Mackinnon Sabrina R, Zarganes-Tzitzikas Tryfon, Adams Cassandra J, Brennan Paul E, Yue Wyatt W
Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK.
Alzheimer's Research UK Oxford Drug Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, Oxford, UK.
Biochimie. 2025 Jan;228:71-81. doi: 10.1016/j.biochi.2024.08.011. Epub 2024 Aug 14.
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.
乙醇酸氧化酶（HAO1）催化乙醛酸的合成，乙醛酸是一种常见的代谢中间体，如果积累会导致肾衰竭。抑制HAO1是治疗原发性高草酸尿症的一种新兴疗法，原发性高草酸尿症是一种罕见的乙醛酸代谢紊乱疾病。在此，我们通过基于纳米荧光素酶互补和发光读数的细胞热位移分析（CETSA），首次对抑制剂与HAO1的摄取和结合进行了基于细胞的测量。通过分析HAO1与完整和裂解的HEK293T细胞中四种特征明确的抑制剂之间的相互作用，我们表明我们的CETSA方法能够区分低渗透性/高结合性和高渗透性/低结合性配体，并且能够根据重组蛋白方法和先前报道的间接细胞分析对HAO1抑制剂进行排名。我们的方法满足了对一种强大、灵敏且可扩展的细胞分析方法的需求，以指导HAO1抑制剂的开发，更广泛地说，该方法可以快速适用于其他靶点，以同时监测化合物亲和力和细胞渗透性。
