dnmt2 pilot screening  (Tocris)

Journal: Communications Chemistry

Article Title: A microscale thermophoresis-based enzymatic RNA methyltransferase assay enables the discovery of DNMT2 inhibitors

doi: 10.1038/s42004-025-01439-9

Figure Lengend Snippet: A MST traces of the DNMT2 enzyme reaction (250 nM DNMT2, 5 µM tRNA, 0.9 µM SAM) incubated for variable durations (0–120 min) and yielded significant thermophoresis shifts. B Substrate conversion plots (F norm vs incubation time) reveal steady-state DNMT2 kinetics. C Dose-response curves of aptaSAH1&2 (both 50 nM) in the presence of SHO108/SH112 inhibitors (0–100 µM) indicated these SAH-analogs are not binding to the aptamer at relevant concentrations. D – F Inhibitor characterization of SHO108 from left to right: MST traces at variable inhibitor concentrations; MST-derived dose-response curves; orthogonal 3 H-incorporation assay-derived dose-response curves from detected counts per minute (CPMA). G – I Inhibitor characterization of SH112 analogous to subplot ( D – F ). All inhibitor characterizations were performed in triplicates (mean ± SD, n = 3).

Article Snippet: A compound library for the DNMT2 pilot screening (including Cpd 1–10 ) was commercially obtained (Tocriscreen Epigenetics 3.0, Cat. No. 7578).

Techniques: Incubation, Binding Assay, Derivative Assay

Journal: Communications Chemistry

Article Title: A microscale thermophoresis-based enzymatic RNA methyltransferase assay enables the discovery of DNMT2 inhibitors

doi: 10.1038/s42004-025-01439-9

Figure Lengend Snippet: A Screening a library including 160 drug-like compounds resulted in the identification of ten potential hit compounds (green) with an F norm > 1000‰ (equals >80% inhibition). Initial hits were confirmed by triplicate validation (mean ± SD, n = 3). Positive reaction control: aptamer spiked with 1 µM SAH; negative control: aptamer mock-treated with DMSO. B Exemplary MST traces of the library’s first 80 compounds. C Orthogonal screening of the library by FP displacement experiments using FTAD as the fluorescent tracer. FP experiments reveal that only alexidine ( Cpd 3 , green) is binding to the SAH-binding site. D An inhibition selectivity panel was established by MST aptamer assays using the MTase assays described in this study and 500 µM of the respective hit compound. Only DNMT2 was inhibited significantly. E Alexidine’s ( Cpd 3 ) dose-response curves and K D determination by DNMT2 FP assays (mean ± SD, n = 3). F FP assays to confirm the reversibility of alexidine binding to DNMT2. The polarization of the DNMT2-FTAD complex (96 mP) is displaced in the presence of alexidine (5 mP) and can be effectively restored by analytical size-exclusion chromatography (92 mP). A second treatment with alexidine leads to a repeated FP displacement (4 mP). G Chemical structures of the identified DNMT2 hits.

Article Snippet: A compound library for the DNMT2 pilot screening (including Cpd 1–10 ) was commercially obtained (Tocriscreen Epigenetics 3.0, Cat. No. 7578).

Techniques: Inhibition, Biomarker Discovery, Control, Negative Control, Binding Assay, Size-exclusion Chromatography

Journal: Communications Chemistry

Article Title: A microscale thermophoresis-based enzymatic RNA methyltransferase assay enables the discovery of DNMT2 inhibitors

doi: 10.1038/s42004-025-01439-9

Figure Lengend Snippet: A Screening a library including 80 cysteine-focused covalent compounds resulted in the identification of one single hit compound (green) with an F norm > 1000‰ (equals >90% inhibition). Initial hits were confirmed by triplicate validation (mean ± SD, n = 3). Positive reaction control: aptamer spiked with 1 µM SAH; negative control: aptamer mock treated with DMSO. B Orthogonal screening of the library by fluorescence polarization displacement experiments using FTAD as the fluorescent tracer. FP experiments reveal that only adamantanyl-acryloylurea ( Cpd 11 , green) is binding to the SAH-binding site. C MST-derived dose-response curve for adamantanyl-acryloylurea ( Cpd 11 ) including the chemical structure. All inhibitor characterizations were performed in triplicates (mean ± SD, n = 3). D FP assay with Cpd 11 showing time-dependent DNMT2 inhibition with hyperbolic FP displacement plots. E Covalent dose-response analysis of subfigure D: k obs vs. [I] for the determination of covalent inhibition constants ( K I , k inact ) , .

Article Snippet: A compound library for the DNMT2 pilot screening (including Cpd 1–10 ) was commercially obtained (Tocriscreen Epigenetics 3.0, Cat. No. 7578).

Techniques: Inhibition, Biomarker Discovery, Control, Negative Control, Fluorescence, Binding Assay, Derivative Assay, FP Assay