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AIM:To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor(EGFR).METHODS:A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790 M,which is resistant to small molecular weight tyrosine kinase inhibitors(TKIs) for EGFR in nonsmall cell lung cancers(NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858 R in NCI-H1975 cells,an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound,Janus kinase 3(JAK3) inhibitor Ⅵ,on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and-negative lung cancer cell lines. JAK3 inhibitor Ⅵ was modeled into the ATP-binding pocket of EGFR T790M/L858 R. Potential physical interactions between the compound and kinase domains of wild-type(WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790 M and JAKs. RESULTS:We found that JAK3 inhibitor Ⅵ,a known inhibitor for JAK3 tyrosine kinase,selectively inhibits EGFR T790M/L858 R,but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor Ⅵ also specifically reduced autophosphorylation of EGFR T790M/L858 R in NCI-H1975 cells upon EGF stimulation,but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore,JAK3 inhibitor Ⅵ suppressed the proliferationof NCI-H1975 cells,but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549.A docking simulation between JAK3 inhibitor Ⅵ and the ATP-binding pocket of EGFR T790M/L858 R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor Ⅵ to EGFR T790M/L858 R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT,similar to EGFR T790 M,suggesting that TKIs for JAKs may also be effective for EGFR T790 M. CONCLUSION:Our findings demonstrate that JAK3 inhibitor Ⅵ is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790 M inhibitors.
AIM: To identify non-quinazoline kinase inhibitors effective against drug-resistant mutants of epidermal growth factor receptor (EGFR). METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to in vitro kinase activation of EGFR with the gatekeeper mutation T790 M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in nonsmall cell lung cancers (NSCLCs). This inhibitory effect was was by measuring autophosphorylation of EGFR T790M / L858 R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor Ⅵ, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and-negative lung cancer cell lines. JAK3 inhibitor Ⅵ was modeled into the ATP-binding pocket of EGFR T790M / L858 R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 we re estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790 M and JAKs. RESULTS: We found that JAK3 inhibitor Ⅵ, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M / L858 R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor Ⅵ also specifically reduced autophosphorylation of EGFR T790M / L858 R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor Ⅵ suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor Ⅵ and the ATP-binding pocket of EGFR T790M / L858 R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor Ⅵ to EGFR T790M / L858 R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignme nts revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790 M, suggesting that TKIs for JAKs may also be effective for EGFR T790 M. CONCLUSION: Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790 M inhibitors.