Osimertinib (AZD-9291) is an EGFR third-generation inhibitor that inhibits the T790M resistance mutation produced by second-generation EGFR inhibitors with irreversible and oral activity. Osimertinib has antitumor activity for the treatment of EGFR-mutated non-small-cell lung cancer.

EGFR (WT):493.8 nM, EGFR (L858R/T790M):11.44 nM, EGFR (exon 19 deletion):12.92 nM

In the EGFRm+ (PC9) and EGFRm+/T790M (H1975) tumor models, oral administration of AZD9291 (5 mg/kg) effectively inhibits AKT and ERK signaling pathways as well as EGFR phosphorylation in tumors, leading to tumor regression.

In mutant EGFR cell lines, AZD9291 effectively inhibits cell proliferation.

ACY-1215 is dissolved and subsequently diluted in assay buffer [50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, and 20 μM tris(2-carboxyethyl)phosphine] to 6-fold the final concentration. HDAC enzymes are diluted to 1.5-fold of the final concentration in assay buffer and pre-incubated with ACY-1215 for 10 minutes before the addition of the substrate. The amount of FTS (HDAC1, HDAC2, HDAC3, and HDAC6) or MAZ-1675 (HDAC4, HDAC5, HDAC7, HDAC8, and HDAC9) used for each enzyme is equal to the Michaelis constant (Km), as determined by a titration curve. FTS or MAZ-1675 is diluted in assay buffer to 6-fold the final concentration with 0.3 μM sequencing grade trypsin. The substrate/trypsin mix is added to the enzyme/compound mix and the plate is shaken for 60 seconds and then placed into a SpectraMax M5 microtiter plate reader. The enzymatic reaction is monitored for release of 7-amino-4-methoxy-coumarin over 30 minutes, after deacetylation of the lysine side chain in the peptide substrate, and the linear rate of the reaction is calculated[1].

AZD-9291 is dissolved in DMSO and stored, and then diluted with appropriate medium before use[1]. PC-9 cells are seeded into T75 flasks (5×105 cells/flask) in RPMI growth media and incubated at 37°C, 5% CO2. The following day the media is replaced with media supplemented with a concentration of EGFR inhibitor equal to the EC50 concentration predetermined in PC-9 cells. Media changes are carried out every 2-3 days and resistant clones allowed to grow to 80% confluency prior to the cells being trypsinised and reseeded at the original seeding density in media containing twice the concentration of EGFR inhibitor. Dose escalations are continued until a final concentration of 1.5 μM Gefitinib, 1.5 μM Afatinib, 1.5 μM WZ4002 or 160 nM AZD-9291 are achieved[1].