A RaPID Macrocyclic Peptide Inhibitor of ROR1/2 Strongly Reduces Proliferation of Diffuse Intrinsic Pontine Glioma Cells

We report the de novo discovery of a cyclic peptide inhibitor of ROR1/2 from the RaPID (random nonstandard peptides integrated discovery) system. Multiple independent affinity selection campaigns converged on a highly conserved family of sequences exhibiting single-digit nanomolar binding affinities to ROR1. One peptide from this family containing three nonproteinogenic residues, including a key cyclic β-amino acid, displayed pronounced activity in cellular assays. Strikingly, this peptide induced potent growth inhibition across multiple Diffuse Intrinsic Pontine Glioma (DIPG) cell lines, whereas other family members had no measurable cellular effects. Mechanistic analysis revealed that the active peptide uniquely competes with Wnt5a, the endogenous ligand of ROR1, for receptor binding. To our knowledge, this report provides the first evidence implicating ROR1/2 as a viable therapeutic target in DIPG and establishes macrocyclic peptides as a promising modality for its pharmacological inhibition.