Kinase experiment:

In vitro inhibitory enzyme kinetic assays are carried out in triplicate using the ATP/NADH coupled assay system in a 96-well format. The final reaction mixture contains 0.5mg/mL Bovine Serum Albumin (BSA), 2mM MnCl2, 1mM phospho(enol) pyruvic acid, 1mM TCEP, 0.1M Hepes 7.4, 2.5mM poly-[Glu4Tyr1] peptide, 1/50 of the final reaction mixture volume of pyruvate kinase/lactic dehydrogenase enzymes from rabbit muscle, 0.5mM NADH, 0.5μM EGFR kinase, 100μM ATP and varied amount of inhibitors. Inhibitors and ATP are mixed and made separate stock from the mixture with all other ingredients and added last to the latter to start the reaction. Steady state initial velocity data are drawn from the slopes of the A340 curves[1].

Animal experiment:

Growth and inhibition of growth is assessed by MTS assay. Ba/F3 cells are exposed to WZ3146 treatment for 72 hours. Growth and inhibition of growth is assessed by MTS assay[1].

WZ3146 is a novel, irreversible inhibitor that specifically inhibits phosphorylation of EGFR(L858R0) and EGFR(E746_A750) with IC50 values of 2nM each.

WZ3146 has been reported to inhibit the phosphorylation of EGFR in the Non–Small-Cell Lung Cancer (NSCLC) cell lines [1]. WZ3146 shows to suppress the growth of EGFR T790M containing cell lines. Besides, Analysis of recombinant EGFR T790M kinase incubated with WZ3146 by electrospray mass spectrometry revealed stoichiometric addition of one inhibitor molecule to the protein. Analysis of a pepsin digest of the modified protein by tandem MS identified Cys 797 as the site of modification thus verifying covalent bond formation between WZ3146 and EGFR [2].

References:
[1] Thanyanan Reungwetwattana, Saravut J. Weroha, Julian R. Molina. Oncogenic Pathways, Molecularly Targeted Therapies, and Highlighted Clinical Trials in Non–Small-Cell Lung Cancer (NSCLC). Clinical Lung Cancer, Volume 13, Issue 4, July 2012, Pages 252-266
[2] Zhou W1, Ercan D, Chen L, Yun CH, Li D, Capelletti M, Cortot AB, Chirieac L, Iacob RE, Padera R, Engen JR, Wong KK, Eck MJ, Gray NS, Janne PA. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature. 2009 Dec 24;462(7276):1070-4.