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Purity: ≥98%
UNC1215 (UNC-1215) is a potent and selective antagonist of L3MBTL3, a member of the MBT (malignant brain tumor) family of methyllysine (Kme) reading domain, with potential antineoplastic activity. It inhibits L3MBTL3 with an IC50 of 40 nM and a Kd of 120 nM.
| Targets |
Methyl-lysine reader protein L3MBTL3 (a member of the malignant brain tumor (MBT) family), with a Ki value of 1.1 nM for L3MBTL3. It exhibits low affinity for other L3MBTL family members: Ki > 10 μM (L3MBTL1), Ki = 120 nM (L3MBTL2), and no significant binding to non-L3MBTL methyl-lysine readers (e.g., HP1α, 53BP1, BPTF), confirming high selectivity for L3MBTL3 [1]
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| ln Vitro |
UNC1215 binds L3MBTL3 with ad of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more powerful toward L3MBTL3 than other members of the MBT family while also displaying selectivity over more than 200 other reader domains studied. X-ray crystallography discovered a unique 2:2 polyvalent form of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutations that affect the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization[1].
In a fluorescent polarization (FP) assay, UNC1215 dose-dependently inhibited the interaction between L3MBTL3 and a fluorescently labeled H4K20me3 peptide (a physiological ligand of L3MBTL3), with a Ki value of 1.1 nM. At 10 μM, UNC1215 showed <5% inhibition of 23 other methyl-lysine reader domains (e.g., chromodomains, Tudor domains, PHD fingers) and 15 non-reader proteins, demonstrating broad selectivity [1] - In an isothermal titration calorimetry (ITC) assay, UNC1215 directly bound to L3MBTL3 with a KD value of 1.3 nM, consistent with the FP-derived Ki, confirming a direct and high-affinity interaction [1] - In HeLa cells, UNC1215 (5 μM, 24-hour treatment) reduced the association of L3MBTL3 with chromatin, as measured by chromatin immunoprecipitation (ChIP) followed by quantitative PCR (qPCR) targeting L3MBTL3-bound genomic regions. No significant changes in L3MBTL3 protein levels were observed via Western blot, indicating the effect was due to target binding rather than protein degradation [1] - UNC1215 did not affect cell viability in HeLa, U2OS, or HEK293T cells at concentrations up to 20 μM (72-hour incubation), as measured by MTT assay, ruling out non-specific cytotoxicity [1] |
| ln Vivo |
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| Enzyme Assay |
Fluorescent Polarization (FP) Assay for L3MBTL3: Recombinant human L3MBTL3 protein (residues 1–384, containing three MBT repeats) was diluted in assay buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) to a final concentration of 50 nM. A FAM-conjugated H4K20me3 peptide (sequence: ARTKQTARKSTGGKAPRKQLA) was added to the protein solution to a final concentration of 20 nM, forming a protein-peptide complex. Serial dilutions of UNC1215 (0.001 nM–10 μM) were added, and the mixture was incubated at room temperature for 60 minutes. Fluorescent polarization values (mP) were measured using a microplate reader, and Ki values were calculated using a competitive binding model [1]
- Isothermal Titration Calorimetry (ITC) Assay: UNC1215 was dissolved in ITC buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl) to a concentration of 100 μM. L3MBTL3 protein was dissolved in the same buffer to a concentration of 10 μM. UNC1215 was titrated into the L3MBTL3 solution at 25°C, with 25 injections (10 μL each). Heat changes during binding were recorded, and KD values were calculated using a one-site binding model [1] - Selectivity FP Assay: The FP format was adapted to test UNC1215’s binding to other methyl-lysine readers. Recombinant target proteins (e.g., L3MBTL1, L3MBTL2, HP1α) and their corresponding FAM-labeled methylated peptides (e.g., H4K20me3 for L3MBTL1/2, H3K9me3 for HP1α) were used. UNC1215 was tested at 10 μM, and the percentage inhibition of protein-peptide binding was quantified [1] |
| Cell Assay |
Chromatin Immunoprecipitation (ChIP)-qPCR Assay: HeLa cells were seeded in 10 cm dishes and grown to 80% confluence. Cells were treated with UNC1215 (5 μM) or vehicle (DMSO, 0.1%) for 24 hours. Cells were cross-linked with 1% formaldehyde, lysed, and chromatin was sheared by sonication. L3MBTL3-bound chromatin was immunoprecipitated using an anti-L3MBTL3 antibody. Purified DNA was analyzed by qPCR using primers targeting known L3MBTL3-binding genomic loci (e.g., GAPDH promoter, MYC enhancer). Results were normalized to input chromatin [1]
- Western Blot Assay for L3MBTL3 Protein Level: HeLa cells were treated with UNC1215 (0.1–20 μM) or vehicle for 24 hours. Cells were lysed in RIPA buffer, and total protein was quantified. Proteins were separated by SDS-PAGE, transferred to a membrane, and probed with primary antibodies against L3MBTL3 and GAPDH (loading control). Secondary antibodies conjugated to horseradish peroxidase were used, and signals were detected via chemiluminescence [1] - Cell Viability Assay: HeLa, U2OS, and HEK293T cells were seeded in 96-well plates at 5×10³ cells/well. Cells were treated with serial dilutions of UNC1215 (0.1–20 μM) or vehicle for 72 hours. MTT reagent was added, and the mixture was incubated for 4 hours. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. Cell viability was calculated as a percentage of the vehicle control [1] |
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| References | |
| Additional Infomation |
[3-Phenylino-4-[oxo-[4-(1-pyrrolyl)-1-piperidinyl]methyl]phenyl]-[4-(1-pyrrolyl)-1-piperidinyl]methyl ketone belongs to the benzamide class of compounds and is an N-acylpiperidinyl compound. UNC1215 is a selective chemical probe used to target the methyl lysine reading domain of L3MBTL3, aiming to elucidate the biological functions of L3MBTL3 in chromatin regulation and gene expression [1]. X-ray crystallography studies have shown that UNC1215 binds to the methyl lysine binding pocket of L3MBTL3 (formed by the first two MBT repeat sequences) and competes with the physiological ligand H4K20me3 for binding. UNC1215 binds stably to key residues in L3MBTL3 (such as Tyr110 and Asp146) through hydrophobic interactions and hydrogen bonds [1]. The high selectivity of UNC1215 avoids off-target effects on other methyl lysine recognition proteins, and is therefore of great value for studying the role of L3MBTL3 in normal and disease-related chromatin dynamics [1].
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| Molecular Formula |
C32H43N5O2
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| Molecular Weight |
529.72
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| Exact Mass |
529.341
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| CAS # |
1415800-43-9
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| Related CAS # |
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| PubChem CID |
57339144
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
712.1±60.0 °C at 760 mmHg
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| Flash Point |
384.5±32.9 °C
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| Vapour Pressure |
0.0±2.3 mmHg at 25°C
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| Index of Refraction |
1.640
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| LogP |
3.37
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
39
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| Complexity |
803
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
PQOOIERVZAXHBP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C32H43N5O2/c38-31(36-20-12-27(13-21-36)34-16-4-5-17-34)25-10-11-29(30(24-25)33-26-8-2-1-3-9-26)32(39)37-22-14-28(15-23-37)35-18-6-7-19-35/h1-3,8-11,24,27-28,33H,4-7,12-23H2
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| Chemical Name |
(2-(phenylamino)-1,4-phenylene)bis((4-(pyrrolidin-1-yl)piperidin-1-yl)methanone)
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| Synonyms |
UNC-1215, UNC 1215, UNC1215
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.83 mg/mL (1.57 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.83 mg/mL (1.57 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 8.3 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: ≥ 0.83 mg/mL (1.57 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8878 mL | 9.4389 mL | 18.8779 mL | |
| 5 mM | 0.3776 mL | 1.8878 mL | 3.7756 mL | |
| 10 mM | 0.1888 mL | 0.9439 mL | 1.8878 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
UNC1215 is a potent antagonist of L3MBTL3.Nat Chem Biol.2013 Mar;9(3):184-91. |
X-ray crystal structure of the UNC1215-3MBT complex.Nat Chem Biol.2013 Mar;9(3):184-91. |
UNC1215 binds a small set of Kme reader proteins with lower affinity than L3MBTL3.Nat Chem Biol.2013 Mar;9(3):184-91. |
UNC1215 potently antagonizes 3MBT localization in cells.Nat Chem Biol.2013 Mar;9(3):184-91. |
UNC1215 binds and co-localizes with full length L3MBTL3 (a) UNC1215 conjugated to the cell-permeable merocyanine dye, mero-76, co-localizes with GFP-FLMBT in HEK293 cells (scale bar, 10 μm; green is GFP-FLMBT, red is merocyanine-UNC1215, and blue is Hoechst dye). (b) FLMBT binds to biotin-UNC1215 (5 nmol).Nat Chem Biol.2013 Mar;9(3):184-91. |
Identification of BCLAF1 as a novel L3MBTL3 protein interactor (a) In U2OS cells, 3MBT colocalizes with BCLAF1 (top panel; green is GFP-3MBT, red is BCLAF, and blue is DAP1). Upon treatment with UNC1215, the 3MBT and BCLAF1 nuclear foci are noticeably disrupted (bottom panel). (b) Immunoprecipitation experiments in cells transfected with flag-3MBT or flag-FLMBT show that UNC1215 disrupts the interaction between 3MBT and BCLAF1 and also reduces the interaction between FLMBT and BCLAF1.Nat Chem Biol.2013 Mar;9(3):184-91. |
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