Assay Report Card

Basic Information

ID: ALA3889256

Type: Binding

Description: Biological Assay: TAK1-TAB1 Binding Inhibitory Potency: The ability of candidate compounds to interact with TAK1-TAB1 is quantitated by a competitive binding assay using the LanthaScreen technology developed by Life Technologies. This assay is based on the binding of a proprietary, Alexa Fluor 647-labeled, ATP-competitive kinase inhibitor (kinase tracer-236) to the TAK1-TAB1 construct in presence of a europium-conjugated antibody, resulting in a FRET (fluorescence resonance energy transfer) signal. Displacement of the kinase tracer by compound results in a lower emission ratio upon excitation of the europium chelate. Candidate compounds are designed as potential irreversible inhibitors of TAK1-TAB1, capable of ligating to an active site cysteine residue. The time dependent nature of irreversible inhibition is investigated by performing the binding assay with and without a pre-incubation of compound and TAK1-TAB1. An increase in potency in the pre-incubated assay suggests the candidate compound could b of 10 nM TAK1-TAB1, 2 nM Eu-anti-his antibody, and 100 nM kinase tracer-236 using a 384-well plate format. Background signal is defined in the absence of TAK1-TAB1 and uninhibited signal is defined in the presence of vehicle (2% DMSO) alone. Compounds were evaluated in an 11 point dose-response ranging from 20 uM to 0.34 nM. The binding assays are performed under two conditions to evaluate time dependence of inhibition. For the pre-incubation assay, TAK1-TAB1 and Eu-anti-his antibody are preincubated with compound or vehicle for two hours prior to the addition of kinase tracer. The non-preincubated assay is run in which TAK1-TAB1 and Eu-anti-his antibody are added to a mixture of compound and kinase tracer. IC50 values of compounds are determined using a 4 parameter logistical fit of emission ratio as a function of the concentration of compound.

Activity Charts

Compound Summary

Parent Molecular WeightALogPPolar Surface Area
294.312.042.04
273.342.992.99
242.282.532.53
276.300.610.61
309.322.722.72
278.291.791.79
242.282.532.53
260.301.631.63
216.241.861.86
318.381.501.50
286.211.401.40
280.292.252.25
233.272.202.20
267.722.412.41
246.271.251.25
242.282.532.53
276.303.073.07
274.321.581.58
322.322.412.41
260.301.631.63
258.280.830.83
274.321.751.75
274.321.881.88
288.352.142.14
288.352.092.09
275.312.182.18
246.271.241.24
289.342.572.57
314.392.532.53
300.362.282.28
318.381.501.50
260.301.491.49
259.312.592.59
312.302.172.17
294.742.062.06
261.282.092.09