Development of a model for the mu opioid receptor pharmacophore.

A series of conformationally restricted analogs of the cyclic $\mu$ opioid receptor selective tetrapeptide Tyr-c(D-Cys-Phe-D-Pen) NH$\sb2$(S-Et-S) (JOM-6) were prepared in order to elucidate the structural and conformational features of the $\mu$ opioid receptor pharmacophore. Since the 3-dimensional structure of the three types of opioid receptors ($\mu,\ \delta,$ and $\kappa)$ remains unsolved, pharmacophore modeling has been an invaluable method for characterizing the molecular mechanism of opioid activity. The structural and conformational parameters of the pharmacophore model described in this thesis provides the foundation for opioid-based drug design and optimization--a process which relies upon the understanding of opioid agonist and antagonist function on the molecular level. Prior to the development of the JOM-6 series, other $\mu$ receptor pharmacophore models retained uncertainty due to the conformational lability of the parent peptide or alkaloid ligand. This has undermined attempts to determine specific $\mu$ receptor bound conformations among a multitude of energetically favorable conformations. This dissertation describes the development of a precise model for the $\mu$ opioid receptor bound pharmacophore and the conformational factors involved in the selective recognition of $\mu$ receptors. A combination of solid- and solution-phase chemistry was employed to synthesize the JOM-6 derivatives. All of the analogs were tested for opioid receptor binding affinity using a radioligand competition binding assay. In addition, several analogs were evaluated in the guinea pig ileum and mouse vas deferens bioassays and shown to be $\mu$ receptor agonists. A particularly noteworthy result was the opioid activity of the Phe$\sp1$ analog of JOM-6. This analog displayed extraordinarily high $\mu$ selective binding which suggests that the phenolic hydroxyl of Tyr$\sp1$ may not be a required pharmacophore group at $\mu$ receptor sites, but may be essential at the $\delta$ receptor. Molecular mechanics computations were employed to study the conformational properties of JOM-6 and several of its high and moderate affinity residue 1 and 3 analogs. These residue 1 and 3 analogs, which incorporated covalently modified derivatives of Tyr and Phe, respectively, displayed reduced conformational lability as compared with the parent tetrapeptide JOM-6. Comparison of the JOM-6 and residue 1 and 3 analog conformations allowed for the determination of the probable binding conformation of the $\mu$ receptor pharmacophore groups. This receptor bound conformation requires a $\chi\sp1$ orientation of trans ($\sim$180$\sp\circ)$ for the sidechains of both Tyr$\sp1$ and Phe$\sp3.$ By integrating the structure- and conformation-affinity relationships of these tetrapeptide derivatives, a complete $\mu$ receptor pharmacophore model was derived.Ph.D.Organic chemistryPharmacy sciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/130253/2/9721994.pd