In vitro activity: AD80 prevents the phosphorylation of RET as well as of extracellular signa-regulated kinase (ERK), AKT, and S6K at low nanomolar concentrations in kinesin family member 5B (KIF5B)-RET-expressing Ba/F3 cells. Treatment with AD80 results in up-regulation of genes that are typically repressed by active KRAS. On the contrary, genes that are activated by KRAS were down-regulated.

Kinase Assay: AD80 is a novel multikinase inhibitor that shows strong activity against human RET, BRAF, S6K, and SRC but were much less active than either AD57 or AD58 against mTOR. AD57 was identified using a Ret-kinase-driven Drosophila model of multiple endocrine neoplasia type 2 and kinome-wide drug profiling, AD57 rescues oncogenic Ret-induced lethality, whereas related Ret inhibitors imparted reduced efficacy and enhanced toxicity. The IC50 value for RET is 4 nM. On the basis of in vitro human kinase profiles, AD80 and AD81 inhibit RET, RAF, SRC and S6K, with greatly reduced mTOR activity relative to AD57 and AD58. AD80 inhibits proliferation of MZ-CRC-1 and TT thyroid cancer cells in culture, probably through the induction of apoptosis.

Cell Assay: Immunoblotting is performed in LC-2/AD cells treated with AD80, cabozantinib, or vandetanib (4 hours). MZ-CRC-1 (MEN2B) and TT (MEN2A) cells are treated with AD80 (0.2 nM to 20 μM) for 7 days and cell viability is quantitated by MTT assay