Poziotinib (NOV120101)(NOV120101; HM781-36B) is an irreversible HER1/2/4 inhibitor (IC50s: 3/5/23 nM).

HER1:3 nM, HER2:5 nM, HER4:23 nM

在负荷N87人胃癌异种移植物的裸鼠体内,Poziotinib（0.5 mg/kg,p.o.）可明显抑制肿瘤生长,Poziotinib 与 5-FU联用可更有效地抑制肿瘤生长.

Poziotinib对EGFR磷酸化和下游信号级联放大的关键组分有抑制作用,如STAT3,AKT和ERK,且对HER2扩增的胃癌细胞生长有特异性抑制效果。经激活HER2扩增的胃癌细胞中线粒体途径,Poziotinib还可诱导G1细胞周期阻滞和细胞凋亡。此外,在HER2诱发的和HER2非扩增的胃癌细胞中,Poziotinib与化疗剂有协同效果。

Enzyme activity assay: To determine the IC50 values of HM781-36B for kinase inhibition, enzymes of EGFR, HER2, and HER4 are expressed as recombinant proteins in Sf9 insect cells. Enzyme selectivity screening is then performed using a tyrosine kinase assay kit. Briefly, the reactions are performed in 96 well polystyrene round-bottomed plates containing kinase buffer composed of 100 mM HEPES (pH 7.4), 25 mM MgCl2, 10 mM MnCl2 and 250 μM Na3VO4. The reactions are initiated by the addition of 100 ng/assay enzyme, 100 μM ATP, and 10 ng/mL poly(Glu, Tyr). After 1 h of incubation at room temperature, the reactions are terminated by adding 6 mM EDTA solution and then anti-phosphotyrosine antibody, PTK Green Tracer, and FP dilution buffer mixtures. The fluorescence polarization values are then measured after 30 min at room temperature using a Victor3 microplate reader. Finally, the IC50 values were calculated using the following equation: Y = bottom + (top–bottom)/(1 + 10(X-logIC50)).

Viable cell growth is determined by an MTT reduction assay. Briefly, all cell lines are seeded at a density of 3 × 103 per well in 96-well culture plates and then incubated at 37 °C for 24 h. The cells are then treated with 0.001, 0.01, 0.1, or 10 μM of HM781-36B. Three days later, 50 μg of MTT are added to each well and the samples are then incubated for 4 h to reduce the dye. Next, the samples are treated with DMSO, after which the absorbance of the converted dye in the living cells is measured at a wavelength of 540 nm. Six replicate wells are used for each analysis, and at least three independent experiments are conducted. Data points shown represent the mean while bars represent the SE.(Only for Reference)