A rebinding-assay for measuring extreme kinetics using label-free biosensors

Abstract In vitro kinetic measurements allow mechanistic characterization of binding interactions and are particularly valuable throughout drug discovery, from confirmation of on-target binding in early discovery to fine-tuning of drug-binding properties in pre-clinical development. Early chemical m...

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Autor principal:	John G. Quinn
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ecb365e6b26849afba262a4b5282f867
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spelling	oai:doaj.org-article:ecb365e6b26849afba262a4b5282f8672021-12-02T15:51:14ZA rebinding-assay for measuring extreme kinetics using label-free biosensors10.1038/s41598-021-87880-x2045-2322https://doaj.org/article/ecb365e6b26849afba262a4b5282f8672021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-87880-xhttps://doaj.org/toc/2045-2322Abstract In vitro kinetic measurements allow mechanistic characterization of binding interactions and are particularly valuable throughout drug discovery, from confirmation of on-target binding in early discovery to fine-tuning of drug-binding properties in pre-clinical development. Early chemical matter often exhibits transient kinetics, which remain challenging to measure in a routine drug discovery setting. For example, characterization of irreversible inhibitors has classically relied on the alkylation rate constant, yet this metric fails to resolve its fundamental constituent rate constants, which drive reversible binding kinetics and affinity complex inactivation. In other cases, extremely rapid association processes, which can approach the diffusion limit, also remain challenging to measure. To address these limitations, a practical kinetic rebinding assay is introduced that may be applied for kinetic screening and characterization of compounds. The new capabilities afforded by this probe-based assay emerge from mixed-phase partitioning in a flow-injection configuration and have been implemented using label-free biosensing. A finite element analysis-based biosensor model, simulating inhibition of rebinding within a crowded hydrogel milieu, provided surrogate test data that enabled development and validation of an algebraic model for estimation of kinetic interaction constants. An experimental proof-of-principle demonstrating estimation of the association rate constant, decoupled from the dissociation process, provided further validation.John G. QuinnNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q John G. Quinn A rebinding-assay for measuring extreme kinetics using label-free biosensors
description	Abstract In vitro kinetic measurements allow mechanistic characterization of binding interactions and are particularly valuable throughout drug discovery, from confirmation of on-target binding in early discovery to fine-tuning of drug-binding properties in pre-clinical development. Early chemical matter often exhibits transient kinetics, which remain challenging to measure in a routine drug discovery setting. For example, characterization of irreversible inhibitors has classically relied on the alkylation rate constant, yet this metric fails to resolve its fundamental constituent rate constants, which drive reversible binding kinetics and affinity complex inactivation. In other cases, extremely rapid association processes, which can approach the diffusion limit, also remain challenging to measure. To address these limitations, a practical kinetic rebinding assay is introduced that may be applied for kinetic screening and characterization of compounds. The new capabilities afforded by this probe-based assay emerge from mixed-phase partitioning in a flow-injection configuration and have been implemented using label-free biosensing. A finite element analysis-based biosensor model, simulating inhibition of rebinding within a crowded hydrogel milieu, provided surrogate test data that enabled development and validation of an algebraic model for estimation of kinetic interaction constants. An experimental proof-of-principle demonstrating estimation of the association rate constant, decoupled from the dissociation process, provided further validation.
format	article
author	John G. Quinn
author_facet	John G. Quinn
author_sort	John G. Quinn
title	A rebinding-assay for measuring extreme kinetics using label-free biosensors
title_short	A rebinding-assay for measuring extreme kinetics using label-free biosensors
title_full	A rebinding-assay for measuring extreme kinetics using label-free biosensors
title_fullStr	A rebinding-assay for measuring extreme kinetics using label-free biosensors
title_full_unstemmed	A rebinding-assay for measuring extreme kinetics using label-free biosensors
title_sort	rebinding-assay for measuring extreme kinetics using label-free biosensors
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/ecb365e6b26849afba262a4b5282f867
work_keys_str_mv	AT johngquinn arebindingassayformeasuringextremekineticsusinglabelfreebiosensors AT johngquinn rebindingassayformeasuringextremekineticsusinglabelfreebiosensors
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A rebinding-assay for measuring extreme kinetics using label-free biosensors

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