LDN-57444

Catalog No.S7135 Batch:S713501

Technical Data

Formula

C₁₇H₁₁Cl₃N₂O₃

Molecular Weight

397.64

CAS No.

668467-91-2

Solubility (25°C)*

In vitro

DMSO

11 mg/mL (27.66 mM)

Water

Insoluble

Ethanol

Insoluble

In vivo (Add solvents to the product individually and in order)

Clear solution	5%DMSO 1 Corn oil	6.0mg/ml	Taking the 1 mL working solution as an example, add 50 μL of 120 mg/ml clear DMSO stock solution to 950 μL of corn oil and mix evenly. The mixed solution should be used immediately for optimal results.
Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O	0.5mg/ml	Taking the 1 mL working solution as an example, add 50 μL of 10 mg/ml clarified DMSO stock solution to 400 μL of PEG300, mix evenly to clarify it; add 50 μL of Tween80 to the above system, mix evenly to clarify; then continue to add 500 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.

* <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.)

Preparing Stock Solutions

Biological Activity

Description

LDN-57444 is a reversible, competitive proteasome inhibitor for Uch-L1 with IC50 of 0.88 μM, 28-fold selectivity over isoform Uch-L3.

Targets

UCH-L1 ^[1]	UCH-L1 ^[1]	UCH-L3 ^[1]
0.4 μM(Ki)	0.88 μM	25 μM

In vitro

Treatment with 50 μM LDN-57444 for 24 h leads to 70% inhibition of the proteasome activity. LDN-57444 causes a significant and concentration-dependent decrease in cell viability at concentrations above 25 μM and the cell viability reduced to 61.81% at 50 μM. LDN-57444 is able to cause cell death through the apoptosis pathway by decreasing the activity of ubiquitin proteasome system and increasing the levels of highly ubiquitinated proteins, both of which can activate unfolded protein response. The apoptosis induced by LDN-57444 may be triggered by the activation of endoplasmic reticulum stress (ERS). ^[4]

In vivo

LDN-57444 causes dramatic alterations in synaptic protein distribution and spine morphology in vivo. Treatment with LDN also results in a rapid fall of Uch-L1 activity, but proteasome inhibition has no effect on cAMP levels over a period of several hours. ^[3]

Protocol (from reference)

Kinase Assay:^{^[1]}	HTS screen To start an assay, 0.5 μL of 5 mg/mL test compound (about 50 μM final reaction concentration) or DMSO control is aliquoted into each well. Both enzyme and substrate are prepared in UCH reaction buffer (50 mM Tris-HCl [pH 7.6], 0.5 mM EDTA, 5 mM DTT, and 0.5 mg/mL ovalbumin). 25 μL of 0.6 nM UCH-L1 is then added to each well except substrate control wells, followed by plate shaking for 45–60 s on an automatic shaker. The enzyme/compound mixture is incubated at room temperature for 30 min before 25 μL of 200 nM Ub-AMC is added to initiate the enzyme reaction. The reaction mixture (300 pM UCH-L1, 100 nM Ubiquitin-AMC with 2.5 μg test compound) is incubated at room temperature for 30 additional minutes prior to quenching the reaction by the addition of 10 μL 500 mM acetic acid per well. The fluorescence emission intensity is measured on a LJL Analyst using a coumarin filter set (ex = 365 nm, em = 450 nm) and is subtracted by the intrinsic compound fluorescence to reveal the enzyme activity. A DMSO control (0.5 μL of DMSO, 25 μL of UCH-L1, 25 μL of ubiquitin-AMC, 10 μL of acetic acid), enzyme control (25 μL of UCH-L1, 25 μL of buffer, 10 μL of acetic acid), substrate control (25 μL of buffer, 25 μL of ubiquitin-AMC, 10 μL of acetic acid), and inhibitor control (0.5 μL of ubiquitin aldehyde [100 nM stock], 25 μL of UCH-L1, 25 μL of ubiquitin-AMC, 10 μL of acetic acid) are also performed in each assay plate to ensure quality and reproducibility. Potential UCH-L1 inhibitors are selected if the compounds demonstrated greater than 60% inhibition compared to the controls. The UCH-L1 enzymatic reactions are manually repeated twice using the same protocol to confirm the results for the hit compounds from the primary robot-assisted screen.
Cell Assay:^{^[1]}	Cell lines H1299 Concentrations ~5 μM Incubation Time 24 h Method MTT assay

Kinase Assay:^{^[1]}

HTS screen
To start an assay, 0.5 μL of 5 mg/mL test compound (about 50 μM final reaction concentration) or DMSO control is aliquoted into each well. Both enzyme and substrate are prepared in UCH reaction buffer (50 mM Tris-HCl [pH 7.6], 0.5 mM EDTA, 5 mM DTT, and 0.5 mg/mL ovalbumin). 25 μL of 0.6 nM UCH-L1 is then added to each well except substrate control wells, followed by plate shaking for 45–60 s on an automatic shaker. The enzyme/compound mixture is incubated at room temperature for 30 min before 25 μL of 200 nM Ub-AMC is added to initiate the enzyme reaction. The reaction mixture (300 pM UCH-L1, 100 nM Ubiquitin-AMC with 2.5 μg test compound) is incubated at room temperature for 30 additional minutes prior to quenching the reaction by the addition of 10 μL 500 mM acetic acid per well. The fluorescence emission intensity is measured on a LJL Analyst using a coumarin filter set (ex = 365 nm, em = 450 nm) and is subtracted by the intrinsic compound fluorescence to reveal the enzyme activity. A DMSO control (0.5 μL of DMSO, 25 μL of UCH-L1, 25 μL of ubiquitin-AMC, 10 μL of acetic acid), enzyme control (25 μL of UCH-L1, 25 μL of buffer, 10 μL of acetic acid), substrate control (25 μL of buffer, 25 μL of ubiquitin-AMC, 10 μL of acetic acid), and inhibitor control (0.5 μL of ubiquitin aldehyde [100 nM stock], 25 μL of UCH-L1, 25 μL of ubiquitin-AMC, 10 μL of acetic acid) are also performed in each assay plate to ensure quality and reproducibility. Potential UCH-L1 inhibitors are selected if the compounds demonstrated greater than 60% inhibition compared to the controls. The UCH-L1 enzymatic reactions are manually repeated twice using the same protocol to confirm the results for the hit compounds from the primary robot-assisted screen.

Cell Assay:^{^[1]}

Cell lines
H1299
Concentrations
~5 μM
Incubation Time
24 h
Method
MTT assay

References

[4] Tan YY, et al. Mol Cell Biochem, 2008, 318(1-2), 109-115.

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Customer Product Validation

: Data from [Data independently produced by , , J Cell Mol Med, 2017, 1-16]

Selleck's LDN-57444 has been cited by 12 publications

Stem Cell Properties of Gastric Cancer Stem-Like Cells under Stress Conditions Are Regulated via the c-Fos/UCH-L3/β-Catenin Axis [ Mol Cells, 2023, 46(8):476-485]	PubMed: 37460253
Stem Cell Properties of Gastric Cancer Stem-Like Cells under Stress Conditions Are Regulated via the c-Fos/UCH-L3/β-Catenin Axis [ Mol Cells, 2023, 46(8):476-485]	PubMed: 37460253
UCHL1 regulates inflammation via MAPK and NF-κB pathways in LPS-activated macrophages [ Cell Biol Int, 2021, 10.1002/cbin.11662]	PubMed: 34288216
Long-term inhibition of UCHL1 decreases hypertension and retinopathy in spontaneously hypertensive rats [ J Int Med Res, 2021, 49(6):3000605211020641]	PubMed: 34130526
The Deubiquitinating Enzyme UCHL1 Promotes Resistance to Pemetrexed in Non-Small Cell Lung Cancer by Upregulating Thymidylate Synthase [ Theranostics, 2020, 15;10(13):6048-6060]	PubMed: 32483437
Deubiquitination of CD36 by UCHL1 promotes foam cell formation [ Cell Death Dis, 2020, 11(8):636]	PubMed: 32801299
Blockage of UCHL1 Activity Attenuates Cardiac Remodeling in Spontaneously Hypertensive Rats [ Hypertens Res, 2020, 10.1038/s41440-020-0486-1]	PubMed: 32541849
UCH-L1 inhibitor LDN-57444 hampers mouse oocyte maturation by regulating oxidative stress and mitochondrial function and reducing ERK1/2 expression [ Biosci Rep, 2020, 40(10)BSR20201308]	PubMed: 33030206
The deubiquitinating enzyme UCHL1 is a favorable prognostic marker in neuroblastoma as it promotes neuronal differentiation. [ J Exp Clin Cancer Res, 2018, 37(1):258]	PubMed: 30359286
The deubiquitinating enzyme UCHL1 negatively regulates the immunosuppressive capacity and survival of multipotent mesenchymal stromal cells [ Cell Death Dis, 2018, 9(5):459]	PubMed: 29686406

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SHIPPING AND STORAGE
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