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Monoclonal Antibody-Based Screening Assay for Factor Inhibiting Hypoxia-Inducible Factor Inhibitors

Systemic Proteomics Research Center, KRIBB, Yuseong, Daejeon, Korea

Jeong Hee Moon

Systemic Proteomics Research Center, KRIBB, Yuseong, Daejeon, Korea

Eun Ah Cho

Omics and Integration Research Center, KRIBB, Yuseong, Daejeon, Korea

Seong-Eon Ryu

Systemic Proteomics Research Center, KRIBB, Yuseong, Daejeon, Korea

Myung Kyu Lee

Omics and Integration Research Center, KRIBB, Yuseong, Daejeon, Korea, mklee{at}kribb.re.kr

The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1 (HIF-1), and the modification abrogates the transcriptional activity of HIF-1. Because FIH is more active on HIF-1 than prolyl hydroxylase domain proteins under hypoxic conditions, its inhibitors have potential to be developed as anti-ischemic drugs targeting normal cells stressed by hypoxia. In this study, the authors developed the first monoclonal antibody, SHN-HIF1, specifically to Asn803 hydroxylated HIF-1 and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1 showed 740 times higher affinity to the Asn803 hydroxylated HIF-1 peptide than the unmodified one. An enzyme-linked immunosorbent assay—based system using SHN-HIF1 displayed at least 30 times more sensitivity than previous methods for screening FIH inhibitors and was easily applicable to develop a high-throughput screening system. SHN-HIF1 also showed an Asn803 hydroxylation-dependent specificity to HIF-1 in cells. Taken together, the results suggest that it may be used to analyze the in vivo and in vitro activities of FIH inhibitors. (Journal of Biomolecular Screening 2008:494-503)

Key Words: hypoxia-inducible factor-1 • factor-inhibiting hypoxia-inducible factor • monoclonal antibody • ELISA • inhibitor screening

This version was published on July 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 6, 494-503 (2008)
DOI: 10.1177/1087057108318800


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