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Purity: ≥98%
LDN-57444 (LDN57444; LDN 57444) is a potent, reversible, and competitive proteasome inhibitor for Uch-L1 (ubiquitin C-terminal hydrolase-L1) with the potential to treat PD-Parkinson's disease. It inhibits Uch-L1 with an IC50 of 0.88 μM, and exhibted 28-fold selectivity over closely related isoform Uch-L3. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is an intracellular protein abundantly expressed in neurons, and a mutation in UCH-L1 has been identified in familial Parkinson's disease. Besides Uch-L1, LDN 57444 also inhibits Uch-L3 with a higher IC50 value of 25μM.
| Targets |
UCH-L1(IC50=0.88 μM);UCH-L3(IC50=25 μM);UCH-L1(Ki=0.40 μM)
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| ln Vitro |
With an IC50 of 0.88 μM, LDN-57444 is a reversible, competitive inhibitor of UCH-L1 and also inhibits the activity of UCH-L3, with an IC50 of 25 μM[1].In mouse brain slices of the hippocampus, 70% of Uch activity is inhibited by LDN-57444 (LDN, 5 μM for 1 hour). After being exposed to 200 nM Aβ for two hours, LDN-57444 (5 μM) does not further reduce potentiation in APP/PS1 slices or in wt slices[2]. In SK-N-SH cells, ubiquitin-proteasome activity is dose-dependently inhibited by LDN-57444 (25-100 μM). Additionally, LDN-57444 (50 μM) causes endoplasmic reticulum stress, apoptosis, and spliced XBP-1 (XBP-1s, 48KD) expression in SK-N-SH cells[3].
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| ln Vivo |
When the mice are exposed to the context at 1, 7, 14, and 21 days after training, LDN-57444 (0.4 mg/kg, i.p.) reduces the positive effect of V-Uch-L1 and impairs contextual conditioning performance.
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| Enzyme Assay |
Initiating an assay involves aliquoting 0.5 μL of a 5 mg/mL test compound (such as LDN-57444, which has a final reaction concentration of approximately 50 μM) or DMSO control into each well. The UCH reaction buffer (50 mM Tris-HCl [pH 7.6], 0.5 mM EDTA, 5 mM DTT, and 0.5 mg/mL ovalbumin) is used to prepare the enzyme and substrate. Next, add 25 μL of 0.6 nM UCH-L1 to every well (apart from the substrate control wells) and shake the plate on an automatic shaker for 45–60 seconds. The enzyme reaction is started by adding 25 μL of 200 nM Ub-AMC after the enzyme/compound mixture has been incubated for 30 minutes at room temperature.After 30 more minutes of room temperature incubation, the reaction mixture (300 pM UCH-L1, 100 nM Ubiquitin-AMC with 2.5 μg test compound) is quenched by adding 10 μL of 500 mM acetic acid per well. Using a coumarin filter set (ex = 365 nm, em = 450 nm) on an LJL Analyst, the fluorescence emission intensity is measured. The intrinsic compound fluorescence is then subtracted to determine the enzyme activity.To ensure quality and reproducibility, each assay plate is also performed with a DMSO control (0.5 μL of DMSO, 25 μL of UCH-L1, 25 μL of ubiquitin-AMC, 10 μL of acetic acid), an enzyme control (25 μL of UCH-L1, 25 μL of buffer, 10 μL of acetic acid), a substrate control (25 μL of buffer, 25 μL of ubiquitin-AMC, 10 μL of acetic acid), and an 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). To validate the findings for the hit compounds from the primary robot-assisted screen, the UCH-L1 enzymatic reactions are manually repeated twice using the same protocol[1].
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| Cell Assay |
Using MTT, a quantitative colorimetric assay is used to measure cell viability. Following medication administration, SK-N-SH cells are cultured with 5 g/L MTT for 4 hours, and then 15 minutes are spent with DMSO added. At 570 nm, the absorption is measured with a micro-plate reader[3].
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| Animal Protocol |
Every animal is put into the conditioning chamber on its own. The shock intensity is increased by 0.1 mA to 0.7 mA over a 30-second interval as the electric current is progressively increased. The first obvious reaction to the shock (flinch), the first strong motor response (run/jump), and the first vocalized distress (scream) are used to assess an animal's behavior. The threshold for each animal's flinching, jumping, and screaming is measured by taking the average of the shock intensity at which that animal exhibits that type of behavioral response to the foot shock. During visible platform training, participants' visual, motor, and motivational skills are also put to the test by timing how long it takes to get to a visible platform submerged in a water-filled pool.A video tracking system records and analyzes the swimming speed as well as the time it takes to reach the platform. In the experiments where fear conditioning is tested in the presence of both LDN-57444 (LDN) and TAT fusion proteins, no differences are seen between the groups of mice. In order to determine when LDN-57444 should be administered, a number of pilot studies are conducted in which the inhibitor is injected intraperitoneally at various times (4 hours prior to, 1 hour prior to, 1 hour following, and 4 hours following) from the electric shock. There is no difference in the freezing of mice injected with vehicle or LDN-57444 during the training phase.[2].
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| References |
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| Molecular Formula |
C17H11CL3N2O3
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| Molecular Weight |
397.64
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| Exact Mass |
395.98
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| Elemental Analysis |
C, 51.35; H, 2.79; Cl, 26.75; N, 7.04; O, 12.07
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| CAS # |
668467-91-2
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| Related CAS # |
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| Appearance |
Solid powder
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| SMILES |
ClC1C([H])=C([H])C2=C(C=1[H])C(C(N2C([H])([H])C1C([H])=C(C([H])=C([H])C=1Cl)Cl)=O)=NOC(C([H])([H])[H])=O
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| InChi Key |
OPQRFPHLZZPCCH-PGMHBOJBSA-N
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| InChi Code |
InChI=1S/C17H11Cl3N2O3/c1-9(23)25-21-16-13-7-12(19)3-5-15(13)22(17(16)24)8-10-6-11(18)2-4-14(10)20/h2-7H,8H2,1H3/b21-16-
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| Chemical Name |
(Z)-3-(acetoxyimino)-5-chloro-1-(2,5-dichlorobenzyl)indolin-2-one.
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| Synonyms |
LDN57444; LDN 57444; LDN-57444
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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 : 11~25 mg/mL ( 27.66~62.87 mM)
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| Solubility (In Vivo) |
5% DMSO+Corn oil: 6mg/ml (Please use freshly prepared in vivo formulations for optimal results.)
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5148 mL | 12.5742 mL | 25.1484 mL | |
| 5 mM | 0.5030 mL | 2.5148 mL | 5.0297 mL | |
| 10 mM | 0.2515 mL | 1.2574 mL | 2.5148 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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