In vitro activity: TPX-0005 is an orally available and potent ATP-competitive inhibitor against ALK, ROS1, TRKA, TRKB and TRKC recombinant kinases and their corresponding clinical resistant mutants.TPX-0005 inhibits H2228 cell migration in a wound healing assay with similar activity to saracatinib. It can not only inhibit the wild-type and a broad spectrum of mutant ALKs, but also overcome primary resistance and suppress metastatic features by inhibiting SRC.

Kinase Assay: TPX-0005 is a novel, rationally-designed, highly potent inhibitor of ALK/ROS1/TRK with IC50 of 5.3 nM, 1.01 nM, 1.26 nM and 1.08 nM for SRC, WT ALK, ALK G1202R and ALK L1196M, respectively. It has potential anticancer activity. It effectively overcomes this primary resistance (IC50 100 nM in cell proliferation assay) with strong inhibition of the phosphorylation of EML4-ALK (IC50 13 nM) and the SRC substrate paxillin (IC50 107 nM). PX-0005 inhibits H2228 cell migration in a wound healing assay with similar activity to saracatinib. Overall, TPX-0005 has a highly favorable profile with ability to overcome multiple ALK resistance mechanisms including secondary mutations, bypass signaling activation, EMT, and warrants clinical investigation

Cell Assay: TPX-0005 is also a potent SRC inhibitor (IC50 5.3 nM). The elevated SRC kinase activity in H2228 lung cancer cell line confers resistance to crizotinib (IC50 1200 nM) and ceritinib (IC50 1000 nM) in cell proliferation assays. TPX-0005 effectively overcame this primary resistance (IC50 100 nM in cell proliferation assay) with strong inhibition of the phosphorylation of EML4-ALK (IC50 13 nM) and the SRC substrate paxillin (IC50 107 nM), along with other downstream signaling targets. TPX-0005 inhibited H2228 cell migration in a wound healing assay with similar activity to saracatinib.