A chromatographic method for determining the interaction between a drug and two target proteins by fabricating a...

Characterization of the drug-target interactions is pivotal throughout the whole procedure of drug development. Most of the current assays, particularly, chromatographic methods lack the capacity to reveal drug adsorption on the muti-target surface. To this end, we derived a reliable and workable mathematical equation for revealing drug bindings to dual targets on the heterogeneous surface starting from the mass balance equation. The derivatization relied on the correlation of drug injection amounts with their retention factors. Experimental validation was performed by determining the binding parameters of three canonical drugs on a heterogeneous surface, which was fabricated by fusing angiotensin receptor type I and type II receptors (AT₁R and AT₂R) at the terminuses of circularly permuted HaloTag (cpHaloTag) and immobilizing the whole fusion protein onto 6-bromohexanoic acid modified silica gel. We proved that immobilized AT₁R-cpHalo-AT₂R maintained the original ligand- and antibody-binding activities of the two receptors in three weeks. The association constants of valsartan, candesartan, and telmisartan to AT₁R were (6.26±0.14) × 10⁵, (9.66±0.71) × 10⁵, and (3.17±0.03) × 10⁵ L/mol. In the same column, their association constants to AT₂R were (1.25±0.04) × 10⁴, (2.30±0.08) × 10⁴, and (8.51±0.06) × 10³ L/mol. The patterns of the association constants to AT₁R/AT₂R (candesartan>valsartan>telmisartan) were in good line with the data by performing nonlinear chromatography on control columns containing immobilized AT₁R or AT₂R alone. This provided proof of the fact that the derivatization allowed the determination of drug bindings on the heterogeneous surface with the utilization of a single series of injections and linear regression. We reasoned that is simple enough to model the bindings of drug adsorption on commercially available adsorbents in fundamental or industrial fields, thus having the potential to become a universal method for analyzing the bindings of a drug to the heterogeneous surface containing multiple targets.