Table 1.
Profiling of Four Screening Hit Compounds by In Vitro Methyltransferase Assays and Assays for Cellular H3K4me2 and Cell Viability
| Methyltransferase or cell-based assay | Compound IC50s, μMb (IC50s<25 μM) | |||||
|---|---|---|---|---|---|---|
| Substratea | RBC-1 | RBC-2 | RBC-3 | RBC-4 | SAH | |
| Assay for cellular H3K4me2c | N/A | 3.4f | 7.2 | 24.3 | 3.9 | ND |
| 9.2 | 23.4 | 1.7 | ||||
| 7.7 | 20.5 | 2.0 | ||||
| Cell viability assayd | N/A | 9.8 | >200 | 85.1 | 30.6 | ND |
| MLL1-WRAD2 | Core histones | 3.7 | 8.4 | 17.6 | 3.0 | 1.3 |
| 3.4 | 11.4 | 17.8 | 3.2 | 0.69 | ||
| MLL1-WRAD2 | Nucleosomes | 2.6 | 33.4 | 10 | 1.8 | 4.7 |
| SET1B-WRAD2 | Core histones | 43.8 | 64.4 | 9.3 | 5.2 | 16 |
| 49.3 | 87 | 9.7 | 5.7 | 7.1 | ||
| MLL2-WRAD2 | Core histones | 132.0 | 179.0 | 11.1 | 13.9 | 14.5 |
| MLL3-WRAD2 | Core histones | 109.0 | >200 | 11.5 | 16.8 | 13.1 |
| MLL4-WRAD2 | Core histones | 84.6 | 75.0 | 8.8 | 5.7 | 9.8 |
| DOT1L | Nucleosomes | >200 | 186.0 | 8.4 | 6.3 | 0.8 |
| G9a | Histone H3 | >200 | >200 | >200 | >200 | ND |
| GLP | Histone H3 | 96.9 | >200 | 24.7 | 12.1 | 0.4 |
| SET7/9 | Core histones | 179.0 | 136.0 | 39.8 | 17.9 | 110.0 |
| SMYD2 | Histone H3 | >200 | 64.5 | 11.6 | 10.7 | 0.7 |
| SUV39H2 | Histone H3 | >200 | >200 | 49.4 | 26.5 | 27.5 |
| SET8 | Histone H4 | >200 | >200 | 27.2 | 12.9 | ND |
| NSD2 | Nucleosomes | >200 | >200 | 3.0 | 4.1 | 3.6 |
| EZH1 complexe | Core histones | >200 | >200 | >200 | 28.8 | 75.4 |
| EZH2 complexe | Core histones | 16.7 | 22.8 | 9.1 | 7.7 | 7.7 |
| 18.4 | 26.6 | 10.4 | 6.5 | 14 | ||
| PRMT1 | Histone H4 | 184.0 | >200 | >200 | >200 | 0.3 |
| PRMT3 | Histone H3 | 17.4 | 185.0 | >200 | >200 | 0.4 |
| PRMT4 | Histone H3 | 117.0 | 188.0 | >200 | >200 | 0.1 |
| PRMT5 | Histone H4 | 14.4 | >200 | >200 | >200 | 0.4 |
| PRMT6 | Histone H4 | 12.6 | 86.5 | >200 | >200 | 0.1 |
a
Substrate concentrations: Histone H3, Histone H4, and H3 Peptide—5 μM; core histones—0.05 mg/mL; nucleosomes (HeLa oligonucleosomes)—0.05 mg/mL as DNA.
b
All IC50s represent 10 dose studies (100 μM top concentration) with singlicate data points, 60-min reaction times. Curves were fitted to a four-parameter model with GraphPad Prism.
c
AlphLISA® H3K4 H3K4Me2 Cell Based Assay for Methyl Modifications.
d
CellTiter-Glo®Luminescent Cell Viability Assay
e
Includes AEBP2, EED, RbAp48, and SUZ12 subunits.
f
IC50 values less than 25 μM are italicized.
EZH, enhancer of zeste homolog; N/A, not applicable; ND, not determined.