Scientific Publications
Structure-activity relationship for BAY-1797 at human P2X4 and P2X7 receptors reveals insights into selectivity and dual antagonism
British Journal of Pharmacology
Background and Purpose: This study aims to better understand the structure-activity relationship for BAY-1797 at human P2X4 receptors using saturation transfer difference (STD) NMR spectroscopy on living cells to structurally elucidate protein-ligand interactions for different analogues.
Experimental Approach: We used HEK-293 cells heterologously expressing either hP2X4 or hP2X7 to determine antagonist activity of BAY-1797 analogues using a fura-2AM calcium influx assay. On-cell saturation transfer difference NMR spectroscopy was used to probe ligand contact points with hP2X4 or hP2X7. In total, 35 analogues of BAY-1797 were synthesized and tested.
Key Results: Screening experiments revealed 11 analogues with antagonist activity at hP2X4 including 2 analogues demonstrating higher % inhibition and retaining potency in the nanomolar range. BAY-1797 was shown to inhibit hP2X7 responses by 50% (IC50 436 nM) and chemical modifications were discovered that could improve antagonist activity at hP2X7 with many analogues showing a higher percentage inhibition to BAY-1797. On-cell STD NMR spectroscopy revealed the major contact points between BAY-1797 and either hP2X4 or hP2X7 proteins in living cells. Site-directed mutagenesis of the identified negative allosteric modulator site on hP2X4 revealed W84, E307 and I312 as residues affecting potency and efficacy. We discovered that BAY-1797 and several analogues demonstrated flipped pharmacological activity at the M105A-hP2X7 mutant showing positive modulator activity. This suggests that certain ligand interactions within this allosteric modulator pocket could have differential pharmacological effects.
British Journal of Pharmacology
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