The ‘ABC’ of split-nanoluciferase HIF heterodimerization bioassays: Applications, Benefits &amp; Considerations

Janssens, Liesl K.; Stove, Christophe P.

doi:10.1016/j.bcp.2024.116478

Biochemical Pharmacology

2024

DOI: 10.1016/j.bcp.2024.116478

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The ‘ABC’ of split-nanoluciferase HIF heterodimerization bioassays: Applications, Benefits & Considerations

Liesl K. Janssens,

Christophe P. Stove

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Untargeted Detection of HIF Stabilizers in Doping Samples: Activity-Based Screening with a Stable In Vitro Bioassay

Janssens,

De Wilde,

Van Eenoo

et al. 2023

Anal. Chem.

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Hypoxia-inducible factor (HIF) stabilizers are listed in the World Anti-Doping Agency’s prohibited list as they can increase aerobic exercise capacity. The rapid pace of emergence of highly structurally diverse HIF stabilizers could pose a risk to conventional structure-based methods in doping control to detect new investigational drugs. Therefore, we developed a strategy that is capable of detecting the presence of any HIF stabilizer, irrespective of its structure, by detecting biological activity. Previously developed cell-based HIF1/2 assays were optimized to a stable format and evaluated for their screening potential toward HIF stabilizers. Improved pharmacological characterization was established by the stable cell-based formats, and broad specificity was demonstrated by pharmacologically characterizing a diverse set of HIF stabilizers (including enarodustat, IOX2, IOX4, MK-8617, JNJ-42041935). The methodological (in solvent) limit of detection of the optimal HIF1 stable bioassay toward detecting the reference compound roxadustat was 100 nM, increasing to 50–100 ng/mL (corresponding to 617–1233 nM in-well) in matching urine samples, owing to strong matrix effects. In a practical context, a urinary limit of detection of 1.15 μg/mL (95% detection rate) was determined, confirming the matrix-dependent detectability of roxadustat in urine. Pending optimization of a universal sample preparation strategy and/or a methodology to correct for the matrix effects, this untargeted approach may serve as a complementing method in antidoping control, as theoretically, it would be capable of detecting any unknown substance with HIF stabilizing activity.

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Untargeted Detection of HIF Stabilizers in Doping Samples: Activity-Based Screening with a Stable In Vitro Bioassay

Janssens,

De Wilde,

Van Eenoo

et al. 2023

Anal. Chem.

View full text Add to dashboard Cite

show abstract

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The ‘ABC’ of split-nanoluciferase HIF heterodimerization bioassays: Applications, Benefits & Considerations

Cited by 1 publication

References 49 publications

Untargeted Detection of HIF Stabilizers in Doping Samples: Activity-Based Screening with a Stable In Vitro Bioassay

Untargeted Detection of HIF Stabilizers in Doping Samples: Activity-Based Screening with a Stable In Vitro Bioassay

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