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Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease.

ABSTRACT: Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene. Missense mutations result in reduced enzyme activity that may be due to misfolding, raising the possibility of small-molecule chaperone correction of the defect. Screening large compound libraries by quantitative high-throughput screening (qHTS) provides comprehensive information on the potency, efficacy, and structure-activity relationships (SAR) of active compounds directly from the primary screen, facilitating identification of leads for medicinal chemistry optimization. We used qHTS to rapidly identify three structural series of potent, selective, nonsugar glucocerebrosidase inhibitors. The three structural classes had excellent potencies and efficacies and, importantly, high selectivity against closely related hydrolases. Preliminary SAR data were used to select compounds with high activity in both enzyme and cell-based assays. Compounds from two of these structural series increased N370S mutant glucocerebrosidase activity by 40-90% in patient cell lines and enhanced lysosomal colocalization, indicating chaperone activity. These small molecules have potential as leads for chaperone therapy for Gaucher disease, and this paradigm promises to accelerate the development of leads for other rare genetic disorders.

SUBMITTER: Zheng W

PROVIDER: S-EPMC1936979 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease.

Zheng Wei W Padia Janak J Urban Daniel J DJ Jadhav Ajit A Goker-Alpan Ozlem O Simeonov Anton A Goldin Ehud E Auld Douglas D LaMarca Mary E ME Inglese James J Austin Christopher P CP Sidransky Ellen E

Proceedings of the National Academy of Sciences of the United States of America 20070801 32

Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene. Missense mutations result in reduced enzyme activity that may be due to misfolding, raising the possibility of small-molecule chaperone correction of the defect. Screening large compound libraries by quantitative high-throughput screening (qHTS) provides comprehensive information on the potency, efficacy, and structure-activity relationships (SAR) of active compounds directly ...[more]

PMID: 17670938

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Project description:Glucocerebrosidase (GCase) is implicated in both a rare, monogenic disorder (Gaucher disease, GD) and a common, multifactorial condition (Parkinson's disease, PD); hence, it is an urgent therapeutic target. To identify correctors of severe protein misfolding and trafficking obstruction manifested by the pathogenic L444P-variant of GCase, we developed a suite of quantitative, high-throughput, cell-based assays. First, we labeled GCase with a small proluminescent HiBiT peptide reporter tag, enabling quantitation of protein stabilization in cells while faithfully maintaining target biology. TALEN-based gene editing allowed for stable integration of a single HiBiT-GBA1 transgene into an intragenic safe-harbor locus in GBA1-knockout H4 (neuroglioma) cells. This GD cell model was amenable to lead discovery via titration-based quantitative high-throughput screening and lead optimization via structure-activity relationships. A primary screen of 10,779 compounds from the NCATS bioactive collections identified 140 stabilizers of HiBiT-GCase-L444P, including both pharmacological chaperones (ambroxol and noninhibitory chaperone NCGC326) and proteostasis regulators (panobinostat, trans-ISRIB, and pladienolide B). Two complementary high-content imaging-based assays were deployed to triage hits: The fluorescence-quenched substrate LysoFix-GBA captured functional lysosomal GCase activity, while an immunofluorescence assay featuring antibody hGCase-1/23 directly visualized GCase lysosomal translocation. NCGC326 was active in both secondary assays and completely reversed pathological glucosylsphingosine accumulation. Finally, we tested the concept of combination therapy by demonstrating synergistic actions of NCGC326 with proteostasis regulators in enhancing GCase-L444P levels. Looking forward, these physiologically relevant assays can facilitate the identification, pharmacological validation, and medicinal chemistry optimization of small molecules targeting GCase, ultimately leading to a viable therapeutic for GD and PD.

Dataset Information

Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease.

Publications

Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease.

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