title: High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.
abstract: Gaucher disease (GD), the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase). Previously, wildtype GCase was used for high throughput screening (HTS) of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.
authors.affiliation: Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America. goldine@mail.nih.gov
authors.forename: Ehud
authors.initials: E
authors.lastname: Goldin
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
authors.forename: Omid
authors.initials: O
authors.lastname: Motabar
authors.forename: Noel
authors.initials: N
authors.lastname: Southall
authors.forename: Jae Hyuk
authors.initials: JH
authors.lastname: Choi
authors.forename: Juan
authors.initials: J
authors.lastname: Marugan
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: Ellen
authors.initials: E
authors.lastname: Sidransky
mesh_terms: D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000302:isolation & purification / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D005776:Gaucher Disease / Q000201:enzymology* / Q000235:genetics / Q000517:prevention & control; D005962:Glucosylceramidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D013154:Spleen / Q000201:enzymology / Q000378:metabolism; D014020:Tissue Extracts / Q000378:metabolism
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D014020:Tissue Extracts; D005962:Glucosylceramidase