Abstract
2-Oxoglutarate (2OG) oxygenases are validated agrochemical and human drug targets. The potential for modulating their activity with 2OG derivatives has not been explored, possibly due to concerns regarding selectivity. We report proof-of-principle studies demonstrating selective enhancement or inhibition of 2OG oxygenase activity by 2-oxo acids. The human 2OG oxygenases studied, factor inhibiting hypoxia-inducible transcription factor HIF-α (FIH) and aspartate/asparagine-β-hydroxylase (AspH), catalyze C3 hydroxylations of Asp/Asn-residues. Of 35 tested 2OG derivatives, 10 enhance and 17 inhibit FIH activity. Comparison with results for AspH reveals that 2OG derivatives selectively enhance or inhibit FIH or AspH. Comparison of FIH structures complexed with 2OG derivatives to those for AspH provides insight into the basis of the observed selectivity. 2-Oxo acid derivatives have potential as drugs, for use in biomimetic catalysis, and in functional studies. The results suggest that the in vivo activity of 2OG oxygenases may be regulated by natural 2-oxo acids other than 2OG.
Highlights
2-Oxoglutarate (2OG) oxygenases are validated agrochemical and human drug targets
The ability of 35 synthetic C3/C4-substituted 2OG derivatives, which were prepared via a reported procedure (Supplementary Fig. 1 and Supplementary Methods)[21], to sustain catalysis by purified recombinant FIH:11 structure (FIH) in the absence of 2OG was examined using an assay directly monitoring substrate depletion and product formation (i.e., a +16 Da mass shift) by solid-phase extraction (SPE) coupled to mass spectrometry (SPE-MS)[44,45]
Two peptides, based on the sequence of (1) the HIF-1α C-terminal transactivation domain fragment (i.e., HIF-1α788–822)[37] and (2) a “consensus” FIH substrate ankyrin repeat sequence[46] (i.e. CA1–20)[47], were used as FIH substrates to test for potential substratedependent differences in terms of cosubstrate analog selectivity
Summary
2-Oxoglutarate (2OG) oxygenases are validated agrochemical and human drug targets. The potential for modulating their activity with 2OG derivatives has not been explored, possibly due to concerns regarding selectivity. We report proof-of-principle studies demonstrating selective enhancement or inhibition of 2OG oxygenase activity by 2-oxo acids. The human 2OG oxygenases studied, factor inhibiting hypoxia-inducible transcription factor HIF-α (FIH) and aspartate/asparagine-β-hydroxylase (AspH), catalyze C3 hydroxylations of Asp/Asnresidues. Comparison with results for AspH reveals that 2OG derivatives selectively enhance or inhibit FIH or AspH. 2-Oxo acid derivatives have potential as drugs, for use in biomimetic catalysis, and in functional studies. We developed an efficient synthesis of C3/C4-substituted 2OG derivatives, some of which are naturally occurring[21] These 2OG derivatives were shown to enhance and/or inhibit the catalytic activity of the human 2OG oxygenase aspartate/asparagine-β-hydroxylase (AspH)[21]. AspH is a medicinal chemistry target for human cancer therapeutics and diagnostics[22,23,24,25,26]; it catalyzes the post-translational oxidation of Asp- and Asnresidues in specific macrocyclic disulfide isomers of epidermal growth factor-like domains (EGFDs) to give the β-erythro hydroxylated product (Fig. 1a)[27,28,29,30,31]
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