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. Author manuscript; available in PMC: 2021 Mar 1.
Published in final edited form as: Eur J Med Chem. 2020 Jan 2;189:112023. doi: 10.1016/j.ejmech.2019.112023

Figure 3. DOT1L inhibitors bind to the SAM binding site.

Figure 3.

NMR spectra of 200 μM 2 in the presence of 5 μM GST-DOT1L. (A) 1H NMR of 2. Compound protons numbered according to corresponding 1H NMR signal. (B) STD NMR of 2 demonstrates binding to DOT1L. (C) 1H NMR with addition of 2 mM SAH. Additional peaks in spectra that do not correspond to 2 are from SAH. (D) STD NMR with addition of 2 mM SAH. Loss of STD NMR signal from 2 indicates that SAH competes with 2 for binding to the SAM binding site. (E) Thermal stability shift assay of 1μM GST-DOT1L in the presence of DMSO or 100 μM adenosine based DOT1L inhibitors (filled bars) or 200 μM non-adenosine DOT1L inhibitors (empty bars) showing increased melting temperature in the presence of compounds indicating binding to DOT1L. (F) 2 inhibits cellular H3K79 dimethylation in MLL-AF9 murine model cells over the time course of 3, 6 and 9 days.