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EGFR signaling and pharmacology in oncology revealed with innovative BRET-based biosensors.

ABSTRACT: Mutations of receptor tyrosine kinases (RTKs) are associated with the development of many cancers by modifying receptor signaling and contributing to drug resistance in clinical settings. We present enhanced bystander bioluminescence resonance energy transfer-based biosensors providing new insights into RTK biology and pharmacology critical for the development of more effective RTK-targeting drugs. Distinct SH2-specific effector biosensors allow for real-time and spatiotemporal monitoring of signal transduction pathways engaged upon RTK activation. Using EGFR as a model, we demonstrate the capacity of these biosensors to differentiate unique signaling signatures, with EGF and Epiregulin ligands displaying differences in efficacy, potency, and responses within different cellular compartments. We further demonstrate that EGFR single point mutations found in Glioblastoma or non-small cell lung cancer, impact the constitutive activity of EGFR and response to tyrosine kinase inhibitor. The BRET-based biosensors are compatible with microscopy, and more importantly characterize the next generation of therapeutics directed against RTKs.

SUBMITTER: Gross F

PROVIDER: S-EPMC10907714 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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EGFR signaling and pharmacology in oncology revealed with innovative BRET-based biosensors.

Gross Florence F Mancini Arturo A Breton Billy B Kobayashi Hiroyuki H Pereira Pedro Henrique Scarpelli PHS Le Gouill Christian C Bouvier Michel M Schann Stephan S Leroy Xavier X Sabbagh Laurent L

Communications biology 20240301 1

Mutations of receptor tyrosine kinases (RTKs) are associated with the development of many cancers by modifying receptor signaling and contributing to drug resistance in clinical settings. We present enhanced bystander bioluminescence resonance energy transfer-based biosensors providing new insights into RTK biology and pharmacology critical for the development of more effective RTK-targeting drugs. Distinct SH2-specific effector biosensors allow for real-time and spatiotemporal monitoring of sig ...[more]

PMID: 38429428

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Project description:The 3C-like protease (3CLpro) of SARS-CoV-2 is an attractive drug target for developing antivirals against SARS-CoV-2. A few small molecule inhibitors of 3CLpro are in clinical trials for COVID-19 treatments, and more inhibitors are under development. One limiting factor for 3CLpro inhibitors development is that the cellular activities of such inhibitors should be evaluated in Biosafety Level 3 (BSL-3) laboratories. Here, we design DNA-coded biosensors that can be used in BSL-2 laboratories to set up cell-based assays for 3CLpro inhibitor discovery. The biosensors were constructed by linking a green fluorescent protein (GFP2) to the N-terminus and a Renilla luciferase (RLuc8) to the C-terminus of SARS-CoV-2 3CLpro, with the linkers derived from the cleavage sequences of 3CLpro. After overexpression of the biosensors in human embryonic kidney (HEK) 293T cells, 3CLpro can be released from GFP2 and RLuc by self-cleavage, resulting in a decrease of the bioluminescence resonance energy transfer (BRET) signal. Using one of these biosensors, pBRET-10, we evaluated the cellular activities of several 3CLpro inhibitors. These inhibitors restored the BRET signal by blocking the proteolysis of pBRET-10, and their relative activities measured using pBRET-10 were consistent with their previously reported anti-SARS-CoV-2 activities. We conclude that the biosensor pBRET-10 is a useful tool for SARS-CoV-2 3CLpro inhibitor discovery. IMPORTANCE The virus proteases 3CLpro are validated drug targets for developing antivirals to treat coronavirus diseases, such as COVID-19. However, the development of 3CLpro inhibitors relies heavily on BSL-3 laboratories. Here, we report a series of BRET-based self-cleaving biosensors that can be used to set up cell-based assays to evaluate the cell permeability and cellular activity of SARS-CoV-2 3CLpro inhibitors in BSL-2 laboratories. The cell-based assay is suitable for high-throughput screening for 3CLpro inhibitors because of the simplicity and good reproducibility of our biosensors. The design strategy can also be used to design biosensors for other viral proteases for which the activation processes involve the self-cleavage of polyproteins.

Dataset Information

EGFR signaling and pharmacology in oncology revealed with innovative BRET-based biosensors.

Publications

EGFR signaling and pharmacology in oncology revealed with innovative BRET-based biosensors.

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