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First published on June 19, 2008, doi:10.1177/1087057108318800

Journal of Biomolecular Screening 2008;13:494.

A more recent version of this article appeared on July 1, 2008

Article

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

Sang-Hyeup Lee, Jeong Hee Moon, Eun Ah Cho, Seong-Eon Ryu, and Myung Kyu Lee*

Korea Research Institute of Bioscience and Biotechnology

* To whom correspondence should be addressed. E-mail: mklee{at}kribb.re.kr.


  Abstract
The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1{alpha} (HIF-1{alpha}), and the modification abrogates the transcriptional activity of HIF-1{alpha}. Because FIH is more active on HIF1{alpha} 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{alpha}, specifically to Asn803 hydroxylated HIF-1{alpha} and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1{alpha} showed 740 times higher affinity to the Asn803 hydroxylated HIF-1{alpha} peptide than the unmodified one. An enzyme-linked immunosorbent assay–based system using SHN-HIF1{alpha} 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. SHNHIF1{alpha} also showed an Asn803 hydroxylation-dependent specificity to HIF-1{alpha} 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 XXXX:xx-xx)


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