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| Targets |
- DPH targets c-Abl kinase (binds to its myristoyl binding site as an activator), with an EC50 of ~1.2 μM for c-Abl activation and a dissociation constant (KD) of ~0.8 μM. [1]
- DPH targets Abl2/Arg kinase (acts as an activator). [2] |
|---|---|
| ln Vitro |
In contrast to the previously discovered allosteric c-Abl with GNF-2, DPH binds to the myristoyl binding site and uses steric hindrance to stop the αI helix from forming its bent conformation. The myristyl acyl binding site is another area in which Silhouette interacts. The first tool chemical for c-Abl activation that penetrates cells is DPH [1].
- c-Abl Kinase Activation (Recombinant Enzyme & K562 Cells): 1. Recombinant c-Abl assay: DPH (0.1–10 μM) dose-dependently activated c-Abl kinase. At 1.2 μM, it achieved 50% maximum activation, confirmed by increased phosphorylation of substrate CrkL via [γ-³²P]ATP autoradiography. At 10 μM, DPH enhanced CrkL phosphorylation by ~2.5-fold vs. vehicle control. [1] 2. K562 cell assay: DPH (1–10 μM) treatment for 30 minutes increased CrkL phosphorylation (Tyr207) by ~20%–80% (western blot) without altering total c-Abl protein levels. [1] - Abl2/Arg-Mediated Neuroprotection (Primary Cortical Neurons): 1. Dendrite loss prevention: Primary cortical neurons were pretreated with DPH (0.5–5 μM) for 1 hour, then exposed to dexamethasone (1 μM) for 48 hours. DPH dose-dependently reversed dendrite loss: 5 μM DPH increased total dendrite length by ~60% and branch points by ~50% (MAP2 immunofluorescence) vs. dexamethasone-only group. [2] 2. Neuronal viability: DPH (0.5–5 μM) alone had no effect on neuron viability (MTT assay), but restored dexamethasone-induced viability reduction (from ~60% to ~90% of control). [2] |
| ln Vivo |
- Neuroprotection in Dexamethasone-Treated Mice:
1. 8-week-old C57BL/6 mice were divided into 3 groups: Control, Dexamethasone (DEX), DEX + DPH. [2] 2. DEX group: Intraperitoneal (i.p.) injection of DEX (10 mg/kg) once daily for 5 days. [2] 3. DEX + DPH group: I.p. injection of DPH (5 mg/kg) 30 minutes before each DEX injection (same DEX schedule). [2] 4. Behavioral tests: Open Field Test showed DPH increased central zone time by ~40% (reduced anxiety); Novel Object Recognition Test showed DPH increased discrimination index by ~35% (improved memory) vs. DEX group. [2] 5. Brain analysis: DPH increased hippocampal CA1 neuron dendrite length by ~50% and branch points by ~45% (MAP2 staining), and restored Abl2/Arg activity (increased phospho-Abl substrate levels by ~60%, western blot) vs. DEX group. [2] |
| Enzyme Assay |
- c-Abl Kinase Activity Activation Assay: Recombinant human c-Abl kinase (wild-type) was incubated with ATP (10 μM), CrkL peptide substrate (50 μM), and DPH (0.1–10 μM) in kinase buffer (pH 7.5) at 30°C for 30 minutes. The reaction was terminated with SDS sample buffer, and phosphorylated CrkL was quantified via [γ-³²P]ATP autoradiography to measure kinase activity. [1]
- c-Abl Myristoyl Binding Site Interaction Assay: Isothermal Titration Calorimetry (ITC) was used to measure DPH binding to c-Abl’s myristoyl site. Recombinant c-Abl kinase domain (10 μM) was titrated with DPH (100 μM) in buffer at 25°C. A KD of ~0.8 μM confirmed direct, specific binding to the target site. [1] - Abl2/Arg Kinase Activity Activation Assay: Recombinant human Abl2/Arg kinase was incubated with ATP (10 μM), Abl peptide substrate (50 μM), and DPH (0.5–5 μM) in kinase buffer (pH 7.5) at 30°C for 30 minutes. Phosphorylated substrate was detected via Fluorescence Resonance Energy Transfer (FRET), showing 5 μM DPH increased Abl2/Arg activity by ~70% vs. vehicle control. [2] |
| Cell Assay |
- c-Abl Activation in K562 Cells:
1. K562 cells were seeded in 6-well plates (2×10⁶ cells/well) and cultured in RPMI 1640 medium (10% FBS) at 37°C (5% CO₂) for 24 hours. [1] 2. Cells were treated with DPH (1–10 μM) for 30 minutes (vehicle: DMSO). [1] 3. Cells were lysed, total protein was extracted, and western blot was performed with anti-phospho-CrkL (Tyr207) and anti-total c-Abl antibodies to assess c-Abl activation. [1] - Neuroprotection in Primary Cortical Neurons: 1. Primary cortical neurons (E18 mouse embryos) were seeded on poly-L-lysine-coated coverslips (5×10⁴ cells/coverslip) and cultured in neurobasal medium (B27 supplement) for 7 days. [2] 2. Neurons were pretreated with DPH (0.5–5 μM) for 1 hour, then treated with dexamethasone (1 μM) for 48 hours (controls: vehicle or dexamethasone alone). [2] 3. Neurons were fixed, immunostained with anti-MAP2 (dendrite marker) and DAPI (nuclei), imaged via confocal microscopy, and dendrite length/branch points were quantified with image analysis software. [2] |
| Animal Protocol |
- Dexamethasone-Induced Neurotoxicity Mouse Model:
1. Animal preparation: 8-week-old male C57BL/6 mice (20–22 g) were acclimated for 1 week (free access to food/water). [2] 2. Drug preparation: DPH was dissolved in DMSO (10% v/v) and diluted with saline; DEX was dissolved in saline. [2] 3. Administration: - Control: I.p. injection of saline (equal volume to drug groups) once daily for 5 days. [2] - DEX: I.p. injection of DEX (10 mg/kg) once daily for 5 days. [2] - DEX + DPH: I.p. injection of DPH (5 mg/kg) 30 minutes before DEX (10 mg/kg) once daily for 5 days. [2] 4. Sample collection: Mice were euthanized 24 hours after the last injection; hippocampi were dissected for immunofluorescence (dendrites) and western blot (Abl2/Arg activity). [2] |
| References |
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| Additional Infomation |
c-Abl activation mechanism: DPH binds to the myristoyl binding site of c-Abl, which normally maintains the autoinhibitory conformation of c-Abl. This binding disrupts the autoinhibition and induces c-Abl activation and phosphorylation of its downstream substrates (e.g., CrkL). [1]
- Abl2/Arg-mediated neuroprotective mechanism: DPH activates Abl2/Arg, promoting actin cytoskeleton remodeling—essential for maintaining dendrites. This reverses dexamethasone-induced actin depolymerization, prevents dendritic loss, and improves neuronal function/behavior. [2] - Cell permeability: DPH is cell permeable, as demonstrated by its ability to activate intracellular c-Abl (K562 cells) and Abl2/Arg (primary neurons) without transfection or membrane permeability. [1][2] |
| Molecular Formula |
C18H13N4O2F
|
|---|---|
| Molecular Weight |
336.31982
|
| Exact Mass |
336.102
|
| CAS # |
484049-04-9
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| PubChem CID |
660311
|
| Appearance |
White to off-white solid powder
|
| LogP |
2.474
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
4
|
| Rotatable Bond Count |
3
|
| Heavy Atom Count |
25
|
| Complexity |
517
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
MPQWYPLPWGUMJE-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C18H13FN4O2/c19-12-8-6-11(7-9-12)15-14(16-17(24)21-18(25)20-16)10-23(22-15)13-4-2-1-3-5-13/h1-10,16H,(H2,20,21,24,25)
|
| Chemical Name |
5-[3-(4-fluorophenyl)-1-phenylpyrazol-4-yl]imidazolidine-2,4-dione
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~50 mg/mL (~148.67 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.43 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.43 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (7.43 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9734 mL | 14.8668 mL | 29.7336 mL | |
| 5 mM | 0.5947 mL | 2.9734 mL | 5.9467 mL | |
| 10 mM | 0.2973 mL | 1.4867 mL | 2.9734 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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