Phd in PHARMACEUTICAL SCIENCES

Thesis title: Chemical Modulation of the Epigenetic Network: Targeting Writers, Erasers, and Readers

Epigenetic and epitranscriptomic modifications finely tune gene expression through the coordinated action of writers, erasers, and readers. Pharmacological modulation of these machineries not only represents a promising strategy for targeting complex diseases, but also enables the development of chemical tools to probe the biochemical pathways and functional frameworks in which these proteins operate. In this work, selective small-molecule ligands against epigenetic and epitranscriptomic regulators across the three functional classes have been developed, integrating rational design, medicinal chemistry, structural biology, and cellular validation. The first chapter focuses on the modulation of the epitranscriptomic writer METTL3/METTL14, responsible for installing the m⁶A modification that governs RNA splicing, export, translation, and decay. Guided by the catalytic role of METTL3 (DPPW motif) and the structural/RNA-binding function of METTL14, a library of 30 non-nucleoside quinazoline derivatives was designed and synthesized. Approximately two-thirds of the compounds showed inhibitory activity, with sub-micromolar potency (IC₅₀ 120–740 nM, TR-FRET) and low-nanomolar inhibition in orthogonal assays (1.5–2.2 nM, HotSpot), and no detectable inhibition of other epigenetic writers (EZH2, G9a, PRMT1, SET8). In cells, the most active analogues preserved viability in non-tumorigenic models (>80% in CD34⁺ and MCF10A) while selectively reducing cancer cell proliferation (−60/70% in MDA-MB-231). In MOLM-13 leukemia cells, the compounds displayed low-micromolar IC₅₀ values and induced marked reductions in global m⁶A (−55/65%) and c-MYC levels (−65%), confirming on-target inhibition and underscoring the therapeutic and mechanistic relevance of targeting the epitranscriptome. The second chapter describes the development of selective inhibitors of the eraser SIRT6. Starting from a crystallographically identified fragment hits, an NAD⁺-dependent binding mode was elucidated and the series optimized to afford MC4637, a selective sub-micromolar spirocyclic inhibitor in deacetylation assay. MC4637 increased H3K9/H3K56 acetylation in cells, exhibited single-digit micromolar antiproliferative activity across lung, breast, and colon cancer cell lines, and enhanced gemcitabine efficacy in pancreatic cancer models, highlighting both therapeutic potential and combinatorial synergy. The third chapter extends epigenetic targeting to Schistosoma mansoni, addressing the need for new therapies beyond Praziquantel, whose limitations include emerging resistance and poor activity against juvenile worms. A series of tranylcypromine-based (TCP) LSD1 inhibitors demonstrated potent activity against schistosomula (NTS), and although activity decreased against adult worms, compound 3.5 retained efficacy (IC₅₀ = 2.23 μM). Several analogues (3.15, 3.18, 3.19) showed low cytotoxicity in human cell lines, and speed-of-action assays confirmed antiparasitic effects within 24–72 h, supporting further in vivo progression and consolidating LSD1 as a pharmacologically relevant target in parasitology. Finally, the fourth chapter reports the identification of pyrimidine-based ligands for the epigenetic readers MBD2 and MeCP2, which recognize methylated cytosines (5-mC) at promoter regions and drive context-dependent transcriptional programs. Several compounds produced significant thermal-shift effects (ΔTₘ) in TSA and nanoDSF assays, indicating direct and selective protein engagement. Recurrent molecular features, such as p-methoxyphenylbutyl substituents and ribose-mimetic motifs, proved critical for anchoring within the methyl-CpG recognition pocket. These ligands constitute chemical probes for interrogating methyl-DNA readout and serve as a foundation for SAR-driven optimization. Moreover, a DUBTAC-based strategy is proposed to stabilize MeCP2, offering a conceptual framework for therapeutic intervention in Rett syndrome and enabling future evolution toward optimized warheads and CNS-penetrant deubiquitinase recruiters. Collectively, this thesis delivers selective chemical probes and molecular leads targeting epigenetic and epitranscriptomic writers, erasers, and readers, demonstrating that precise modulation of these pathways can yield powerful tools for chemical biology and unlock therapeutic opportunities across oncology, parasitology, and neuroepigenetics.