Identification of Bioactive SNM1A Inhibitors

Abstract

SNM1A is a nuclease required to repair DNA interstrand cross-links (ICLs) caused by some anticancer compounds, including cisplatin. Unlike other nucleases involved in ICL repair, SNM1A is not needed to restore other forms of DNA damage. As such, SNM1A is an attractive target for selectively increasing the efficacy of ICL-based chemotherapy. Using a fluorescence-based exonuclease assay, we screened a bioactive library of compounds for inhibition of SNM1A. Of the 52 compounds initially identified as hits, 22 compounds showed dose-response inhibition of SNM1A. An orthogonal gel-based assay further confirmed nine small molecules as SNM1A nuclease activity inhibitors with IC₅₀ values in the mid-nanomolar to low micromolar range. Finally, three compounds showed no toxicity at concentrations able to significantly potentiate the cytotoxicity of cisplatin. These compounds represent potential leads for further optimization to sensitize cells toward chemotherapeutic agents inducing ICL damage.

Figure 1

HTS assay for SNM1A inhibitors. (A) Schematic of the HTS oligonucleotide substrate (5P-FQ listed in Figure S1) with a fluorophore–quencher pair and expected products with or without an inhibitor. (B) Purified SNM1A after final cation exchange chromatography. The SNM1A-containing sample was immobilized to S-Seph (GE Healthcare) at 300 mM and eluted with a linear salt gradient (0.3–1 M). Eluted SNM1A-containing fractions were pooled and concentrated. Fractions were resolved with 12% SDS-PAGE containing trichloroethanol. Gel was visualized using stain-free enabled GelDoc (Bio-Rad). (C) K_M determination of purified SNM1A and 5P-FQ. Fluorescence was measured every minute for 2 h at 26 °C at 526 nm using the BioTek Synergy 4 Hybrid microplate reader. The assay was performed in triplicate, and K_M was determined using GraphPad Prism. (D) Z′ score determination for the HTS SNM1A inhibition assay. SNM1A (3 nM) was incubated with 8 nM HTS substrate with and without zinc control for 40 min. Fluorescence was measured with an EnVision plate reader (PerkinElmer) at 535 nm.

Figure 2

Putative HTS hits of SNM1A inhibitors. (A) Schematic of the HTS assay. (B) Replica plot of the HTS campaign of 3941 compounds. Percent activity was calculated as the difference between readings after 80 and 0 min. Diagonal lines indicate standard deviation of high controls. Boxed area indicates assay cutoff for possible inhibitors. Red dots indicate HTS assay hits and potential SNM1A inhibitors. Green dots indicate compounds that do not meet the cutoff and do not inhibit SNM1A.

Figure 3

Validation of SNM1A inhibitors. (A) HTS hit validation with dose-dependent inhibition screen. Hits from Figure 2B were tested for dose–response inhibition in duplicate. Only compounds exhibiting dose–response inhibition in at least the low micromolar range are shown. All other curves are shown in Figure S3. (B) Gel-based secondary screen of putative SNM1A inhibitors. SNM1A (3 nM) was assayed with 25 and 6.25 μM compound, as noted. Inhibited and uninhibited reactions utilized zinc acetate (1 mM) and DMSO, respectively. A single-stranded 30mer oligonucleotide substrate (50 nM) containing a 3′ fluorophore is shown and listed in Figure S1 as 5P-3F. Products from nonprocessive exonuclease activity of SNM1A result in a shortening of the substrate. Products were resolved using 20% denaturing PAGE and imaged with the Typhoon imager (GE Healthcare) at 526 nm. Compounds in red were excluded from further characterization. The dagger symbol refers to compounds exhibiting intrinsic internal fluorescence.

Figure 4

Gel-based dose–response assays for IC₅₀ determination. (A) Representative results for the gel-based assay for exonuclease IC₅₀ determination. Schematic of the single-strand exonuclease substrate (5P-1F in Figure S1) is shown. The product is a single 5′ nucleotide with a fluorophore. Lanes 2–13 represent reactions with an inhibitor, increasing 2-fold per lane. Each assay was designed such that the IC₅₀ values lay between lanes 6 and 9. (B) Representative gel for the gel-based assay for endonuclease IC₅₀ determination. Gapped endonuclease substrate schematic is shown (5F-gap in Figure S1). The product is a 35mer oligonucleotide with a 5′ fluorophore. Lanes 2–7 represent reactions with a compound, increasing 4-fold per lane. Each assay was designed such that the IC₅₀ values lay between lanes 3 and 5. SNM1A (0.2 nM, 200 nM) was incubated with a substrate (110 nM, 30 nM) for 60 or 150 min for exonuclease and endonuclease inhibitions, respectively. Products were resolved using 23% denaturing PAGE and imaged with the ChemiDoc at 526 nm. Products were quantified with ImageLab (Bio-Rad). All assays were performed in triplicate. (C) Summary of IC₅₀ values of SNM1A exonuclease and endonuclease activities. IC₅₀ was determined using GraphPad Prism. F denotes the fluorophore, P denotes the phosphorylation, and SEM denotes the standard error of the mean.

Figure 5

SNM1A inhibitors potentiate cisplatin toxicity. (A) Schematic of the cisplatin potentiation assay. (B) Cisplatin potentiation with SNM1A inhibitors (25 μM). Cell survival is reported for the LD₁₀ value of cisplatin (dashed line) as well as with cisplatin (LD₁₀, 15 μM, in dark gray) or without (light blue). Relative % survival is expressed as percent normalized to cells incubated with control vehicle (DSMO) only. Assays were performed in duplicate, where error bars represent SEM. One asterisk symbol denotes a t test significance of p < 0.05, and two asterisk symbols denote p < 0.01 of inhibitor alone vs inhibitor and cisplatin.

Figure 6

Inhibitors of SNM1A.