Abstract
2-Oxoglutarate (2OG) oxygenases have important roles in human biology and are validated medicinal chemistry targets. Improving the selectivity profile of broad-spectrum 2OG oxygenase inhibitors may help enable the identification of selective inhibitors for use in functional assignment work. We report the synthesis of F- and CF3-substituted derivatives of the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylate (2,4-PDCA). Their inhibition selectivity profile against selected functionally distinct human 2OG oxygenases was determined using mass spectrometry-based assays. F-substituted 2,4-PDCA derivatives efficiently inhibit the 2OG oxygenases aspartate/asparagine-β-hydroxylase (AspH) and the JmjC lysine-specific Nε-demethylase 4E (KDM4E); The F- and CF3-substituted 2,4-PDCA derivatives were all less efficient inhibitors of the tested 2OG oxygenases than 2,4-PDCA itself, except for the C5 F-substituted 2,4-PDCA derivative which inhibited AspH with a similar efficiency as 2,4-PDCA. Notably, the introduction of a F- or CF3-substituent at the C5 position of 2,4-PDCA results in a substantial increase in selectivity for AspH over KDM4E compared to 2,4-PDCA. Crystallographic studies inform on the structural basis of our observations, which exemplifies how a small change on a 2OG analogue can make a substantial difference in the potency of 2OG oxygenase inhibition.
Highlights
2-Oxoglutarate (2OG) oxygenases have important roles in human biology and are validated medicinal chemistry targets
2,4-PDCA derivatives bearing F- or CF3-substituents at the C3 or C5 position were synthesized from commercially sourced F- or CF3substituted isonicotinic acid derivatives in three or two steps, respec tively, employing an analogous strategy to that used for the synthesis of C3 aminoalkyl-substituted 2,4-PDCA derivatives [15]
4.2 ± 1.0 d)inactive d)inactive d)inactive a) Mean of two independent runs (n = 2; mean ± standard deviation (SD)); b) using 0.05 μM His6-AspH315–758 and 1.0 μM of a thioether-linked cyclic peptide based on human coagulation factor X [14, 28]; c) using 0.15 μM His6-FIH and 5.0 μM hypoxia inducible transcription factor-1α (HIF-1α) C-terminal transactivation domain fragment (HIF-1α CAD, amino acids 788–822) [29]; d) 2,4-PDCA derivatives were termed inactive if 2OG oxygenase inhibition was not observed in the tested concentration range (100 – 0.002 μM) as shown in Supporting Figure S3; e) using 0.15 μM KDM4E and 10.0 μM of a variant of a histone 3 fragment (H31–15K9me3, amino acids 1–15) [31]; f) using 0.15 μM His6-RIOX226–465 and 5.0 μM of RPL27A31–49 peptide (Supplementary Information Section 5) [30]
Summary
2-Oxoglutarate (2OG)-dependent oxygenases couple two electron substrate oxidations with the oxidative decarboxylation of 2OG to give succinate and CO2; they employ Fe(II) as a cofactor [1]. 2OG dependent hydroxylases have validated functions in human biology (Fig. 1a); for example, they act as sensors in the hypoxic response, i.e. the hypoxia inducible transcription factor-1α (HIF-1α) prolyl hydroxylases PHD1–3 together with the asparaginyl hydroxylase factor inhibiting the hypoxia inducible transcription factor-1α (FIH) catalyze the hydroxylation of HIF-α substrates in an O2-dependent manner [2]. Broad-spectrum 2OG oxygenase inhibitors are described, including the natural product N-oxalylglycine (NOG, 1; Fig. 1b) [13] and pyridine-2,4-dicarboxylate (2,4-PDCA, 2; Fig. 1c) [4]. These broad-spectrum inhibitors show distinct selectivity profiles; for example, 2,4-PDCA efficiently inhibits AspH and some JmjC KDMs [14, 15]. 2,4-PDCA is only a weak inhibitor of the PHDs and of FIH [15], which catalyzes the stereoselective C3 hydroxylation of Asn/Asp/His/Ser/Leu-residues (Supporting Figure S1) [16, 17]. We present proof-of-concept studies on how the introduction of substituents at the C5 position of 2, 4-PDCA, which has not previously been investigated in structure activ ity relationship studies on 2OG oxygenases, increases the selectivity of 2, 4-PDCA for AspH
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