Inhibition of the ERCC1–XPF structure-specific endonuclease to overcome cancer chemoresistance

Abstract

ERCC1-XPF is a structure-specific endonuclease that is required for the repair of DNA lesions, generated by the widely used platinum-containing cancer chemotherapeutics such as cisplatin, through the Nucleotide Excision Repair and Interstrand Crosslink Repair pathways. Based on mouse xenograft experiments, where ERCC1-deficient melanomas were cured by cisplatin therapy, we proposed that inhibition of ERCC1-XPF could enhance the effectiveness of platinum-based chemotherapy. Here we report the identification and properties of inhibitors against two key targets on ERCC1-XPF. By targeting the ERCC1-XPF interaction domain we proposed that inhibition would disrupt the ERCC1-XPF heterodimer resulting in destabilisation of both proteins. Using in silico screening, we identified an inhibitor that bound to ERCC1-XPF in a biophysical assay, reduced the level of ERCC1-XPF complexes in ovarian cancer cells, inhibited Nucleotide Excision Repair and sensitised melanoma cells to cisplatin. We also utilised high throughput and in silico screening to identify the first reported inhibitors of the other key target, the XPF endonuclease domain. We demonstrate that two of these compounds display specificity in vitro for ERCC1-XPF over two other endonucleases, bind to ERCC1-XPF, inhibit Nucleotide Excision Repair in two independent assays and specifically sensitise Nucleotide Excision Repair-proficient, but not Nucleotide Excision Repair-deficient human and mouse cells to cisplatin.

Unexpected Growth-Promoting Effect of Oxaliplatin in Excision Repair Cross-Complementation Group 1 Transfected Human Colon Cancer Cells

“…Within this latter part, the precise understanding on mechanisms of action and resistance is of major interest in order to develop new and innovative strategies based on these mechanisms. For platinum derivatives, this is what we and others have done with the search of compounds inhibiting nucleotide excision repair through various approaches [19][20][21][22]. With the main involvement of ERCC1 in the activity of platinum drugs, we here performed an in vitro and in vivo study on cells transfected with a plasmid coding ERCC1, with particular interest in the cellular response to oxaliplatin.…”

“…The ERCC1/XPF complex has also been shown to interact with the protein hub SLX4 with a subsequent involvement in double strand break repair [17,18]. Based on these observations, we and others developed a strategy to increase the activity of platinum derivatives and double strand break inducers through the inhibition of the interaction of ERCC1 and XPF proteins or of the enzymatic nuclease activity of XPF [19,20], as well as the inhibition of the interaction between ERCC1 and XPA [21,22]. During this work, we developed cell models expressing interacting domains of ERCC1, XPA and XPF proteins, and the characterization of these models allowed a better insight into the role of the interaction between these proteins in cancer cells [23].…”

Catechols and 3-hydroxypyridones as inhibitors of the DNA repair complex ERCC1-XPF

“…The catechol motif is present in known nuclease inhibitors 4 and although the dibromo-substitution and hydrazone motif were considered undesirable, 1 showed good selectivity for ERCC1-XPF in counterscreen assays against the nucleases FEN-1 and DNase I. 3 We therefore sought to explore whether it was possible to design out the unwanted features and gain additional potency while retaining good selectivity for ERCC1-XPF. Testing of initial analogues showed that the catechol group was required for inhibitory activity since mono-or di-methylation led to complete loss of activity.…”

“…2 We sought to identify novel inhibitors of ERCC1-XPF using a high-throughput fluorescencebased in vitro endonuclease assay 3 and one of the hits obtained was compound 1 (Figure 1) with an IC50 value of approximately 30 µM and modest ligand efficiency. The catechol motif is present in known nuclease inhibitors 4 and although the dibromo-substitution and hydrazone motif were considered undesirable, 1 showed good selectivity for ERCC1-XPF in counterscreen assays against the nucleases FEN-1 and DNase I.…”

“…Neither compound showed binding to a control protein included in the study. Compound 13 was profiled in three cell-based assays using A375 melanoma cells to measure 1) direct nucleotide excision repair (NER) using a recombinant GFP reporter assay, 3,9 2) potentiation of cisplatin induced cell toxicity 10 and 3) immunofluorescence detection of H2AX, a marker of DNA damage.…”