METTL3 inhibitors for epitranscriptomic modulation of cellular processes

Abstract The methylase METTL3 is the writer enzyme of the N 6 -methyladenosine (m 6 A) modification of RNA. Using a structure-based drug discovery approach, we identified a METTL3 inhibitor ( UZH1a ) with potency in a biochemical assay of 280 nM, while its enantiomer UZH1b is 100 times less active. The crystal structure of the complex of METTL3 with UZH1a illustrates the interactions that make it selective against protein methyltransferases. We observed a dose-dependent reduction in m 6 A methylation level of mRNA in several cell lines treated with UZH1a already after 16 h of exposure, as determined by triple-quadrupole LC mass spectrometry, while its enantiomer UZH1b was essentially inactive at concentrations up to 100 µM. Interestingly, the kinetics of m 6 A level reduction in mRNAs followed a first-order reaction model, with a half-decay time τ of 1.8 h and a maximum m 6 A inhibition level of 70%, which is in line with the previously observed shorter half-life of m 6 A-modified mRNAs. Notably, treatment with the compounds did not alter cellular METTL3 levels, ruling out indirect effects on m 6 A levels. The effect of the m 6 A level depletion by UZH1a directly translated into growth inhibition of MOLM-13 leukemia cells, under short-term and long-term culture. Incubation of the MOLM-13 cells with UZH1a , but not with UZH1b , resulted in increased cell apoptosis and cell cycle arrest already after 16 h of incubation. Interestingly, other cell lines sensitive to METTL3 level (U2Os, HEK293T) did not reveal statistically significant differences between UZH1a and UZH1b in a cell viability assay, confirming that the degree of reliance on m 6 A signalling for survival can vary between cancers/cell types.