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. 2023 Mar 24;24(7):6109.
doi: 10.3390/ijms24076109.

Chemical Space Virtual Screening against Hard-to-Drug RNA Methyltransferases DNMT2 and NSUN6

Robert A Zimmermann  1 , Tim R Fischer  1 , Marvin Schwickert  1 , Zarina Nidoieva  1 , Tanja Schirmeister  1 , Christian Kersten  1
Affiliations

Chemical Space Virtual Screening against Hard-to-Drug RNA Methyltransferases DNMT2 and NSUN6

Robert A Zimmermann et al. Int J Mol Sci. .

Abstract

Targeting RNA methyltransferases with small molecules as inhibitors or tool compounds is an emerging field of interest in epitranscriptomics and medicinal chemistry. For two challenging RNA methyltransferases that introduce the 5-methylcytosine (m5C) modification in different tRNAs, namely DNMT2 and NSUN6, an ultra-large commercially available chemical space was virtually screened by physicochemical property filtering, molecular docking, and clustering to identify new ligands for those enzymes. Novel chemotypes binding to DNMT2 and NSUN6 with affinities down to KD,app = 37 µM and KD,app = 12 µM, respectively, were identified using a microscale thermophoresis (MST) binding assay. These compounds represent the first molecules with a distinct structure from the cofactor SAM and have the potential to be developed into activity-based probes for these enzymes. Additionally, the challenges and strategies of chemical space docking screens with special emphasis on library focusing and diversification are discussed.

Keywords: DNMT2; NSUN6; RNA methyltransferases; chemical spaces; molecular docking; ultra-large molecular libraries; virtual screening.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Molecular structures of the DNMT2 and NSUN6 cofactor SAM, the reaction product and auto-inhibitor SAH and the pan-methyltransferase inhibitor SFG.
Figure 2
Figure 2
Virtual screening workflow for DNMT2 performing first molecular docking and then clustering (A), and NSUN6 starting from a diversity subset (first clustering) followed by docking and analog search in the whole chemical space (indicated by the green arrow) prior hit selection and testing (B).
Figure 3
Figure 3
Binding modes of SAH-bound to DNMT2 (A) (PDB-ID 1G55) and SFG bound to NSUN6 (B) (PDB-ID 5WWR). Enzymes are shown with white surface and carbon atoms, ligands with green carbon atoms. Polar contacts are shown as yellow dashed lines, water molecules as red spheres. For clear view only residues forming polar contacts with the ligands are shown as lines and labeled as well as C-72 (light blue carbon atoms) and the catalytic Cys-residues 326 and 373 in the NSUN6-tRNA-SFG complex (B).
Figure 4
Figure 4
Predicted binding modes of 1.4 in complex with DNMT2 (A), 1.18 in complex with DNMT2 (B), 2.5 in complex with NSUN6 (C), and 2.8 in complex with NSUN6 (D). Docking poses are depicted with green carbon atoms, enzymes with white carbon atoms, and transparent surfaces. For a clear view, only residues forming polar interactions (yellow dashed lines) are shown and labeled. For orientation, the crystallographic reference ligands SAH (DNMT2, PDB-ID 1G55) and SFG (NSUN6, PDB-ID 5WWR) are shown with magenta carbon atoms. In NSUN6, C-72 is depicted with light blue carbon atoms for orientation, but tRNA was removed during molecular docking.
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