| Assay Method Information | |
| | Inhibition of LRRK2 and LRRK2 G2019S |
| Description: | The compounds were evaluated in LRRK2 and in the mutated form LRRK2 G2019S. This mutation is more frequent in the familial forms of Parkinson's Disease and has a significant increase in kinase activity. The experimental determination of the inhibition of both enzymes was carried out using the Adapta method, which is an evaluation method of fluorescent kinase activity that determines ADP in a highly sensitive manner. The methodology can be divided into two stages: kinase reaction and ADP determination. In the first stage, all the components for the kinase reaction are added to the well and incubated for 60 min. After the reaction, the ADP detection solution which contains a Europium-labeled anti-ADP antibody (Alexa Fluor 647 labeled ADP tracer) and EDTA, to stop the kinase reaction, are added to the reaction well. The ADP formed in the kinase reaction without inhibitor will displace the Alexa Fluor 647 labeled ADP tracer of the antibody, resulting in the TR-FRET signal decrease. In the presence of the inhibitor, the amount of ADP formed is reduced, which does not modify the antibody-tracer interaction and, therefore, has a higher TR-FRET signal.The assay is performed in 384-well plates. Adding 100 nL of the solution with the compound to be evaluated in 1% DMSO, 2.4 μL of HEPES solution, 2.5 μL of ATP solution, 4.5 μL of substrate solution. The 10 μl of the kinase reaction contain: 75-70 ng LRRK2 and 200 μM ERM (LRRKtide) in 25 mM Tris/7.5 mM HEPES pH 8.2, 0.005% BRIJ-35, 5 mM MgCl2, 0.5 mM EGTA, 0.01% NaN3 or 3-12 ng LRRK2 G2019S and 200 μM ERM (LRRKtide) in 25 mM Tris/7.5 mM HEPES pH 8.2, 0.005% BRIJ-35, 5 mM MgCl2, 0.5 mM EGTA, 0.01% NaN3. The plate is stirred for 30 s on a stirring plate and centrifuged for 1 min in a centrifuge at 1,000 g. The reaction is incubated at room temperature for 60 min. After this time period, 5 μL of the detection mixture is added, the plate is stirred for 30 s on a stirring plate and centrifuged for 1 min at 1,000 g. Fluorescence is determined in a plate reader and the data are analysed. |
| Affinity data for this assay | |
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