Emanuele Fabbrizi

OC26 – Emanuele Fabbrizi

Department of Drug Chemistry and Technologies, Sapienza University of Rome, Italy

Targeting m6A Epitranscriptomic Machinery: Design and Biological Characterization of Potent Quinazoline-Derived METTL3–METTL14 Inhibitors
Fabbrizi Emanuele¹, Fiorentino Francesco ², Mancini Andrea¹, Caflisch Amedeo³, Fatica Alessandro², Rotili Dante⁴, and Antonello Mai¹ ¹ Department of Drug Chemistry and Technologies, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy; ² Department of Biochemical Sciences, Sapienza University of Rome, 00185 Rome, Italy; ³ Department of Biochemistry, University of Zurich, Zurich, Switzerland; ⁴ Department of Science, Roma Tre University, 00146 Rome, Italy.
Abstract Epitranscriptomics involves dynamic, reversible RNA modifications regulating splicing, nuclear export, stability, and translation without altering sequence. N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNA and is installed by a multicomponent methyltransferase complex whose catalytic core is the METTL3/METTL14 heterodimer [1]. METTL3 contains the SAM-dependent catalytic domain with the conserved DPPW motif, whereas METTL14 acts as an RNA-binding scaffold enabling substrate recognition and positioning [2]. Dysregulation of this complex promotes tumorigenesis by enhancing stability and translation of oncogenic transcripts such as c-MYC, supporting malignant growth, thus representing a therapeutic target [3]. We report the design, synthesis, and biochemical characterization of 30 non-nucleosidic quinazoline derivatives as METTL3/METTL14 inhibitors. The quinazoline scaffold mimics adenine interactions in the catalytic pocket and enables SAR exploration via substitutions at C2, C4, C6, and C7. Orthogonal biochemical assays identified sub-micromolar inhibitors. In HTRF assays, IC₅₀ values ranged from 120 to 740 nM, with MC4774 as the most potent (119 nM). In the HotSpot radiometric assay ([³H]-SAM incorporation), potency increased markedly: MC4905 (1.56 nM), MC4118 (1.86 nM), MC4009 (2.15 nM), and MC4774 (2.22 nM) (Figure 1). Compounds showed >1000-fold selectivity over EZH2, G9a, PRMT1, and SET8, with no inhibition up to 10 µM. Cell assays showed a favorable therapeutic index: MC4905 and MC4009 preserved viability in CD34⁺ cells (88–92%) and MCF10A cells (85–90%), while reducing MDA-MB-231 viability to ~25–30% at 5 µM (72 h), outperforming STM2457 [3]. Combination with cisplatin or olaparib enhanced antiproliferative effects. In MOLM-13 AML cells, IC₅₀ values were 1.47 and 1.59 µM, with 55–65% m6A reduction and ~65% c-MYC downregulation, confirming on-target activity.
References [1] Saletore Y, Meyer K, Korlach J, Vilfan ID, Jaffrey S, Mason CE. The birth of the Epitranscriptome: deciphering the function of RNA modifications. Genome Biol. 2012 Oct 31;13(10):175. [2] Wang X, Feng J, Xue Y, Guan Z, Zhang D, Liu Z, Gong Z, Wang Q, Huang J, Tang C, Zou T, Yin P. Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex. Nature. 2016 Jun 23;534(7608):575-8. [3] Fiorentino F, Menna M, Rotili D, Valente S, Mai A. METTL3 from Target Validation to the First Small-Molecule Inhibitors: A Medicinal Chemistry Journey. J Med Chem. 2023 Feb 9;66(3):1654-1677. doi: 10.1021/acs.jmedchem.2c01601. Epub 2023 Jan 24. PMID: 36692498; PMCID: PMC9923689.