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APS-2-79 (APS-279) is a potent antagonist of MAPK (mitogen-activated protein kinase) with antitumor activity. It influences KSR-dependent MAPK signaling by inhibiting RAF heterodimerization and the conformational alterations necessary for phosphorylating and activating KSR-bound MEK. KSR-induced phosphorylation of MEK and ERK was found to be suppressed by APS-2-79. The direct targeting of KSR as an active site mutant by APS-2-79, which has been shown to stimulate KSR-based MAPK outputs independently of ATP-binding, was necessary for the MAPK signaling suppression that APS-2-79 caused. This direct targeting of KSR could significantly reduce the activity of APS-2-79. Additionally, the addition of APS-2-79 significantly decreased the RAF-caused KSR-stimulated MEK phosphorylation. APS-2-79 may be used as a treatment option for Ras-driven cancers and has the potential to enhance the effectiveness of current MAPK inhibitors.
| Targets |
MEK1; KSR2 (IC50 = 120 nM)
KSR2 (IC₅₀ values not provided in the literature, but it binds to the ATP-binding pocket of KSR2) [1] |
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| ln Vitro |
APS-2-79 (5 μM) suppresses KSR-stimulated MEK and ERK phosphorylation in 293H cells[1].
APS-2-79 (1 μM) enhances the clinical MEK inhibitor trametinib's effectiveness in cancer cell lines with K-Ras mutations.[1] APS-2-79 stabilizes the inactive state of KSR, antagonizes RAF heterodimerization, and inhibits the conformational changes required for phosphorylation and activation of KSR-bound MEK [1] In ATPᵇⁱᵒᵗⁱⁿ probe-labelling assays, APS-2-79 competes with ATP for binding to KSR2 in purified KSR2–MEK1 complexes [1] APS-2-79 inhibits BRAF and CRAF-mediated phosphorylation of MEK in a KSR-dependent manner; it effectively suppresses phosphorylation of KSR2-bound MEK but has little effect on free MEK [1] In 293H cells co-expressing full-length KSR–Flag and MEK1–GFP, APS-2-79 (5 μM, 2 h treatment) impedes KSR-stimulated MAPK signalling, reducing the phosphorylation levels of MEK (Ser218/Ser222) and ERK (Thr202/Tyr204); this inhibitory effect is not observed in cells expressing the KSR(A690F) mutant [1] APS-2-79 does not affect BRAF(V600E)-induced MAPK activation in 293H cells [1] When combined with MEK inhibitors (trametinib, binimetinib, PD0325901, AZD6244), APS-2-79 shows synergistic effects in Ras-mutant cell lines (HCT116, A549, LOVO, CALU-6, SW620, SK-MEL-2, HEPG2, MEWO) as indicated by positive Bliss scores; no significant synergy is observed in RAF-mutant cell lines (A375, SK-MEL-239, COLO-205, H2087, SW1417) [1] In HCT116 cells, co-treatment with APS-2-79 (250 nM, 1 μM) and trametinib enhances downregulation of Ras-MAPK signalling, reducing phospho-ERK levels and lowering the IC₉₀ value of trametinib [1] APS-2-79 (1 μM) inhibits 246 kinases in kinome-wide assays, with the most inhibited kinases including YES1, ERBB4, FGR, CSK, HCK, and MERTK [1] |
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| ln Vivo |
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| Enzyme Assay |
APS-2-79 acts as an antagonist of RAF-mediated MEKphosphorylation and a kinase suppressor of Ras (KSR)-dependent. With an IC50 for KSR2 of 120±23 nM, APS-2–79 binds specifically to KSR2 within the KSR2-MEK1 complex.
Purify the KSR2–MEK1 complex from insect cells through freeze-thaw lysis, sonication, cobalt resin affinity chromatography, TEV protease and λ-phosphatase treatment, ion-exchange chromatography (Sp-HP), and gel-filtration chromatography [1] Perform ATPᵇⁱᵒᵗⁱⁿ probe-labelling assays by incubating purified KSR2–MEK1 complexes with 2 μM ATPᵇⁱᵒᵗⁱⁿ in the presence of increasing concentrations of APS-2-79 or free ATP; detect probe-labelling of KSR2 and MEK1 using western blot with biotin-specific antibodies [1] Conduct in vitro RAF phosphorylation assays by incubating BRAF (200 nM) or CRAF (10 nM) with KSR2–MEK1 complexes, KSR2(A690F)–MEK1 complexes, or free MEK1 (500 nM) in the presence of 1 mM ATP and different concentrations of APS-2-79; measure MEK phosphorylation levels using western blot with phospho-MEK (Ser218/Ser222) antibodies [1] Perform bio-layer inferometry (BLI) assays by immobilizing BRAF or BRAF mutants (F667E, R509H) on sensor heads, then incubating with different concentrations of KSR2–MEK1 complexes or free MEK1 (625 nM to 10 μM) in the presence or absence of 25 μM APS-2-79; monitor association (0–660 s) and dissociation (660–1500 s) phases to calculate binding affinity (Kd), association rate (Kon), and dissociation rate (Koff) [1] |
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| Cell Assay |
In 96-well plates, cell viability assays are carried out. In order to achieve linear growth over the course of the assays, the optimal cell densities for 96-well plate assays are identified. A549, HCT-116, A375, SK-MEL-239, COLO-205, LOVO, SK-MEL-2, CALU-6, MEWO, SW620, and SW1417 cells are plated at 500 cells per well and treated for 72 hours with inhibitors (e.g., APS-2-79; 100-3,000 nM) before viability measurements are taken. 2000 H2087 and HEPG2 cells are plated in wells, and they are then exposed to inhibitors (such as APS-2-79; 100–3,000 nM) for 72 hours. Resazurin is used to measure cell viability, and the percent of viable cells is calculated by comparing inhibitor-treated samples to DMSO controls[1].
Transfect 293H cells with full-length KSR–Flag and MEK1–GFP (or MEK(AAAA)–GFP) plasmids; 24 h after transfection, treat cells with 5 μM APS-2-79, APS-3-77, or dabrafenib for 2 h; collect cells and perform western blot to detect phosphorylated MEK (Ser218/Ser222), phosphorylated ERK (Thr202/Tyr204), total MEK, and total KSR [1] Transfect 293H cells with MEK–GFP and KSR–Flag or KSR(R718H)–Flag plasmids; 24 h after transfection, treat cells with increasing concentrations of trametinib (0.13–100 nM, threefold dilutions) for 48 h; collect cells and perform western blot to analyze MAPK signalling pathway activity [1] Treat Ras-mutant (HCT116, A549, etc.) and RAF-mutant (A375, SK-MEL-239, etc.) cell lines with APS-2-79 (100 nM–3 μM, threefold dilutions) in combination with MEK inhibitors (trametinib: 0.01–100 nM, threefold dilutions; binimetinib, PD0325901, AZD6244: 0.1–10 μM, threefold dilutions); after incubation, assess cell viability and calculate Bliss scores to evaluate synergy [1] Treat HCT116 and SK-MEL-239 cells with increasing concentrations of trametinib combined with DMSO, 250 nM, or 1 μM APS-2-79 for 48 h; perform western blot to detect phospho-ERK levels and calculate IC₉₀ values of trametinib [1] Treat HCT116 cells with APS-2-79 and/or trametinib for 48 h; perform western blot to detect phospho-AMPK, phospho-ERK, and total MEK levels [1] |
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| Animal Protocol |
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| References | |||
| Additional Infomation |
APS-2-79 is a small molecule compound that mimics the KSR allele and inhibits oncogenic Ras mutations[1]
APS-2-79 binds to the ATP-binding pocket in KSR2; key interactions include contact with Thr802, Phe516, Phe793 and Arg692 of KSR2, which makes it more selective for KSR than for RAF protein[1]. The binding of APS-2-79 to KSR2 induces a conformational change to form an “induced lock” (residue I809–Q814) and alters residues Trp685 and His686 at the KSR–RAF heterodimer interface, thereby hindering the higher-order assembly of the RAF–KSR–MEK complex[1]. APS-2-79 enhances the efficacy of MEK inhibitors by antagonizing the MEKi-induced Ras–MAPK signaling complex, providing a therapeutic strategy for treating Ras-driven cancers by co-targeting enzyme activity and scaffold activity in the Ras-MAPK pathway [1]. The inactive state of KSR is stabilized by APS-2-79. APS-2-79 shifts the equilibrium of the KSR2-MEK1 complex toward the OFF state, antagonizing RAF dimerization and subsequent MEK phosphorylation [1] |
| Molecular Formula |
C23H21N3O3
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|---|---|---|
| Molecular Weight |
387.44
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| Exact Mass |
387.158
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| Elemental Analysis |
C, 71.30; H, 5.46; N, 10.85; O, 12.39
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| CAS # |
2002381-25-9
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| Related CAS # |
APS-2-79 hydrochloride;2002381-31-7
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| PubChem CID |
121499159
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
528.6±50.0 °C at 760 mmHg
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| Flash Point |
273.5±30.1 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.656
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| LogP |
5.36
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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 |
29
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| Complexity |
502
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C(=C(C([H])([H])[H])C=1[H])N([H])C1C2=C([H])C(=C(C([H])=C2N=C([H])N=1)OC([H])([H])[H])OC([H])([H])[H]
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| InChi Key |
PEKZLFZZBGBOPJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H21N3O3/c1-15-11-17(29-16-7-5-4-6-8-16)9-10-19(15)26-23-18-12-21(27-2)22(28-3)13-20(18)24-14-25-23/h4-14H,1-3H3,(H,24,25,26)
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| Chemical Name |
6,7-dimethoxy-N-(2-methyl-4-phenoxyphenyl)quinazolin-4-amine
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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: ≥ 2 mg/mL (5.16 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 20.0 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 mg/mL (5.16 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 20.0 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 mg/mL (5.16 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 | 2.5810 mL | 12.9052 mL | 25.8104 mL | |
| 5 mM | 0.5162 mL | 2.5810 mL | 5.1621 mL | |
| 10 mM | 0.2581 mL | 1.2905 mL | 2.5810 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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