Competitive binding between 4,4′-diphenylmethane-bis(methyl) carbamate and RAGE ligand MG-H1 on human umbilical vein endothelial cell by cell membrane chromatography

The compound 4,4′-diphenylmethane-bis(methyl) carbamate (CM1) has a protective activity on AGEs-induced endothelial dysfunction on human umbilical vein endothelial cell (HUVEC) in our previous study. It suggested that CM1 which may act as a competitive antagonist to the blockade of AGEs to receptor of AGEs (RAGE) and attenuate the HUVEC damage. In order to testify that hypothesis, the cell membrane chromatography (CMC) combined with high performance liquid chromatography (HPLC) was developed for analyzing the competitive binding properties on RAGE of HUVEC between CM1 and MG-H1, the agonist of RAGE. The results from saturation binding of CM1 and MG-H1 on cells demonstrated that dissociation equilibrium constants (Kd) of CM1 and MG-H1 were 3.653 nM and 4.12 nM, respectively; while maximum binding capacity (Bmax) of CM1 and MG-H1 were 30.08 and 18.72 fmol/mg protein, respectively. In competition experiments, IC50 of CM1 with pre-incubation 10−10 M and 10−9 M MG-H1 were 1.37 × 10−9 M and 4.56 × 10−8 M, respectively. The present findings indicated that CM1 conjugated competitively to cells with RAGE ligand MG-H1. The primary study illustrated that CMC combined with HPLC analysis method could be an alternative, rapid and efficient approach for the interaction of drug molecule and receptor, and that CM1 intervene the AGEs inducing HUVEC damage may via the competitively block the AGEs–RAGE path way.

► CM1 competitively bind to RAGE with RAGE ligand MG-H1.
► A HPLC method was established for competition binding of CM1 with MG-H1.
► This method was an alternative way for competitive binding of drug to receptor.
► This binding was performed on intact cells.