Structure-Based Design of a Potent and Selective YTHDC1 Ligand.

N6-Adenosine methylation (m6A) is a prevalent post-transcriptional modification of mRNA, with YTHDC1 being the reader protein responsible for recognizing this modification in the cell nucleus. Here, we present a protein structure-based medicinal chemistry campaign that resulted in the YTHDC1 inhibitor 40, which shows an equilibrium dissociation constant (Kd) of 49 nM. The crystal structure of the complex (1.6 Å resolution) validated the design. Compound 40 is selective against the cytoplasmic m6A-RNA readers YTHDF1-3 and YTHDC2 and shows antiproliferative activity against the acute myeloid leukemia (AML) cell lines THP-1, MOLM-13, and NOMO-1. For the series of compounds that culminated into ligand 40, the good correlation between the affinity in the biochemical assay and antiproliferative activity in the THP-1 cell line provides evidence of YTHDC1 target engagement in the cell. The binding to YTHDC1 in the cell is further supported by the cellular thermal shift assay. Thus, ligand 40 is a tool compound for studying the role of YTHDC1 in AML.

Proteolysis Targeting Chimera Degraders of the METTL3-14 m6A-RNA Methyltransferase.

Methylation of adenine N6 (m6A) is the most frequent RNA modification. On mRNA, it is catalyzed by the METTL3-14 heterodimer complex, which plays a key role in acute myeloid leukemia (AML) and other types of blood cancers and solid tumors. Here, we disclose the first proteolysis targeting chimeras (PROTACs) for an epitranscriptomics protein. For designing the PROTACs, we made use of the crystal structure of the complex of METTL3-14 with a potent and selective small-molecule inhibitor (called UZH2). The optimization of the linker started from a desfluoro precursor of UZH2 whose synthesis is more efficient than that of UZH2. The first nine PROTAC molecules featured PEG- or alkyl-based linkers, but only the latter showed cell penetration. With this information in hand, we synthesized 26 PROTACs based on UZH2 and alkyl linkers of different lengths and rigidity. The formation of the ternary complex was validated by a FRET-based biochemical assay and an in vitro ubiquitination assay. The PROTACs 14, 20, 22, 24, and 30, featuring different linker types and lengths, showed 50% or higher degradation of METTL3 and/or METTL14 measured by Western blot in MOLM-13 cells. They also showed substantial degradation on three other AML cell lines and prostate cancer cell line PC3.

1,4,9-Triazaspiro[5.5]undecan-2-one Derivatives as Potent and Selective METTL3 Inhibitors.

N6-methyladenosine (m6A) is the most frequent of the 160 RNA modifications reported so far. Accumulating evidence suggests that the METTL3/METTL14 protein complex, part of the m6A regulation machinery, is a key player in a variety of diseases including several types of cancer, type 2 diabetes, and viral infections. Here we report on a protein crystallography-based medicinal chemistry optimization of a METTL3 hit compound that has resulted in a 1400-fold potency improvement (IC50 of 5 nM for the lead compound 22 (UZH2) in a time-resolved Förster resonance energy transfer (TR-FRET) assay). The series has favorable ADME properties as physicochemical characteristics were taken into account during hit optimization. UZH2 shows target engagement in cells and is able to reduce the m6A/A level of polyadenylated RNA in MOLM-13 (acute myeloid leukemia) and PC-3 (prostate cancer) cell lines.

METTL3 Inhibitors for Epitranscriptomic Modulation of Cellular Processes.

The methylase METTL3 is the writer enzyme of the N6 -methyladenosine (m6 A) modification of RNA. Using a structure-based drug discovery approach, we identified a METTL3 inhibitor with potency in a biochemical assay of 280 nM, while its enantiomer is 100 times less active. We observed a dose-dependent reduction in the m6 A methylation level of mRNA in several cell lines treated with the inhibitor already after 16 h of treatment, which lasted for at least 6 days. Importantly, the prolonged incubation (up to 6 days) with the METTL3 inhibitor did not alter levels of other RNA modifications (i. e., m1 A, m6 Am , m7 G), suggesting selectivity of the developed compound towards other RNA methyltransferases.