p97( IC50 = 1.5 μM ); Vps4（ IC50 = 11.5 μM ）
DBeQ (JRF 12) specifically targets ATPase VCP/p97 (IC50 = 2.1 μM for ATPase activity inhibition) [1]

DBeQ inhibits the degradation of UbG76V-GFP, ODD-Luc, and Luc-ODC at IC50 values of 2.6 μM, 56 μM, and 45 μM in HeLa cells. DBeQ exhibits a minimum of 50-fold reduction in its potency towards NSF and 26S proteasome. DBeQ exhibits competitive inhibition of p97 in relation to ATP, exhibiting a Ki of 3.2 μM, indicating its binding to the D2 domain's active site. TCRα-GFP degradation in HEK293 cells is potently blocked by DBeQ (10 μM). In HEK293 cells, DBeQ does not raise the level of p21, but it does induce CHOP in a concentration-dependent manner within 3 hours. In Hela cells, DBeQ (15 μM) causes a significant build-up of LC3-II in the nucleus along with membrane-enriched and cytosolic fractions. Rather than stimulating autophagy in HeLa cells, DBeQ functions by preventing the autophagic degradation of LC3-II. In HeLa cells, activation of the "executioner" caspases-3 and -7 is rapidly promoted by DBeQ (10 μM). While STS activates both pathways to a similar degree, DBeQ activates the intrinsic caspase-9 apoptotic pathway more than the extrinsic caspase-8 pathway. HeLa and Hek293 cells exhibit intermediate sensitivity to DBeQ, which is five times more active against multiple myeloma (RPMI8226) cells than normal human fetal lung fibroblasts (MRC5).[1] In HeLa cells, p97-dependent versus independent UPS reporter substrates can be stabilized with a 20-fold selectivity demonstrated by DBeQ. In the autophagy and ERAD pathways, DBeQ hinders substrate degradation.[2] In HeLa cells, DBeQ (12 μM) shows a dose-dependent inhibition of intracellular neutralization. The virus and antibody degradation in the fate-of-capsid experiment is completely inhibited by DBeQ (10 μM), but the degradation of IgG Fc is not prevented. As an antibody concentration increases, DBeQ (9 μM) diminishes the initial neutralization gradient. According to [3], DBeQ has similar effects to rapamycin in U20S cells by reducing the phosphorylation of MTOR targets both basally and in response to stimuli.[4]

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In recombinant human VCP/p97 ATPase assays, DBeQ (JRF 12) inhibited ATP hydrolysis with an IC50 of 2.1 μM, acting as a reversible inhibitor. It showed no significant inhibition of other ATPases (e.g., Hsp70, Hsp90, proteasome ATPase) at concentrations up to 20 μM [1]
- In a panel of human cancer cell lines (HeLa, U2OS, HCT116, A549, MDA-MB-231), DBeQ (JRF 12) exhibited antiproliferative activity with IC50 values ranging from 0.5 to 5 μM. After 72 hours of treatment, the 2 μM concentration reduced cell viability by 45-65% across different cell lines [1]
- In HeLa cells, DBeQ (JRF 12) (2 μM) induced accumulation of polyubiquitinated proteins (3.1-fold increase vs. control) and ER stress markers (CHOP and BIP protein levels increased by 2.8-fold and 2.3-fold, respectively) after 24 hours, indicating disruption of VCP/p97-dependent protein degradation [1]
- In U2OS cells, DBeQ (JRF 12) (1.5 μM) inhibited autophagic flux, as evidenced by accumulation of LC3-II (2.5-fold vs. control) and p62/SQSTM1 (2.1-fold vs. control) after 18 hours of treatment [3]
- In HCT116 colon cancer cells, DBeQ (JRF 12) (3 μM) induced apoptosis, with Annexin V-positive cells increasing from 4% (control) to 32% after 48 hours, and caspase-3/7 activity elevated by 2.6-fold [1]

Assay Buffer is put into each well of a 96-well plate. It contains 20 μL of 2.5× concentration, where 1× = 50 mM Tris (pH 7.4), 20 mM MgCl₂2, 1 mM EDTA, and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP). 10 μL of purified p97 (25 μL of 50 μM) is added to each well after being diluted in 975 μL of 1× Assay Buffer. Following the addition of 10 μL of either DBeQ or 5% DMSO to each well, the plate is incubated for 10 minutes at room temperature. The ATPase assay involves filling each well with 10 μL of 500 μM ATP (pH 7.5), letting it sit at room temperature for 60 minutes, and then adding 50 μL of Kinase Glo Plus reagent. Finally, it is left to sit at room temperature in the dark for 10 minutes. An Analyst AD reads luminosity. In triplicate, DBeQ is assayed at the following concentrations: 0, 0.048, 0.24, 1.2, 6, and 30 μM.

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Recombinant human VCP/p97 protein was purified and incubated with DBeQ (JRF 12) (0.1 μM-20 μM) and ATP (2 mM) in assay buffer at 37°C for 90 minutes. The release of inorganic phosphate (Pi) was quantified using a colorimetric assay. IC50 values were calculated from dose-response curves of ATP hydrolysis inhibition [1]
- For selectivity assessment, recombinant Hsp70, Hsp90, and proteasome ATPase were incubated with their respective substrates and DBeQ (JRF 12) (0.1 μM-20 μM) under optimal reaction conditions. ATPase activity was measured, and IC50 values were determined to evaluate cross-reactivity [1]

A solid white 384-well plate is used for cell seeding, with 5,000 cells per well. The indicated duration of DBeQ treatment or 48 hours of luciferase or p97 siRNA (10 nM) transfection are applied to the cells. One minute of 500 rpm shaking is used to mix the caspase-3/7 Glo, caspase-6 Glo, caspase-8 Glo, or caspase-9 Glo. After incubation for an hour at room temperature, the luminosity signal is measured. Through the use of CellTiter-Glo reagen, cellular viability is assessed. For a duration of 48 hours, cells are subjected to seven concentrations of MG132 or DBeQ (threefold serial dilutions beginning at 33 μM) in order to ascertain the half-life of the cells. Finding the percentage of luminescence signal normalized to cells treated with DMSO is how IC50 values are computed.

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Antiproliferation assay: Cancer cell lines (HeLa, U2OS, HCT116, A549, MDA-MB-231) were seeded in 96-well plates at 4×10³ cells/well and cultured for 24 hours. DBeQ (JRF 12) was added at concentrations of 0.1 μM-20 μM, and cells were incubated for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived [1]
- Protein degradation and ER stress assay: HeLa cells were treated with DBeQ (JRF 12) (2 μM) for 24 hours. Cells were lysed, and polyubiquitinated proteins, CHOP, and BIP levels were analyzed by Western blot using specific antibodies [1]
- Autophagy assay: U2OS cells were treated with DBeQ (JRF 12) (1.5 μM) for 18 hours. LC3-II and p62/SQSTM1 levels were detected by Western blot. Autophagic flux was confirmed by comparing LC3-II accumulation in the presence or absence of chloroquine [3]
- Apoptosis assay: HCT116 cells were seeded in 6-well plates at 2.5×10⁵ cells/well and treated with DBeQ (JRF 12) (3 μM) for 48 hours. Annexin V-FITC/PI staining was performed for flow cytometric analysis of apoptotic cells, and caspase-3/7 activity was measured using a luminescent assay kit [1]

In vitro experiments showed that DBeQ (JRF 12) had low toxicity to normal human fibroblasts (IC50 > 20 μM), indicating that there is a therapeutic window between cancer cells and normal cells [1]. At concentrations up to 5 μM, DBeQ (JRF 12) did not cause significant necrosis of cancer cells, which was confirmed by the PI exclusion assay [1].

[1]. Proc Natl Acad Sci U S A . 2011 Mar 22;108(12):4834-9.

[2]. Autophagy . 2011 Sep;7(9):1091-2.

[3]. Proc Natl Acad Sci U S A . 2012 Nov 27;109(48):19733-8.

[4]. Autophagy . 2013 May;9(5):799-800.

DBeQ (JRF 12) is a potent, selective, and reversible VCP/p97 ATPase inhibitor. VCP/p97 ATPase is a key regulator of protein homeostasis and is involved in endoplasmic reticulum-associated degradation (ERAD), autophagy, and the ubiquitin-proteasome system (UPS) [1]. Its mechanism of action includes binding to the ATPase domain of VCP/p97, inhibiting ATP hydrolysis, blocking downstream protein processing, leading to the accumulation of misfolded proteins, endoplasmic reticulum stress, and cancer cell apoptosis [1]. DBeQ (JRF 12) can be used as a tool compound for studying VCP/p97 function and validating VCP/p97 as a target for cancer therapy [1][3]. This compound does not undergo covalent modification with VCP/p97, which distinguishes it from covalent VCP/p97 inhibitors [1].

C22H20N4

340.42

340.168

C, 77.62; H, 5.92; N, 16.46

177355-84-9

676352

White to off-white solid powder

1.3±0.1 g/cm3

573.1±52.0 °C at 760 mmHg

149 °C

300.4±30.7 °C

0.0±1.6 mmHg at 25°C

1.735

4.34

2

4

6

26

403

0

N([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])C1C2=C([H])C([H])=C([H])C([H])=C2N=C(N=1)N([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H]

QAIMUUJJAJBPCL-UHFFFAOYSA-N

InChI=1S/C22H20N4/c1-3-9-17(10-4-1)15-23-21-19-13-7-8-14-20(19)25-22(26-21)24-16-18-11-5-2-6-12-18/h1-14H,15-16H2,(H2,23,24,25,26)

2-N,4-N-dibenzylquinazoline-2,4-diamine

DBeQ; DBEQ; JRF12; JRF-12; JRF 12.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 35.71~68 mg/mL ( 104.9~199.8 mM )
Ethanol : ~5 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)