Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways

Abstract

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.

Fig. 1.

DBeQ (1) is a reversible and selective inhibitor of p97. (A) Western blot assays to evaluate specificity of the top 10 hits to emerge from HTS. HeLa cells that stably expressed Ub^G76V-GFP and ODD-Luc were treated with MG132, washed, and then incubated in the presence of CHX, plus test compound for 2 h before harvest. Samples were immunoblotted to detect ODD-Luc. The Ponceau S-stained filter serves as a loading control. The high level of remaining antigen in lane 2 indicates that MG132 largely blocked ODD-Luc degradation, whereas the relatively low level in lane 3 indicates that 20 μM DBeQ failed to prevent ODD-Luc proteolysis. (B) Inhibition of ATPase activity of p97 (diamonds), NSF (circles), or the ATP-dependent chymotryptic activity of 26S proteasome (triangles) by DBeQ. (C) Reversibility of compound inhibition was determined by first accumulating Ub^G76V-GFP in the presence of MG132 (4 μM for 1 h), washing out MG132 and exposing cells to CHX plus test compound for 2 h, and then washing out test compound and monitoring decay of GFP signal in CHX for 0–4 h. (D) Lineweaver-Burk plot of the competitive inhibition of p97 ATPase activity by DBeQ.

Fig. 2.

DBeQ impairs the ERAD pathway. (A) Hek293 cells stably expressing the ERAD reporter TCRα-GFP were used to determine effect of DBeQ on the ERAD pathway. Cells were treated with drug as described in Fig. 1A, and GFP intensity was determined by flow cytometry at the indicated time points. (B) HeLa cells were transfected with negative control siRNA (NC) or p97 siRNA (10 nM) for 72 h (lanes 1–4) or with cDNAs encoding wild-type (WT) or ATPase mutant (QQ) p97 for 24 h (lanes 5–8), and the levels and distributions of the indicated proteins were determined by immunoblotting cytosolic (Cyto) and nuclear plus membrane (NM) fractions. (C) HeLa cells were incubated with 10 μM of MG132, 25 μM of CHX, or 5, 10, or 15 μM of DBeQ for 3 h, and the levels of CHOP and LC3 in the nuclear plus membrane (NM) fractions were determined by immunoblotting.

Fig. 3.

DBeQ impairs the autophagy pathway. (A) Cells were incubated with 20 μM of MG132 or DBeQ for 3 h, and cytosolic LC3 level was determined by immunoblotting. The Ponceau S-stained filter serves as a loading control. (B) Degradation of LC3-II was monitored in Earle's balanced salt solution (EBSS) nutrient-starvation medium for 0, 30, 120, or 240 min (lanes 1–4). DBeQ (15 μM) completely stabilized LC3-II (lanes 5–7), whereas 2.5 μM DBeQ had little effect (lanes 8–10). (C) Cells were incubated with DMSO (lane 1) or Baf (0.25 μM, 5 h; lane 2) or Baf plus DBeQ (lanes 3 and 4) for 5 h or first treated with Baf for 2.5 h then exposed to DBeQ for an additional 2.5 h (lanes 5 and 6). The same experiments were carried out at 10 μM Baf (lanes 7–11). Lane 12 contains sample from cells treated with DBeQ (10 μM, 5 h) alone. All treatments were evaluated by immunoblotting cell lysates with antibodies to detect the indicated proteins. (D) Cells were treated with Z-VAD (25 μM), Z-VAD plus DBeQ (15 μM), CHQ (20 μM), or CHQ plus DBeQ, followed by immunoblotting cell extracts to detect the indicated proteins.

Fig. 4.

DBeQ and depletion of p97 induce activation of caspases. (A) HeLa cells were incubated with the indicated concentrations of 1541, DBeQ, or STS for 4 h before determination of caspases-3 plus -7 activities in the cell extract. (B) HeLa cells were incubated with 20 μM of 1541, 10 μM of DBeQ, or 2.5 μM of STS for 2, 4, or 6 h before determination of caspases-3 plus -7 activities in the cell extract. (C–E) Same as B, except caspases-6, -8, and -9 activities were determined, respectively. (F) HeLa cells were transfected with negative control siRNA (NC) or p97 siRNA (10 nM) for 72 h, after which the activities of the indicated caspases were measured in total cell lysate. (G) HeLa cells were incubated with the indicated concentrations of drugs for 3 h before determination of caspases-3 plus -7 activities in total cell lysate. DBeQ plus CHX were included at 10 μM and 100 μM, respectively. (H) HeLa cells were incubated with the indicated concentrations of drugs for 1, 2, 4, 6, 8, 9, or 21 h before determination of caspases-3 plus -7 activities in total cell lysate. (I) Same as H, except cellular viability was determined using CellTiter-Glo after 3, 6, 8, or 20 h.