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Purity: ≥98%
b-AP15 (formerly known as NSC687852; b-AP15; USP14 Inhibitor III; b-AP-15; NSC-687852) is a novel, potent and specific deubiquitinase (DUB) inhibitor of 19S proteasomes activity of Ub-AMC cleavage with potential antineoplastic activity. It inhibits deubiquitinase with an IC50 of 2.1 μM. b-AP15 displays antitumor activity in several preclinical solid tumor models. b-AP15 also triggers time- and dose-dependent apoptosis of the human multiple myeloma (MM) cell lines RPMI8226 and U266, as determined by phosphatidylserine exposure. Furthermore, b-AP15 triggered processing of pro-caspase-3 and cleavage of poly (ADP-ribose) polymerase in MM cells. b-AP15 also induced caspase-independent apoptosis in primary human natural killer cells. Taken together, b-AP15 may have potential for treatment of multiple myeloma patients.
| Targets |
UCHL5/Usp14
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| ln Vitro |
The indicated concentrations of b-AP15 are added to purified 19S proteasomes (5 nM), and the cleavage of Ub-AMC is used to measure DUB activity. Using non-linear regression analysis, the IC50 value (2.1±0.411 μM) is ascertained from log concentration curves in Graph Pad Prism. b-AP15 is a proteasome inhibitor of a hitherto undiscovered class that prevents the 19S regulatory particle from deubiquitinating. Polyubiquitin accumulated as a result of b-AP15'sinhibitionof the activity of two 19S regulatory-particle-associated deubiquitinases, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14). Tumor cell apoptosis induced by b-AP15 is not affected by TP53 status or overexpression of the apoptosis inhibitor BCL2[1].Using Ub-AMC as the substrate, the capacity of b-AP15 to inhibit proteasome deubiquitinase activity is ascertained. An observed IC50 is 16.8±2.8 μM[2].A particular USP14 and UCHL5 inhibitor called b-AP15 stops the growth of MM cells and causes them to undergo apoptosis[3].
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| ln Vivo |
In syngenic mouse models, b-AP15 (2.5 mg/kg) inhibits tumor growth with less frequent administration schedules. We used a 2-d-on, 2-d-off schedule to administer b-AP15 to C57BL/6J mice with Lewis lung carcinomas (LLCs) and a 1-d-on, 3-d-off schedule to BALB/c mice with orthotopic breast carcinoma (4T1). In the C57BL/6J mice model, T/C=0.16 (P≤0.01) and in the BALB/c mice model, T/C=0.25 (P≤0.001), respectively, b-AP15 significantly inhibited tumor growth. A decrease in the quantity of lung metastases is also noted in the group of mice treated with b-AP15 for 4T1 breast carcinomas[1].
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| Enzyme Assay |
In tests for the inhibition of deubiquitinase, 19S regulatory particle (5 nM), 26S (5 nM). Using a Wallac VICTOR Multilabel counter or a Tecan Infinite M1000 fitted with 380 nm excitation and 460 nm emission filters, researchers observed the cleavage of ubiquitin-AMC (1,000 nM) in UCH-L1 (5 nM), UCH-L3 (0.3 nM), USP2CD (5 nM), USP7CD (5 nM), USP8CD (5 nM), or BAP1 (5 nM) after they were incubated with DMSO or b-AP15.[1].
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| Cell Assay |
Cell viability is monitored by either the fluorometric microculture cytotoxicity assay or the MTT assay. Using DMSO as the control, cells are seeded into 96-well flat-bottomed plates for the MTT assay, and they are then exposed to medications for an entire night. Each well receives 10 µl of a stock solution containing 5 mg/mL MTT at the conclusion of the incubations, and the plates are then incubated for 4 hours at 37°C. Overnight at 37°C, formazan crystals are dissolved in a 100 µL 10% SDS/10 mM HCl solution. An enzyme-linked immunosorbent assay (ELISA) plate reader is used to measure absorbance at 590 nm[2].
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| Animal Protocol |
Mice[1]
In the squamous carcinoma model, female SCID mice are given a subcutaneous injection of 1×10^6 FaDu cells into their right rear flank. The formula for measuring tumor growth is length×width^2×0.44. 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 after tumors have grown to a size of about 200 mm^3 (defined as day 0).In order to create a colon carcinoma model, we gave female nude mice subcutaneous injections of 2.5×10^6 HCT-116 colon carcinoma cells overexpressing Bcl2 into their right flank. We administered intraperitoneal injections of 5 mg of b-AP15 per kg of body weight to the mice. We subcutaneously injected 2×10^5 LLC cells into the right rear flank of female C57/B6 mice to create the lung carcinoma model. We randomized mice to receive either vehicle (n = 4) or b-AP15 (n = 4) intraperitoneally at 5 mg per kg of body weight after tumors had grown to a size of about 50 mm^3 (defined as day 0). A treatment cycle consisted of 2 days of treatment followed by 2 days of rest (2 days on, 2 days off) for a duration of 2 weeks. |
| References |
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| Molecular Formula |
C22H17N3O6
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| Molecular Weight |
419.39
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| Exact Mass |
419.11
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| Elemental Analysis |
C, 63.01; H, 4.09; N, 10.02; O, 22.89
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| CAS # |
1009817-63-3
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| Appearance |
Yellow solid powder
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| SMILES |
O=C1/C(=C(\[H])/C2C([H])=C([H])C(=C([H])C=2[H])[N+](=O)[O-])/C([H])([H])N(C(C([H])=C([H])[H])=O)C([H])([H])/C/1=C(/[H])\C1C([H])=C([H])C(=C([H])C=1[H])[N+](=O)[O-]
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| InChi Key |
GFARQYQBWJLZMW-JYFOCSDGSA-N
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| InChi Code |
InChI=1S/C22H17N3O6/c1-2-21(26)23-13-17(11-15-3-7-19(8-4-15)24(28)29)22(27)18(14-23)12-16-5-9-20(10-6-16)25(30)31/h2-12H,1,13-14H2/b17-11+,18-12+
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| Chemical Name |
(3E,5E)-1-acryloyl-3,5-bis(4-nitrobenzylidene)piperidin-4-one.
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| Synonyms |
b-AP15; b-AP15; b-AP-15; USP14 Inhibitor III; UCHL5UCH37 Inhibitor II; NSC687852.
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : 20~48 mg/mL ( 47.69~114.45 mM )
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (4.96 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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. Solubility in Formulation 2: 4% DMSO + Corn oil: 1mg/ml (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 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 |
*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.
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.
(2) Be sure to add the solvent(s) in order.
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