Split-enzyme immunoassay to monitor EGFR-HER2 heterodimerization on cell surfaces.

Autor: Kim SJ; Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, United States., Dixon AS; Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, United States., Owen SC; Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, United States; Department of Biomedical Engineering, Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, United States. Electronic address: shawn.owen@hsc.utah.edu.
Jazyk: angličtina
Zdroj: Acta biomaterialia [Acta Biomater] 2021 Nov; Vol. 135, pp. 225-233. Date of Electronic Publication: 2021 Sep 05.
DOI: 10.1016/j.actbio.2021.08.055
Abstrakt: Over 30,000 protein-protein interactions with pathological implications have been identified; yet, discovering and investigating drugs that target these specific interactions is greatly limited by the inability to monitor native protein-protein interactions (PPIs) efficiently. The two most frequently used tools to monitor PPIs, resonance-energy transfer (RET) assays and protein complementation assays (PCA), face significant limitations. RET assays have a narrow working range of 10 to 50 Å, while PCA require permanent attachment of a reporter probe to a protein of interest by chemical conjugation or genetic engineering. We developed a non-invasive assay platform to measure PPIs without modifications to the proteins of interest and is functional at a greater working range than RET assays. We demonstrate our approach by monitoring the EGFR-HER2 heterodimerization on relevant cell surfaces, utilizing various EGFR- and HER2-specific binders (e.g., Fab, DARPin, and VHH) fused with small fragments of a tri-part split-luciferase derived from NanoLuc®. Following independent binding of the binder fusions to their respective targets, the dimerization of EGFR and HER2 induces complementation of the luciferase fragments into a functional native structure, producing glow-type luminescence. We have confirmed the functionality of the platform to monitor EGFR-HER2 dimerization induction and inhibition. STATEMENT OF SIGNIFICANCE: We describe a platform technology for rapid monitoring of protein-protein interactions (PPIs). Our approach is uses a luciferase split into three parts - two short peptide "tags" and a large third fragment. Each of the short peptides can be fused to antibodies which bind to domains of a target antigens which orients the two tags and facilitates refolding of an active enzyme. To our knowledge this is the first example of a split-enzyme used to monitor PPIs without requiring any modification of the target proteins. We demonstrate our approach on the important PPI of HER2 and EGFR. Significantly, we quantify stimulation and inhibition of these partners, opening the possibility of using our approach to assess potential drugs without engineering cells.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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