Rational design of a covalent ACE2 decoy receptor that broadly neutralizes SARS-CoV-2 variants

Hino, Nobumasa; Takada, Risa; Suzuki, Kyosuke; Tokunoh, Nagisa; Karaki, Tatsuya; Ohtake, Kazumasa; Ogami, Haruna; Nishiura, Takafumi; Ishimoto, Kenji; Tsukamoto, Tomohito; Ago, Yukio; Okada, Yoshiaki; Okamoto, Toru; Ono, Chikako; Matsuura, Yoshiharu; Obika, Satoshi; Sakamoto, Kensaku; Yoshioka, Yasuo; Nakagawa, Shinsaku

doi:https://doi.org/10.1002/pro.70306

Rational design of a covalent ACE2 decoy receptor that broadly neutralizes SARS-CoV-2 variants

Hino, Nobumasa; Takada, Risa; Suzuki, Kyosuke et al.

Protein Science, 2025, 34(10), e70306

Number of Access:15（2025-11-23 11:06 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/102968

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Title	Rational design of a covalent ACE2 decoy receptor that broadly neutralizes SARS-CoV-2 variants
Creator	NRID 1000090469916 1000090469916 ORCID 0000-0002-7072-1183 0000-0002-7072-1183 researchmap permalink crosslinker crosslinker Scopus Author ID 24175680600 24175680600 CreatorName Hino, Nobumasa
	CreatorName Takada, Risa
	CreatorName Suzuki, Kyosuke
	CreatorName Tokunoh, Nagisa
	CreatorName Karaki, Tatsuya
	NRID 1000080593631 1000080593631 CreatorName Ohtake, Kazumasa
	CreatorName Ogami, Haruna
	CreatorName Nishiura, Takafumi
	CreatorName Ishimoto, Kenji
	CreatorName Tsukamoto, Tomohito
	CreatorName Ago, Yukio
	NRID 1000050444500 1000050444500 researchmap permalink magic_roundabout magic_roundabout Scopus Author ID 55721995000 55721995000 CreatorName Okada, Yoshiaki
	NRID 1000080628595 1000080628595 researchmap permalink TOKAMOTO TOKAMOTO Scopus Author ID 10144905800 10144905800 CreatorName Okamoto, Toru
	NRID 1000030626437 1000030626437 researchmap permalink ChikakoOno ChikakoOno Scopus Author ID 54990934700 54990934700 CreatorName Ono, Chikako
	NRID 1000050157252 1000050157252 ORCID 0000-0001-9091-8285 0000-0001-9091-8285 researchmap permalink matsuura_123 matsuura_123 Scopus Author ID 7202988996 7202988996 CreatorName Matsuura, Yoshiharu
	NRID 1000080243252 1000080243252 ORCID 0000-0002-6842-6812 0000-0002-6842-6812 researchmap permalink read0014368 read0014368 Scopus Author ID 7003800550 7003800550 CreatorName Obika, Satoshi
	NRID 1000050240685 1000050240685 CreatorName Sakamoto, Kensaku
	NRID 1000000392308 1000000392308 ORCID 0000-0002-7265-9221 0000-0002-7265-9221 researchmap permalink read0076530 read0076530 Scopus Author ID 55502847200 55502847200 CreatorName Yoshioka, Yasuo
	NRID 1000070207728 1000070207728 researchmap permalink read0042859 read0042859 CreatorName Nakagawa, Shinsaku
Subject	ACE2-Fc
	covalent proteins
	cross-linking
	genetic code expansion
	non-canonical amino acids
	SARS-CoV-2
Description	Hino N., Takada R., Suzuki K., et al. Rational design of a covalent ACE2 decoy receptor that broadly neutralizes SARS-CoV-2 variants. Protein Science 34, e70306 (2025); https://doi.org/10.1002/pro.70306.
Abstract	The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of variants that evade existing vaccines and antibody therapies necessitate novel, potent, and broad-spectrum antiviral strategies. Enhancing therapeutic proteins with additional covalent binding capabilities, such as minibinders and nanobodies, reportedly potentiates their antiviral efficacy by irreversibly capturing the viral cell-entry protein. However, viral mutations that interfere with covalent bonding or reduce viral affinity with therapeutic proteins might compromise the efficacy of this strategy. Therefore, in this study, we aimed to develop a broadly neutralizing covalent angiotensin-converting enzyme 2-Fc (ACE2-Fc) decoy using a rational design strategy that integrates functional genomics with structural information. Using this approach, we targeted a highly conserved and functionally constrained residue on the viral receptor-binding domain (RBD) and identified tyrosine 473 (Y473) as an optimal target. We engineered ACE2-Fc constructs by replacing glutamate 23 (E23) and threonine 27 (T27) with the non-canonical amino acid—fluorosulfate-L-tyrosine (FSY), generating E23FSY and T27FSY constructs. These constructs formed a specific and efficient covalent bond with Y473 of the RBD. Notably, this covalent capture was retained against the highly mutated Omicron BA.5 RBD. In pseudovirus neutralization assays, both E23FSY and T27FSY exhibited markedly enhanced potency against both wild-type-like (D614G) and Omicron variants compared to their non-covalent counterparts. These results demonstrate that using an inherently escape-resistant decoy receptor to covalently target evolutionarily constrained residues on the viral RBD is a highly efficient strategy for creating potent, broad-spectrum covalent inhibitors against rapidly evolving viruses such as SARS-CoV-2.
Publisher	John Wiley and Sons Inc
Source Title	Protein Science
Volume (Issue)	34(10)
Page	e70306
Date of Issued	2025-09-13
Language	English
Handle URL	https://hdl.handle.net/11094/102968
PISSN	09618368
eISSN	1469896X
Relation.isIdenticalTo	DOI (Publisher Version) https://doi.org/10.1002/pro.70306
Access Rights	open access
CopyRight	This article is licensed under a Creative Commons Attribution 4.0 International License.
CopyRight	© 2025 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
oaire:version	Version of Record
Category	学術雑誌論文 Journal Article

Resource Type	journal article
Local Type	Journal Article
DCMI Type	Text
DCTERMS.bibliographicCitation	Protein Science. 2025, 34(10), p. e70306
DC.title	Rational design of a covalent ACE2 decoy receptor that broadly neutralizes SARS-CoV-2 variants
DC.creator	Hino, Nobumasa
	Takada, Risa
	Suzuki, Kyosuke
	Tokunoh, Nagisa
	Karaki, Tatsuya
	Ohtake, Kazumasa
	Ogami, Haruna
	Nishiura, Takafumi
	Ishimoto, Kenji
	Tsukamoto, Tomohito
	Ago, Yukio
	Okada, Yoshiaki
	Okamoto, Toru
	Ono, Chikako
	Matsuura, Yoshiharu
	Obika, Satoshi
	Sakamoto, Kensaku
	Yoshioka, Yasuo
	Nakagawa, Shinsaku
DC.publisher	John Wiley and Sons Inc
DC.language' scheme='DCTERMS.RFC1766	English
DC.type' scheme='DCTERMS.DCMIType	Text
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2025-09-13
DC.identifier	https://doi.org/10.1002/pro.70306
DC.subject	ACE2-Fc
	covalent proteins
	cross-linking
	genetic code expansion
	non-canonical amino acids
	SARS-CoV-2
DC.description	Hino N., Takada R., Suzuki K., et al. Rational design of a covalent ACE2 decoy receptor that broadly neutralizes SARS-CoV-2 variants. Protein Science 34, e70306 (2025); https://doi.org/10.1002/pro.70306.
DCTERMS.abstract	The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of variants that evade existing vaccines and antibody therapies necessitate novel, potent, and broad-spectrum antiviral strategies. Enhancing therapeutic proteins with additional covalent binding capabilities, such as minibinders and nanobodies, reportedly potentiates their antiviral efficacy by irreversibly capturing the viral cell-entry protein. However, viral mutations that interfere with covalent bonding or reduce viral affinity with therapeutic proteins might compromise the efficacy of this strategy. Therefore, in this study, we aimed to develop a broadly neutralizing covalent angiotensin-converting enzyme 2-Fc (ACE2-Fc) decoy using a rational design strategy that integrates functional genomics with structural information. Using this approach, we targeted a highly conserved and functionally constrained residue on the viral receptor-binding domain (RBD) and identified tyrosine 473 (Y473) as an optimal target. We engineered ACE2-Fc constructs by replacing glutamate 23 (E23) and threonine 27 (T27) with the non-canonical amino acid—fluorosulfate-L-tyrosine (FSY), generating E23FSY and T27FSY constructs. These constructs formed a specific and efficient covalent bond with Y473 of the RBD. Notably, this covalent capture was retained against the highly mutated Omicron BA.5 RBD. In pseudovirus neutralization assays, both E23FSY and T27FSY exhibited markedly enhanced potency against both wild-type-like (D614G) and Omicron variants compared to their non-covalent counterparts. These results demonstrate that using an inherently escape-resistant decoy receptor to covalently target evolutionarily constrained residues on the viral RBD is a highly efficient strategy for creating potent, broad-spectrum covalent inhibitors against rapidly evolving viruses such as SARS-CoV-2.
DC.rights	This article is licensed under a Creative Commons Attribution 4.0 International License.
DC.rights	© 2025 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
DC.format	102968
citation_title	Rational design of a covalent ACE2 decoy receptor that broadly neutralizes SARS-CoV-2 variants
citation_author	Hino, Nobumasa
	Takada, Risa
	Suzuki, Kyosuke
	Tokunoh, Nagisa
	Karaki, Tatsuya
	Ohtake, Kazumasa
	Ogami, Haruna
	Nishiura, Takafumi
	Ishimoto, Kenji
	Tsukamoto, Tomohito
	Ago, Yukio
	Okada, Yoshiaki
	Okamoto, Toru
	Ono, Chikako
	Matsuura, Yoshiharu
	Obika, Satoshi
	Sakamoto, Kensaku
	Yoshioka, Yasuo
	Nakagawa, Shinsaku
citation_publisher	John Wiley and Sons Inc
citation_language	English
citation_date	2025-09-13
citation_journal_title	Protein Science
citation_volume	34
citation_issue	10
citation_firstpage	e70306
citation_doi	https://doi.org/10.1002/pro.70306
citation_public_url	https://hdl.handle.net/11094/102968

Rational design of a covalent ACE2 decoy receptor that broadly neutralizes SARS-CoV-2 variants

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