Article

Design and Discovery of new pyrimidine coupled nitrogen aromatic rings as chelating groups of JMJD3 Inhibitors

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Abstract

The histone methylation on lysine residues is one of the most studied post-translational modifications, and its aberrant states have been associated with many human diseases. In 2012, Kruidenier et al. reported GSK-J1 as a selective Jumonji H3K27 demethylase (JMJD3 and UTX) inhibitor. However, there is limited information on the structure–activity relationship of this series of compounds. Moreover, there are few scaffolds reported as chelating groups for Fe(II) ion in Jumonji demethylase inhibitors development. To further elaborate the structure–activity relationship of selective JMJD3 inhibitors and to explore the novel chelating groups for Fe(II) ion, we initialized a medicinal chemistry modification based on the GSK-J1 structure. Finally, we found that several compounds bearing different chelating groups showed similar activities with respect to GSK-J1 and excellent metabolic stability in liver microsomes. The ethyl ester prodrugs of these inhibitors also showed a better activity than GSK-J4 for inhibition of TNF-α production in LPS-stimulated murine macrophage cell line Raw 264.7 cells. Taking together, the current study not only discovered alternative potent JMJD3 inhibitors, but also can benefit other researchers to design new series of Jumonji demethylase inhibitors based on the identified chelating groups.

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... Compound 2 showed higher potency and selectivity for KDM4C over other KDM subfamilies (Korczynska et al., 2016). Analogs of GSK-J1 (3) (Kruidenier et al., 2012) preserving the β-amino acid and changing the 2-(pyridine-2-yl)pyrimidine scaffold were developed and found to exhibit better activity at TNF-α production than parent 3 (Hu et al., 2016). Benzoxazole (4) was shown to inhibit KDM6 subfamily member JMJD3 and to induce the cell cycle arrest in the S-phase on A375 melanoma cells (Giordano et al., 2019). ...
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Aberrant epigenetic modifications are involved in cancer development. Jumonji C domain-containing histone lysine demethylases (KDMs) are found mainly up-regulated in breast, prostate, and colon cancer. Currently, growing interest is focusing on the identification and development of new inhibitors able to block the activity of KDMs and thus reduce tumor progression. KDM4A is known to play a role in several cellular physiological processes, and was recently found overexpressed in a number of pathological states, including cancer. In this work, starting from the structure of purpurogallin 9aa, previously identified as a natural KDM4A inhibitor, we synthesized two main sets of compound derivatives in order to improve their inhibitory activity against KDM4A in vitro and in cells, as well as their antitumor action. Based on the hypothetical biogenesis of the 5-oxo-5H-benzo[7]annulene skeleton of the natural product purpurogallin (Salfeld, 1960; Horner et al., 1961; Dürckheimer and Paulus, 1985; Tanaka et al., 2002; Yanase et al., 2005) the pyrogallol and catechol units were first combined with structural modifications at different positions of the aryl ring using enzyme-mediated oxidative conditions, generating a series of benzotropolone analogs. Two of the synthetic analogs of purpurogallin, 9ac and 9bc, showed an efficient inhibition (50 and 80%) of KDM4A in enzymatic assays and in cells by increasing levels of its specific targets, H3K9me3/2 and H3K36me3. However, these two compounds/derivatives did not induce cell death. We then synthesized a further set of analogs of these two compounds with greater structural diversification. The most potent of these analogs, 9bf, displayed the highest KDM4A inhibitory enzymatic activity in vitro (IC50 of 10.1 and 24.37 μM) in colon cancer cells, and the strongest antitumor action in several solid and hematological human cancer cell lines with no toxic effect in normal cells. Our findings suggest that further development of this compound and its derivatives may lead to the identification of new therapeutic antitumor agents acting through inhibition of KDM4A.
... Indeed, concerning JMJD3, up to date, only one JMJD3/UTX binder has been reported, GSK-J1, 1 along with a series of its derivatives showing a similar or lower activity. 18 Recently, we identified the quinoline-5,8 dicarboxylic acid scaffold as new potential lead compound for the development of JMJD3 inhibitors, using a fragment-based approach. 19 This chemical core is endowed with an unprecedented selectivity toward the proposed biological target and a relevant inhibition potency in the micromolar range. ...
Article
JMJD3 is a member of the KDM6 subfamily and catalyzes the demethylation of lysine 27 on histone H3 (H3K27). This protein was identified as a useful tool in understanding the role of epigenetics in inflammatory conditions and in cancer as well. Guided by a virtual fragment screening approach, we identified the benzoxazole scaffold as a new hit suitable for the development of tighter JMJD3 inhibitors. Compounds were synthesized by a microwave-assisted one-pot reaction under catalyst and solvent-free conditions. Among these, compound 8 presented the highest inhibitory activity (IC 50 = 1.22 ± 0.22 μM) in accordance with molecular modeling calculations. Moreover, 8 induced the cycle arrest in S-phase on A375 melanoma cells.
... 13 Structure−activity relationship (SAR) studies of GSK-J1 led to the identification of thiazolo-, pyrazolo-, and triazolopyrimidine derivatives, which showed similar or reduced potency. 14 As claimed in a recent article by Nowak et al., 15 although potent inhibitors have been identified for most KDM subfamilies, the identification of selective and cell-permeable inhibitors remains a challenge. McAllister et al. 16 have reviewed KDM inhibitors developed since 2014 and pointed out that the vast majority of the identified inhibitors are OGA competitors and are all derivatives of a reduced number of metal-chelating fragments. ...
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... Administration of 66b significantly reduced the expression of 16 in 34 LPS-driven cytokines by PCR array. Xiong et al. designed and synthesized a series of derivatives of 66a to study the detail of SAR of this scaffold of selective KDM6 inhibitors 130 . The representative compound 67a had an equivalent potency against 66a (IC 50 ¼ 0.15 lM versus 0.15 lM). ...
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Histone demethylation is a vital process in epigenetic regulation of gene expression. A number of histone demethylases are present to control the methylated states of histone. Among these enzymes, KDM4s are one subfamily of JmjC KDMs and play important roles in both normal and cancer cells. The discovery of KDM4s inhibitors is a potential therapeutic strategy against different diseases including cancer. Here, we summarize the development of KDM4s inhibitors and some related pharmaceutical information to provide an update of recent progress in KDM4s inhibitors.
... [1] Recently,aseries of GSK-J1 derivatives was reported, showing activity similar to or lower than that of the reference compound. [10] Thus, the discovery of small molecules that are able to selectively modulate the biological functiono f JMJD3 is very attractive, in that they will shed light on itsr ole, both in normal biological processes and under diseasec onditions, expanding the cancert herapy toolkit. ...
Article
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The quinoline-5,8 dicarboxylic acid scaffold has been identified by a fragment-based approach as new potential lead compound for the development of JMJD3 inhibitors. Among them, 3-(2,4-dimethoxypyrimidin-5-yl)quinoline-5,8-dicarboxylic acid (compound 3) shows low micromolar inhibitory activity against Jumonji domain-containing protein 3 (JMJD3). The experimental evaluation of inhibitory activity against seven related isoforms of JMJD3 highlighted an unprecedented selectivity toward the biological target of interest.
... Likely candidates to establish demethylation of H3K4me1/2 to establish the unmethylated state are limited, and we previously showed that expression of LSD1 (KDM1), a H3K4me2/1 demethylase [45], did indeed alter TRIM24-regulated gene expression in MCF7 cells [7]. Recent studies linking ER and enzymes that modulate histone methylation, UTX, LSD1 and MLL2 in ER-positive breast cancer cells suggest that histone methylation and demethylation is an important regulatory node that may yield to recently developed inhibitors of these enzymes [46][47][48][49]. Multiple histone acetyltransferases, including CBP/p300 [50] and GCN5 [51], may acetylate H3K23, and ERa has been shown to promote chromatin acetylation by recruiting CBP/p300 [52,53]. ...
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... In vivo administration of GSK-J4 killed the TAL1-positive primary human leukemia cells in a patientderived xenotransplant (PDX) model [139]. Novel KDM6B inhibitors modified from the previously identified pan-KDM6 inhibitor GSK-J1 have been reported recently and the ethyl ester prodrugs of these inhibitors showed better activity than GSK-J4 in a cell-based functional assay [141]. However, it is noteworthy that GSK-J1/J4 may also inhibit KDM5B and KDM5C at higher concentrations [142]. ...
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... 682 A study that explored the SAR by replacing the pyridine ring of GSK-J1 with other potential chelating heterocycles was also published. 703 Several compounds featuring a pyrazole or triazole ring instead of the pyridine ring with IC 50 values of 0.15−0.27 μM were identified. ...
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... Recent studies linking ER and enzymes that modulate histone methylation, UTX, LSD1 and MLL2 in ER-positive breast cancer cells suggest that histone methylation and demethylation is an Drug Discovery Today: Technologies | Vol. 19,2016 important regulatory node that may yield to recently developed inhibitors of these enzymes [46][47][48][49]. Multiple histone acetyltransferases, including CBP/p300 [50] and GCN5 [51], may acetylate H3K23, and ERa has been shown to promote chromatin acetylation by recruiting CBP/p300 [52,53]. ...
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... Due to the limited information on the structureactivity relationship of GSK-J1 and its isomers, Hu et al., initialized a medicinal chemistry modification based on GSK-J1 structure, and they finally identified several compounds which exhibited similar activities as with GSK-J1. The inhibition of these compounds on LPS-stimulated genes was also proven in macrophages [80]. Some other inhibitors of JmjC demethylases have been identified including the α-ketoglutaric acid mimics N-oxalylglycine [81], methylstat [82], and catechols [83]. ...
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Selective inhibitors of Jumonji domain-containing protein (JMJD) histone demethylases are candidate anticancer agents as well as potential tools for elucidating the biological functions of JMJDs. On the basis of the crystal structure of JMJD2A and a homology model of JMJD2C, we designed and prepared a series of hydroxamate analogues bearing a tertiary amine. Enzyme assays using JMJD2C, JMJD2A, and prolyl hydroxylases revealed that hydroxamate analogue 8 is a potent and selective JMJD2 inhibitor, showing 500-fold greater JMJD2C-inhibitory activity and more than 9100-fold greater JMJD2C-selectivity compared with the lead compound N-oxalylglycine 2. Compounds 17 and 18, prodrugs of compound 8, each showed synergistic growth inhibition of cancer cells in combination with an inhibitor of lysine-specific demethylase 1 (LSD1). These findings suggest that combination treatment with JMJD2 inhibitors and LSD1 inhibitors may represent a novel strategy for anticancer chemotherapy.
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For a physiologically realistic joint range of motion and therefore range of muscle fiber lengths, only part of the whole muscle force-length curve can be used in vivo; that is, only a section of the force-length curve is expressed. Previous work has determined that the expressed section of the force-length curve for individual muscles can vary between subjects; however, the degree of intersubject variability is different for different muscles. This study determined the expressed section of both the rectus femoris and gastrocnemius--muscles with very different ratios of tendon slack length to muscle fiber optimum length--for 28 nonspecifically trained subjects to test the hypothesis that the value of this ratio affects the amount of variability in the expressed section. The force-length curves of the two muscles were reconstructed from moment-angle data using the method of Herzog & ter Keurs (1988). There was no relationship between the expressed sections of the force-length curve for the two muscles. Less variability was found in the expressed section of the gastrocnemius compared with the rectus femoris, supporting the hypothesis. The lack of relationship between the expressed sections of the two muscles has implications for motor control and for training muscle for rehabilitation.
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Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of N(epsilon)-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of N-oxalyl-d-tyrosine derivatives to explore the extent of a subpocket at the JMJD2 active site. Some of the inhibitors were shown to be selective for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2. A crystal structure of JMJD2A in complex with one of the potent inhibitors was obtained; modeling other inhibitors based on this structure predicts interactions that enable improved inhibition for some compounds.
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Epigenetics is rising to prominence in biology as a mechanism by which environmental factors have intermediate-term effects on gene expression without changing the underlying genetic sequence. This can occur through the selective methylation of DNA bases and modification of histones. There are wide-ranging implications for the gene-environment debate and epigenetic mechanisms are causing a reevaluation of many traditional concepts such as heritability. The reversible nature of epigenetics also provides plausible treatment or prevention prospects for diseases previously thought hard-coded into the genome. Here, we consider how growing knowledge of epigenetics is altering our understanding of biology and medicine, and its implications for future research.
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Recent studies indicate that histone lysine methylation is subject to enzyme-catalyzed reversion, and jumonji C (JmjC) domain-containing proteins have been identified as one of the members of histone demethylases. Although an increasing number of histone demethylases have been identified and biochemically characterized, their biological functions are poorly characterized. To elucidate the physiological functions, we generated the knockout mouse model of dimethylated or monomethylated histone 3 lysine 9 (H3K9me2/1)-specific JmjC domain-containing histone demethylase 2A (JHDM2A; also known as JMJD1A and KDM3A) and showed that JHDM2A is essential for spermatogenesis. Jhdm2a-deficient mice exhibited impaired postmeiotic chromatin condensation, which caused infertility, even though the hormonal levels were maintained. Further molecular and biochemical analysis revealed that JHDM2A directly bound to the core promoter regions of transition nuclear protein 1 (Tnp1) and protamine 1 (Prm1) genes, and it induced the transcriptional activation of these genes by removing H3K9 methylation, which is known as a silencing marker of gene transcription. This work uncovered a role for JHDM2A in spermatogenesis and identified 2 downstream genes that are critical for sperm nuclear condensation. In addition, we also showed that JHDM2A plays a role in regulating fat metabolic gene expression in muscle and brown fat tissue, and the knockout mice exhibited obesity and hyperlipidemia. Thus, JHDM2A possesses organ/tissue-specific target genes, and impairment of this molecule cannot be compensated by other JmjC-containing histone demethylases, suggesting the importance of this molecule in vivo.
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Posttranslational modifications of histone N-terminal tails impact chromatin structure and gene transcription. While the extent of histone acetylation is determined by both acetyltransferases and deacetylases, it has been unclear whether histone methylation is also regulated by enzymes with opposing activities. Here, we provide evidence that LSD1 (KIAA0601), a nuclear homolog of amine oxidases, functions as a histone demethylase and transcriptional corepressor. LSD1 specifically demethylates histone H3 lysine 4, which is linked to active transcription. Lysine demethylation occurs via an oxidation reaction that generates formaldehyde. Importantly, RNAi inhibition of LSD1 causes an increase in H3 lysine 4 methylation and concomitant derepression of target genes, suggesting that LSD1 represses transcription via histone demethylation. The results thus identify a histone demethylase conserved from S. pombe to human and reveal dynamic regulation of histone methylation by both histone methylases and demethylases.