Abstract：
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.
摘要：
缺氧诱导因子（HIF）是多种治疗适应症的有趣靶标。虽然HIF激活是治疗贫血相关和缺血性疾病所需的，HIF抑制对抗癌药物开发具有极大的兴趣。HIF途径中的不同信号事件正被药物发现计划所靶向。对HIF选择性(也可能是HIF1/2同工型选择性)化合物特别感兴趣。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异源二聚化试验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，相似或不同的测定方案的应用允许检测对HIF异源二聚化的各种影响，作为氧感测途径中的关键信号事件：增加的HIF异源二聚化（roxadustat，MG-132),降低HIF异二聚化(PX-478,布洛芬)和直接(HIF同工型选择性)异二聚化抑制作用(PT-2385)。处理时间和测定方案的变化允许评估对HIFα-HIFβ异二聚化的直接和间接影响。除了评估这些新的生物测定有关药理学表征的应用外，讨论了与使用蜂窝相关的好处和注意事项，基于发光的生物测定。简而言之，好处包括生物读出的双向性质，上游检测机制，HIF1和HIF2效应之间的区别以及各种条件的模拟。具体和一般的考虑包括基于细胞，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广泛的应用中提供了各种治疗药物的药物发现和药理学表征的好处，应用相同或优化的实验方案。
