Label-free target identification reveals oxidative DNA damage as the mechanism of a selective cytotoxic agent

Abstract

Phenotypic screening can not only identify promising first-in-class drug candidates, but can also reveal potential therapeutic targets or neomorphic functions of known proteins. In this study, we identified target proteins of SB2001, a cytotoxic agent that acts specifically against HeLa human cervical cancer cells. Because SB2001 lacks chemical modification sites, label-free target identification methods including thermal stability shift-based fluorescence difference in two-dimensional gel electrophoresis (TS-FITGE) and thermal proteome profiling (TPP) were applied to characterize its mechanism of action. Owing to their differences, the two label-free target identification methods uncovered complementary target candidates. Candidates from both methods were prioritized according to their selective lethality upon the knockdown of those genes in HeLa cells, compared to CaSki cells which were used as a negative control cell line from the human cervix. LTA4H was identified only by TS-FITGE, but not by TPP, because only one isoform was stabilized by SB2001. Furthermore, it was implied that a non-canonical function of LTA4H was involved in the SB2001 activity. MTH1 was identified by both TS-FITGE and TPP, and SB2001 inhibited the function of MTH1 in hydrolyzing oxidized nucleotides. Compared to CaSki cells, HeLa cells displayed downregulated DNA mismatch repair pathways, which made HeLa cells more susceptible to the oxidative stress caused by SB2001, resulting in increased 8-oxoG concentrations, DNA damage, and subsequent cell death.

Fig. 1. Workflow and characteristics of TS-FITGE and TPP. In both TS-FITGE and TPP, cells were treated with either DMSO or drug, and heated to various temperatures. After cell lysis, the remaining proteins in the soluble fraction were collected. TS-FITGE: soluble proteins were conjugated with fluorescence dyes (Cy3 for the DMSO-treated group and Cy5 for the drug-treated group) and then pooled, followed by separation on a 2D gel. The Cy5 to Cy3 fluorescence ratio for each proteoform was quantified. The distribution of the ratio was plotted on a box plot to select outliers as hits with significant thermal stability shifts. TPP: soluble proteins were digested with trypsin into peptides, which were conjugated with isobaric mass tags (a different tag was used for each temperature). The resulting peptides were pooled and analyzed by liquid chromatography-tandem mass spectrometry. The reporter ions of each peptide were quantified, and the melting temperatures (T_m) of the corresponding proteins were calculated. Proteins with significant T_m changes between the DMSO- and drug-treated groups were chosen as the target candidates.

Fig. 2. SB2001, a HeLa cell-specific cytotoxic agent, and the identification of its target candidates. (a) The chemical structure of SB2001. (b) Cell viability after treatment with SB2001 for 2 days in HeLa and CaSki cells. Data are presented as the mean ± SEM (n = 2). (c) Target candidates from TS-FITGE and TPP in HeLa cells. Candidate proteins were prioritized according to the lethality ratio of HeLa cells over CaSki cells after the gene knockdown of each candidate protein.

Fig. 3. SB2001 stabilizes one isoform of LTA4H and inhibits a non-canonical function of LTA4H. (a) Representative images of TS-FITGE (pH 3–10) in HeLa cells. Images of the Cy3 channel (green, DMSO-treated) and Cy5 channel (red, 10 μM of SB2001-treated) are overlaid. The area in the white box is magnified. (b) Box plot showing the distribution of the Cy5/Cy3 fluorescence ratio of each spot in the 55 °C gel. Center line denotes median, and the upper and lower box limits are the first and third quartiles, respectively. The whiskers indicate the 1st–99th percentiles. The spot denoted by a triangle in (a) is indicated by the red arrow. (c) Melting curves of LTA4H from TPP. (d) CETSA in HeLa cells with the LTA4H antibody (n = 5). (e) LTA4H isoform spots in the 2D gel were detected by anti-LTA4H immunoblotting, and the spots in TS-FITGE are indicated by triangles. (f) TS-FITGE 55 °C gel with SC-57461A, a known LTA4H inhibitor. (g) In vitro hydrolase activity of LTA4H upon treatment with SB2001 or SC-57461A (n = 2). (h) In vitro aminopeptidase activity of LTA4H upon treatment with SB2001 or SC-57461A (n = 2). (i) Viability of HeLa cells upon treatment with SB2001 or SC-57461A. (j) Sensorgrams of surface plasmon resonance (SPR) assay showing the binding kinetics of SB2001 (0.625 to 10 μM) to immobilized LTA4H. The dissociation constant (K_D) was calculated as the ratio of rate constants (k_d/k_a). The figure below shows the steady-state response against various concentrations of SB2001. Representative data from two independent experiments. (k) DMSO-normalized cell viability upon treatment with SB2001 after gene knockdown of LTA4H in HeLa cells (n = 4). Data are presented as the mean ± SEM.

Fig. 4. SB2001 stabilizes MTH1 and inhibits its function of hydrolyzing oxidized nucleotides. (a) Representative images of TS-FITGE (pH 4–7) in HeLa cells. Images of the Cy3 channel (green, DMSO-treated) and Cy5 channel (red, 10 μM of SB2001-treated) are overlaid. The area in the white box is magnified. (b) Box plot showing the distribution of the Cy5/Cy3 fluorescence ratios for each spot in the 52 °C gel. Center line denotes median, and the right and left box limits are the first and third quartiles, respectively. The whiskers indicate the 1st–99th percentiles. The a and b spots denoted in (a) are pointed by the red arrows. (c) Melting curves of MTH1 from TPP. (d) CETSA in HeLa cells with the MTH1 antibody (n = 3). (e) Isothermal dose–response stabilization of MTH1 by various concentrations of SB2001 at 52 °C. (f) Inhibition of the in vitro hydrolytic activity of MTH1 by SB2001 using 8-oxo-dGTP or 2-OH-dATP as a substrate. (g) DMSO-normalized cell viability upon treatment with SB2001 after MTH1 knockdown in HeLa cells (n = 4). (h) SPR sensorgrams showing the kinetics of SB2001 (0.16 to 40 μM) to immobilized MTH1. The dissociation constant (K_D) was calculated as the ratio of rate constants (k_d/k_a). The inset shows the steady-state response against various concentrations of SB2001. Representative data from two independent experiments. Data are presented as the mean ± SEM.

Fig. 5. HeLa cell-specific cytotoxicity of SB2001 originates from the downregulated mismatch repair (MMR) pathway. (a) CETSA in CaSki cells with the MTH1 antibody upon treatment of SB2001 (10 μM). (b) HeLa to CaSki ratio of protein expression levels acquired using quantitative mass spectrometry. MTH1 protein is indicated by a red arrow. (c) Differential expression of the MMR pathway in HeLa cells compared to that in CaSki cells. Undetected proteins (PMS2 and EXO1) are shown in gray color. (d) Viability of HeLa and CaSki cells upon treatment with an oxidant, KBrO₃, for 2 d (n = 2). (e) Cytotoxicity of SB2001 in HeLa cells upon co-treatment with an antioxidant, N-acetylcysteine, for 2 d (n = 2). (f) GI₅₀ of SB2001 after the knockdown of MMR genes in HeLa cells (n = 2). Data are presented as the mean ± SEM.

Fig. 6. SB2001 selectively induces oxidative DNA damage in HeLa cells. (a) Fluorescence imaging of 8-oxoG in live cells. Intensities in the nuclear region were quantified (n > 11). Representative data from three biological replicates. (b) Silver-stained images of the comet assay measuring DNA damage. Tail moments of each comet were quantified (n > 6). Representative data from two biological replicates. (c) Immunoblot of ATM-p53 DNA damage response and apoptosis markers. (d) Schematic model of the selective cytotoxicity of SB2001 toward HeLa cells compared to CaSki cells. Data are presented as the mean ± SEM. The P value was derived from a two-tailed Student's t-test. ***P < 0.001, **P < 0.01, and *P < 0.05.