Studies on the Interaction of the Histone Demethylase KDM5B with Tricarboxylic Acid Cycle Intermediates

Abstract

Methylation of lysine-4 of histone H3 (H3K4men) is an important regulatory factor in eukaryotic transcription. Removal of the transcriptionally activating H3K4 methylation is catalyzed by histone demethylases, including the Jumonji C (JmjC) KDM5 subfamily. The JmjC KDMs are Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenases, some of which are associated with cancer. Altered levels of tricarboxylic acid (TCA) cycle intermediates and the associated metabolites D- and L-2-hydroxyglutarate (2HG) can cause changes in chromatin methylation status. We report comprehensive biochemical, structural and cellular studies on the interaction of TCA cycle intermediates with KDM5B, which is a current medicinal chemistry target for cancer. The tested TCA intermediates were poor or moderate KDM5B inhibitors, except for oxaloacetate and succinate, which were shown to compete for binding with 2OG. D- and L-2HG were moderate inhibitors at levels that might be relevant in cancer cells bearing isocitrate dehydrogenase mutations. Crystallographic analyses with succinate, fumarate, L-malate, oxaloacetate, pyruvate and D- and L-2HG support the kinetic studies showing competition with 2OG. An unexpected binding mode for oxaloacetate was observed in which it coordinates the active site metal via its C-4 carboxylate rather than the C-1 carboxylate/C-2 keto groups. Studies employing immunofluorescence antibody-based assays reveal no changes in H3K4me3 levels in cells ectopically overexpressing KDM5B in response to dosing with TCA cycle metabolite pro-drug esters, suggesting that the high levels of cellular 2OG may preclude inhibition. The combined results reveal the potential for KDM5B inhibition by TCA cycle intermediates, but suggest that in cells, such inhibition will normally be effectively competed by 2OG.

Cancer-associated 2-oxoglutarate analogues modify histone methylation by inhibiting histone lysine demethylases

“…To evaluate the turnover rate and substrate specificity, we calculated the kcat and kcat/Km values for all enzymes and substrates ( Table 2). The kcat values were highly similar for all enzymes ranging from 0.004 to 0.04 s -1 ( Table 2) and in the same range than the 0.015 s -1 reported earlier for KDM5B 18 . For KDM4A and KDM5B there was no difference in the kcat values between H3K9me3 and H3K27me3 and H3K4me3 and H3K4me2 substrates, respectively ( Table 2).…”

“…This setting provides a basis for comparing the results between the enzymes and for gaining an understanding of which enzymes are actually inhibited by the 2-OG-analogue oncometabolites and are causally linked to the transformed phenotype. Previous reports have concentrated on kinetic and inhibitory data for the catalytic domains of human KDM4A and KDM5B and full-length KDM5B, these data having been generated by methods that detect substrate demethylation by MALDI-TOF-MS or antibody-based AlphaScreen or formaldehyde dehydrogenase (FDH) assay, which detects formaldehyde generated by substrate hydroxylation and subsequent demethylation 2,18,[23][24][25][26][27][28][29] . Of the available methods MALDI-TOF-MS is the only direct method available to detect the demethylated product.…”

Metformin directly targets the H3K27me3 demethylase KDM6A/UTX

“…On the basis of the similar property principle (Nigsch & Mitchell, 2008), which states that structural similar molecules are more likely to have similar properties and biological activities, we used 2D and 3D molecular fingerprints to disentangle whether biguanides functioned as KDM inhibitors by mimicking KDM crystallographic ligands and/or tricarboxylic acid cycle (TCA) intermediates that have been shown to interact with KDMs (Tarhonskaya et al., 2017) and extend lifespan (Mishur et al., 2016). When pairwise similarity calculations were measured using Dice and Tanimoto coefficients, we failed to observe a significant 2D structural resemblance between biguanides and KDM‐targeted metabolites (Figure 5, top panels).…”

“…Thus, the unique side chains that are known to determine the pharmacological differences of biguanides including mitochondrial accumulation and mCI inhibition (Boukalova et al., 2016; Bridges, Sirviö, Agip & Hirst, 2016; Bridges et al., 2014) might also influence the ability of the biguanides to interfere with KDM6A/UTX activity. Our discovery that biguanides do not exhibit either 2D structural‐topological analogies or 3D nonanalogous bioisosteric physicochemical relationships with KDM‐targeted metabolites (Mishur et al., 2016; Tarhonskaya et al., 2017) strongly suggest that different biguanides might be viewed as a new family of pharmacologically active KDM6A/UTX regulators.…”

In Vitro Enzyme Assays for JmjC‐Domain‐Containing Lysine Histone Demethylases (JmjC‐KDMs)

“…The K i values should therefore be determined whenever possible. See prior reports (Chowdhury et al., ; Rose et al., ; Tarhonskaya et al., ) for further examples of IC 50 and K i value determination for 2OG competitive inhibitors of JmjC‐KDMs.…”