HIF-1/2α-IN-2 is a potent inhibitor of HIF-1/2α, which effectively reduces the levels of HIF-1/2α. This compound elicits an iron starvation response by specifically targeting ISCA2, a key protein involved in Iron Sulfur Cluster Assembly 2. [1]

HIF-1/2α-IN-2 (compound #1) (0-25 μM; 24 h) decreases HIF-2α translation instead of transcription, with a downward trend in VEGFA and POU5F1 (HIF-2α target genes) transcription and insignificant effect on EPAS1 (HIF-2α) [1]. HIF-1/2α-IN-2 (0-100 μM, 24 h) inhibits the production of luciferase driven by the HIF-2α Iron-Responsive Element (IRE) luciferase reporter with an IC 50 value of 3.9 μM, to block IRE-dependent translation of HIF-2α [1]. HIF-1/2α-IN-2 (0, 10, 50 μM) protects ISCA2 from Pronase-mediated (4 μg/mL) degradation, and (1.5 μM; 24 h) targets ISCA2 to induce iron and metals accumulation in normoxia 786-0 cells [1]. HIF-1/2α-IN-2 (0-100 μM; 24 h) results ISCA2 inhibition and promotes cell death via ferroptosis [1]. Western Blot Analysis [1] Cell Line: Normoxia 786-0 cells (20%O 2 ) and hypoxic ACHN cells (or 1%O 2 for final 24 h to induce HIF expression) Concentration: 0, 1, 5, 10, 25 μM Incubation Time: 24 hours Result: Decreased the protein level of HIF-2α, FTH1, ISCA2, and increased IRP2 protein level at high concentrations over 10 μM in normoxia 786-0 cells. Dcreased the HIF-2α and FTH1 levels at 1 μM in hypoxic ACHN cells. Cell Viability Assay [1] Cell Line: Normoxia 786-0 cells Concentration: 0-100 μM Incubation Time: 24 hours Result: Inhibited 786-0 cells viability by inducing ferroptosis, with an IC 50 value of 22.0 μM.