WZ4002

Biological Activity

WZ4002 is a novel, selective EGFR kinase inhibitor against EGFR T790M (mutation of the gatekeeper T790 residue) with IC50 of 8 nM. This agent is 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wildtype EGFR, than quinazoline-based EGFR inhibitors (HKI-272 and CL-387,785) in vitro. WZ4002 inhibits EGFR, AKT and ERK1/2 phosphorylation in NSCLC cell lines and WZ4002 prevents of EGFR phosphorylation in NIH-3T3 cells expressing different EGFR T790M mutant alleles. In addition, the phosphorylated EGFR of Src TKI-resistant H1975 cells, as well as HCC827 cells, is completely suppressed by the third generation EGFR TKI, WZ4002. In a 2-week efficacy study, WZ4002 treatment resulted in significant tumour regressions compared with vehicle alone in both T790M-containing murine models.

Protocol

Cell Experiment
Cell lines	NSCLC or Ba/F3 cells
Preparation method	Cell proliferation and growth assays NSCLC or Ba/F3 cells were exposed to treatment for 72 hours and the number of cells used per experiment determined empirically and has been previously established2,7. All experimental points were set up in six to twelve wells and all experiments were repeated at least three times. The data was graphically displayed using GraphPad Prism version 5.0 for Windows, (GraphPad Software; www.graphpad.com). The curves were fitted using a non-linear regression model with a sigmoidal dose response.
Concentrations	0~10 μM
Incubation time	72 h

Animal Experiment
Animal models	EGFR-TL (T790M/L858R) mice and EGFR exon19 Deletion-T790M (TD) inducible bitransgenic mice
Formulation	NMP (10% 1-methyl-2-pyrrolidinone: 90% PEG-300)
Dosages	25mg/kg daily
Administration	gavage

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Chemical Information

Customer Product Validations & Biological Datas

	Source	Clin Cancer Res (2017). Figure 4. WZ4002 (Abmole Bioscience)
	Method	vitro observations
	Cell Lines	Mice harboring H1975 ER-TWIST+4-OHT tumors
	Concentrations	25mg/kg
	Incubation Time	5 d
	Results	"Consistent with our in vitro observations, while single-agent WZ4002 was sufficient to markedly slow the growth of the tumors, the addition of ABT-263 was necessary to shrink tumors (Fig. 4D)."
	Rating

	Source	Clin Cancer Res (2017). Figure 3. WZ4002 (Abmole Bioscience)
	Method	Western blotting
	Cell Lines	H1975 parental cells (par), H1975 R1 cells (R1), and H1975 R2 cells (R2)
	Concentrations	1 μM
	Incubation Time	72 h
	Results	Similarly, in the H1975 EMT model induced by ER-TWIST, knockdown of ZEB1 led to de-repression of BIM protein and RNA (Fig. 3I and Sup. Fig. 5B, right panel) and restored EGFRi-induced apoptosis and re-sensitized these EMT cancers to WZ4002 (Fig. 3J and 3K).
	Rating

	Source	Clin Cancer Res (2017). Figure 2. WZ4002 (Abmole Bioscience)
	Method	apoptosis quantified
	Cell Lines	HCC4006 and H1975 cells
	Concentrations	1 μM
	Incubation Time	72 h
	Results	We found directly inducing EMT by exogenous TWIST activation (Fig. 2) or chronic TGF- β treatment (Sup. Fig. 2) led to depressed levels of BIM resulting in suppression of EGFRi-induced apoptosis and resistance to EGFRi (WZ4002 and gefitinib).
	Rating

Product Citations

• 

  Clin Cancer Res. 2017 Oct 19; pii: clincanres.1577.2017.

  Epithelial-to-mesenchymal transition antagonizes response to targeted therapies in lung cancer by suppressing BIM
  WZ4002 purchased from AbMole

References

Combined therapy with mutant-selective EGFR inhibitor and Met kinase inhibitor for overcoming erlotinib resistance in EGFR mutant lung cancer.
Nakagawa et al. Mol Cancer Ther. 2012 Jul 25. PMID: 22844075.

NF-κB signaling is activated and confers resistance to apoptosis in three-dimensionally cultured EGFR-mutant lung adenocarcinoma cells.
Sakuma et al. Biochem Biophys Res Commun. 2012 Jul 13;423(4):667-71. PMID: 22695117.

WZ4002, a third-generation EGFR inhibitor, can overcome anoikis resistance in EGFR-mutant lung adenocarcinomas more efficiently than Src inhibitors.
Sakuma et al. Lab Invest. 2012 Mar;92(3):371-83. PMID: 22157722.

Novel mutant-selective EGFR kinase inhibitors against EGFR T790M.
Zhou W, et al. Nature. 2009 Dec 24;462(7276):1070-4. PMID: 20033049.