Next-Generation TRIDs journey: From Rational Drug Design to in vivo Validation

Genetic diseases result from alterations in the DNA sequence that impact either single genes orentire chromosomes. In monogenic disorders specifically, various mutation types can lead to aloss of gene function. Among these, nonsense mutations account for ~11% of hereditarydisorders, for which available treatments remain largely symptomatic rather than curative.Beyond genetic diseases, about 12% of tumour suppressor gene defects are also caused bynonsense mutations.This doctoral research taking as nonsense models Cystic fibrosis transmembrane conductanceregulator (CFTR) gene and TP53, combined computational drug discovery, chemical synthesis,biological validation in cellular models, and proteomic analyses in murine models to identifynovel translational readthrough inducers (TRIDs), assess their therapeutic potential and furtheranalyze the impact of the readthrough in vivo.A ligand-based pharmacophore model enabled the identification of new scaffolds within the1,2,4-triazole and diaryl urea families. Several derivatives promoted translational readthrough,and structure–activity relationship analyses highlighted extended aromatic groups, hydrogenbond acceptors, and asymmetric core substitutions as key topological features. Four triazoliccompounds (IP14, IP15, IP17, IP18) showed no cytotoxicity and successfully restored full-length p53 in TP53 nonsense mutant cells, with correct nuclear localization, transcriptionalreactivation, and favourable in silico ADMET profiles.Proteomic studies involving cystic fibrosis mouse model carrying nonsense mutations furtherrevealed CFTR’s central role in mitochondrial bioenergetics and proteostasis. CFTR deficiencywas associated with basal inflammation and tissue-specific mechanisms, including disruptedhepatic glycogen–lipid coordination and altered neuronal signalling. Both validated TRIDs,NV848 and PTC124, remodelled proteomic profiles, with NV848 showing deeper corrections,particularly in liver, and greater convergence toward wild-type proteome profile.Overall, this work demonstrates that an integrated strategy of computational modeling, targetedsynthesis, and multi-level validation can yield promising TRIDs. The study not only highlightsthe therapeutic relevance of TRIDs for nonsense mutation–derived diseases, such as cysticfibrosis and p53 related cancer, but also expands our understanding of the systemicconsequences of CFTR loss, paving the way for innovative treatments for a broad range ofnonsense mutation disorders.