The hypoxia-inducible factor (HIF) transcription factors are members of the basic-helix-loop-helix (bHLH) family of transcription factors and play important roles in maintaining cellular oxygen homeostasis. HIF-1α has emerged as an important drug target in breast and prostate cancer, cardiovascular disease, and ischemia. ML-228 is an activator of the HIF signaling pathway, as demonstrated by HIF response element (HRE) reporter assay (EC50 = 1.2 μM), HIF-1α nuclear translocation assay (EC50 = 1.3 μM), and increased VEGF expression (EC50 = 1.6 μM). Its activity in the HRE assay is blocked by excess iron, suggesting that ML-228 can chelate iron. Molecular modeling indicates that ML-228 does not modulate HIF signaling by binding to prolyl hydroxyases. ML-228 also significantly inhibits ligand binding to several channels, receptors, and transporters, including ERG potassium channel, 5-HT2B and A3 adenosine receptors, and dopamine transporter.