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.
Systemic Proteomics Research Center, KRIBB, Yuseong, Daejeon, Korea.
Sang-Hyeup
SH
Lee
Jeong Hee Moon
Eun Ah Cho
Seong-Eon
SE
Ryu
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