UNC1215, an effective and specific MBT (malignant brain tumor) antagonist, binds L3MBTL3 (IC50/Kd: 40/120 nM). The selectivity of UNC1215 for L3MBTL3 is 50-fold higher versus other members of the human MBT family.

L3MBTL3:120 nM(Kd), L3MBTL3:40 nM

UNC1215与L3MBTL3结合,竞争性置换含单或二甲基赖氨酸的肽段。UNC1215对L3MBTL3的选择性比L3MBTL1高约75倍。UNC1215无细胞毒性,通过MBT域的Kme结合口袋,直接结合到L3MBTL3。UNC1215使GFP-L3MBTL3融合蛋白的细胞移动性与点突变增加,干扰GFP-L3MBTL3表型模拟的Kme结合功能,影响UNC1215的定位。UNC1215（30 μM）不影响UHRF1的串联Tudor域、CBX7的染色质域及JARID1A的PHD域。

AlphaScreen assay: Compound plates (1 μL at 10 or 30 mM highest concentration) are diluted in 1×assay buffer (20 mM Tris pH 8.0, 25 mM NaCl, 2 mM DTT and 0.05% Tween-20) over 2 steps using a Multimek robotic pipettor and 1 μL is spotted into the wells of 384-well assay Proxiplates. To these plates 9 μL of protein- peptide mix in 1× assay buffer is added by Multidrop and incubated for 30 min at room temperature. At this point 2 μL of streptavidin-conjugate donor and nickel-chelate acceptor beads (45 μg/mL in 1× assay buffer) are added, the plates are allowed to incubate for an additional 30 min in the dark at room temperature. After incubation the plates are read on EnVision mulilabel reader equipped with HTS alpha screen laser. The screens reported are performed up to 10 or 30 μM, and therefore it should be noted that those compounds referred to as inactive are indeed inactive only within the concentration range tested. PHF23 and JARID1A are GST tagged and consequently for these assays GST-acceptor beads are used. It should be noted that any positive binding curves for L3MBTL4 that are generated yielded curves with very shallow slopes, suggesting a nonspecific interaction. The data for the IC50 values is calculated from replicate runs in that the datapoints for each compound concentration are averaged and plotted using 4-parameters curve fitting.

CellTiter-Glo luminescent cell viability assay(Only for Reference)