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

Abstract

Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins. tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes, such as mitotic spindle formation, telomere homeostasis and Wnt/β-catenin signalling. In recent years, tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis. Further, it has become clear that tankyrases are not only enzymes, but also act as scaffolding proteins forming large cellular signalling complexes. While many potent and selective tankyrase inhibitors of the poly-ADp-ribosylation function exist, the inhibition of tankyrase scaffolding functions remains scarcely explored. In this work we present a robust, simple and costeffective high-throughput screening platform based on FRET for the discovery of small molecule probes targeting the protein-protein interactions of tankyrases. Validatory screening with the platform led to the identification of two compounds with modest binding affinity to the tankyrase 2 ARC4 domain, demonstrating the applicability of this approach. The platform will facilitate identification of small molecules binding to tankyrase ARC or SAM domains and help to advance a structure-guided development of improved chemical probes targeting tankyrase oligomerization and substrate protein interactions. Poly(ADP-ribosyl) polymerases (PARPs) called tankyrases (TNKSs) are key regulators of diverse cellular processes such as mitotic spindle formation, telomere homeostasis, Wnt/β-catenin signalling and glucose metabolism 1-5. In humans, two tankyrases with overlapping functions exist and are termed TNKS1 and TNKS2 6,7. Like other enzymes of the PARP family, tankyrases catalyse the transfer of multiple ADP-ribose units to their protein substrates 8-10 , thus leaving them poly-ADP-ribosylated. In many cases this serves as a signal for subsequent ubiquitination and thereby proteasomal degradation 11,12. Prominent targets of tankyrases include Axin1/2 13,14 , a major regulator of β-catenin levels, TRF1 7,9,15,16 , a telomere binding protein that inhibits telomere extension and NuMA 17-19 , a protein involved in the formation of spindle-poles during mitosis. The major role of tankyrases in the regulation of β-catenin levels has led to the development of multiple TNKS inhibitors 2,20-22. These inhibitors function by binding to the NAD + binding pocket of the catalytic ARTD domain (ADP-ribosyl-transferases diphtheria toxin-like), inhibiting the poly-ADP-ribosylation function. However, it has been shown that tankyrases also contribute through oligomerization and mediation of protein-protein interactions to the Wnt-signalling pathway 23. We refer to these non-catalytic functions of tankyrases as scaffolding functions. By being able to specifically target different domains involved in the scaffolding function with a tool compound, it could be possible to inves...

A molecular toolbox for ADP-ribosyl binding proteins

“…The measurements were done as previously described (Sowa et al, 2020). Briefly, the samples were excited at 410 nm and emission at 477 nm and 527 nm wavelengths were measured.…”

Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimer formation and enzymatic function

“…Expression vectors pNH-TrxT (#26106), pNIC-CH (#26117), pFastBac1 His-MBP (#30116) were procured from Addgene. pNIC28-MBP was constructed by replacement of TrxT with MBP (Sowa et al, 2020). Throughout the study, Uniprot IDs corresponding to DTX3L (#Q8TDB6-1) and PARP9 (#Q8IXQ6-1) were used.…”

Analogs of TIQ-A as inhibitors of human mono-ADP-ribosylating PARPs

“…17 TNKS2 constructs were cloned into pNIC-MBP expression vectors and an additional purification on an MBPTrap HP 5 ml column (GE Healthcare) prior to cleavage with TEV protease was performed. 20 Details of the constructs for each PARP enzyme are listed in Table S1.…”