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) at concentrations ranging from 0 to 25 μM over 24 hours primarily reduces HIF-2α translation rather than its transcription, leading to decreased transcription of VEGFA and POU5F1, which are HIF-2α target genes, without significantly affecting EPAS1 (HIF-2α) [1]. At concentrations of 0 to 100 μM, the same compound over 24 hours inhibits luciferase production in an Iron-Responsive Element (IRE) luciferase reporter driven by HIF-2α with an IC 50 value of 3.9 μM, effectively blocking IRE-dependent HIF-2α translation [1]. Additionally, HIF-1/2α-IN-2 at 0, 10, and 50 μM protects ISCA2 from Pronase-mediated degradation at 4 μg/mL, and at 1.5 μM over 24 hours, it causes accumulation of iron and metals in normoxia 786-0 cells by targeting ISCA2 [1]. This compound, at concentrations up to 100 μM over 24 hours, inhibits ISCA2 and induces cell death via ferroptosis [1]. Western blot analysis with normoxia 786-0 cells and hypoxic ACHN cells exposed to 0, 1, 5, 10, and 25 μM for 24 hours reveals a decrease in the protein levels of HIF-2α, FTH1, ISCA2, and an increase in IRP2 protein levels at higher concentrations over 10 μM in 786-0 cells, and a decrease in HIF-2α and FTH1 levels at 1 μM in ACHN cells [1]. Cell viability assays show that HIF-1/2α-IN-2 inhibits 786-0 cell viability by inducing ferroptosis, with an IC 50 value of 22.0 μM [1].