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

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

In nude mice bearing N87 human gastric carcinoma xenografts, Poziotinib (0.5 mg/kg, orally administered) significantly inhibits tumor growth. The combined use of Poziotinib and 5-FU further enhances the efficacy in suppressing tumor growth.

Poziotinib inhibits the phosphorylation of EGFR and key components of downstream signaling cascades, such as STAT3, AKT, and ERK, and exhibits specific inhibitory effects on the growth of gastric cancer cells with HER2 amplification. It can induce G1 cell cycle arrest and apoptosis in gastric cancer cells with activated HER2 amplification through the mitochondrial pathway. Furthermore, Poziotinib demonstrates synergistic effects with chemotherapeutic agents in both HER2-amplified and non-amplified gastric cancer cells.

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)