Monoclonal antibody-based screening assay for factor inhibiting hypoxia-inducible factor inhibitors.
The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1alpha (HIF-1alpha), and the modification abrogates the transcriptional activity of HIF-1alpha. Because FIH is more active on HIF-1alpha 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-HIF1alpha, specifically to Asn803 hydroxylated HIF-1alpha and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1alpha showed 740 times higher affinity to the Asn803 hydroxylated HIF-1alpha peptide than the unmodified one. An enzyme-linked immunosorbent assay-based system using SHN-HIF1alpha 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-HIF1alpha also showed an Asn803 hydroxylation-dependent specificity to HIF-1alpha in cells. Taken together, the results suggest that it may be used to analyze the in vivo and in vitro activities of FIH inhibitors.
Lee
Sang-Hyeup
SH
Systemic Proteomics Research Center, KRIBB, Yuseong, Daejeon, Korea.
Jeong Hee Moon
Eun Ah Cho
Ryu
Seong-Eon
SE
Myung Kyu Lee
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism; D001681:Biological Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005620:Freund's Adjuvant / Q000494:pharmacology; D006900:Hydroxylation; D000860:Hypoxia / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D000276:Adjuvants, Immunologic; D000911:Antibodies, Monoclonal; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012097:Repressor Proteins; D001216:Asparagine; D005620:Freund's Adjuvant