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| | LRRK2-G2019S Kinase activity assay |
| Description: | The measurements of the activities were made using a LRRK2-G2019S(human) Kinase activity assay as described hereunder. The potency of compounds was evaluated in vitro using the Lanthascreen technology (ThermoFisher Scientific). The biological assay requires the DYKDDDDK-tagged full-length LRRK2 G2019S protein, the fluorescent peptide substrate named LRRKtide or ERM (RLGRDKYKTLRQIRQ supμLied by PolyPeptide Group), the ATP substrate, and different concentrations of test compounds. In a typical experiment, the reaction assay buffer contains 50mM Tris pH8.2, 1mM EGTA, 2.5mM MgCI2, 0.05% Triton X100, 0.15% BSA and 1 mM DTT. The detection of the assay is performed using 1 nM Terbium labeled anti-phospho-LRRKtide (ThermoFisher Scientific) diluted in a TR-FRET buffer (ThermoFisher Scientific). The experimental procedure was performed with Greiner small volume 384-well. The assay is started by a pre-incubation step of full-length LRRK2 G2019S mutant protein with reaction assay buffer and a dilution series of test compound for at least 30 minutes at room temperature. The working solution of compounds was prepared in a separate Greiner microμLate. The kinase reaction was initiated by addition of the ATP/LRRKtide substrate mix diluted in the reaction assay buffer. The reaction mix was incubated for at least 10 minutes at room temperature (initial velocity conditions). Reaction was stopped by addition of Terbium labeled anti-phospho-LRRKtide diluted into the detection buffer supμLemented with EDTA (ThermoFisher Scientific). Plates are incubated for at leastn30 minutes at room temperature and read on an Envision™ multimode μLate reader (PerkinElmer) with LanthaScreen™. The TR-FRET 520:495 nm emission ratio was calculated and used to determine the IC50 value from a dose response curve fit to the 4-parameter logistic equation. The activity with respect to LRRK2 kinase in this test is given by the concentration which inhibits 50% of the LRRK2 activity (or IC50) in nM. |
| Affinity data for this assay | |
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