Fragment-based screening identifies molecules targeting the substrate-binding ankyrin repeat domains of tankyrase

Katie Pollock,Mirco Meniconi,Mariola Zaleska,Ian Collins,Manjuan Liu,Sebastian Guettler,Mark Pfuhl

doi:10.1038/s41598-019-55240-5

Abstract

The PARP enzyme and scaffolding protein tankyrase (TNKS, TNKS2) uses its ankyrin repeat clusters (ARCs) to bind a wide range of proteins and thereby controls diverse cellular functions. A number of these are implicated in cancer-relevant processes, including Wnt/β-catenin signalling, Hippo signalling and telomere maintenance. The ARCs recognise a conserved tankyrase-binding peptide motif (TBM). All currently available tankyrase inhibitors target the catalytic domain and inhibit tankyrase’s poly(ADP-ribosyl)ation function. However, there is emerging evidence that catalysis-independent “scaffolding” mechanisms contribute to tankyrase function. Here we report a fragment-based screening programme against tankyrase ARC domains, using a combination of biophysical assays, including differential scanning fluorimetry (DSF) and nuclear magnetic resonance (NMR) spectroscopy. We identify fragment molecules that will serve as starting points for the development of tankyrase substrate binding antagonists. Such compounds will enable probing the scaffolding functions of tankyrase, and may, in the future, provide potential alternative therapeutic approaches to inhibiting tankyrase activity in cancer and other conditions.

Highlights

The PARP enzyme and scaffolding protein tankyrase (TNKS, TNKS2) uses its ankyrin repeat clusters (ARCs) to bind a wide range of proteins and thereby controls diverse cellular functions
Given the anticipated impairment of cell permeability by the N-terminal tankyrase-binding peptide motif (TBM) arginine, we investigated whether the guanidine group could be substituted
We explored the stabilisation of TNKS2 ARC5 by the abovementioned TBM peptide derivatives and found a good correlation between the differential scanning fluorimetry (DSF) data and the fluorescence polarisation (FP) data obtained with TNKS2 ARC4, further demonstrating the suitability of the DSF assay (Supplementary Fig. 2B)

Summary

Introduction

The PARP enzyme and scaffolding protein tankyrase (TNKS, TNKS2) uses its ankyrin repeat clusters (ARCs) to bind a wide range of proteins and thereby controls diverse cellular functions. We identify fragment molecules that will serve as starting points for the development of tankyrase substrate binding antagonists Such compounds will enable probing the scaffolding functions of tankyrase, and may, in the future, provide potential alternative therapeutic approaches to inhibiting tankyrase activity in cancer and other conditions. The accumulation of tankyrase and its substrates may further accentuate catalysis-independent functions of tankyrase, which have been emerging recently One such example is our surprising observation that tankyrase can promote Wnt/β-catenin signalling independently www.nature.com/scientificreports of its catalytic PARP activity, at least when tankyrase levels are high[9]. Under these conditions, tankyrase catalytic inhibitors do not completely block tankyrase-driven β-catenin-dependent transcription, pointing to both catalytic and non-catalytic (scaffolding) functions of tankyrase. We set out to identify and characterise small molecule fragments that bind to the tankyrase ARC domains, as a first step towards the discovery of compounds capable of blocking the interaction of tankyrase binders and substrates with the ARC domains of tankyrase

Methods

Results

Conclusion