UCHL5/Usp14
b-AP15 (NSC 687852) specifically targets two 19S proteasome-associated deubiquitinating enzymes: USP14 (IC50 = 0.6 μM) and UCHL5 (IC50 = 0.8 μM) [1][3]
b-AP15 (NSC 687852) shows no significant inhibition of other DUBs (USP1, USP2, USP5, USP7, UCH-L1: IC50 > 50 μM) or proteasomal catalytic subunits (IC50 > 100 μM) [1][3]

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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In recombinant USP14 and UCHL5 enzyme assays, b-AP15 (NSC 687852) dose-dependently inhibited their deubiquitinating activity with IC50 values of 0.6 μM (USP14) and 0.8 μM (UCHL5), acting as reversible inhibitors [1][3]
- In a panel of human cancer cell lines (multiple myeloma: RPMI 8226, U266, MM.1S; solid tumors: HeLa, A549, HCT116), b-AP15 (NSC 687852) exhibited potent antiproliferative activity with IC50 values ranging from 0.3 to 2.5 μM. After 72 hours of treatment, 1 μM concentration reduced cell viability by 60-80% across different cell lines [3]
- In RPMI 8226 multiple myeloma cells, b-AP15 (NSC 687852) (0.5 μM) induced rapid accumulation of polyubiquitinated proteins (3.5-fold vs. control) and ER stress, with CHOP and BIP protein levels increasing by 3.2-fold and 2.8-fold, respectively, after 12 hours [1][3]
- In bortezomib-resistant MM cell lines (RPMI 8226/Bort, U266/Bort), b-AP15 (NSC 687852) maintained antiproliferative activity with IC50 values of 0.7 μM and 1.1 μM, respectively, compared to 0.4 μM and 0.8 μM in parental sensitive cells [3]
- In HeLa cells, b-AP15 (NSC 687852) (1 μM) induced apoptosis within 24 hours, with Annexin V-positive cells increasing from 3% (control) to 42% and caspase-3/7 activity elevated by 3.8-fold [2]
- In HCT116 colon cancer cells, b-AP15 (NSC 687852) (0.8 μM) inhibited colony formation by 75% compared to control, indicating long-term antiproliferative effects [3]

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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In NOD/SCID mice bearing RPMI 8226 multiple myeloma xenografts, intraperitoneal administration of b-AP15 (NSC 687852) (5 mg/kg, once daily for 21 days) significantly inhibited tumor growth. Tumor volume was reduced by 72% compared to vehicle-treated mice, and tumor weight decreased by 68% [3]
- In the same xenograft model, b-AP15 (NSC 687852) (5 mg/kg) treatment led to accumulation of polyubiquitinated proteins (2.9-fold vs. vehicle) and activation of caspase-3 (cleaved caspase-3 levels increased by 3.1-fold) in tumor tissues, confirming on-target DUB inhibition [3]
- In nude mice bearing HeLa cervical cancer xenografts, intraperitoneal administration of b-AP15 (NSC 687852) (8 mg/kg, twice weekly for 3 weeks) reduced tumor volume by 65% compared to vehicle controls [2]

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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USP14/UCHL5 deubiquitinating activity assay: Purified recombinant human USP14 or UCHL5 was incubated with ubiquitin-AMC (fluorogenic substrate) and b-AP15 (NSC 687852) (0.01-10 μM) in assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT) at 37°C for 60 minutes. Fluorescence intensity (excitation 360 nm, emission 460 nm) was measured to quantify deubiquitination. IC50 values were calculated from dose-response inhibition curves [1][3]
- DUB selectivity assay: Recombinant USP1, USP2, USP5, USP7, UCH-L1, and proteasomal 20S subunits were incubated with their respective substrates and b-AP15 (NSC 687852) (0.1-100 μM) under optimal conditions. Enzyme activity was quantified to evaluate cross-reactivity [1][3]

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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Antiproliferation assay: Cancer cell lines (RPMI 8226, U266, MM.1S, HeLa, A549, HCT116) and bortezomib-resistant derivatives were seeded in 96-well plates at 3×10³ cells/well and cultured for 24 hours. b-AP15 (NSC 687852) was added at concentrations of 0.01-20 μM, and cells were incubated for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived [3]
- Polyubiquitination and ER stress assay: RPMI 8226 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with b-AP15 (NSC 687852) (0.5 μM) for 12 hours. Cells were lysed, and polyubiquitinated proteins, CHOP, and BIP levels were analyzed by Western blot [1][3]
- Apoptosis assay: HeLa cells were treated with b-AP15 (NSC 687852) (1 μM) for 24 hours. Annexin V-FITC/PI staining was performed for flow cytometric analysis of apoptotic cells, and caspase-3/7 activity was measured by luminescent assay [2]
- Colony formation assay: HCT116 cells were seeded in 6-well plates at 500 cells/well and treated with b-AP15 (NSC 687852) (0.8 μM) or vehicle. After 14 days of culture, colonies were stained with crystal violet and counted to calculate inhibition rate [3]

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.

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NOD/SCID mice (RPMI 8226 xenograft model): 6-8 weeks old NOD/SCID mice were subcutaneously inoculated with RPMI 8226 multiple myeloma cells (5×10⁶ cells/mouse). When tumors reached a volume of ~100 mm³, mice were randomly divided into vehicle and b-AP15 (NSC 687852) groups. b-AP15 (NSC 687852) was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered intraperitoneally at 5 mg/kg once daily for 21 days. Vehicle-treated mice received DMSO/saline mixture. Tumor volume was measured every 3 days, and body weight was monitored weekly. Tumors were excised for Western blot analysis [3]
- Nude mice (HeLa xenograft model): 6-8 weeks old nude mice were subcutaneously inoculated with HeLa cells (5×10⁶ cells/mouse). When tumors reached ~120 mm³, mice were treated with b-AP15 (NSC 687852) (8 mg/kg, ip, twice weekly for 3 weeks) or vehicle. Tumor volume was measured every 3 days, and tumors were excised for protein analysis [2]

In vitro experiments showed that b-AP15 (NSC 687852) had reduced toxicity to normal human fibroblasts (IC50 > 30 μM) and peripheral blood mononuclear cells (IC50 > 25 μM), suggesting that it has a therapeutic window [3]. In vivo experiments showed that at the test dose (5-8 mg/kg, intraperitoneal injection), b-AP15 (NSC 687852) did not cause significant weight loss (≤7% change from baseline) or obvious toxicity in mice [2][3]. Compared with the vector control group, there were no significant changes in liver function (ALT, AST) or kidney function (creatinine, BUN) in mice treated with b-AP15 (NSC 687852) [2][3]. The plasma protein binding rate of b-AP15 (NSC 687852) was lower than that of the control group. 687852)In mice, it was 93-95% (in vitro plasma binding assay)[3]

[1]. Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nat Med. 2011 Nov 6;17(12):1636-40.

[2]. The 19S Deubiquitinase Inhibitor b-AP15 is Enriched in Cells and Elicits Rapid Commitment to Cell Death. Mol Pharmacol. 2014 Jun;85(6):932-45.

[3]. A novel small molecule inhibitor of deubiquitylating enzyme USP14 and UCHL5 induces apoptosis in multiple myeloma and overcomes bortezomib resistance. Blood. 2014 Jan 30;123(5):706-16.

B-AP15 belongs to the piperidinone class of compounds. Its structure is piperidin-4-one, with a 3-oxopropyl-1-en-3-yl substitution at position 1 and a 4-nitrobenzyl substitution at positions 3 and 5. It is an inhibitor of ubiquitin-specific processing protein 14 (USP14) and ubiquitin carboxyl-terminal hydrolase isoenzyme L5 (UCHL5). It exhibits apoptosis-inducing, antitumor, anti-inflammatory, and proteasome-inhibiting effects. It is a nitrophenol belonging to the piperidinone and acrylamide classes of compounds.

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b-AP15 (NSC 687852) is a potent and selective inhibitor of USP14 and UCHL5, two deubiquitinating enzymes (DUBs) associated with 19S proteasome regulatory particles [1][3] - Their mechanisms of action include inhibiting USP14/UCHL5-mediated deubiquitination, blocking proteasome degradation of polyubiquitinated proteins, and inducing endoplasmic reticulum stress and apoptosis in cancer cells [1][2] - b-AP15 (NSC 687852) overcomes bortezomib resistance in multiple myeloma cells both in vitro and in vivo by targeting upstream DUBs rather than proteasome catalytic subunits [3] - This compound has been used as a tool compound to study the function and cancer biology of 19S DUBs in the proteasome and has the potential to be used as an anticancer drug for bortezomib-resistant tumors [1][2][3]

C22H17N3O6

419.39

419.111

C, 63.01; H, 4.09; N, 10.02; O, 22.89

1009817-63-3

5351435

Yellow solid powder

1.4±0.1 g/cm3

670.4±55.0 °C at 760 mmHg

359.3±31.5 °C

0.0±2.0 mmHg at 25°C

1.702

4.24

0

6

3

31

750

0

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-]

GFARQYQBWJLZMW-JYFOCSDGSA-N

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+

(3E,5E)-1-acryloyl-3,5-bis(4-nitrobenzylidene)piperidin-4-one.

b-AP15; b-AP15; b-AP-15; USP14 Inhibitor III; UCHL5UCH37 Inhibitor II; NSC687852.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO : 20~48 mg/mL ( 47.69~114.45 mM )

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.

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Solubility in Formulation 2: 4% DMSO + Corn oil: 1mg/ml

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