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

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.

A FRET-based high-throughput screening platform for the discovery of chemical probes targeting the scaffolding functions of human tankyrases

“…While the development of TNKS inhibitors mainly focussed on the inhibition of the catalytic domain, the function of tankyrases is coupled to many regulatory processes beyond its catalytic function 3 , 23 , 25 . With newer findings about the scaffolding function of tankyrases and its contribution to the regulation of the catalytic activity, several reports have suggested that the inhibition of multimerization or protein-binding function may represent an effective way for the specific inhibition of tankyrases 23 , 34 – 36 , 43 . Such inhibitors would also be valuable chemical probes aiding the investigation of TNKS mechanism of function in the cell.…”

“…In the future, the assay will help to identify small chemical inhibitors of TNKS ARC-protein binders and SAM-SAM-interactions. With only a few reported small chemical binders for TNKS ARCs to date 34 – 36 , we could already expand the number with our validatory screening efforts appreciably. Last, X-ray crystallographic studies of ARCs with these molecules would provide important insights into the binding modes and could lead to a structure guided design of novel chemical probes with higher affinity.…”

“…So far, inhibitors of SAM domain interactions have not been reported. While there are recent reports on the early development of small chemical molecules targeting TNKS ARCs 34 – 36 , a suitable assay system for high-throughput screening is still missing. In our work, we developed in vitro assay systems based on fluorescence resonance energy transfer (FRET) to screen for inhibitors of TNKS scaffolding functions.…”

“…These cell-permeable peptides were shown to compete with Axin for the binding of tankyrases and thus led to suppression of Wnt-signalling in cells. More recently, small molecules binding to TNKS ARC domains have been discovered by either virtual screening 35 or fragment-based screening using differential scanning fluorimetry combined with an NMR-based approach 36 .…”

Poly(ADP-Ribose) Glycohydrolase (PARG) vs. Poly(ADP-Ribose) Polymerase (PARP) – Function in Genome Maintenance and Relevance of Inhibitors for Anti-cancer Therapy

“…Often but not exclusively, tankyrase-mediated PARylation is recognized and ubiquitinated by the E3 ligase RNFL146 (Zhang et al, 2011), this targets substrates for proteasomal degradation and thus regulation is commonly via alteration of protein stability. However, evidence is also emerging that tankyrases can have catalytic independent functions (Pollock et al, 2019). As tankyrase biology is relatively less well understood than that of PARP1-3, it is likely that further, unknown tankyrase functions exist.…”