ABSTRACT
Hydroxylation of hypoxia-inducible factor, a nuclear transcription factor, is catalysed by iron and 2-oxoglutarate dependent hydroxylases. Various analogues of the 2-oxoglutarate cosubstrate were synthesised and shown to inhibit the activity of human hypoxia-inducible factor-1alpha prolyl hydroxylases in cell-free extracts.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Ketoglutaric Acids/chemical synthesis , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Transcription Factors/metabolism , Catalysis , Cell-Free System , Enzyme Inhibitors/pharmacology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Iron/chemistry , Iron Chelating Agents/pharmacology , Isoenzymes/antagonists & inhibitors , Ketoglutaric Acids/pharmacology , Molecular StructureABSTRACT
Analogues of the naturally occurring cyclic hydroxamate dealanylalahopcin, which is an inhibitor of procollagen prolyl-4-hydroxylase, were synthesised and shown to be inhibitors of the human hypoxia-inducible factor prolyl hydroxylases.
Subject(s)
Aminobutyrates/chemistry , Aminobutyrates/pharmacology , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Transcription Factors/metabolism , Aminobutyrates/chemical synthesis , Aminobutyrates/metabolism , Animals , Autoradiography , Binding Sites , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Iron/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Procollagen-Proline Dioxygenase/metabolism , Tumor Suppressor Proteins/analysis , Tumor Suppressor Proteins/metabolismABSTRACT
AlkB is one of four proteins involved in the adaptive response to DNA alkylation damage in Escherichia coli and is highly conserved from bacteria to humans. Recent analyses have verified the prediction that AlkB is a member of the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family of enzymes. AlkB mediates repair of methylated DNA by direct demethylation of 1-methyladenine and 3-methylcytosine lesions. Other members of the Fe(II) and 2OG-dependent oxygenase family, including those involved in the hypoxic response, are targets for therapeutic intervention. Assays measuring 2OG turnover were used to investigate the selectivity of AlkB. 1-Methyladenosine, 1-methyl-2'-deoxyadenosine, 3-methylcytidine, and 3-methyl-2'-deoxycytidine all stimulated 2OG turnover by AlkB but were not demethylated indicating an uncoupling of 2OG and prime substrate oxidation and that oligomeric DNA is required for hydroxylation and subsequent demethylation. In contrast the equivalent unmethylated nucleosides did not stimulate 2OG turnover indicating that the presence of a methyl group in the substrate is important in initiating oxidation of 2OG. Stimulation of 2OG turnover by 1-methyladenosine was highly dependent on the presence of a reducing agent, ascorbate or dithiothreitol. Following the observation that AlkB is inhibited by high concentrations of 2OG, analogues of 2OG, including 2-mercaptoglutarate, were found to specifically inhibit AlkB. The flavonoid quercetin inhibits both AlkB and the 2OG oxygenase factor-inhibiting hypoxia-inducible factor (FIH) in vitro. FIH inhibition by quercetin occurs in the presence of excess iron indicating a specific interaction, while the inhibition of AlkB by quercetin is, predominantly, due to nonspecific iron chelation.
Subject(s)
Escherichia coli Proteins/antagonists & inhibitors , Mixed Function Oxygenases/antagonists & inhibitors , Ascorbic Acid/pharmacology , Escherichia coli Proteins/metabolism , Hydroxylation , Hypoxia-Inducible Factor 1, alpha Subunit , Ketoglutaric Acids/metabolism , Mixed Function Oxygenases/metabolism , Quercetin/pharmacology , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolismABSTRACT
The activity of the transcription factor hypoxia-inducible factor (HIF) is regulated by oxygen-dependent hydroxylation. Under normoxic conditions, hydroxylation of proline residues triggers destruction of its alpha-subunit while hydroxylation of Asn(803) in the C-terminal transactivation domain of HIF-1 alpha (CAD) prevents its interaction with p300. Here we report crystal structures of the asparagine hydroxylase (factor-inhibiting HIF, FIH) complexed with Fe((II)), 2-oxoglutarate cosubstrate, and CAD fragments, which reveal the structural basis of HIF modification. CAD binding to FIH occurs via an induced fit process at two distinct interaction sites. At the hydroxylation site CAD adopts a loop conformation, contrasting with a helical conformation for the same residues when bound to p300. Asn(803) of CAD is buried and precisely orientated in the active site such that hydroxylation occurs at its beta-carbon. Together with structures with the inhibitors Zn((II)) and N-oxaloylglycine, analysis of the FIH-CAD complexes will assist design of hydroxylase inhibitors with proangiogenic properties. Conserved structural motifs within FIH imply it is one of an extended family of Fe((II)) oxygenases involved in gene regulation.
