamics (MD) simulations, accumulating 78.35 μs, were employed to refine candidate selection. In vitro evaluations included STAT3 luciferase reporter assays, western blotting, flow cytometry, cell proliferation assays, cytotoxicity assays, surface plasmon resonance (SPR), and biolayer interferometry (BLI). Site-directed mutagenesis and long-timescale MD simulations validated the binding site. RESULTS: Nineteen compounds were experimentally evaluated, identifying Z1268663363 and Z1268663684 as potent IL-6 signaling inhibitors. Both compounds suppressed IL-6-induced STAT3, JAK1, JAK2, ERK1/2, and gp130 phosphorylation and inhibited IL-6-dependent TF-1 cell proliferation. SPR and BLI confirmed direct binding to IL-6R (Kd = 14.83 μM and 8.27 μM) and disruption of IL-6/IL-6R complex formation. Mutagenesis validated residues Y249 and E296 as critical for binding. Two additional analogs, Z1268663546 and Z1268663944, also exhibited inhibitory activity against IL-6 signaling. CONCLUSION: This study delineates a druggable small-molecule binding site on IL-6R and identifies four novel inhibitors with validated on-target activity, providing a critical foundation for developing next-generation oral therapies for IL-6-driven diseases.

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