Dysferlinopathies are a class of muscular dystrophies caused by recessive mutations in dysferlin (DYSF). Dysferlin mediates the repair of plasma membrane wounds resulting from normal muscle contractile forces. In patients and in mice where Dysferlin protein is reduced or absent, there is progressive degeneration of skeletal muscle that leads to loss of ambulation. The orphan nature of dysferlinopathy has hampered drug discovery efforts. There are no therapeutic treatments for dysferlinopathy patients, despite evidence of genetic disease modifiers that could be targeted for drug discovery purposes. Moreover, despite efforts, no in vitro high-throughput DYSF(-) muscle membrane repair assay has been successfully developed to identify therapeutics for in vivo testing. To address this unmet need, and as a proof-of-concept demonstration of the utility of lower model organisms in drug discovery, we have developed a high-throughput screening platform to identify small molecule and genetic suppressors of dysferlinopathy in Caenorhabditis elegans. Dysferlin and C. elegans FER-1 are conserved homologs within the Ferlin protein family. C. elegans fer-1 mutants are infertile due to defective sperm development. The biological mechanism of this infertility is similar in nature to the plasma membrane repair defect found in mammalian dysferlinopathy, namely an inability to fuse vesicles to plasma membrane. Using a conditional fer-1 missense mutant, we performed compound and mutagenic screens and identified novel small molecules and genetic suppressors capable of restoring fertility to otherwise sterile animals. Interestingly, the suppressors do not restore fertility to a recently obtained fer-1 null allele, suggesting that they operate by restoring FER-1 function rather than bypassing the need for FER-1. In this proposal we seek to optimize the current leads from our existing screening platform, and further improve our platform's selectivity and power in identifying therapeutic candidates by: 1) Determining the mechanisms of action of the fer-1misense mutation suppressors in order to evaluate their therapeutic potential for the one-third of patients who have missense mutations. 2) Identifying genetic suppressors of the fer-1 null mutant that restore fertility in the absence of FER-1. Mammalian counterparts of such suppressors may be targets to aid therapeutic discovery for ALL dysferlinopathy patients. 3) Determining if transgenic expression of human Dysferlin in the germ line of fer-1 deficient animals is sufficient to restore fertility. The creation of a humanized C. elegans dysferlinopathy model will have far reaching implications for therapeutic discovery as it could be used to screen for compound suppressors of specific patient mutations or mutational classes. C. elegans fer-1 mutants form the basis of our multimodal screening platform, which seeks to address difference among patient mutation classes in searching for therapies. As such, this approach will likely hasten the preclinical route to in vivo testing of drugs to benefit dysferlinopathy patients with muscular dystrophy.
Mutations in the human gene dysferlin result in a number of progressively degenerative muscular dystrophies, collectively called dysferlinopathies. The proposed research plan exploits the infertility phenotype associated with mutations in the Caenorhabditis elegans dysferlin homolog, fer-1, using it as a proxy for dysferlinopathy in screening for drugs and genetic mutations that can restore fertility to otherwise sterile animals. The simple and yet powerful selectivity of this whole-animal screening platform holds promise as a rapid and cost-effective method of identifying safe and efficacious small molecules and/or conserved drug screening targets, with the ultimate goal of identifying treatments for muscular dystrophy patients.
