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A drug discovery platform to identify compounds that inhibit EGFR triple mutants

Abstract

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein–protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.

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Fig. 1: Overview of MaMTH-DS platform and top candidates identified in pilot screen.
Fig. 2: Validation of midostaurin and gilteritinib as EGFR ex19del/T790M/C797S and EGFR L858R/T790M/C797S activating mutant inhibitors.
Fig. 3: Validation of EMI1 as an EGFR ex19del/T790M/C797S and EGFR L858R/T790M/C797S activating mutant inhibitor.
Fig. 4: Investigating effect of EMI1 on activated EGFR L858R/T790M/C797S endosomal trafficking.
Fig. 5: Generation and testing of EMI1 chemical analogs.

Data availability

All data supporting the findings presented in this study are available in the paper and its Supplementary Information files.

Code availability

All R code used in the analysis of the presented drug screening data is available from the authors on request.

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Acknowledgements

We thank L. Riley for valuable discussions and editing of this manuscript. We also thank P.A. Jänne and M.J. Eck (both at Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA) for providing us with the PC9-T790M and PC9-C797S cells used in our study, and the Center for Information Services and High Performance Computing of the TU Dresden for providing support and computational resources. This research was supported by funding from Consortium Québécois sur la Découverte du Médicament (CQDM) (Explore) and OCE (no. 23929). In addition, work in the Stagljar laboratory is supported by the Canadian Cancer Society Research Institute (no. 703889), Genome Canada via Ontario Genomics (grant nos, 9427 and 9428), Ontario Research fund (grant nos. ORF/DIG-501411 and RE08-009), CQDM (Quantum Leap) and Brain Canada (Quantum Leap, and Cancer Research Society (grant no. 23235)). J.H.P.’s work in the laboratory of Mark Lemmon at Yale was supported by National Institutes of Health grant nos. R01-CA198164 and R35-GM122485. G.P. acknowledges the support of the Ontario Institute for Cancer Research and its funding from the Government of Ontario.

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Authors

Contributions

P.S. performed preliminary MaMTH testing and validation/functional analysis of identified compounds, managed the project and wrote the manuscript. J.S. generated MaMTH reporter constructs and cells lines, performed MaMTH-DS screening and data analysis, managed the project and wrote the manuscript. Y.K. and M.Z. performed and analyzed the high-throughput EGFR microscopy data. K.W., L.D., A.L., N.V., A.V., S.P., F.A., Z.Y. and V.W. performed construct generation, preliminary MaMTH validation and were involved in the validation/functional analysis of compounds. N.R., N.A.P., Y.W., A.S. and M.S.T. generated and validated organoids and provided cell lines. A.R. and A.A. performed and analyzed microtubule dynamics data. L.E.W.G., A.D. and J.W. performed MaMTH-DS screening. B.T., A.A., M.P., B.J., R.M., D.U. and R.A. performed medicinal chemistry and assisted technically throughout the whole project. G.P. performed computational chemistry, molecular docking and hit expansion. J.H.P. assisted with computational modeling and undertook some biochemical studies. R.M.S., M.J., M.S., M.F.M., N.L. and F.A.S. provided clinical expertise and support. All authors discussed the results and commented on the manuscript. I.S. initiated and supervised the project and wrote the manuscript.

Corresponding author

Correspondence to Igor Stagljar.

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Competing interests

I.S., P.S. and J.S. (in conjunction with the University of Toronto) are listed as inventors on a patent (publication no. 20190091205) for the use of EMI1 (and structurally related analogs), midostaurin, gilteritinib and AZD7762 (and structurally related analogs) in the treatment of mutant EGFR-mediated NSCLC.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–32, Tables 1 and 2 and Notes 1 and 2

Reporting Summary

Supplementary Dataset 1

Collection of compounds used in MaMTH-DS pilot screening.

Supplementary Dataset 2

Results of MaMTH-DS screening of EGFR L858R/T790M/C797S in the presence of ShcI (round 1).

Supplementary Dataset 3

Results of MaMTH-DS screening of EGFR L858R/T790M/C797S in the presence of ShcI (round 2).

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Saraon, P., Snider, J., Kalaidzidis, Y. et al. A drug discovery platform to identify compounds that inhibit EGFR triple mutants. Nat Chem Biol 16, 577–586 (2020). https://doi.org/10.1038/s41589-020-0484-2

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