Macrocyclized Extended Peptides: Inhibiting the Substrate-Recognition Domain of Tankyrase

Abstract

We report a double-click macrocyclization approach for the design of constrained peptide inhibitors having non-helical or extended conformations. Our targets are the tankyrase proteins (TNKS), poly(ADP-ribose) polymerases (PARP) that regulate Wnt signaling by targeting Axin for degradation. TNKS are deregulated in many different cancer types, and inhibition of TNKS therefore represents an attractive therapeutic strategy. However, clinical development of TNKS-specific PARP catalytic inhibitors is challenging due to off-target effects and cellular toxicity. We instead targeted the substrate-recognition domain of TNKS, as it is unique among PARP family members. We employed a two-component strategy, allowing peptide and linker to be separately engineered and then assembled in a combinatorial fashion via click chemistry. Using the consensus substrate-peptide sequence as a starting point, we optimized the length and rigidity of the linker and its position along the peptide. Optimization was further guided by high-resolution crystal structures of two of the macrocyclized peptides in complex with TNKS. This approach led to macrocyclized peptides with submicromolar affinities for TNKS and high proteolytic stability that are able to disrupt the interaction between TNKS and Axin substrate and to inhibit Wnt signaling in a dose-dependent manner. The peptides therefore represent a promising starting point for a new class of substrate-competitive inhibitors of TNKS with potential for suppressing Wnt signaling in cancer. Moreover, by demonstrating the application of the double-click macrocyclization approach to non-helical, extended, or irregularly structured peptides, we greatly extend its potential and scope, especially given the frequency with which such motifs mediate protein-protein interactions.

Figure 1

Structure of TNKS1 and TNKS2. (a) Domain architecture, comprising a homopolymeric run of histidine, proline and serine (HPS) residues, the ankyrin repeat cluster (ARC), sterile alpha motifs (SAM), and catalytic PARP domains. (b) Structure of human TNKS2 ARC4 (gray cartoon) in complex with substrate peptide LPHLQRSPPDGQSFRS (purple; PDB ID: 3TWR); for clarity, only the central part of the peptide (in bold) is labeled. (c) Structure of mouse TNKS1 ARC2–3 (gray cartoon) in complex with mouse Axin1_1–80; only residues 18–30 (green) and 60–78 (yellow) of mAxin1 are visible in the crystal structure (PDB ID: 3UTM).

Figure 2

Macrocyclic peptide design. (a, left) Sequences of the synthesized peptides with the UAAs denoted as ‘X’; (right) chemical structures of the different UAA side chains, where n2, n3, and so forth indicate the number of CH₂ units in the side chain. (b) Chemical structures of the linkers, where m3–m5 indicate the number of carbon/nitrogen atoms between the two alkynyl groups. (c) Schematic of the click reaction between the peptides in (a) and linkers in (b); see Figure S2 for full chemical structures of representative peptides.

Figure 3

Determining the optimal peptide-linker combination. Dissociation constants were determined by competitive FP assay using labeled T-pep1 bound to ARC4 as the tracer for the first panel of macrocyclized peptides in which we varied the positions of the two UAAs (denoted X) in the peptide sequence, the lengths of the side chains, and the length of the linker. “n” is the number of methylene (CH₂) groups in the side chain of the UAAs; ‘m’ is the number of carbon/nitrogen atoms between the two triazole groups in the linker. Special linkers are m5c, a rigid linker based on napthalene, and m7N, a linear linker made from a urea derivative described earlier. A dash in the “m” box denotes uncross-linked peptide. The dissociation constant of the unlabeled linear peptide, pep1, is shown for comparison. Uncross-linked cp4n4 and cp5n4 were measured as comparisons to their cross-linked counterpart. A full list of dissociation constants is given in Table S1.

Figure 4

(a) Crystal structure of TNKS2 ARC4 (gray surface) in complex with the macrocyclized peptide cp4n4m5 (orange carbons; linker in red). (b) 2F_obs – F_calc electron density (blue mesh) of the linker and of the central residues of the peptide (orange; linker in red) contoured at 1σ, rotated approximately 90° along the horizontal axis compared to (a).

Figure 5

(a) Direct FP measurements for a subset of the second panel of TAMRA-labeled peptides based on the cp4 sequence (TAMRA-Ahx-REXDGDXE) with (b) their binding affinities listed in a table (see Table S2 for the full list of peptides and their binding affinities). The K_d for T-cp4n1m3 is not detectable (n.d.). (c) Crystal structure of TNKS2 ARC4 (gray surface) in complex with macrocyclized peptide cp4n2m3 (cyan; linker in green). Superposition of cp4n2m3 (cyan/green) and cp4n4m5 (orange/red) in the two structures of the complexed peptides; TNKS2 ARC4 is shown in gray. (d) 2F_obs – F_calc electron density, contoured at 1σ, of the linker and of the central residues of peptide cp4n2m3. (e) Overlay of the three structures: the complex with the natural substrate peptide LPHLQRSPPDGQSFRS is shown (PDB ID: 3TWR) (protein: pink; peptide: purple). Ankyrin repeats 4 and 5 of TNKS2 ARC4-cp4n2m3 (cyan) move by 2–3 Å compared to those in the complex with cp4n4m5 (orange) and the linear peptide (pink). (f) CD spectra of linear peptides pep1 (green) and cp4n4 (red) and of cross-linked peptides cp4n4m5 (orange), cp4n2m3 (cyan), and the non-binding cross-linked peptide cp4n1m3 (purple).

Figure 6

Thermodynamic parameters obtained for the binding of the peptides to TNKS2 ARC4 and to TNKS1 ARC2–3, as measured by ITC. Error bars are those obtained from standard derivation of the mean from duplicate measurements.

Figure 7

Competition of peptides with GST-tagged human Axin1_1–80 for binding to TNKS2 ARC4. The TNKS2 ARC4-Axin1_1–80 complex, immobilized on glutathione beads, was incubated with increasing concentrations of peptide. After washing, the protein remaining bound to the resin was run on SDS-PAGE. The TNKS2 ARC4 band was quantified using densitometry and normalized against the GST-Axin1_1–80 band. The data are plotted relative to the sample without peptide incubation (which was set at a value of 1).

Figure 8

Effect on Wnt-activated HEK 293T cells of treatment with representative peptides. Error bars are those obtained from triplicate sample measurements from two independent experiments.