Neratinib

Biological Activity

Neratinib is a potent irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2 with IC50 of 92 nM and 59 nM, respectively. Neratinib inhibits the proliferation of EGFR-dependent A431 cells with an IC50 of 81 nM. Neratinib (HKI-272) also weakly inhibits tyrosine kinases KDR and Src with IC50 of 0.8 μM and 1.4 μM, respectively. Likewise, Neratinib inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner (IC50 = 0.24 μM). Neratinib enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that Neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Moreover, Neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib (HKI-272) stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations.

Product Citations

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  Cell Death Dis. 2021 Feb 15;12(2):179.

  Targeting transcription of MCL-1 sensitizes HER2-amplified breast cancers to HER2 inhibitors
  Neratinib purchased from AbMole

• 

  Int J Clin Exp Med. 2019 Aug 30;12(8):10373-10379.

  Neratinib inhibits the malignant phenotype via inhibiting the HER2 in osteosarcoma cells
  Neratinib purchased from AbMole

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  ACS Chem Biol. 2016 May 20;11(5):1245-54.

  Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors
  Neratinib purchased from AbMole

Customer Product Validations & Biological Datas

	Source	ACS Chem Biol (2016). Figure 5. Neratinib (Abmole Bioscience)
	Method	FECH inhibition assay
	Cell Lines	K562 Cells
	Concentrations	1 μM
	Incubation Time	6 days
	Results	In line with the Kd values obtained from kinobead experiments, Vemurafenib, CUDC 101, Cyc-116, Linsitinib, MK-2461, Neratinib, GSK-690693, and Crenolanib decreased FECH binding to the PPIX beads by 50% or more compared to the DMSO control.

	Source	ACS Chem Biol (2016). Figure 4. Neratinib (Abmole Bioscience)
	Method	FECH inhibition assay
	Cell Lines	K562 Cells
	Concentrations	1 μM
	Incubation Time	6 days
	Results	We then subjected MK-2461, Neratinib, and Linsitinib to the same assay (1 μM for 6 days), and each drug clearly led to reduced heme levels indicating inhibition of FECH activity in K562 cells with Neratinib showing the most potent effect (60% reduction).

	Source	ACS Chem Biol (2016). Figure 3. Neratinib (Abmole Bioscience)
	Method	FECH binding
	Cell Lines	K562 Cells
	Concentrations
	Incubation Time
	Results	The basis of this assay is that proteins bound to a drug molecule are stabilized against heat induced unfolding and aggregation, and the extent of this protection is dose dependent.

	Source	ACS Chem Biol (2016). Figure 2. Neratinib (Abmole Bioscience)
	Method	FECH binding
	Cell Lines
	Concentrations
	Incubation Time
	Results	Figure 2b−f show the full dose response data for five selected inhibitors, and the complete set of these plots can be found in Supporting Information Figure S1 and Table S1.

Protocol

Cell Experiment
Cell lines	3T3, 3T3/neu, A431, SK-Br-3, BT474, MDA-MB-435, and SW62 cells
Preparation method	Cell Proliferation Assays. Cells were plated in 96-well tissue culture plates (3T3, 3T3/neu, 5000 cells/well; A431, SK-Br-3, BT474, MDA-MB-435, and SW620, 10,000 cells/well). The following day, dilutions of compound (0.5 ng/ml–5 μg/ml) were added, and cells were cultured for 2 days (6 days for BT474). Cell proliferation was determined using sulforhodamine B, a protein binding dye. Briefly, cells were fixed with 10% trichloroacetic acid and washed extensively with water. Cells were then stained with 0.1% sulforhodamine B (Sigma-Aldrich) and washed in 5% acetic acid. Protein-associated dye was solubilized in 10 mm Tris, and absorbance was measured at 450 nm (Victor2). Inhibition of cell proliferation was calculated using the formula: percentage of inhibition = 100 − 100 (Td − To/Tc − To), where Td is the absorbance of drug treated cells, Tc is the absorbance of untreated cells, and To is the absorbance at the time of drug addition. To values were determined by plating cells separately and fixing them at the time of drug addition. The concentration of compound which inhibits cell proliferation by 50% (IC50) was determined from inhibition curves.
Concentrations	0.5 ng/ml-5 μg/ml
Incubation time	2 days

Animal Experiment
Animal models	BT474, MCF-7, and SK-OV-3 tumour xenografts model in female athymic (nude) mice
Formulation	0.5% methocellulose-0.4% polysorbate-80 (Tween 80)
Dosages	40 or 60mg/kg daily for 20 days
Administration	oral 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 Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 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 Compound A of 10 mg/kg.

Chemical Information

References

[1] Zhao XQ, et al. Mol Pharmacol. Neratinib reverses ATP-binding cassette B1-mediated chemotherapeutic drug resistance in vitro, in vivo, and ex vivo.

[2] Rabindran SK, et al. Cancer Res. Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase.