Pharmacological chaperones to cure genetic diseases: development of drugs and identification of new targets

Different mutations of the gene encoding lysosomal alpha-galactosidase (AGAL) result in a wide phenotypic spectrum, with respect to age at onset, rate of disease progression, severity of clinical manifestations of Fabry disease (OMIM: 30150). Patients with the late onset or atypical form retain some AGAL activity and are asymptomatic until adult age when they develop cardiac and kidney problems. There exists an approved therapy for the cure of Fabry disease that requires infusions of recombinant wild type AGAL. In order to prevent irreversible damages to organs, this therapy, which is known as enzyme replacement therapy (ERT) should start early. This poses a problem because ERT is expensive and imposes an unreasonable burden for patients with milder disease variants. For these patients there is an alternative therapy with small drugs, the so called pharmacological chaperones (PC), that are, in principle, less expensive. We correlate the flexibility of the sites where AGAL mutations occur with the residual activity in the cells. This result is useful for the evaluation of severity and for the choice of a personalized therapy. Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for PC that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease. We are screening in silico large databases of chemicals in order to find new PC for Fabry, and we are testing the most promising hits in vitro and in the cells. We are trying to extend the approach with PC to a congenital disorder of glycosylation caused by mutations in the gene encoding phosphomannomutase2(PMM2) that is known as CDG-PMM2 (MIM: 212065). We have characterized in vitro the mutants that are most commonly found in European population, we have demonstrated that the genotype R141H/F119L is eligible for the therapy with PC. Unfortunately in silico screening was not possible because the enzyme structure had been solved in non native conditions and in open conformation. Exploiting non-biased ligand migration and protein dynamics, we have produced the first accurate model of a ligand bound complex for PMM2 which will be used for high throughput screening to find PC for CDG-PMM2.