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. 2014 Jul 18;9(7):e102092.
doi: 10.1371/journal.pone.0102092. eCollection 2014.

The pharmacological chaperone AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha-glucosidase, and promotes glycogen reduction in a transgenic mouse model of Pompe disease

Richie Khanna  1 , Allan C Powe Jr  1 , Yi Lun  1 , Rebecca Soska  1 , Jessie Feng  1 , Rohini Dhulipala  1 , Michelle Frascella  1 , Anadina Garcia  1 , Lee J Pellegrino  1 , Su Xu  1 , Nastry Brignol  1 , Matthew J Toth  1 , Hung V Do  1 , David J Lockhart  1 , Brandon A Wustman  1 , Kenneth J Valenzano  1
Affiliations

The pharmacological chaperone AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha-glucosidase, and promotes glycogen reduction in a transgenic mouse model of Pompe disease

Richie Khanna et al. PLoS One. .

Abstract

Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER)-retained P545L mutant form of human GAA (P545L GAA). AT2220 increased the specific activity of P545L GAA toward both natural (glycogen) and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO) and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As such, AT2220 may warrant further evaluation as a treatment for Pompe disease.

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Conflict of interest statement

Competing Interests: Richie Khanna, Yi Lun, Rebecca Soska, Jessie Feng, Michelle Frascella, Anadina Garcia, Lee J. Pellegrino, Su Xu, Nastry Brignol, Hung V. Do, and Kenneth J. Valenzano are employed by Amicus Therapeutics and are shareholders in the company. Allan C. Powe, Jr., Rohini Dhulipala, Mathew J. Toth, David J. Lockhart, and Brandon A. Wustman are former employees of Amicus Therapeutics. Amicus Therapeutics funded the research and any publication fees. The recombinant human GAA (Lumizyme) used in this study is a product of Genzyme. There are no further patents, products in development, or marketed products to declare. This does not alter the authors' adherence to all of the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. AT2220 increases the specific activity, processing, and lysosomal delivery of multiple mutant forms of GAA.
(A) AT2220-mediated increases in GAA activity and protein levels in lysates from COS-7 cells transiently transfected with different mutant forms of GAA. Transfected cells were incubated with 100 µM AT2220 for 3 days, harvested, and lysed for activity and protein measurements. GAA activity was measured using the natural substrate glycogen (green) or the artificial substrate 4-methylumbelliferyl-α-glucopyranoside (4-MUG; blue). GAA (all isoforms) were detected through immunoblotting using the rabbit polyclonal antisera FL059 (see also, Fig. 1C). GAA protein levels were then quantified by densitometry using known amounts of rhGAA as calibration standards. The red dotted line indicates baseline (i.e., no change). (B) AT2220-mediated increases in the relative specific activities of multiple mutant forms of GAA calculated as the ratio of GAA enzyme activity against glycogen or 4-MUG to the GAA protein levels in transfected COS-7 cells. Enzyme activity and protein levels were measured as described in Fig. 1A. The red dotted line indicates no change from baseline. (C) AT2220-dependent increases in the processing and maturation of different mutant forms of GAA. Transfected cells were incubated with or without 100 µM AT2220, harvested, lysed, and immunoblotted as described in Fig. 1A. Dashes on the left of the Western blot represent the molecular weights (kDa) of the different GAA isoforms; arrows on the right indicate the presence of the mature, lysosomal 76 and 70 kDa forms of GAA. (D and E) Human skin fibroblasts from normal and Pompe subjects were incubated for 5 days with or without 100 µM AT2220. Cells were then harvested, lysed, and assayed for GAA activity using 4-MUG (D), and for GAA protein levels by immunoblotting (E). Dashes on the left of the Western blot represent the molecular weights (kDa) of the different GAA isoforms. WT, wild-type.
Figure 2
Figure 2. AT2220 increases the specific activity of mutant GAA precursors.
(A) Enzymatic activity of mutant GAA secreted from COS-7 cells transiently expressing GAA mutants and incubated with or without 100 µM AT2220. GAA in conditioned media was collected by incubation with Concanavalin A-Sepharose (Con-A) beads followed by centrifugation for GAA enrichment and removal of AT2220. Enzymatic activity was measured on half of the beads using the artificial substrate 4-MUG. (B) Immunoblotting of conditioned media from COS-7 cells transfected with different GAA mutants. GAA precursor protein (the 110 kDa species) in the remaining half of the Con-A pulldowns was denatured with sample buffer, separated by SDS-PAGE, immunoblotted, and quantified as described in Fig. 1. WT, wild-type.
Figure 3
Figure 3. AT2220 promotes the export of P545L GAA from the ER.
COS-7 cells were transiently transfected with wild-type or P545L GAA, immediately followed by 3-day incubation with and without 100 µM AT2220. Cells were then fixed and stained with antibodies against GAA (green) and calnexin, a marker for the endoplasmic reticulum (ER; red) as described in ‘Materials and Methods’. Co-localization of the GAA and calnexin signals appears yellow. Puncta (indicative of lysosomal localization) were seen throughout wild-type cells, as well as in P545L cells incubated with AT2220 (arrowheads). Scale bars: 50 µm.
Figure 4
Figure 4. AT2220 increases the stability of mature P545L GAA in lysosomes of Pompe fibroblasts.
(A and B) P545L GAA fibroblasts were incubated with 100 µCi 35S-cysteine/methionine for 3 days, chased for 0 to 4 days, then harvested and lysed. Incubation with 100 µM AT2220 during the pulse only, the pulse and the chase, or neither was performed as indicated. Isoforms of GAA (denoted as the 110, 95, 76, and 70 kDa species) were immunoprecipitated from cell lysates, separated by SDS-PAGE, and imaged with a phosphorimager. As controls, immunoprecipitates from metabolically labeled wild-type (denoted as ‘WT’ in the figure) and R854× fibroblasts (both in the absence of AT2220) were also evaluated. (C) Levels of the mature 76 kDa isoform of P545L GAA were quantified, normalized against the levels present at the 1-day chase time point, and plotted as a function of time. The data were fitted with an exponential function to estimate the half-life of the 76 kDa isoform of P545L GAA in the presence or absence of AT2220. The 1-day chase time point was chosen as time 0 for the exponential fit in order to eliminate the potential contribution of labeled 110 kDa precursor to the 76 kDa pool.
Figure 5
Figure 5. hP545L GAA Tg/KO mice have reduced tissue GAA activity, and elevated glycogen and LAMP1 levels.
(A) GAA and glycogen levels were assessed in tissue lysates prepared from heart and gastrocnemius of 12-week old male Gaa KO, hP545L GAA Tg/KO, and wild-type (denoted as ‘WT’ in the figure) mice. Significant differences in GAA activity and glycogen levels in hP545L GAA Tg/KO and Gaa KO mice compared to wild-type mice are reported as *p<0.05, t-test. Significant differences in GAA activity and glycogen levels in hP545L GAA Tg/KO mice compared to Gaa KO mice are reported as #p<0.05, t-test. Each bar represents the mean ± SEM from four mice per group analyzed in triplicate. Glycogen accumulation (B) and lysosomal proliferation (assessed by the quantity of LAMP1) (C) were assessed histochemically in Gaa KO, hP545L GAA Tg/KO, and wild-type (denoted as ‘WT’ in the figure) mouse cardiomyocytes and skeletal muscles fibers of the gastrocnemius. Glycogen staining is represented as dark pink spots, and LAMP1 staining as dark brown spots (each denoted with black arrows). The data shown are representative photomicrographs from four mice using a 40× (scale bar: 50 µm) or 20× (scale bar: 100 µm) objective in (B) and (C), respectively.
Figure 6
Figure 6. Oral AT2220 administration increases GAA activity in hP545L GAA Tg/KO mouse tissues.
Eight-week old male hP545L GAA Tg/KO mice were administered AT2220 ad libitum in drinking water for four weeks at the indicated doses. GAA activity was measured in homogenates of heart, gastrocnemius, soleus, diaphragm, triceps, and skin as described in ‘Materials and Methods’. The data presented represent the mean ± SEM of 7 mice/group analyzed in triplicate. Significant increases in GAA activity are reported as *p<0.05 vs. untreated, t-test. The effect was also significant for a linear trend, indicating a dose-dependent increase in GAA activity (post-hoc analysis, p<0.05). Insets. Tissue GAA protein levels were determined by Western blotting. The upper band represents the 110 kDa precursor isoform. The appearance of the 76 kDa mature lysosomal form is indicated by the dark arrow. Each lane on each Western blot contains the tissue homogenate from a single animal. The Western blot shown is representative of two experiments, each of which included a different mouse from each group.
Figure 7
Figure 7. Oral AT2220 administration reduces tissue glycogen levels in hP545L GAA Tg/KO mice.
(A) Eight-week old male hP545L GAA Tg/KO mice were administered 100 mg/kg per day AT2220 ad libitum in drinking water for four weeks either daily or less-frequently using four cycles of a ‘3 on/4 off’ or a ‘5 on/2 off’ regimen. Mice were euthanized 24 hours after AT2220 withdrawal in the daily regimen, and after 4 or 2 days off in the ‘3 on/4 off’ and ‘5 on/2 off’ regimens, respectively. The data presented represent the mean ± SEM of 7 mice/group analyzed in triplicate. Significant reductions in glycogen levels are reported as *p<0.05 vs. untreated, t-test, or #p<0.05 daily vs. less-frequent, t-test. WT, wild-type. (B) Cell type-specific reduction of glycogen in gastrocnemius, triceps, quadriceps, and heart of hP545L GAA Tg/KO mice administered 100 mg/kg/day AT2220 ad libitum in drinking water either daily or less-frequently (5 on/2 off’). The glycogen signal is represented as dark blue spots denoted with black arrows. The data shown are representative photomicrographs from 7 mice/group (magnification: 20X). Scale bars: 100 µm for gastrocnemius, triceps, and quadriceps; 50 µm for heart.
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Amicus Therapeutics funded the research. The funder provided support in the form of salaries for all authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.