Isofagomine- and 2,5-Anhydro-2,5-imino-d-glucitol-Based Glucocerebrosidase Pharmacological Chaperones for Gaucher Disease Intervention

Abstract

SummaryGaucher 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 GC folding by binding to the variants active site, stabilizing the native conformation of GC in the neutral pH environment of the endoplasmic reticulum (ER), enabling their 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 patientderived 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.

Isofagomine Induced Stabilization of Glucocerebrosidase

“…Treatment of N370S/N370S and F213I/L444P fibroblasts with 25 μM IFG for five days enhanced GCase activity 2.5-and 4.3-fold over non-pretreated cells, respectively (see Figure 1C). The increased activity of the F213I mutation extends the panel of GCase mutations (N370S [8] and G202R [9]) whose activity can be enhanced by IFG.…”

“…[8] This likely reflects the fact that the efficacy of a PC not only depends on its affinity for the target enzyme but also on its bioavailability in terms of membrane permeability, subcellular distribution and metabolism. [9] Thus, the actual intracellular concentration of IFG after treatment is likely much less than the extracellular concentration. The actual concentration and any residual inhibitory effects IFG might have on lysosomal GCase are difficult to determine, as the in vitro assays for GCase enhancement include washing of cells prior to lysis and dilution of the intracellular IFG by the addition of lysis and assay buffers (>100-fold).…”

Multi-system disorders of glycosphingolipid and ganglioside metabolism

“…The fi rst generation of these agents for the GSL storage diseases are primarily iminosugars that are either 5-or 1-deoxy or 3,4-dideoxy glycoside derivatives of glucose or galactose, and they are potent reversible competitive inhibitors of specifi c lysosomal hydrolases. The current agents, isofagomine [3,4-dideoxy-5-(hydroxymethyl) piperidine] and DGJ (1-deoxy galactonojirimycin), have shown signifi cant ability to enhance the activity of a great variety of different mutant GCases or ␣ -galactosidases A, respectively, in tissue culture and in peripheral blood leukocytes of affected patients with Gaucher or Fabry disease, respectively (309)(310)(311). Clinical trials in Fabry disease are ongoing with DGJ, but the development of isofagomine has been stopped due to the lack of clinical effect in all but one patient with Gaucher disease type 1 (http://fi les.…”

Synthesis of N-substituted iminosugars from 2′-carbonyl-C-glycofuranosides

“…These compounds are therefore of growing interest as new therapeutic leads, and two simple iminosugar derivatives have already been approved for therapeutic purposes: GlysetTM (N-hydroxyethyl-1-deoxynojirimycin, 2) for the treatment of complications associated with type II diabetes, 8,9 and ZavescaTM (N-butyl-1-deoxynojirimycin, 3) for the treatment of Gaucher disease. 10 The initial results were fairly promising as patients have shown clear improvement in organ volume as well as in haematological variables, which necessitated further exploration and investigation of N-alkylated iminosugars. [11][12][13] Considering the high potential of iminosugars as carbohydrate mimics, the design of a general and efficient approach to synthesize N-alkylated iminosugars appears to be an important issue yet to be solved.…”