Table 4. Molecular Targets from the Pull-down Assay Using the Bisamide Probes in SK-OV-3 Cell Lysate^a.

protein	protein EC50 (μM)b	26 AC50 (μM)c	26Kiapp (μM)d	17 AC50 (μM)c	17 Kiapp (μM)d	28 AC50 (μM)c
GSK3β	1.3	1.2	NA	10	NAe	NAb
Pirin	1.3	1.5	0.028	10	0.19	NAb

^a(A) SK-OV-3 cell lysate protein binding curves for immobilized 29. (B) Protein displacement curves for pyrrolidine 26. Curves are an average of two different mixing conditions. EC₅₀ and AC₅₀ are determined from analysis of the curves without limits.

^bEC₅₀ is the apparent immobilized bisamide probe concentration at which 50% of the available protein is bound. The probe bisamide is assumed to be >10-fold molar excess over proteins in the lysate.

^cAC₅₀ is the apparent bisamide concentration at which 50% of the protein is displaced from the immobilized bisamide probe. At least 2-fold enrichment of bound proteins when compared to the matrix.

^dK_i^app values are estimated using the Cheng–Prusoff equation, apparent [29-immobilized] = 67 μM,⁵² assuming no mass transport limitation, interactions are purely competitive and all interactions are direct.

^eNot applicable (NA). K_i^app cannot be calculated if the interaction with the probe is indirect.