Development of an in vivo plant-based screen for identifying pharmacological chaperones for treatment of human lysosomal storage diseases

Small-molecule- enzyme enhancement therapy has emerged as an attractive approach for the treatment of lysosomal storage diseases (LSDs), a broad group of genetic diseases caused by mutations in genes encoding lysosomal enzymes or proteins required for lysosomal function. Missense mutant lysosomal enzymes normally subjected to rapid disposal by ER-associated degradation (ERAD) can be stabilized by small molecule chaperones that increase residual enzyme activity largely by increasing the transport and maturation of the mutant enzyme. Mucopolysaccharidosis I (MPS I) and Gaucher disease were my research targets – two LSDs caused by a deficiency of alpha-L-iduronidase (IDUA) and β-glucocerebrosidase (GCase), respectively. My goals were two-fold: (1) To determine the proteostasis of a severely defective mutant lysosomal enzyme in plant cells. (2) To develop a plant-cell-based screening system to identify putative LSD therapeutics. For the former goal, post-ER trafficking of the severely malfolded L444P GCase protein, and some aspects of cellular homeostasis, were restored to different degrees by ERAD inhibitors and proteostasis regulators, which increased the steady-state levels of the mutant protein inside the plant cells and rescued a proportion of protein from proteolysis. For goal 2, I developed a plant-cell-screening tool for identifying putative small molecule therapeutics based on selecting for library molecules capable of enhancing the post-ER transport of missense mutant lysosomal enzymes. Since the recombinant variants were equipped with a signal peptide, and the expression cells - transgenic tobacco BY2 cells - possess no lysosomes, the assay was based on increased lysosomal enzyme activity in the secretion media. I first established the proof-of-principle for the assay (i.e. its selectivity and specificity) based on recombinant N370S GCase, and two characterized chaperones - ambroxol and N-(n-nonyl) deoxynojirimycin. Two IDUA mutant proteins that underlie MPS I disease (P533R- and R383H- IDUA), formed the basis of the plant-cell-based assay that was used to screen a library of 1,040 Food and Drug Administration-approved drugs. Downstream validation of the hits identified in the primary screening by secretion and heat denaturation assays resulted in the identification of a potential candidate molecule (‘X-372’) for P533R IDUA. Further development of this molecule may yield a therapeutic for MPS I disease.