FROM BENCH TO BIOACTIVITY: DESIGN, SYNTHESIS, AND PRECLINICAL EVALUATION OF NOVEL NONSENSE MUTATION SUPPRESSORS

Premature termination codons (PTCs) in mRNA disrupt normal protein synthesis and can cause genetic disorders such as Duchenne muscular dystrophy (DMD), an X-linked, progressive neuromuscular disease. DMD is caused by mutations in the DMD gene, which encodes the dystrophin protein. The absence of functional dystrophin in muscle leads to progressive muscle wasting, physical inactivity, respiratory depression, cardiac insufficiency, and premature mortality. Approximately 13% of DMD gene mutations are nonsense mutations that generate PTCs in the mRNA sequence, resulting in premature termination of translation and the production of a truncated, non-functional protein.Despite the significant clinical impact of these mutations, there are currently no highly effective or standardized treatments available, making the development of targeted therapies an urgent medical need. One emerging strategy involves the use of translational readthrough-inducing drugs (TRIDs), which aim to override PTCs and enable the production of full-length functional proteins. TRIDs hold great therapeutic potential for treating genetic nonsense disorders, including DMD.Published data from our lab revealed that a new TRID compound, NV848 (N-(5-methyl-1,2,4-oxadiazol-3-yl) acetamide), demonstrated a favorable pharmacokinetic profile and was able to rescue target protein expression in nonsense-related in vitro assays and in Cystic Fibrosis (CF) nonsense murine model. Furthermore, metabolic stability evaluations suggested lower degradation rates compared to PTC124 (ataluren), indicating improved bioavailability and offering a strong reason for its assessment as a therapeutic candidate for treatment in an animal model of DMD.The thesis work investigated the translational readthrough potential of NV848 in improving skeletal and cardiac muscle structural integrity and function, as well as in restoring DMD mRNA and dystrophin protein expression in mdx mice, the most common murine model of DMD, which carries a spontaneous nonsense mutation in the DMD gene. Additionally, this thesis also described the design, synthesis, characterization, and biological evaluation pathway of new NV compounds, structurally related to NV848 and Ataluren, following a pharmacophore model, to identify optimized small molecules having enhanced TRIDs activities and improved pharmacological properties.In the first part of the thesis, mdx mice were administered with NV848 or the reference readthrough compound PTC124 through oral gavage at a dosage of 60 mg/kg for 14 consecutive days. The forelimb grip strength test was conducted to assess muscle function in wild-type and mdx mice before, during, and after the administration period. After behavioral testing, mice were sacrificed, and organs were collected for further biochemical assay. Real-time RT-qPCR was performed to quantify the expression of mRNA in skeletal quadriceps muscle. Similarly, Hematoxylin and Eosin staining was performed to observe morphological improvement, accompanied by an immunohistochemistry (IHC) assay for skeletal muscle and an immunofluorescence (IF) assay for cardiac muscle to perceive the expression of dystrophin protein after chronic treatment of NV848.In the second part of the project, new NV analogues were synthesized in a multi-step procedure. The products were purified by column chromatography and characterized by HPLC/MS and 1H NMR spectra analysis. Preliminary biological evaluation employed luciferase reporter and cell viability assays for confirmation of readthrough efficacy and cytotoxicity safety.Our results demonstrated that chronic administration of the compound, NV848, significantly improved skeletal muscle strength, reflecting improvements in muscle function and endurance. Similarly, quadriceps muscles exhibited significant structural repair, characterized by reduced fiber necrosis and diminished inflammatory infiltrates. RT-qPCR results showed that NV848-treated mice exhibited increased DMD mRNA levels. The molecular data corresponded with the protein findings as NV848-treated mice exhibited dystrophin re-expression at the sarcolemma in femoral quadriceps sections, in contrast to untreated mdx muscles, which showed no such expression. Although immunofluorescence analyses of cardiac tissues revealed minimal and no statistically significant dystrophin signals, an organized γ-sarcoglycan alignment at the membrane signified improved membrane integrity and partial restoration of the dystrophin-associated protein complex.Furthermore, NV930 and its derivatives (NV3261, NV3262, NV3263, NV3264, and NV3265) were successfully synthesized after identification through computational pharmacophore modeling design. The structures were characterized by HPLC-MS and 1H NMR analysis and demonstrated favorable yields. Moreover, the newly synthesized NV compounds slightly enhanced luminescence intensity, indicating their potential to facilitate translational readthrough of premature termination codons. Also, in the cell viability assay, the cells exposed to 12 μM concentration of the new compounds for 24, 48, and 72 hours maintained cell survival over 90% without any morphological changes, indicating minimal cellular toxicity.In conclusion, our study contributes to the development of targeted approaches for DMD by offering important insights into the therapeutic efficacy of the novel TRID compound NV848 and by making preclinical data available for future drug development. Similarly, our new compound chemical scaffolds achieved minimal cellular toxicity, reinforcing their potential as safe candidates for future preclinical investigations.