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. Author manuscript; available in PMC: 2023 Dec 22.
Published in final edited form as: J Med Chem. 2022 Dec 2;65(24):16510–16525. doi: 10.1021/acs.jmedchem.2c01380

Figure 5. Competition assay.

Figure 5.

ACC 1 μM was incubated with 1 μM of 17 for 1 h at 22 °C after which either 100 μM of 1 or 3 alone, or 50 μM of 1 and 3 were added. Samples were subject to gel filtration using SpinTrap columns at different times after addition or the monovalent compounds, and the concentration of 17 in the flow through was quantified by LC/MSMS after addition of buffer. No dissociation of 17 is observed when 1 or 3 alone is incubated with the ACC:17 complex (blue and purple). However, rapid dissociation of 17 was observed when 1 and 3 were incubated together with the ACC:17 complex (red). Fitting of the data to a single exponential function in GraphPad Prism gave a rate of inhibitor dissociation of 0.3 min−1 when both 1 and 3 were used, giving a residence time of 17 on ACC of 3.3 min.