Lck (IC50 = 0.2 nM); VEGF2 (IC50 = 1 nM); p38α (IC50 = 3 nM); Jak3 (IC50 = 72 nM); MLR (IC50 = 30 nM); IL-2 (IC50 = 21 nM)
Lck (IC50 = 0.0002 μM) [1]
Src (IC50 = 0.002 μM) [1]
Syk (IC50 = 0.292 μM) [1]
Jak3 (IC50 = 0.072 μM) [1]
KDR (IC50 = 0.001 μM) [1]
p38α (IC50 = 0.003 μM) [1]
Other kinases with IC50 >0.1 μM: JNK2 (0.173 μM), JNK3 (0.145 μM), Tyk2 (1.83 μM), Itk (>25 μM), Btk (0.545 μM), Aurora1 (>5 μM), Aurora2 (>25 μM), CDK5 (>25 μM), MSK1 (>25 μM), PKBα (>25 μM), Pak2 (>25 μM), PKAβ (>25 μM), Zap-70 (>25 μM) [1]

Secondary analysis of hit compounds AMG-47a and Ponatinib
Two of the strongest hits were Ponatinib, a pan BCR-ABL kinase inhibitor, and AMG-47a, a potential Lck kinase inhibitor. In the primary screen these compounds decreased EGFP-KRASG12V signal by ∼40% at 1 µM. Concentrations of AMG-47a above 1 uM increased EGFP-KRASG12V signal, possibly because of higher concentrations of this compound lead to more apoptotic cells with higher autofluorescence. We thus decided to move forward with doses of AMG-47a and Ponatinib at near the maximally effective concentrations in validation assays. We first confirmed that these compounds decreased fluorescence signal in the HeLa EGFP-KRASG12V cells by flow cytometry. A 48-hour treatment of cells by AMG-47a and Ponatinib led to a 30–40% decrease in EGFP signal in these cells ; treating cells for 3 and 5 days yielded similar results . To test for the selectivity of these compounds, we generated HeLa cells expressing EGFP from the same inducible vector as controls and used these cells in a counter-screen. We reasoned that the HeLa EGFP cells would be sensitive to compounds that show non-specific inhibitory activities against the doxycycline-inducible promoter, against general RNA transcription and protein translation, or against the fluorescence or stability of EGFP. Using flow cytometry, we observed that Torin-1 indeed decreased the fluorescence signal in the HeLa EGFP cells, likely through its inhibition of general protein translation. On the other hand, Ponatinib and AMG-47a did not affect EGFP levels in these cells . We further assessed the effect of Ponatinib, AMG-47a, and Torin-1 on the fluorescence of HeLa cells expressing EGFP-KRASWT and found that they also decreased EGFP-KRASWT signal . Thus these compounds do not appear to discriminate between mutant and WT KRAS proteins.
Lastly, we tested the loss of EGFP-KRASG12V proteins directly by western blot. As a positive control, we transfected a KRAS siRNA into HeLa EGFP-KRASG12V cells and observed a dose-dependent reduction in EGFP-KRASG12V protein levels. Both AMG-47a and Ponatinib had a modest effect on EGFP-KRASG12V protein levels after 3 days. In the primary screen, the loss of EGFP-KRASG12V signal plateaued at ∼50% for AMG-47a and ∼55% for Ponatinib, though we were only able to consistently detect a 20–30% reduction in western blot protein levels. Although this decrease was small, both compounds had no effect on the levels of the control EGFP protein . Together these results support the notion that AMG-47a and Ponatinib selectively affect the levels of EGFP-KRASG12V protein in the cell.[2]

---

AMG-47a potently inhibited Lck kinase with an IC50 of 0.2 nM. It also inhibited KDR (IC50 = 17 nM), p38α (IC50 = 9 nM), and Jak3 (IC50 = 868 nM) in enzymatic assays. In cellular assays, it inhibited anti-CD3/CD28-induced IL-2 production in human whole blood with an IC50 of 0.272 μM. It showed marginal inhibition of LPS-induced TNFα production in human whole blood (IC50 >2.5 μM). In the absence of whole blood, the IL-2 IC50 was 21 nM. The compound exhibited improved solubility (>200 μg/mL in fasted state simulated intestinal fluid, pH 6.8) and oral bioavailability relative to earlier analogs. AMG-47a also inhibited the human mixed lymphocyte reaction (MLR) but the exact IC50 was not specified in the text [1].

In an anti-CD3-induced IL-2 production mouse model, oral administration of AMG-47a produced a dose-dependent inhibition of IL-2 production in female BALB/c mice. The ED50 was determined to be 11 mg/kg. At the ED50, the mean terminal plasma concentration (EC50) was ~630 nM. When adjusted for free fraction based on mouse protein binding (97.1% bound), the free EC50 was ~18 nM, consistent with the in vitro inhibitory activity in the absence of whole blood (IL-2 IC50 = 21 nM) [1].

Lck HTRF (Homogeneous Time-Resolved Fluorescence) kinase assay: The assay measures ATP-dependent phosphorylation of a biotinylated gastrin peptide substrate. Final concentrations: gastrin 1.2 μM, ATP 0.5 μM (Km value ~0.6 μM), Lck (GST-kinase domain fusion, AA 225-509) 250 pM. Buffer: 50 mM HEPES pH 7.5, 50 mM NaCl, 20 mM MgCl2, 5 mM MnCl2, 2 mM DTT, 0.05% BSA. The reaction was quenched with detection reagent containing streptavidin-allophycocyanin (SA-APC, 0.0004 mg/mL final) and europium-labeled anti-phosphotyrosine antibody (Eu-anti-PY, 0.025 mM final) in 50 mM Tris pH 7.5, 100 mM NaCl, 3 mM EDTA, 0.05% BSA, 0.1% Tween-20. Plates were read on a fluorescence plate reader with excitation at 320 nm and emission at 615 nm and 655 nm. Assays for other kinases were performed similarly, with varying concentrations of enzyme, peptide substrate, and ATP depending on the specific activity and measured Km values [1].

Human whole-blood anti-CD3/CD28-induced IL-2 secretion assay: Whole blood from normal volunteers diluted 50% in tissue culture medium was preincubated with compound, then stimulated with anti-CD3 and anti-CD28 antibodies. After ~20 h at 37°C, 5% CO2, secreted IL-2 in supernatants was quantified by ELISA.
Human whole-blood LPS-induced TNFα assay: Heparinized human whole blood diluted 50% in RPMI 1640 with L-glutamine, 10% human serum AB, and 1% pen/strep was preincubated with compound for 1 h at 37°C, then challenged with LPS (0.1 μg/mL final) and incubated overnight (18 h). Secreted TNFα was measured by ELISA.
Purified T cell IL-2 assay (no whole blood): Human T cells purified from peripheral blood lymphocytes (1×10^5/well) were preincubated with or without compound, then stimulated with anti-CD3 and anti-CD28 antibodies in 96-well plates for ~20 h at 37°C, 5% CO2. Supernatant IL-2 was quantified by ELISA. The IL-2 IC50 for AMG-47a in this assay was 21 nM [1].

Anti-CD3 induced IL-2 production in mice: Female BALB/c mice (12 weeks old, ~20 g) were dosed orally with AMG-47a suspended in 2% hydroxypropylmethylcellulose with 1% Tween-80 at indicated doses (10, 30, 100 mg/kg) 1 h prior to challenge. Mice were then challenged intravenously with anti-mouse CD3 monoclonal antibody (3 μg/mouse in PBS). After 90 min, blood was collected via cardiac puncture, and serum IL-2 levels were measured by ELISA. Each group had 5 animals [1].

Solubility: AMG-47a exhibited solubility >200 μg/mL in fasted state simulated intestinal fluid (SIF) at pH 6.8 containing 5 mM sodium taurocholate, 1.5 mM lecithin, 2.9 mM KH2PO4, and 0.22 M KCl [1].

Plasma protein binding: AMG-47a showed 95.0% binding in human plasma, 97.1% in mouse plasma, and 95.3% in rat plasma (at 5 μg/mL, ~12.9 μM) as determined by ultracentrifugation.
Selectivity: AMG-47a exhibited marginal inhibition of LPS-induced TNFα production in human whole blood (IC50 >2.5 μM), indicating lack of significant p38α-mediated proinflammatory cytokine suppression in this assay [1].

[1]. Discovery of aminoquinazolines as potent, orally bioavailable inhibitors of Lck: synthesis, SAR, and in vivo anti-inflammatory activity. J Med Chem. 2006 Sep 21;49(19):5671-86.

[2]. A high-throughput assay for small molecule destabilizers of the KRAS oncoprotein. PLoS One. 2014 Aug 5;9(8):e103836.

AMG-47a is a selective Lck inhibitor developed for the treatment of T-cell-mediated autoimmune and inflammatory diseases. It exhibited excellent selectivity (>1000-fold) over a wide range of structurally diverse kinases including Syk, Tyk2, Itk, Btk, CDK5, Msk1, Pak2, Jnk1, Aurora, PKBα, PKAβ, and Zap-70, but not over other Src-family members such as Src. The compound is an aminoquinazoline derivative with a 2-morpholinoethylamino substituent, which improved solubility and oral bioavailability relative to earlier leads [1].

C29H28F3N5O2

535.5601

535.219

C, 65.04; H, 5.27; F, 10.64; N, 13.08; O, 5.97

882663-88-9

882663-88-9

16086114

Light yellow to yellow solid powder

1.3±0.1 g/cm3

1.637

5.38

2

9

7

39

795

0

O=C(C1C=C(C2C=C3C(N=C(NCCN4CCOCC4)N=C3)=CC=2)C(C)=CC=1)NC1C=C(C(F)(F)F)C=CC=1

DVRSTRMZTAPMKO-UHFFFAOYSA-N

InChI=1S/C29H28F3N5O2/c1-19-5-6-21(27(38)35-24-4-2-3-23(17-24)29(30,31)32)16-25(19)20-7-8-26-22(15-20)18-34-28(36-26)33-9-10-37-11-13-39-14-12-37/h2-8,15-18H,9-14H2,1H3,(H,35,38)(H,33,34,36)

4-methyl-3-[2-(2-morpholin-4-ylethylamino)quinazolin-6-yl]-N-[3-(trifluoromethyl)phenyl]benzamide

AMG-47a; AMG47; AMG-47; AMG 47; AMG 47a; AMG47a

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ≥ 34 mg/mL (~63.5 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)