Identification and Characterization of Ambroxol as an Enzyme Enhancement Agent for Gaucher Disease

Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243–249, 310–312, and 386–400 near the active site of GCase are stabilized. Consistent with its mixed-type inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes. Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243–249, 310–312, and 386–400 near the active site of GCase are stabilized. Consistent with its mixed-type inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes. Gaucher disease (GD), 3The abbreviations used are: GDGaucher diseaseABXambroxolEETenzyme enhancement therapyERendoplasmic reticulumERTenzyme enhancement therapyFDAFood and Drug AdministrationFZNfluphenazineGCglucosylceramideGCaseglucocerebrosidaseGD-1type 1 Gaucher diseaseGD-2type 2 Gaucher diseaseGD-3type 3 Gaucher diseaseGdnHClguanidine hydrochlorideHexβ-hexosaminidaseH/D-Exhydrogen/deuterium exchangeIFGisofagomineLSDlysosomal storage diseaseLamp-1 or -2lysosomal associated membrane protein-1 or -2MUbGlc4-methylumbelliferyl-β-d-glucopyranosideMUG4-methylumbelliferyl-β-N-acetylglucosaminidePDIprotein-disulfide isomerasePCpharmacological chaperoneSRTsubstrate reduction therapySUPREXStability of Unpurified Proteins from Rates of hydrogen/deuterium ExchangeTDCsodium taurodeoxycholic acidHPLChigh pressure liquid chromatographyIFGisofagomineFBSfetal bovine serumCBEconduritol B epoxideMAP2-amino-2-methyl-1-propanolrGCasehuman recombinant GCaseMALDImatrix-assisted laser desorption ionization.3The abbreviations used are: GDGaucher diseaseABXambroxolEETenzyme enhancement therapyERendoplasmic reticulumERTenzyme enhancement therapyFDAFood and Drug AdministrationFZNfluphenazineGCglucosylceramideGCaseglucocerebrosidaseGD-1type 1 Gaucher diseaseGD-2type 2 Gaucher diseaseGD-3type 3 Gaucher diseaseGdnHClguanidine hydrochlorideHexβ-hexosaminidaseH/D-Exhydrogen/deuterium exchangeIFGisofagomineLSDlysosomal storage diseaseLamp-1 or -2lysosomal associated membrane protein-1 or -2MUbGlc4-methylumbelliferyl-β-d-glucopyranosideMUG4-methylumbelliferyl-β-N-acetylglucosaminidePDIprotein-disulfide isomerasePCpharmacological chaperoneSRTsubstrate reduction therapySUPREXStability of Unpurified Proteins from Rates of hydrogen/deuterium ExchangeTDCsodium taurodeoxycholic acidHPLChigh pressure liquid chromatographyIFGisofagomineFBSfetal bovine serumCBEconduritol B epoxideMAP2-amino-2-methyl-1-propanolrGCasehuman recombinant GCaseMALDImatrix-assisted laser desorption ionization. caused by deficiency of a lysosomal enzyme glucocerebrosidase (GCase), an acidic β-glucosidase (EC 3.2.1.45), encompasses a continuum of clinical spectrum from a perinatal lethal disorder to an asymptomatic form. Three major clinical subtypes (1.Sidransky E. Ginns E.I. J. Med. Genet. 1997; 34: 876-877Crossref PubMed Google Scholar, 2.Sidransky E. Adv. Pediatr. 1997; 44: 73-107PubMed Google Scholar, 3.Zhao H. Grabowski G.A. Cell. Mol. Life Sci. 2002; 59: 694-707Crossref PubMed Scopus (83) Google Scholar) have been broadly defined, primarily based on the degree of neurological involvement. These are useful in determining prognosis and management (1.Sidransky E. Ginns E.I. J. Med. Genet. 1997; 34: 876-877Crossref PubMed Google Scholar, 2.Sidransky E. Adv. Pediatr. 1997; 44: 73-107PubMed Google 1 as the is by the of disease, and 2 and 3 and as are by the of neurological the and were by of and rate of disease are H. Grabowski G.A. Cell. Mol. Life Sci. 2002; 59: 694-707Crossref PubMed Scopus (83) Google patients with have an of from to with a neurological with have 2 of have a neurological and the or of GD are to have an increased of disease J. J. E. PubMed Scopus Google Scholar, E. J. Med. Genet. PubMed Scopus Google Scholar, PubMed Scopus Google from patients of GCase The of is to the of GCase associated with GD are H. Sci. PubMed Scopus Google have been in the to GD. are E. PubMed Scopus Google The most prevalent found are N370S in patients and in patients E. J. Genet. PubMed Scopus Google be found in patients E. E. J. Med. Genet. PubMed Scopus Google of are to the of GCase in the endoplasmic the protein to the The that have been to the of GCase are and F213I H. H. H. E. PubMed Scopus Google Scholar, Mol. PubMed Scopus Google Scholar, E. Sci. 2002; PubMed Scopus Google Scholar, J. PubMed Scopus Google GD with an N370S or F213I in a of or the of GCase, has been to levels of lysosomal GCase activity H. Sci. PubMed Scopus Google Scholar, H. H. H. E. PubMed Scopus Google Scholar, E. Sci. 2002; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google These as by the of the mutant GCase in the molecules to and the by the Cell. PubMed Scopus Google in increased in the is that the the of the be GCase to the J. PubMed Scopus Google the for lysosomal storage disease bind at the neutral pH of the and at the acidic pH of enzyme therapy for GD is to the treatment of of the of the enzyme to the G.A. PubMed Scopus Google approach is and be molecules are to the are to and be of the small identified as for GCase, are that have been in and are by the drug The screen of of Food and Drug has to be a and to small molecules that as agents for lysosomal in J. J. PubMed Scopus Google to library have an assay at of the enzyme J. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. E. J. E. Sci. PubMed Scopus Google the identification of as an agent for GCase a thermal denaturation assay to screen the of library of 1,040 that have been used to treat the biochemical of ABX treatment on GD and the with lysosomal GCase activity and protein Additionally, storage was as by liquid mass spectrometry of treatment of an with has been used to treat patients Genet. PubMed Scopus Google Scholar, PubMed Scopus Google as a of lysosomal enzyme activity is in most to storage of J. PubMed Scopus Google based on small molecules that the of the the of the mutant are attractive and with the to treat the neurological forms of of have in clinical and J. PubMed Scopus Google Scholar, J. Grabowski G.A. J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google has been to a with in and a of disease Mol. PubMed Scopus Google Scholar) and to the of GCase in PubMed Scopus Google by the storage of mutant enzyme in the the levels of the protein to has been associated with the of Mol. Genet. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, H. Mol. Genet. PubMed Scopus Google of a drug library has been to small molecules that as agents for different J. J. PubMed Scopus Google as as agents for PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, H. J. PubMed Scopus Google assay based on the of to GCase identified as the major was in a screen to as a of GCase thermal in the assay to mutant GCase The of to stabilize GCase in was confirmed and modeling The in of to as a have been the of or the of the drug by These be the for the to of to the levels of enhancement of mutant GCase activity in as ABX, an inhibitor of GCase in H. Sci. PubMed Scopus Google These that the of a to as a be on the of its in binding inhibitory be to be effective in mutant enzyme activity in to thermal denaturation as the assay for the library to that with the that to the active site of GCase. ABX was identified in screen in of inhibitory was found to be a type inhibitor of GCase 2 and A to and Google the of ABX were with maximal inhibition at the neutral is an for an at the pH of the pH near that of ABX the enzyme and an of its activity at the maximal in type of pH dependence for a GCase inhibitor was for an was found to as a in GD as with ABX, to the levels of inhibition at neutral pH Mol. Genet. PubMed Scopus Google The pH dependence of ABX binding to GCase was confirmed in of its binding was to a for ABX at pH the a its to bind GCase a pH in inhibition of the enzyme the lysosomal and major of ABX as an agent is that for ABX has been used to treat for airway mucus and has been used as an treatment for hyaline membrane disease in newborns, increases in the E. E. J. PubMed Scopus Google Scholar, H. J. 2002; PubMed Scopus Google Scholar, H. H. J. has been to have and H. J. 2002; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Drug PubMed Scopus Google both of be useful in of the of H. Mol. Genet. PubMed Scopus Google the of of ABX has to be Drug PubMed Scopus Google the and studies of ABX in J. J. J. Google Scholar, PubMed Scopus Google ABX in clinical to as an agent for GD be ABX that the GCase in and and are the from clinical studies J. 44: PubMed Scopus Google Scholar, Drug PubMed Scopus Google ABX enhancement of GCase activity in and a the drug in with GD is on of ABX in a in with was to in patients and the of ABX in the is E. J. Scopus Google studies in have that ABX is in and H. H. J. a ABX was found at of and of in the ABX is most to be in in be an in the treatment of the forms of is the of ABX as a drug PubMed Scopus Google the or of in levels were significantly in lymphoblasts were is that the treatment and the that the GCase activity levels found in the GD Additionally, a in levels is the most used for the of for of levels was 2 of or Grabowski G.A. Pediatr. PubMed Scopus Google Scholar, PubMed Scopus Google that or of in the in lymphoblasts that treatment increased levels the that be in cells, enhancement of GCase in the and inhibition of the enzyme in the treatment of with a to be ABX to be an inhibitor of GCase pH and its enhancement of GCase in the be by its in the of the in the of segments from the wild type GCase that upon binding of that of the and PubMed Scopus Google as as of small identified by of a library PubMed Scopus Google stabilize segments and of the of was the to stabilize and the from the studies that both ABX and with GCase by as is the with G.A. PubMed Scopus Google and These with non-active site the mixed-type inhibition for ABX and has a thermal denaturation assay as an for small for ABX, a drug with a of in was identified as a of GCase and as a pH-dependent, inhibitor of the small was to as an agent for with common GCase associated with and The treatment of in increased levels of GCase activity and protein in and the reduction of These that ABX be developed as an effective agent for as an or an to or Gaucher disease (GD), 3The abbreviations used are: GDGaucher diseaseABXambroxolEETenzyme enhancement therapyERendoplasmic reticulumERTenzyme enhancement therapyFDAFood and Drug AdministrationFZNfluphenazineGCglucosylceramideGCaseglucocerebrosidaseGD-1type 1 Gaucher diseaseGD-2type 2 Gaucher diseaseGD-3type 3 Gaucher diseaseGdnHClguanidine hydrochlorideHexβ-hexosaminidaseH/D-Exhydrogen/deuterium exchangeIFGisofagomineLSDlysosomal storage diseaseLamp-1 or -2lysosomal associated membrane protein-1 or -2MUbGlc4-methylumbelliferyl-β-d-glucopyranosideMUG4-methylumbelliferyl-β-N-acetylglucosaminidePDIprotein-disulfide isomerasePCpharmacological chaperoneSRTsubstrate reduction therapySUPREXStability of Unpurified Proteins from Rates of hydrogen/deuterium ExchangeTDCsodium taurodeoxycholic acidHPLChigh pressure liquid chromatographyIFGisofagomineFBSfetal bovine serumCBEconduritol B epoxideMAP2-amino-2-methyl-1-propanolrGCasehuman recombinant GCaseMALDImatrix-assisted laser desorption ionization.3The abbreviations used are: GDGaucher diseaseABXambroxolEETenzyme enhancement therapyERendoplasmic reticulumERTenzyme enhancement therapyFDAFood and Drug AdministrationFZNfluphenazineGCglucosylceramideGCaseglucocerebrosidaseGD-1type 1 Gaucher diseaseGD-2type 2 Gaucher diseaseGD-3type 3 Gaucher diseaseGdnHClguanidine hydrochlorideHexβ-hexosaminidaseH/D-Exhydrogen/deuterium exchangeIFGisofagomineLSDlysosomal storage diseaseLamp-1 or -2lysosomal associated membrane protein-1 or -2MUbGlc4-methylumbelliferyl-β-d-glucopyranosideMUG4-methylumbelliferyl-β-N-acetylglucosaminidePDIprotein-disulfide isomerasePCpharmacological chaperoneSRTsubstrate reduction therapySUPREXStability of Unpurified Proteins from Rates of hydrogen/deuterium ExchangeTDCsodium taurodeoxycholic acidHPLChigh pressure liquid chromatographyIFGisofagomineFBSfetal bovine serumCBEconduritol B epoxideMAP2-amino-2-methyl-1-propanolrGCasehuman recombinant GCaseMALDImatrix-assisted laser desorption ionization. caused by deficiency of a lysosomal enzyme glucocerebrosidase (GCase), an acidic β-glucosidase (EC 3.2.1.45), encompasses a continuum of clinical spectrum from a perinatal lethal disorder to an asymptomatic form. Three major clinical subtypes (1.Sidransky E. Ginns E.I. J. Med. Genet. 1997; 34: 876-877Crossref PubMed Google Scholar, 2.Sidransky E. Adv. Pediatr. 1997; 44: 73-107PubMed Google Scholar, 3.Zhao H. Grabowski G.A. Cell. Mol. Life Sci. 2002; 59: 694-707Crossref PubMed Scopus (83) Google Scholar) have been broadly defined, primarily based on the degree of neurological involvement. These are useful in determining prognosis and management (1.Sidransky E. Ginns E.I. J. Med. Genet. 1997; 34: 876-877Crossref PubMed Google Scholar, 2.Sidransky E. Adv. Pediatr. 1997; 44: 73-107PubMed Google 1 as the is by the of disease, and 2 and 3 and as are by the of neurological the and were by of and rate of disease are H. Grabowski G.A. Cell. Mol. Life Sci. 2002; 59: 694-707Crossref PubMed Scopus (83) Google patients with have an of from to with a neurological with have 2 of have a neurological and the or of GD are to have an increased of disease J. J. E. PubMed Scopus Google Scholar, E. J. Med. Genet. PubMed Scopus Google Scholar, PubMed Scopus Google Gaucher disease enzyme enhancement therapy endoplasmic enzyme enhancement therapy Food and Drug glucosylceramide glucocerebrosidase type 1 Gaucher disease type 2 Gaucher disease type 3 Gaucher disease guanidine hydrochloride hydrogen/deuterium exchange lysosomal storage disease lysosomal associated membrane protein-1 or reduction therapy of Unpurified Proteins from Rates of hydrogen/deuterium taurodeoxycholic acid pressure liquid bovine B recombinant GCase laser desorption ionization. Gaucher disease enzyme enhancement therapy endoplasmic enzyme enhancement therapy Food and Drug glucosylceramide glucocerebrosidase type 1 Gaucher disease type 2 Gaucher disease type 3 Gaucher disease guanidine hydrochloride hydrogen/deuterium exchange lysosomal storage disease lysosomal associated membrane protein-1 or reduction therapy of Unpurified Proteins from Rates of hydrogen/deuterium taurodeoxycholic acid pressure liquid bovine B recombinant GCase laser desorption ionization. from patients of GCase The of is to the of GCase associated with GD are H. Sci. PubMed Scopus Google have been in the to GD. are E. PubMed Scopus Google The most prevalent found are N370S in patients and in patients E. J. Genet. PubMed Scopus Google be found in patients E. E. J. Med. Genet. PubMed Scopus Google of are to the of GCase in the endoplasmic the protein to the The that have been to the of GCase are and F213I H. H. H. E. PubMed Scopus Google Scholar, Mol. PubMed Scopus Google Scholar, E. Sci. 2002; PubMed Scopus Google Scholar, J. PubMed Scopus Google GD with an N370S or F213I in a of or the of GCase, has been to levels of lysosomal GCase activity H. Sci. PubMed Scopus Google Scholar, H. H. H. E. PubMed Scopus Google Scholar, E. Sci. 2002; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google These as by the of the mutant GCase in the molecules to and the by the Cell. PubMed Scopus Google in increased in the is that the the of the be GCase to the J. PubMed Scopus Google the for lysosomal storage disease bind at the neutral pH of the and at the acidic pH of lysosomes. The enzyme therapy for GD is to the treatment of of the of the enzyme to the G.A. PubMed Scopus Google approach is and be molecules are to the are to and be of the small identified as for GCase, are that have been in and are by the drug The screen of of Food and Drug has to be a and to small molecules that as agents for lysosomal in J. J. PubMed Scopus Google to library have an assay at of the enzyme J. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. E. J. E. Sci. PubMed Scopus Google the identification of as an agent for GCase a thermal denaturation assay to screen the of library of 1,040 that have been used to treat the biochemical of ABX treatment on GD and the with lysosomal GCase activity and protein Additionally, storage was as by liquid mass spectrometry of treatment of an with has been used to treat patients Genet. PubMed Scopus Google Scholar, PubMed Scopus Google as a of lysosomal enzyme activity is in most to storage of J. PubMed Scopus Google based on small molecules that the of the the of the mutant are attractive and with the to treat the neurological forms of of have in clinical and J. PubMed Scopus Google Scholar, J. Grabowski G.A. J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google has been to a with in and a of disease Mol. PubMed Scopus Google Scholar) and to the of GCase in PubMed Scopus Google by the storage of mutant enzyme in the the levels of the protein to has been associated with the of Mol. Genet. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, H. Mol. Genet. PubMed Scopus Google of a drug library has been to small molecules that as agents for different J. J. PubMed Scopus Google as as agents for PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, H. J. PubMed Scopus Google assay based on the of to GCase identified as the major was in a screen to as a of GCase thermal in the assay to mutant GCase The of to stabilize GCase in was confirmed and modeling The in of to as a have been the of or the of the drug by These be the for the to of to the levels of enhancement of mutant GCase activity in as ABX, an inhibitor of GCase in H. Sci. PubMed Scopus Google These that the of a to as a be on the of its in binding inhibitory be to be effective in mutant enzyme activity in to thermal denaturation as the assay for the library to that with the that to the active site of GCase. ABX was identified in screen in of inhibitory was found to be a type inhibitor of GCase 2 and A to and Google the of ABX were with maximal inhibition at the neutral is an for an at the pH of the pH near that of ABX the enzyme and an of its activity at the maximal in type of pH dependence for a GCase inhibitor was for an was found to as a in GD as with ABX, to the levels of inhibition at neutral pH Mol. Genet. PubMed Scopus Google The pH dependence of ABX binding to GCase was confirmed in of its binding was to a for ABX at pH the a its to bind GCase a pH in inhibition of the enzyme the lysosomal and major of ABX as an agent is that for ABX has been used to treat for airway mucus and has been used as an treatment for hyaline membrane disease in newborns, increases in the E. E. J. PubMed Scopus Google Scholar, H. J. 2002; PubMed Scopus Google Scholar, H. H. J. has been to have and H. J. 2002; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Drug PubMed Scopus Google both of be useful in of the of H. Mol. Genet. PubMed Scopus Google the of of ABX has to be Drug PubMed Scopus Google the and studies of ABX in J. J. J. Google Scholar, PubMed Scopus Google ABX in clinical to as an agent for GD be ABX that the GCase in and and are the from clinical studies J. 44: PubMed Scopus Google Scholar, Drug PubMed Scopus Google ABX enhancement of GCase activity in and a the drug in with GD is on of ABX in a in with was to in patients and the of ABX in the is E. J. Scopus Google studies in have that ABX is in and H. H. J. a ABX was found at of and of in the ABX is most to be in in be an in the treatment of the forms of is the of ABX as a drug PubMed Scopus Google the or of in levels were significantly in lymphoblasts were is that the treatment and the that the GCase activity levels found in the GD Additionally, a in levels is the most used for the of for of levels was 2 of or Grabowski G.A. Pediatr. PubMed Scopus Google Scholar, PubMed Scopus Google that or of in the in lymphoblasts that treatment increased levels the that be in cells, enhancement of GCase in the and inhibition of the enzyme in the treatment of with a to be ABX to be an inhibitor of GCase pH and its enhancement of GCase in the be by its in the of the in the of segments from the wild type GCase that upon binding of that of the and PubMed Scopus Google as as of small identified by of a library PubMed Scopus Google stabilize segments and of the of was the to stabilize and the from the studies that both ABX and with GCase by as is the with G.A. PubMed Scopus Google and These with non-active site the mixed-type inhibition for ABX and has a thermal denaturation assay as an for small for ABX, a drug with a of in was identified as a of GCase and as a pH-dependent, inhibitor of the small was to as an agent for with common GCase associated with and The treatment of in increased levels of GCase activity and protein in and the reduction of These that ABX be developed as an effective agent for as an or an to or has been used to treat patients Genet. PubMed Scopus Google Scholar, PubMed Scopus Google as a of lysosomal enzyme activity is in most to storage of J. PubMed Scopus Google based on small molecules that the of the the of the mutant are attractive and with the to treat the neurological forms of of have in clinical and J. PubMed Scopus Google Scholar, J. Grabowski G.A. J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google has been to a with in and a of disease Mol. PubMed Scopus Google Scholar) and to the of GCase in PubMed Scopus Google by the storage of mutant enzyme in the the levels of the protein to has been associated with the of Mol. Genet. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, H. Mol. Genet. PubMed Scopus Google The of a drug library has been to small molecules that as agents for different J. J. PubMed Scopus Google as as agents for PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, H. J. PubMed Scopus Google assay based on the of to GCase identified as the major was in a screen to as a of GCase thermal in the assay to mutant GCase The of to stabilize GCase in was confirmed and modeling The in of to as a have been the of or the of the drug by These be the for the to of to the levels of enhancement of mutant GCase activity in as ABX, an inhibitor of GCase in H. Sci. PubMed Scopus Google These that the of a to as a be on the of its in binding inhibitory be to be effective in mutant enzyme activity in to thermal denaturation as the assay for the library to that with the that to the active site of GCase. ABX was identified in screen in of inhibitory was found to be a type inhibitor of GCase 2 and A to and Google the of ABX were with maximal inhibition at the neutral is an for an at the pH of the pH near that of ABX the enzyme and an of its activity at the maximal in type of pH dependence for a GCase inhibitor was for an was found to as a in GD as with ABX, to the levels of inhibition at neutral pH Mol. Genet. PubMed Scopus Google The pH dependence of ABX binding to GCase was confirmed in of its binding was to a for ABX at pH the a its to bind GCase a pH in inhibition of the enzyme the lysosomal and major of ABX as an agent is that for ABX has been used to treat for airway mucus and has been used as an treatment for hyaline membrane disease in newborns, increases in the E. E. J. PubMed Scopus Google Scholar, H. J. 2002; PubMed Scopus Google Scholar, H. H. J. has been to have and H. J. 2002; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Drug PubMed Scopus Google both of be useful in of the of H. Mol. Genet. PubMed Scopus Google the of of ABX has to be Drug PubMed Scopus Google the and studies of ABX in J. J. J. Google Scholar, PubMed Scopus Google ABX in clinical to as an agent for GD be The ABX that the GCase in and and are the from clinical studies J. 44: PubMed Scopus Google Scholar, Drug PubMed Scopus Google ABX enhancement of GCase activity in and a the drug in with GD is on of ABX in a in with was to in patients and the of ABX in the is E. J. Scopus Google studies in have that ABX is in and H. H. J. a ABX was found at of and of in the ABX is most to be in in be an in the treatment of the forms of is the of ABX as a drug PubMed Scopus Google the or of in levels were significantly in lymphoblasts were is that the treatment and the that the GCase activity levels found in the GD Additionally, a in levels is the most used for the of for of levels was 2 of or Grabowski G.A. Pediatr. PubMed Scopus Google Scholar, PubMed Scopus Google The that or of in the in lymphoblasts that treatment increased levels the that be in cells, enhancement of GCase in the and inhibition of the enzyme in the treatment of with a to be ABX to be an inhibitor of GCase pH and its enhancement of GCase in the be by its in the of the in the of segments from the wild type GCase that upon binding of that of the and PubMed Scopus Google as as of small identified by of a library PubMed Scopus Google stabilize segments and of the of was the to stabilize and The the from the studies that both ABX and with GCase by as is the with G.A. PubMed Scopus Google and These with non-active site the mixed-type inhibition for ABX and has a thermal denaturation assay as an for small for ABX, a drug with a of in was identified as a of GCase and as a pH-dependent, inhibitor of the small was to as an agent for with common GCase associated with and The treatment of in increased levels of GCase activity and protein in and the reduction of These that ABX be developed as an effective agent for as an or an to or the of and with for and with for for with of the in for for and and for of the and of for for The and were from the by the of of of was by an from the with with

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