Identification of selective ligands targeting two GPCRs by receptor-affinity chromatography coupled with high-throughput sequencing techniques.

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 (ETAR) based on RAC were utilized for lead screening from a DNA-encoded library. The specific ligands of AT1R (ligand A1, A2) and ETAR (ligand B1, B2) were synthesized after decoding by high-throughput sequencing techniques. The dissociation rate constants (kd) of ligand A1, A2 to AT1R and B1, B2 to ETAR were 9.65 × 10-4, 31.1 × 10-4 and 0.66, 1.22 s-1 by peak profiling assay. The association constant (KA) 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.

Design, synthesis and evaluation of 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acid derivatives as ETA receptor selective antagonists using FRET assay.

The endothelin axis and in particular the two receptor subtypes, ETA and ETB, are under investigation for the treatment of various diseases such as pulmonary arterial hypertension, fibrosis, renal failure and cancer. Previous work in our lab has shown that 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acid derivatives exhibit noteworthy endothelin receptor antagonist activity. A series of analogues with modifications centered around position 6 of the heterocyclic quinolone core and replacement of the aryl carboxylic acid group with an isosteric tetrazole ring was designed and synthesized to further optimize the structure activity relationship. The endothelin receptor antagonist activity was determined by in vitro Förster resonance energy transfer (FRET) using GeneBLAzer® assay technology. The most potent member of this series exhibited ETA receptor antagonist activity in the subnanomolar range with an IC50 value of 0.8nM, and was 1000-fold selective for the ETA receptor compared to the ETB receptor. Its activity and selectivity profile resembles that of the most recently approved drug, macitentan.