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| Targets |
EphA4 receptor ligand-binding domain (LBD) (Kd = 0.42 ± 0.02 μM by ITC; Ki = 0.65 μM by FPA) [1]
EphA3 LBD (Kd = 4.55 ± 1.09 μM by ITC) [1] EphA2 LBD (No binding, N.B.) [1] |
|---|---|
| ln Vitro |
In primary cortical neurons, 123C4 induced growth cone collapse in a concentration-dependent manner. At 500 nM, 10 μM, and 100 μM, the percentages of collapsed growth cones were significantly increased compared to control. Co-treatment with 100 μM 123C4 plus ephrinA1-Fc also showed effect. [1]
In primary hippocampal neurons, 123C4 stimulated EphA4 phosphorylation. Western blot analysis showed that 10 μM and 100 μM 123C4 increased the pEphA4/total EphA4 ratio. [1] In primary neurons, FITC-labeled 123C4 (10 μM and 100 μM) was internalized and co-localized with the early endosomal marker EEA1, indicating receptor endocytosis. The co-localization was dose-dependent. [1] In HEK293 cells stably transfected with EphA4, 123C4 did not cause receptor phosphorylation, but it antagonized ephrin-A5-induced phosphorylation. [1] |
| ln Vivo |
In SOD1(G93A) mutant mice (ALS model), daily intraperitoneal injection of 123C4 at 30 mg/kg starting from postnatal day 60 improved average lifespan from 134.3 ± 7.2 days (control) to 142.8 ± 6.9 days (p < 0.01, Student's t-test). [1]
Disease duration (from onset to endpoint) was prolonged from 28.2 ± 4.2 days (control) to 38.6 ± 5.7 days (p < 0.01) in 123C4-treated mice. [1] Kaplan-Meier survival plots showed increased survival of 123C4-treated mice compared to control mice. [1] |
| Enzyme Assay |
Fluorescence polarization competition assay (FPA): The EphA4 LBD was pre-incubated with test compounds at various concentrations in PBS (pH 7.2) for 10 min at room temperature, then FITC-labeled KYL peptide (KYLPYWPVLSSL) was added. After 30 min incubation, polarization values were measured at excitation/emission 480/535 nm. Ki values were determined by sigmoidal dose-response nonlinear regression. [1]
Isothermal titration calorimetry (ITC): Direct dissociation constants between 123C4 and EphA4 LBD (or mutants) were measured using an ITC200 instrument. Thermodynamic parameters (ΔH, -TΔS) were also determined. For wild-type EphA4 LBD, Kd = 0.42 ± 0.02 μM, ΔH = -11.06 ± 0.06 kcal/mol. [1] NMR spectroscopy: 2D [13C,1H]-HSQC spectra of 13C-methionine labeled EphA4 LBD (50 μM) were acquired at 700 MHz. Upon titration of 123C4 (10-50 μM), cross-peaks of Met60 and Met164 disappeared and new peaks appeared, indicating slow exchange on the NMR timescale. Off-rate estimated < 30 s⁻¹, giving Kd < 300 nM. Displacement of ephrin-B2 by 123C4 was also shown by NMR. [1] Molecular docking: Docking of 123C4 into EphA4 LBD (PDB ID 4M4R) was performed using Gold software. A low-energy binding pose showed the ligand adopting a type I β-turn with intramolecular hydrogen bond. [1] |
| Cell Assay |
Primary cortical or hippocampal neurons were isolated from C57BL/6 mice (P0-P1 or E18-E18). Tissues were dissected and treated with papain/DNase I solution for 20 min at 37°C, then mechanically dissociated and plated on poly-D-lysine/laminin-coated coverslips or plates. Cells were maintained in Neurobasal media with B27 supplement under 5% CO2/10% O2 at 37°C. [1]
Immunocytochemistry: Neurons were fixed with 4% PFA, permeabilized with 0.1% Triton X, blocked with 5% BSA. Axon growth cones were visualized with phalloidin-rhodamine, dendrites with anti-MAP2 antibody, early endosomes with anti-EEA1 antibody. Images were captured by confocal microscopy. [1] Western blot: Cells were lysed in lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 1 mM sodium pervanadate, protease inhibitors). Lysates were immunoprecipitated with anti-EphA4 antibody and protein-A agarose, then run on SDS-PAGE, transferred to nitrocellulose, blotted with HRP-conjugated anti-phosphotyrosine antibody, and developed with ECL reagent. Blots were stripped and re-probed for total EphA4. [1] Image analysis: For growth cone collapse, 100 images per treatment group (2-3 neurons/image) were collected. Axon was identified as longest MAP-2 positive neurite. Collapsed growth cones were counted. Co-localization of FITC-123C4 and EEA1 was quantified using ImageJ Colocalization Threshold plugin. Statistical analysis used two-tailed unpaired Student's t-test or ANOVA. [1] |
| Animal Protocol |
SOD1(G93A) mutant mice (B6.Cg-Tg(SOD1(G93A))1Gur/J) were bred. Equal number of same gender littermates were randomly divided into two groups (n=12 each, 6 male and 6 female). Mice were treated from postnatal day 60 until endpoints by intraperitoneal (i.p.) injections with either 123C4 or saline control. 123C4 was dissolved in normal saline at 2 mg/mL and sterilized by 0.2 μm filter. Dose: 30 mg/kg body weight daily. Animals were assessed daily for hind limb tremor and loss of splay reflex. Endpoint: mouse could not roll over within 10 s after being pushed onto its side. Disease onset and survival were compared using Kaplan-Meier method. [1]
Preliminary PK study: Single dose of 123C4 (5 mg/kg) administered intravenously in BalbC mice. Compound concentration in blood and brain was measured after 30 minutes. [1] Pilot toxicity study: Two SOD1(G93A) mice were treated with 30 mg/kg 123C4 daily i.p. from postnatal day 60 for several weeks. No adverse signs of toxicity were observed compared to vehicle control. [1] |
| ADME/Pharmacokinetics |
Solubility: 123C4 has solubility of about 100 μM in PBS. [1]
Plasma stability: Half-life (t1/2) > 60 min in rat plasma, significantly longer than KYL peptide (t1/2 ~10 min). [1] Brain penetration: After single intravenous dose of 5 mg/kg in BalbC mice, approximately 60% of 123C4 reached the brain at 30 min, with concentrations similar to its Kd value for EphA4 LBD. [1] |
| Toxicity/Toxicokinetics |
In a pilot study, SOD1(G93A) mice treated with daily i.p. injections of 123C4 at 30 mg/kg for several weeks showed no adverse signs of toxicity compared to control mice receiving vehicle only. [1]
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| References | |
| Additional Infomation |
123C4 is a potent and selective EphA4 LBD ligand developed for ALS (amyotrophic lateral sclerosis). It acts as an EphA4 agonist in primary cortical neurons, inducing receptor phosphorylation and endocytosis. The proposed mechanism may involve reverse signaling and internalization of EphA4, preventing engagement with ephrin-B2 on reactive astrocytes. The compound also shows efficacy in delaying ALS progression in SOD1(G93A) mice. Potential applications in Alzheimer's disease, spinal cord injury, brain injury, and certain cancers are suggested. 123C4 is more stable in plasma and has lower molecular weight compared to previously reported peptides. [1]
|
| Molecular Formula |
C43H47CLN8O6
|
|---|---|
| Molecular Weight |
807.336288690567
|
| Exact Mass |
806.33
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| CAS # |
2034159-30-1
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| PubChem CID |
122661600
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| Appearance |
White to off-white solid powder
|
| LogP |
4.3
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| Hydrogen Bond Donor Count |
8
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
19
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| Heavy Atom Count |
58
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| Complexity |
1330
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| Defined Atom Stereocenter Count |
3
|
| SMILES |
ClC1C=CC(=CC=1)C[C@@H](C(N[C@H](C(NCCC1=CNC2C=CC(=CC1=2)OC)=O)CC1C=CN=CC=1)=O)NC([C@H](CC1=CNC2C=CC(=CC1=2)O)NC(CCCN)=O)=O
|
| InChi Key |
SRCCZHZOKZJHOK-IGMOWHQGSA-N
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| InChi Code |
InChI=1S/C43H47ClN8O6/c1-58-32-9-11-36-34(23-32)28(24-48-36)14-18-47-41(55)37(20-27-12-16-46-17-13-27)51-42(56)38(19-26-4-6-30(44)7-5-26)52-43(57)39(50-40(54)3-2-15-45)21-29-25-49-35-10-8-31(53)22-33(29)35/h4-13,16-17,22-25,37-39,48-49,53H,2-3,14-15,18-21,45H2,1H3,(H,47,55)(H,50,54)(H,51,56)(H,52,57)/t37-,38-,39-/m0/s1
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| Chemical Name |
4-amino-N-[(2S)-1-[[(2S)-3-(4-chlorophenyl)-1-[[(2S)-1-[2-(5-methoxy-1H-indol-3-yl)ethylamino]-1-oxo-3-pyridin-4-ylpropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(5-hydroxy-1H-indol-3-yl)-1-oxopropan-2-yl]butanamide
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 : ~100 mg/mL (~123.86 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.10 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 (3.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2386 mL | 6.1932 mL | 12.3864 mL | |
| 5 mM | 0.2477 mL | 1.2386 mL | 2.4773 mL | |
| 10 mM | 0.1239 mL | 0.6193 mL | 1.2386 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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