Quantification of PARP7 Protein Levels and PARP7 Inhibitor Target Engagement in Cells Using a Split Nanoluciferase System

Duell, Anna K; Sanderson, Daniel J.; Cohen, Michael S.

doi:10.1007/978-1-0716-2891-1_24

Cited by 2 publications

(1 citation statement)

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“…Since its original characterisation 15 and application to CETSA 12 , HiBiT-LgBiT complementation has been used primarily as a reporter system, where addition of LgBiT is used to locate and/or quantify HiBiT-tagged target protein, in studies monitoring cell processes such as binding and import of viral proteins 16,17 , protein degradation 18,19 and changes in protein translation 20 . More recently its application in CETSA has been presented 12 , and reported for inhibitor development campaigns 13,[21][22][23][24] for a handful of therapeutic targets [25][26][27][28][29][30][31] .…”

Section: Introductionmentioning

confidence: 99%

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

Mackinnon,

Zarganes-Tzitzikas,

Adams

et al. 2024

Preprint

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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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