Structureactivity Relationship

Despite a common N-terminal tripeptide (Tyr-D-Xaa-Phe), the two groups of opioid peptides, dermorphins and deltorphins, differ enormously in receptor selectivities but bind to their own receptors with similar affinities. The N-terminal domain contains the minimum sequence essential for binding to opioid receptors whereas the C-terminal domain contains the address requisites for receptor selectivity.

The N-terminal tetrapeptides of D-Met-deltorphin and D-Ala-deltor-phins did not show preference for delta receptors over mu receptors. The common determinants concurring to the remarkably efficient targeting of deltorphins towards the delta receptors were identified through structure-activity relationship studies conducted on an extensive series of synthetic analogues. The following structural requirements explain why the deltorphins are such potent and selective delta agonists: a phenolic side chain (Tyr) and a protonated nitrogen at the N-terminus; a D-isomer in the second position, which restricts peptide conformation and confers biological stability; a second aromatic center (Phe), common to all opioid peptides; an anionic residue (Asp7 in D-Met-deltorphin; Asp4 and Glu4 in D-Ala-deltorphins) in the C-terminal tetrapeptide, which increases electrostatic ligand repulsion by the negatively charged mu receptor and electrostatic binding to the positively charged site of the delta receptor; and a hydrophobic region associated with residues in the C-terminal address domain [1,24].

Recent site-directed mutagenesis and chimeric receptor molecules indicated that specific 5-opiate receptor binding requires receptor amino acid sequences 291-300, namely the terminal portion of extracellular loop 3 and the initial sequence of transmembrane segment VII, where Arg292 could actively participate in electrostatic binding to ligand anionic residues [25]. The hydroxyl group of Tyr in the peptide could form a hydrogen bond with transmembrane II Asp95 [26] or transmembrane III Asp128 [27].

The tertiary structure of deltorphin assessed by combined use of nuclear magnetic resonance (2D NMR) and spectroscopy in DMSO, indicated a common S-shaped arrangement in the deltorphin N-terminal peptide. This structure contains a type II'h-turn in which D-Xaa2 lies sandwiched between h Tyrl and Phe3 in a trans configuration. The folded C-terminal tail comes into close contact with the tripeptide amino end (unlike the linear, flexible C-terminus of the mu agonist dermorphins) and places the Tyrl and Phe3 aromatic rings in definite orientations that are best suited for the delta-receptor. Moreover, Bryant et al. indicated a similar extended tertiary architecture for D-Met-deltorphin and D-Ala-deltorphin-I but unique compact topographies for D-Ala-deltorphin-II [28-31].

In the D-Met-deltorphin molecule, substitution of the positively charged His4 by a variety of amino acids is generally detrimental. An aliphatic side chain and L-isomer at the fifth residue appear critical for activity, but the C-terminal residue in the sixth and seventh position can generally be replaced by other amino acids with only marginal effects [32].

While D-Ala-deltorphins have delta-binding affinity similar to D-Met-deltorphin (0.3-2.0 nM), they consistently have the highest delta-opiate selectivity. The rank order of selectivity (Ki5/Ki|i) is D-Ala-deltorphin-I = D-Ala-deltorphin-II (3000-4000)>D-Met-deltorphin (700) >>D-Ile-deltor-phin (100) >> D-Leu-deltorphin heptadecapeptide or its N-terminal deca-peptide fragment.

The high delta-selectivity of D-Ala-deltorphins can be attributed to their C-terminal tetrapeptide sequence in which the anionic residue plays an important role. Elimination of the charge at the fourth position normally results in opiates that have similar delta and mu affinity and generally lack selectivity. Substitution of Gly in the fourth position would permit D-Ala-

deltorphins to assume a more extended conformation, dramatically increasing their A-affinity and potency. The hydrophobic qualities of the residues at the fifth and the sixth positions (Val5-Val6) are crucial in maintaining the affinity and selectivity of D-Ala-deltorphins, as evidenced in peptide analogues in which the aliphatic quality of the side chain was enhanced [33-36].

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