The pharmacological chaperone isofagomine increases the activity of the Gaucher disease L444P mutant form of beta-glucosidase

Abstract

Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid beta-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.

Figure 1. IFG increases N370S and L444P GCase activity in Gaucher patient-derived cells

Panel A. N370S fibroblasts (DMN89.45) were incubated with the indicated concentrations of IFG tartrate for five days and GCase activity was directly measured in lysed cells as described in ‘Materials and Methods’. In the experiment shown, a concentration-dependent increase of approximately 2.5-fold was seen in GCase activity. The increase in GCase activity was found significant for a linear trend (one-way ANOVA), indicating a concentration-dependent effect. Panel B. L444P fibroblasts (GM07968) and LCLs (GS0501) were incubated with the indicated concentrations of IFG tartrate for five days and GCase activity was directly measured in lysed cells. In the experiments shown, a small but reproducible 1.3-fold increase in GCase activity was seen in fibroblast lysates (left panel), and a 3.5-fold increase was seen in LCL lysates (right panel). The increase in GCase activity measured in LCLs was found significant for a linear trend (one-way ANOVA). Summary data from the fibroblast and LCL cell lines shown here, as well as others, are presented in Table 1. Insets, GCase protein levels were increased in Gaucher fibroblasts and LCLs after five-day incubation with IFG, as directly measured by Western blotting (50 μg total protein per lane). Blots were probed with an anti-human GCase antibody and a β-actin antibody (loading control). The data shown are representative of three independent experiments. Panel C. Gaucher fibroblasts homozygous for L444P GCase (GM07968) were incubated for five days with the indicated concentrations of IFG tartrate. Cell lysates were then subjected to either glycoprotein- or GCase-enrichment using Con A- and immunocapture, respectively, as described in ‘Materials and Methods’. GCase activity was measured on the precipitated beads. In the experiments shown, concentration-dependent increases (approximately 2-fold) were seen in GCase activity. The increases were found significant for a linear trend (one-way ANOVA). Panel D. Gaucher fibroblasts homozygous for L444P GCase (GM07968) were incubated for five days with the indicated concentrations of IFG tartrate, followed by 24-hour washout (media only). GCase activity was measured directly in lysed cells. In the experiments shown, an approximately 1.7-fold increase was seen in L444P GCase activity after 24-hour washout. This increase was found significant for a linear trend (one-way ANOVA). In all panels, the data have been normalized to baseline (untreated) values and are representative of three or six independent experiments as indicated in Tables 1 and 2, with each point the mean±SEM of triplicate determinations. Statistically significant differences from untreated were determined using a two-tailed, unpaired student’s t-test with ^*p<0.05, ^**p<0.01, and ^***p<0.001.

Figure 2. IFG increases the lysosomal pool of L444P GCase in Gaucher patient-derived fibroblasts

Fibroblasts derived from healthy volunteers (WT; CRL2097) and Gaucher patients homozygous for the N370S (DMN89.45) or L444P (GM07968, GM00877, GM10915) mutant forms of GCase were incubated in the absence or presence of 100 μM IFG tartrate for 14 days. GCase (green) and the lysosomal marker LAMP-1 (red) were visualized by confocal microscopy after indirect immunofluorescence staining as described in ‘Materials and Methods’. In the merged images, yellow denotes co-localization of the two proteins, indicative of their lysosomal localization. Nuclei are stained with DAPI (blue). IFG treatment increased the lysosomal pool of GCase in wild-type as well as N370S and L444P GCase fibroblasts (as shown by the increased amount of yellow in the merged images). Representative cells are shown to demonstrate the degree of co-localized GCase and LAMP-1. Magnification: 63x.

Figure 3. IFG reduces GC levels in Gaucher fibroblasts and LCLs

Fibroblasts (GM07968, left side of panel) and LCLs (GS0501, right side of panel) homozygous for L444P GCase were incubated in the absence or presence of 30 μM IFG for 7 days followed by a 3-day washout (‘7 on/3 off’). Parallel cultures of these cell lines were incubated for 10 days with 30 μM IFG or 500μM NB-DNJ (‘10 on’). GC levels were then measured as described in ‘Materials and Methods’ as well as in normal control fibroblasts (CRL2076) or LCLs (WT0003). The data are expressed as the mean±SEM from 3 flasks for each condition tested. Statistically significant differences from untreated in GC levels were determined using a two-tailed, unpaired student’s t-test with ^*p<0.05, ^**p<0.01, and ^***p<0.001, or #p<0.05 for untreated versus ‘10 on’. Similar results were seen in two other L444P GCase cell lines (GM10915 fibroblasts and GS0505 lymphoblasts; see Table 3).

Figure 4. Tissue distribution pharmacokinetics of IFG

Eight-week old male Sprague-Dawley rats were fasted overnight prior to administration of IFG tartrate (600 mg/kg, equivalent to 300 mg/kg free base) by oral gavage. Tissue and blood samples were drawn as a function of time. IFG levels were assessed by LC-MS/MS in plasma and tissue homogenates as described in ‘Materials and Methods’. Each point represents the mean±SEM from 3 rats.

Figure 5. IFG increases tissue L444P GCase activity in vivo

Panel A. Two-month old male L444P GCase mice were administered IFG HCl (3, 10, or 30 mg/kg per day, equivalent to 2.5, 8.2, and 25 mg/kg free base, respectively) ad libitum in drinking water for two weeks. GCase activity in liver lysates was measured as described in ‘Materials and Methods’. Significant increases in GCase activity were seen at all three doses. Each bar represents the mean±SEM of GCase activity from 4 mice/group analyzed in triplicate. The treatment was also found significant for a linear trend (one-way ANOVA), indicating a dose-dependent effect. Panel B. Two-month old male L444P GCase mice were administered IFG tartrate (20 mg/kg per day, equivalent to 10 mg/kg free base) ad libitum for four weeks. GCase activity was measured in tissue lysates as described in ‘Materials and Methods’. Significant increases in GCase activity were seen in liver (4-fold), spleen (4-fold), lung (5-fold), and brain (2-fold). Tissue GCase activity from untreated wild-type C57BL/6 mice are also shown. Each bar represents the mean±SEM of GCase activity from 4 mice/group analyzed in triplicate. Inset. Six-month old male L444P GCase mice were administered IFG tartrate (20 mg/kg per day, equivalent to 10 mg/kg free base) ad libitum for 24 weeks and GCase activity was measured in mineralized bone and bone marrow lysates as described in ‘Materials and Methods’. Significant increases in L444P GCase activity (up to 2-fold) were seen with IFG administration. Each bar represents the mean±SEM of GCase activity from 7–8 mice/group analyzed in triplicate. Panel C. GCase protein levels in the tissue samples (50 μg) used in panel B were directly measured by Western blotting using anti-mouse GCase and β-actin (loading control) antibodies as described in ‘Materials and Methods’. IFG tartrate administration increased GCase activity in liver (3-fold), spleen (2-fold), lung (4-fold), and brain (1.2-fold). Each lane represents one mouse from each group and is representative of two experiments with two different mice from each group. Panel D. Primary cultures of mouse liver macrophages were derived from two-month old untreated male L444P GCase mice and incubated with IFG tartrate for five days at the concentrations indicated as described in ‘Materials and Methods’. In the experiment shown, a significant and concentration-dependent increase (approximately 2-fold) in L444P GCase activity was seen in macrophage lysates. The increase was also found significant for a linear trend (one-way ANOVA). The data shown have been normalized to untreated values and are representative of three independent experiments, with each point the mean±SEM of triplicate determinations. In panels A, B, and D, statistically significant differences from untreated were determined using a two-tailed, unpaired student’s t-test with ^*p<0.05, ^**p<0.01, and ^***p<0.001.

Figure 6. Time course for decay of increased L444P GCase activity after IFG withdrawal

Four-month old male L444P GCase mice were administered drinking water (dotted lines) or IFG tartrate (20 mg/kg per day, equivalent to 10 mg/kg free base (solid line)) ad libitum in drinking water for four weeks followed by a washout period (drinking water only) for up to eight days. Groups of mice were then euthanized on Days 0, 2, 4, or 8 after IFG tartrate withdrawal and GCase activity was measured in tissue lysates. Statistically significant increases above baseline were maintained in liver, spleen, and lung GCase activity for up to four days, and in brain for up to two days. Each data point represents the mean±SEM of tissue GCase activity from 6 mice/group analyzed in triplicate. Statistically significant differences from untreated were determined using a two-tailed, unpaired student’s t-test with ^*p<0.05, ^**p<0.01, and ^***p<0.001.

Figure 7. IFG reduces organ weights and the levels of plasma markers for Gaucher disease in L444P GCase mice

Panel A. One-month old male L444P GCase mice were administered IFG HCl (10 mg/kg per day, equivalent to 8.2 mg/kg free base) ad libitum for up to 24 weeks. Mice (4–6/time point) were euthanized after 4, 12, or 24 weeks of administration and total body, liver, and spleen weights were recorded and compared to those of age-matched untreated L444P GCase and wild-type C57BL/6 mice (4 mice/time point). Statistically significant reductions in organ weights compared to untreated L444P GCase mice were seen in spleen (upper panel) and liver (lower panel). Organ weights are expressed as a percentage of body weight. Panel B. Four-month old male L444P GCase mice were administered IFG tartrate (20 mg/kg per day, equivalent to 10 mg/kg free base) ad libitum for eight weeks (11 mice/time point). Chitin III (upper panel) and IgG (lower panel) levels were measured in plasma samples and compared to those of age-matched untreated L444P GCase (11 mice/time point) and wild-type C57BL/6 mice (6 mice/time point). Statistically significant reductions in both markers compared to untreated L444P GCase mice were seen. Insets. One-month old male L444P GCase mice were administered drinking water (dotted lines) or IFG HCl (10 mg/kg per day, equivalent to 8.2 mg/kg free base (solid lines)) ad libitum for up to 24 weeks. Mice were euthanized after 4, 12, or 24 weeks of treatment (4–6 mice/time point). A trend of reduction in both chitin III and IgG levels was seen with time compared to age-matched untreated L444P GCase mice. Statistically significant differences from untreated were determined using a two-tailed, unpaired student’s t-test with ^*p<0.05.