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. 2020 Dec 7;2(2):146–161. doi: 10.1158/2643-3230.BCD-20-0173

Figure 2.

Figure 2.

Lead optimization and efficacy enhancement of TETi. A, Consensus binding site for known pseudosubstrate NOG. B, 2D TETi pharmacophore. C, TET2−/− leukemia cell killing effect of 20 compounds. TET2-deficient SIG-M5 AML cells at 105/mL were treated with 25 μmol/L compounds for 3 days, and viable cells were determined by trypan blue exclusion method. D, LD50 and TET-inhibitory effect of 11 selected compounds. SIG-M5 cells at 105/mL were treated with increasing concentrations of TETi for 3 days, and surviving cells were counted by trypan blue exclusion on automated Vi-CELL counter and LD50 was calculated from viable cell number in GraphPad Prism. TET dioxygenase activity was assessed by measuring 5hmC in a dot blot assay. SIG-M5 cells at 3 × 105/mL were treated with 25 μmol/L compounds for 12 hours in the presence of 100 μmol/L sodium ascorbate and the relative 5hmC/5mC quantification using dot blot was performed on genomic DNA. E, LD50 of TETi against normal bone marrow–derived mononuclear cells (NBM) and TET2-mutant and TET2-deficient leukemia cells (SIG-M5 and MOLM13). Cells were treated with increasing doses of indicated TETi for 3 days, and LD50 was calculated from viable cell number in GraphPad Prism. F, Structure of ester and acid forms of TETi76. Acid forms were used for cell-free assays, whereas diethyl esters were used in cell culture. G and H, Dose-dependent inhibition of TET activity by TETi76 in cell-free condition. Catalytic domains of recombinant TET1, TET2, and TET3 were incubated with TETi76 separately in the presence of different concentrations of cofactors (αKG and Fe2+), and the 5hmC was monitored by ELISA using anti 5hmC antibodies. I, Measurement of direct interaction of TETi76 with TET2CD by MST. Binding interaction of TETi76 with TET2CD was monitored using label-free MST on Monolith NT.LabelFree instrument. The normalized change in MST signal ΔFnorm = (Fbound − Ffree)/Response amplitude at different TETi76 concentrations is plotted. Data were analyzed using MO.Affinity analysis V2.3 software using built-in Kd model for data fitting and calculation of dissociation constant. J, Testing inhibitory effect of TETi76 on αKG- and Fe2+-dependent demethylases. Purified enzymes were used with their substrate in the presence or absence of 15 μmol/L TETi76 in alpha screen (https://bpsbioscience.com), and the relative activity was determined for a vehicle control. Each assay was accompanied by a positive control (see Supplementary Table S6). Data are representative for experiments performed in triplicates at least twice (C–E and G–I). Data, mean ± SEM.