Sgx 523

PD0325901, sorafenib tosylate, dasatinib (LC Laboratories, Woburn, MA, USA), rapamycin doxorubicin, Nutlin-3a, SGX-523 (Selleck, Houston, TX, USA), and KT5270 (Santa Cruz Biotechnology, Dallas, TX, USA) were prepared in DMSO (Sigma-Aldrich, St. Louis, MO, USA). Cells were seeded into a 96-well plate using an epMotion 5075 pipetting system (Eppendorf, Hamburg, Germany). Treatments began when cells reached 30% confluency. Cells were treated for 72 h. Cell viability was measured using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Assays were measured using a Benchmark Plus microplate spectrophotometer (Bio-Rad, Hercules, CA, USA) at 490 and 700 nm reference wavelengths. Cell viability was performed twice in duplicate wells. Treated wells were normalized to non-treated wells and then were normalized to DMSO-treated plates. The concentration of compound required to cause 50% inhibition of cell viability (IC₅₀) was calculated (23 (link)). A combinatorial index was calculated following Chou and Talalay (23 (link)). If a drug combination resulting in synergy (CI<1) that combination was repeated for a total of three separate experiments in duplicate wells. The IC₅₀ and CI were then calculated as previously described (23 (link)).

PC-9 cells (EGFR Ex19 del E746_A750) were purchased from the European Collection of Cell Cultures in 2014. RPC-9 cells (Gefitinib-resistant; EGFR Ex19 del E746_A750 and Ex20 T790M) were established from a parental PC-9 cell line in our laboratory^{18 (link)}. HCC827 cells (EGFR Ex19 del E746_A750)^{5 (link)} and PC-9/BRc1 cells (afatinib-resistant; EGFR Ex19 del E746_A750 and Ex20 T790M)^{19 (link)} were kindly provided by Dr. William Pao (Vanderbilt University, Nashville, TN, USA). Cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin in a tissue culture incubator at 37 °C under 5% CO₂.
Naquotinib was provided by Astellas Pharma Inc. (Tokyo, Japan) under a material transfer agreement. Gefitinib, afatinib, osimertinib, crizotinib, SGX-523, selumetinib, and trametinib were purchased from Selleck Chemicals (Houston, TX, USA). UNC569 was purchased from Merck Millipore (Billerica, MD, USA). All compounds were dissolved in dimethyl sulfoxide for in vitro studies.
Growth inhibition was measured using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay^{20 (link)}. Briefly, cells were plated onto 96-well plates at a density of 2,000–3,000 per well and continuously exposed to each drug for 96 h.

Gefitinib (#S1025, Selleck Chem) was dissolved in DMSO at concentration of 100 mM, and diluted further to 1 μM in KSF medium (#17005042, Thermo Fisher Scientific) without supplements for final use. SGX-523 (#S1112, Selleck Chem) was dissolved in DMSO and diluted to 200 nM in KSF medium without supplements for final use. DMSO was used as control and diluted in the same manner. The anti-EGFR antibody (Erbitux, Bristol-Myers Squibb) was diluted to 10 μg/ml in KSF medium without supplements for use. EGF (#10450–013, Thermo Fisher Scientific) was diluted in PBS containing 0.1% BSA at a concentration of 2 μg/ml, and diluted to a final concentration of 20 or 40 ng/ml in KSF medium without supplements.

FS-93 was synthesized as previously described [29 (link)]. SGX-523, gefetinib and NVP-AUY922 were obtained from Selleck Chemicals (Houston, USA). All these compounds were dissolved to 10 mM with DMSO as a stock solution and stored at −20°C.