2022
DOI: 10.1021/acsptsci.2c00175
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An Optimized Microscale Thermophoresis Method for High-Throughput Screening of DNA Methyltransferase 2 Ligands

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 applicabi… Show more

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Cited by 13 publications
(7 citation statements)
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“…Yet the 4-Cl/Br-3-NO 2 substituted sulfonamide-based derivatives 78 and 80 as well as the 4chloropyrazine derivative 91 showed the highest inhibition (IC 50 < 2.5 μM). The results also highlight the quality of our FTAD-based MST screening method 20 because the screening results showed high consistency with the measured inhibition in the 3 H-assay.…”
supporting
confidence: 54%
See 1 more Smart Citation
“…Yet the 4-Cl/Br-3-NO 2 substituted sulfonamide-based derivatives 78 and 80 as well as the 4chloropyrazine derivative 91 showed the highest inhibition (IC 50 < 2.5 μM). The results also highlight the quality of our FTAD-based MST screening method 20 because the screening results showed high consistency with the measured inhibition in the 3 H-assay.…”
supporting
confidence: 54%
“…To determine the binding affinity of the compounds toward hDNMT2, a screening was performed with full-length hDNMT2 (if not described otherwise, full-length protein was used) based on a microscale thermophoresis (MST) displacement method using the fluorescent ligand FTAD. 20 Potent binders were defined as compounds that were able to displace FTAD appoximately to the same extent as SFG (MST shift ≥13‰; cut-off ≥10‰). For all potent binders an apparent K D value (K D app ) was determined using MST.…”
mentioning
confidence: 99%
“…In an effort to establish an MST displacement binding assay with SAM like molecules with an attached fluorophore, we obtained a crystal structure of nsp10-16 with the fluorescent RNA methyltransferase probe 5-FAM-triazolyl-adenosyl-Dab (FTAD) (Zimmermann et al 2022) (figure S 12). More detailed information on binding assays and the FTAD crystal structure can be found in the supplementary material.…”
Section: Resultsmentioning
confidence: 99%
“…All virtual screening hits were subjected to a microscale thermophoresis (MST) pre-screening at three different concentrations of 300, 100, and 33.3 µM ( Tables S2 and S3 ). MST proved to be especially suitable as a primary binding assay due to its high sensitivity and robustness that allowed the application for very weak binders or even fragments [ 65 ] and was demonstrated to be highly accurate for DNMT2 and NSUN6 ligand identification, previously [ 25 , 66 ]. For the literature known reference ligand SAH, K D values of 11.8 µM and 9.1 µM for DNMT2 and NSUN6, respectively, were determined ( Table 2 ).…”
Section: Resultsmentioning
confidence: 99%