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Luminescence-based complementation assay to assess target engagement and cell permeability of glycolate oxidase (HAO1) inhibitors

Lookup NU author(s): Dr Sabrina MackinnonORCiD, Professor Wyatt YueORCiD

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

© 2024 The Authors. 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.


Publication metadata

Author(s): Mackinnon SR, Zarganes-Tzitzikas T, Adams CJ, Brennan PE, Yue WW

Publication type: Article

Publication status: Published

Journal: Biochimie

Year: 2024

Pages: ePub ahead of Print

Online publication date: 14/08/2024

Acceptance date: 13/08/2024

Date deposited: 02/09/2024

ISSN (print): 0300-9084

ISSN (electronic): 6183-1638

Publisher: Elsevier BV

URL: https://doi.org/10.1016/j.biochi.2024.08.011

DOI: 10.1016/j.biochi.2024.08.011

PubMed id: 39151880


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Funding

Funder referenceFunder name
Alzheimer's Research UK: ARUK-2021DDI-OX
Fund For Cures UK, Ltd
NIHR Oxford Biomedical Research Centre (BRC), University of Oxford
Wellcome Trust: 092809/Z/10/Z
Wellcome Trust: 106169/ZZ14/Z

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