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)

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]

[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.

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.)