Engineering an autonomous VH domain to modulate intracellular pathways and to interrogate the eIF4F complex

Yuri Frosi, Yen-Chu Lin, Jiang Shimin, Siti Radhiah Ramlan, Kelly Hew, Alf Henrik Engman, Anil Pillai, Kit Yeung, Yue Xiang Cheng, Tobias Cornvik, Par Nordlund, Megan Goh, Dilraj Lama, Zachary P. Gates, Chandra S. Verma, Dawn Thean, David P. Lane, Ignacio Asial () and Christopher J. Brown ()
Additional contact information
Yuri Frosi: p53 Laboratory (A*STAR)
Yen-Chu Lin: p53 Laboratory (A*STAR)
Jiang Shimin: p53 Laboratory (A*STAR)
Siti Radhiah Ramlan: p53 Laboratory (A*STAR)
Kelly Hew: DotBio Pte. Ltd., 1 Research Link
Alf Henrik Engman: DotBio Pte. Ltd., 1 Research Link
Anil Pillai: DotBio Pte. Ltd., 1 Research Link
Kit Yeung: DotBio Pte. Ltd., 1 Research Link
Yue Xiang Cheng: DotBio Pte. Ltd., 1 Research Link
Tobias Cornvik: Nanyang Technological University
Par Nordlund: DotBio Pte. Ltd., 1 Research Link
Megan Goh: p53 Laboratory (A*STAR)
Dilraj Lama: Karolinska Institutet
Zachary P. Gates: Institute of Molecular and Cell Biology, A*STAR
Chandra S. Verma: Nanyang Technological University
Dawn Thean: p53 Laboratory (A*STAR)
David P. Lane: p53 Laboratory (A*STAR)
Ignacio Asial: DotBio Pte. Ltd., 1 Research Link
Christopher J. Brown: p53 Laboratory (A*STAR)

Nature Communications, 2022, vol. 13, issue 1, 1-22

Abstract: Abstract An attractive approach to target intracellular macromolecular interfaces and to model putative drug interactions is to design small high-affinity proteins. Variable domains of the immunoglobulin heavy chain (VH domains) are ideal miniproteins, but their development has been restricted by poor intracellular stability and expression. Here we show that an autonomous and disufhide-free VH domain is suitable for intracellular studies and use it to construct a high-diversity phage display library. Using this library and affinity maturation techniques we identify VH domains with picomolar affinity against eIF4E, a protein commonly hyper-activated in cancer. We demonstrate that these molecules interact with eIF4E at the eIF4G binding site via a distinct structural pose. Intracellular overexpression of these miniproteins reduce cellular proliferation and expression of malignancy-related proteins in cancer cell lines. The linkage of high-diversity in vitro libraries with an intracellularly expressible miniprotein scaffold will facilitate the discovery of VH domains suitable for intracellular applications.

Date: 2022
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DOI: 10.1038/s41467-022-32463-1

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