"DAKLI": a multipurpose ligand with high affinity and selectivity for dynorphin (kappa opioid) binding sites.

Abstract

We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as '25I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (I opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.To label one type of receptor binding site in the presence of related types, a radioligand should (i) have very high affinity for the site to be labeled, (ii) be highly selective for that site, (iii) have high enough specific radioactivity to be usable at a low concentration to maximize its binding at the preferred site relative to that at the next-preferred site, (iv) for convenience incorporate a radioisotope such as 125I with a high-energy mode of decay.Dynorphin A (Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-ProLys-Leu-Lys-Trp-Asp-Asn-Gln) is a 17-residue endogenous opioid peptide with selectivity for the K opioid receptor (1-3). Dynorphin A-(1-13) is as potent and as selective for the K binding site as is the natural full-length peptide (4), so we surmised that this fragment could be modified safely by extension at the C terminus. For flexibility of functionalization with various reporter groups, we used a primary amino function at the C terminus as a handle. Lysine-11 and lysine-13 (especially the former) play essential roles in determining K affinity and selectivity (5). To abolish the reactive E-amino groups, yet retain the charges at these two positions, we replaced lysine with arginine. Our C-terminal extension consisted of glycine-14, followed by 1,5-diaminopentane. The resulting peptide is called "DAKLI" (Dynor-After synthesis of the peptide by the solid-phase method and its removal from the resin by aminolysis with 1,5-diaminopentane, the free a-amino group of tyrosine-1 remains blocked by the t-butoxycarbonyl (Boc) group and the only reactive amine is at the extended C terminus. This feature allows the facile production, from [Boc] 25 -22°(c 0.4, AcOH). Reversed-phase analytical HPLC (as above but 4 x 250 mm; particle size, 5 ,um; gradient, 10-50% CH3CN; 0.03 M in NH4OAc, pH 4.5; A = 280 nm) showed the product to be 90% pure, and the amino acid analysis after 6 M HCl hydrolysis (18 hr, 110°C) gave ratios in accord with the desired structure: Pro, 1.1 (1); Gly, 2.9 (3); Ile, 0.9 (1); Leu, 2.0 (2); Tyr, 1.3 (1); Phe, 1.1 (1); Arg, 4.9 (5). The expected amino acid sequence was obtained on an Applied Biosystems (Foster City, CA) 470A microsequencer (K. Jarnagin, Syntex Research).Preparation of lodinated Peptides DAKLII and DAKLII**. BH (Sigma) was iodinated by the chloramine-T method as follows: to 5 mg of dry BH was added 50 mg of NaI in 40 ,ul of 50 mM sodium phosphate buffer, followed immediately by 0.5 ml of chloramine T (30 mg/ml in 250 mM sodium phosphate buffer). The reaction was terminate...

Microinjection of Dynorphin into the Supraoptic and Paraventricular Nuclei Produces Antidiuretic Effects through Vasopressin Release

“…In this study, we observed that microinjection of the three K-agonists, DYN, U50,488H and DAKLI, into the PVN produced antidiuretic effects with the potency order of DAKLI > DYN > U50,488H. This order is consistent with the one for K-receptor binding affinity (30). In the PVN, the DYN-induced antidiureses was partially inhibited by a high dose of NAL.…”

“…This is probably explained by the low selectivity and affinity for K-receptors of NAL. DAKLI is one of the highest affinity ligands for K-receptors (30).…”

Chimeric opioid peptides: tools for identifying opioid receptor types.

“…Goldstein et al (6) demonstrated that dynorphin peptides, at low concentrations (<1 nM), were completely degraded by guinea pig brain membranes (10 mg/ml, based on initial wet weight of brain) in a very short incubation time. Diluting the membranes could reduce the extent of degradation, but this procedure may be impractical in a binding assay.…”

“…It has been known that the N-terminal 13 residues of the endogenous K ligand dyn A are necessary and sufficient for affinity and specificity (9). dyn A-analogue kappa ligand (DAKLI), a dyn A-derived peptide ligand designed on this principle, has high affinity and selectivity for K opioid receptors; and biotinylated DAKLI, even after it is bound to the receptors, can bind avidin (6). In the present study, the C-terminal amide group of dermorphin, deltorphins, and DSLET, as well as the alcohol group of DAGO were removed, and a 23-residue C-terminal extension was added.…”

“…[D-Pen2, D-Pen5]enkephalin (DPDPE) (3) and deltorphin I and II (4) have similar properties with respect to 8 sites, as do dynorphin A (dyn A) (5) and its derivatives (6) for K sites. Each of these peptides has a subnanomolar dissociation constant at its preferred binding site, at least two orders of magnitude greater affinity than at its next-preferred binding site.…”