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

David R Spring,Yu Heng Lau,Wenshu Xu,Chandra Verma,Anasuya Chattopadhyay,Yaw Sing Tan,Marko Hyvönen,Gerhard Fischer,Laura S Itzhaki,Marc De La Roche

doi:10.1021/jacs.6b10234

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.

Highlights

The development of effective strategies for modulating protein−protein interactions (PPIs) has the potential to vastly expand the range of druggable proteins
Computational alanine scanning was first carried out on structures extracted from three independent 50 ns molecular dynamics (MD) simulations of the complex between TNKS2 ARC4 and the 8-residue consensus sequence to assess which positions would be amenable to replacement by an unnatural amino acid (UAA) having azido functionality for conjugation to the linker
A first panel of macrocyclized peptides was synthesized with azido-functionalized UAAs at positions 2 or 3 and 7 or 8 (Figure 2a), and they were acetylated at the Nterminus to neutralize the terminal charge

Summary

■ INTRODUCTION

The development of effective strategies for modulating protein−protein interactions (PPIs) has the potential to vastly expand the range of druggable proteins. For the higher-affinity binder cp4n2m3, its CD spectrum resembles that of a random coil with a minimum at 196 nm, it has a small positive maximum at 217−218 nm similar to that of a collagen;[59] this distinctive secondary structure is presumably due to the tighter constraint restricting the flexibility of the peptide backbone It is consistent with our goal of designing the macrocycles to restrict the conformations of the peptides to reduce the entropy cost of binding as further evidenced in the isothermal titration calorimetry data shown below. For the Arg[9] cell-penetrating sequence, when we used the two Ahx spacers we observed a degree of non-specific binding to TNKS2 ARC4, likely due to the arginine residues attached via the long flexible spacers competing with the binding peptide for a hotspot interaction on the protein, and this conjugate was not used further.

■ SUMMARY

■ ACKNOWLEDGMENTS

■ REFERENCES