PT-2385

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.

High-altitude polycythemia (HAPC) is a common chronic altitude disease caused by living in low-pressure and low-oxygen environment. At present, there is still no effective cure for HAPC. HIF-2α may play an important role in the development of HAPC in regulating the increased red blood cell excessively induced by HIF-EPO and the blood vessel formation induced by VEGF-VEGFR. Here, we established a rat HAPC model and treated it with the HIF-2α inhibitor PT2385. We mainly evaluated the therapeutic effect of PT2385 on HAPC rats by observing the changes in rat phenotype, tissue and organ damage, red blood cell and hemoglobin content, angiogenesis, lipid peroxidation reaction, and inflammatory factors. The results showed that PT2385 treatment improved the congestion phenotype characteristics, inhibited increased erythrocytes and hemoglobin, reduced blood vessel formation, lipid peroxidation, and inflammation, and reduced tissue and organ damage in HAPC rats. This study preliminarly explains the physiological, pathological, and immunological effects of PT2385 treatment for HAPC. It provides a new idea, a reliable experimental basis, and theoretical support for the clinical prevention and treatment of HAPC.