Home Proteases DUB inhibitor LDN-57444

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LDN-57444 DUB inhibitor

Cat.No.S7135

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

Chemical Structure

Molecular Weight: 397.64

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Quality Control

Batch: S713501 DMSO]11 mg/mL]false]Water]Insoluble]false]Ethanol]Insoluble]false Purity: 99.71%
99.71

Chemical Information, Storage & Stability

Molecular Weight 397.64 Formula

C17H11Cl3N2O3

 Storage (From the date of receipt)
CAS No. 668467-91-2 Download SDF Storage of Stock Solutions

Synonyms N/A Smiles CC(=O)ON=C1C2=C(C=CC(=C2)Cl)N(C1=O)CC3=C(C=CC(=C3)Cl)Cl

Solubility

In vitro
Batch:

DMSO : 11 mg/mL ( (27.66 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Water : Insoluble

Ethanol : Insoluble

Molarity Calculator

Mass Concentration Volume Molecular Weight

In vivo
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Mechanism of Action

Targets/IC50/Ki
UCH-L1 [1]
0.4 μM(Ki)
UCH-L1 [1]
0.88 μM
UCH-L3 [1]
25 μM
In vitro
Treatment with 50 μM LDN-57444 for 24 h leads to 70% inhibition of the proteasome activity. This compound 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. It 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 this chemical may be triggered by the activation of endoplasmic reticulum stress (ERS). [4]
Kinase Assay
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.
In vivo
LDN-57444 causes dramatic alterations in synaptic protein distribution and spine morphology in vivo. Treatment with this compound 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]
References

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