In silico identification and characterization of potential orthosteric and allosteric pharmacological chaperones of the NAGLU enzyme and evaluation of their chaperone effect in vitro

Mucopolysaccharidosis type IIIB is produced by mutations in the NAGLU gene, which encodes for the lysosomal enzyme N-alpha-acetylglucosaminidase (NAGLU). This leads to a reduced NAGLU activity that prevents it to catalize the hydrolysis of α-N-acetylglucosamine from heparan sulfate, causing the built up of heparan sulfate in the lysosomes of several tissues, especially in the brain. Currently, there is no approved treatment for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC), small molecules that can bind to the defective enzyme and aid in their propper folding. Until recently, no PCs had been described for MPS IIIB. In this study we identified and characterized novel potential PCs for the NAGLU enzyme. For this purpose, we characterized the orthosteric site and predicted potential allosteric sites in silico. We performed a virtual screening for both the orthosteric and the two most energetically favorable allosteric sites against a database of 11,422 molecules. Then, considering factors such as binding affinity, predicted and/or reported biological function, blood brain barrier permeability and gastrointestinal absorption, 5 potential PCs were selected for further in silico characterization. Considering the neuropathological aspects of the disease and the non-invasiveness of a gastrointestinal delivery we selected the highest-ranking PCs in the screenings that had both blood brain barrier permeability and gastrointestinal absorption. We later performed characterizations through molecular dynamics simulations. Based on the in silico results, we selected atovaquone and piperaquine for the in vitro assays. The in vitro evaluation allowed us to confirm the binding to the NAGLU enzyme, and the ability of the PCs to restore the enzyme activity of mutant enzyme in diseased cell lines. These results represent novel PCs described for MPS IIIB and show the possibility to design novel therapeutic alternatives for this disease and other protein deficiency related diseases.