RESUMEN
Duchenne Muscular Dystrophy is an inherited muscle degeneration disease for which there is still no efficient treatment. However, compounds active on the disease may already exist among approved drugs but are difficult to identify in the absence of cellular models. We used the Caenorhabditis elegans animal model to screen a collection of 1000 already approved compounds. Two of the most active hits obtained were methazolamide and dichlorphenamide, carbonic anhydrase inhibitors widely used in human therapy. In C. elegans, these drugs were shown to interact with CAH-4, a putative carbonic anhydrase. The therapeutic efficacy of these compounds was further validated in long-term experiments on mdx mice, the mouse model of Duchenne Muscular Dystrophy. Mice were treated for 120 days with food containing methazolamide or dichlorphenamide at two doses each. Musculus tibialis anterior and diaphragm muscles were histologically analyzed and isometric muscle force was measured in M. extensor digitorum longus. Both substances increased the tetanic muscle force in the treated M. extensor digitorum longus muscle group, dichlorphenamide increased the force significantly by 30%, but both drugs failed to increase resistance of muscle fibres to eccentric contractions. Histological analysis revealed a reduction of centrally nucleated fibers in M. tibialis anterior and diaphragm in the treated groups. These studies further demonstrated that a C. elegans-based screen coupled with a mouse model validation strategy can lead to the identification of potential pharmacological agents for rare diseases.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Modelos Animales de Enfermedad , Distrofina/deficiencia , Distrofia Muscular Animal/prevención & control , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Inhibidores de Anhidrasa Carbónica/metabolismo , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Diclorfenamida/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Distrofina/genética , Humanos , Metazolamida/farmacología , Ratones , Ratones Endogámicos mdx , Actividad Motora , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatología , Interferencia de ARN , Factores de TiempoRESUMEN
Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.