M A R C B L

4. Quantitation of the Potency of Competing Agents

In the competition-binding assay, the ability of various concentrations of the test compound (6-12 concentrations differing by log, 1/2 log or increments of double) in competing a single fixed concentration of ligand (1-2 X Kd) at a particular receptor concentration is determined. The potency of compounds is calculated as the concentration of competitor that effectively competes for 50% of the specific ligand binding (IC50) and is calculated from the competition-binding curve (Fig. 6). The IC50 values of competitors can be obtained by indirect Hill plots, logit-log analysis, computer-based nonlinear regression analysis, or visual inspection. The relationship used in the indirect Hill plots is log BJ(B - Bl) = n log [I] + n log IC50 (7)

where B is the amount of binding in the absence of competitor, Bx is the amount of binding in the presence of competitor I, and [I] is the concentration of competitor. When percent control specific activity is plotted on the y axis against logw competitor concentration on the x axis, the IC50 is given by the competitor concentration corresponding to 50% control specific binding, and the slope gives the apparent Hill coefficient. A slope of - 1 suggests that the radioligand and the competitor interact with a single receptor population. The IC50 determined in the competition-binding studies depends on the radioligand concentration [L], and

Figure 6 Competition of ligand receptor binding by drug compounds. IC50, the concentration of compound required to inhibit binding activity by 50%, is determined by plotting the percentage control activity against log concentration of the compound. IC50 determined for the compound is 0.81 nM. The data is fitted with nonlinear regression curve fit analysis-sigmoidal fit.

Figure 6 Competition of ligand receptor binding by drug compounds. IC50, the concentration of compound required to inhibit binding activity by 50%, is determined by plotting the percentage control activity against log concentration of the compound. IC50 determined for the compound is 0.81 nM. The data is fitted with nonlinear regression curve fit analysis-sigmoidal fit.

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