Gaucher disease results from mutations in the lysosomal enzyme acid beta-glucosidase (GCase). Although enzyme replacement therapy has improved the health of some affected individuals, such as those with the prevalent N370S mutation, oral treatment with pharmacological chaperones may be therapeutic in a wider range of tissue compartments by restoring sufficient activity of endogenous mutant GCase. Here we demonstrate that isofagomine (IFG, 1) binds to the GCase active site, and both increases GCase activity in cell lysates and restores lysosomal trafficking in cells containing N370S mutant GCase. We also compare the crystal structures of IFG-bound GCase at low pH with those of glycerol-bound GCase at low pH and apo-GCase at neutral pH. Our data indicate that IFG induces active GCase, which is secured by interactions with Asn370. The design of small molecules that stabilize substrate-bound conformations of mutant proteins may be a general therapeutic strategy for diseases caused by protein misfolding and mistrafficking.

Regulation of decapping is a critical determinant of mRNA stability. We recently identified hDcp2 as a human decapping enzyme with intrinsic decapping activity. This activity is specific to N 7 -methylated guanosine containing RNA. The hDcp2 enzyme does not function on the cap structure alone and is not sensitive to competition by cap analog, suggesting that hDcp2 requires the RNA for cap recognition. We now demonstrate that hDcp2 is an RNA-binding protein and its recognition and hydrolysis of the cap substrate is dependent on an initial interaction with the RNA moiety. A biochemical characterization of hDcp2 revealed that a 163 amino acid region containing two evolutionarily conserved regions, the Nudix fold hydrolase domain and the adjacent Box B region contained methyl-cap-specific hydrolysis activity. Maximum decapping activity for wild-type as well as truncation mutants of hDcp2 required Mn 2+ as a divalent cation. The demonstration that hDcp2 is an RNA-binding protein with an RNA-dependent decapping activity will now provide new approaches to identify specific mRNAs that are regulated by this decapping enzyme as well as provide novel avenues to control mRNA decapping and turnover by influencing the RNA-binding property of hDcp2.

Lysosomal enzymes are responsible for the degradation of a wide variety of glycolipids, oligosaccharides, proteins, and glycoproteins. Inherited mutations in the genes that encode these proteins can lead to reduced stability of newly synthesized lysosomal enzymes. While often catalytically competent, the mutated enzymes are unable to efficiently pass the quality control mechanisms of the endoplasmic reticulum, resulting in reduced lysosomal trafficking, substrate accumulation, and cellular dysfunction. Pharmacological chaperones (PCs) are small molecules that bind and stabilize mutant lysosomal enzymes, thereby allowing proper cellular translocation. Such compounds have been shown to increase enzyme activity and reduce substrate burden in a number of preclinical models and clinical studies. In this Perspective, we review several of the lysosomal diseases for which PCs have been studied and the SAR of the various classes of molecules.

Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid b-glucosidase (GCase). We have shown previously that the small molecule pharmacological chaperone isofagomine (IFG) binds and stabilizes N370S GCase, resulting in increased lysosomal trafficking and cellular activity. In this study, we investigated the effect of IFG on L444P GCase. Incubation of Gaucher patient-derived lymphoblastoid cell lines (LCLs) or fibroblasts with IFG led to approximately 3.5-and 1.3-fold increases in L444P GCase activity, respectively, as measured in cell lysates. The effect in fibroblasts was increased approximately 2-fold using glycoprotein-enrichment, GCase-immunocapture, or by incubating cells overnight in IFG-free media prior to assay, methods designed to maximize GCase activity by reducing IFG carryover and inhibition in the enzymatic assay. IFG incubation also increased the lysosomal trafficking and in situ activity of L444P GCase in intact cells, as measured by reduction in endogenous glucosylceramide levels. Importantly, this reduction was seen only following three-day incubation in IFG-free media, underscoring the importance of IFG removal to restore lysosomal GCase activity. In mice expressing murine L444P GCase, oral administration of IFG resulted in significant increases (2-to 5-fold) in GCase activity in disease-relevant tissues, including brain. Additionally, eight-week IFG administration significantly lowered plasma chitin III and IgG levels, and 24-week administration significantly reduced spleen and liver weights. Taken together, these data suggest that IFG can increase the lysosomal activity of L444P GCase in cells and tissues. Moreover, IFG is orally available and distributes into multiple tissues, including brain, and may thus merit therapeutic evaluation for patients with neuronopathic and non-neuronopathic Gaucher disease.

Many human diseases result from mutations in specific genes. Once translated, the resulting aberrant proteins may be functionally competent and produced at near-normal levels. However, because of the mutations, the proteins are recognized by the quality control system of the endoplasmic reticulum and are not processed or trafficked correctly, ultimately leading to cellular dysfunction and disease. Pharmacological chaperones (PCs) are small molecules designed to mitigate this problem by selectively binding and stabilizing their target protein, thus reducing premature degradation, facilitating intracellular trafficking, and increasing cellular activity. Partial or complete restoration of normal function by PCs has been shown for numerous types of mutant proteins, including secreted proteins, transcription factors, ion channels, G protein-coupled receptors, and, importantly, lysosomal enzymes. Collectively, lysosomal storage disorders (LSDs) result from genetic mutations in the genes that encode specific lysosomal enzymes, leading to a deficiency in essential enzymatic activity and cellular accumulation of the respective substrate. To date, over 50 different LSDs have been identified, several of which are treated clinically with enzyme replacement therapy or substrate reduction therapy, although insufficiently in some cases. Importantly, a wide range of in vitro assays are now available to measure mutant lysosomal enzyme interaction with and stabilization by PCs, as well as subsequent increases in cellular enzyme levels and function. The application of these assays to the identification and characterization of candidate PCs for mutant lysosomal enzymes will be discussed in this review. In addition, considerations for the successful in vivo use and development of PCs to treat LSDs will be discussed.