Ex Vivo and in Vivo Effects of Isofagomine on Acid β-Glucosidase Variants and Substrate Levels in Gaucher Disease

Ying Sun,Benjamin Liou,You-Hai Xu,Brian Quinn,Wujuan Zhang,Rick Hamler,Kenneth D.R Setchell,Gregory A Grabowski

doi:10.1074/jbc.m111.280016

Abstract

Isofagomine (IFG) is an acid β-glucosidase (GCase) active site inhibitor that acts as a pharmacological chaperone. The effect of IFG on GCase function was investigated in GCase mutant fibroblasts and mouse models. IFG inhibits GCase with K(i) ∼30 nM for wild-type and mutant enzymes (N370S and V394L). Fibroblasts treated with IFG at μM concentrations showed enhancement of WT and mutant GCase activities and protein levels. Administration of IFG (30 mg/kg/day) to the mice homozygous for GCase mutations (V394L, D409H, or D409V) led to increased GCase activity in visceral tissues and brain extracts. IFG effects on GCase stability and substrate levels were evaluated in a mouse model (hG/4L/PS-NA) that has doxycycline-controlled human WT GCase (hGCase) expression driven by a liver-specific promoter and is also homozygous for the IFG-responsive V394L GCase. Both human and mouse GCase activity and protein levels were increased in IFG-treated mice. The liver-secreted hGCase in serum was stabilized, and its effect on the lung and spleen involvement was enhanced by IFG treatment. In 8-week IFG-treated mice, the accumulated glucosylceramide and glucosylsphingosine were reduced by 75 and 33%, respectively. Decreases of storage cells were correlated with >50% reductions in substrate levels. These results indicate that IFG stabilizes GCase in tissues and serum and can reduce visceral substrates in vivo.

Highlights

Isofagomine (IFG) is an acid ␤-glucosidase (GCase) active site inhibitor that acts as a pharmacological chaperone
IFG effects on GCase stability and substrate levels were evaluated in a mouse model that has doxycycline-controlled human WT GCase expression driven by a liver-specific promoter and is homozygous for the IFG-responsive V394L GCase
IFG Inhibition of GCases—The inhibition properties of IFG were tested with purified recombinant human WT GCase (hGCase) and the recombinant human N370S and V394L mutant GCases in media from insect cells expressing these specific GCases [22]

Summary

EXPERIMENTAL PROCEDURES

Materials—The following were from commercial sources: 4-methylumbelliferyl ␤-D-glucopyranoside (Biosynth AG, Switzerland); sodium taurocholate (Calbiochem); mouse anti␤-actin monoclonal antibody (Sigma); NuPAGE 4 –12% BisTris gel, NuPAGE MES SDS running buffer, and DMEM (Invitrogen); rat anti-mouse CD68 monoclonal antibody (Serotec, Oxford, UK); M-PER Mammalian Protein Extraction Reagent and BCA Protein Assay Reagent (Pierce); HybondTM-ECLTM nitrocellulose membrane and ECL detection reagent (Amersham Biosciences); and ABC Vectastain and Alkaline Phosphatase kit II (Black) (Vector Laboratories, Burlingame, CA). The IC50 values for IFG with each GCase were determined by using various concentrations of IFG that were incubated with homogeneous hGCase (imiglucerase) or the purified human mutant GCase (N370S or V394L) from insect cell medium. 10 ␮l of each sample was removed to a new vial and diluted by 4 ϫ 104-fold with reaction buffer (0.25% sodium taurocholate, 0.25% Triton X-100, 25 mM citrate, 50 mM phosphate, pH 5.6) to achieve [IFG] Ͻ0.65 or 1.25 nM, and GCase activity was determined as described [22]. Tissue homogenate (50 ␮l) or serum (50 ␮l) was diluted with 100 ␮l of acetonitrile followed by mixing with 100 ␮l of 100 ng/ml [13C2,15N]IFG (internal standard) dissolved in acetonitrile/methanol and 0.50% formic acid (70:30, v/v). A low limit of quantitation was set at 8 ng/g of tissue and 1 ng/ml for serum

RESULTS

DISCUSSION

IFG treatment