DBeQ (JRF 12)

Alias: DBeQ; DBEQ; JRF12; JRF-12; JRF 12.
Cat No.:V1319 Purity: ≥98%
DBeQ (JRF-12; JRF 12; JRF12) is a reversible and ATP-competitive p97 inhibitor with potential antineoplastic activity.
DBeQ (JRF 12) Chemical Structure CAS No.: 177355-84-9
Product category: p97
This product is for research use only, not for human use. We do not sell to patients.
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Purity & Quality Control Documentation

Purity: ≥98%

Product Description

DBeQ (JRF-12; JRF 12; JRF12) is a reversible and ATP-competitive p97 inhibitor with potential antineoplastic activity. Its IC50 value for p97 inhibition is 1.5 μM. By interfering with the ubiquitin-dependent and autophagic protein clearance pathways, DBeQ can be used to treat cancer.

Biological Activity I Assay Protocols (From Reference)
Targets
p97( IC50 = 1.5 μM ); Vps4( IC50 = 11.5 μM )
ln Vitro
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]
ln Vivo

Enzyme Assay
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.
Cell Assay
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.
Animal Protocol


References

[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.

These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C22H20N4
Molecular Weight
340.42
Exact Mass
340.17
Elemental Analysis
C, 77.62; H, 5.92; N, 16.46
CAS #
177355-84-9
Appearance
Solid powder
SMILES
C1=CC=C(C=C1)CNC2=NC(=NC3=CC=CC=C32)NCC4=CC=CC=C4
InChi Key
QAIMUUJJAJBPCL-UHFFFAOYSA-N
InChi Code
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)
Chemical Name
2-N,4-N-dibenzylquinazoline-2,4-diamine
Synonyms
DBeQ; DBEQ; JRF12; JRF-12; JRF 12.
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
DMSO : 35.71~68 mg/mL ( 104.9~199.8 mM )
Ethanol : ~5 mg/mL
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 2.9375 mL 14.6877 mL 29.3755 mL
5 mM 0.5875 mL 2.9375 mL 5.8751 mL
10 mM 0.2938 mL 1.4688 mL 2.9375 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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Note: Chemical formula is case sensitive: C12H18N3O4  c12h18n3o4
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Calculation results

Working concentration mg/mL;

Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
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Biological Data
  • DBeQ impairs the ERAD pathway.Proc Natl Acad Sci U S A.2011 Mar 22;108(12):4834-9.
  • DBeQ impairs the autophagy pathway.Proc Natl Acad Sci U S A.2011 Mar 22;108(12):4834-9.
  • VCP is essential for TRIM21-mediated neutralization of AdV by antibody.Proc Natl Acad Sci U S A.2012 Nov 27;109(48):19733-8.
  • VCP is required for degradation of virus and antibody in ADIN but not for TRIM21-mediated degradation of cytosolic IgG Fc.Proc Natl Acad Sci U S A.2012 Nov 27;109(48):19733-8.
  • DBeQ (1) is a reversible and selective inhibitor of p97.Proc Natl Acad Sci U S A.2011 Mar 22;108(12):4834-9.
  • VCP is essential for efficient and potent ADIN.Proc Natl Acad Sci U S A.2012 Nov 27;109(48):19733-8
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