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. 2022 Oct 19;5(11):1079-1085.
doi: 10.1021/acsptsci.2c00175. eCollection 2022 Nov 11.

An Optimized Microscale Thermophoresis Method for High-Throughput Screening of DNA Methyltransferase 2 Ligands

Robert Alexander Zimmermann  1 , Marvin Schwickert  1 , J Laurenz Meidner  1 , Zarina Nidoieva  1 , Mark Helm  1 , Tanja Schirmeister  1
Affiliations

An Optimized Microscale Thermophoresis Method for High-Throughput Screening of DNA Methyltransferase 2 Ligands

Robert Alexander Zimmermann et al. ACS Pharmacol Transl Sci. .

Abstract

Developing methyltransferase inhibitors is challenging, since most of the currently used assays are time-consuming and cost-intensive. Therefore, efficient, fast, and reliable methods for screenings and affinity determinations are of utmost importance. Starting from a literature-known fluorescent S-adenosylhomocysteine derivative, 5-FAM-triazolyl-adenosyl-Dab, developed for a fluorescence polarization assay to investigate the histone methyltransferase mixed-lineage leukemia 1, we herein describe the applicability of this compound as a fluorescent tracer for the investigation of DNA-methyltransferase 2 (DNMT2), a human RNA methyltransferase. Based on these findings, we established a microscale thermophoresis (MST) assay for DNMT2. This displacement assay can circumvent various problems inherent to this method. Furthermore, we optimized a screening method via MST which even indicates if the detected binding is competitive and gives the opportunity to estimate the potency of a ligand, both of which are not possible with a direct binding assay.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1. Synthesis of FTAD (6)
Reagents and conditions: (i) NaN3, H2O, 85 °C, 24 h, 67%; (ii) 5-FAM, TPTU, DIPEA, DMF, 0–20 °C, 48 h, 96%; (iii) CuSO4, sodium ascorbate, MeOH/H2O, 16 h, 61%; (iv) 1. TFA, DCM, 5 °C; 2. TFA, H2O, 5 °C, 99%.
Scheme 2
Scheme 2. Establishment of DNMT2 Microscale Thermophoresis Assay
Steps: (1) Unlabeled DNMT2 cannot be observed by MST. (2) DNMT2 is pre-incubated with FTAD; the FTAD-DNMT2 complex can be observed by MST. (3) Displacement of FTAD with ligand of interest; free FTAD can be observed by MST.
Figure 1
Figure 1
Microscale thermophoresis assay for FTAD against a dilution series of human DNMT2. (A) Raw traces of runs. (B) Normalized fluorescence plotted against concentration of human DNMT2. Data is given as mean ± SD of triplicates. KD of FTAD against DNMT2 was found to be 1.8 μM ± 0.1 μM. (C) Z-factor determination of microscale thermophoresis assay. In orange, free FTAD without DNMT2; in blue, FTAD in the presence of 2 μM human DNMT2.
Figure 2
Figure 2
Displacement of FTAD by literature known inhibitors in a microscale thermophoresis assay. Data is given as mean ± SD of quadruplicates. (A) SAH (7), EC50 = 15.0 ± 0.7 μM; (B) SFG (8), EC50 = 20.9 ± 0.7 μM; (C) compound 9, EC50 = 12.4 ± 0.5 μM; and (D) compound 10, EC50 = > 100 μM.
Figure 3
Figure 3
Correlation of MST shifts at 20 μM compound concentrations and inhibition at 100 μM in a tritium incorporation assay for literature-known compounds: (A) for direct MST assay and (B) for MST displacement assay.
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