Opioid peptide receptor studies, 11: Involvement of Tyr148, Trp318 and His319 of the rat ?-opioid receptor in binding of ?-selective ligands

Abstract

Previous data obtained with the cloned rat µ opioid receptor demonstrated that the ''super-potent'' opiates, ohmefentanyl (RTI-4614-4) and its four enantiomers, differ in binding affinity, potency, efficacy, and intrinsic efficacy. Molecular modeling (Tang et al., 1996) of fentanyl derivatives binding to the µ receptor suggests that Asp147, Tyr148, Trp318, and His319 are important residues for binding. According to this model, Asp147 interacts with the positively charged opiate agonist to form potent electrostatic and hydrogen-bonding interactions. In this study, the role of weak electrostatic and hydrogen-bonding ''-'' interactions of the O atom of the carbonyl group and the phenyl ring structures of RTI-4614-4 and its four enantiomers with residues Tyr148, Trp318, and His319 were explored via site-directed mutagenesis. Tyr148 (in transmembrane helix 3 5TMH36), Trp318 (TMH7), and His319 (TMH7) were individually replaced with phenylalanine or alanine. Receptors transiently expressed in COS-7 cells were labeled with [ 125 I]IOXY according to published procedures. Mutation of Tyr148 to phenylalanine reduced the binding affinities of some µ-selective agonists (2-7 fold) but did not alter the affinities of DAMGO, naloxone, and the non-selective opiates etorphine and buprenorphine. In contrast, this mutation significantly increased the binding affinities (decreased the Kd values) of [D-Ala 2 ,D-Leu 5 ]enkephalin, IOXY, and dermorphin. Mutation of Trp318 decreased opioid receptor binding to almost undetectable levels. Substitution of alanine for His319 significantly reduced binding affinities for the opioid ligands tested (1.3-to 48-fold), but did not alter the affinities of naloxone and bremazocine. These results indicate the importance of Tyr148 and His319 for the binding of fentanyl derivatives to the µ receptor. Functional studies using the mutant receptors will provide additional insight into the mechanism of action of RTI-4614-4 and its four enantiomers.

Molecular recognition of opioid receptor ligands

“…24 , 72 For example, the ␦ -selective ligand 1 shows enhanced activity for the receptor when a phenethyl moiety replaces the propyl-containing lead. 72 Additionally, it has been shown that N-phenethylmorphine shows greater potency than its parent compound morphine. 73 Also important to note is that this pocket contains several residues that can hydrogen-bond with the hydroxyl group of ohmefentanyl, specifi cally a threonine in TM II.…”

“…In fact, confl icting pharmacophores have been proposed for each ligand class. [24][25][26][27][28][29][30] For the ␦ -selective SNC 80 derivatives, controversy exists over whether the pharmacophoric determinants of SNC 80 (and analogs) are the same as those for the ␦ -selective opiates NTI and SIOM. [57][58][59] For the fentanyls and arylacetamides, different yet important discrepancies exist.…”

“…Unfortunately, fentanyl ' s fl exibility makes such methods very diffi cult to interpret. [24][25][26] Potential bioactive conformations have been analyzed, yet the fi ndings have not been conclusive. 30 , 68-70 In particular, the conformation of the phenethyl group has been the topic of numerous discussions.…”

“…71 However, when this residue was mutated to phenylalanine, only a minor difference in binding was observed. 72 Another study suggests that the phenethyl group points up toward Lys III:02. 30 A third study suggests that the phenethyl moiety is projected deep within the cavity of TM II, III, and VII.…”

“…[24][25][26][27][28][29][30] Thus, this review is broken up into sections based on ligand class. For each class, molecular recognition will be discussed in terms of how specifi c interactions assist ligand binding.…”