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JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M.

ABSTRACT: 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 T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R 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/L858R. 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 T790M and JAKs. RESULTS:We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R 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/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R 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 T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M. CONCLUSION:Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.

SUBMITTER: Nishiya N

PROVIDER: S-EPMC4657120 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M.

Nishiya Naoyuki N Sakamoto Yasumitsu Y Oku Yusuke Y Nonaka Takamasa T Uehara Yoshimasa Y

World journal of biological chemistry 20151101 4

<h4>Aim</h4>To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR).<h4>Methods</h4>A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measur ...[more]

PMID: 26629323

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Project description:IntroductionTargeted therapy using specific inhibitors against tyrosine kinases (TKs) is a paradigm in non-small-cell lung cancer management. However, the success of TK inhibitor (TKI) therapy depends on certain activating or acquired mutations, which render sensitivity or resistance to TKIs in the patients. The acquisition of epidermal growth factor receptor (EGFR) T790M point mutation is the most common mechanism of resistance to TKI in non-small cell lung cancer. A number of molecular strategies are now available for molecular testing of non-small cell lung cancers. However, almost all of them are cost-intensive and laborious and require high-end advanced equipment. Thus, assays that are rapid, simple, and cost-effective, yet sensitive, are most ideal in clinical settings for screening such therapeutically relevant mutations.Materials and methodsAllele-specific loop-mediated isothermal amplification assay (AS-LAMP), which is a variant of the original LAMP assay, is a promising diagnostic technique for screening single-nucleotide polymorphisms. Using commercially available plasmid constructs as template DNA, AS-LAMP assay for EGFR T790M mutation was optimized with six different sets of reaction mixture containing varying concentrations of buffer and primers. The results of AS-LAMP assay were further validated by ultrasensitive droplet digital polymerase chain reaction.ResultsOnly one of the six sets of reaction mixture could accurately distinguish between wild type and mutated DNA, indicating that the primers and buffer are the two most critical components that determine the accuracy of AS-LAMP. The optimized AS-LAMP assay was further used to screen germ line and somatic T790M mutations in non-small cell lung cancer using blood and tissue samples collected from patients.ConclusionDevelopment of an accurate and rapid diagnostic assay that can detect resistant mutations without the need for sequencing is highly useful for clinicians in deciding on the eligibility of patients for TKI therapy. Considering its several inherent advantages, AS-LAMP assay could become an effective molecular tool for screening baseline or acquired EGFR T790M mutations in non-small cell lung cancer patients.

Dataset Information

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M.

Publications

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M.

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