RaPID macrocyclic peptide inhibitor potently reduces Diffuse Intrinsic Pontine Glioma cell proliferation by targeting ROR1/2.

Diffuse Intrinsic Pontine Glioma (DIPG) is an aggressive pediatric brain tumor with a dismal prognosis, largely due to its infiltrative nature and resistance to conventional therapies. Current treatments offer limited efficacy, highlighting an urgent need for novel therapeutic strategies. Receptor tyrosine kinase-like orphan receptors ROR1 and ROR2 are emerging as attractive targets in various cancers, playing critical roles in cell proliferation, survival, and migration, often through non-canonical Wnt signaling pathways. This study explores ROR1/2 inhibition as a potential therapeutic avenue for DIPG, addressing a significant unmet medical need.

Researchers employed the RaPID (random nonstandard peptides integrated discovery) system to identify novel cyclic peptide inhibitors of ROR1/2. Multiple independent affinity selection campaigns were conducted, leading to the discovery of a conserved family of sequences. One specific peptide from this family, containing three nonproteinogenic residues including a key cyclic β-amino acid, was then tested in cellular assays. Its activity was evaluated across multiple Diffuse Intrinsic Pontine Glioma (DIPG) cell lines for growth inhibition, with other family members serving as controls. Mechanistic analysis further investigated how the active peptide interacts with ROR1, specifically examining its competition with the endogenous ligand Wnt5a.

The RaPID system successfully identified a family of cyclic peptides exhibiting high affinity for ROR1, with binding affinities in the single-digit nanomolar range. Crucially, only one specific macrocyclic peptide from this family, distinguished by its unique nonproteinogenic residues, demonstrated significant cellular activity. This active peptide induced potent growth inhibition across multiple Diffuse Intrinsic Pontine Glioma (DIPG) cell lines, a striking effect not observed with other closely related family members. Mechanistic studies revealed a unique mode of action for the active peptide: it directly competes with Wnt5a, the natural ligand for ROR1, for receptor binding. This competitive inhibition effectively disrupts the Wnt5a-ROR1 signaling axis, which is implicated in DIPG pathogenesis. The findings represent the first evidence supporting ROR1/2 as a viable therapeutic target in DIPG. Furthermore, this work establishes macrocyclic peptides as a promising modality for pharmacological inhibition of these receptors.

The active macrocyclic peptide uniquely competes with Wnt5a for ROR1 binding, leading to potent growth inhibition across multiple DIPG cell lines.