Macrocyclic peptides that inhibit Wnt signalling via interaction with Wnt3a.

Here we report de novo macrocyclic peptide binders to Wnt3a, a member of the Wnt protein family. By means of the Random non-standard Peptides Integrated Discovery (RaPID) system, we have performed in vitro selection against the complex of mouse Wnt3a (mWnt3a) with human afamin (hAFM) to discover macrocyclic peptides that bind mWnt3a with K D values as tight as 110 nM. One of these peptides, WAp-D04 (Wnt-AFM-peptide-D04), was able to inhibit the receptor-mediated signaling process, which was demonstrated in a Wnt3a-dependent reporter cell-line. Based on this initial hit, we applied a block-mutagenesis scanning display to identify a mutant inhibitor, WAp-D04-W10P, with 5-fold greater potency in a reporter assay. This work represents the first instance of molecules capable of inhibiting Wnt signaling through direct interaction with a Wnt protein, a molecular class for which targeting has been challenging due its highly hydrophobic nature.

Discovery of Functional Macrocyclic Peptides by Means of the RaPID System.

Flexizymes, highly flexible tRNA aminoacylation ribozymes, have enabled charging of virtually any amino acid (including non-proteogenic ones) onto tRNA molecules. Coupling to a custom-made in vitro translation system, namely the flexible in vitro translation (FIT) system, has unveiled the remarkable tolerance of the ribosome toward molecules, remote from what nature has selected to carry out its elaborate functions. Among the very diverse molecules and chemistries that have been ribosomally incorporated, a plethora of entities capable of mediating intramolecular cyclization have revolutionized the design and discovery of macrocyclic peptides. These macrocyclization reactions (which can be spontaneous, chemical, or enzymatic) have all served as tools for the discovery of peptides with natural-like structures and properties. Coupling of the FIT system and mRNA display techniques, known as the random non-standard peptide integrated discovery (RaPID) system, has in turn allowed for the simultaneous screening of trillions of macrocyclic peptides against challenging biological targets. The macrocyclization methodologies are chosen depending on the structural and functional characteristics of the desired molecule. Thus, they can emanate from the peptide's N-terminus or its side chains, attributing flexibility or rigidity, or even result in the installation of fluorescent probes.

Structural Features and Binding Modes of Thioether-Cyclized Peptide Ligands.

Macrocyclic peptides are an emerging class of bioactive compounds for therapeutic use. In part, this is because they are capable of high potency and excellent target affinity and selectivity. Over the last decade, several biochemical techniques have been developed for the identification of bioactive macrocyclic peptides, allowing for the rapid isolation of high affinity ligands to a target of interest. A common feature of these techniques is a general reliance on thioether formation to effect macrocyclization. Increasingly, the compounds identified using these approaches have been subjected to x-ray crystallographic analysis bound to their respective targets, providing detailed structural information about their conformation and mechanism of target binding. The present review provides an overview of the target bound thioether-closed macrocyclic peptide structures that have been obtained to date.