Glycosphingolipid Depletion in Fabry Disease Lymphoblasts with Potent Inhibitors of Glucosylceramide Synthase

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Kidney International, Vol. 57 (2000), pp. 446–454

Glycosphingolipid depletion in Fabry disease lymphoblasts with potent inhibitors of glucosylceramide synthase

AKIRA ABE,LOIS J. AREND,LIHSUEH LEE,CLIFFORD LINGWOOD,ROSCOE O. BRADY, and JAMES A. SHAYMAN

Nephrology Division, Department of Internal Medicine and Department of Pathology, University of Michigan Medical Center, Ann Arbor, Michigan, USA; Hospital for Sick Children, Toronto, Ontario, Canada; and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA

Glycosphingolipid depletion in Fabry disease lymphoblasts glycosphingolipids with ␣-galactosyl linkages, including with potent inhibitors of glucosylceramide synthase. galabiosylceramide and globotriaosylceramide, as well Background. Fabry disease is an inherited X-linked disorder as the blood group B and B1 antigenic glycosphingolipids resulting in the loss of activity of the lysosomal hydrolase ␣-galactosidase A and causing the clinical manifestations of [2]. Globotriaosylceramide is also the receptor for vero- renal failure, cerebral vascular disease, and myocardial in- toxin [3]. Globotriaosylceramide accumulates in the af- farction. The phenotypic expression of this disorder is manifest fected tissues of Fabry disease, including the kidney, by the accumulation of glycosphingolipids containing ␣-gal- vascular endothelium, peripheral nerves, and heart. The actosyl linkages, most prominently globotriaosylceramide. major clinical manifestations of Fabry disease include Methods. Based on quantitative structure activity studies, we recently reported two newly designed glucosylceramide renal failure, cerebral vascular disease, myocardial in- synthase inhibitors based on 1-phenyl-2-palmitoylamino-3-pyr- farction, neuropathic pain, and skin lesions termed angi- rolidino-1-propanol (P4). These inhibitors, 4Ј-hydroxy-P4 and okeratomas. ethylenedioxy-P4, were evaluated for their ability to deplete Glucosylceramide synthase catalyzes the conversion globotriaosylceramide and other glucosylceramide-based lipids of ceramide to glucosylceramide using uridine diphospho in Fabry lymphocytes and were compared with N-butyldeoxynojirimycin, another reported glucosylceramide synthase inhib- (UDP)-glucose as a glucose donor [4]. This glycosyltrans- itor. ferase is a key enzyme in the biosynthesis of glycosphin- Results. Concentrations as low as 10 nmol/L of 4Ј-hydroxy- golipids because most glycosphingolipids are glucosylc- P4 and ethylenedioxy-P4 resulted in 70 and 80% depletion, eramide based. A potential strategy for the treatment of respectively, of globotriaosylceramide, with maximal depletion Fabry disease (or indeed of any other glycosphingolipid occurring at three days of treatment. There was no impairment of cell growth. In contrast, N-butyldeoxynojirimycin only mini- storage disease in which glucosylceramide is the base mally lowered globotriaosylceramide levels, even at concentra- cerebroside) is therefore the inhibition of glucosylcera- tions as high as 10 ␮mol/L. Globotriaosylceramide depletion mide synthase, the earliest glycosylation step in globotri- was confirmed by the loss of binding of FITC-conjugated vero- aosylceramide synthesis (Fig. 1). toxin B subunit to the lymphoblasts. The prototypical glucosylceramide synthase inhibitor is Conclusions. These findings suggest that selective glucosylceramide synthase inhibitors are highly effective in the deple- d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-pro- tion of globotriaosylceramide from Fabry cell lines. We suggest panol (PDMP) [5]. Using PDMP as a lead compound, that these compounds have potential therapeutic utility in the we previously observed that empiric substitutions of a treatment of Fabry disease. palmitoyl group for the fatty acid in amide linkage and of a pyrrolidino function for the morpholino group resulted in a glucosylceramide synthase inhibitor of sig- Fabry disease is an inherited X-linked disorder re- nificantly greater potency [6]. This compound 1-phenyl- sulting in a deficiency of the lysosomal ␣-galactosidase 2-palmitoylamino-3-pyrrolidino-1-propanol (P4) was ap- A [1]. This disorder is manifest by the accumulation of proximately 20 times more potent than PDMP when assayed against a crude cellular homogenate. More re- Key words: X-linked disorder, globotriaosylceramide, ␣-galactosidase cently, we have identified two highly active glucosylcer- A, Fabry cell lines, verotoxin. amide synthase inhibitors based on simple quantitative Received for publication July 13, 1999 structure activity relationships. Phenyl group substitu- and in revised form September 15, 1999 tions yielded 4Ј-hydroxy-1-phenyl-2-palmitoylamino-3- Accepted for publication September 28, 1999 pyrrolidino-1-propanol (p-OH-P4) and ethylenedioxy-  2000 by the International Society of Nephrology 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol

446 Abe et al: Glycosphingolipid depletion in Fabry disease 447

previously described [8]. The B subunit of verotoxin (VT1B) was purified by affinity chromatography and labeled with fluorescein and was dialyzed to remove free fluorescein, also as described [9]. Glucosylceramide synthase inhibitors were synthe- sized by the Mannich reaction from 2-N-acylaminoaceto- phenone, paraformaldehyde, and pyrrolidine, followed by reduction with sodium borohydride, as detailed previously [5]. Four enantiomers are produced during the synthesis. Because only the d-threo enantiomers are active in inhibiting the glucosylceramide synthase, resolu- tion of the active d-threo inhibitors is performed by chiral chromatography [7].

Cell treatment Epstein-Barr virus-transformed normal and Fabry lymphoblasts were thawed and dispersed into a 50-fold volume of RPMI-1640 medium supplemented with 10% bovine fetal serum, penicillin (100 U/mL), and streptomycin (100 U/mL). The cells were collected by centrifugation at 600 ϫ g for 10 minutes, resuspended in the same medium, and grown in a 25 cm2 bent-necked flask (Falcon, Meylan, France) containing 10 mL of the medium. For a typical study, one-million Fabry lymphoblasts were plated in a 75 cm2 of bent-necked flask containing 30 mL of the medium and were incubated for 24 hours before treatment with glucosylceramide synthase inhibitors. The cells were then treated with or without d-threo- 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), d-threo-4Ј-hydroxy-P4 (p-OH-P4), d-threo-3Ј,4Ј-ethylene- dioxyphenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4), or N-butyldeoxynojirimycin (NBDN) for a defined time period. Each P4 compound was added into the culture medium as a bovine serum albumin (BSA) complex, as described previously [5]. In both control and NBDN treatments, identical amounts of BSA as either BSA inhibitor or BSA alone were added to the culture medium. Following inhibitor treatment, the cells were Fig. 1. Pathway for globotriaosylceramide metabolism. The synthesis of glucosylceramide is blocked by P4, the P4 homologues, and NBDN. collected by centrifugation at 600 ϫ g for 10 minutes and ␣-Galactosidase A is deficient in Fabry disease resulting in globotriao- washed with 30 mL of cold phosphate-buffered saline sylceramide accumulation. (PBS). The cells were collected by centrifugation, resuspended in 5 mL of cold PBS, and transferred into a glass tube. The cell suspension was centrifuged for 10 minutes (EtDO-P4) [7]. These inhibitors are approximately 2000 at 600 ϫ g. The resultant cell pellet was fixed with 3 mL times more potent than PDMP when similarly assayed. of methanol and 3 mL of chloroform. The cells were In this study, we used these novel glucosylceramide syn- then disrupted in a bath sonicator. After dispersion, the thase inhibitors to test whether globotriaosylceramide mixture stood for 30 minutes at room temperature and levels could be depleted in a transformed lymphoblast was then centrifuged for 30 minutes at 2000 ϫ g. The cell line derived from a patient with Fabry disease. supernatant was transferred into a second glass tube. The remaining residue was mixed with 1 mL of methanol and 1 mL of chloroform and was briefly sonicated. After METHODS a repeat centrifugation, the supernatant was combined Reagents with the first supernatant. The residue was air dried Immortalized B cells, generated and maintained fol- and used for determination of the protein content. The lowing Epstein-Barr virus transformation, were used as pooled supernatants were mixed with 3.6 mL of 0.9% 448 Abe et al: Glycosphingolipid depletion in Fabry disease

(wt/vol) of NaCl and centrifuged for ﬁve minutes at 800 ϫ g. The chloroform layer was washed with 2 mL of methanol plus 1.6 mL of 0.9% (wt/vol) NaCl and centrifuged for ﬁve minutes at 800 ϫ g. The resultant lower layer was transferred into a glass tube and dried under a stream of nitrogen. The dried lipids were kept at Ϫ20ЊC before analysis.

Lipid analysis The dried lipids were dissolved in chloroform/methanol (95:5), and a fraction was used for the determination of total phospholipid [10]. The remaining lipids were dissolved in 2 mL of chloroform and 1 mL 0.21 mol/L NaOH in methanol and incubated for one hour at room temperature. The reaction was terminated by the addition of 0.8 mL of 0.25 N HCl. After centrifugation for ﬁve minutes at 800 ϫ g, the lower layer was transferred into a glass tube and mixed with 4 mL of methanol plus

1.6 mL of 0.05 N HCl containing 25 mmol/L HgCl2. The reaction mixture was incubated for 15 minutes at 37ЊC and then mixed with 2 mL of chloroform plus 1.6 mL water. After centrifugation for five minutes at 800 ϫ g, the lower layer was washed with 2 mL of methanol plus 1.6 mL of 30 mmol/L ethylenediaminetetraacetic acid (EDTA; pH 8.0). The lower layer was washed twice with 2 mL of methanol plus 1.6 mL of water. The resultant lower layer was transferred into another glass tube and dried down under a stream of nitrogen. The dried hy- drolyzed lipids were kept at Ϫ20ЊC before analysis. The dried lipids were dissolved in chloroform/methanol (95:5) and applied to a high-performance thin layer chromatography plate (Merck, Darmstadt, Germany) to determine the level of each neutral glycosphingolipid. Fifty to 70 nmol of total phospholipid were applied to the plate. The plate was first developed in a solvent system consisting of chloroform/methanol (98:2) and was air dried. The plate was then developed in one of two solvent systems consisting of either chloroform/methanol/acetic acid/water (61:33:3:3) or chloroform/metha- Fig. 2. ␣-Galactosidase and ␤-hexaminidase activities in Epstein-Barr nol/water (60:35:8). These systems separated ceramide virus-transformed normal and Fabry lymphoblasts. Both enzyme activi- dihexoside (CDH), globotriaosylceramide [ceramide tri- ties were measured as described in the Methods section. (A and B) hexoside (CTH)], and globoside (Gb ) and ceramide These show ␣-galactosidase and ␤-hexosaminidase activities, respec- 4 tively. Symbols are: (᭡) buffer; (᭹) normal; (᭿) Fabry. monohexoside (CMH) and CDH, respectively. Follow- ing the second development, the plates were dried and sprayed with 8% (wt/vol) CuSO pentahydrate in water/ 4 sion of the band corresponding to CTH to a new band, methanol/concentrate H PO (60:32:8) and charred for 3 4 which had the same Rf as that of CDH. 15 minutes at 150ЊC. Densitometry was measured with a Kodak digital camera connected to a computer and Protein assay scanned by NIH 1.49 image program. Known amounts of The residue after lipid extraction was dissolved in 5 glucosylceramide or Gb4 were used to obtain a standard to 10 mL of 0.1 N NaOH by using a heating water bath. curve in each thin layer chromatography analysis. The The protein concentration was determined by use of the correspondence of individual bands to those of neutral bicinchoninic acid reagent (Pierce, Rockford, IL, USA). glycosphingolipids was confirmed by orcinol staining. In addition, the treatment of the lipid extract with ␣-gal- ␣-Galactosidase assay actosidase from green coffee beans (Sigma Chemical Lymphoblasts were collected by centrifugation for 10 Co., St. Louis, MO, USA) resulted in a complete conver- minutes at 600 ϫ g and washed twice with chilled PBS. Abe et al: Glycosphingolipid depletion in Fabry disease 449

Table 1. Effect of P4, P4 homologues, and NBDN on glycosphingolipid levels in Epstein-Barr virus-transformed Fabry lymphoblasts

CMH CDH CTH Gb4 Inhibitor ng/nmol phospholipid None 24 hours 2.77, 2.74 5.21, 5.68 1.44, 1.76 n.d. 48 hours 3.79, 2.83 5.33, 4.26 3.81, 3.75 2.08, 1.81 72 hours 3.49, 3.41 5.40, 5.51 3.80, 4.37 1.65, 2.17 D-t-p-OH-P4 10 nmol/L 24 hours n.d. 4.37, 3.94 1.32, 1.27 n.d. 48 hours n.d. 1.94, 2.40 2.18, 2.18 1.31, 2.47 72 hours n.d. 1.19, 1.35 1.42, 1.32 1.09, 1.01 D-t-p-OH-P4 100 nmol/L 24 hours n.d. 3.72, 4.67 1.04, 1.34 n.d. 48 hours n.d. 1.68, 2.10 1.45, 1.30 1.05, 0.94 72 hours n.d. 1.18, 1.39 0.45, 1.01 0.67, 0.58 D-t-p-EtDO-P4 10 nmol/L 24 hours n.d. 3.86, 4.06 1.07, 1.34 n.d. 48 hours n.d. 1.71, 1.31 1.28, 1.23 1.05, 0.83 72 hours n.d. 0.38, 0.35 0.53, 0.41 0.48, 0.40 D-t-p-EtDO-P4 100 nmol/L 24 hours n.d. 3.99, 3.82 1.15, 1.13 n.d. 48 hours n.d. 1.13, 1.42 0.99, 1.06 0.82, 0.83 72 hours n.d. 0.38, 0.19 0.55, 0.51 0.46, 0.61 NBDN 10 nmol/L 24 hours 2.47, 2.59 3.93, 3.85 1.58, 1.45 n.d. 48 hours 2.89, 2.73 4.64, 3.34 3.19, 2.68 1.87, 1.43 72 hours 2.90, 3.19 4.12, 4.56 3.42, 3.62 1.56, 1.44 NBDN 10 ␮mol/L 24 hours 1.30, 1.47 2.91, 2.88 1.55, 1.38 n.d. 48 hours 2.24, 1.73 2.50, 2.88 2.48, 2.15 1.52, 1.44 72 hours 1.83, 2.69 2.34, 3.84 2.43, 3.45 1.44, 1.60 The lipids were extracted, subjected to alkaline methanolysis and acid hydrolysis, and dried under nitrogen as previously employed. The dried lipids were dissolved in chloroform:methanol (95:5) and applied to a high performance thin layer chromatography plate in an amount equivalent to 50 to 70 nmol of phospholipid phosphate. The plate was ﬁrst developed in a solvent system consisting of chloroform:methanol (98:2) and air dried. For analysis of CDH, CTH and Gb4 plates were developed in a second solvent system consisting of chloroform:methanol:acetic acid:water (61:33:3:3). CMH was analyzed by development in cloroform:methanol:water (60:35:8). Plates were sprayed with cupric sulfate reagent, charred, and the lipids quantitated by densitometry. n.d. denotes not detectable.

The cellular pellet was suspended in 3 mg/mL sodium of 250 ␮L of 0.2 mol/L Na2CO3. The product p-nitrophe- taurocholate and 28 mmol/L citric acid/44 mmol/L nol was measured as described previously in this article.

Na2HPO4 (pH 4.4) and sonicated for 10 seconds three times with a probe-type sonicator in a ice water bath. Labeling Epstein-Barr virus transformed Fabry The cell homogenate was centrifuged for 30 minutes at lymphoblasts with FITC-conjugated verotoxin 20,000 ϫ g at 4ЊC. The resultant supernatant was used Fabry lymphoblasts were treated with or without 50 as an enzyme source for ␣-galactosidase [11] and nmol/L p-OH-P4 for three days as described earlier in ␤-hexosaminidase assays [11]. this article. After inhibitor treatment, the cells were col- ␣-Galactosidase was assayed in a total volume of 600 lected by centrifugation at 600 ϫ g for 10 minutes, sus- ␮L. The reaction mixture consisted of 5 mmol/L p-nitro- pended in 10 mL of chilled PBS, and mixed with 25 mL phenyl-␣-galactopyranoside, 28 mmol/L citric acid/44 of chilled PBS. The cells were collected by centrifugation mmol/L Na2HPO4 (pH 4.4), 5 mg/mL BSA, and 500 at 600 ϫ g for 10 minutes, resuspended in 3 mL of chilled ng/mL of protein of the supernatant. The reaction was PBS, and counted with a hemacytometer. One million initiated by the addition of the cell supernatant, kept at cells in 150 ␮L of PBS were incubated with 50 ␮L of PBS 37ЊC for 10, 30, and 90 minutes, and terminated by adding or 50 ␮L of PBS containing 200 ␮g/mL FITC-conjugated

600 ␮L of 0.2 mol/L Na2CO3. The product, p-nitrophenol, verotoxin B (FITC-VTB) for 40 minutes at room temper- was measured by the absorbence at 400 nm (⑀400: 18,300 ature. The reaction tube was wrapped with aluminum at pH 9 to 10). ␤-Hexosaminidase was assayed in a total foil and inverted five times gently every five minutes. volume of 250 ␮L. The reaction mixture consisted of 1 The cells were washed three times with 3 mL of PBS mmol/L p-nitro-phenyl-␤-glucosamine, 30 mmol/L so- and centrifuged at 4000 r.p.m. for five minutes. The pellet dium citrate (pH 4.5), 0.1% (wt/vol) Triton 100-X, and was resuspended in 0.5 mL of PBS for subsequent cyto- 24 ng/mL of supernatant protein. The reaction was initi- metric analysis on a Coulter Elite ESP. Unlabeled cells ated by the addition of the supernatant, kept at 37ЊC for were used for initial light scatter analysis, and the per- 5, 10, 20, and 30 minutes, and terminated by the addition centage of cells with specific staining by FITC-VTB was 450 Abe et al: Glycosphingolipid depletion in Fabry disease

Fig. 3. Effects of P4 derivatives and NBDN on neutral glycosphingolipid level in Epstein- Barr virus-transformed Fabry lymphoblasts. (A) This shows a representative thin layer chro- matogram of lymphoblast following inhibitor treatment for two days. The plate was developed in a two-step solvent system consisting of chloroform/methanol (98:2) as the ﬁrst development and chloroform/methanol/acetic acid/ water (61:33:3:3) as the second development. The lipids were visualized by charring, as described in the Methods section. Abbreviations are: CMH, ceramide monohexoside; CDH, ceramide dihexoside; CTH, ceramide trihexo-

side; Gb4, globoside; and SM, sphingomyelin.

defined by positioning cursors on the basis of the un- are much greater than that of P4 [7]. Recently NBDN, stained control cells. The data were collected on a 4 a nonspecific inhibitor of glucosylceramide synthase, has decade log 10 scale of increasing fluorescence intensity. been reported to have limited efficacy in decreasing gly- Ten thousand cells were analyzed for each sample. cosphingolipid levels in tissues of knockout mice with Tay-Sachs and Sandhoff diseases phenotypes [12, 13]. RESULTS We therefore compared the effects of these inhibitors on protein, phospholipid, and neutral glycosphingolipid Epstein-Barr virus transformed normal and Fabry lymphoblasts contents of Fabry lymphocytes. There was no significant difference in protein and Lysosomal enzyme activities were measured in control phospholipid content between untreated and treated ␣ and Fabry-transformed lymphoblasts. The total -gal- cells with any of the inhibitors at each time point studied. actosidase activity in Epstein-Barr virally transformed Under all conditions, lymphocytic aggregations were Fabry lymphoblasts was significantly lower than that of preserved and were microscopically similar, indicating transformed normal lymphoblasts (Fig. 2A). By contrast, that these inhibitors do not inhibit cell growth under the ␤-hexosaminidase activity, a typical lysosomal enzyme, conditions employed. was detected and comparable in both cell lines (Fig. 2B). Because the assay employed for lysosomal ␣-galacto- Ceramide dihexoside, globotriaosylceramide (CTH), sidase measures both A and B isoforms, most of the lyso- and Gb4 contents in Fabry lymphocytes were significantly somal ␣-galactosidase activity in the normal lymphoblasts reduced by treatment of the cells with p-OH-P4 and is of the A isoform. Therefore, the lysosomal ␣-galac- EtDO-P4 over three days (Table 1 and Fig. 3A). Most tosidase deficiency was specific to the Fabry lymphoblast. of glucosylceramide in the Fabry cells disappeared within These cells were thus used for the subsequent studies. 24 hours of treatment with both P4 homologues. CTH levels, reflective of globotriaosylceramide, decreased by Effects of P4, P4 derivatives, and approximately 70 and 80% of control values, with 10 N-butyldeoxynojirimycin on Epstein-Barr nmol/L and 100 nmol/L p-OH-P4, respectively, by the virus-transformed Fabry lymphoblasts third day of treatment. A 90% decrease in CTH levels A previous study showed that inhibitory effects of was observed following treatment with 10 or 100 nmol/L p-OH-P4 and EtDO-P4 on glucosylceramide synthase EtDO-P4. These results indicate EtDO-P4 is slightly Abe et al: Glycosphingolipid depletion in Fabry disease 451

Fig. 3. Continued. (B) This denotes the time- dependent changes in ceramide dihexoside (᭺), ceramide trihexoside (globotriaosylceramide; ᭹), and globoside (᭝) as a percentage of control glycosphingolipids. Glucosylceramide levels (CMH, ᮀ) are only shown for NBDN in which both 10 nmol/L and 10 ␮mol/L incubations only partially depleted the cerebroside.

more potent than p-OH-P4 as an inhibitor of neutral In addition, a reduction in CDH or CTH was observed. glycosphingolipid synthesis (Fig. 3B). However, the depletion of the cerebroside and more On the other hand, NBDN was significantly less potent highly glycosylated lipids was obviously weaker than in the reduction of neutral glycosphingolipids when com- that observed in the presence of 50 nmol/L p-OH-P4 pared with P4 or the P4 homologues. Concentrations (Table 2). as high as 10 ␮mol/L NBDN were less potent than 10 To confirm that globotriaosylceramide was signifi- nmol/L of the P4 homologues and, surprisingly, did not cantly depleted, cells were labeled with FITC-conjugated completely deplete the lymphoblasts of their glucosylcer- verotoxin and analyzed by flow cytometry. Two popula- amide. A second set of determinations was therefore tions of cells were identified, those with high affinity performed with higher NBDN concentrations. When the binding and those with binding, which was only slightly Fabry lymphoblasts were treated with 100 ␮mol/L greater than the autofluorescence observed in the ab- NBDN, the glucosylceramide content was undetectable. sence of FITC-verotoxin. Treatment for 48 hours with 452 Abe et al: Glycosphingolipid depletion in Fabry disease

Table 2. Effects of high dose NBDN and D-threo-p-OH-P4 on neutral glycosphingolipid levels in Fabry lymphoblasts CMH CDH CTH Inhibitor ng/nmol phospholipid None 3.48, 4.07, 3.72 8.31, 8.20, 6.72 7.62, 7.66, 6.53 NBDN 10 ␮mol 1.96, 2.31 5.00, 4.65 5.09, 5.38 100 ␮mol n.d., n.d. 2.95, 3.26 4.01, 2.83 D-threo-p-PH-P4 50 nmol n.d. 1.77 2.13 Fabry lymphoblasts were treated for 72 hours with each inhibitor. ND denotes not detectable.

50 nmol/L p-OH-P4 totally eliminated the high-affinity binding of the fluorescent marker to the lymphoblasts (Fig. 4). However, the low-affinity binding was not af- fected.

DISCUSSION Although other inherited sphingolipidoses such as Gaucher disease have been the focus of intense programs in therapeutics, Fabry disease remains comparatively un- derstudied. As a result, only limited reports have been published on potential enzyme replacement therapy or gene therapy [8, 14]. An alternative strategy for treating sphingolipid storage diseases is the pharmacological inhibition of key enzymes in the synthesis of the Fabry glycosphingolipids. Recent reports have provided some support for approaching glycosphingolipid storage disorders Fig. 4. Effect of D-threo-p-OH-P4 on globotriaosylceramide expres- with substrate depletion. sion by Fabry lymphocytes. Flow cytometric analysis of human Fabry N-butyldeoxynojirimycin, originally developed as a lymphocytes treated with 50 nmol/L D-threo-p-OH-P4 (p-OH-P4) for potential antiviral agent for HIV infections based on its three days. (A) Unstained control lymphocytes. Specific staining with FITC-VTB was assigned as a fluorescence intensity greater than 95% ability to inhibit ␣-glucosidase, was also identified as a of the unstained control cells. (B) Untreated lymphocytes incubated weak inhibitor of glucosylceramide synthase [15]. The with FITC-VTB for 40 minutes. Approximately 80% of cells were IC for the glucosylceramide synthase inhibition occurs stained with VTB, which is used as a marker of globotriaosylceramide 50 expression. Of the total stained cells, 47% are in the second peak with in the mid-micromolar range. Nevertheless, when fed to a log greater fluorescence intensity than the control unstained cells. knockout mice lacking ␤-hexosaminidase A (Tay-Sachs (C) Lymphocytes treated for three days with p-OH-P4 and incubated with FITC-VTB. The fluorescence of the second, high-intensity peak phenotype) or ␤-hexosaminidase B (Sandhoff pheno- of cells is completely eliminated by p-OH-P4, demonstrating inhibition type), NBDN partially corrected the phenotype in each of globotriaosylceramide expression. of these models and prevented ganglioside GM2 accumulation [12, 13]. The use of “experimental epistasis” pro- vides additional proof of concept for the substrate-depletion strategy. When ␤-hexosaminidase B-deficient mice duces cell growth arrest and, at sufficiently high concen- were crossed with those lacking GalNAc transferase, trations, apoptosis. The growth inhibitory effects have GM2 accumulation was prevented and a normal pheno- been demonstrated to be the result of ceramide accumu- type ensued [16]. lation [17]. Although originally thought to be the result The low inhibitory activity against the glucosylceramide of substrate accumulation, the PDMP-induced increase synthase and its potential toxicities limit the potential use in cell ceramide results from the inhibition of a second of NBDN in substrate-depletion therapy. PDMP, the par- metabolic pathway. This pathway is the acylation of cera- ent compound of the inhibitors used in these studies, is mide at the 1-hydroxyl of ceramide and is catalyzed by comparably active when compared with NBDN. Al- an acidic phospholipase A2 with transacylase activity though it lacks activity against ␣-glucosidase, PDMP in- [18, 19]. Abe et al: Glycosphingolipid depletion in Fabry disease 453

Although the inhibitory effects of PDMP on the gluco- In summary, we have begun to evaluate the potential sylceramide synthase and transacylase are comparable of applying a substrate depletion strategy to the treat- with regard to their IC50s, P4 is significantly more active ment of Fabry disease. By using transformed lympho- against the glucosylceramide synthase. Thus, it has been blasts from a patient with Fabry disease, it can be shown possible to design and screen new inhibitors that can that short-term incubations with potent inhibitors of glu- block glucosylceramide formation in the mid- to low- cosylceramide synthase result in a significant depletion nanomolar range, but that are relatively inactive against of globotriaosylceramide. More convincing proof of con- the transacylase and therefore do not inhibit cell growth. cept will require the use of suitable in vivo models. The p-OH-P4 and EtDO-P4 homologues are two such inhibitors. The IC s of these compounds are 90 and 100 Reprint requests to James A. Shayman, M.D., Nephrology Division, 50 Department of Internal Medicine, University of Michigan, Box 0676, nmol/L, respectively, when glucosylceramide synthase is Room 1560 MSRBII, 1150 West Medical Center Drive, Ann Arbor, assayed in a cellular homogenate. However, when stud- Michigan 48109-0676, USA. ied in intact Madin-Darby canine kidney cells, these com- E-mail: [email protected] pounds decrease glucosylceramide to 80% of control levels at concentrations as low as 11 nmol/L. Only at REFERENCES concentrations of 1 ␮mol/L or greater were changes in 1. Brady RO, Gal AE, Bradley RM, Martensson E, Warshaw cell ceramide levels observed. Thus, 10 and 100 nmol/L AL, Laster L: Enzymatic defect in Fabry’s disease: Ceramide were the concentrations chosen for this study. trihexosidase deficiency. N Engl J Med 276:1163–1167, 1967 The absence of growth inhibition of the Fabry lympho- 2. Desnick RJ, Ioannou YA, Eng CM, Scriver CR (eds): ␣-Galac- tosidase A Deficiency: Fabry Disease in the Metabolic and Molecular blasts when exposed to P4 or the P4 homologues is con- Bases of Inherited Disease, edited by Desnick RJ, Ioannou YA, sistent with this pharmacological profile. In this study, Eng CM, Serwar CR, New York, McGraw-Hill, 1995, pp 2741– none of the inhibitors studied, including NBDN, signifi- 2784 3. Nyholm PG, Magnusson G, Zheng Z, Norel R, Bingington- cantly impaired cell growth. Although NBDN did cause Boyd B, Lingwood CA: Two distinct binding sites for globotriaosyl moderate depletion of glucosylceramide, only the P4 ceramide on verotoxins: Identification by molecular modeling and compounds significantly reduced the more highly glyco- confirmation using deoxy analogues and a new glycolipid receptor for all verotoxins. Chem Biol 3:263–275, 1996 sylated lipids. However, at 100 ␮mol/L concentrations, 4. 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Rohrer J, Schweitzer A, Johnson KF, Kornfeld S: A determi- verotoxin receptor. These data also provide independent nant in the cytoplasmic tail of the cation-dependent mannose support for the depletion of globotriaosylceramide by 6-phosphate receptor prevents trafficking to lysosomes. J Cell Biol 130:1297–1306, 1995 P4 homologues because verotoxin is highly specific for 12. Platt FM, Neises GR, Reinkensmeier G, Townsend MJ, Perry this glycosphingolipid receptor. VH, Proia RL, Winchester B, Dwek RA, Butters TD: Prevention EtDO-P4 was superior to the other compounds evalu- of lysosomal storage in Tay-Sachs mice treated with N-butyldeoxynojirimycin. Science 276:428–431, 1997 ated. In our original description of the phenyl-substituted 13. Jeyakumar M, Butters TD, Cortina-Borja M, Hunnam V, Proia homologues, p-OH-P4 had superior inhibitory activity RL, Perry VH, Dwek RA, Platt FM: Delayed symptom onset against glucosylceramide synthase when compared with and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin. Proc Natl Acad Sci USA 96:6388–6393, the ethylenedioxy compound [7]. That comparison, how- 1999 ever, was conducted in crude cell homogenates. This 14. Desnick RJ, Dean KJ, Grabowski G, Bishop DF, Sweeley CC: study compares activity in intact cells. Therefore, it is Enzyme therapy in Fabry disease: Differential in vivo plasma clear- ance and metabolic effectiveness of plasma and splenic alpha- likely that EtDO-P4 partitions across the cell membranes galactosidase A isozymes. Proc Natl Acad Sci USA 76:5326–5330, more favorably than does p-OH-P4. 1996 454 Abe et al: Glycosphingolipid depletion in Fabry disease

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