Novel ligands lacking a positive charge for the delta- and mu-opioid receptors

Recently we reported using minilibraries to replace Lys(9) [somatostatin (S RIF) numbering] of the potent somatostatin agonist L-363,301 (c[-Pro-Phe-D- Trp-Lys-Thr-Phe-]) to generate the potent neurokinin receptor (NK-I) antago nist c[-Pro-Phe-D-Trp-p-F-Phe-Thr-Phe-]. This novel cyclic hexapeptide did not bind the SRIF receptor. Thus, a single mutation converted L-363,301, a SRIF agonist with potency ca. 2-8 times the potency of SRIF in laboratory a nimals,(24) into a selective NK-1 receptor antagonist with an IC50 of 2 nM in vitro. During the screening of the same libraries for ligands of the del ta-opioid receptor, we identified four compounds (1-4) which represent a ne w class of d-opioid antagonists, some of which were also NK-1 receptor anta gonists. The most potent delta-opioid antagonist, c[-Pro-1-Nal-D-Trp-Tyr-Th r-Phe-] (2), showed a K-e value of 128 nM in the mouse vas deferens assay a nd a delta-receptor binding affinity constant of 152 nM in the rat brain me mbrane binding assay. These results are of interest because they represent a novel class of delta-opioid antagonists and, like two previously reported delta-opioid antagonists, they lack a positive charge. To examine further the requirement for a positive charge in the delta-opioid ligands, we prepa red two analogues of the beta-casomorphin-derived mixed mu-agonist/delta-an tagonist, H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-] (7), in which we eliminated the positive charge either through formylation of the primary amino group (5) o r by the deletion of this N-terminal amino group (6). These latter compound s proved to be delta-opioid antagonists with K-e values in the 16-120 nM ra nge, as well as fairly potent mu-opioid antagonists (K-e approximate to 200 nM). These six compounds provide the most convincing evidence to date that there is no requirement for a positive charge in mu- and delta-opioid rece ptor antagonists. In addition, cyclic hexapeptide 4 lacks a phenolic hydrox yl group. Taken together, these data suggest that the prevailing assumption s about delta- and mu-opioid receptor binding need revision and that the re ceptors for these opioid ligands have much in common with the NK-I and soma tostatin receptors.