Target protein identification in live cells and organisms with a non-diffusive proximity tagging system

Published: Dec. 6, 2024

Identifying target proteins for bioactive molecules is essential understanding their mechanisms, developing improved derivatives, and minimizing off-target effects. Despite advances in identification (target-ID) technologies, significant challenges remain, impeding drug development. Most target-ID methods use cell lysates, but maintaining an intact cellular context vital capturing specific drug-protein interactions, such as those with transient protein complexes membrane-associated proteins. To address these limitations, we developed POST-IT (Pup-On-target Small molecule Target Identification Technology), a non-diffusive proximity tagging system live cells, orthogonal to the eukaryotic system. utilizes engineered fusion of proteasomal accessory factor A (PafA) HaloTag transfer Pup proximal upon directly binding small molecule. After optimization eliminate self-pupylation polypupylation, minimize depupylation, optimize chemical linkers, successfully identified known targets discovered new binder, SEPHS2, dasatinib, VPS37C hydroxychloroquine, enhancing our drugs’ mechanisms action. Furthermore, demonstrated application zebrafish embryos, highlighting its potential broad biological research

Language: Английский

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Target protein identification in live cells and organisms with a non-diffusive proximity tagging system

eLife, Journal Year: 2024, Volume and Issue: 13

Published: Dec. 27, 2024

Identifying target proteins for bioactive molecules is essential understanding their mechanisms, developing improved derivatives, and minimizing off-target effects. Despite advances in identification (target-ID) technologies, significant challenges remain, impeding drug development. Most target-ID methods use cell lysates, but maintaining an intact cellular context vital capturing specific drug–protein interactions, such as those with transient protein complexes membrane-associated proteins. To address these limitations, we developed POST-IT (Pup-On-target Small molecule Target Identification Technology), a non-diffusive proximity tagging system live cells, orthogonal to the eukaryotic system. utilizes engineered fusion of proteasomal accessory factor A HaloTag transfer Pup proximal upon directly binding small molecule. After optimization eliminate self-pupylation polypupylation, minimize depupylation, optimize chemical linkers, successfully identified known targets discovered new binder, SEPHS2, dasatinib, VPS37C hydroxychloroquine, enhancing our drugs’ mechanisms action. Furthermore, demonstrated application zebrafish embryos, highlighting its potential broad biological research

Language: Английский

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Painting cell-cell interactions by horseradish peroxidase and endogenously generated hydrogen peroxide

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 13, 2024

ABSTRACT Cell-Cell interactions are fundamental in biology for maintaining physiological conditions, with direct contact being the most straightforward mode of interaction. Recent advancements have led to development various chemical tools detecting or identifying these interactions. However, use exogenous cues, such as toxic reagents, bulky probes, and light irradiations, can disrupt normal cell physiology. For example, toxicity hydrogen peroxide (H 2 O ) limits applications peroxidases proximity labeling field. In this study, we aimed address limitation by demonstrating that membrane-localized Horseradish Peroxidase (HRP-TM) efficiently utilizes endogenously generated extracellular H . By harnessing endogenous , observed HRP-TM-expressing cells effectively label contacting without need treatment. Furthermore, confirmed HRP-TM labels proximal an interaction-dependent manner. These findings offer a novel approach studying cell-cell under more confounding effects stimuli. Our study contributes elucidating interaction networks model organisms, providing valuable insights into dynamic interplay between their native network.

Language: Английский

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Target protein identification in live cells and organisms with a non-diffusive proximity tagging system

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 8, 2024

Abstract Identifying target proteins for bioactive molecules is essential understanding their mechanisms, developing improved derivatives, and minimizing off-target effects. Despite advances in identification (target-ID) technologies, significant challenges remain, impeding drug development. Most target-ID methods use cell lysates, but maintaining an intact cellular context vital capturing specific drug-protein interactions, such as those with transient protein complexes membrane-associated proteins. To address these limitations, we developed POST-IT (Pup-On-target Small molecule Target Identification Technology), a non-diffusive proximity tagging system live cells, orthogonal to the eukaryotic system. utilizes engineered fusion of proteasomal accessory factor A (PafA) HaloTag transfer Pup proximal upon directly binding small molecule. After optimization eliminate self-pupylation polypupylation, minimize depupylation, optimize chemical linkers, successfully identified known targets discovered new binder, SEPHS2, dasatinib, VPS37C hydroxychloroquine, enhancing our drugs’ mechanisms action. Furthermore, demonstrated application zebrafish embryos, highlighting its potential broad biological research

Language: Английский

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Laccase-catalyzed tyrosine click reaction with 1-methyl-4-arylurazole: rapid labeling on protein surfaces

Chemical Communications, Journal Year: 2024, Volume and Issue: 60(96), P. 14208 - 14211

Published: Jan. 1, 2024

Our study shows efficient tyrosine labeling using 1-methyl-4-arylurazole (MAUra) with laccase under mild conditions. This method achieves a high efficiency ( k cat / K m = 7.88 × 10 4 M −1 s ), selectively targeting exposed sites on proteins.

Language: Английский

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Painting Cell–Cell Interactions by Horseradish Peroxidase and Endogenously Generated Hydrogen Peroxide

ACS Chemical Biology, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 18, 2024

Cell-cell interactions are fundamental in biology for maintaining physiological conditions with direct contact being the most straightforward mode of interaction. Recent advancements have led to development various chemical tools detecting or identifying these interactions. However, use exogenous cues, such as toxic reagents, bulky probes, and light irradiation, can disrupt normal cell physiology. For example, toxicity hydrogen peroxide (H

Language: Английский

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