0 0 PubChem PubChem PubChem sample assay, Glucocerebrosidase-p1 Beta-glucocerebrosidase catalyzes the hydrolysis of beta-glucocerebroside to glucose and ceramide. The inherited deficiency of beta-glucocerebrosidase results in Gaucher disease, which is characterized by a wide variety of symptoms including hepatosplenomegaly, anemia, thrombocytopenia, bony lesions and bone marrow infiltration with characteristic storage cells, known as Gaucher cells. There are also forms of the disorder affecting the central nervous system. Patients with the same genotypes can manifest with diverse clinical presentations and it is believed that improper folding and trafficking of beta-glucocerbrosidase may contribute to the phenotypes observed. Low molecular weight molecules, acting as chaperones, may potentially restore trafficking of misfolded beta-glucocerebrosidase from the endoplasmic reticulum to the lysosomes, thereby enhancing functional lysosomal beta-glucocerebrosidase activity. Using normal beta-glucocerebrosidase, an assay was developed to screen for small molecule inhibitors that could potentially act as molecular chaperones on the mutant forms. Two pro-fluorescent substrates were chosen for two screens against a small molecule library. One beta-glucocerebrosidase assay used Resorufin beta-D-Glucopyranoside (Marker Gene Technology Inc.), generating a red fluorescent product. The second assay used Fluorescein di-beta-D-Glucopyranoside, generating a blue fluorescent product. The Km for the Resorufin beta-D-Glucopyranoside and Fluorescein di-beta-D-Glucopyranoside were determined to be 71.2 uM and 855 uM, respectively. The IC50 values of Conduritol B Epoxide, a known glucocerebrosidase inhibitor, were determined to be 87.5 uM and 117 uM, respectively. The use of two difference pro-fluorescent substrates in the screens helped to eliminate false positives. All compounds were screened in a titration series resulting in AC50s (e.g., IC50s or EC50s). Data normalization and AC50 calculations were accomplished using GeneData software. Both enzyme and substrate were diluted in buffer composed of 50mM citric acid/KH2PO4, 10mM sodium taurocholate and 0.01% tween-20 at pH 5.9. The final concentration of the enzyme was 0.2 nM in both assays. The compound library was titrated in 7 to 15 concentrations for the primary screens with a final concentration ranging from 0.6 nM to 50 uM. This particular assay protocol is for the "red" substrate, and it was performed in 1536-well plate format as follows: Red fluorescence substrate assay: 2 uL enzyme 20 nL compound 1 uL substrate (final concentration of 30 uM) Incubate at room temperature (RT) for 20 minutes Read the plate in Viewlux plate reader (Ex=548(7) and Em=600(10)). Comments, keywords, assay descriptions.. 32888 8163674 12434014 9331372 12587096 606463 230800 231000 http://yoursite.com/glucocerebrosidase-p1.html http://yoursite.com 1 Qualified AC50 Qualified AC50; can be an IC50 or EC50. 1 254 2 Activity Direction Indicates direction of activity: inactive, decreasing, increasing. 4 254 3 Activity Qualifier Indicates if AC50 is "=" to value or if it is infinite activity, ">". 4 254 4 Log of AC50 Log of AC50. 1 254 5 Standard Error Standard error for Log AC50. 1 254 6 Model Name Curve fitting model used. 4 254 7 Validation Flag AC50 validation flag; true if preliminary data modified. 3 254 8 Number of Points Number of valid points used for fitting. 2 254 9 Activity at 3.658nM % Activity at given concentration. 1 15