Discovery, Structure-Activity Relationship, and Biological Evaluation of Noninhibitory Small Molecule Chaperones of Glucocerebrosidase

NIH Chemical Genomic Center, National Center for Advancing Translation Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland, United States.
Journal of Medicinal Chemistry (Impact Factor: 5.45). 05/2012; 55(12):5734-48. DOI: 10.1021/jm300063b
Source: PubMed


A major challenge in the field of Gaucher disease has been the development of new therapeutic strategies including molecular chaperones. All previously described chaperones of glucocerebrosidase are enzyme inhibitors, which complicates their clinical development because their chaperone activity must be balanced against the functional inhibition of the enzyme. Using a novel high throughput screening methodology, we identified a chemical series that does not inhibit the enzyme but can still facilitate its translocation to the lysosome as measured by immunostaining of glucocerebrosidase in patient fibroblasts. These compounds provide the basis for the development of a novel approach toward small molecule treatment for patients with Gaucher disease.

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    • "However, its high cost and inability to cross the blood–brain barrier have prompted the search for chemical chaperones that can enhance GCase levels and reduce lysosomal storage in GD (Suzuki, 2013). We identified a noninhibitory chaperone molecule NCGC00188758, a pyrazolo (N-(4-ethynylphenyl)- 5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxamide) (Patnaik et al., 2012), referred to here as NCGC758, that can reverse the disease phenotype of GMs by facilitating the translocation of GCase from the endoplasmic reticulum to the lysosome (Aflaki et al., 2014). In this study, we show that this noninhibitory chaperone can induce autophagy and reduce IL-1b secretion in GMs, confirming that deficient GCase accounts for the phenomena observed. "
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    ABSTRACT: Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylcer-amide macrophages, the accumulation of glucosylceramide in lysosomes and the secretion of inflammatory cytokines. However, the connection between this lysosomal storage and inflammation is not clear. Studying macrophages derived from peripheral monocytes from patients with type 1 Gaucher disease with genotype N370S/N370S, we confirmed an increased secretion of interleukins IL-1β and IL-6. In addition, we found that activation of the inflammasome, a multiprotein complex that activates caspase-1, led to the maturation of IL-1β in Gaucher macrophages. We show that inflammasome activation in these cells is the result of impaired autophagy. Treatment with the small-molecule glucocerebrosidase chaperone NCGC758 reversed these defects, inducing autophagy and reducing IL-1β secretion, confirming the role of the deficiency of lysosomal glucocerebrosidase in these processes. We found that in Gaucher macrophages elevated levels of the autophagic adaptor p62 prevented the delivery of inflammasomes to autophagosomes. This increase in p62 led to activation of p65-NF-kB in the nucleus, promoting the expression of inflammatory cytokines and the secretion of IL-1β. This newly elucidated mechanism ties lysosomal dysfunction to inflammasome activation, and may contribute to the massive organomegaly, bone involvement and increased susceptibility to certain malignancies seen in Gaucher disease. Moreover, this link between lysosomal storage, impaired autophagy, and inflammation may have implications relevant to both Parkinson disease and the aging process. Defects in these basic cellular processes may also provide new therapeutic targets.
    Aging cell 10/2015; DOI:10.1111/acel.12409 · 6.34 Impact Factor
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    • "Measuring the thermophoretic behaviour of a protein in the presence of differing ligand concentrations by MST allows quantitative analysis of molecular interactions in solution on the microlitre scale. The MST technique has previously been used to investigate protein-protein2122, small organic molecule-protein232425 and antibody-protein interactions26. "
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    ABSTRACT: Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA.
    Scientific Reports 04/2014; 4:4765. DOI:10.1038/srep04765 · 5.58 Impact Factor
    • "There is a vast amount of publications on the use of chaperones to enhance lysosomal GCase activity [13–23]. One such chaperone is ambroxol [24–26]. "
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    ABSTRACT: Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients.
    Blood Cells Molecules and Diseases 11/2012; 50(2). DOI:10.1016/j.bcmd.2012.10.007 · 2.65 Impact Factor
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