2-Oxoglutarate derivatives can selectively enhance or inhibit the activity of human oxygenases

Nakashima, Yu; Brewitz, Lennart; Tumber, Anthony; Salah, Eidarus; Schofield, Christopher J.

doi:10.1038/s41467-021-26673-2

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Published: 10 November 2021

2-Oxoglutarate derivatives can selectively enhance or inhibit the activity of human oxygenases

Nature Communications volume 12, Article number: 6478 (2021) Cite this article

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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.

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Introduction

2-Oxoglutarate (2OG) and Fe(II)-dependent oxygenases couple substrate oxidation with 2OG decarboxylation to give succinate and CO₂; they have important functions in human biology, e.g., in hypoxia signaling, extracellular matrix biosynthesis, lipid metabolism, and DNA/RNA damage repair¹. Nature appears to have evolved complex mechanisms to regulate the activity of, at least, some of the human 2OG oxygenases, including sometimes by the use of small-molecules^2,3,4,5,6. In cancer cells, normal regulation of 2OG oxygenase activity can be dysregulated, inter alia owing to elevated levels of tricarboxylic acid (TCA) cycle and related metabolites, such as (R)-2-hydroxyglutarate (2HG)^6,7,8. The presence of 2HG has been correlated with ten–eleven-translocation (TET) and JmjC lysine-specific N^ε-demethylases (JmjC KDMs) inhibition; in vitro studies revealed that 2HG inhibits these 2OG oxygenases in a 2OG-competitive manner^4,9. By contrast with the extensive studies on 2HG- and TCA cycle intermediate-mediated inhibition of 2OG oxygenases, only one human metabolite other than 2OG itself, i.e., 2-oxoadipate (2OA), has been reported to enable catalysis by wild-type 2OG oxygenases by acting as a cosubstrate^10,11,12,13.

Despite the importance of 2-oxo acids in biology, medicine, and synthetic chemistry^{14,15,16,17,18}, few studies have been directed at identifying 2-oxo acids other than 2OA which retain the catalytic activity of human 2OG oxygenases. C4-alkyl-substituted 2OG derivatives have been investigated as alternative cosubstrates for human JmjC lysine-specific N^ε-demethylase 4 A (KDM4A), however, activity was observed with active site mutated, but not wildtype, KDM4A¹⁹. 2OG analogs have been shown to restore the activity of clinically observed variants of phytanoyl-CoA 2-hydroxylase (PAHX), but do not enable wild-type PAHX catalysis²⁰.

Recently, we developed an efficient synthesis of C3/C4-substituted 2OG derivatives, some of which are naturally occurring²¹. 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 Asn-residues 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}.

**Fig. 1: Comparison of AspH and FIH catalysis and steady-state kinetic parameters for FIH-catalyzed oxidative decarboxylation of 2OG derivatives from initial hydroxylation rates.**

Like AspH, human factor inhibiting the hypoxia-inducible transcription factor HIF-α (FIH) catalyzes the post-translational oxidation of Asp- and Asn-residues in a 2OG-dependent manner^32,33,34, however, to give the β-threo hydroxylated product (Fig. 1b)³⁵. FIH was initially identified as catalyzing hydroxylation of an Asn-residue in the C-terminal transactivation domain of HIF-α isoforms (Asn803 in HIF-1α), a reaction that suppresses HIF-mediated transcription³⁶. FIH also accepts substrates other than HIF-α³⁷; in particular, FIH catalyzes post-translational hydroxylations of Asp- and Asn-residues in ankyrin repeat domain proteins (e.g., iKB³⁸, Notch³⁹, and ASB4⁴⁰). The current evidence suggests that FIH is biochemically more promiscuous with respect to its substrate requirements than AspH as it also catalyzes the hydroxylation of residues other than Asp and Asn (Fig. 1b)^41,42,43.

The observation of AspH cosubstrate activity using C3/C4-substituted 2OG derivatives raises the possibility of selective enhancement or inhibition of 2OG oxygenases by 2-oxo acid derivatives. Here, we describe proof-of-principle studies with two isolated human 2OG oxygenases, i.e., FIH and AspH, that validate this concept. We chose to work with FIH and AspH since because both accept Asp- and Asn-residues as hydroxylation substrates, they represent a challenge in terms of selectivity. The combined crystallographic and mass spectrometry (MS)-based studies with FIH and AspH demonstrate the potential for selective enhancement or inhibition of specific 2OG oxygenase activity by 2-oxo acid derivatives.

Results

Synthetic 2OG derivatives are FIH cosubstrates

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 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-1α_788–822)³⁷ and (2) a “consensus” FIH substrate ankyrin repeat sequence⁴⁶ (i.e. CA_1–20)⁴⁷, were used as FIH substrates to test for potential substrate-dependent differences in terms of cosubstrate analog selectivity. Initial screening assays were performed in the absence of 2OG, but in the presence of high concentrations (330 μM) of the synthetic C3/C4-substituted 2OG derivatives to facilitate the identification of those 2OG derivatives which sustain FIH catalysis.

In general, FIH-catalyzed hydroxylation of CA_1–20 appeared to be more efficient than of HIF-1α_788–822 when using 2OG as the control cosubstrate (Table 1, entry 1). High levels of FIH substrate hydroxylation were observed for 3-methyl-2OG (1) and 4-methyl-2OG (12), both of which are natural products^48,49,50,51, regardless of the substrate, indicating that these 2OG derivatives can efficiently replace 2OG (Table 1, entries 2 and 13). NMR studies reveal that FIH converts 1 and 12 into 2-methylsuccinate with a similar efficiency to which it converts 2OG into succinate (Fig. 1c–d, Supplementary Figs. 2–4). FIH-catalyzed substrate hydroxylation was clearly apparent using other 2OG derivatives, i.e., 2, 3, 13-16, 22, and 28 (Entries 3, 4, 14–16, 23, and 29); however, in all these cases, conversions were lower compared to 2OG, 1 or 12 (Table 1).

Table 1 Synthetic C3/C4-substituted 2OG derivatives sustain FIH catalysis in the absence of 2OG.

Full size table

The results reveal that FIH activity decreases with both substrates with the increasing length of the 2OG C3/C4-alkyl substituent (i.e., Me > Et > Pr). The cosubstrate activity, albeit at low levels, for 22 and 28 is remarkable as their cyclic/aromatic carbon scaffolds deviate substantially from the acyclic 2OG carbon scaffold (Entries 23 and 29). Under these conditions, none of the analogs appeared to promote HIF-1α (HIF-1α_788–822) hydroxylation relative to ankyrin (CA_1–20) hydroxylation, suggesting the use of 2OG derivatives to modulate FIH activity with respect to particular substrates may be challenging (see below). All the other 2OG derivatives investigated did not show cosubstrate activity under the reaction conditions, in accord with the lack of FIH cosubstrate activity of the 2OG derivative 19, reported using an LC-MS assay⁵².

Comparison of these initial results for FIH with those reported for AspH²¹ revealed the potential of selectively promoting FIH and/or AspH catalysis by using specific 2OG derivatives as cosubstrates. For example, the 2OG derivatives 2, 3, and 13–15 are efficient cosubstrates for FIH, but not for AspH, whereas 35 and 29–33 showed activity with AspH, but not with FIH (Table 1)²¹. Notably, 3-methyl-2OG (1) is an efficient cosubstrate for both FIH and AspH (Table 1, entry 2), whereas its isomer 4-methyl-2OG (12) is apparently more efficient in maintaining FIH compared with AspH activity (Table 1, entry 13). 4-Carboxyphenylglyoxylic acid (28) is an efficient AspH cosubstrate but shows only low FIH activity with CA_1–20 and none with HIF-1α_788–822 as a substrate (Table 1, entry 29). Note that our initial screening conditions differed when investigating FIH and AspH cosubstrate activity; i.e., the enzyme and substrate concentrations were different (0.15 μM and 5.0 μM for FIH; 0.1 μM and 2.0 μM for AspH) and different buffers were used (FIH: 50 mM Tris, 50 mM NaCl, pH 7.5; AspH: 50 mM HEPES, pH 7.5); these differences might, in part, reflect observed differences in activity with the 2OG derivatives. We, therefore, carried out more detailed kinetic FIH studies to better compare the results with those reported for AspH²¹.

Kinetic analyses

To quantify FIH activity with the 2OG derivatives, kinetic analyses of selected compounds (i.e., 2OG, 1, 12, 14, and 22) were performed using HIF-1α_788–822³⁷ and CA_1–20⁴⁷ (Fig. 1e–h; Supplementary Figs. 5 and 6). Maximum velocities (\({v}_{{{{{{\rm{max }}}}}}}^{{{{{{\rm{app}}}}}}}\)) and Michaelis constants (\({K}_{m}^{{{{{{\rm{app}}}}}}}\)) were determined using 2OG as a control. The \({v}_{{{{{{\rm{max }}}}}}}^{{{{{{\rm{app}}}}}}}\)-values reveal that the FIH-catalyzed hydroxylation of CA_1–20 is faster than that of HIF-1α_788–822 for all the cosubstrates analyzed, including 2OG (Table 2, entry 1).

Table 2 Comparison of the steady-state kinetic parameters of FIH for 2OG and the synthetic 2OG derivatives 1, 12, 14, and 22 with those reported for AspH^a,b.

Full size table

The FIH \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-values for 2OG are lower than those reported using different assays (25–110 μM)^37,53, but in the range of those determined for other human 2OG oxygenases using SPE-MS assays (0.5–5 μM)^27,54,55, likely reflecting the high sensitivity of SPE-MS assays. The FIH \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-value for 4-methyl-2OG (12) is similar to that of 2OG, indicating a similar affinity; by contrast, the FIH \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-value for 1, the 3-methyl regioisomer of 12, is higher (Table 2). Increasing the size of the alkyl substituent at C4 from methyl (12) to ethyl (14) results in a higher \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-value when using CA_1–20, whereas only a marginal effect on its \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-value was observed when using HIF-1α_788–822. In general, the FIH \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-values for 2OG, 1, 12, and 14 are in the same range (~0.5 to ~4.0 μM; Table 2, entries 1–4), whereas the \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-values are substantially higher for 22 (15 and 39 μM, respectively; Table 2, entry 5). All the determined \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-concentrations are in the range of reported physiological 2OG levels in human plasma (9–12 μM 2OG)⁵⁶, but lower than reported 2OG concentrations in healthy cells (~1 mM)^57,58.

An active site titration, performed using N-oxalyl-d-phenylalanine⁵² (NOFD) as an inhibitor, revealed the apparent quantitative activity of FIH (Supplementary Fig. 7). Based on the concentration of active sites, turnover numbers (catalytic constants, \({k}_{{{{{{\rm{cat}}}}}}}^{{{{{{\rm{app}}}}}}}\)) and specificity constants (\({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)) were calculated. The \({k}_{{{{{{\rm{cat}}}}}}}^{{{{{{\rm{app}}}}}}}\)-values decrease in the order of 2OG > 12 > 1 > 14 ≥ 22 (Table 2). The FIH \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-values for the 2OG derivatives are higher when CA_1–20 was used instead of HIF-1α_788–822. The highest FIH \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-value was observed for 2OG when using CA_1–20 (~383 mM⁻¹ s⁻¹; Table 2, entry 1), which is about eightfold higher than the value for 2OG when using HIF-1α_788–822 (~48 mM⁻¹ s⁻¹; Table 2, entry 1) and at least one order of magnitude higher than the values observed for the 2OG derivatives investigated, with the notable exception of the \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-value with 12 using CA_1–20, compared with which it is approximately threefold higher (~132 mM⁻¹ s⁻¹; Table 2, entry 3). The results reveal that the FIH \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-values for the 2OG derivatives vary, to some extent, with the nature of the substrate. Considering the high number of reported FIH substrates, this observation suggests that for some substrates, the \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-values of FIH for 2OG derivatives might actually be higher than for 2OG. They also reveal that the \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-values of FIH for the 2OG derivatives might be higher for a specific substrate (i.e., when using 12 and CA_1–20; Table 2, entry 3) than the \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-values of FIH for the natural cosubstrate 2OG when using different substrates (i.e., HIF-1α_788–822; Table 2, entry 1).

The FIH \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-values for the 2OG derivatives are in the range of those reported for AspH, which were also obtained using SPE-MS assays (Table 2)²¹. Notably, the FIH \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-value for 2OG with CA_1–20 is about threefold higher than the one of AspH for 2OG (~383 and ~130 mM⁻¹ s⁻¹, respectively; Table 2). By contrast, the FIH \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-value for 1 with CA_1–20 is an order of magnitude lower than that for AspH (~25 and ~296 mM⁻¹ s⁻¹, respectively; Table 2). The FIH \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-values for 22 are similar to the reported one of AspH for 28 (~1 mM⁻¹ s⁻¹; Table 2, entries 5 and 6)²¹, which, however, is not an efficient FIH cosubstrate.

FIH and AspH competition studies

Comparisons of the effects of the synthetic 2OG derivatives on FIH catalysis with their reported effect on AspH catalysis²¹ indicated their potential to selectively enhance FIH or AspH activity. However, the FIH and AspH data were obtained under different conditions (different enzyme/(co-)substrate/cofactor concentrations, enzyme/substrate ratios, and buffers), possibly perturbing the results of their direct comparison. It is an advantage of MS-based turnover assays that the hydroxylation of different substrate peptides can be monitored in parallel in a single reaction vessel. Thus, the FIH-catalyzed hydroxylation of two different substrates as well as FIH- and AspH-catalyzed substrate hydroxylations were assessed simultaneously in the same vessel, complementing prior kinetic and turnover studies using a single isolated enzyme and substrate.

An equimolar mixture of the HIF-1α_788–822³⁷ (blue) and CA_1–20⁴⁷ (orange) peptides in a single reaction vessel was submitted to FIH-catalyzed modification at saturating 2OG and Fe(II) concentrations (Fig. 2a). The observed turnovers reflect the results of the kinetic experiments with individual substrates (Table 2). In agreement with an approximate eightfold difference observed in the FIH \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-values for 2OG, >90% conversion of CA_1–20 was observed after 5 min, while only ~60% conversion of HIF-1α_788–822 was observed after 12 min. This observation raises the possibility of the use of 2OG derivatives for selective enhancement of the oxidation of specific sets of substrates for a 2OG oxygenase.

**Fig. 2: 2OG derivatives selectively sustain the catalytic activities of 2OG oxygenases in vitro.**

Next, an equimolar amount of the FIH substrate CA_1–20 and the AspH substrate hFX-EGFD1_86–124-4Ser (5.0 μM each), the latter based on the sequence of the reported AspH substrate human coagulation factor X^27,31,59, was submitted to equimolar amounts of FIH and AspH (0.15 μM each) in the presence of 2OG (20 μM) and Fe(II) (10 μM) in a single reaction vessel. The presence of two 2OG oxygenases did not result in substantial peptide ionization suppression and high-quality time course data were obtained: >95% AspH-catalyzed hFX-EGFD1_86–124-4Ser hydroxylation was observed within about 1 min, whereas >80% FIH-catalyzed CA_1–20 hydroxylation was observed after 4 min (Fig. 2b). By contrast to the analysis of the FIH and AspH kinetic parameters for 2OG with individual isolated enzymes (Table 2, entry 1), the results reveal that, when using 2OG as a cosubstrate under the tested conditions, AspH catalysis is more efficient than FIH catalysis. This might reflect the dimeric nature of FIH in solution⁶⁰ and/or the different assay conditions used to obtain the kinetic parameters for isolated FIH and AspH; for example, a shorter substrate than the hFX-EGFD1_86–124-4Ser peptide was used when determining the AspH kinetic parameters²¹.

In a similar single vessel experiment, an approximately twofold decrease in FIH efficiency was observed when 2OG was substituted for 1 (20 μM), keeping other reaction parameters same, i.e., >80% CA_1–20 hydroxylation was observed after about 8 min rather than 4 min (Fig. 2c), whereas no detrimental effect on AspH catalysis was observed (>95% hFX-EGFD1_86–124-4Ser hydroxylation within ~2 min Fig. 2c), in agreement with the kinetics of the individual enzymes (Table 2).

As the kinetic data with isolated FIH and AspH suggests (Table 2), using a saturating concentration of 12 (20 μM) as a cosubstrate instead of 2OG or 1, reversed the reactivity profile (Fig. 2d). Thus, with 12, FIH catalysis was more efficient than AspH catalysis, reaching ~90% CA_1–20 hydroxylation after ~5 min, whereas AspH-catalyzed hFX-EGFD1_86–124-4Ser hydroxylation reached ~90% after ~10 min in the same reaction vessel. Interestingly, the presence of both 2OG (100 μM) and 12 (20 μM) in the same reaction vessel reversed this effect, i.e., AspH catalysis is faster than FIH catalysis (Fig. 2e). Complete AspH-catalyzed hydroxylation of hFX-EGFD1_86–124-4Ser was observed after ~2 min, which is only marginally slower than in the absence of any 12 (Fig. 2b). The FIH-catalyzed hydroxylation of CA_1–20 was not affected; it reaches ~90% conversion in the presence of saturating 2OG or 12 concentrations, and also in the presence of both cosubstrates (Fig. 2e).

Comparing FIH and AspH activities in a single reaction vessel reveals that only FIH-catalyzed hydroxylation is observed when using 2OG derivative 14 (100 μM) in the absence of 2OG (Fig. 2f). However, FIH catalysis was less efficient for 14 than for 2OG, 1 or 12, which were used at lower concentrations (20 μM). AspH-catalyzed hydroxylation of hFX-EGFD1_86–124-4Ser was not observed, in agreement with the reported lack of cosubstrate activity with 14 (Table 1)²¹. Similarly, selective enhancement of AspH activity was observed without 2OG using the phenyl-ring containing 2OG derivative 28 (Supplementary Fig. 8), which showed negligible amounts of cosubstrate activity for FIH (Table 1).

Crystallography

Crystallographic studies were initiated to investigate the binding of the 2OG derivatives to FIH and to compare the results with those reported for AspH²¹. Initially, FIH was crystallized in the presence of Zn(II), as a substitute for Fe(II), and in the presence of the C3-alkyl-substituted 2OG derivatives 1 and 3. In the absence of substrate, the FIH complexes crystallized in the P4₁2₁2 space group (FIH:1, 2.29 Å resolution; FIH:3, 2.18 Å resolution; Supplementary Figs. 9–12). The structures were solved by molecular replacement (MR) using a reported FIH structure (PDB ID: 1H2K)⁶¹ as a search model. The superimposition of both structures with the reported FIH:2OG:HIF-1α_786–826 structure (PDB ID: 1H2L)⁶¹ reveals that the conformation of 2OG does not substantially change upon introducing substituents at C3, albeit the conformation of the C3–C4 ethylene unit of 1 and 3 slightly differs from that of 2OG (Fig. 3a). The C3-alkyl substituents apparently align to interact with the hydrophobic sidechains of Leu186 and Leu188.

**Fig. 3: The synthetic 2OG derivatives 1, 3, 14, 15, and 22 bind FIH similarly to 2OG.**

FIH co-crystallized with 1, Zn, and CA_1–20 in the P4₁2₁2 space group (FIH:1:CA_1–20, 2.01 Å resolution; Supplementary Fig. 13). All FIH:substrate structures were solved by MR using a reported FIH structure (PDB ID: 1H2K)⁶¹ as a search model. Superimposition of the FIH:1:CA_1–20 structure with the reported FIH:2OG:HIF-1α_786–826 (PDB ID: 1H2L; Fig. 3b)⁶¹ and the FIH:1 structures (Fig. 3c) reveals that the conformation of 1 does not substantially change upon substrate binding (Supplementary Fig. 14). The nature of the FIH substrate does not seem to influence the conformation of 1, as revealed by co-crystallization with a fragment of the ankyrin repeat-containing validated human FIH substrate tankyrase-2⁶² (FIH:1:TNKS2_691–710; 2.01 Å resolution; Supplementary Figs. 15–17).

FIH was also crystallized in the presence of CA_1–20 and the C4-alkyl-substituted 2OG derivatives 14 and 15 (FIH:14:CA_1–20, 2.21 Å resolution; FIH:15:CA_1–20, 1.76 Å resolution; Supplementary Figs. 18–21). The conformations of 14 and 15 were very similar to that of 2OG with their C4-alkyl substituents facing towards the hydrophobic Leu186 and Leu188 sidechains (Fig. 3d), as observed for the C3-alkyl 2OG derivatives. The observation that the conformations of C3- and C4-alkyl 2OG derivatives are very similar to that of 2OG in complex with FIH is in agreement with their similar \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-values (Table 2). The difference in \({k}_{{{{{{\rm{cat}}}}}}}^{{{{{{\rm{app}}}}}}}\) is the major factor for the observed differences in the \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-values for 14 and 2OG; the lower \({k}_{{{{{{\rm{cat}}}}}}}^{{{{{{\rm{app}}}}}}}\) for 14 may reflect the lower conformational/rotational flexibility of the substituted 2OG derivatives due to stronger hydrophobic interactions with the Leu186 and Leu188 sidechains and/or the enhanced binding to and thus slower release of the corresponding alkyl-substituted succinate reaction product from FIH.

In the FIH:22:CA_1–20 structure (1.75 Å resolution; Supplementary Figs. 22–23), 22 occupies a different conformation than that observed for 2OG, possibly to compensate for its rigid cyclic carbon-backbone (Fig. 3e). The FIH \({K}_{m}^{{{{{{\rm{app}}}}}}}\) for 22 reflects this conformational difference as it is about tenfold higher compared with the \({K}_{m}^{{{{{{\rm{app}}}}}}}\) for 2OG, 1, and 14, regardless of the substrate employed (Table 2), indicative of a lower affinity of FIH for 22.

The hydrophobic interactions of the C3/C4-alkyl 2OG derivatives with the Leu186 and Leu188 sidechains in FIH resemble those of the C3/C4-alkyl-substituted 2OG derivatives with the Met670, Val676, and Val727 sidechains in AspH structures (Fig. 3f and Supplementary Figs. 24–25)²¹. In addition to differences in the 2OG C5 carboxylate binding motif, the interaction of aliphatic C3/C4-substituents of the 2OG derivatives with hydrophobic residues and/or hydrophobic pockets (as observed for AspH) in the active sites of 2OG oxygenases, appears to influence the conformation of the 2OG derivatives. Although the C3/C4-alkyl 2OG derivatives were used as racemic mixtures, the crystallographic analyses reveal that, at least predominantly, the (S)-enantiomers of the 2OG derivatives bind to FIH, potentially owing to their ability to engage in more efficient hydrophobic interactions with the hydrophobic Leu186 and Leu188 sidechains (Supplementary Figs. 10, 12, 14, 16, 19, and 21). A similar observation has been made for AspH, however, in this case, the (R)-enantiomers of the C3/C4-alkyl-substituted 2OG derivatives engage in hydrophobic interactions with AspH²¹. Interestingly, the unusual geometry of the AspH Fe(II)-binding site^21,31, i.e., only two AspH histidine residues coordinate the Fe(II) cofactor (e.g., H₆₇₉XG₆₈₁…H₇₂₅) rather than the typical triad of ligands (HXD/E…H) found in the active site of other human 2OG hydroxylases including FIH (H₁₉₉XD₂₀₁…H₂₇₉)¹, appears not to directly affect 2OG derivative binding to AspH nor AspH/FIH cosubstrate selectivity based on the superimposition of AspH and FIH substrate structures complexed with 1 (Fig. 3f).

Synthetic 2OG derivatives inhibit FIH

To further investigate the utility of the 35 synthetic C3/C4-substituted 2OG derivatives for selectively modulating FIH or AspH activity, their inhibitory effect on FIH activity was investigated^44,45. As the crystallographic studies revealed that the conformations of the CA_1–20 and HIF-1α_786–826 peptides align well at the FIH active site (Fig. 3b, d, and e), i.e. the predicted binding site of the 2OG derivatives, the inhibition assays were performed exclusively with HIF-1α_788–822³⁷ owing to its reduced reactivity, facilitating the experimental set-up. Half maximum inhibitory concentrations (IC₅₀-values) of the 2OG derivatives were determined (comprehensive analysis in Supplementary Table 1), using NOFD⁵² as a positive inhibition control. The IC₅₀ for NOFD (~0.2 μM; Table 3, entry 1) is substantially lower in the SPE-MS assay compared to a previously employed derivatization assay (\({K}_{i}^{{{{{{\rm{app}}}}}}}\) ~ 93 μM)⁵², highlighting the better sensitivity of this direct assay (0.15 μM FIH was used in the SPE-MS assay and 4.0 μM FIH in the derivatization assay).

Table 3 Synthetic 2OG derivatives inhibit FIH.

Full size table

The increase in length of the 2OG C3/C4-substituent from methyl via ethyl to propyl appears to correlate with enhanced FIH inhibition (Table 3). Thus, although the C3/C4 methyl-substituted 2OG derivatives 1 and 12 do not inhibit FIH (Table 3, entries 2 and 7), 3-propyl-2OG (3) inhibits FIH efficiently (IC₅₀ ~2.7 μM; Table 3, entry 4). The potencies of the synthetic 2OG derivatives as a function of substituent length appear to inversely correlate with their cosubstrate efficiency (Tables 1 and 3). For example, high levels of FIH cosubstrate activity, but not FIH inhibition, were observed for 1 and 12 (Table 1, entries 2 and 13), whereas the C4 ethyl and propyl-substituted 2OG derivatives 14 and 15 are more efficient FIH cosubstrates, but less efficient FIH inhibitors compared to the corresponding C3 regioisomers 2 and 3 (Tables 1 and 3).

C4-Benzyl-2OG (19) inhibits FIH efficiently in the SPE-MS assay (IC₅₀ ~ 3.6 μM; Table 3, entry 12), whereas only weak inhibition has been reported when employing a 2-oxo acid derivatization assay⁵². Notably, 19 inhibits FIH more than one order of magnitude less efficiently than the structurally related, but conformationally more rigid, NOFD (note that NOFD was used in the enantiopure form, while 19 was used as a racemic mixture). However, the potency of 19 was increased when replacing its C4 benzyl- with a 3-phenylpropyl-substituent (IC₅₀ ~0.9 μM; Table 3, entry 11). The C4 benzyl- and C4 3-phenylpropyl-substituted 2OG derivatives 19 and 18 are two- and sixfold, respectively, more efficient FIH inhibitors than their corresponding C3 regioisomers (Table 3, entries 5, 10, 11, and Supplementary Table 1). However, the potency of C3 benzyl-derivative 6 could be substantially enhanced when introducing a sterically bulkier CH₂(9,9-dimethyl-9H-fluoren-2-yl-substituent at C3 (IC₅₀ ~ 1.9 μM; Table 3, entry 6).

In general, the 2OG derivatives inhibit FIH less efficiently than AspH, which might, at least to some extent, reflect the higher 2OG concentration used in the FIH inhibition assays (10 μM 2OG in the FIH assays, 3 μM 2OG in the AspH assays). Thus, the C4 ethyl- and propyl-substituted 2OG derivatives 14 and 15 are highly efficient AspH inhibitors, consistent with their reported lack of AspH cosubstrate activity²¹, whereas the same compounds are more than an order of magnitude less efficient in inhibiting FIH (Table 3, entries 9 and 10). Inhibition selectivity is even higher for 4,4-dimethyl-2OG (13), which efficiently inhibits AspH (IC₅₀ ~ 0.4 μM²¹) but not at all FIH (Table 3, entry 8). This result highlights the importance of apparently minor structural modifications on inhibitor selectivity as 4-methyl-2OG (12) neither inhibits AspH nor FIH (Table 3, entry 7) and also reveals the potential of 2OG-competitive 2-oxo acids for selective 2OG oxygenase inhibition (the mode of AspH inhibition by 13 has previously been shown to be 2OG-competitive²¹).

Interestingly, varying the substitution pattern of the 2OG derivatives can result in a reversal of inhibition selectivity. For example, 3-ethyl 2OG (2) inhibits AspH more efficiently than FIH (IC₅₀ ~ 1.2 μM for AspH and ~4.6 μM for FIH; AspH:FIH inhibitor selectivity ~4:1; Table 3, entry 3), however, substituting its C3 ethyl with a propyl group reverses inhibition selectivity; the corresponding 2OG derivative 3 inhibits FIH more efficiently than AspH (IC₅₀ ~ 5.7 μM for AspH and ~2.7 μM for FIH; AspH:FIH inhibitor selectivity ~1:2; Table 3, entry 4). Similarly, the substitution of a 2OG CH₂-unit by an NH-unit can result in a reversal of inhibition: While 4-benzyl-2OG (19) is more efficient in inhibiting AspH compared to FIH (AspH:FIH inhibitor selectivity: ~7:1; Table 3, entry 12), its NH-containing derivative NOFD is substantially more efficient in inhibiting FIH rather than AspH (AspH:FIH inhibitor selectivity: ~1:50; Table 3, entry 1).

2OG derivative 28 neither inhibits AspH nor FIH catalysis (Table 3, entry 13) and also derivatives of 28 bearing substituents at the aromatic core do not inhibit the two oxygenases (Supplementary Table 1). In principle, selective 2OG oxygenase inhibition by 2OG derivatives bearing an aromatic or heteroaromatic core appears to be feasible, as revealed by the inhibition profile of 2OG derivative 35, which bears a thiophene moiety and selectively inhibits AspH over FIH (Table 3, entry 14).

The inhibition results suggest that the ratio of FIH to AspH activity may be perturbed by using an AspH-selective inhibitor in the presence of the natural cosubstrate 2OG, a strategy that is complementary to the one previously investigated relying on the use of an FIH-selective cosubstrate (i.e., 14; Fig. 2f). AspH was most selectively inhibited by 4,4-dimethyl-2OG (13), which binds to AspH efficiently and is slowly converted into 2,2-dimethyl succinate (Table 3, entry 8)²¹; therefore, FIH- and AspH-catalyzed substrate hydroxylations were simultaneously assessed in a single reaction vessel using SPE-MS in the presence of a physiological relevant 2OG concentration (0.5 mM) and varied concentrations of 13 (Fig. 4). Increasing the concentration of 13 from 0 to 2.0 mM reveals selective AspH inhibition in a dose-dependent manner, with no effect on FIH activity being observed. In the absence of the inhibitor 13, AspH catalysis is substantially faster than FIH catalysis, with substrate hydroxylation reaching apparent completion after ~1 min; ~80% FIH-catalyzed substrate turnover was only observed after 4 min (Fig. 4a). Using equimolar amounts of 2OG and 13 (0.5 mM each), ~80% conversion is observed for FIH after 4 min while only ~60% conversion is observed for AspH (Fig. 4d). In the presence of a fourfold excess of 13 with respect to 2OG, <20% of AspH-catalyzed substrate hydroxylation was observed after 4 min (Fig. 4f). A comparable result was obtained in a titration experiment with 2OG and 4-ethyl-2OG (14), which is an alternative FIH cosubstrate and an AspH inhibitor (Fig. 2f and Table 3, entry 9)²¹. The titration revealed that a tenfold excess of 14 over 2OG substantially slows down AspH catalysis while showing only a marginal effect on FIH catalysis (Supplementary Fig. 27). The results reveal the potential of 2-oxo acids for selective 2OG oxygenase inhibition at physiologically relevant 2OG concentrations.

**Fig. 4: 4,4-Dimethyl-2OG selectively inhibits AspH in the presence of FIH at physiological 2OG concentrations.**

Mechanism of FIH inhibition

To investigate the mechanism by which the synthetic 2OG derivatives inhibit FIH, the concentration-dependent effect of the potent FIH inhibitor 18 on the 2OG \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-value was investigated using HIF-1α_788–822 (Supplementary Fig. 28). The 2OG \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-value decreases in a linear fashion with ascending inhibitor concentrations, while the \({v}_{{{{{{\rm{max }}}}}}}^{{{{{{\rm{app}}}}}}}\)-values converge, indicating a linear 2OG-competitive inhibition mechanism (Fig. 5a). Using non-linear regression, the \({K}_{i}\)-value of 18 was determined to be ~0.2 μM. Analysis of a Cornish-Bowden plot⁶³ further supports a 2OG-competitive inhibition mode, i.e., parallel lines for different 2OG concentrations are obtained when plotting [2OG]/v against [18] and their intercept with the y axis increases on increasing the 2OG concentration (Fig. 5b).

**Fig. 5: The 2OG derivatives 11 and 18 inhibit FIH by different modes.**

FIH was crystallized in the presence of Zn(II) and the 2OG derivatives 11 or 18 to investigate their binding modes. FIH crystallized in the P4₁2₁2 space group (FIH:11, 1.99 Å resolution; FIH:18, 1.90 Å resolution; Supplementary Figs. 29 and 31). Clear electron density corresponding to 18 or 11, respectively, was observed in the active site (Fig. 5c and d); 11 and 18 bind FIH in a similar manner as 2OG (Fig. 5e). The analysis of the electron density maps for the 2OG derivatives indicates that only (S)-11 and (S)-18 bind FIH, even though both were present as racemic mixtures (Supplementary Figs. 30 and 32).

The conformation of (S)-18 in the FIH:18 structure is reminiscent of the NOFD conformation in the reported FIH:NOFD⁵² structure, indicating that these molecules inhibit FIH by a similar mechanism: their bulky phenyl substituents extent into the FIH substrate-binding pocket and appear to shield the active site metal from the substrate and/or O₂ (Fig. 5e). By contrast, the rigid and relatively bulky fluorenyl substituent of (S)-11 orients in a different manner than the substituents of (S)-18 and NOFD, triggering a conformational change in the Tyr102_FIH sidechain. In the FIH:11 structure, the Tyr102_FIH sidechain faces into the substrate-binding position, likely to avoid a steric clash with the fluorenyl substituent of (S)-11, and thus blocks the substrate from binding to the FIH active site (red arrow in Fig. 5f). Movement of the Tyr102_FIH sidechain has been previously observed in crystal structures of FIH complexed with different substrates (Supplementary Fig. 33)⁴³. In agreement with the proposal that the coordination of (S)-11 and (S)-18 to the FIH active site impairs substrate binding, FIH crystals with both inhibitor and substrate-bound could not be obtained. Even though inhibitors 11 and 18 display no substantial selectivity between AspH and FIH, the crystallographic analyses may help in the design of selective FIH inhibitors.

Discussion

Different strategies can be envisaged in which 2OG derivatives might be used to alter the normal reaction outcomes of one or more 2OG oxygenases (Fig. 6). In one case, a 2OG derivative might selectively enhance and/or inhibit the activity of one 2OG oxygenase in the presence of others (strategy 1, Fig. 6a). The application of reaction-selective 2OG oxygenase cosubstrates could also enable control of reaction outcomes for sequential reactions catalyzed by a 2OG oxygenase (strategy 2, Fig. 6b). A 2OG oxygenase may also be envisaged to convert its substrate to a different product with a 2OG derivative compared to 2OG (strategy 3, Fig. 6c). In the current work, we describe proof-of-principle studies that 2OG derivatives can be used to enable strategy 1 (Fig. 6a), i.e., to selectively modulate the activity of one or both of two human 2OG oxygenases, which both catalyze the hydroxylation of Asn- and Asp-residues, i.e., FIH and AspH.

**Fig. 6: 2OG derivatives may be used to selectively control reaction outcomes of 2OG oxygenase catalysis.**

Our results of screens of 2OG derivatives with FIH and AspH led to the identification of two complementary approaches to selectively modulate the activities of FIH and AspH (strategy 1, Fig. 6a). FIH activity can be selectively enhanced in the presence of AspH using either an FIH-selective cosubstrate (i.e., 14; Fig. 2f) in the absence of 2OG, or an AspH-selective inhibitor in the presence of a physiologically relevant concentration of 2OG (i.e., 13; Fig. 4). AspH activity can be selectively enhanced in the presence of FIH using a partially AspH-selective cosubstrate (i.e., 28; Supplementary Fig. 8) in the absence of 2OG. High levels of cosubstrate/inhibitor selectivity were observed in all cases, revealing the potential of 2-oxo acids for the selective enhancement and/or inhibition of 2OG oxygenase catalysis. This potential is supported by the reported lack of cosubstrate/inhibition activity of some of the tested C4-alkyl-substituted 2OG derivatives with wild-type KDM4A, which is a 2OG-dependent JmjC KDM¹⁹.

The application of MS-based assays to simultaneously assess FIH and AspH activity in the same reaction vessel complements kinetic studies with isolated 2OG oxygenases and enables the direct comparison of 2OG oxygenase activities. Importantly, the simultaneous turnover assays show that natural products can have a profound impact on the reactivity profiles of isolated 2OG oxygenases. For example, 3-methyl-2OG (1), which is an ingredient of honey⁴⁸, promotes AspH catalysis relative to FIH catalysis, whereas its regioisomer, 4-methyl-2OG (12), which is present in plants^49,50 and in wine⁵¹, promotes FIH catalysis relative to AspH catalysis in the absence of 2OG (Fig. 2; strategy 1, Fig. 6a). Considering that cancer-associated mutations of the human isocitrate-dehydrogenase complex result in a 2OG deficient phenotype which, along with elevated 2-hydroxyglutarate levels, can correlate with decreased 2OG oxygenase activity^64,65, the result that naturally occurring 2OG derivatives can enable 2OG oxygenase activity could be of in vivo relevance and medicinal value, e.g., to treat side effects arising from reduced 2OG oxygenase activity.

Some proteins, such as human Notch, are substrates of both AspH and FIH: AspH catalyzes the hydroxylation of extracellular Notch EGFDs⁶⁶ and FIH catalyzes the hydroxylation of ankyrin repeats in the Notch intracellular domain³⁹. Nature likely controls the FIH and AspH-catalyzed post-translational Notch modifications by spatial separation of the enzymes (FIH is predominantly present in the cytosol⁶⁷, whereas the oxygenase domain of AspH faces into the ER⁶⁸). The use of a selective cosubstrate may enable the selective enhancement of AspH or FIH catalysis acting on the same substrate, thus complementing natural control by spatial separation. Although there is likely scope for improvement of the synthetic 2OG derivatives reported by us, our studies provide proof-of-principle for this concept, i.e., 4-carboxyphenylglyoxylic acid (28) is a substantially more efficient AspH than FIH cosubstrate (Supplementary Fig. 8), whereas 4-ethyl-2OG (14) is an FIH cosubstrate but an AspH inhibitor (Fig. 2f).

The comparison of FIH:2OG derivative structures with reported AspH:2OG derivative structures reveals that the unusual Fe(II)-binding geometry of AspH, which has only two Fe(II) binding ligands compared to the typical triad present in other 2OG hydroxylases (including in FIH and KDM4A)²¹, does not appear to underpin the observed differences in cosubstrate selectivity patterns. In near all the FIH:2OG derivative and AspH:2OG derivative structures, the 2OG derivatives occupy the 2OG-binding site and display similar conformations as 2OG (Fig. 3); a notable exception is the structurally distinct 2OG derivative 22, whose conformation differs owing to its cyclic structure (Fig. 3e). Together with the reported lack of cosubstrate activity of certain C4-substituted 2OG derivatives with KDM4A¹⁹, these observations suggest that the interactions of the C3/C4-substituents of the 2OG derivatives with sidechains of (hydrophobic) residues flanking the 2OG-binding site, which form hydrophobic regions, determine the efficiency of cosubstrate binding and coproduct release (Fig. 3f). In general, the FIH \({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)-values for the 2OG derivatives appear to decrease upon increasing the length of the 2OG C4 substituent from hydrogen to methyl and ethyl, independent from the nature of the substrate (Table 2). As the \({K}_{m}^{{{{{{\rm{app}}}}}}}\)-values remain in the same range within experimental error (0.5~3.1 μM, Table 2), this observation indicates that the 2OG derivatives bind FIH with similar affinities, but likely undergo subsequent oxidative decarboxylation reactions less efficiently than 2OG, resulting in apparent inhibition. Although they employ the same general strategies for 2OG binding⁶⁹, the 2OG-binding sites of different 2OG oxygenases vary substantially and thus may be particularly suited for the binding of specific 2OG derivatives resulting either in selective inhibition or activation as evidenced by our work.

The results presented here highlight the potential of 2-oxo acids, including 2OG derivatives and natural products, to inhibit 2OG oxygenases and related enzymes. The potential of 2-oxo acids for the inhibition of 2OG oxygenases is supported by the (partially) selective FIH inhibitor NOFD, a derivative of the broader spectrum 2OG oxygenase inhibitor NOG⁵². The inhibition selectivity of NOG derivatives has also been studied for other subclasses of 2OG oxygenases including the JmjC KDMs and TET enzymes^70,71,72,73. The observation that dimethyl N-oxalylglycine (DMOG), a cell-wall penetrable NOG precursor, inhibits 2OG oxygenases in vivo and in cellular studies, supports the potential utility of corresponding 2OG derivatives for cellular functional assignment studies, regardless of the high 2OG concentrations in cells (~1 mM 2OG^57,58). The potential of 2-oxo acids to inhibit 2OG oxygenases is shown by the results for 4,4-dimethyl-2OG (13), which efficiently and selectively inhibits AspH (IC₅₀ ~ 0.4 μM²¹), even in the presence of physiologically relevant 2OG concentrations, i.e., 0.5 mM 2OG (Fig. 4).

The involvement of 2-oxo acids in multiple metabolic pathways may be considered to limit the utility of 2OG derivatives for their development as human therapeutics. Indeed, the presence of 2OG derivatives with substituents at the C3/C4 position may modulate the activities of 2OG-dependent enzymes other than 2OG oxygenases. For example, it has been reported that 4,4-dimethyl-2OG (13) is a substantially less-efficient substrate of glutamic oxaloacetic aminotransferase compared with the natural substrate 2OG or 4-methyl-2OG (12), suggesting that 13 might not interfere with the catalytic activities of human aminotransferases⁷⁴. However, from a safety perspective, it is notable that some of the 2OG derivatives described here are present in human nutrition^48,49,50,51 and cells appear to tolerate reasonably high levels of DMOG with no obvious toxicity⁷⁵. Further, considering the pleiotropic effects that NOG and its derivatives exhibit in cellular studies, application of related 2-oxo acid derivatives to cells may trigger phenotypes useful in identifying previously overlooked 2OG oxygenase/2OG-utilizing enzyme functions. FIH⁷⁶ and AspH^{22,23,24,25,26} are proposed medicinal chemistry targets and the use of (partially) selective 2OG derivatives, such as 13 or corresponding esters in cellular studies may help to improve the currently limited understanding on their biochemical roles and complement similar approaches using heterocyclic small-molecules^{24,76,77,78,79,80,81}.

In summary, the combined results with FIH and AspH, 2OG oxygenases that both catalyze Asp- and Asn-residue hydroxylations, demonstrate the potential of 2-oxo acid derivatives to selectively enhance or inhibit one of the enzyme reactions in the presence of the other, in some cases in the presence of 2OG. From a set of 35 2OG derivatives, we identified 10 as enhancing and 17 as inhibiting FIH activity. Although further mechanistic studies are required to analyze the details of why some 2-oxo acid derivatives inhibit and others enable 2OG oxygenase catalysis, our structural studies provide insight into the active site regions into which the C3/C4 2OG substituents bind; this information should be useful in the identification of 2-oxo acid derivatives, including natural products present in food, that enable selective modulation of 2OG oxygenase reaction outcomes in vivo. Analogous strategies described here for modulation of 2OG oxygenase activity by cosubstrate derivatives should be applicable to other enzyme families employing modified cosubstrates.

Methods

Protein production and purification

Wildtype N-terminally His₆-tagged human FIH was produced and purified by an adaption of a literature procedure⁸². In brief, DNA encoding for wild-type human FIH with a N-terminal His₆-tag was cloned into a pNIC28-Bsa4 plasmid. After transformation of the plasmid into E. coli BL21 (DE3) cells, expression was performed at 37 °C in LB media supplemented with 37.5 µg/mL kanamycin. FIH-production was induced at an OD₆₀₀ of ~0.6 at 20 °C by adding isopropyl β-d-thiogalactopyranoside (final concentration of 0.5 mM). Cultures were grown overnight. Cells were spun at 5000 rpm for 10 mins and the resulting cell pellets were stored at −80 °C.

The cell pellets were suspended in buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP) and lysed by high-pressure homogenization (three passages). Following cell lysis, the insoluble cell debris was removed by centrifugation (36,000 × g, 1 h, 4 °C). His₆-tagged FIH was purified by sequential Ni(II)-affinity chromatography (using 50 mM HEPES, pH 7.4, 1 M NaCl, 0.5 mM TCEP with increasing imidazole concentrations: 20–300 mM) and size-exclusion chromatography (HiLoad 16/600 Superdex 75 pg column; buffer: 20 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP) using an ÄKTAxpress machine. Fractions containing FIH were pooled and concentrated; FIH was >95% pure by SDS-PAGE and MS analysis. Purified FIH was stored at a concentration of 42 μM (20 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP) at −78 °C, fresh aliquots were used for all FIH assays (cleavage of the His₆-tag did not show beneficial effects on FIH activity).

A truncated construct of wild-type N-terminally His₆-tagged human AspH, comprising the catalytic oxygenase domain and the tetratricopeptide repeat domain (His₆-AspH_315-758), was produced and purified as previously reported^27,31.

Substrate peptides

FIH substrates were based on the sequence of the two reported human FIH substrate proteins HIF-1α^34,36 (HIF-1α C-terminal transactivation domain fragment amino acids 788–822, HIF-1α_788–822: DESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRAL; FIH catalyzes hydroxylation of Asn803^34,37) and tankyrase-2⁶² (tankyrase-2 amino acids 691–710, TNKS2_691–710: NLEVAEYLLQHGADVNAQDK; FIH catalyzes hydroxylation of Asn706⁶²) and of a reported synthetic consensus ankyrin repeat⁴⁶ (CA_1–20:⁴⁷ HLEVVKLLLEAGADVNAQDK; FIH catalyzes hydroxylation of Asn16⁴⁷). The AspH substrate peptide, i.e., hFX-EGFD1_86–124-4Ser^27,31, was based on the sequence of the EGFD1 (amino acids 86–124) of the reported AspH substrate human coagulation factor X (hFX), four hFX cysteine residues (Cys90_hFX, Cys95_hFX, Cys112_hFX, Cys121_hFX) have been substituted for serine residues to avoid disulfide scrambling. Peptides were synthesized by solid-phase peptide synthesis and purified by GL Biochem (Shanghai) Ltd (Shanghai, China); all peptides were prepared with C-terminal amides.

FIH inhibition assays

FIH inhibition assays were performed as described⁴⁴, using cosubstrate/cofactor stock solutions (l-ascorbic acid, LAA: 50 mM in MQ-grade water; 2-oxoglutarate, 2OG: 10 mM in MQ-grade water; ammonium iron(II) sulfate hexahydrate, FAS, (NH₄)₂Fe(SO₄)₂·6H₂O: 400 mM in 20 mM HCl diluted to 1 mM in MQ-grade water), which were freshly prepared from commercially-sourced solids (Sigma Aldrich).

Dimethyl sulfoxide (DMSO) solutions of the 2OG derivatives were dry dispensed across 384-well polypropylene assay plates (Greiner) in an approximately threefold and 11-point dilution series (100 μM top concentration; the final DMSO assay concentration was kept constant at 0.5%_v/v) using an ECHO 550 acoustic dispenser (Labcyte). DMSO and NOFD⁵² were used as negative and positive inhibition controls, respectively. Each reaction was performed in technical duplicates in adjacent wells of the assay plates; additionally, assays were performed in two independent duplicates on different days using different inhibitor solutions.

The Enzyme Mixture (25 μL/well), containing 0.3 μM His₆-FIH in buffer (50 mM Tris, 50 mM NaCl, pH 7.5), was dispensed across the inhibitor-containing 384-well assay plates with a multidrop dispenser (ThermoFischer Scientific) at 20 °C under an ambient atmosphere. The plates were subsequently centrifuged (1000 rpm, 15 s) and incubated for 15 min at 20 °C. The Substrate Mixture (25 μL/well), containing 10.0 μM HIF-1α_788–822³⁷ substrate, 200 μM LAA, 20.0 μM 2OG, and 20.0 μM FAS in buffer (50 mM Tris, 50 mM NaCl, pH 7.5), was added using the multidrop dispenser. The plates were centrifuged (1000 rpm, 15 s) and after incubating for 15 min, the enzyme reaction was stopped by the addition of 10%_v/v aqueous formic acid (5 μL/well). The plates were then centrifuged (1000 rpm, 30 s) and analyzed by MS.

MS-analyses were performed using a RapidFire RF 365 high-throughput sampling robot (Agilent) attached to an iFunnel Agilent 6550 accurate-mass quadrupole time-of-flight mass spectrometer operated in the positive ionization mode. Assay samples were aspirated under vacuum for 0.6 s and loaded onto a C4 SPE cartridge. After loading, the C4 SPE cartridge was washed with 0.1%_v/v aqueous formic acid to remove non-volatile buffer salts (5.5 s, 1.5 mL/min). The peptide was eluted from the SPE cartridge with 0.1%_v/v aqueous formic acid in 85/15 _v/v acetonitrile/water into the mass spectrometer (5.5 s, 1.25 mL/min) and the SPE cartridge re-equilibrated with 0.1%_v/v aqueous formic acid (0.5 s, 1.25 mL/min). The mass spectrometer was operated using the MassHunter Workstation B.08.00 software (Agilent), the mass spectrometer parameters were: capillary voltage (4000 V), nozzle voltage (1000 V), fragmentor voltage (365 V), gas temperature (280 °C), gas flow (13 L/min), sheath gas temperature (350 °C), sheath gas flow (12 L/min). The m/z + 2 (for CA_1–20) or +3 (for HIF-1α_788–822) charge states of the peptide (substrate) and the hydroxylated peptide (product) were used to extract ion chromatogram data, peak areas were integrated using RapidFire Integrator 4.3.0 (Agilent). Data were exported into Microsoft Excel and used to calculate the % conversion of the hydroxylation reaction using the equation: % conversion = 100 × (integral product peptide)/(integral substrate peptide+integral product peptide). Normalized dose–response curves (NOFD and DMSO controls) were obtained from the raw data by non-linear regression (GraphPad Prism 5) and used to determine IC₅₀ values. The standard deviation (SD) of two independent IC₅₀ determinations (n = 2) was calculated using GraphPad Prism 5. Z′-factors were calculated according to the cited literature using Microsoft Excel (Supplementary Fig. 26)⁸³.

Determination of kinetic parameters

Maximum velocities (\({v}_{{{{{{\rm{max }}}}}}}^{{{{{{\rm{app}}}}}}}\)) and Michaelis constants (\({K}_{m}^{{{{{{\rm{app}}}}}}}\)) of FIH were determined in independent triplicates for 2OG and the synthetic 2OG derivatives by SPE-MS monitoring HIF-1α_788–822³⁷ or CA_1–20⁴⁷ turnover. His₆-FIH (1.8 μL, 42 μM) was added at 20 °C to a substrate mixture containing 5.0 μM substrate, 100 μM LAA, 20 μM FAS, and 2OG/2OG derivative in 0.5 mL buffer (50 mM Tris, 50 mM NaCl, pH 7.5). Final 2OG/2OG derivative concentrations are given in Supplementary Figs. 5 and 6. Reactions were monitored with a rate of ~1 sample/40 s using the same SPE-MS configuration as described above. Data were analyzed as described above and the slopes of the initial reaction rates (Supplementary Figs. 5 and 6) fitted to a Michaelis–Menten plot using non-linear regression (GraphPad Prism 5). The total concentration of active FIH was determined by an active site titration (Supplementary Fig. 7) and used to calculate turnover numbers (\({k}_{{{{{{\rm{cat}}}}}}}^{{{{{{\rm{app}}}}}}}\)) and specificity constants (\({k}_{{{{{{\rm{cat}}}}}}}\)/\({K}_{m}\)) for 2OG and the 2OG derivatives.

Competition assays

To a Substrate Mixture containing 5.0 μM of each of the requisite substrates (for FIH: HIF-1α_788–822³⁷ and/or CA_1–20⁴⁷; for AspH: hFX-EGFD1_86–124-4Ser^27,31), 100 μM LAA, 10 μM FAS, and 20 μM 2OG/2OG derivative in 1.0 mL buffer (50 mM Tris, 50 mM NaCl, pH 7.5) was added 0.15 μM of His₆-FIH and His₆-AspH_315-758 (when appropriate). Peptide turnover was monitored by SPE-MS using the same instrument configuration (with the exception that the peptides were eluted from the SPE cartridge with 0.1%_v/v aqueous formic acid in 80/20 _v/v acetonitrile/water) as described above.

Crystallography

Crystallization experiments were performed in 96-well, three-subwell, low profile Intelliplates (Art Robbins Instruments) using a Phoenix RE liquid dispensing robot (Art Robbins Instruments) with 1.6 M ammonium sulfate, 6%_w/v PEG400, and 0.1 M HEPES buffer (pH 7.5) as the mother liquid. N-Terminally His₆-tagged FIH (20 mg/mL) was mixed with ZnCl₂ (1 mM), 2OG derivative (2 mM), and, when appropriate, a synthetic consensus ankyrin repeat peptide (5 mM; CA_1–20⁴⁷) or a tankyrase-2 fragment peptide⁶² (5 mM; TNKS2_691–710) as FIH substrate. Crystals were grown using the vapor diffusion method at 20 °C in 200 nL of 300 nL sitting drops with 2:1, 1:1, or 1:2 sample:well solution ratios; precipitants are listed in Supplementary Tables 2 and 3. Crystals were cryo-protected using mother liquor supplemented with 25%_v/v glycerol before cryo-cooling in liquid N₂. Data were collected at 100 K using synchrotron radiation at diamond light source beamlines I03 and I04. Data were indexed, integrated, and scaled using the autoPROC⁸⁴, STARANISO⁸⁵, or Xia2⁸⁶ strategy of the beamline auto-processing pipeline (Supplementary Tables 2 and 3).

The FIH crystal structures were determined by MR using the AutoMR (PHASER⁸⁷) subroutine in PHENIX (version 1.18.2)⁸⁸. The search model used for MR was based on PDB ID 1H2K⁶¹ for FIH crystal structures. The structural model was improved by iterative cycles of manual re-building in Coot (version 0.8.6.1)⁸⁹ and crystallographic refinement in phenix.refine⁹⁰ (refinement details are summarized in Supplementary Tables 2 and 3). The crystal structure data for FIH complexed to Zn, 2OG derivative, and, in some cases, substrate (i.e. CA_1–20 or TANK2_691–710) have been deposited in the protein data bank (see Data availability section). PyMOL (version 4.6)⁹¹ was used for the generation of graphical representations; polder omit maps were calculated using Polder Maps⁹² in PHENIX (version 1.18.2)⁸⁸.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The crystal structure data for FIH complexed to Zn, 2OG derivative (3-methyl-2OG, 1; 3-propyl-2OG, 3; 3-(9,9-dimethyl-9H-fluoren-2-yl)methyl-2OG, 11; 4-ethyl-2OG, 14; 4-propyl-2OG, 15; 4-(3-phenylpropyl)-2OG, 18; 3-(carboxycarbonyl)cyclopentane-1-carboxylic acid, 22), and, in some cases, substrate (i.e. CA_1–20 or TANK2_691–710) have been deposited in the protein data bank under PDB accession codes: 7A1L (FIH:1), 7A1M (FIH:3), 7A1J (FIH:18), 7A1K (FIH:11), 7A1N (FIH:1:CA_1–20), 7A1O (FIH:14:CA_1–20), 7A1P (FIH:15:CA_1–20), 7A1Q (FIH:22:CA_1–20), and 7A1S (FIH:1:TANK2_691–710). In addition, data of the following reported crystal structure have been used: 1H2L⁶¹, 1H2K⁶¹, 1YCI⁵², and 6YYX²¹. Source data are provided with this paper.

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Acknowledgements

This research was funded in part by the Wellcome Trust (106244/Z/14/Z). For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. We thank Cancer Research UK (C8717/A18245) and the Biotechnology and Biological Sciences Research Council (BB/J003018/1 and BB/R000344/1) for funding. Y.N. thanks JSPS for an Overseas Research Fellowship (2020060219) and the Daiichi Sankyo Foundation of Life Science. L.B. thanks the Deutsche Forschungsgemeinschaft for a fellowship (BR 5486/2-1). We thank the Diamond Light Source and staff for the allocation of beam time and support.

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Yu Nakashima
Present address: Institute of Natural Medicine, University of Toyama, 2630-Sugitani, 930-0194, Toyama, Japan
These authors contributed equally: Yu Nakashima, Lennart Brewitz.

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Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, University of Oxford, 12 Mansfield Road, OX1 3TA, Oxford, UK
Yu Nakashima, Lennart Brewitz, Anthony Tumber, Eidarus Salah & Christopher J. Schofield

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Yu Nakashima
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Anthony Tumber
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Eidarus Salah
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Y.N. performed crystallizations and solved and refined the FIH crystal structures. L.B. and A.T. performed assays. L.B. synthesized 2OG derivatives. E.S. and L.B. produced and purified enzymes. All authors analyzed data. L.B., Y.N., and C.J.S. wrote the manuscript.

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Nakashima, Y., Brewitz, L., Tumber, A. et al. 2-Oxoglutarate derivatives can selectively enhance or inhibit the activity of human oxygenases. Nat Commun 12, 6478 (2021). https://doi.org/10.1038/s41467-021-26673-2

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Received: 02 June 2021
Accepted: 12 October 2021
Published: 10 November 2021
DOI: https://doi.org/10.1038/s41467-021-26673-2

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A human protein hydroxylase that accepts D-residues

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Introduction

Results

Synthetic 2OG derivatives are FIH cosubstrates

Kinetic analyses

FIH and AspH competition studies

Crystallography

Synthetic 2OG derivatives inhibit FIH

Mechanism of FIH inhibition

Discussion

Methods

Protein production and purification

Substrate peptides

FIH inhibition assays

Determination of kinetic parameters

Competition assays

Crystallography

Reporting summary

Data availability

References

Acknowledgements

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Supplementary information

Supplementary Information

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