The EC50 of Tracer-6908 with ACVR1-c-nanoLuc and ideal conditions for the target engagement assay have been determined in the previous post. However, it is still necessary to verify that the IC50 values determined in the assay are closed enough approximation to the actual IC50 values of the compounds. If the IC50 values are strongly influenced by the concentration of Tracer-6908 used, multiple assays with progressively lower Tracer-6908 will need to be performed. The actual IC50 of the compounds can then be estimated by plotting the Tracer-6908 concentrations against the corresponding IC50 values and regressing to no Tracer-6908. To address this, multiple nanoBRET target engagement assays were setup with increasing concentrations of Tracer-6908 with the same serial dilution of M4K1962 (starting from the EC50 of 65nM).