Identification of selective ligands targeting two GPCRs by receptor-affinity chromatography coupled with...

The rapid growth of demands for drug discovery has necessitated the ongoing pursuit of new methods for specific ligands screening and identification. This work combined receptor-affinity chromatography (RAC) with high-throughput sequencing techniques to rapidly screen and identify the specific ligands. By this method, immobilized angiotensin II type I receptor (AT₁R) and endothelin receptor A (ET_AR) based on RAC were utilized for lead screening from a DNA-encoded library. The specific ligands of AT₁R (ligand A₁, A₂) and ET_AR (ligand B₁, B₂) were synthesized after decoding by high-throughput sequencing techniques. The dissociation rate constants (k_d) of ligand A₁, A₂ to AT₁R and B₁, B₂ to ET_AR were 9.65 × 10^-4, 31.1 × 10^-4 and 0.66, 1.22 s^-1 by peak profiling assay. The association constant (K_A) to the receptors of four ligands was 5.4 × 10⁶, 3.3 × 10⁶ and 1.6 × 10⁶, 2.2 × 10⁵ by injection amount dependent method. The kinetic and thermodynamic parameters of the four specific ligands are similar to those of the positive drugs. This indicates that they are promising to drug candidates. The druggability of the four ligands through pharmacokinetic investigation by HPLC-MS/MS presented desired pharmacokinetic behavior including the fast absorption, the relatively slow elimination. These results, taking together, indicated that the RAC combined with high-throughput sequencing techniques can screen and identify the specific ligands according to various proteins, thus creating a general strategy for rapid discovery of promising drug candidates.