Identification of novel inhibitors of tyrosine kinase receptor RET

Germline and somatic point mutations of RET receptor tyrosine kinase cause multiple endocrine neoplasia (MEN) type 2 syndromes and sporadic medullary thyroid carcinoma (MTC). Moreover, RET gene rearrangements are associated to papillary thyroid carcinoma (PTC), lung adenocarcinoma (ADC) and chronic myelomonocytic leukemia (CMML). Recently Vandetanib (ZD6474), a multiple kinase inhibitor (KI) targeting RET, has been approved for MTC treatment. We tested 22 novel KIs with different structure and specificity for their ability to inhibit RET activity in NIH3T3 fibroblasts expressing MTC-associated RET C634R and M918T oncogenic mutants. Among them, we selected three structurally similar type II tyrosine kinase inhibitors, ALW-II-41-27, HG-6-63-01 and XMD15-44, that were able to significantly reduce RET phosphorylation at 10 nM dose. ALW-II-41-27, HG-6-63-01 and XMD15-44 blocked RET-mediated signaling and proliferation with a half maximal inhibitory concentration (IC50) of less than 50 nM in rat fibroblasts transformed by RET/C634R and RET/M918T, while they were poorly effective on parental RAT1 cells (IC50 >200nM). Although with different efficacy, the three compounds inhibited various MTC-associated RET intracellular mutants (RET E768D, L790F, Y791F, S891A, V804L/M and A883F) and RET chimeric oncogenes (RET/PTC1, RET/PTC3, KIF5B-RET and FGFR1OP-RET). In addition, ALW-II-41-27, HG-6-63-01 and XMD15-44 inhibited RET activity and signaling in human cell lines (TT, MZCRC1 and TPC1) carrying oncogenic RET alleles (C634W, M918T and RET/PTC1, respectively) and blocked the growth of TT and MZCRC1 cells with an IC50 of 1-5 nM and of TPC1 cells with an IC50 of 10-50 nM. Proliferation of non-tumoral human thyroid follicular cells (Nthy-ori 3-1) growth was virtually unaffected (IC50 350-1000 nM). Finally, in nude mice, ALW-II-41-27 (40 mg/kg i.p once per day) reduced growth of RET/C634Y-fibroblast xenografts by more than 50% (1,280 mm3 drug-treated vs 2,810 mm3 vehicle-treated). In conclusion, we have identified a pharmacophore (3-triflouromethyl-4-methylpiperazinepheny), shared by ALW-II-41-27, HG-6-63-01 and XMD15-44, that may be optimized in order to develop potent and selective RET inhibitors for the treatment of human cancers sustaining oncogenic activation of RET.