NF-56-EJ40

NF-56-EJ40 is bound deep inside the hydrophobic pocket, with the acid group coordinated by the hydroxyl groups of the conserved residues Y83 ^2.64 and Y30 ^1.39 on one side, and R281 ^7.39 on the other side. The conserved E18 ^1.27 is predicted to form an additional hydrogen bond to the piperazine ring of NF-56-EJ40. E22 ^1.31 and N274 ^7.32 in human SUCNR1 are replaced by K181.31 and K269 ^7.32 in rat SUCNR1. These two amino acid exchanges could prevent the binding of NF-56-EJ40 to rat SUCNR1 owing to steric hindrance. Radioligand-binding studies with human SUCNR1 showed partial agreement with our homology model: the Y30 ^1.39 F mutant of human SUCNR1, showing reduced binding of NF-56-EJ40. Similar effects are observed with the E18 ^1.27 K and E18 ^1.27 R mutants, probably owing to steric clashes of the Lys and Arg residues with NF-56 -EJ40 and the loss of a h ydrogen bond to its piperazine ring.
Human SUCNR1 residues are introduced into rat SUCNR1 to form the double mutant K18 ^1.31 E/K269 ^7.32 N (hereafter denoted humanized rat SUCNR1) ( K _i of 17.4 nM and 33.5 nM for human and humanized rat SUCNR1, respectively). NF-56-EJ40 increases the thermal stability of both humanized rat SUCNR1 and human SUCNR1, but not that of rat SUCNR1.

SUCNR1 (GPR91)