In vitro activity: AG-1478 (also known as Tyrphostin AG-1478) is a potent and selective EGFR (epidermal growth factor receptor) inhibitor with IC50 of 3 nM in cell-free assays. It reversibly inhibits rat brain Kv1.5 potassium channels with IC50 of 9.8 μM which is independent of protein tyrosine kinase (PTK) activity. AG-1478 also inhibits the growth of leiomyoma and myometrium cell cultures with IC50 values of 5.6 and 5.7 μM, respectively. Previous studies suggest that EGFR antagonism may be effective for the treatment of various diseases such as cancer, angiotensin II-induced cardiac hypertrophy and diabetic cardiomyopathy. Therefore, AG-1478 has the potential to be used as therapeutics for these disorders.

Kinase Assay: AG-1478 is high selective over ErbB2 and PDGFR with IC50 of>100 μM. AG-1478 preferentially inhibits U87MG cells expressing truncated EGFR with IC50 of 8.7 μM, compared to those expressing endogenous wt EGFR or overexpressing exogenous wt EGFR with IC50 of 34.6 μM and 48.4 μM, respectively, and inhibits the DNA synthesis with IC50 of 4.6 μM, 19.67 μM, and 35.2 μM, respectively. AG-1478 also preferentially inhibits the tyrosine kinase activity and autophosphorylation of the ΔEGFR compared to endogenous or overexpressed exogenous wt EGFR. AG-1478 (0.25 μM) abolishes the MAPK activation induced by Ang II, a Ca2+ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. AG-1478 inhibits EGF-induced mitogenesis of the BaF/ERX and LIM1215 cells with IC50 of 0.07 μM and 0.2 μM, respectively. AG1478 is able to inhibit the function of ATP-binding cassette (ABC) transporters such as ABCB1 and ABCG2, with a more pronounced effect on ABCG2.

Cell Assay: Cells are exposed to different concentrations of AG-1478 for 72 hours in 96-well plates. The effects of AG-1478 on cell growth are examined using an Alamar Blue assay. A 20-μL aliquot of Alamar Blue is added to each well, and its absorbance is determined using a Spectromax Scanning Micro plate Reader. The effects of AG-1478 are expressed as percentage of growth inhibition using untreated cells as the control (0% inhibition). Cellular DNA synthesis is determined using a [3H]thymidine incorporation assay.