Histone demethylase KDM2/7

TC-E 5002/compound 9 was 20-fold more active than 6 in KDM7B in MALDI assays. Docking of compound 9 in the active site of KDM7B suggested that the cyclopropyl group of compound 9 interacts with the phenyl-group of Phe 250 through CH−π or cyclopropyl−π interactions (Figure 2). Furthermore, compound 9 inhibited KDM2A (also known as JHDM1A, FBXL11), KDM7A (also known as JHDM1D, KIAA1718), and KDM7B, demonstrating inhibition of all KDM2/7 subfamily members tested (Tables 2 and 3). Compound 9 displayed selectivity for KDM2/7 over KDM4A (IC50 > 120 μM), KDM4C (IC50 = 83 μM), KDM5A (IC50 = 55 μM), and KDM6A (also known as UTX) (IC50 > 100 μM); note however different assays conditions were used (see Experimental Section). Thus, compound 9 was the most potent KDM7B inhibitor identified in the enzyme assays. In addition, compound 13 showed comparatively high selectivity for KDM2A over KDM7B and the other isozymes tested. Because the molecular modeling suggest that the space around the cyclopropane ring of 9 is not so large in the hydrophobic pocket of KDM7B (Figure 2), it may be difficult for compound 13 bearing a phenyl ring to have a conformation which can efficiently interact with Phe of the pocket. On the other hand, KDM2A has a hydrophobic pocket more spacious than KDM7B (Supporting Information Figure S2), in which the phenyl ring can efficiently interact with hydrophobic amino acid residues of KDM2A. These may be the reason that compound 13 shows selectivity for KDM2A over KDM7B.[1]

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To investigate whether TC-E 5002/compound 9 is active as an inhibitor of KDM7A and KDM7B in cells, we performed a cellular assay with Western blot analysis. Because KDM7 is known to catalyze the demethylation of H3K27me2,10,11 the methylation level of H3K27 in cells was analyzed. In this study, we used N2a cells because it has been reported that KDM7 is expressed in the cells.11 As Figure 3 shows, the level of H3K27me2 was dose-dependently elevated in the presence of 9. Although the interpretation of changes in global histone N-methylation status can be complex, the elevation in the H3K27me2 levels is consistent with KDM7 inhibition. These results suggest that H3K27me2 demethylation is inhibited by compound 9 in cells, and it looks to be useful as a tool for probing the biological role of KDM7.[1]

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Although it has been reported that RNAi-mediated knockdown of KDM7B suppresses the growth of some cancer cells,6 it remains unclear whether the demethylase function of KDM7B is responsible for the suppression because RNAi-mediated knockdown of KDM7B should cause loss of not only the demethylase function but also other functions of KDM7B,12 including those relating to noncatalytic binding domains. Initially, we investigated the N2a cell growth inhibition activity of TC-E 5002/compound 9. N2a cell growth suppression by compound 9 was observed (GI50 = 86 μM) (Figure 4) at the concentration range in which distinct H3K27 methylation was detected on Western blot analysis (Figures 3). Thus, this may suggest the demethylase function of KDM7 is involved in this cell growth, however, more potent and selective compounds will be needed to fully elucidate this fact. Next, we carried out cell growth inhibition assays of compound 9 as well as prodrugs of 1, 2, and 3 using HeLa cells and KYSE150 cells (Table 3). It has also been reported that knockdown of KDM4C decreases cell proliferation,7a however, the KDM4C inhibitors 2, 3 (Table 3), and their methyl ester prodrugs (GI50 > 500 μM) did not show any effects on the growth inhibition of tested cancer cells although they are cell membrane permeable.7d,13 These results suggest that the demethylase activity of KDM4C is not directly associated with cancer cell growth, at least in some cell types. On the other hand, cell growth suppression by the KDM2/7-subfamily inhibitor 9 was observed for KYSE150 and HeLa cell lines (Figure 5). Furthermore, compound 9 caused H3K27 methylation both in HeLa cells and in KYSE150 cells at the concentration range in which the cell growth inhibition was observed (Figure 6). The data shown in Table 3 indicate that KDM2/7 inhibitors are worthy of evaluation as candidate anticancer agents.[1]

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A recent study reported that KDM7B activates the transcription of the E2F1 transcription factor in HeLa cells, which promotes cell cycle progression.14 Because TC-E 5002/compound 9 decreases the growth of HeLa cells with H3K27me2 accumulation (Table 3; Figures 5 and 6), we examined whether compound 9 down-regulates the expression of E2F1 in HeLa cells by quantitative RT-PCR. As shown in Figure 7, compound 9 significantly decreases the mRNA level of E2F1 at 80 μM in which the growth of HeLa cells was affected. These data suggest that the KDM7B-mediated regulation of E2F1 gene expression may be one of the mechanisms of growth regulation in some cancer cells.[1]

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We also investigated the effect of TC-E 5002/compound 9on cell cycle progression by FACS analysis. HeLa cells and KYSE150 cells incubated with 10 or 100 μM of compound 9 for 24 h showed a dose-dependent reduction in G2–M phase, whereas there was a dose-dependent increase in G0–G1 phase (Figure 8). These results revealed that HeLa cells and KYSE150 cells cultured with compound 9 arrested in the G0/G1 phase of the cell cycle, which is consistent with the down-regulation of E2F1 by compound 9 (Figure 7). [1]

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KDM7B Inhibition Assay[1]
KDM7B (0.5 mg/mL) was incubated with 150 mM KCl, 2.5% glycerol, 0.5 mM dithiothreitol, 0.05 mM PMSF, 2.5 mM glutathione reduced form, 20 μM (+)-Fe(II)-l-ascorbic acid, 20 μM ZnCl2, 0.5 mM ascorbic acid, 0.5 mM 2-oxoglutarate, and 5 μM H3K4me3K9me2 (ART(Kme3)QTAR(Kme2)STGGKAPRKQL-Cys) for 1 h at 37 °C in 8 μL of 10 mM Tris-HCl buffer (pH 8.0). The reaction was stopped by adding 75 μL of matrix-solution (5 mg/mL α-cyano-4-hydroxycinnamic acid, 37% acetonitrile, and 0.12% trifluoroacetic acid) and then sonicated for 30 s. Then 1 μL of the reaction mixture was spotted on the sample plate, dried, and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using Voyager-DE PRO. The KDM7B inhibition activity of the test compounds was calculated from the remaining amount of H3K9me2. The 50% inhibitory concentration (IC50) of the test compounds was calculated as the concentration at which the half amount of H3K9me2 was removed compared to that removed when the enzyme was added (Supporting Information Figure S7).

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KDM4C Inhibition Assay[1]
The KDM4C-inhibitory activity was measured using 0.6 mg/mL enzyme. The compounds were dissolved in DMSO. The final concentrations of DMSO in the reaction mixtures were less than 3.3%, and it was confirmed that 3.3% DMSO did not affect the KDM4C activity. Reaction with DMSO alone was also done as a control. Reaction mixtures (94.6 μL), containing all of the materials except H3K9me3 peptide and 2-oxoglutarate, were preincubated for 5 min. Then the reactions were started by the addition of 5.4 μL of a solution of 0.93 mM H3K9me3 peptide and 3.7 mM 2-oxoglutarate . The enzyme activity was determined as described above. The ratio of the enzyme activity measured in the presence of inhibitor to that of the control was plotted against log [Inhibitor]. To confirm that the reduction of the KDM4C activity by test compounds was not due to inhibition of the coupled enzyme FDH, we examined the effects of the test compounds on FDH activity. The reaction mixture (0.1 mL) contained 20 mM HEPES-KOH, pH 7.5, 50 μM formaldehyde, 1 mM 3-acetylpyridine adenine dinucleotide, 1 mM reduced glutathione, 0.1 mg/mL BSA, 0.1 mg/mL FDH, and a fixed concentration of 123 μM test compound. The FDH activity was measured by monitoring APADH formation as described above. The FDH activity in the presence of test compounds was similar to that in the absence of the compounds.

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KDM4A Inhibition Assay[1]
The KDM4A activity was measured by the FDH-coupled assay as described for KDM4C except that reactions were performed in a final volume of 30 μL in 384-well plate (Nunc) and a final concentration of the KDM4A was 0.37 mg/mL.

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KDM5A Inhibition Assay[1]
The KDM5A activity was measured by the FDH-coupled assay as described for KDM4C except that reactions were performed with H3K4me3 peptide in a final volume of 30 μL in 384-well plate (Nunc) and a final concentration of the KDM5A was 0.64 mg/mL.

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KDM2A Inhibition Assay[1]
The inhibitory activities of test compounds against KDM2A was assayed according to the method reported in ref (4).

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KDM6A Inhibition Assay[1]
The Epigenase JMJD3/UTX demethylase activity/inhibition assay kit was used for KDM6A enzyme assay. The materials supplied with the kit, 100 μM of test compounds and KDM6A (300 ng/well) were added to wells coating trimethylated histone substrate, according to the supplier’s protocol. The resulting mixtures were incubated at 37 °C for 120 min. After enzyme reaction, each well was reacted with capture antibody for 60 min and with detection antibody for 30 min. Finally, developer solution and stop solution were added to wells in sequence and the absorbance (450 nm) in each well was measured with an ARVO X3 microplate reader. The KDM6A inhibition activity of test compounds was calculated from the absorbance readings.

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KDM7A Inhibition Assay[1]
The KDM7A assay was run using MALDI and K27me2 peptide as a substrate under the same conditions as used in ref (4).

RNA Isolation and Semi-qRT-PCR[1]
HeLa cells were treated for 48 h with 0.238% DMSO or TC-E 5002/compound 9 at the concentration of 30 and 80 μM, respectively. Total RNA was isolated from HeLa cells using RNAzol following the manufacturer’s protocol. First-strand cDNA synthesis from total RNA was carried out using ReverTra Ace. Resulting cDNA was then analyzed by semiquantitative PCR (semi-qPCR) using 2720 thermal cycler. Primers are specific for genes tested, and their sequences are as follows:
GAPDH 450bp Primer(F): 5′-TCCACCACCCTGTTGCTGTA-3′ (20mer) Primer(R): 5′-ACCACAGTCCATGCCATCAC-3′ (20mer)
E2F1 435bp Primer(F): 5′-ACTCCTCGCAGATCGTCATCATCT-3′(24mer) Primer(R): 5′-GGACGTTGGTGATGTCATAGATGCG-3′(25mer)
Cycle parameters were 94 °C for 2 min, followed by 28 (E2F1), 20 (GAPDH) cycles of 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 30 s, with a final extension at 68 °C for 1 min

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FACS Analysis[1]
Cells (5 × 105) were treated for 24 h with TC-E 5002/compound 9 at the indicated concentrations in RPMI 1640 with 10% fetal bovine serum, then harvested by trypsinization. The cells were collected by centrifugation, fixed with ice-cold 70% ethanol, washed with phosphate-buffered saline, and resuspended in 0.5 mL of phosphate-buffered saline containing propidium iodide (10 μg/mL) and RNase A (0.2 mg/mL). After a final incubation at 25 °C for 30 min, the cells were analyzed using a JSAN flow cytometer. A total of 30000 events were counted for each sample. Data were analyzed using FlowJo software

[1]. Identification of the KDM2/7 histone lysine demethylase subfamily inhibitor and its antiproliferative activity. J Med Chem. 2013 Sep 26;56(18):7222-31.

Histone N(ε)-methyl lysine demethylases KDM2/7 have been identified as potential targets for cancer therapies. On the basis of the crystal structure of KDM7B, we designed and prepared a series of hydroxamate analogues bearing an alkyl chain. Enzyme assays revealed that compound 9 potently inhibits KDM2A, KDM7A, and KDM7B, with IC50s of 6.8, 0.2, and 1.2 μM, respectively. While inhibitors of KDM4s did not show any effect on cancer cells tested, the KDM2/7-subfamily inhibitor 9 exerted antiproliferative activity, indicating the potential for KDM2/7 inhibitors as anticancer agents.

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We have identified a novel KDM2/7 subfamily inhibitor 9, which should be useful as a lead structure in the development of more potent and selective KDM2/7 inhibitors. Such inhibitors are candidates for anticancer agents as well as tools for studying the biological roles of KDM2/7 subfamily in cells.[1]

C15H27NO4

285.38

285.194

C, 63.13; H, 9.54; N, 4.91; O, 22.42

1453071-47-0

71735843

White to off-white solid powder

87 °C

3.209

2

4

12

20

302

0

NEHSERYKENINRH-UHFFFAOYSA-N

InChI=1S/C15H27NO4/c17-14(16(20)12-11-15(18)19)8-6-4-2-1-3-5-7-13-9-10-13/h13,20H,1-12H2,(H,18,19)

3-[9-cyclopropylnonanoyl(hydroxy)amino]propanoic acid

TCE 5002; TC E 5002; 1453071-47-0; TC-E 5002; N-(9-Cyclopropyl-1-oxononyl)-N-hydroxy-beta-alanine; 3-(9-CYCLOPROPYL-N-HYDROXYNONANAMIDO)PROPANOIC ACID; KDM2/7-IN-1; CHEMBL2424812; NCDM-64; MFCD28166486; TC-E 5002

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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