Covalent and Noncovalent Targeting of the Tcf4/β-Catenin Strand Interface with β-Hairpin Mimics

Abstract

β-Strands are a fundamental component of protein structure, and these extended peptide regions serve as binding epitopes for numerous protein-protein complexes. However, synthetic mimics that capture the conformation of these epitopes and inhibit selected protein-protein interactions are rare. Here we describe covalent and noncovalent β-hairpin mimics of an extended strand region mediating the Tcf4/β-catenin interaction. Our efforts afford a rationally designed lead for an underexplored region of β-catenin, which has been the subject of numerous ligand discovery campaigns.

Figure 1.

Tcf4 utilizes both helical and extended β-strand regions (blue) to recognize the armadillo repeat domains of β-catenin (grey) (A). The Tcf4 extended strand utilizes four key binding, or hotspot, residues - D16, L18, I19, and F21 (shown as green spheres) - to engage the β-catenin surface (B). The strand binding surface of β-catenin features two cysteine residues (magenta, C429 and C466) providing an opportunity to convert Tcf4 strand mimics into covalent ligands for β-catenin.

Figure 2.

Rational design of covalent and noncovalent β-catenin ligands. We began our design efforts by identifying a minimal linear Tcf4 peptide that recognizes β-catenin with micromolar affinity (Step 1). We next stabilized the strand conformation as part of a cyclic β-hairpin using the hydrogen bond surrogate (HBS) approach (Step 2). Finally, we modified the hairpin with an electrophile to covalently engage neighboring β-catenin cysteine residues (Step 3).

Figure 3.

(A) Chemical Structure of minimal Tcf4 mimic Strand-A. Analysis of related sequences is included in Supporting Information Table S3. (B) Overlay of Tcf4 strands in complex with β-catenin. The conformation of Tcf4 differs between crystal structures with PDB code 1JPW (pink strand) suggesting a curved conformation as compared to an extended motif in PDB code 1JDH (blue strand). (C) Molecular modeling with idealized dipeptide turn motif (PDB codes 1LE0 (yellow ribbon) and 1LE1) suggests that type II’ β-turns may mimic Tcf4 geometry in 1JPW. (D) Design of constrained and unconstrained β-hairpin mimics. The unconstrained hairpins (Unc-βtTcf1 and Unc-βtTcf2) consist of the Tcf4 strand (blue arrow) linked to dipeptide β-turn inducers (D-Pro-Gly or Gly-Asn) and a de novo partner strand (TZTRA, gray arrow) designed to stabilize a β-hairpin conformation. Residue Z is designed to be either hydrophobic (tryptophan) or polar (glutamine). The constrained β-hairpins (HBS-βtTcf1 and HBS-βtTcf2) contain an HBS bridge along with the turn inducers and the partner β-strand.

Figure 4.

The inhibitory potential of Tcf4-derived covalent β-hairpins. (A) Replacement of L18 with a diaminopropionic acid (Dap) linked chloroacetamide group provides β-hairpin covalent ligands. K_i values are compared after 6 hours incubation at 25 °C. (B) Replacement of chloroacetyl in βtCov1 with electrophiles spanning a range of reactivities demonstrates a trend in K_i values that follows electrophile reactivity: mimics with more reactive electrophiles such as bromoacetamide βtCov1B are more potent than mimics with weakly reactive electrophiles such as acrylamide βtCov1A.

Figure 5.

MALDI mass spectrometry analysis of β-catenin crosslinking to covalent Tcf4 β-hairpins. (A) β-Catenin samples were treated with chloracetyl-containing β-hairpin peptides and analyzed by MALDI MS. Integrated MALDI peak areas were used to calculate relative amounts of labeled adducts. The results suggest that βtCov1 provides the lowest amount of labeling, but that the ratio of mono- to multi-labeled adducts is the highest for this sequence (B). (C) Mono-, di-, and multi-labeling by βtCov1 derivatives with different electrophilic warheads follows expected electrophile reactivity trends. (D) The ratio of mono- to multi-labeled adducts suggests that the chloroacetyl group yields the highest selectivity. (E) MS analysis of chymotrypsin-digested samples suggests that the major βtCov1 adducts form at C429 (Site 1) and C520 (Site 2).