Methodology for the identification of small molecule inhibitors of the Fanconi Anaemia ubiquitin E3 ligase complex

Michael F Sharp,Sylvie Van Twest,Winnie Tan,Jennii Lui,Wayne Crismani,Kaylene J Simpson,Andrew J Deans,Vince J Murphy

doi:10.1038/s41598-020-64868-7

Abstract

DNA inter-strand crosslinks (ICLs) threaten genomic stability by creating a physical barrier to DNA replication and transcription. ICLs can be caused by endogenous reactive metabolites or from chemotherapeutics. ICL repair in humans depends heavily on the Fanconi Anaemia (FA) pathway. A key signalling step of the FA pathway is the mono-ubiquitination of Fanconi Anaemia Complementation Group D2 (FANCD2), which is achieved by the multi-subunit E3 ligase complex. FANCD2 mono-ubiquitination leads to the recruitment of DNA repair proteins to the site of the ICL. The loss of FANCD2 mono-ubiquitination is a common clinical feature of FA patient cells. Therefore, molecules that restore FANCD2 mono-ubiquitination could lead to a potential drug for the management of FA. On the other hand, in some cancers, FANCD2 mono-ubiquitination has been shown to be essential for cell survival. Therefore, inhibition of FANCD2 mono-ubiquitination represents a possible therapeutic strategy for cancer specific killing. We transferred an 11-protein FANCD2 mono-ubiquitination assay to a high-throughput format. We screened 9,067 compounds for both activation and inhibition of the E3 ligase complex. The use of orthogonal assays revealed that candidate compounds acted via non-specific mechanisms. However, our high-throughput biochemical assays demonstrate the feasibility of using sophisticated and robust biochemistry to screen for small molecules that modulate a key step in the FA pathway. The future identification of FA pathway modulators is anticipated to guide future medicinal chemistry projects with drug leads for human disease.

Highlights

DNA inter-strand crosslinks (ICLs) threaten genomic stability by creating a physical barrier to DNA replication and transcription
Simultaneous loss of USP1 – the protein required for deubiquitination of FANCD218 – is synthetic lethal with loss of BRCA119
The purified proteins in the mono-ubiquitination assays used for the high-throughput screen are biotinylated-ubiquitin (b-ubiquitin), His-UBE1, FANCT, Flag-FANCB, FANCL, FAAP100, MBPFANCC, FANCE, FANCF, GST-XlFANCD2 and Flag-XlFANCI

Summary

Introduction

DNA inter-strand crosslinks (ICLs) threaten genomic stability by creating a physical barrier to DNA replication and transcription. The core complex contains FANCA, FANCG and FAAP20 (FANC-A-G-20), of which FANCA is mutated in most cases of FA and is required for FANCD2 mono-ubiquitination in cells. The first biochemical study used a fragment library and a biophysical approach to identify inhibitors of FANCT which resulted in three compounds that were able to inhibit FANCD2 ubiquitination reactions with recombinant proteins.

Results

Conclusion