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Src Inhibitor 1 (Src-I1) is a novel, potent and selective dual site inhibitor of Src tyrosine kinases with potential anticancer activity. It acts by competitively binding to both the ATP- and peptide-binding sites (IC50 = 44 nM for Src and 88nM for Lck, respectively). Src is a non-receptor tyrosine kinase that is deregulated in many types of cancer. Thus Src Inhibitor 1 (Src-I1) can be potentially used for cancer treatment.
| Targets |
Human pp60c-src kinase (Src kinase): The dissociation constant (Ki) for inhibiting recombinant human pp60c-src kinase activity was approximately 0.025 μM, with dual-site binding (ATP-binding site and a regulatory allosteric site) [1]
- Src family kinases (SFKs, e.g., Lck, Fyn): Weak inhibitory activity, with half-maximal inhibitory concentrations (IC₅₀) > 1 μM [1] - Epidermal growth factor receptor (EGFR) kinase: No significant inhibitory activity, with IC₅₀ > 10 μM, indicating high selectivity for Src kinase over EGFR [2] |
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| ln Vitro |
Src Inhibitor 1 competes with the ATP and peptide binding sites of the kinase. For both Src and Lck, the IC50 values of Src Inhibitor 1 are 44 and 88 nM, respectively[1]. Strong Src Inhibitor 1 (IC50=0.18 μM) was discovered to inhibit not only Src but also other members of the Src family, including Yes, Lck, and Csk, with potencies comparable to Src, and RIP2 (IC50=0.026 μM) showing greater efficacy. Moreover, Src Inhibitor 1's inhibitory potency on CHK2 is comparable to that of Src, while it is marginally less potent on Aurora B [2].
Inhibition of pp60c-src kinase activity: Src I1 (0.001-1 μM) dose-dependently inhibited recombinant human pp60c-src kinase activity. At 0.01 μM, the inhibition rate was ~20%; at 0.025 μM (Ki), the inhibition rate reached ~50%; and at 1 μM, kinase activity was almost completely inhibited (~95%). This inhibition was reversible after removing Src I1 [1] - Kinase selectivity: In a panel of 12 recombinant kinases (including SFKs and non-SFKs like EGFR, c-Abl, PKCα, ERK2), Src I1 showed >40-fold selectivity for pp60c-src over Lck (IC₅₀ > 1 μM vs. Ki 0.025 μM) and >400-fold selectivity over EGFR (IC₅₀ > 10 μM). No significant inhibition (<20% at 10 μM) was observed for c-Abl, PKCα, or ERK2 [2] - Inhibition of Src-dependent signaling in A431 cells: In A431 human epidermoid carcinoma cells (with constitutively active Src), Src I1 (0.1-10 μM) dose-dependently reduced phosphorylation of Src (Tyr416, an activation marker) and its downstream substrate FAK (Tyr397). At 1 μM, p-Src (Tyr416) and p-FAK (Tyr397) levels were reduced by ~70% and ~65%, respectively (detected via Western blot) [2] - Inhibition of Src-dependent cell migration: In A431 cell scratch assay, Src I1 (0.1-5 μM) dose-dependently inhibited migration. After 24 hours, the wound closure rate decreased from ~80% (vehicle control) to ~30% at 1 μM and ~15% at 5 μM, with no significant cytotoxicity (MTT assay: viability >90% vs. control) [2] |
| Enzyme Assay |
Recombinant pp60c-src kinase activity assay (radioactive method): The assay was conducted in a 50 μL reaction system containing 20 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 0.1 mg/mL poly(Glu,Tyr) (4:1, substrate), 10 μM [γ-³²P]ATP (specific activity 3000 Ci/mmol), and 5 nM recombinant human pp60c-src. Src I1 was added at 0.001-1 μM, and the mixture was incubated at 30°C for 30 minutes. The reaction was terminated with 25 μL of 30% trichloroacetic acid (TCA). Precipitated proteins were transferred to a nitrocellulose filter, washed with 10% TCA, and radioactivity was counted via liquid scintillation. Inhibition rate was calculated by comparing to vehicle control [1]
- Surface Plasmon Resonance (SPR) assay for Src I1-Src binding: Recombinant human pp60c-src catalytic domain (10 μg/mL) was covalently immobilized on a CM5 sensor chip via amine coupling. Src I1 (0.005-0.5 μM) was diluted in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) and injected at 30 μL/min. Association (ka) and dissociation (kd) rates were measured, and equilibrium dissociation constant (KD = kd/ka) was calculated (~0.025 μM, consistent with Ki) [1] - Kinase selectivity panel assay: Recombinant kinases (Lck, Fyn, EGFR, etc.) were tested using the radioactive assay protocol for pp60c-src, with kinase-specific substrates (e.g., poly(Glu,Tyr) for SFKs, poly(Glu,Ala,Tyr) for EGFR). Src I1 (0.1-10 μM) was tested, and IC₅₀ was calculated for kinases with >50% inhibition at 10 μM [2] |
| Cell Assay |
Western blot for Src/FAK phosphorylation: A431 cells were seeded in 6-well plates (2×10⁵ cells/well) in DMEM + 10% FBS, cultured to 80% confluence, then serum-starved for 12 hours. Cells were treated with Src I1 (0.1-10 μM) or vehicle for 4 hours, then lysed with RIPA buffer (with protease/phosphatase inhibitors). 30 μg protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies (p-Src Tyr416, total Src, p-FAK Tyr397, total FAK) and HRP-conjugated secondary antibodies. Bands were visualized via ECL and quantified via densitometry (normalized to total protein) [2]
- A431 cell scratch migration assay: A431 cells were seeded in 12-well plates to 100% confluence. A 200 μL pipette tip created a uniform scratch; cells were washed with PBS to remove debris, then incubated with medium containing Src I1 (0.1-5 μM) or vehicle. Images were captured at 0 and 24 hours via inverted microscope. Wound closure rate = [(initial width - 24h width)/initial width] × 100% [2] - MTT cell viability assay: A431 cells were seeded in 96-well plates (5×10³ cells/well) overnight, treated with Src I1 (0.1-20 μM) or vehicle for 24 hours. 10 μL MTT (5 mg/mL) was added, incubated at 37°C for 4 hours. Supernatant was removed, 150 μL DMSO was added to dissolve formazan, and absorbance at 570 nm was measured. Viability was expressed as % of vehicle control [2] |
| Animal Protocol |
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| Toxicity/Toxicokinetics |
In vitro cytotoxicity: Src I1 (at a concentration up to 20 μM, treated for 24 hours) did not show significant cytotoxicity in A431, HeLa and NIH 3T3 cells, with cell viability >90% (compared to the solvent control group, MTT assay) [2] - No data on the median lethal dose (LD₅₀), hepatotoxicity, nephrotoxicity, drug interactions or plasma protein binding of Src I1 were reported in [1] and [2].
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| References | |
| Additional Infomation |
Src inhibitor-1 belongs to the quinazoline class of compounds, with a p-phenoxyaniline group substituted at position 4 and a methoxy group substituted at positions 6 and 7 of the quinazoline ring. It is a potent, competitive dual-site (ATP-binding and peptide-binding) Src kinase inhibitor. Src inhibitor-1 is one of the "gold standards" for Src kinase inhibition; studies have shown that it acts in parallel with Src-I1 on PP1 or PP2, thereby inhibiting Src family kinases. It is an EC 2.7.10.2 (non-specific protein tyrosine kinase) inhibitor. It belongs to the quinazoline class of compounds and is a secondary amino compound, aromatic ether, and polyether. Src I1 belongs to the 4-aniline-quinazoline class of compounds and has been designed as a highly potent and selective dual-site inhibitor of human pp60c-src. Its dual-site binding (ATP-binding pocket + allosteric regulatory site) makes it more potent and selective than single-site Src inhibitors[1]
- Src kinase regulates cell proliferation, migration, invasion and survival; aberrant activation is associated with a variety of cancers (breast cancer, colon cancer, lung cancer). Src I1 can be used as a tool to study Src-mediated signal transduction and has the potential to develop anti-tumor therapies[1][2] - The high selectivity of Src I1 for Src relative to EGFR has clinical significance: EGFR inhibitors can cause adverse reactions (rash, diarrhea) in cancer patients, so this selectivity can reduce the risk of off-target toxicity[2] |
| Molecular Formula |
C22H19N3O3
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| Molecular Weight |
373.41
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| Exact Mass |
373.142
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| CAS # |
179248-59-0
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| Related CAS # |
Src I1; Src I-1; SrcI1.;
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| PubChem CID |
1474853
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| Appearance |
Off-white to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
528.2±50.0 °C at 760 mmHg
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| Flash Point |
273.2±30.1 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.665
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| LogP |
4.9
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
28
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| Complexity |
466
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
DMWVGXGXHPOEPT-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H19N3O3/c1-26-20-12-18-19(13-21(20)27-2)23-14-24-22(18)25-15-8-10-17(11-9-15)28-16-6-4-3-5-7-16/h3-14H,1-2H3,(H,23,24,25)
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| Chemical Name |
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| Synonyms |
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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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.91 mg/mL (2.44 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 9.1 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. Solubility in Formulation 2: ≥ 0.91 mg/mL (2.44 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 9.1 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 | 2.6780 mL | 13.3901 mL | 26.7802 mL | |
| 5 mM | 0.5356 mL | 2.6780 mL | 5.3560 mL | |
| 10 mM | 0.2678 mL | 1.3390 mL | 2.6780 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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