IU1

Catalog No.S7134 Batch:S713402

Technical Data

Formula	C₁₈H₂₁FN₂O
Molecular Weight	300.37	CAS No.	314245-33-5
Solubility (25°C)*	In vitro	Ethanol	60 mg/mL (199.75 mM)
		DMSO	6 mg/mL (19.97 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

IU1 is a cell-permeable, reversible and selective proteasome inhibitor of human USP14 with IC50 of 4.7 μ M, 25-fold selective to IsoT. IU1 induces autophagy.

Targets

USP14 ^[1]
(cell-free assay)

4.7 μM

In vitro

IU1 binds specifically to the activated form of USP14. IU1 can potentially inhibit USP14 by preventing its docking on the proteasome, exhibiting little or no activity toward 8 other DUBs, IsoT, UCH37, BAP1, UCH-L1, UCH-L3, USP15, USP2, USP7. USP14 inhibition is rapidly established upon addition of IU1 and rapidly reversed upon its removal. IU1 inhibits USP14 induced chain trimming and decreases electrophoretic mobility of Ub-CCNB species. IU1 enhances proteasomal degradation of Ub-CCNB in the presence of USP14. IU1 promots degradation of tau and depletes TDP-43, ATXN3, and glial fibrillary acidic protein (GFAP) in proteotoxic mechanisms. ^[1]

Protocol (from reference)

Kinase Assay:^{^[1]}	High-throughput screening Screening is conducted at the ICCB-Longwood screening facility. 10 μL of recombinant USP14 protein are dispensed into each well of a 384-well low volume plate in duplicate, using a Wellmate plate dispenser. 33.3 nL of compound from the library are pin-transferred into the wells using a Seiko pin transfer robotic system, followed by pre-incubation for about 30 min. The last two columns of each plate are used for positive and negative controls for the assay. To initiate the enzyme reaction, 10 μL of VS-proteasome plus Ub-AMC mixture are added to each well, using a Wellmate dispenser. Samples are then incubated for another 45 min. Ub-AMC hydrolysis is measured at Ex355/Em460 using an Envision plate reader. The final concentrations of USP14, VS-proteasome and Ub-AMC are 15 nM, 1 nM and 0.8 μM, respectively. The final concentration of test compound is approximately 17 μM. Enzymes and substrates are prepared in Ub-AMC assay buffer (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM ATP, 5 mM MgCl₂, 1 mM DTT, and 1 mg/Ml ovalbumin).
Cell Assay:^{^[1]}	Cell lines MEF cells Concentrations ~1.5 mM Incubation Time 48 hours Method MTT

Kinase Assay:^{^[1]}

High-throughput screening
Screening is conducted at the ICCB-Longwood screening facility. 10 μL of recombinant USP14 protein are dispensed into each well of a 384-well low volume plate in duplicate, using a Wellmate plate dispenser. 33.3 nL of compound from the library are pin-transferred into the wells using a Seiko pin transfer robotic system, followed by pre-incubation for about 30 min. The last two columns of each plate are used for positive and negative controls for the assay. To initiate the enzyme reaction, 10 μL of VS-proteasome plus Ub-AMC mixture are added to each well, using a Wellmate dispenser. Samples are then incubated for another 45 min. Ub-AMC hydrolysis is measured at Ex355/Em460 using an Envision plate reader. The final concentrations of USP14, VS-proteasome and Ub-AMC are 15 nM, 1 nM and 0.8 μM, respectively. The final concentration of test compound is approximately 17 μM. Enzymes and substrates are prepared in Ub-AMC assay buffer (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM ATP, 5 mM MgCl₂, 1 mM DTT, and 1 mg/Ml ovalbumin).

Cell Assay:^{^[1]}

Cell lines
MEF cells
Concentrations
~1.5 mM
Incubation Time
48 hours
Method
MTT

References

[1] Byung-Hoon L, et al. Nature, 2010, 467(7312), 179-184.

Customer Product Validation

: Data from [Data independently produced by , , Glia, 2018, 66(2):379-395]

: Data from [Data independently produced by , , Eur J Immunol, 2018, doi:10.1002/eji.201847603]

Selleck's IU1 has been cited by 20 publications

PKCiota Inhibits the Ferroptosis of Esophageal Cancer Cells via Suppressing USP14-Mediated Autophagic Degradation of GPX4 [ Antioxidants (Basel), 2024, 13(1)114]	PubMed: 38247539
USP14 promotes tryptophan metabolism and immune suppression by stabilizing IDO1 in colorectal cancer [ Nat Commun, 2022, 13-1:5644]	PubMed: 36163134
USP14 maintains HIF1-α stabilization via its deubiquitination activity in hepatocellular carcinoma [ Cell Death Dis, 2021, 12(9):803]	PubMed: 34420039
Development of potent and selective inhibitors targeting the papain-like protease of SARS-CoV-2 [ Cell Chem Biol, 2021, S2451-9456(21)00213-0]	PubMed: 33979649
The m6A Reader YTHDF1 Facilitates the Tumorigenesis and Metastasis of Gastric Cancer via USP14 Translation in an m6A-Dependent Manner [ Front Cell Dev Biol, 2021, 9:647702]	PubMed: 33791305
Qing-Fei-Pai-Du decoction and wogonoside exert anti-inflammatory action through down-regulating USP14 to promote the degradation of activating transcription factor 2 [ FASEB J, 2021, 35(9):e21870]	PubMed: 34436790
USP14 negatively regulates RIG-I-mediated IL-6 and TNF-α production by inhibiting NF-κB activation [ Mol Immunol, 2021, 130:69-76]	PubMed: 33360745
USP14 is a deubiquitinase for Ku70 and critical determinant of non-homologous end joining repair in autophagy and PTEN-deficient cells. [ Nucleic Acids Res, 2020, 48(2):736-747]	PubMed: 31740976
Inhibition of USP14 suppresses the formation of foam cell by promoting CD36 degradation. [ J Cell Mol Med, 2020, 10.1111/jcmm.15002]	PubMed: 31970862
Inhibition of USP14 enhances the sensitivity of breast cancer to enzalutamide [ J Exp Clin Cancer Res, 2019, 38(1):220]	PubMed: 31126320

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SHIPPING AND STORAGE
Selleck products are transported at room temperature. If you receive the product at room temperature, please rest assured, the Selleck Quality Inspection Department has conducted experiments to verify that the normal temperature placement of one month will not affect the biological activity of powder products. After collecting, please store the product according to the requirements described in the datasheet. Most Selleck products are stable under the recommended conditions.

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