缺氧诱导因子（HIF）是多种治疗适应症的有趣靶点。虽然 HIF 激活是治疗贫血相关和缺血性疾病所必需的，但 HIF 抑制对于抗癌药物的开发具有巨大的意义。 HIF 通路内的不同信号传导事件正在成为药物发现计划的目标，特别关注 HIF 选择性（也可能是 HIF1/2 同工型选择性）化合物。在这项研究中，我们应用最近开发的基于细胞的分裂纳米荧光素酶 HIF 异二聚化测定来研究化合物的作用，通过各种作用机制靶向 HIF 活性。这项研究表明，应用相似或不同的检测方案可以检测对 HIF 异二聚化作为氧传感途径中关键信号事件的各种影响：HIF 异二聚化增加（roxadustat，MG-132），HIF 异二聚化减少（PX-478，布洛芬）和直接（HIF 亚型选择性）异二聚化抑制作用 (PT-2385)。治疗时间和检测方案的变化可以评估对 HIFα-HIFβ 异二聚化的直接和间接影响。除了评估这些新生物测定法在药理学特征方面的应用之外，还讨论了与细胞发光生物测定法的使用相关的益处和注意事项。简而言之，好处包括生物读数的双向性质、检测的上游机制、HIF1 和 HIF2 效应之间的区分以及各种条件的模拟。具体和一般考虑因素包括基于细胞、技术和疾病/药物相关方面（例如非特异性效应、颜色干扰）。总之，这些生物测定的多功能性在药物发现和各种治疗的药理学表征方面的广泛应用中提供了好处，应用相同或优化的实验方案。版权所有 © 2024。由爱思唯尔公司出版。

Hypoxia-inducible factors (HIF) are interesting targets for multiple therapeutic indications. While HIF activation is desired for the treatment of anemia-related and ischemic diseases, HIF inhibition is of tremendous interest to anti-cancer drug development. Different signaling events within the HIF pathway are being targeted by drug discovery programs, with a special interest in HIF-selective (possibly also HIF1/2 isoform-selective) compounds. In this study, we applied recently developed cell-based split-nanoluciferase HIF heterodimerization assays to study the effects of compounds, targeting HIF activity by various mechanisms of action. This study shows that the application of similar or diverse assay protocols allows to detect various influences on HIF heterodimerization as a key signaling event in the oxygen sensing pathway: increased HIF heterodimerization (roxadustat, MG-132), decreased HIF heterodimerization (PX-478, ibuprofen) and direct (HIF isoform-selective) heterodimerization inhibiting effects (PT-2385). Changes in treatment time and in the assay protocol allowed to assess direct and indirect effects on HIFα-HIFβ heterodimerization. In addition to the evaluation of applications of these new bioassays regarding pharmacological characterizations, benefits and considerations are discussed related to the use of cellular, luminescent-based bioassays. Briefly, benefits include the bidirectional nature of the biological readout, the upstream mechanism of detection, the differentiation between HIF1 and HIF2 effects and the simulation of various conditions. Specific and general considerations include cell-based, technical and disease/drug-related aspects (e.g., non-specific effects, color interference). In summary, the versatility of these bioassays offers benefits in widespread applications regarding drug discovery and pharmacological characterization of various therapeutics, applying either the same or optimized experimental protocols.Copyright © 2024. Published by Elsevier Inc.