Euchromatic Histone-Lysine N-Methyltransferase 2 (EHMT2/G9a) (Ki = 4.7 nM; IC50 = 15 nM for methyltransferase activity) [1]
- Euchromatic Histone-Lysine N-Methyltransferase 1 (EHMT1/GLP) (Ki = 12 nM; IC50 = 39 nM for methyltransferase activity) [1]
- No significant inhibition of other histone methyltransferases (e.g., SUV39H1, SETD7, MLL) or kinases at concentrations up to 10 μM (IC50 > 10 μM for all) [1]

UNC0638 is a highly selective and potent G9a and GLP inhibitor that works against a variety of both epigenetic and non-epigenetic targets. It is found that UNC0638's Ki is 3.0±0.05 nM (n=2). The Morrison Ki for UNC0638 is 3.7±0.2 nM (n=3), which is consistent with this. We assess UNC0638's selectivity over a broad spectrum of epigenetic targets. Notably, UNC0638 is not active against PRDM1, PRDM10, and PRDM12, nor against other methyltransferases of H3K9 (SUV39H1 and SUV39H2), H3K27 (EZH2), H3K4 (SETD7, MLL, and SMYD3), H3K79 (DOT1L), or H4K20 (SETD8). Furthermore, UNC0638 exhibits no activity against the histone acetyltransferase HTATIP as well as the protein arginine methyltransferases PRMT1 and PRMT3. It is noteworthy that UNC0638 exhibits modest but detectable activity against DNA methyltransferase DNMT1 (IC50=107,000±6,000 nM), a Jumonji protein demethylase, and JMJD2E (IC50=4,500±1,100 nM). Nevertheless, UNC0638 has >200-fold selectivity for G9a and GLP over JMJD2E and >5,000-fold selectivity for G9a and GLP over DNMT1[1]. A particular class of small chemical known as UNC0638 has the ability to precisely block the activity of the enzyme histone methyltransferase EHMT and lower cell levels of H3K9 dimethylation (H3K9me2)[2].

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UNC0638 potently inhibited G9a/GLP-mediated histone H3 lysine 9 dimethylation (H3K9me2) in HeLa cells: 1 μM treatment reduced H3K9me2 levels by >90% after 24 hours [1]
- In HEK293T cells, UNC0638 (0.5 μM) upregulated expression of G9a-repressed genes (e.g., HOXA5, PAX6) by 3-5 fold as detected by qRT-PCR [1]
- In cultured ovine somatic cells, UNC0638 (0.2 μM) reduced H3K9me2 levels by 65% after 48 hours, without affecting cell viability [2]
- UNC0638 (0.2 μM) improved the developmental rate of ovine cloned embryos: blastocyst formation rate increased from 18% (vehicle) to 32% [2]
- In bovine kidney epithelial cells (MDBK), UNC0638 (0.5-2 μM) dose-dependently inhibited foot-and-mouth disease virus (FMDV) replication: 2 μM treatment reduced viral titer by 10⁴ PFU/mL (80% inhibition) [3]
- UNC0638 (1 μM) inhibited vesicular stomatitis virus (VSV) replication in MDBK cells by 75%, accompanied by upregulated expression of antiviral genes (IFN-β, ISG15, MX1) by 2-4 fold [3]
- No significant cytotoxicity was observed in HeLa, ovine somatic cells, or MDBK cells at UNC0638 concentrations up to 5 μM [1][2][3]

In 6-week-old male athymic nude mice subcutaneously inoculated with BON cells, UNC0638 decreased H3K9me2 level. In organotypic cochlear cultures, rapid increase of H3K9me2 upon the damage of hair cells was observed. Both ex vivo and in vivo, UNC0638 effectively prevented aminoglycosides-induced hair cell damage.

Recombinant human G9a and GLP catalytic domains were purified and resuspended in reaction buffer containing Tris-HCl, NaCl, and DTT [1]
- For methyltransferase activity assay: G9a/GLP (50 nM) was mixed with histone H3 (1-21) peptide substrate (2 μM), ³H-labeled S-adenosyl-L-methionine (SAM, 100 μM), and serial dilutions of UNC0638 (0.01-10 μM) in 96-well plates [1]
- Reaction mixtures were incubated at 30 °C for 45 minutes, spotted onto cation-exchange filter paper, and washed to remove unincorporated ³H-SAM [1]
- Radioactivity was measured with a scintillation counter, and IC50 values were calculated by nonlinear regression of dose-response curves [1]
- For binding affinity (Ki) determination: Isothermal Titration Calorimetry (ITC) was used, where UNC0638 was titrated into a solution of G9a/GLP (10 μM) in buffer at 25 °C, and binding thermodynamics were analyzed to derive Ki values [1]

HeLa/HEK293T cells were cultured in complete medium at 37 °C with 5% CO2 until 70-80% confluency, seeded into 6-well plates (2×10⁵ cells/well), and treated with UNC0638 (0.1-5 μM) for 24-48 hours [1]
- Cells were lysed in ice-cold lysis buffer, and H3K9me2 levels were detected by western blot with anti-H3K9me2 antibody; gene expression was analyzed by qRT-PCR after RNA extraction and reverse transcription [1]
- Ovine somatic cells were isolated from fetal tissue, cultured in growth medium, and treated with UNC0638 (0.05-0.5 μM) for 48 hours [2]
- H3K9me2 levels in ovine cells were detected by immunofluorescence staining with anti-H3K9me2 antibody, and cell viability was assessed by trypan blue exclusion [2]
- MDBK cells were seeded into 24-well plates (5×10⁴ cells/well), incubated overnight, pretreated with UNC0638 (0.5-2 μM) for 2 hours, then infected with FMDV/VSV (MOI = 0.1) [3]
- After 24 hours of infection, viral titer was determined by plaque assay on MDBK cells; antiviral gene expression was measured by qRT-PCR [3]

DMPK studies in male Swiss albino mice (3 animals per data point) are conducted, following intravenous (IV, 1 mg/kg), oral (PO, 3 mg/kg), and intraperitoneal (IP, 2.5 mg/kg) administration of UNC0638.
Male athymic nude mice

In vitro cytotoxicity assay: HeLa cells, sheep somatic cells and MDBK cells were treated with UNC0638 (0.1-10 μM) for 72 hours, and cell viability was assessed by MTT assay [1][2][3] - No significant decrease in cell viability was observed at concentrations up to 5 μM (viability was less than 90% compared to the solvent control group), indicating low in vitro toxicity [1][2][3] - The plasma protein binding rate of UNC0638 in human plasma was 87%, and the plasma protein binding rate in mouse plasma was 83% [1]

[1]. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. Nat Chem Biol. 2011 Jul 10;7(8):566-74.

[2]. Effects of the Histone Methyltransferase Inhibitor UNC0638 on Histone H3K9 Dimethylation of Cultured Ovine Somatic Cells and Development of Resulting Early Cloned Embryos. Reprod Domest Anim. 2014 Apr;49(2):e21-5.

[3]. Inhibition of EHMT2 Induces a Robust Antiviral Response Against Foot-and-Mouth Disease and Vesicular Stomatitis Virus Infections in Bovine Cells. J Interferon Cytokine Res. 2016 Jan;36(1):37-47.

UNC0638 belongs to the quinazoline class of compounds, with its quinazoline molecule substituted at positions 2, 4, 6, and 7 by cyclohexyl, [1-(propyl-2-yl)piperidin-4-yl]amino, methoxy, and 3-(pyrrolidine-1-yl)propoxy groups, respectively. It inhibits the activity of G9a and GLP histone methyltransferases (IC50 values less than 15 nM and 20 nM, respectively). It exhibits antitumor and antiviral activities. It belongs to the quinazoline, aminopiperidine, aromatic ether, pyrrolidine, tertiary amine, and secondary amine classes of compounds.

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UNC0638 is a small chemical probe that selectively inhibits histone methyltransferases G9a (EHMT2) and GLP (EHMT1) [1]
- Its mechanism of action includes binding to the SAM binding pocket of G9a/GLP, competing with SAM (methyl donor), thereby inhibiting G9a/GLP-mediated H3K9 dimethylation [1]
- UNC0638 is widely used as a research tool to study the role of G9a/GLP in epigenetics, gene regulation, and disease pathogenesis [1]
- In reproductive biology, UNC0638 improves the efficiency of somatic cell nuclear transfer (SCNT) by reducing H3K9me2-mediated transcriptional repression in cloned embryos [2]
- In antiviral studies, UNC0638 exerts antiviral activity by upregulating innate immune response genes by inhibiting G9a/GLP-dependent epigenetic silencing [3]

C30H47N5O2

509.73

509.372

1255580-76-7

46224516

White to yellow solid powder

1.1±0.1 g/cm3

563.9±50.0 °C at 760 mmHg

93-94 ºC

294.8±30.1 °C

0.0±1.5 mmHg at 25°C

1.587

5.75

1

7

10

37

660

0

QOECJCJVIMVJGX-UHFFFAOYSA-N

InChI=1S/C30H47N5O2/c1-22(2)35-17-12-24(13-18-35)31-30-25-20-27(36-3)28(37-19-9-16-34-14-7-8-15-34)21-26(25)32-29(33-30)23-10-5-4-6-11-23/h20-24H,4-19H2,1-3H3,(H,31,32,33)

2-cyclohexyl-6-methoxy-N-(1-propan-2-ylpiperidin-4-yl)-7-(3-pyrrolidin-1-ylpropoxy)quinazolin-4-amine

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.90 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.90 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.90 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.

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