Screening of protein-based inhibitors for the intracellular domain of epidermal growth factor receptor by directed evolution using the yeast Gγ recruitment system

Ririka Asama; Masahiro Tominaga; Sayaka Ito; Yoichiro Ito; Kazuhiro Takemura; Shun Sakuraba; Kohei Katsurada; Nobuo Fukuda; Akihiko Kondo; Jun Ishii

doi:10.1016/j.jbiosc.2024.07.007

Screening of protein-based inhibitors for the intracellular domain of epidermal growth factor receptor by directed evolution using the yeast Gγ recruitment system

J Biosci Bioeng. 2024 Nov;138(5):375-381. doi: 10.1016/j.jbiosc.2024.07.007. Epub 2024 Aug 9.

Authors

Affiliations

¹ Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
² Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
³ Ph.D. Program in Biomedical Artificial Intelligence, National Tsing Hua University, Hsinchu 300044, Taiwan.
⁴ Institute for Quantum Life Science, National Institutes for Quantum Science and Technology, 4-9-1 Anagawa, Inage, Chiba 263-8555, Japan; Department of Quantum Life Science, Graduate School of Science, Chiba University, 1-33 Yayoi, Inage, Chiba 265-8522, Japan.
⁵ Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan; Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
⁶ Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; Center for Sustainable Resource Science, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan.
⁷ Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan. Electronic address: [email protected].

PMID: 39122620
DOI: 10.1016/j.jbiosc.2024.07.007

Abstract

Protein-based therapeutics, including antibodies and antibody-like-proteins, have increasingly attracted attention due to their high specificity compared to small-molecular drugs. The Gγ recruitment system, one of the in vivo yeast two-hybrid systems for detecting protein-protein interactions, has been previously developed using yeast signal transduction machinery. In this study, we modified the Gγ recruitment system to screen the protein mutants that efficiently bind to the intracellular domain of the epidermal growth factor receptor L858R mutant (cytoEGFR^L858R). Using the modified platform, we performed in vivo directed evolution for growth factor receptor-bound protein 2 (Grb2) and its truncated variant containing only the Src-homology 2 (SH2) domain, successfully identifying several mutants that more strongly bound to cytoEGFR^L858R than their parental proteins. Some of them contained novel beneficial mutations (F108Y and Q144H) and specifically bound to the recombinant cytosolic phosphorylated EGFR in vitro, highlighting the utility of the evolutionary platform.

Keywords: Directed evolution; EGFR kinase domain; Epidermal growth factor receptor; G protein-coupled receptor signaling; Growth factor receptor-bound protein 2; Phosphotyrosine; Protein-based inhibitor; Protein–protein interaction; Src-homology 2; Yeast two-hybrid system.

MeSH terms

Directed Molecular Evolution*
ErbB Receptors* / genetics
ErbB Receptors* / metabolism
Humans
Mutation
Phosphorylation
Protein Binding
Protein Domains
Saccharomyces cerevisiae / genetics
Saccharomyces cerevisiae / metabolism
Two-Hybrid System Techniques
src Homology Domains

Substances

ErbB Receptors
EGFR protein, human