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Luminescence-based complementation assay to assess target engagement and cell permeability of glycolate oxidase (HAO1) inhibitors.
Mackinnon, Sabrina R; Zarganes-Tzitzikas, Tryfon; Adams, Cassandra J; Brennan, Paul E; Yue, Wyatt W.
Afiliación
  • Mackinnon SR; Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK.
  • Zarganes-Tzitzikas T; Alzheimer's Research UK Oxford Drug Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, Oxford, UK.
  • Adams CJ; Centre for Medicines Discovery, Nuffield Department of Medicine Research Building (NDMRB), University of Oxford, Oxford, UK.
  • Brennan PE; Centre for Medicines Discovery, Nuffield Department of Medicine Research Building (NDMRB), University of Oxford, Oxford, UK. Electronic address: paul.brennan@cmd.ox.ac.uk.
  • Yue WW; Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK. Electronic address: wyatt.yue@newcastle.ac.uk.
Biochimie ; 2024 Aug 14.
Article en En | MEDLINE | ID: mdl-39151880
ABSTRACT
Glycolate oxidase (HAO1) catalyses the synthesis of glyoxylate, a common metabolic intermediate that causes renal failure if accumulated. HAO1 inhibition is an emerging treatment for primary hyperoxaluria, a rare disorder of glyoxylate metabolism. Here we report the first cell-based measurement of inhibitor uptake and engagement with HAO1, by adapting the cellular thermal shift assay (CETSA) based on Nano luciferase complementation and luminescence readout. By profiling the interaction between HAO1 and four well-characterised inhibitors in intact and lysed HEK293T cells, we showed that our CETSA method differentiates between low-permeability/high-engagement and high-permeability/low-engagement ligands and is able to rank HAO1 inhibitors in line with both recombinant protein methods and previously reported indirect cellular assays. Our methodology addresses the unmet need for a robust, sensitive, and scalable cellular assay to guide HAO1 inhibitor development and, in broader terms, can be rapidly adapted for other targets to simultaneously monitor compound affinity and cellular permeability.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Biochimie Año: 2024 Tipo del documento: Article País de afiliación: Reino Unido Pais de publicación: Francia
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Biochimie Año: 2024 Tipo del documento: Article País de afiliación: Reino Unido Pais de publicación: Francia