NF-56-EJ40 is a potent and highly selective antagonist of human SUCNR1 (GPR91), demonstrating high affinity with an IC50 value of 25 nM and a Ki of 33 nM. It exhibits minimal activity against rat SUCNR1. Notably, NF-56-EJ40 possesses significant affinity for humanized rat SUCNR1, with a Ki of 17.4 nM[1].
体外研究
NF-56-EJ40 binds deeply in the hydrophobic pocket, and the acidic group is coordinated by the hydroxyl groups of the conserved residues Y832.64 and Y301.39 and R2817.39 on the other side. The conserved E181.27 is predicted to form additional hydrogen bonds with the piperazine ring of NF-56-EJ40. E221.31 and N2747.32 in human SUCNR1 are replaced by K181.31 and K2697.32 in rat SUCNR1. These two amino acid exchanges can prevent the binding of NF-56-EJ40 to rat SUCNR1 due to steric hindrance. Radioligand binding to human SUCNR1 showed partial consistency with our homologous model: the Y301.39F mutant of human SUCNR1 showed reduced binding to NF-56-EJ40. Similar effects have been observed in the E181.27K and E181.27R mutants, possibly due to the spatial collision of the Lys and Arg residues with NF-56-EJ40 and the loss of hydrogen bonds with their piperazine rings[1].
Residues from human SUCNR1 are incorporated into rat SUCNR1, creating the double mutant K181.31E/K2697.32N (referred to as humanized rat SUCNR1), displaying Ki values of 17.4 nM for human and 33.5 nM for humanized rat SUCNR1, respectively. NF-56-EJ40 enhances the thermal stability of both human and humanized rat SUCNR1, but does not affect the thermal stability of rat SUCNR1[1].
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