RESUMEN

The diverse role of the splicing factor PTBP1 in human cells has been widely studied and was found to be a driver for several diseases. PTBP1 binds RNA through its RNA-recognition motifs which lack obvious pockets for inhibition. A unique transient helix has been described to be part of its first RNA-recognition motif and to be important for RNA binding. In this study, we further confirmed the role of this helix and envisioned its dynamic nature as a unique opportunity to develop stapled peptide inhibitors of PTBP1. The peptides were found to be able to inhibit RNA binding via fluorescence polarization assays and directly occupy the helix binding site as observed by protein crystallography. These cell-permeable inhibitors were validated in cellulo to alter the regulation of alternative splicing events regulated by PTBP1. Our study demonstrates transient secondary structures of a protein can be mimicked by stapled peptides to inhibit allosteric mechanisms.

RESUMEN

WDR5 is an adaptor protein involved in the regulation of various epigenetic modifier complexes. Various inhibitors have been described but only as inhibitors of its protein-protein interactions. Here we describe peptidic macrocycles that act as inhibitors of the interaction between WDR5 and long non-coding RNAs. The findings provide a new strategy to modulate the biological function of WDR5 as an RNA binding epigenetic regulator.

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RESUMEN

RNA-binding proteins are essential regulators of RNA processing and function. Translational repression assays can be used to study how they interact with specific RNA sequences by insertion of such a consensus sequence into the 5' untranslated region of a reporter mRNA and measuring reporter protein translation. The straightforward set-up of these translational repression assays avoids the need for the isolation of the protein or the RNA providing speed, robustness and a low-cost method. Here, we report the optimization of the assay to function with linear RNA sequences instead of the previously reported hairpin type sequences to allow the study of a wider variety of RNA-binding proteins. Multiplication of a consensus sequence strongly improves the signal allowing analysis by both fluorescence intensity measurements and flow cytometry.

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Introduction of unnatural amino acids can significantly improve the binding affinity and stability of peptides. Commercial availability of such amino acids is limited, and their synthesis is a long and tedious process. We here describe a method that allows the functionalization of peptides directly on solid-support by converting lysine residues to Katritzky salts, and subjecting them to a photochemical Giese reaction under mild reaction conditions. The method avoids the need for amino acid synthesis and instead offers a late-stage modification route for rapid peptide diversification. While numerous modification approaches at the lysine amine have been described, this work provides the first example of deaminative functionalization of peptides at lysine. The two-step protocol is compatible with various substrates, lysine analogues, resins, and all proteinogenic amino acids. Finally, by leveraging solid-phase modification, this protocol facilitates the functionalization of longer peptides as was demonstrated using biologically relevant peptides of up to 15 amino acids.

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