DBeQ

Catalog No.S7199 Batch:S719901

Technical Data

Formula	C₂₂H₂₀N₄
Molecular Weight	340.42	CAS No.	177355-84-9
Solubility (25°C)*	In vitro	DMSO	68 mg/mL (199.75 mM)
		Ethanol	5 mg/mL (14.68 mM)
		Water	Insoluble
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description

DBeQ (JRF 12) is a selective, potent, reversible, and ATP-competitive p97 inhibitor with IC50 of 1.5 μM.

Targets

p97 ^[1]

1.5 μM

In vitro

DBeQ blocks UbG76V-GFP, ODD-Luc and Luc-ODC degradation with IC50 of 2.6 μM, 56 μM and 45 μM in HeLa cells. DBeQ is at least 50-fold less potent toward N-ethylmaleimide–sensitive factor (NSF) and 26S proteasome. DBeQ inhibits p97 competitively with respect to ATP, with Ki of 3.2 μM, suggesting that it binds to the active site of the D2 domain. DBeQ (10 μM) potently blocks degradation of TCRα-GFP in HEK293 cells. DBeQ induces CHOP within 3 hours in a concentration-dependent manner but does not increase p21 level in HEK293 cells. DBeQ (15 μM) induces a strong accumulation of LC3-II in the nucleus plus membrane-enriched and cytosolic fractions in Hela cells. DBeQ acts by blocking autophagic degradation of LC3-II instead of inducing autophagy in HeLa cells. DBeQ (10 μM) rapidly promotes activation of the “executioner” caspases-3 and -7 in HeLa cells. DBeQ activates the intrinsic caspase-9 apoptotic pathway more than the extrinsic caspase-8 pathway, whereas STS activates both pathways to a similar extent. DBeQ is fivefold more active against multiple myeloma (RPMI8226) cells than normal human fetal lung fibroblasts (MRC5), with HeLa and Hek293 cells showing intermediate sensitivities. ^[1] DBeQ exhibits 20-fold selectivity for stabilizing p97-dependent vs. independent UPS reporter substrates in HeLa cells. DBeQ impairs degradation of substrates within the ERAD and autophagy pathways. ^[2] DBeQ (12 μM) inhibits intracellular neutralization in a dose-dependent manner in HeLa cells. DBeQ (10 μM), which completely inhibits degradation of virus and antibody in the fate-of-capsid experiment, fails to prevent degradation of IgG Fc. DBeQ (9 μM) reduces the initial gradient of neutralization as a function of antibody concentration. ^[3] DBeQ decreases both basal and nutrient-stimulated phosphorylation of MTOR targets similar to the effects of rapamycin in U20S cells. ^[4]

Features

Rapidly and potently induces activation of executioner caspases and cell death.

Protocol (from reference)

Kinase Assay:^{^[1]}	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. Puriﬁed 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 ﬁnal 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.
Cell Assay:^{^[1]}	Cell lines MRC-5, Hek293, HeLa and RPMI8226 cells Concentrations 33 μM Incubation Time 48 hours Method 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 ﬁtting the percentage of luminescence signal normalized to DMSO treated cells).

Kinase Assay:^{^[1]}

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. Puriﬁed 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 ﬁnal 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.

Cell Assay:^{^[1]}

Cell lines
MRC-5, Hek293, HeLa and RPMI8226 cells
Concentrations
33 μM
Incubation Time
48 hours
Method
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 ﬁtting the percentage of luminescence signal normalized to DMSO treated cells).

References

[4] Ching JK, et al. Autophagy, 2013, 9(5), 799-800.

+ Expand

Selleck's DBeQ has been cited by 10 publications

Elucidating cellular interactome of chikungunya virus identifies host dependency factors [ Virol Sin, 2023, 38(4):497-507]	PubMed: 37182691
Anti-L1 antibody-bound HPV16 pseudovirus is degraded intracellularly via TRIM21/proteasomal pathway [ Virol J, 2022, 19(1):90]	PubMed: 35619167
VCP/p97 regulates Beclin-1-dependent autophagy initiation [ Nat Chem Biol, 2021, 10.1038/s41589-020-00726-x]	PubMed: 33510452
White spot syndrome virus benefits from endosomal trafficking, substantially facilitated by a valosin-containing protein, to escape autophagic elimination and propagate in crustacean Cherax quadricarinatus [ J Virol, 2020, JVI.01570-20]	PubMed: 32967962
ATG8-Binding UIM Proteins Define a New Class of Autophagy Adaptors and Receptors [ Cell, 2019, 177(3):766-781]	PubMed: 30955882
Functional cooperativity of p97 and histone deacetylase 6 in mediating DNA repair in mantle cell lymphoma cells [Vekaria PH Leukemia, 2019, 10.1038/s41375-018-0355-y]	PubMed: 30664664
The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8. [ EMBO J, 2019, 38(21):e102361]	PubMed: 31613024
The Identification of Potential Therapeutic Targets for Cutaneous Squamous Cell Carcinoma. [ J Invest Dermatol, 2019, 10.1016/j.jid.2019.09.024]	PubMed: 31705877
Aggregation of a hepatitis C virus replicase module induced by ablation of p97/VCP. [ J Gen Virol, 2017, 98(7):1667-1678]	PubMed: 28691899
Perturbation of cellular proteostasis networks identifies pathways that modulate precursor and intermediate but not mature levels of frataxin. [ Sci Rep, 2015, 5:18251]	PubMed: 26671574

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