Cell membrane chromatography relative competitive method for the accurate determination of relative K_D values of...

The specific binding of a drug to the receptor is a prerequisite for its action. The equilibrium dissociation constant (K_D) is an important parameter for measuring the strength of drug-receptor interactions. Cell membrane chromatography (CMC) is a powerful way to determine the K_D value; however, the common disadvantage is that the attenuation of biological activity with the analysis process leads to corresponding errors in comparing K_D values of a series of drugs. Therefore, it is of practical significance to analyze the relative K_D values of drugs for the same membrane receptor under the same conditions. We developed a CMC relative competitive method to determine the relative K_D values of drugs through simultaneous injection of the control compound and analyte in each analysis, circumventing the error in K_D values of drugs due to the attenuation of the biological activity of the CMC column. The results showed that the K_D values of CD147 antagonists and MRGPRX2 agonists determined using the CMC relative competitive method correlated well with the K_D values obtained via frontal analysis and stepwise frontal method using the CD147^h (MRGPRX2^h)/CMC system. Critically, the biological activities of the CD147 antagonists and MRGPRX2 agonists were significantly correlated with K_D values measured using the CMC relative competitive method. Therefore, the CMC relative competitive method can accurately and efficiently evaluate the relative K_D values of drugs and effectively predict the differences in pharmacological activity between a series of drugs, which has important guiding significance in the development of targeted drugs.