Discovery of METTL3 Small Molecule Inhibitors by Virtual Screening of Natural Products

Abstract

N6-Methyladenosine (m6A) is the most prevalent mRNA modification in mammalian cells that is mainly catalyzed by the methyltransferase complex of methyltransferase-like 3 and methyltransferase-like 14 (METTL3-METTL14). Many lines of evidence suggest that METTL3 plays important roles in several diseases such as cancers and viral infection. In the present study, 1,042 natural products from commercially available sources were chosen to establish a screening library, and docking-based high-throughput screening was performed to discover potential METTL3 inhibitors. The selected compounds were then further validated by an in vitro methyltransferase inhibition assay in which m6A content was determined by LC-MS/MS. A cellular assay of the inhibition of m6A methylation was performed to determine the METTL3 inhibitory activity of the selected compound. CCK-8 assay was applied to evaluate the effects of the selected compound on tumor cell viability. Additionally, binding mode analysis, molecular dynamics (MD) simulation, and binding free energy analysis were performed to study the process and characteristics of inhibitor binding. Finally, quercetin was identified as a METTL3 inhibitor with an IC50 value of 2.73 μM. The cellular assay of m6A methylation inhibition showed that quercetin decreased m6A level in a dose-dependent manner in MIA PaCa-2 pancreatic cancer cells. CCK-8 assay showed quercetin efficiently inhibited the proliferation of MIA PaCa-2 and Huh7 tumor cells, with IC50 values 73.51 ± 11.22 μM and 99.97 ± 7.03 μM, respectively. Molecular docking studies revealed that quercetin filled the pocket of the adenosine moiety of SAM but not the pocket of the SAM methionine in the METTL3 protein, and hydrogen bonds, hydrophobic interactions, and pi-stacking were formed. The values of the root mean square deviation (RMSD), the root mean square fluctuations (RMSF), and binding free energy suggested that quercetin can efficiently bind to the pocket of the METTL3 protein and form a stable protein-ligand complex. The present study is the first to identify METTL3 inhibitors from natural products, thus providing a basis for subsequent research and facilitating the development of METTL3-targeting drugs for diseases.

Targeting RNA modifications with pharmacological agents: New frontiers in cancer therapy

“…Quercetin, a competitive METTL3 inhibitor with micromolar potency, was recently discovered through the virtual screening of natural products 155 . Quercetin is cell permeable and capable of decreasing mRNA m6A levels in human pancreatic adenocarcinoma cells 155 . Quercetin's anti‐proliferative effects have been confirmed in various cancer cell lines, including liver, lung, breast, and pancreatic cancer cells 155 .…”

“…Quercetin, a competitive METTL3 inhibitor with micromolar potency, was recently discovered through the virtual screening of natural products 155 . Quercetin is cell permeable and capable of decreasing mRNA m6A levels in human pancreatic adenocarcinoma cells 155 .…”

“…Liver cancer, pancreatic cancer [155] Allosteric inhibitor CDIBA Shows dose-dependent antiproliferative activities in multiple AML cell lines.…”

“…Quercetin is cell permeable and capable of decreasing mRNA m6A levels in human pancreatic adenocarcinoma cells 155 . Quercetin's anti‐proliferative effects have been confirmed in various cancer cell lines, including liver, lung, breast, and pancreatic cancer cells 155 . Notably, quercetin has been studied for its anti‐tumour properties for over two decades, and clinical trials have shown no reported toxicity or side effects 156,157 .…”

Interplay between m6A epitranscriptome and epigenome in cancer: current knowledge and therapeutic perspectives

“…To date, the METTL3 inhibitor STC‐15, 152 developed by Storm Therapeutics, is the only epitranscriptomic compound that has entered clinical trials (NCT05584111). Interestingly, natural compounds 153 and AI‐based approaches have also been used to speed up the lengthy process of drug discovery, but with mixed results 87 . Since m 6 A writer and eraser proteins possess a broad range of substrates, it would also be worthwhile to develop molecules specifically targeting m 6 A readers.…”