In vitro activity: Larotrectinib (formerly known as LOXO-101, ARRY-470) is a potent, orally bioactive, highly selective, ATP competitive TRK inhibitor with IC50s in low nanomolar range (2 to 20 nM) for inhibition of all TRK family members in binding and cellular assays. It has 100x selectivity over other kinases, and is with 2 to 20 nM cellular potency against the TRKA, TRKB, and TRKC kinases. LOXO-101 was evaluated for off-target kinase enzyme inhibition against a panel of non-TRK kinases at a concentration of 1,000 nM and ATP concentrations around the Km. Preclinical models of LOXO-101 using TRK-fusion-bearing human-derived cancer cell lines demonstrate inhibition of the fusion oncoprotein and cellular proliferation in vitro, and tumor growth in vivo.

Kinase Assay: LOXO-101 is a small molecule that was designed to block the ATP binding site of the TRK family of receptors, with 2 to 20 nM cellular potency against the TRKA, TRKB, and TRKC kinases. IC50 value: 2 - 20 nM Target: TRKA/B/C in vitro: LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors. LOXO-101 is evaluated for off-target kinase enzyme inhibition against a panel of 226 non-TRK kinases at a compound concentration of 1,000 nM and ATP concentrations near the Km for each enzyme. In the panel, LOXO-101 demonstrates greater than 50% inhibition for only one non-TRK kinase (TNK2 IC50, 576 nM). Measurement of proliferation following treatment with LOXO-101 demonstrates a dose-dependent inhibition of cell proliferation in all three cell lines. The IC50 is less than 100 nM for CUTO-3.29 and less than 10 nM for KM12 and MO-91, consistent with the known potency of this drug for the TRK kinase family.

Cell Assay: Ba/F3 cells expressing MPRIP-NTRK1 (RIP-TRKA) or EV were lysed after 5 h of treatment with the indicated doses of drugs (ARRY-470; G, gefitinib 1,000 nM) or DMSO control. The cell lysate are used for western bolt analysis.