Cell membrane chromatography relative competitive method for the accurate determination of relative KD values of drug-receptor interactions.

Abstract

The specific binding of a drug to the receptor is a prerequisite for its action. The equilibrium dissociation constant (KD) is an important parameter for measuring the strength of drug-receptor interactions. Cell membrane chromatography (CMC) is a powerful way to determine the KD value; however, the common disadvantage is that the attenuation of biological activity with the analysis process leads to corresponding errors in comparing KD values of a series of drugs. Therefore, it is of practical significance to analyze the relative KD values of drugs for the same membrane receptor under the same conditions. We developed a CMC relative competitive method to determine the relative KD values of drugs through simultaneous injection of the control compound and analyte in each analysis, circumventing the error in KD values of drugs due to the attenuation of the biological activity of the CMC column. The results showed that the KD values of CD147 antagonists and MRGPRX2 agonists determined using the CMC relative competitive method correlated well with the KD values obtained via frontal analysis and stepwise frontal method using the CD147h (MRGPRX2h)/CMC system. Critically, the biological activities of the CD147 antagonists and MRGPRX2 agonists were significantly correlated with KD values measured using the CMC relative competitive method. Therefore, the CMC relative competitive method can accurately and efficiently evaluate the relative KD 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.