| Assay Method Information | |
| | MAP4K4 Kinase Activity Assay |
| Description: | Compounds of the present invention were screened for their inhibitory activity against MAP4K4, versus selected off-target hits found with early members of this chemical series. MAP4K4 kinase activity was monitored using the CisBio HTRF Transcreener ADP assay, a competitive immunoassay with a reproducible Z′>0.6. In the detection step, endogenous ADP and d2-labeled ADP compete for binding an anti-ADP monoclonal antibody labelled with Eu3+ cryptate. A ratiometric fluorescent read-out is used at 665 and 620 nm. Reactions were performed in the presence of 1% DMSO with ATP added at Km(10 μM), 0.5 nM human MAP4K4 kinase domain (Invitrogen), 1 M biotin-myelin basic protein as substrate (Invitrogen), and extension of reaction time to 2h. Assays were run in Greiner low volume plates with a final reaction volume of 10 μl. The MAP4K4 inhibition data are provided in Table 33 below for selected compounds of the present invention. |
| Affinity data for this assay | |
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