PMC

Oxidative stress can be induced in NSC34 cells by serum withdrawal. (a) DCF fluorescence in wild-type NSC34 cells under basal conditions and (b) after serum withdrawal. Scale bar, 50 μm. (c) Serum withdrawal induced a threefold increase in DCF fluorescence in wild-type NSC34 cells. Graph shows means ± 1 SEM. (d) Ebselen reduced serum withdrawal-induced oxidative stress, measured by DCF fluorescence, in NSC34 cells in a dose-dependent manner, with a half-maximal effect (EC₅₀) of 4 μM. There was no increase in toxicity, as measured by EthD1 fluorescence.

Summary of the performances of the top molecules in the in vitro screening experiments. (a) NSC34 serum-free dose–response curves. (b) G93A-SOD1 NSC34 basal DCF assay results (mean + SEM). Where a negative result is shown, the DCF fluorescence was reduced to levels below background fluorescence levels in blank wells containing medium and carboxy-H₂DCFDA, but no cells. (c) G93A-SOD1 NSC34 menadione MTT viability assay results (mean + SEM).

Effects of the best-hit molecules on survival of primary mouse motor neurons deprived of growth factors. Results show the mean numbers of motor neurons ± SEM counted in 20 random fields from four cultures. All conditions used medium without growth factors or antioxidants, except “Complete,” which contained complete motor neuron medium, as defined under Materials and methods. ⁎p = 0.020, ⁎⁎p = 0.014 (Wilcoxon test, adjusted for false discovery rate, after significant results from a Kruskal–Wallis test (p = 0.0005)).

Chemical structures of Spectrum library compounds that had a significant effect in all four in vitro screens and can be tolerated in vivo. Compounds are subdivided according to their predicted biochemical properties (). (a) Compounds satisfying Lipinski's Rule of 5 for an orally available drug and with a polar surface area (PSA) of < 70 Å². Compounds (b) with a high PSA or (c) that failed the Rule of 5 were not taken further.

Mutant SOD1 induces oxidative stress in a motor neuron cell culture model. (a) Western blot of NSC34 cell lysates, showing human SOD1 (upper band) and endogenous murine SOD1 (lower band). (b) Basal oxidative stress levels measured by quantification of DCF fluorescence in NSC34 cell lines expressing normal and mutant human SOD1, normalized to cell number. Data shown are means ± SEM. ANOVA discovered significant differences between treatments (p = 1.3 × 10^− 7). There was no significant difference between any of the control cell lines (white bars), but all mutant SOD1-expressing cells (gray bars) have higher intracellular ROS than control cells (p < 0.0001, Student's t test adjusted for false discovery rate).

Summary of screening results in NSC34 cells. (a) Results from a typical 384-well plate of NSC34 cells used to screen the Spectrum Collection. The solid line shows the midpoint (mean) between the negative (vehicle, 0.2% DMSO) and the positive control (10 μM ebselen), and the broken line shows the threshold of the lower bound of a 99% prediction interval for observations on the negative control. All molecules giving a DCF fluorescence value below this broken line were classified as hits (provided no toxicity was observed by EthD1 fluorescence). (b) Representative dose–response curves from two molecules. Dotted lines show the concentration giving the half-maximal effect (EC₅₀). (c) Plotting % reduction in DCF versus minimum toxic dose/EC₅₀ shows which molecules are effective antioxidants with a large window between an effective dose and a toxic dose. Cutoff points of below a 40% reduction in DCF fluorescence and below a toxicity/EC₅₀ of 30 produced 45 hits, shown in the top-right quadrant.

Cellular pathways activated by best-hit molecules. (a) Typical traces from TRAP assay. There was a 1-min delay between each reading of the plate. (b) Summary of TRAP assay results, showing that of the best-hit molecules tested, only esculetin significantly delayed decay of R-PE (p = 0.0051 for both esculetin and Trolox, Wilcoxon test). (c) Typical dose–response curves for the effect of CAPE on induction of 4 × ARE-TK-EGFP (solid line) and TK-EGFP (broken line). (d) Summary of the induction of ARE-TK-EGFP. Only CAPE significantly induced ARE-mediated gene transcription (p = 0.0022; Student's t test). (e) 5-LOX activity assay. Resveratrol, esculetin, and ebselen all significantly reduced LTB₄ synthesis by 5-LOX (p = 0.0028, 0.014, and 0.028, respectively; Student's t test). (f) NF-κB activation assay. Menadione (10 μM) induced translocation of NF-κB to the nucleus (white bar). This translocation could be significantly inhibited by CAPE, esculetin, and ebselen (p = 0.00021, 0.0013, and 0.031, respectively; Wilcoxon test). All results shown are means ± SEM. All pairwise tests were performed only after statistical significance was discovered with the appropriate combined test (ANOVA or Kruskal–Wallis) and were adjusted for multiple comparisons.