Identification of cytoprotective benzodiazepinone by HTS. A, example of HTS screening results is provided. A graphical representation is provided where normalized relative ATP content (y axis) is plotted against well number (1–96 (A1 → H12)) (x axis). Wells A1 to D1 are active compound controls (received salubrinal + TG); wells E1 to H1 are assay negative controls (received DMSO + TG); wells A12 to H12 (column 12) are assay-positive controls (received DMSO without TG). The Z′ for the plate is 0.87. A hit compound is found in well G8 (asterisk). B, 11 benzodiazepinone hit compounds and an inactive analogue (IA-1) (see Table 1) were compared with respect to protection against thapsigargin-induced cell death using undifferentiated CSM14.1 cells. Data represent relative survival measured by ATP content and expressed as % control relative to cells cultured with DMSO and without TG. Sal = salubrinal.

Benzodiazepinones inhibit ER stress-induced JNK and p38 MAPK activation. CSM14.1 cells were cultured with DMSO or with 25 μm hit compounds in DMSO for 2 h, followed by treatment of TG (15 μm). Cell lysates were prepared and analyzed by SDS-PAGE/immunoblotting using antibodies specific for phospho-c-Jun (Ser-73), c-Jun, phospho-p38 MAPK (Thr-180/Tyr-182), p38 MAPK, phospho-eIF2α (Ser-51), eIF2α, ATF6 (showing the cleaved form), CHOP, and α-tubulin. Control samples (left panel) correspond to cells treated with DMSO alone or DMSO plus TG.

Illustration showing site of action of cytoprotective benzodiazepinone compounds in comparison with salubrinal in modulating UPR pathways. The active benzodiazepinones, such as prototype compound CID-2891837, interfere with ASK1 by inhibiting dephosphorylation of Ser-967 residue, leading to increased binding with 14-3-3. Inactive ASK1 cannot propagate stress signals to JNK and p38 MAPK. Salubrinal suppresses dephosphorylation of eIF2α.

Benzodiazepinones selectively protect against cell death induced by ER stress. A, undifferentiated CSM14.1 cells were cultured in 96-well plates (for ATP assay) or in 24-well plates (for annexin-V staining/flow cytometry). The next day, cells were treated with DMSO (0.5%) or hit compounds (25 μm compound with 0.5% DMSO final concentrations) for 2 h, followed by treatment of various cell death-inducing reagents, including 15 μm thapsigargin (TG) for 24 h, 10 μg/ml tunicamycin (TUN) for 72 h, 2.5 μm staurosporine (STS) for 24 h, 50 μm VP16 for 48 h, or 30 ng/ml TNF plus 10 μg/ml cycloheximide (CHX) for 24 h. Cellular ATP content was measured for staurosporine and TNF/CHX samples, normalizing data relative to cells treated with DMSO alone and presented as percentage of control (mean ± S.D., n = 3). For measuring cell viability in cultures treated with tunicamycin or VP16, cells were stained with FITC-annexin V and PI and then analyzed by flow cytometry to identify live cells (annexin V-negative plus PI-negative). Assays were performed at least three times and expressed as % control relative to cells culture with DMSO without TG or compounds (mean ± S.D., n = 3). z, benzyloxycarbonyl. B, pathway selectivity of a hit compound (CID-2891837) is demonstrated as an example. Experiments were performed as in A. Cell viability was assessed by annexin-V-FITC staining, followed by fluorescence-activated cell sorter analysis. Data represent relative fluorescence (x axis) versus cell number (y axis). C, benzodiazepinone compounds protect against TG-induced cytotoxicity in HeLa and PPC-1 cells. Data were generated as in A. D, primary cortical neuron cells were preincubated with DMSO or 25 μm compound CID-2891837 in DMSO for 2 h. TG was treated at 5 μm for 18 h prior to determining the percentage of apoptotic neurons (marked by immunostaining) based on Hoechst dye staining (mean ± S.D., n = 3).

Benzodiazepinone compounds increase Ser-967 phosphorylation of ASK1 and association with 14-3-3. A, phosphorylation of ASK1 at various sites was monitored by phospho-specific antibody immunoblotting. 293T cells were transfected with pcDNA-HA-ASK1, and 1 day later the cells were incubated with DMSO or 100 μm compounds in DMSO for 2 h. Compounds included five active benzodiazepinone hits (CID-2891837, CID-2891533, CID-2891714, CID-2882794, and CID-2879344), an active hit compound representing another chemical class with a different mechanism (EI14), and an inactive benzodiazepinone analogue (CID-2882293). Cell extracts were prepared and subjected to immunoblotting using anti-phospho-ASK1 antibodies specific for Ser-83, Thr-845, or Ser-967 or with anti-HA antibody. B, relative densities of phosphorylated ASK1 bands (gray = Ser-83; black = Thr-845; white = Ser-967) were determined by densitometry mode of ImageJ software and normalized relative to ASK1 protein, calculating intensity relative to cells cultured with DMSO without TG. C, compounds from A were compared with respect to cytoprotective activity against TG-induced killing of undifferentiated CSM14.1 cells. Active benzodiazepinone compounds are represented by black bars, and controls are depicted with gray bars. Relative cell viability was based on measurements of ATP content (mean ± S.D., n = 3). D, 293T cells were transfected with pcDNA-HA-ASK1 and pEBG-GST-14-3-3, and then 1 day later cells were incubated with DMSO, CID-2891837 (active compound) or CID-2882293 (inactive compound) in DMSO for 2 h, before TG (20 μm) treatment for various times as indicated. Cell extracts were prepared, and GST-14-3-3 protein was recovered using glutathione-Sepharose, followed by immunoblotting (IB) analysis using anti-HA antibody to detect HA-ASK1 protein and anti-GST antibody to confirm pulldown of comparable amounts of GST-14-3-3 protein. Aliquots of the same cell lysates (50 μg) were analyzed by immunoblotting using anti-phospho-ASK1(Ser-967) and HA antibodies. E, benzodiazepinone compounds modulate interaction of endogenous ASK1 and 14-3-3 in OVCAR5 and SNB19 cells. Cells (5 × 10⁶) were cultured in 100-mm dishes and incubated overnight, and then compound CID-2891837 (active), CID-2882293 (inactive), or DMSO was added, followed 2 h later by TG (20 μm) treatment for 30 min. Cell extracts were prepared and normalized for total protein content, and endogenous ASK1 protein was immunoprecipitated (IP). Immune complexes were analyzed by immunoblotting using antibodies specific for ASK1, ASK1 (phospho-Ser-967), or 14-3-3. Aliquots of the cell lysates (50 μg) were analyzed directly by immunoblotting using antibodies specific for 14-3-3 to confirm comparable levels of this protein in all samples. F, HeLa cells were transfected with control (Ctrl.) RNAi (scrambled) or two different RNAi reagents targeting ASK1. After 24 h, cells were treated with 15 μm TG for 24 h for analysis of relative cell viability by ATP content (mean ± S.D., n = 3) (bottom). Because of low endogenous levels of ASK1 protein, ASK1 siRNA-transfected cells were transfected with pcDNA-HA-ASK1, and after 24 h, cells were lysed for analysis of HA-ASK1 and β-actin protein levels by immunoblotting (top), to confirm RNAi efficiency.