1. Cell Cycle/DNA Damage
  2. Deubiquitinase
  3. b-AP15

b-AP15 (Synonyms: NSC 687852)

Cat. No.: HY-13989 Purity: 98.93%
Handling Instructions

b-AP15 is a specific inhibitor of the deubiquitinating enzymes UCHL5 and Usp14.

For research use only. We do not sell to patients.

b-AP15 Chemical Structure

b-AP15 Chemical Structure

CAS No. : 1009817-63-3

Size Price Stock Quantity
10 mM * 1 mL in DMSO USD 66 In-stock
Estimated Time of Arrival: December 31
10 mg USD 60 In-stock
Estimated Time of Arrival: December 31
25 mg USD 100 In-stock
Estimated Time of Arrival: December 31
50 mg USD 150 In-stock
Estimated Time of Arrival: December 31
100 mg USD 250 In-stock
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200 mg USD 450 In-stock
Estimated Time of Arrival: December 31
500 mg USD 750 In-stock
Estimated Time of Arrival: December 31
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Based on 3 publication(s) in Google Scholar

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Description

b-AP15 is a specific inhibitor of the deubiquitinating enzymes UCHL5 and Usp14.

IC50 & Target

UCHL5/Usp14[1]

In Vitro

Purified 19S proteasomes (5 nM) are treated with indicated concentrations of b-AP15 and DUB activity is determined by detectionof Ub-AMC cleavage. The IC50 value (2.1±0.411 μM) is determined from log concentration curves in Graph Pad Prism using non linear regression analysis. b-AP15 as a previously unidentified class of proteasome inhibitor that abrogates the deubiquitinating activity of the 19S regulatory particle. b-AP15 inhibited the activity of two 19S regulatory-particle-associated deubiquitinases, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation of polyubiquitin. b-AP15 induced tumor cell apoptosis that is insensitive to TP53 status and overexpression of the apoptosis inhibitor BCL2[1]. The ability of b-AP15 is determined to inhibit proteasome deubiquitinase activity using Ub-AMC as the substrate. An IC50 of 16.8±2.8 μM is observed[2]. b-AP15 is a specific USP14 and UCHL5 inhibitor, which blocks growth and induces apoptosis in MM cells[3].

In Vivo

b-AP15 (2.5 mg/kg) inhibits tumor growth in syngenic mice models with less frequent administration schedules. We administered b-AP15 to C57BL/6J mice with Lewis lung carcinomas (LLCs) using a 2-d-on, 2-d-off schedule and to BALB/c mice with orthotopic breast carcinoma (4T1) using a 1-d-on, 3-d-off schedule. b-AP15 significantly inhibited tumor growth in both models, with T/C=0.16 (P≤0.01) for the C57BL/6J mice and T/C=0.25 (P≤0.001) for the BALB/c mice. A reduction in the number of pulmonary metastases also is observed in the group of mice with 4T1 breast carcinomas treated with b-AP15[1].

Molecular Weight

419.39

Formula

C₂₂H₁₇N₃O₆

CAS No.

1009817-63-3

SMILES

O=C1/C(CN(C(C=C)=O)C/C1=C\C2=CC=C([N+]([O-])=O)C=C2)=C/C3=CC=C([N+]([O-])=O)C=C3

Shipping

Room temperature in continental US; may vary elsewhere

Storage
Powder -20°C 3 years
  4°C 2 years
In solvent -80°C 6 months
  -20°C 1 month
Solvent & Solubility
In Vitro: 

DMSO : ≥ 44 mg/mL (104.91 mM)

*"≥" means soluble, but saturation unknown.

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 2.3844 mL 11.9221 mL 23.8442 mL
5 mM 0.4769 mL 2.3844 mL 4.7688 mL
10 mM 0.2384 mL 1.1922 mL 2.3844 mL
*Please refer to the solubility information to select the appropriate solvent.
In Vivo:
  • 1.

    Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% saline

    Solubility: 2.5 mg/mL (5.96 mM); Suspended solution; Need ultrasonic

*All of the co-solvents are provided by MCE.
References
Kinase Assay
[1]

For deubiquitinase inhibition assays, 19S regulatory particle (5 nM), 26S (5 nM) UCH-L1 (5 nM), UCH-L3 (0.3 nM), USP2CD (5 nM) USP7CD (5 nM) USP8CD (5 nM) or BAP1 (5 nM) is incubated with DMSO or b-AP15 and monitored the cleavage of ubiquitin-AMC (1,000 nM) using a Wallac VICTOR Multilabel counter or a Tecan Infinite M1000 equipped with 380 nm excitation and 460 nm emission filters[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cell Assay
[2]

Cell viability is monitored by either the fluorometric microculture cytotoxicity assay or the MTT assay. For the MTT assay, cells are seeded into 96-well flat-bottomed plates overnight and exposed to drugs, using DMSO as the control. At the end of incubations, 10 µl of a stock solution of 5 mg/mL MTT is added into each well, and the plates are incubated 4 hours at 37°C. Formazan crystals are dissolved with 100 µL 10% SDS/10 mM HCl solution overnight at 37°C. Absorbance is measured using an enzyme-linked immunosorbent assay (ELISA) plate reader at 590 nm[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Administration
[1]

Mice[1]
For the squamous carcinoma model, 1×106 FaDu cells are subcutaneously injected into the right rear flank of female SCID mice. Tumor growth is measured by the formula length×width2×0.44. When tumors have grown to a size of approximately 200 mm3 (defined as day 0), mice are randomized to receive either vehicle (n=10) or b-AP15 (n=15) at 5 mg per kg of body weight by daily subcutaneous injection. For the colon carcinoma model, we subcutaneously injected 2.5×106 HCT-116 colon carcinoma cells overexpressing Bcl2 into the right flank of female nude mice. We treated mice with 5 mg of b-AP15 per kg of body weight by intraperitoneal injection. For the lung carcinoma model, we subcutaneously injected 2×105 LLC cells into the right rear flank of female C57/B6 mice. When tumors had grown to a size of approximately 50 mm3 (defined as day 0), we randomized mice to receive either vehicle (n=4) or b-AP15 (n=4) at 5 mg per kg of body weight intraperitoneally, with a treatment cycle consisting of 2 d of treatment followed by 2 d of rest (2 d on, 2 d off) for 2 weeks.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

References

Purity: 98.93%

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b-AP15
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HY-13989
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