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 diseaseTwo 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. 

Glucosylceramidase Activity Assay Kit (Fluorometric)

K2003-100 Biovision each 705.6 EUR

Proteasome Activity Fluorometric Assay Kit

K2096-100 ApexBio 100 assays 602.4 EUR

GST Fluorometric Activity Assay Kit

K2105-100 ApexBio 100 assays 602.4 EUR

PLTP Activity Fluorometric Assay Kit

K2087-100 ApexBio 100 assays 802.8 EUR

CETP Activity Fluorometric Assay Kit

K2089-100 ApexBio 100 assays 870 EUR

Calpain Activity Fluorometric Assay Kit

K2062-100 ApexBio 100 assays 752.4 EUR

HDAC Activity Fluorometric Assay Kit

K2031-100 ApexBio 100 assays 602.4 EUR

HAT Activity Fluorometric Assay Kit

K2034-100 ApexBio 100 assays 668.4 EUR

DPP4 Activity Fluorometric Assay Kit

K2178-100 ApexBio 100 assays 668.4 EUR

Neuraminidase Activity Fluorometric Assay Kit

K2230-100 ApexBio 100 assays 718.8 EUR

Transketolase Activity Assay Kit (Fluorometric)

K2004-100 Biovision each 744 EUR

Carboxylesterase Activity Assay Kit (Fluorometric)

K2014-100 Biovision each 744 EUR

Lysozyme Activity Assay Kit (Fluorometric)

K236-100 Biovision each 588 EUR

Calpain Activity Fluorometric Assay Kit

K240-100 Biovision each 705.6 EUR

Proteasome Activity Fluorometric Assay Kit

K245-100 Biovision each 574.8 EUR

GST Fluorometric Activity Assay Kit

K260-100 Biovision each 561.6 EUR

Sirtuin Activity Assay Kit (Fluorometric)

K324-100 Biovision each 686.4 EUR

HDAC Activity Fluorometric Assay Kit

K330-100 Biovision each 588 EUR

HAT Activity Fluorometric Assay Kit

K334-100 Biovision each 646.8 EUR

HDAC3 Activity Fluorometric Assay Kit

K343-100 Biovision each 574.8 EUR

HDAC8 Activity Fluorometric Assay Kit

K348-100 Biovision each 588 EUR

Chymotrypsin Activity Assay Kit (Fluorometric)

K352-100 Biovision each 588 EUR

Thrombin Activity Fluorometric Assay Kit

K373-100 Biovision each 836.4 EUR

Plasmin Activity Assay Kit (Fluorometric)

K381-100 Biovision each 548.4 EUR

HDAC6 Activity Assay Kit (Fluorometric)

K466-100 Biovision each 757.2 EUR

Deubiquitinase Activity Assay Kit (Fluorometric)

K485-100 Biovision each 639.6 EUR

Neprilysin Activity Assay Kit (Fluorometric)

K487-100 Biovision each 732 EUR

Chitotriosidase Activity Assay Kit (Fluorometric)

K512-100 Biovision each 744 EUR

?-Glucuronidase Activity Assay Kit (Fluorometric)

K514-100 Biovision each 744 EUR

Methyltransferase Activity Assay Kit (Fluorometric)

K521-100 Biovision each 679.2 EUR

Sphingomyelinase Activity Fluorometric Assay Kit

K574-100 Biovision each 548.4 EUR

Urokinase Activity Fluorometric Assay Kit

K728-100 Biovision each 652.8 EUR

Neuraminidase Activity Fluorometric Assay Kit

K732-100 Biovision each 627.6 EUR

Uricase Activity Assay Kit (Fluorometric)

K734-100 Biovision each 614.4 EUR

DPP4 Activity Fluorometric Assay Kit

K779-100 Biovision each 652.8 EUR

Protease Activity Fluorometric Assay Kit

K781-100 Biovision each 464.4 EUR

Renin Activity Fluorometric Assay Kit

K800-100 Biovision each 627.6 EUR

Lipoxygenase Activity Assay Kit (Fluorometric)

K978-100 Biovision each 783.6 EUR

?-Xylosidase Activity Assay Kit (Fluorometric)

K981-100 Biovision each 705.6 EUR

Alkaline Phosphatase Activity Fluorometric Assay Kit

K2077-500 ApexBio 500 assays 435.6 EUR

Lipase Activity Fluorometric Assay Kit III

K2095-100 ApexBio 100 assays 703.2 EUR

Beta-Secretase Activity Fluorometric Assay Kit

K2043-100 ApexBio 100 assays 769.2 EUR

Beta-Mannosidase Activity Assay Kit (Fluorometric)

K2045-100 Biovision 100 assays 661.2 EUR

Cathepsin B Activity Fluorometric Assay Kit

K2151-100 ApexBio 100 assays 619.2 EUR

Cathepsin K Activity Fluorometric Assay Kit

K2152-100 ApexBio 100 assays 619.2 EUR

Cathepsin L Activity Fluorometric Assay Kit

K2153-100 ApexBio 100 assays 619.2 EUR

Cathepsin D Activity Fluorometric Assay Kit

K2154-100 ApexBio 100 assays 619.2 EUR

Cathepsin S Activity Fluorometric Assay Kit

K2155-100 ApexBio 100 assays 619.2 EUR

Myeloperoxidase (MPO) Fluorometric Activity Assay Kit

K2169-100 ApexBio 100 assays 686.4 EUR

Catalase Activity Colorimetric/Fluorometric Assay Kit

K2177-100 ApexBio 100 assays 501.6 EUR

OxiSelect Catalase Activity Assay Kit, Fluorometric

STA-339 Cell Biolabs 500 assays 908.4 EUR

Cathepsin B Activity Fluorometric Assay Kit

K140-100 Biovision each 614.4 EUR

Cathepsin K Activity Fluorometric Assay Kit

K141-100 Biovision each 614.4 EUR

Cathepsin L Activity Fluorometric Assay Kit

K142-100 Biovision each 614.4 EUR

Cathepsin D Activity Fluorometric Assay Kit

K143-100 Biovision each 614.4 EUR

Cathepsin S Activity Fluorometric Assay Kit

K144-100 Biovision each 614.4 EUR

Cathepsin H Activity Fluorometric Assay Kit

K145-100 Biovision each 574.8 EUR

Cathepsin E Activity Assay Kit (Fluorometric)

K165-100 Biovision each 764.4 EUR

Granzyme B Activity Fluorometric Assay Kit

K168-100 Biovision each 639.6 EUR

cAMP Phosphodiesterase Activity Assay Kit (Fluorometric)

K2013-100 Biovision each 744 EUR

NADH Oxidase Activity Assay Kit (Fluorometric)

K2019-100 Biovision each 705.6 EUR

Adenosine Deaminase Activity Assay Kit (Fluorometric)

K328-100 Biovision each 548.4 EUR

HAT (H4) Activity Fluorometric Assay Kit

K336-100 Biovision each 601.2 EUR

InSitu HDAC Activity Fluorometric Assay Kit

K339-100 Biovision each 574.8 EUR

Factor VIIIa Activity Assay Kit (Fluorometric)

K358-100 Biovision each 646.8 EUR

Beta-Secretase Activity Fluorometric Assay Kit

K360-100 Biovision each 744 EUR

Factor Xa Activity Fluorometric Assay Kit

K361-100 Biovision each 744 EUR

Factor IXa Activity Assay Kit (Fluorometric)

K364-100 Biovision each 588 EUR

Neutrophil Elastase Activity Assay kit (Fluorometric)

K383-100 Biovision each 502.8 EUR

?-Secretase (BACE1) Activity Assay Kit (Fluorometric)

K388-100 Biovision each 724.8 EUR

Phospholipase A2 Activity Assay Kit (Fluorometric)

K400-100 Biovision each 679.2 EUR

Acid Phosphatase Activity Fluorometric Assay Kit

K421-500 Biovision each 418.8 EUR

Alkaline Phosphatase Activity Fluorometric Assay Kit

K422-500 Biovision each 418.8 EUR

DNAse I Activity Assay Kit (Fluorometric)

K429-100 Biovision each 836.4 EUR

Pepsin/Pepsinogen Activity Assay Kit (Fluorometric)

K446-100 Biovision each 561.6 EUR

Cytidine Deaminase Activity Assay Kit (Fluorometric)

K451-100 Biovision each 561.6 EUR

Diamine Oxidase Activity Assay Kit (Fluorometric)

K496-100 Biovision each 679.2 EUR

Oxalate Oxidase Activity Assay Kit (Fluorometric)

K509-100 Biovision each 574.8 EUR

?-L-Fucosidase Activity Assay Kit (Fluorometric)

K542-100 Biovision each 627.6 EUR

Cyclooxygenase (COX) Activity Assay Kit (Fluorometric)

K549-100 Biovision each 502.8 EUR

PLTP Activity Fluorometric Assay Kit II

K593-100 Biovision each 705.6 EUR

CETP Activity Fluorometric Assay Kit II

K595-100 Biovision each 770.4 EUR

Enolase Activity Colorimetric/Fluorometric Assay Kit

K691-100 Biovision each 952.8 EUR

Lipoprotein Lipase Activity Fluorometric Assay Kit

K721-100 Biovision each 646.8 EUR

Lipase Activity Fluorometric Assay Kit III

K724-100 Biovision each 639.6 EUR

Myeloperoxidase (MPO) Fluorometric Activity Assay Kit

K745-100 Biovision each 660 EUR

Asparaginase Activity Colorimetric/Fluorometric Assay Kit

K754-100 Biovision each 660 EUR

Enteropeptidase/Enterokinase Activity Fluorometric Assay Kit

K758-100 Biovision each 732 EUR

PicoProbe? Hexokinase Activity Assay Kit (Fluorometric)

K769-100 Biovision each 764.4 EUR

Peroxidase Activity Colorimetric/Fluorometric Assay Kit

K772-100 Biovision each 464.4 EUR

Catalase Activity Colorimetric/Fluorometric Assay Kit

K773-100 Biovision each 502.8 EUR

MMP-3 Activity Fluorometric Assay Kit

K783-100 Biovision each 614.4 EUR

Rat Renin Activity Fluorometric Assay Kit

K806-100 Biovision each 777.6 EUR

Adenosylhomocysteinase (AHCY) Activity Fluorometric Assay Kit

K807-100 Biovision each 1038 EUR

TEV Protease Activity Assay Kit (Fluorometric)

K842-100 Biovision each 601.2 EUR

Total Phosphodiesterase Activity Assay Kit (Fluorometric)

K927-100 Biovision each 738 EUR

Lysyl Oxidase Activity Assay Kit (Fluorometric)

K928-100 Biovision each 639.6 EUR

PicoProbeTM Glucokinase Activity Assay Kit (Fluorometric)

K969-100 Biovision each 711.6 EUR

Aromatase (CYP19A) Activity Assay Kit (Fluorometric)

K983-100 Biovision each 744 EUR

Cystathionine ? Synthase Activity Assay Kit (Fluorometric)

K998-100 Biovision each 744 EUR

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.

Leave a Comment