| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg | |||
| 250mg | |||
| 500mg | |||
| Other Sizes |
Purity: ≥98%
| Targets |
EphA2 (Kd = 12 nM)
EphA2 (receptor tyrosine kinase); RET (tyrosine kinase) - reported to inhibit RET in other models (REF 25). |
|---|---|
| ln Vitro |
ALW-II-41-27 inhibits Ba/F3 cells transformed with Tel fusions of EphA3, Kit, Fms, KDR, FLT1, FGR, Src, Lyn, Bmx, and Bcr-Abl with an EC50 below 500 nM. Bcr-Abl and ALW-II-41-27 show cross-reactivity. Inhibiting b-raf, CSF1R, DDR1, DDR2, EphA2, EphA5, EphA8, EphB1, EphB2, EphB3, Frk, Kit, Lck, p38α, p38β, PDGFRα, PDGFRβ, Raf1, and numerous other kinases, ALW-II-41-27 shows how the addition of a thiophene group can significantly affect kinase selectivity[1].
ALW-II-41-27 significantly impaired growth of MDA-MB-231 cells in MTT assays in a dose-dependent manner after 72 h in culture (REF 6a). Treatment with ALW-II-41-27 reduced levels of c-Myc and cyclin E2 while elevating p27/KIP1 in MDA-MB-231 relative to control (REF 6b). ALW-II-41-27 significantly reduced EphA2 phosphorylation at tyrosine 588 in MDA-MB-231 cells (REF 6b). Treatment with ALW-II-41-27 reduced c-Myc, P-Rb, cyclin E2 and enhanced p27/KIP1 expression in wild-type C3-TAg tumor cells relative to NG-25 control (REF 6d). ALW-II-41-27 significantly reduced colony size in wild-type C3-TAg three-dimensional spheroid culture, mimicking the effects of EphA2-deficiency; the impact on EphA2-deficient C3-TAg lines was not significant, suggesting minimal off-target effects (REF 6f, 6g). ALW-II-41-27 significantly reduced basal and ephrin-A1-Fc induced tyrosine phosphorylation of EphA2 in C3-TAg cells (REF 6e). |
| ln Vivo |
ALW-II-41-27 treatment significantly reduced tumor volume over time in two independent TNBC PDX lines (HCI-001 and HCI-010) within 3-5 days after treatment, and persisted through day 7 (REF 6i; S7a).
Tumors from animals treated with ALW-II-41-27 displayed a significant decrease in proliferation (Ki67) (REF 6j; S7c) with no change in apoptosis (S7d, S7e). ALW-II-41-27-treated tumors showed decreased protein expression of c-Myc and increased expression of p27/KIP1 in tumor sections versus controls (S7f). PDX tumor lysates (HCC-010) showed a significant decrease in EphA2 phosphorylation in animals that received two doses of ALW-II-41-27 versus control-treated animals (S7g). |
| Enzyme Assay |
For evaluation of EphA2 phosphorylation: C3-TAg cells were starved and stimulated with ephrin-A1-Fc (1 μg/ml) for 0, 15 and 30 min in the presence or absence of ALW-II-41-27 or NG-25 control. EphA2 was immunoprecipitated from 500 μg cell lysate using anti-EphA2 antibodies (C-20/SC-924 and D7) followed by A/G PLUS-Agarose beads. Immunoprecipitated products were probed for tyrosine phosphorylation using mixed PY99 and PY20 antibodies and for EphA2 (REF 6e).
For target validation in PDX tumors: An independent cohort of HCC-010 PDX tumor-bearing animals was treated with two doses (at 0 and 8 h) with vehicle versus ALW-II-41-27. Tumors were collected 1 h following final treatment. Tumor lysates were generated and EphA2 immunoprecipitated. Lysates and IP products were probed for phospho-EphA2 (Tyr588), and blots were stripped and reprobed for total EphA2 (S7g). |
| Cell Assay |
In order to ascertain the function of EPHA2, myogenic precursors were exposed to ALW-II-41-27 (0.5 μM) for a duration of 12 hours in GM, after which they underwent myogenic differentiation.
MTT growth assay: MDA-MB-231 and wild-type C3-TAg tumor cells were grown in the presence of increasing concentrations (0.5-1 μM) of ALW-II-41-27 versus analog control NG-25. Growth was assessed by MTT assay after 72 h in culture. 3×10^3 cells were plated into 96-well plates with at least four replicates in growth media. MTT reagent was added and plates were read using a multi-mode reader. Cell growth or survival was normalized to control cells (REF 6a, 6c). Three-dimensional spheroid culture: Wild-type and EphA2-deficient C3-TAg cells were grown in three-dimensional spheroid culture on Matrigel for 7 days in the presence of 1 μM ALW-II-41-27 versus analog control NG-25. Colony size was measured in photomicrographs using NIH Image J software (REF 6f, 6g). Immunoblot analysis: Cells were treated with ALW-II-41-27 or NG-25 control, lysates (25-50 μg) were collected, and immunoblot analyses were performed using antibodies against P-EphA2 Tyr588, c-Myc, p27/KIP1, cyclin E2, P-Rb, and β-actin. Signals were detected using chemiluminescence or LI-COR Odyssey detection systems (REF 6b, 6d). EphA2 phosphorylation assay in C3-TAg cells: Cells were starved and stimulated with ephrin-A1-Fc (1 μg/ml) for indicated times in the presence or absence of 1 μM NG-25 or ALW-II-41-27. EphA2 was immunoprecipitated and products probed for tyrosine phosphorylation (REF 6e). |
| Animal Protocol |
PDX studies: 2 mm³ portions of cryopreserved tissue (HCI-001 and HCI-010 TNBC-derived lines) were implanted in the surgically cleared inguinal mammary fat pads of 3-4-week-old NOD-SCID recipient female mice. Tumors were allowed to engraft and grow to at least 200 mm³, then randomized into cohorts and injected intraperitoneally twice daily for 1 week with vehicle control or 15 mg/kg ALW-II-41-27 in 10% 1-methyl-2-pyrrolidinone and 90% polyethylene glycol 300. Tumors were measured four times weekly and tumor volume calculated as volume = length × width² × 0.52. At the end of the treatment period, tumors were collected and analyzed for proliferation (Ki67), apoptosis (cleaved caspase 3) and microvascular density (CD31) (REF 6h-6j; S7).
Independent cohort for target validation: HCC-010 PDX tumor-bearing animals were treated with two doses (at 0 and 8 h) with vehicle versus ALW-II-41-27 (15 mg/kg). Tumors were collected 1 h following final treatment to validate EphA2 targeting (S7g). |
| References | |
| Additional Infomation |
ALW-II-41-27 (CAS#: 1186206-79-0) is a small molecule tyrosine kinase inhibitor of EphA2. Previous studies from Nathanael Gray's laboratory tested ALW-II-41-27 for affinity and inhibition of EphA2 kinase activity relative to other Eph RTK family members and other kinases, and exhibited marked specificity for EphA2 relative to other kinases (REF 22, 23).
A more recent study reported that ALW-II-41-27 inhibits RET tyrosine kinase activity and function in RET-transformed fibroblasts, HEK293 cells, and in primary tumor cells and cell lines from human thyroid cancer (REF 25). The authors tested the potential off-target effects of ALW-II-41-27 in an EphA2-null model (EphA2-deficient C3-TAg cells) and found that the impact on EphA2-deficient lines was not significant, suggesting minimal off-target effects for this inhibitor (REF 6f, 6g). ALW-II-41-27 was purchased from MedChem Express (REF Materials and Methods). The inhibitor was previously shown to effectively inhibit EphA2 function in non-small cell lung cancer models (REF 22, 23). |
| Molecular Formula |
C32H32F3N5O2S
|
|---|---|
| Molecular Weight |
607.6890
|
| Exact Mass |
607.223
|
| Elemental Analysis |
C, 63.25; H, 5.31; F, 9.38; N, 11.52; O, 5.27; S, 5.28
|
| CAS # |
1186206-79-0
|
| Related CAS # |
1186206-79-0
|
| PubChem CID |
42628503
|
| Appearance |
White to light yellow solid powder
|
| LogP |
7.423
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
9
|
| Rotatable Bond Count |
8
|
| Heavy Atom Count |
43
|
| Complexity |
933
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
S1C([H])=C([H])C([H])=C1C1C([H])=NC([H])=C(C=1[H])C(N([H])C1=C(C([H])([H])[H])C([H])=C([H])C(=C1[H])C(N([H])C1C([H])=C([H])C(=C(C(F)(F)F)C=1[H])C([H])([H])N1C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])[H])C([H])([H])C1([H])[H])=O)=O
|
| InChi Key |
HYWXBDQAYLPMIX-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C32H32F3N5O2S/c1-3-39-10-12-40(13-11-39)20-23-8-9-26(17-27(23)32(33,34)35)37-30(41)22-7-6-21(2)28(16-22)38-31(42)25-15-24(18-36-19-25)29-5-4-14-43-29/h4-9,14-19H,3,10-13,20H2,1-2H3,(H,37,41)(H,38,42)
|
| Chemical Name |
N-[5-[[4-[(4-ethylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]carbamoyl]-2-methylphenyl]-5-thiophen-2-ylpyridine-3-carboxamide
|
| Synonyms |
ALWII-41-27; ALW-II-41-27; ALW II-41-27; ALW II-4127; ALW-II-4127; ALWII-4127
|
| 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: ~100 mg/mL (~164.6 mM)
Ethanol: ~100 mg/mL (~164.6 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.11 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 (4.11 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 (4.11 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 | 1.6456 mL | 8.2279 mL | 16.4558 mL | |
| 5 mM | 0.3291 mL | 1.6456 mL | 3.2912 mL | |
| 10 mM | 0.1646 mL | 0.8228 mL | 1.6456 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.
|
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
COA
