Binding of norbinaltorphimine (norBNI) congeners to wild-type and mutant mu and kappa opioid receptors: Molecular recognition loci for the pharmacophore and address components of kappa antagonists

Molecular modifications of both the kappa opioid antagonist norbinaltorphim ine (norBNI, 1) and the kappa receptor have provided evidence that the sele ctivity of this ligand is conferred through ionic interaction if its N17' p rotonated amine group (an "address") with a nonconserved acidic residue (Gl u297) on the kappa receptor. In the present study, we have examined the eff ect of structural modifications on the affinity of norBNI analogues for wil d-type and mutant kappa and mu opioid receptors expressed in COS-7 cells. C ompounds 2, 3, and 7, which have an antagonist pharmacophore and basic N17' group in common with norBNI, retained high affinity for the wild-type kapp a but exhibited greatly reduced affinity for mutant kappa receptors (E297K and E297A). Modification of the phenolic or N-substituent groups of the ant agonist pharmacophore (4 and 5) or removal of basicity at the address N17' center (6) led to greatly reduced affinity for the wild-type and mutant rec eptors. The reduced affinity upon modification of the kappa receptor is con sistent with the ionic interaction of the protonated N17' group of kappa an tagonists (1-3, 7) with the carboxylate group of E297 at the top of TM6. Th is was supported by the greatly enhanced affinity of compounds 1-3 for the mutant mu receptor (K303E), as compared to the wild-type mu receptor, given that residue K303 occupies a position equivalent to that of E297 in the ka ppa receptor. In view of the high degree of homology of the seven TM domain s of the kappa and mu opioid receptors, it is suggested that the antagonist pharmacophore is bound within this highly conserved region of the kappa or mutant mu receptor and that an anionic residue at the top of TM6 (E297 or K303E, respectively) provides additional binding affinity.