Design and Evaluation of Stable Cysteine-Modified Monobody Scaffolds for Mirror-Image Synthesis

Mirror-image proteins (d-proteins) are promising therapeutic molecules with high biological stability and low immunogenicity. We recently developed a novel d-monobody scaffold variant with reduced immunogenicity. This variant incorporates two cysteine substitutions that enable the chemical synthesis of d-monobodies via native chemical ligation. In this study, the structure-activity relationship of monobody scaffold variants was investigated to identify more suitable positions for cysteine modifications. Several monobody variants with different cysteine substitution patterns and additional cysteine-selective modifications were designed and synthesized. Comprehensive functional analysis of the synthetic monobody derivatives led to the identification of a favorable monobody scaffold with potent target binding and high thermal stability. The optimized monobody scaffold with a cysteine cross-linker was used to develop d-monobody with additional functional groups.

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Publication Details

Journal
Bioconjugate Chemistry
Published
2025-06-21
DOI
https://doi.org/10.1021/acs.bioconjchem.5c00181
Primary Topic
Monoclonal and Polyclonal Antibodies Research
Type
article
Field-Weighted Citation Impact
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article

Design and Evaluation of Stable Cysteine-Modified Monobody Scaffolds for Mirror-Image Synthesis

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2 links
Bioconjugate Chemistry
Monoclonal and Polyclonal Antibodies Research
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Design and Evaluation of Stable Cysteine-Modified Monobody Scaffolds for Mirror-Image Synthesis

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article en

Abstract

Mirror-image proteins (d-proteins) are promising therapeutic molecules with high biological stability and low immunogenicity. We recently developed a novel d-monobody scaffold variant with reduced immunogenicity. This variant incorporates two cysteine substitutions that enable the chemical synthesis of d-monobodies via native chemical ligation. In this study, the structure-activity relationship of monobody scaffold variants was investigated to identify more suitable positions for cysteine modifications. Several monobody variants with different cysteine substitution patterns and additional cysteine-selective modifications were designed and synthesized. Comprehensive functional analysis of the synthetic monobody derivatives led to the identification of a favorable monobody scaffold with potent target binding and high thermal stability. The optimized monobody scaffold with a cysteine cross-linker was used to develop d-monobody with additional functional groups.

Bioconjugate ChemistryVol. 36(7)
Kyoto University (JP), Kyoto Pharmaceutical University (JP), Medicina (PL), Nagoya University (JP), Graduate School USA (US), Kumamoto University (JP)
Takeda Science Foundation, Japan Agency for Medical Research and Development, Tokyo Biochemical Research Foundation, Astellas Foundation for Research on Metabolic Disorders, Terumo Foundation for Life Sciences and Arts, Japan Society for the Promotion of Science
Openalex Percentile: Top 78%
Monoclonal and Polyclonal Antibodies Research
DOISourceOpenAlex
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