DBEQ (JRF 12) is a selective, potent, reversible, and ATP-competitive p97 inhibitor.

p97:1.5 μM

10 μM DBeQ完全抑制病毒和抗体的降解,但不抑制IgG Fc的降解。10 μM DBeQ快速促进HeLa细胞中caspases-3和-7激活。比激活外在caspase-8通路,DBeQ更有效激活内在caspase-9凋亡途径,而STS激活两种途径程度相似。10 μM DBeQ有效抑制HEK293细胞中TCRα-GFP降解。DBeQ作用于HEK293细胞,3小时内剂量依赖性诱导CHOP,但不增加p21水平。DBeQ作用于U20S细胞,降低基本的和营养刺激的MTOR靶点磷酸化,与雷帕霉素效果类似。15 μM DBeQ 作用于Hela细胞,诱导LC3-II 在在细胞核及浓缩膜和胞质级组分中大量积累。DBeQ作用于HeLa细胞,抑制UbG76V-GFP,ODD-Luc和Luc-ODC 降解,IC50分别为2.6 μM、56 μM 和45 μM。

Manual ATPase Assay: Assay Buffer [20 μL of 2.5× concentration, where 1× = 50 mM Tris (pH 7.4), 20 mM MgCl2, 1 mM EDTA, and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP)] is dispensed into each well of a 96-well plate. Puri?ed p97 (25 μL of 50 μM) is diluted in 975 μL of 1× Assay Buffer, and 10 μL is dispensed in each well. DBeQ (10 μL) or 5% DMSO (10 μL) is then added to each well, and the plate is incubated at room temperature for 10 min. The ATPase assay is carried out by adding to each well 10 μL of 500 μM ATP (pH 7.5), incubating at room temperature for 60 min, and then adding 50 μL Kinase Glo Plus reagent, followed by a ?nal 10-min incubation at room temperature in the dark. Luminescence is read on an Analyst AD. DBeQ is assayed at a range of concentrations (0, 0.048, 0.24, 1.2, 6, and 30 μM) in triplicate.

Cells are seeded on a 384-well solid white plate (5,000 cells/well). Cells are transfected with luciferase siRNA or p97 siRNA (10 nM) for 48 hours or treated with DBeQ for the indicated amount of time. Caspase-3/7 Glo, caspase-6 Glo, caspase-8 Glo, or caspase-9 Glo is added into each well and mixed by shaking at 500 rpm for 1 min. Luminescence signal is determined after incubation at room temperature for 1 hour. Cellular viability is determined with CellTiter-Glo reagen. To determine the IC50 of cellular viability, cells are treated with MG132 or DBeQ at seven concentrations (threefold serial dilutions starting at 33 μM) for 48 hours. IC50 values are calculated from ?tting the percentage of luminescence signal normalized to DMSO treated cells).(Only for Reference)