Enzyme replacement therapy and substrate reduction therapy represent the current available options for the clinical management of Gaucher disease. While both treatments are effective in improving the hematologic and visceral symptoms in patients with mild, non-neuronopathic forms of the disease, their efficacy may be limited by their inability to cross the blood-brain barrier (ERT) or adverse side effects (SRT). Recent studies have demonstrated the ability of sugar analog inhibitors to increase the activity of mutant lysosomal enzymes in patient fibroblasts by acting as pharmacological chaperones [10
]. As active site inhibitors, these compounds bind to the mutant enzymes and stabilize them in the ER, thereby preventing their ER-associated degradation and facilitating their folding and transport out of this compartment to lysosomes. We have recently reported that the iminosugar IFG increases GlcCerase activity via multiple mechanisms in N370S Gaucher fibroblasts [10
]. Besides protecting newly synthesized N370S GlcCerase from ER-associated degradation, IFG treatment also improves the catalytic properties of mutant enzyme. In order to be successful as a therapeutic agent, IFG should selectively increase GlcCerase activity without inhibiting other cellular enzymes that are responsible for the hydrolysis of glucose-containing disaccharides (lactase and sucrase-isomaltase) and oligosaccharides (N-linked oligosaccharides and glycogen). The data presented in this study show that IFG has significantly less inhibitory activity toward a number of glucose-metabolizing enzymes compared to NB-DNJ, as summarized in .
Summary of the Inhibitory Effects of IFG and NB-DNJ on Various Glucose-Binding Enzymes
The sucrase-isomaltase enzyme complex is an enterocyte-specific, brush-border membrane disaccharidase that is required for the hydrolysis of dietary sucrose and some starches. Since only monosaccharides are absorbed, sucrase/isomaltase deficiency via genetic mutations or pharmacological inhibition with sugar analogs (e.g. NB-DNJ) leads to increased luminal disaccharides and subsequent osmotic diarrhea [9
]. In contrast to NB-DNJ, we show that IFG is only a weak inhibitor of these enzymes and is therefore unlikely to cause gastrointestinal problems at clinically relevant doses (). Sierks and colleagues showed that IFG inhibits yeast isomaltase, with a Ki
of 7.2 μM, a value somewhat lower than the 100 μM IC50 value we determined for the human enzyme [26
]. The failure of IFG and NB-DNJ to inhibit lactase is consistent with previous work showing that galactose analogs such as NB-DGJ but not glucose analogs are good inhibitors of this enzyme [9
NB-DNJ has been used to inhibit glucosylceramide synthase and thus reduce substrate accumulation in Gaucher patients. We tested IFG on this enzyme to determine whether this iminosugar would also inhibit glucosylceramide synthase. In contrast to the effect of NB-DNJ on glucosylceramide synthase, we found that IFG is a weak inhibitor inhibition of this enzyme in the in vitro assays, reinforcing the selectivity of the drug. At the present time, it is unclear whether long-term inhibition of glucosylceramide synthase will have any adverse consequences in human patients. Since IFG does not inhibit this enzyme, we predict that chronic administration of IFG would not affect glycolipid biosynthesis.
Several enzymes that bind to glucose are also present in the endoplasmic reticulum including two glucosidases, ER α-glucosidase I and ER α-glucosidase II, involved in the initial processing of N-linked oligosaccharides. Alterations in glucose trimming on newly synthesized glycoproteins can influence the ability of these proteins to fold properly and to be monitored by the ER quality control system [27
]. Chronic inhibition of protein folding and quality control in the ER could subsequently affect the function of several different proteins and may account for some of the side effects observed with prolonged treatment with sugar analogs. Our results demonstrate that IFG does not significantly inhibit α-glucosidase II activity in vitro
or in cultured cells and therefore minimizes these concerns.
IFG has been reported to inhibit glycogen phosphorylase in a dose-dependent manner with IC50 values ranging from 0.7 μM in liver and muscle homogenates [28
] to 1–3.3 μM in brain and astrocyte homogenates [29
]. Waagepetersen and co-workers found that high concentrations of IFG (400 μM) prevented norepinephrine-induced glycogen degradation in intact astrocytes while a lesser concentration of the compound (5 μM) had only a small effect. High concentrations of IFG (400 μM) also caused an accumulation of glycogen in neocortical astrocytes and cultured mouse optic nerves [30
]. In hepatocytes, IFG inhibited both basal and glucagon-induced glucose production at much lower concentrations (IC50 values of 3 and 2 μM, respectively) [28
]. The reason for this difference in sensitivity is not clear. Our studies with human fibroblasts expressing the N370S mutant form of GlcCerase showed that incubation with 30 μM IFG resulted in optimal enhancement of enzyme activity [10
]. Since this is higher than required to inhibit glycogen phosphorylase in hepatocytes, it will be important to determine the effect of this concentration of IFG on glycogen metabolism in rodents.
NB-DNJ and NN-DNJ have also been reported to inhibit glycogen breakdown in vitro
and in mice, but in these instances the mechanism does not involve inhibition of glycogen phosphorylase [32
]. Rather, low micromolar concentrations of these compounds inhibit acid α-glucosidase, the enzyme that mediates the breakdown of glycogen in lysosomes, and the glycogen debranching enzyme, α1,6-glucosidase. Treatment of mice with these agents resulted in the accumulation of glycogen in liver and muscle. The effect of NB-DNJ was transient, but glycogen accumulation persisted for months when NN-DNJ was administered. Further, the NN-DNJ effect was seen at a 10-fold lower dose (250mg/kg/day) than required for the NB-DNJ effect. The difference in the effects of these two iminosugars was attributed to the increased hydrophobicity of NN-DNJ that leads to increased in vivo uptake and tissue retention of the compound [32
]. It should be noted that NN-DNJ, like IFG, has been shown to increase the activity of GlcCerase in some Gaucher patient fibroblasts [15
]. These findings reinforce the need to examine the effect of IFG on glycogen metabolism in rodents in future studies.
In summary, IFG appears to act in a selective fashion to increase the activity of GlcCerase. It does not significantly inhibit the activity of several other glucose-binding enzymes, several of which are inhibited by NB-DNJ (). This selective action of IFG warrants its further evaluation as a potential therapeutic agent in the treatment of some forms of Gaucher disease.