The MaMTH-DS assay detected inhibitors of mutant EGFR in non–small cell lung cancer cells.

  • Major Finding: The MaMTH-DS assay detected inhibitors of mutant EGFR in non–small cell lung cancer cells.

  • Concept: Two inhibitors would not have been identified by standard in vitro kinase or cell-based assays.

  • Impact: MaMTH-DS, which can be adapted for other receptor tyrosine kinases, provided four candidate drugs.

In vitro kinase assays have been successful in identifying inhibitors of cancer-driving mutant receptor tyrosine kinases (RTK) such as mutant EGFR. However, these assays have limitations, including the inability to detect inhibitors that require additional components to mediate their effects and the inability to test for important drug characteristics, such as lack of cellular toxicity. Saraon, Snider, and colleagues describe an adaptation of their previously developed mammalian membrane two-hybrid (MaMTH) assay, originally designed to detect protein–protein interactions between integral membrane proteins in live-cell membranes. In the new assay, termed MaMTH-DS (for MaMTH drug screening), RTKs—which are membrane proteins—are subjected to high-throughput screening for inhibition by small molecules. As a proof of principle, MaMTH-DS was used to test a panel of 2,960 small-molecule candidates for inhibition of an osimertinib-resistant EGFR triple mutant on non–small cell lung cancer (NSCLC) cells. Using this screening method, three new chemicals (midostaurin, AZD7762, and EMI1) emerged as inhibitors of mutant but not wild-type EGFR, and one more candidate compound (gilteritinib) was identified based on functional similarity to one of the inhibitors. Biochemical assays and experiments using EGFR-mutant NSCLC cell lines and organoids validated that these compounds were potent and specific. These results imply that midostaurin and gilteritinib, which were recently approved by the FDA to treat FLT3-mutant acute myeloid leukemia, may be of use in certain EGFR-mutant NSCLCs. Importantly, AZD7762 and EMI1 would not have been identified as mutant-EGFR inhibitors in commonly used in vitro or cell-based assays. Collectively, this work provides new candidate drugs for further investigation and demonstrates the utility of MaMTH-DS as a screening platform—and, notably, the assay could be adapted to identify inhibitors of RTKs other than EGFR as well.

Saraon P, Snider J, Kalaidzidis Y, Wybenga-Groot LE, Weiss K, Rai A, et al. A drug discovery platform to identify compounds that inhibit EGFR triple mutants. Nat Chem Biol 2020 Feb 24 [Epub ahead of print].

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