Human 2-oxoglutarate (2OG) dependent oxygenases (inhibitor) [1]
.
- Specifically inhibits: aspartate/asparagine-β-hydroxylase (AspH), JmjC lysine-specific N-demethylase 4E (KDM4E), factor inhibiting HIF (FIH), ribosomal oxygenase 2 (RIOX2/MINA53) [1]
.
- IC50 values against specific enzymes are detailed in the "In Vitro" section [1]
.

Inhibition of AspH: 2,4-PDCA inhibits human AspH with an IC50 of approximately 0.03 μM in a solid phase extraction coupled to mass spectrometry (SPE-MS) assay using a peptide substrate (hFX-EGFDI86-124-4Ser) [1]
.
- Inhibition of KDM4E: 2,4-PDCA inhibits human KDM4E with an IC50 of approximately 0.29 μM in an SPE-MS assay monitoring demethylation of a histone 3 peptide (H3K9me3) [1]
.
- Inhibition of FIH: 2,4-PDCA inhibits human FIH with an IC50 of approximately 4.7 μM in an SPE-MS assay [1]
.
- Inhibition of RIOX2: 2,4-PDCA inhibits human RIOX2 with an IC50 of approximately 4.0 μM in an SPE-MS assay using the RPL27A31-49 substrate peptide [1]
.
- Effect of F- and CF3-substitution: The F- and CF3-substituted derivatives of 2,4-PDCA (compounds 7, 8, 13, 14) are generally less potent inhibitors of the tested 2OG oxygenases than 2,4-PDCA itself, with the following exceptions and observations:
- The C5 F-substituted derivative (8) inhibits AspH with a similar potency (IC50 ~0.05 μM) to 2,4-PDCA (IC50 ~0.03 μM) [1]
.
- The C3 and C5 F-substituted derivatives (7 and 8) are approximately fourfold less potent against KDM4E (IC50 ~1.12 μM and 1.30 μM, respectively) compared to 2,4-PDCA [1]
.
- The CF3-substituted derivatives (13 and 14) are much less potent inhibitors of AspH and KDM4E, with IC50 values >10 μM for AspH and >20 μM for KDM4E [1]
.
- None of the F- or CF3-substituted derivatives (7, 8, 13, 14) showed significant inhibition of FIH or RIOX2 at the concentrations tested (IC50 >100 μM for FIH; IC50 >100 μM for RIOX2) [1]
.
- Selectivity Profile: The introduction of a F- or CF3-substituent at the C5 position of 2,4-PDCA (compounds 8 and 14) results in a substantial increase in selectivity for AspH over KDM4E compared to unsubstituted 2,4-PDCA. This is most notable for the C5 F-substituted derivative (8), which retains potent AspH inhibition (IC50 0.05 μM) while being much weaker against KDM4E (IC50 1.30 μM) [1]
.
- Crystallographic Studies: X-ray crystallography of AspH in complex with F-substituted derivatives (7 and 8) reveals their binding mode. They coordinate to the active site metal (Mn in the crystal structure) via their N-atom and the 2-carboxylate, similar to unsubstituted 2,4-PDCA. The F-substituents point towards the active site entrance (C3-F) or into a pocket formed by residues Tyr391, Tyr406, and Lys409 (C5-F), explaining the observed differences in potency and selectivity [1]
.

General SPE-MS Inhibition Assay: The inhibitory activity of 2,4-PDCA and its derivatives against human 2OG oxygenases (AspH, FIH, KDM4E, RIOX2) was determined using solid phase extraction coupled to mass spectrometry (SPE-MS) assays. These assays directly monitor the mass shift of a peptide substrate due to enzyme catalysis: hydroxylation (+16 Da) for AspH, FIH, and RIOX2, or demethylation (-14 Da and -28 Da) for KDM4E. Assays were performed in 384-well plates. Compounds (in DMSO) were dispensed using an acoustic dispenser to create a 3-fold, 11-point dilution series (top concentration 100 μM). The final DMSO concentration was kept constant at 0.5%. Enzyme mixture (containing the recombinant human 2OG oxygenase in buffer) was added to the plates and incubated. Then, a substrate mixture (containing the peptide substrate, 2OG, L-ascorbic acid, and ammonium iron(II) sulfate) was added to initiate the reaction. After incubation, the reaction was stopped with formic acid. The plates were analyzed using a high-throughput sampling robot coupled to a Q-TOF mass spectrometer. Samples were loaded onto a C4 SPE cartridge, washed, and eluted for MS analysis. The peak areas for substrate and product peptides were integrated, and the % conversion was calculated. IC50 values were determined by fitting normalized dose-response curves using GraphPad Prism [1]
.
- Specific RIOX2 Assay Details: For RIOX2, the enzyme mixture contained 0.3 μM His-RIOX2-465 in reaction buffer (50 mM HEPES, 50 mM NaCl, pH 7.5). After a 15-minute incubation with inhibitor, the substrate mixture containing 10 μM RPL27A31-49 substrate peptide, 200 μM L-ascorbic acid, 20 μM 2OG, and 20 μM ammonium iron(II) sulfate in reaction buffer was added. The reaction was stopped after 30 minutes with 10% aqueous formic acid. The m/z +4 charge states of the substrate and hydroxylated product peptides were used for quantitation [1]
.
- Assay Quality: The assays were of high quality with Z' factors >0.5. The Hill slopes of the inhibition curves for 2,4-PDCA and its active derivatives were close to the theoretical value of -1, consistent with competition with 2OG for binding to the active site [1]
.

The text mentions potential applications, but does not provide experimental data on ADME properties. It suggests that hydrophobic F- or CF3-substituents on the 2,4-PDCA scaffold might increase cell-wall permeability. It also notes that 2,4-PDCA dimethylesters have been used in cell-based and in vivo studies, implying that esterification is a strategy to enhance cellular penetration [1]
.
- It is also suggested that F-substituted derivatives could be used as 19F NMR probes, and that radiolabeled 18F analogues might be developed for Positron Emission Tomography (PET) studies [1]

[1]. Fluorinated derivatives of pyridine-2,4-dicarboxylate are potent inhibitors of human 2-oxoglutarate dependent oxygenases. J Fluor Chem. 2021 Jul;247:109804.

[2]. Bioconversion of lignin-derived aromatics into the building block pyridine 2,4-dicarboxylic acid by engineering recombinant Pseudomonas putida strains. Bioresour Technol. 2022 Feb;346:126638.

Lutidinic acid is a pyridine dicarboxylic acid with carboxyl groups at the 2 and 4 positions. It is the conjugate acid of Lutidinic acid (1-).

---

Background and Role: 2,4-PDCA (pyridine-2,4-dicarboxylate) is a well-known, broad-spectrum inhibitor of 2-oxoglutarate (2OG) dependent oxygenases. It acts by competing with the cosubstrate 2OG for binding to the active site Fe(II) [1]
.
- Selectivity Profile: While a broad inhibitor, 2,4-PDCA shows a distinct selectivity profile. It efficiently inhibits AspH and some JmjC KDMs (like KDM4E) but is only a weak inhibitor of the PHDs and FIH [1]
.
- Derivatives for Enhanced Selectivity: This study explores F- and CF3-substituted derivatives of 2,4-PDCA at the C3 and C5 positions to improve inhibitor selectivity. The results show that introducing a substituent at the C5 position, particularly a fluorine atom, can dramatically increase selectivity for AspH over KDM4E while maintaining potency against AspH. This is a significant improvement over previous attempts to modify the C3 position [1]
.
- Potential Applications of Derivatives: The F-substituted 2,4-PDCA derivatives are proposed to have additional applications, such as:
- Tools for validating AspH as a cancer therapeutic target (AspH is upregulated on some cancer cells) [1]
.
- Use as electron-deficient substrates for nucleophilic aromatic substitution to label active site cysteine residues in some 2OG oxygenases (e.g., TET enzymes) [1]
.
- Use as 19F NMR probes for protein binding studies [1]
.
- Potential scaffolds for developing 18F-labeled Positron Emission Tomography (PET) tracers [1]
.

C7H5NO4

167.12

167.021

499-80-9

10365

Off-white to light yellow solid powder

1.6±0.1 g/cm3

574.8±35.0 °C at 760 mmHg

243-246 °C

301.4±25.9 °C

0.0±1.7 mmHg at 25°C

1.628

-0.19

2

5

2

12

204

0

MJIVRKPEXXHNJT-UHFFFAOYSA-N

InChI=1S/C7H5NO4/c9-6(10)4-1-2-8-5(3-4)7(11)12/h1-3H,(H,9,10)(H,11,12)

pyridine-2,4-dicarboxylic acid

2,4-PDCA 2,4PDCA 2,4 PDCA

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~598.37 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (14.96 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (14.96 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (14.96 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)