1. Cell Cycle/DNA Damage
  2. Deubiquitinase
  3. LDN-57444

LDN-57444 

Cat. No.: HY-18637 Purity: 97.73%
Handling Instructions

LDN-57444 is a reversible, competitive and site-directed inhibitor of ubiquitin C-terminal hydrolase L1 (UCH-L1), with an IC50 of 0.88 μM and a Ki of 0.40 μM; LDN-57444 also suppresses UCH-L3 activity, with an IC50 of 25 μM.

For research use only. We do not sell to patients.

LDN-57444 Chemical Structure

LDN-57444 Chemical Structure

CAS No. : 668467-91-2

Size Price Stock Quantity
Free Sample (0.5-1 mg)   Apply now  
10 mM * 1 mL in DMSO USD 125 In-stock
Estimated Time of Arrival: December 31
10 mg USD 114 In-stock
Estimated Time of Arrival: December 31
50 mg USD 384 In-stock
Estimated Time of Arrival: December 31
100 mg USD 624 In-stock
Estimated Time of Arrival: December 31
200 mg   Get quote  
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Top Publications Citing Use of Products

    LDN-57444 purchased from MCE. Usage Cited in: J Surg Oncol. 2017 Jun;115(8):932-940.

    p-ERK1/2 and ERK1/2 P-gp protein level are assessed by Western blot.

    LDN-57444 purchased from MCE. Usage Cited in: Biochem Biophys Res Commun. 2018 Mar 4;497(2):726-733.

    Drug screen of UCHL1 and the function of inhibitors in autophagy. Both LDN and NSC could inhibit UCHL1 hydrolase activity. mRFP-GFP-LC3 stable cell line is treated with 5 μM LDN or 10 μM NSC in the condition of DMSO or Torin1 respectively for 6 h. The number of LC3 puncta remarkably is raised both in LDN and NSC treatment cells compared to the control.

    LDN-57444 purchased from MCE. Usage Cited in: Biochem Biophys Res Commun. 2018 Mar 4;497(2):726-733.

    Drug screen of UCHL1 and the function of inhibitors in autophagy. Both LDN and NSC could inhibit UCHL1 hydrolase activity. Each cell lysate is analyzed by immunoblotting with anti p62 and LC3 antibodies.
    • Biological Activity

    • Protocol

    • Technical Information

    • Purity & Documentation

    • References

    Description

    LDN-57444 is a reversible, competitive and site-directed inhibitor of ubiquitin C-terminal hydrolase L1 (UCH-L1), with an IC50 of 0.88 μM and a Ki of 0.40 μM; LDN-57444 also suppresses UCH-L3 activity, with an IC50 of 25 μM.

    IC50 & Target

    IC50: 0.88 μM (UCH-L1), 25 μM (UCH-L3)[1]
    Ki: 0.40 μM (UCH-L1)[1]

    In Vitro

    LDN-57444 is a reversible, competitive inhibitor of UCH-L1, with an IC50 of 0.88 μM, and also suppresses UCH-L3 activity, with an IC50 of 25 μM[1]. LDN-57444 (LDN, 5 μM for 1 hr) inhibits 70% of Uch activity in hippocampal slices of the mouse brain. LDN-57444 (5 μM for 2 hr) does not reduce potentiation further in APP/PS1 slices or in wt slices exposed to 200 nM Aβ[2]. LDN-57444 (25-100 μM) inhibits ubiquitin-proteasome activity dose-dependently in SK-N-SH cells. LDN-57444 (50 μM) also induces apoptotic cell death, causes the endoplasmic reticulum stress and results in expression of spliced XBP-1(XBP-1s, 48KD) in SK-N-SH cells[3].

    In Vivo

    LDN-57444 (0.4 mg/kg, i.p.) blocks the beneficial effect of V-Uch-L1, and worsens contextual conditioning performance as the mice are exposed to the context at 1, 7, 14, and 21 days after training[2].

    Storage
    Powder -20°C 3 years
      4°C 2 years
    In solvent -80°C 6 months
      -20°C 1 month
    Solvent & Solubility
    In Vitro: 

    DMSO : 25 mg/mL (62.87 mM; Need ultrasonic)

    H2O : < 0.1 mg/mL (insoluble)

    Preparing
    Stock Solutions
    Concentration Solvent Mass 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
    *Please refer to the solubility information to select the appropriate solvent.
    In Vivo:
    • 1.

      Add each solvent one by one:  10% DMSO    90% corn oil

      Solubility: ≥ 2.5 mg/mL (6.29 mM); Clear solution

    *All of the co-solvents are provided by MCE.
    References
    Kinase Assay
    [1]

    To start an assay, 0.5 μL of 5 mg/mL test compound (including LDN-57444, 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. 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[1].

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    Cell Assay
    [3]

    Cell viability is measured by a quantitative colorimetric assay with MTT. After drug treatment SK-N-SH cells are incubated for 4 h with 5 g/L MTT and then DMSO is added for 15 min. The absorption is quantified at 570 nm using a micro-plate reader[3].

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    Animal Administration
    [2]

    Each animal is placed individually into the conditioning chamber. The electric current is gradually increased (0.1 mA for 1 sec. at 30 sec. intervals increasing the shock intensity by 0.1 mA to 0.7 mA). Animal behavior is evaluated for the first visible response to the shock (flinch), the first extreme motor response (run/jump), and the first vocalized distress (scream). Threshold to flinching, jumping, and screaming is quantified for each animal by averaging of the shock intensity at which each animal manifests a behavioral response of that type to the foot shock. Visual, motor, and motivation skills are also tested with visible platform training by measuring the time and the speed to reach a visible platform placed within a pool filled with water. Both time to reach the platform and swimming speed are recorded and analyzed with a video tracking system. No difference is observed among different groups of mice in the experiments in which fear conditioning is tested both in the presence of LDN-57444 (LDN) and TAT fusion proteins. To decide the time of administration of LDN-57444, a series of preliminary experiments are performed in which the inhibitor is injected intra-peritoneally at different intervals (4 hrs before, 1 hr before, 1 hr after and 4 hrs after) from the electric shock. During the training phase, there is no difference in the freezing of LDN-57444- or vehicle-injected mice[2].

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    References
    Molecular Weight

    397.64

    Formula

    C₁₇H₁₁Cl₃N₂O₃

    CAS No.

    668467-91-2

    SMILES

    O=C1N(CC2=CC(Cl)=CC=C2Cl)C3=C(C=C(Cl)C=C3)/C1=N/OC(C)=O

    Shipping

    Room temperature in continental US; may vary elsewhere

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    Product Name:
    LDN-57444
    Cat. No.:
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    Cat. No.: HY-18637