Proc. Natl. Acad. Sci. USA Vol. 78, No. 10, pp. 6543-6547, October 1981 Neurobiology Specific receptor for the opioid peptide dynorphin: Structure activity relationships (myenteric plexus/opiate/endorphin/multiple opiate receptors/neuropeptide) CHARLES CHAVKIN AND AVRAM GOLDSTEIN Addiction Research Foundation and Stanford University, Palo Alto, California 94304 Contributed by Avram Goldstein, July 1, 1981 ABSTRACT The structural features responsible for the high MATERIALS AND METHODS potency and opiate receptor specificity of the opioid peptide dy- norphin in the guinea pig ileum myenteric plexus were examined. The following opioid peptides were obtained from Peninsula Successive removal of'COOH-terminal amino acids from dynor- Laboratories (San Carlos, CA): dynorphin-(1-13), dynorphin- phin-(1-13) demonstrated important contributions of lysine-13, (1-9), dynorphin-(6-13), a-N-acetyldynorphin-(6-13), lysine-11, and arginine-7 to the potency. Removal ofthe NH2-ter- [DAla2]dynorphin-(1-11), [DAla2]dynorphin-(1-13)NH2, a-N- minal tyrosine abolished the biologic activity. Several other struc- methyldynorphin-(1-13)NH2, endo-Gly'-dynorphin-(1-13), and tural modifications were shown to affect potency: substitution of [Arg8]a-neo-endorphin-(1-8). For each of these peptides, pu- D-alanine for glycine-2 reduced the potencies ofdynorphin-(1-13) rity was shown to be >98% by thin-layer chromatography, elec- amide, -(1-11), and -(1-10); and methyl esterification ofthe COOH trophoresis, and amino acid analysis, by the criteria described terminus enhanced the potencies of dynorphin-(1-12), 41-10), (1). [Leu]Enkephalin, [DAla2, Leu5]enkephalin, and -(1-9), -(1-8), and -(1-7). Within the dynorphin sequence, lysine- [Leu]enkephalin-Arg6 were obtained from Biosearch (San Ra- 11 and arginine-7 were found to be important for selectivity ofin- fael, CA). Dynorphin-(3-13) and dynorphin-(2-13)-Ser-Asp- teraction with the dynorphin receptor, which is distinguishable Asn-Gln were obtained from Peninsula Laboratories as crude from the ja receptor in this tissue. products after cleavage from the synthesizing resin. These two peptides were purified to homogeneity on C18 reverse-phase The-opioid peptide dynorphin was recently purified from por- high-pressure liquid chromatography (HPLC) as described be- cine pituitary extracts and its sequence was partially deter- low, and composition was confirmed by amino acid analysis. mined. The 13-residue peptide at its NH2 terminus, Tyr-Gly- A series of fragments was prepared with COOH-terminal Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys, was synthe- amino acids successively removed from dynorphin-(1-13) and sized and shown to contain the pharmacologically active portion from analogues with amino acid substitutions. Carboxypepti- ofthe natural material (1). In the opiate receptor bioassay using dase B (Sigma, type I, diisopropyl fluorophosphate-treated) was the guinea pig ileum myenteric plexus-longitudinal muscle used to prepare dynorphin-(1-12) and dynorphin-(1-11) from preparation, the differences between dynorphin-(1-13) and dynorphin-(1-13) and dynorphin-(1-8) from dynorphin-(1-9). [Leu]enkephalin are particularly interesting because the NH2 Trypsin (Sigma, type XI, diphenylcarbamoyl chloride-treated) terminus of dynorphin corresponds to the [Leu]enkephalin was used to prepare dynorphin-(1-7) and dynorphin-(1-6) from pentapeptide sequence. First, dynorphin-(1-13) is about 700 dynorphin-(1-13), [DA1a2]dynorphin-(1-7) and [DAa2]dynorphin- times more potent than [Leu]enkephalin; and second, the ag- (1-6) from [DAla2]dynorphin-(1-13)NH2, and endo-Gly -d onist effects ofdynorphin-(1-13) are 1/'3th as sensitive to nalox- norphin-(1-6) and endo-Gly'-dynorphin-(1-7) from endo-Gly - one antagonism (1). The higher potency of dynorphin-(1-13) dynorphin-(1-13). Prolyl endopeptidase purified from porcine indicates that the COOH-terminal extension ofthe enkephalin brain by M. Orlowski (6) was used to prepare dynorphin-(1-10) sequence [dynorphin-(6-13)] might provide additional points from and of receptor interaction. The differences in sensitivity to nalox- dynorphin-(1-11) [DAla ]dynorphin-(1-10) from one antagonism suggest that the two agonists have different [DAla2]dynorphin-(1-11). opiate receptor specificity (2); thus, the lower sensitivity ofdy- Peptides from reaction mixtures were purified by repeated norphin-(1-13) to naloxone antagonism suggested the existence passage through a C18 reverse-phase HPLC column (0.39 x 30 of a separate dynorphin receptor (1). cm, uBondapak, Waters, 15-35% linear gradient ofacetonitrile Evidence for a separate dynorphin receptor distinct from the in 5 mM trifluoroacetic acid, 1.5 ml/min) with monitoring of morphine (A) and enkephalin (8) receptors has been provided eluate at two wavelengths, 220 and 254 nm, until a single sym- by Wfister et al. (3, 4) who used the mouse vas deferens opiate metrical absorbance peak was obtained. Purity was also con- receptor bioassay. We have recently demonstrated (5) that the firmed in two different thin-layer chromatography systems dynorphin receptor is physically distinct from the ,u receptor (chloroform/methanoVacetic acid/water, 15:10:2:3, Brink- that mediates the effects ofenkephalin and normorphine in the mann Polygram 0.25-mm sil G plates; n-butanoVpyridine/ guinea pig ileum myenteric plexus. acetic acid/water, 21:12:2:15, Eastman cellulose). Concentra- In this paper we present an analysis ofpotency and receptor tions of final products were determined at 274.5 nm by using specificity in a series ofdynorphin analogues in the guinea pig Abbreviations: IC50, concentration for 50% inhibition of guinea pig ileum myenteric plexus-longitudinal muscle bioassay. ileum myenteric plexus-longitudinal muscle contraction; endo-Gly'- dynorphin-(1-13) has glycine inserted after leucine-5 in the dynorphin- The publication costs ofthis article were defrayed in part by page charge (1-13) sequence; HPLC, high-pressure liquid chromatography. payment. This article must therefore be hereby marked "advertise- Throughout this paper, unless otherwise indicated, peptide abbrevia- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. tions signify free NH2 and COOH termini. 6543 Downloaded by guest on September 26, 2021 6544 Neurobiology: Chavkin and Goldstein Proc. Nad Acad. Sci. USA 78 (1981) the tyrosine extinction coefficient, 1340, and confirmed by chain length, however, has the additional effect of introducing molar yields in amino acid analysis. Identities offragments were a negatively charged COOH-terminal acidic group into a novel confirmed by amino acid composition analysis (Dionex analyzer position in the binding site. To eliminate this confounding con- with fluorescence detection) after hydrolysis at 110°C for 24 hr tribution, we removed the negative charge from each of the in 6 M HG1 (sealed tube with argon). Mole ratios were computed peptide fragments by converting the acids to their methyl relative to arginine, with a hydrolysate. of dynorphin(1-13) or esters. [DAla2]dynorphin-(1-13)NH2 prepared and analyzed concur- The IC50 values (and confidence intervals) of the methyl-es- rently, used as standard. terified dynorphin fragments are shown at the left of Table 1. Methyl esters were prepared readily for several fragments, Potency was significantly decreased after the removal oflysine- because the terminal a-COOH is the only acidic group in the 13, lysine-il, and arginine-7. The successive reductions in po- dynorphin-(1-13) sequence. Peptide was dissolved in anhy- tency were about 50%, 25%, and nearly 99%, respectively. drous methanol equilibrated with dry HG1 gas. The reaction Because removal of the other amino acids in the sequence did mixture was fractionated on a C18 reverse-phase HPLC column not significantly affect the potency, this analysis suggests these with the gradient conditions described above. In each case the three amino acids are largely responsible for the potency en- HPLC eluate contained two absorbance peaks, one in the po- hancement, with arginine-7 making the greatest contribution. sition of the parent peptide and a second retained 1-5 min The potencies of the nonesterified dynorphin fragments are longer. This second peak, eluting as expected for the more hy- also shown in Table 1. In this series, removal ofthe COOH-ter- drophobic ester, was purified to homogeneity as before by re- minal amino acid affected potency both by removing the amino peated HPLC. The resulting peptides were shown to be methyl acid functional group and by repositioning the COOH-terminal ester derivatives by saponification; with 90% or greater recov- negative charge. As with the methyl ester dynorphin fragments, ery, the parent (free carboxylic acid) peptide was generated. relative potency was significantly decreased by removal of lys- Potencies ofthe peptides were evaluated by using the guinea ine-13, lysine-li, and arginine-7. Here, however, removal of pig ileum myenteric plexus-longitudinal muscle bioassay as leucine-12 and arginine-6 had the opposite effect of increasing described (7). The concentration of each peptide required to the potency, an effect not seen in the methyl ester series. inhibit the muscle twitch by 50% (IC50) was determined by test- The contribution of the COOH-terminal negative charge to ing at three or more concentrations giving 20-80% inhibition these potency changes can be estimated by comparing the po- and then interpolating ICG5 by log-linear regression analysis. tencies of the COOH-terminal acids and methyl esters of each Agonist effects of all the peptides were reversed by naloxone. peptide. As shown in the last column ofTable 1, methyl ester- Naloxone sensitivity was determined by measuring IC50 in the ification did not significantly change the IC50 of dynorphin- presence and absence of 100 nM naloxone. Peptides were di- (1-13), -(1-11), -(1-6), or -(1-5). However, the potencies ofdy- luted in MeOH/0. 1 M HCl, 1:1 (vol/vol), which, in doses less norphin-(1-12), -(1-10), -(1-9), -(1-8), and -(1-7) were enhanced than 20 ,ul in the 5-ml bath, has no effect on the amplitude of 7- to 13-fold, indicating that the COOH-terminal negative contraction.