Clinically approved HIF-PHIs modulate redox metabolism, cell growth, and angiogenesis independent of HIF-1α/HIF-2α

HIF-prolyl hydroxylase inhibitors are used to treat anemia in chronic kidney disease. These drugs stabilize hypoxia-inducible factors HIF-1α and HIF-2α, which activate erythropoiesis and iron metabolism pathways. Clinically approved HIF-PHIs including roxadustat and molidustat exhibit distinct molecular structures and selectivity profiles, yet their HIF-independent effects remain poorly understood. Here we show that roxadustat and molidustat modulate mitochondrial function, oxidative stress, lysosomal activity, and lipid accumulation, resulting in distinct cellular phenotypes in HIF-null cells. Notably, roxadustat exhibited anti-proliferative and anti-angiogenic activity in HIF-null cells, contradicting expectations of VEGF-driven angiogenesis via HIF stabilization. RNA sequencing and pathway analysis revealed compound-specific off-target gene regulation affecting cellular processes beyond canonical hypoxia responses including energy metabolism and immune signaling. These findings illuminate mechanisms underlying potential adverse effects- such as thrombosis- and identify alternative therapeutic pathways, providing a framework for optimizing HIF-PHI safety profiles and expanding their clinical applications in oncology and metabolic disorders.