Discovery, Structure-Activity Relationship, and Biological Evaluation of Noninhibitory Small Molecule Chaperones of Glucocerebrosidase
ABSTRACT 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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- "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. "
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
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- "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]. "
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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ABSTRACT: Heterozygous mutations in the glucocerebrosidase gene (GBA1) are associated with increased risk for α-synuclein aggregation disorders ('synucleinopathies'), which include Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Homozygous GBA1 mutations lead to reduced GBA1 lysosomal activity underlying three variants of Gaucher disease (GD). Despite the wealth of clinical and genetic evidence supporting the association between mutant genotypes and synucleinopathy risk, the precise mechanisms by which GBA1 mutations lead to PD and DLB remain unclear. Here, we summarize recent findings that highlight the complexity of this pathogenetic link. In neural cells, both gain and loss of function mechanisms, as conferred by mutant GBA1 expression and activity loss, respectively, seem to promote aberrant α-synuclein processing. In addition, we draw attention to recent insights gleaned from GD animal models regarding axonal pathology, brain inflammation and memory dysfunction. From a translational perspective, we discuss the concepts of neural enzyme replacement therapy and pharmacological agents as potential treatment strategies for GBA1-associated synucleinopathies. Finally, we touch on the issue whether aberrant α-synuclein species may coregulate GBA1 activity in the vertebrate brain, thereby providing a reverse link, i.e., between an important synucleinopathy risk factor and the enzyme's lysosomal function. In summary, several leads connecting GBA1 mutations with α-synuclein misprocessing have emerged as potential targets for the treatment of GBA1-related synucleinopathies, and possibly, for non-GBA1-associated neurodegenerative diseases.Neurodegenerative Diseases 02/2012; 10(1-4):195-202. DOI:10.1159/000335038 · 3.51 Impact Factor