Characterizing inhibitors of human AP endonuclease 1

AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds (MLS000552981, MLS000419194) inhibit APE1 in vitro with low micromolar IC50 and do not appear to aggregate in this concentration range. However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme

Compounds studied in this work.

1, 7-nitroindole-2-carboxylic acid or CRT0044876; 2, 5-fluoroindole-2-carboxylic acid; 3, 5-nitroindole-2-carboxylic acid; 4, 6-bromoindole-2-carboxylic acid; 5, myricetin; 6, AR03 or MLS000552981 of the NIH MLSMR library; 7, APE1 Inhibitor III, MLS000419194, or N-(3-(benzo[d]- thiazol-2-yl)-6-isopropyl-4,5,6,7-tetrahydrothieno[2,3-c]- pyridin-2-yl)acetamide. For compounds 1–4, the 2-carboxyl is in the deprotonated form that likely predominates at pH 7.5.</p

APE1 chemical shift perturbations induced by MgCl₂ (0.25 mM).

(a) 15N-TROSY spectra for APE1 (0.10 mM) in the absence (black) or presence (red) of MgCl2 (0.25 mM). Spectra were also collected for APE1 with [MgCl2] at 0.063 and 0.125 mM. Two residues near the Mg2+-binding site (69, 308) exhibit peaks for apo APE1 but not APE1 with MgCl2 (≥0.063 mM). (b) CSPs (Δδ) induced by MgCl2 (0.25 mM) as a function of amino acid residue. Labels with one or more stars denote residues for which a peak is not seen in spectra collected for APE1 with 0.25 mM MgCl2; for these residues, Δδ values were calculated using spectra for APE1 with the highest [MgCl2] for which the peak is observed (*, 0.063 mM; **, 0.125 mM). Residues exhibiting Δδ ≥ 0.015 ppm are labeled in both figures. (TIF)</p

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(PDB)</p

APE1 chemical shift perturbations induced by MgCl₂ (1.0 mM).

(a) 15N-TROSY spectra for APE1 (0.10 mM) in the absence (black) or presence (red) of MgCl2 (1.0 mM). Spectra were also collected for APE1 with lower MgCl2 concentrations (0.063, 0.125, 0.25, 0.50. 0.75 mM). Two residues near the Mg2+-binding site (69, 308) exhibit peaks for apo APE1 but not APE1 with MgCl2 (≥0.063 mM). (b) CSPs (Δδ) induced by MgCl2 (1.0 mM) versus amino acid residue. Labels with stars mark residues for which a peak is not seen in spectra of APE1 with 1.0 mM MgCl2; for these residues, Δδ values were calculated using spectra for APE1 with the highest [MgCl2] for which that peak is observed (*, 0.063 mM; **, 0.125 mM; ***, 0.25 mM; ****, 0.50 mM; *****, 0.75 mM). Residues exhibiting Δδ ≥ 0.015 ppm are labeled. (TIF)</p