Interrogating the fate of dopamine transporter mutants by pharmacochaperones

Mutations in genes cause diseases. Some of these mutations affect the folding of the protein encoded by the gene. Misfolded proteins are non-functional and thus give rise to diseases. Individual folding diseases are rare, but collectively folding diseases account for a large fraction of hereditary and acquired disorders. In fact, folding diseases are at the very root of the molecular revolution in medicine: the term molecular medicine was coined some 75 years ago to highlight the paradigm shift, which arose from the study of a folding disease, i.e. sickle cell anemia. Folding diseases also arise from mutations of solute carriers. There are >400 solute carriers (SLCs) in the human genome. They are required to transport hydrophilic small molecules across the lipid bilayer, which envelops the cells and which also defines cellular organelles. The transporters of serotonin (SERT/SLC6A4) and dopamine (DAT/SLC6A3) are closely related, they belong to the solute carrier 6 (SLC6) family and are arguably the most extensively studied transporters. Point mutations in DAT cause a syndrome of infantile dystonia and Parkinsonism. The mutations result in a folding-deficient protein, which does not reach the cell surface. If DAT is not delivered to the presynaptic membrane of dopaminerig neurons, it cannot replenish the vesicular stores of dopamine. This gives rise to the symptoms. DAT and SERT are targets for both, therapeutic and illicit drugs; thus, market forces have combined to explore the chemical space resulting in a rich pharmacology. The working hypothesis underlying this project posited that there were small molecules (=pharmacochaperones), which corrected the folding defect. In our search for these pharmacochaperones, we pursued a two-pronged approach: screening of candidate compounds and a rational search based on an improved understanding of conformational changes, binding modes and the folding trajectory. Disease-relevant mutations also occur in other SLC6 transporters, in particular the human creatine transporter-1 (CrT-1/SLC6A8); the resulting misfolding is associated with intellectual disability and epilepsy. We showed that it was possible to rescue some mutants of DAT and of CrT-1 by pharmacochaperones: a major breakthrough of this project was the identification of compound 9b, which eclipsed noribogaine, the best hitherto available pharmacochaperone, based on both, its spectrum (rescuing 6 of the 13 disease-associated DAT mutants) and its efficacy (up to 4-fold higher than that of noribogaine). We also corrected misfolding of several CrT-1 mutants by 4-phenylbutyrate. This is of interest, because 4-phenylbutyrate is an approved paediatric drug. Thus, translation of our insights into a clinically applicable treatment by ought to be simplified. In our second approach, major advances were made in understanding the transport cycle of SLC6 transporters, the associated conformational transitions and the underlying energy landscape. Combined with the analysis of thermal unfolding of SLC6 transporters, these insights provide an indirect glimpse on the folding trajectory. We are confident that our findings will eventually lead to a treatment strategy, which allows for correcting the folding deficit in affected individuals. In fact, we teamed up with a company to explore avenues for translation of our insights into phase I/II clinical trials.