Identification of Targets of the HIF‑1 Inhibitor IDF-11774 Using Alkyne-Conjugated Photoaffinity Probes

• Published in: Bioconjugate chemistry 2016-08, Vol.27 (8), p.1911-1920
• Main Authors: Ban, Hyun Seung, Naik, Ravi, Kim, Hwan Mook, Kim, Bo-Kyung, Lee, Hongsub, Kim, Inhyub, Ahn, Heechul, Jang, Yerin, Jang, Kyusik, Eo, Yumi, Song, Kyung Bin, Lee, Kyeong, Won, Misun
• Format: Article
• Language: English
• Subjects: Adamantane - analogs & derivatives; Adamantane - pharmacology; Adenosine Triphosphate - biosynthesis; Alkynes - chemistry; Allosteric Site - drug effects; Benzoic Acid - pharmacology; Binding sites; Cancer; Cell Respiration - drug effects; Fluorescence; HCT116 Cells; Heat shock proteins; HSP70 Heat-Shock Proteins - antagonists & inhibitors; HSP70 Heat-Shock Proteins - chemistry; HSP70 Heat-Shock Proteins - metabolism; Humans; Hypoxia; Hypoxia-Inducible Factor 1 - antagonists & inhibitors; Mitochondria - drug effects; Mitochondria - metabolism; Models, Molecular; Oxygen; Piperazines - pharmacology; Protein Conformation; Protein Domains; Staining and Labeling
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/acs.bioconjchem.6b00305

We developed a hypoxia-inducible factor-1 (HIF-1) inhibitor, IDF-11774, as a clinical candidate for cancer therapy. To understand the mechanism of action of IDF-11774, we attempted to isolate target proteins of IDF-11774 using bioconjugated probes. Multifunctional chemical probes containing sites for click conjugation and photoaffinity labeling were designed and synthesized. After fluorescence and photoaffinity labeling of proteins, two-dimensional electrophoresis (2DE) was performed to isolate specific molecular targets of IDF-11774. Heat shock protein (HSP) 70 was identified as a target protein of IDF-11774. We revealed that IDF-11774 inhibited HSP70 chaperone activity by binding to its allosteric pocket, rather than the ATP-binding site in its nucleotide-binding domain (NBD). Moreover, IDF-11774 reduced the oxygen consumption rate (OCR) and ATP production, thereby increasing intracellular oxygen tension. This result suggests that the inhibition of HSP70 chaperone activity by IDF-11774 suppresses HIF-1α refolding and stimulates HIF-1α degradation. Taken together, these findings indicate that IDF-11774-derived chemical probes successfully identified IDF-11774’s target molecule, HSP70, and elucidated the mode of action of IDF-11774 in inhibiting HSP70 chaperone activity and stimulating HIF-1α degradation in cancer cells.