Discovery of Macrocyclic Peptide Binders, Covalent Modifiers, and Degraders of a Structured RNA by mRNA Display

RNA targeting represents
a compelling strategy for addressing
challenging
therapeutic targets that are otherwise intractable through traditional
protein targeting. Revolutionary approaches in RNA-focused small molecule
libraries have successfully identified RNA-binding ligands but generally
remain limited in diversity and impeded by a dearth of structural
insight into RNA and RNA complexes. Cyclic peptides are potential
structural mimics of evolutionary RNA-protein interacting motifs and
can be massively diversified and selected via genetically encoded
libraries, offering a complementary approach. This study introduces
genetically encoded thioether cyclic peptide libraries constructed
through mRNA display using a dibromoxylene linker and its fluorosulfonyl
derivative that can covalently engage RNA nucleophiles. Using an optimized
mRNA display workflow for RNA binders, we discovered high affinity,
covalent and noncovalent binders for SNCA 5′
UTR IRE, the upstream iron-responsive element that post-transcriptionally
regulates the expression of α-synuclein, an intrinsically disordered
protein implicated in Parkinsonism and related neurodegenerative diseases.
Notably, a stringent selection strategy employing “base-paired”
target analog counterselection enhanced specificity by deenriching
nonspecific electrostatic interactions mediated by polycationic residues.
Further engineering hit peptides with an imidazole tag yielded selective
RNA degraders in which covalent degraders showed noticeably improved
potency from noncovalent counterparts. This work provides a prototype
framework for evolution-driven, high-throughput, RNA-targeted drug
discovery using cyclic peptides.