Pharmacologic chaperoning as a strategy to treat Gaucher disease.

We briefly introduce the most common lysosomal storage disorder, Gaucher disease, concisely describe the Food and Drug Administration approved strategies to ameliorate Gaucher disease, and then outline the emerging pharmacologic chaperone strategy that offers the promise to remedy this malady.

Isofagomine- and 2,5-anhydro-2,5-imino-D-glucitol-based glucocerebrosidase pharmacological chaperones for Gaucher disease intervention.

Gaucher disease, resulting from deficient lysosomal glucocerebrosidase (GC) activity, is the most common lysosomal storage disorder. Clinically important GC mutant enzymes typically have reduced specific activity and reduced lysosomal concentration, the latter due to compromised folding and trafficking. We and others have demonstrated that pharmacological chaperones assist variant GC folding by binding to the active site, stabilizing the native conformation of GC in the neutral pH environment of the endoplasmic reticulum (ER), enabling its trafficking from the ER to the Golgi and on to the lysosome. The mutated GC fold is generally stable in the lysosome after pharmacological chaperone dissociation, owing to the low pH environment for which the fold was evolutionarily optimized and the high substrate concentration, enabling GC to hydrolyze glucosylceramide to glucose and ceramide. The hypothesis of this study was that we could combine GC pharmacological chaperone structure-activity relationships from distinct chemical series to afford potent novel chaperones comprising a carbohydrate-like substructure that binds in the active site with a hydrophobic substructure that binds in a nearby pocket. We combined isofagomine and 2,5-anhydro-2,5-imino-D-glucitol active site binding substructures with hydrophobic alkyl adamantyl amides to afford novel small molecules with enhanced ability to increase GC activity in patient-derived fibroblasts. The cellular activity of N370S and G202R GC in fibroblasts is increased by 2.5- and 7.2-fold with isofagmine-based pharmacological chaperones N-adamantanyl-4-((3R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)piperidin-1-yl)-butanamide (3) and N-adamantanyl-4-((3R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)piperidin-1-yl)pentanamide (4), respectively, the best enhancements observed to date.

Chemical chaperones and permissive temperatures alter localization of Gaucher disease associated glucocerebrosidase variants.

Point mutations in the lysosomal hydrolase, glucocerebrosidase (GC), can cause Gaucher disease, a common lysosomal storage disease. Several clinically important GC mutations impede folding in the endoplasmic reticulum (ER) and target these enzymes for ER-associated degradation (ERAD). The removal of these misfolded proteins decreases the lysosomal concentration of GC, which results in glucosylceramide accumulation. The most common GC variant, N370S, and other clinically relevant variants, G202R and L444P, exhibit different cellular localization patterns in patient-derived fibroblasts. We show that these distributions can be altered by manipulation of the ER folding environment, either by chemical chaperones or by temperature shifts. N370S, L444P, and G202R GC are destabilized in the neutral pH environment of the ER, rendering them prone to ERAD. Fibroblasts harboring the G202R and L444P GC mutations grown at 30 degrees C localize the mutant proteins to the lysosome, and this increases total GC activity. Both of these temperature-sensitive mutants appear to be stable at 37 degrees C once they are trafficked to the low pH environment of the lysosome. Chemical chaperones correct the ER instability and significant ER retention of G202R GC. N370S is also destabilized under ER simulating conditions, a deficiency that is corrected by chemical chaperone binding. These data clearly show manipulating the ER environment with chemical chaperones increases the lysosomal concentration of partially active GC variants and suggest that small molecules could be used to treat Gaucher disease.

Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles.

Gaucher disease is a lysosomal storage disorder caused by deficient glucocerebrosidase activity. We have previously shown that the cellular activity of the most common Gaucher disease-associated glucocerebrosidase variant, N370S, is increased when patient-derived cells are cultured with the chemical chaperone N-nonyl-deoxynojirimycin. Chemical chaperones stabilize proteins against misfolding, enabling their trafficking from the endoplasmic reticulum. Herein, the generality of this therapeutic strategy is evaluated with other glucocerebrosidase variants and with additional candidate chemical chaperones. Improved chemical chaperones are identified for N370S glucocerebrosidase. Moreover, we demonstrate that G202R, a glucocerebrosidase variant that is known to be retained in the endoplasmic reticulum, is also amenable to chemical chaperoning. The L444P variant is not chaperoned by any of the active site-directed molecules tested, likely because this mutation destabilizes a domain distinct from the catalytic domain.

X-ray structure of human acid-beta-glucosidase covalently bound to conduritol-B-epoxide. Implications for Gaucher disease.

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-beta-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 A resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep.4, 704-709) and have now solved the structure of Glc-Cerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.

The biological and chemical basis for tissue-selective amyloid disease.

Factors controlling the onset and progression of extracellular amyloid diseases remain largely unknown. Central to disease etiology is the efficiency of the endoplasmic reticulum (ER) machinery that targets destabilized mutant proteins for degradation and the enhanced tendency of these variants to aggregate if secreted. We demonstrate that mammalian cells secrete numerous transthyretin (TTR) disease-associated variants with wild-type efficiency in spite of compromised folding energetics. Only the most highly destabilized TTR variants are subjected to ER-associated degradation (ERAD) and then only in certain tissues, providing insight into tissue selective amyloidosis. Rather than a "quality control" standard based on wild-type stability, we find that ER-assisted folding (ERAF), based on global protein energetics, determines the extent of export. We propose that ERAF (influenced by the energetics of the protein fold, chaperone enzyme distributions, and metabolite chaperones) in competition with ERAD defines the unique secretory aptitude of each tissue.

Chemical chaperones increase the cellular activity of N370S beta -glucosidase: a therapeutic strategy for Gaucher disease.

Gaucher disease is a lysosomal storage disorder caused by deficient lysosomal beta-glucosidase (beta-Glu) activity. A marked decrease in enzyme activity results in progressive accumulation of the substrate (glucosylceramide) in macrophages, leading to hepatosplenomegaly, anemia, skeletal lesions, and sometimes CNS involvement. Enzyme replacement therapy for Gaucher disease is costly and relatively ineffective for CNS involvement. Chemical chaperones have been shown to stabilize various proteins against misfolding, increasing proper trafficking from the endoplasmic reticulum. We report herein that the addition of subinhibitory concentrations (10 microM) of N-(n-nonyl)deoxynojirimycin (NN-DNJ) to a fibroblast culture medium for 9 days leads to a 2-fold increase in the activity of N370S beta-Glu, the most common mutation causing Gaucher disease. Moreover, the increased activity persists for at least 6 days after the withdrawal of the putative chaperone. The NN-DNJ chaperone also increases WT beta-Glu activity, but not that of L444P, a less prevalent Gaucher disease variant. Incubation of isolated soluble WT enzyme with NN-DNJ reveals that beta-Glu is stabilized against heat denaturation in a dose-dependent fashion. We propose that NN-DNJ chaperones beta-Glu folding at neutral pH, thus allowing the stabilized enzyme to transit from the endoplasmic reticulum to the Golgi, enabling proper trafficking to the lysosome. Clinical data suggest that a modest increase in beta-Glu activity may be sufficient to achieve a therapeutic effect.

Discovery of a 3-amino-6-phenyl-pyridazine derivative as a new synthetic antineuroinflammatory compound.

Excessive glial activation, with overproduction of cytokines and oxidative stress products, is detrimental and a hallmark of neurodegenerative disease pathology. Suppression of glial activation is a potential therapeutic approach, and protein kinases are targets of some antiinflammatory drugs. To address an unmet need for selective inhibitors of glial activation, we developed a novel 3-amino-6-phenylpyridazine derivative that selectively blocks increased IL-1 beta, iNOS, and NO production by activated glia, without inhibition of potentially beneficial glial functions.