Glucosylceramidase-Aktivitäts-Assay-Kit (fluorometrisch)

Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency in lysosomal glucocerebrosidase due to genetic mutations leads to Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available to treat type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are not yet treatable.

 New therapeutic approaches for Gaucher disease are small molecule drugs that can cross the blood-brain barrier, such as pharmacological chaperones and enzyme activators. 

Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development to treat Gaucher disease. However, the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex Red enzyme reporting system. This assay is in a homogeneous assay format and has been miniaturized in a 1,536-well plate format for high-throughput screening. The sensitivity and robustness of the assay is similar to other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high-throughput screening. 536-well plate format for high-throughput screening. The sensitivity and robustness of the assay is similar to other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high-throughput screening. 536-well plate format for high-throughput screening. The sensitivity and robustness of the assay is similar to other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high-throughput screening.

introduction

Gaucher disease is an inherited lysosomal storage disease with an estimated incidence of 1 in 40,000–60,000 individuals in the general population. This disease is caused by deficiency of the lysosomal enzyme glucocerebrosidase (EC 3.2.1.45), resulting in accumulation of glucosylceramide in macrophages of the reticuloendothelial system. 

• To date, 271 mutations in the glucocerebrosidase gene have been identified, including 212 missense/nonsense changes that may result in misfolding, reduced stability, and/or mis-tightening of this lysosomal protein. 
• Patients with Gaucher disease can present at any age and often have organomegaly, anemia, thrombocytopenia, and bone crisis. Some patients have neuronopathic forms of the disorder.  Enzyme replacement therapy (ERT) has been used successfully to treat type 1 or non-neuropathy Gaucher disease, but it is extremely expensive and not effective in treating CNS manifestations.
• Efforts in recent years have focused on the development of small molecule drugs that can penetrate the blood-brain barrier and treat all types of Gaucher disease, including the neuronopathic forms of the disease.
•  Compared to protein-based drugs like ERT, small molecule drugs are advantageous because they can be administered orally, do not induce autoimmune reactions, and are relatively easy to manufacture. Several approaches for the development of new drugs to treat Gaucher disease have emerged, including pharmacological chaperone therapy, activity enhancement of mutated enzymes, and substrate reduction therapy.
• Pharmacological chaperones are small molecules that can bind to mutant proteins and aid in their proper folding, maturation, and transport to functional sites such as lysosomes. Isofagomine, an iminosugar analogue developed by Amicus Therapeutics, was in clinical trials for the treatment of Gaucher disease. Two closely related pharmacological chaperones are also in clinical trials for the treatment of Fabry disease and Pompe disease, other lysosomal storage diseases.
• Substrate reduction therapy for Gaucher disease uses a small molecule enzyme inhibitor of ceramide glucosyltransferase to block the synthesis of glucosylceramide, which accumulates in the lysosomes of patients with Gaucher disease. The long-term clinical efficacy and drug safety of substrate reduction therapy are still under investigation.
•  In addition, small molecule activators that can enhance residual mutant enzyme activity in lysosomes could be therapeutically useful for the treatment of Gaucher disease, particularly those with chaperone capability.

Enzyme assays are widely used for lead compound discovery in drug development. 

To simplify enzyme assay methods and detection methods, artificial chromogenic and fluorogenic substrates are commonly used, allowing for a homogeneous assay format to increase compound screening throughput. Enzyme assays using natural substrates are physiologically more relevant, but often not suitable for high-throughput screening (HTS) of large compound libraries due to complicated assay protocols and limited screening throughput. Several fluorogenic enzyme substrates are available for measuring glucocerebrosidase activity. The most commonly used are the blue-fluorogenic substrate 4-methylumbelliferyl-β-D-glucopyranoside (4MU-β-glc)  and the chromogenic substrate,  p -Nitrophenyl-β-D-glucopyranoside [  10  ]. Both substrates are used in assays for the clinical diagnosis of Gaucher disease and in composite screens. The red fluorogenic substrate, resorufin-β-D-glucopyranoside (res-β-glc) , is advantageous for compound screenings as it is less prone to compound autofluorescence and thus produces fewer false-positive results . Additionally, a green fluorogenic substrate, fluorescein-di-β-D-glucopyranoside (Flu-β-glc), is occasionally used [  14  ,  15 ]. However, these substrates are man-made and consist of a glucose linked to a fluorophore that is not naturally present in cells. 

Although these fluorogenic substrates are sensitive and easy to use in the assay, they may not reflect the physiological conditions in cells. In addition, the specificity of fluorogenic substrates could be an issue in assays that use cell or tissue preparations instead of purified recombinant enzymes, as they can be cleaved by other cellular enzymes present in these crude preparations   [  16-18]. Alternatively, the natural substrate glucosylceramide can be used in the glucocerebrosidase assay to more accurately measure glucocerebrosidase activity in enzyme preparations from cells and tissues. It has been reported that glucocerebrosidase activity can be measured with a fluorophore-labeled natural substrate, bodipyglucosylceramide, but an additional step using high-performance liquid chromatography (HPLC) is required. Using this fluorophore-labeled glucosylceramide as a substrate, the specific glucocerebrosidase activity can be measured in crude cell and tissue preparations by separating the Bodipy-labeled product from the substrate using an HPLC step. 

This assay allows the measurement of glucocerebrosidase activity using the true natural substrate glucosylceramide, which is more relevant to the physiological conditions in cells and tissues. We describe herein the development of a natural substrate glucocerebrosidase assay using an Amplex Red glucose oxidase reporting system in a homogeneous assay format. We also compare the advantages and disadvantages of this assay in compound screens to those performed with three other available glucocerebrosidase enzyme assays using different substrates, including red and blue fluorogenic substrates.