Small Molecule Antagonists of the DNA Repair ERCC1/XPA Protein-Protein Interaction

Abstract

The DNA excision repair protein ERCC1 and the DNA damage sensor protein, XPA are highly overexpressed in patient samples of cisplatin-resistant solid tumors including lung, bladder, ovarian, and testicular cancer. The repair of cisplatin-DNA crosslinks is dependent upon nucleotide excision repair (NER) that is modulated by protein-protein binding interactions of ERCC1, the endonuclease, XPF, and XPA. Thus, inhibition of their function is a potential therapeutic strategy for the selective sensitization of tumors to DNA-damaging platinum-based cancer therapy. Here, we report on new small-molecule antagonists of the ERCC1/XPA protein-protein interaction (PPI) discovered using a high-throughput competitive fluorescence polarization binding assay. We discovered a unique structural class of thiopyridine-3-carbonitrile PPI antagonists that block a truncated XPA polypeptide from binding to ERCC1. Preliminary hit-to-lead studies from compound 1 reveal structure-activity relationships (SAR) and identify lead compound 27 o with an EC₅₀ of 4.7 μM. Furthermore, chemical shift perturbation mapping by NMR confirms that 1 binds within the same site as the truncated XPA_67-80 peptide. These novel ERCC1 antagonists are useful chemical biology tools for investigating DNA damage repair pathways and provide a good starting point for medicinal chemistry optimization as therapeutics for sensitizing tumors to DNA damaging agents and overcoming resistance to platinum-based chemotherapy.

Keywords: DNA damage and repair, cisplatin, chemotherapy; ERCC1, XPA, XPF; NMR structure; high-throughput screening (HTS); nucleotide excision repair (NER); protein-protein interaction (PPI); small molecule inhibitor.

Figure 1.

a) Fluorescence polarization (FP)/anisotropy competitive binding inhibition assay with FAM-XPA_67-80; b) Identification of screening hits from compound libraries which inhibit ERCC1/XPA_67-80 heterodimerization at 20 μM concentration.

Figure 2.

Confirmed HTS hits of ERCC1/XPA 14-mer.

Figure 3.

Backbone chemical shift perturbation mapping of compound 1 binding to ERCC1. a) Plot of combined ¹H and ¹⁵N amide chemical shift differences for non-proline residues. Ratios of the ERCC1 to compound 1 used in ¹⁵N-¹H HSQC experiments are shown as the P/S value. Combined ¹⁵N and ¹H shift differences values were calculated as $\Delta \delta =\sqrt{\left\{\frac{1}{2}\left[{({\Delta \delta }_H)}^2+{(\frac{{\Delta \delta }_N}{5})}^2\right]\right\}}$. Blue, magenta, yellow and green horizontal bars include residues shown on the molecular surface in the same color scheme. b) Proposed model of compound 1 bound to ERCC1 placing in XPA-binding site.

Scheme 1.. Synthesis and initial SAR of nitrile and alcohol.

Reagents and conditions: (a) 3-nitrophenacyl bromide, 10% KOH, DMF; (b) NaBH4, THF/MeOH (1:1)

Scheme 2.. Evaluation of pyridine ring linker heteroatom.

Reagents and conditions: (a) NaBH4, THF/MeOH (1:1); (b) K2CO3, DMF; (c) i. KOtBu, THF, ii. 3-nitrophenacyl bromide

Scheme 3.. Reduction to aniline derivatives of 1, 12, 13.

Reagents and conditions: (a) H2, 10% Pd/C, DCM/MeOH (1:1); (b) NaBH4, THF/MeOH (1:1); (c) Fe, NH4Cl, DCM/MeOH (2:1), water (1% v/v), 40 °C

Scheme 4.. Amide substitution of 17a and 17b.

Reagents and conditions: (a) HATU, RCO2H, DIPEA, DMF