β-Endorphin. Biological activity of synthetic analogs with analgesia inhibiting property in rat vas deferens and guinea pig ileum assays

Three synthetic analogs of human β-endorphin (β_h-EP) (I, [Gln⁸, Gly³¹]-β_h-EP-Gly-Gly-NH₂; II, [Arg^{9,12,24,28,29}]-β_h-EP and III, [Cys^11,26, Phe²⁷, Gly³¹]-β_h-EP), which have been shown to possess potent inhibiting activity to β_h-EP-induced analgesia, were assayed in rat vas deferens and guinea pig ileum bioassay systems. In the rat vas deferens assay, relative potencies of these analogs were β_h-EP, 100; I, 30; II, 40; III, 1, whereas in the guinea pig ileum assay: β_h-EP, 100; I, 184; II, 81; III, 163. From previous studies on their analgesia potency in mice and opiate receptor-binding activity in rat brain membranes, their activity in rat vas deferens correlates well with the analgesic potency and the activity from guinea pig ileum assay shows good correlations with that from the opiate receptor-binding assay.     

HUMAN β-ENDORPHIN: SYNTHESIS AND BIOLOGICAL ACTIVITY OF ANALOGS WITH SUBSTITUTIONS IN POSITIONS 2, 9, 18, 27 AND 31

Four analogs of the opioid peptide human β-endorphin (β_h-EP) have been synthesized: [d -Lys⁹,Phe²⁷,Gly³¹]-β_h-EP, [d -Phe¹⁸,Phe²⁷,Gly³¹)-β_h-EP, [d -Thr²,d -Lys⁹,Phe²⁷,Gly³¹]-β_h-EP, and [d -Thr²,d -Phe¹⁸,Phe²⁷,Gly³¹]-β_h-EP. All are practically indistinguishable from β_h-EP in the guinea pig ileum assay. All show diminished analgesic potency in the mouse tail-flick assay.     

β-ENDORPHIN: SYNTHESIS OF ANALOGS WITH EXTENSION AT THE CARBOXYL TERMINUS WITH HIGH RADIORECEPTOR BINDING ACTIVITY

Four analogs of human β-endorphin (β_h-EP) have been synthesized: [Gly³¹]-β_h-EP-Gly-NH₂, [CH₃(CH₂)₄NH³¹₂]-β_h-EP, [Gly³¹]-β_h-EP-Gly-Gly-NH₂, and [Gln⁸, Gly³¹]-β_h-EP-Gly-Gly-NH₂. All are more active than β_h-EP in an opiate receptor binding assay. Stepwise extension at the COOH-terminus shows a progressive increase in binding activity. The last analog, which combines extension at the COOH-terminus with elimination of the remaining anionic charge in β_h-EP, is nine times more active than the parent molecule.     

Human β-endorphin

Three analogs of human β-endorphin (β_h-EP) were synthesized by the solidphase method: [Gln⁸, Trp²⁷]-β_h-EP (I), [Gln⁸,Arg⁹,Trp²⁷]-β_h-EP (II), and [Gln⁹,Arg¹¹,Trp²⁷]-β-EP (III). Radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: β_h-EP, 100; I, 778; II, 467; III, 449. Relative potencies in an analgesic assay were: β-EP, 100; I, 114; II, 165; III, 83. The 8–11 segment of β_h-EP can tolerate a net increase in charge of + 2 without diminishing analgesic potency. The substitution of Glu⁸ may be one of the more dependable means of designing β-endorphin antagonists.     

β-Endorphin: Synthesis and properties of analogs with replacement of lysine residues by arginine

Human β-endorphin analogs, [Arg^{9,19,24,28,29}]-β-endorphin (I) and [Arg^24,28,29]-β-endorphin (II), have been synthesized by the solid-phase method. Peptide II had 86% of the analgesic potency and 216% of the receptor binding activity of the parent molecule. Peptide I had only 18% analgesic potency but its binding activity was more than three time greater than that of human β-endorphin.     

Synthesis and properties of dermorphin and an analog of β-endorphin containing the dermorphin sequence

Dermorphin (I) and [D-Ala², Phe³, Gly⁴, Tyr⁵, Pro⁶]-β_c-EP (II) have been synthesized by the solid-phase method (β-EP, camel β-endorphin). Positions I through 7 of II correspond to the sequence of I. Relative potencies of synthetic peptides in the mouse tail-flick test for analgesia by the intracerebroventricular route were: human β-endorphin, 100; camel β-endorphin, 164; I, 450; II, 440. The dermorphin was about 670 times more potent than morphine in the assay. Peptide II represents a rare instance where the enkephalin moiety of β-endorphin has been altered to produce a more potent analgesic.     

β-Endorphin: synthesis and properties of analogs modified in positions 8 and 31

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: [Gln^8.31]-β_h-EP(I), [Arg⁸, Gln³¹] -β_h-EP(II), [Ala⁸, Gln³¹] -β_h-EP (III), and [Val⁸, Gln³¹]-β_h-EP(IV). Radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: β_h-EP, 100; I, 200; II, 150; III, 150; IV, 120. Relative potencies in an analgesic assay were: β_h-EP, 100; 1,236; II, 254;III, 116; IV, 121. The side-chain of Glu-8 in β_h-EP can be replaced by a variety of structures without diminishing biological activity.     

Preparation and properties of tritiated human [Tyr 18, Trp 27]-β -endorphin

Tritiated [Tyr 18, Trp 27]-βh-EP was prepared from the corresponding diiodotyrosine derivative by catalytic reduction in the presence of carrier free tritium gas. A photoaffinity probe for β-endorphin (β-EP) receptors was prepared by selective modification of [Tyr¹⁸, Trp²⁷]-β_h-endorphin with 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) under acidic conditions to yield [Trp¹⁸-2,4-NAPS-Trp²⁷]-β_h-endorphin (NAPS-β-EP). NAPS-β-EP was purified by high performance liquid chromatography and characterized by ultraviolet absorption spectroscopy and peptide mapping. Tritiated NAPS-β-EP was prepared from tritiated [Tyr¹⁸, Trp²⁷]-β_h-endorphin with 2,4-NAPS-Cl. The ability of NAPS-β-EP to form covalent bonds to macromolecules due to photolysis was established using bovine serum albumin. The efficiency of photolytic cross-linking was 15% and the equilibrium dissociation constant was 1.3 times 10^-5 M.     

Synthesis and properties of human β-endorphin-(1–17) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–17) (I), [D-Ala²]-β_h-EP-(1–17) (II), [Gln⁸]-β_h-EP-(1–17) (III) and [D-Ala², Gln⁸]-β_h-EP-(1–17) (IV). Measurement in a radio-receptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 33; II, 47; III, 889; IV, 123; β_h-endorphin, 2253.     

Chemical synthesis of human β-endorphin(1–27) analogs by peptide segment coupling

[Gly⁸]β_hEP(1–27)NH₂ and [l -Leu⁸]β_hEP(1–27)NH₂, two analogs of human β-endorphin, were synthesized by both all-stepwise solid phase synthesis and peptide segment coupling. For the peptide segment coupling method, two thiocarboxyl peptides, Msc-[Gly⁸]β_hEP(1–8)SH and Msc-[l -Leu⁸]β_hEP(1–8)SH, were synthesized by standard solid phase method on 4-[α-(Boc-Gly-S)benzyl]phenoxyacetamidomethy-resin and 4-[α-(Boc-l -Leu-S)benzyl]phenoxyacetamidomethy-resin. These two thiocarboxyl peptides were coupled to H-[Lys(Cit)^{9, 19, 24}]-β_hEP(9–27)NH₂ [Gly⁸]β_hEP(1–27)NH, and [l -Leu⁸]β_hEP(1–27)NH₂ were obtained after removal of Msc groups and citraconyl groups from products of the segment coupling reaction. The yields of both [Gly⁸]β_hEP(1–27)NH₂ and [l -Leu⁸]β_hEP(1–27)NH₂ in the segment coupling reaction were approximately 18%. Less than 1 % of racemization of Leu-8 occurred during coupling of Msc-[l -Leu⁸]β_hEP(1–8)SH to H-[Lys(Cit)^{9, 19, 24}]-β_h EP(9–27)NH₂. Results of amino acid composition analysis, analysis by reverse phase high pressure liquid chromatography and receptor binding activity assays of the analogs showed that peptide analogs prepared by segment coupling method and those prepared by all-stepwise solid phase synthesis were identical. Results of receptor binding activity assays suggested that the molecular charge properties of β-endorphin(1–27) and its analogs influenced the receptor binding activity.     

THE SYNTHESIS AND OPIATE ACTIVITY OF HUMAN β-ENDORPHIN ANALOGS

Four analogs of human β-endorphin have been synthesized and their opiate activities have been determined. One of the analogs, [27-phenylalanine, 31-glycine]-β_h-endorphin, was shown to possess an analgesic potency greater than that of the natural peptide.     

β-Endorphin

Three β_h-EP analogs which show different extents of alteration in analgesic potency by substitution of a single amino acid residue were assayed for their peripheral opioid activity and the binding to opioid μ-receptor to determine the relationships among the opioid activities obtained from different assays. In the guinea pig ileum assay, [Gln⁸]-β_h-EP showed a higher inhibitory activity than the parent peptide. [Tyr³¹]-analog had the same potency as β_h-EP, while [Trp²⁷]-analog retained only one fourth the potency of β_h-EP. Assayed on the vas deferens of the mouse and the rat, all three substituted β_h-EP analogs exhibited a lower potency than their parent peptide. Receptor binding assay using [³H]-dihydromorphine as the primary ligand showed that [Gln⁸]-analog had a binding potency 1.5-fold that of β_h-EP, while the potencies of [Tyr³¹]- and [Trp²⁷]-analogs were not significantly different from that of the parent peptide. No correlation in relative potency was found between vas deferens assays and their μ-receptor binding or analgesic activity. However, the relative potencies of binding to μ-receptor in [Gln⁸]- and [Tyr³¹]-analogs were found to be consistent with those of analgesic and guinea pig ileum assays, whereas the binding to β-EP receptor of all analogs appeared to be related to the charge properties of β-EP molecule.     

β-Endorphin: synthesis and properties of human β-endorphin-(1–28) and its analogs

Three analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–28) (II), [D-Ala², Gln⁸] - β_h-EP-(1–28) (III). Radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: β_h-EP, 100; I, 85; II, 380; III, 146. Relative potencies in an analgesic assay were: β_h-EP; 100; I, 18; II, 36; III, 13.     

Synthesis and properties of human β-endorphin-(1–9) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–9) (I), [D-Ala²]-β_h-EP-(1–9) (II), [Gln⁸]-β_h-EP-(1–9) (III), and [D-Ala², Gln⁸]-β_h-EP-(1–9) (IV). Measurement in a radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 76; II, 100; III, 200; IV, 200. Two new amino acid derivatives were prepared and used for synthesis of the analogs: N^α-t-butyloxycarbonyl-O-(cyclopentyl) -tyrosine and N^α-t-butyloxycarbonyl-γ-(cyclopentyl)-glutamic acid.     

Differential involvement of disulfide bridges on the folding of a scorpion toxin

Leiurotoxin I is a neurotoxin, blocker of Ca²⁺-activated apamin-sensitive K⁺ channel, purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. It is a 31-residue polypeptide reticulated by three disulfide bridges, i.e. Cys³-Cys²¹, Cys⁸-Cys²⁶ and Cys¹²-Cys²⁸. To investigate the role of these disulfide bridges in the folding of this toxin, analogs lacking one disulfide bridge were synthesized. The structures of two analogs in which two half-cystines were replaced by α-aminobutyrate residues to suppress one disulfide bridge, were analyzed by ¹H NMR. The NMR studies reveal a three-dimensional structure identical with the native toxin for the analog lacking disulfide bridge Cys³-Cys²¹ and a loss of organized structure for another analog lacking disulfide bridge Cys¹²-Cys²⁸. These analogs are, respectively, fully active and only weakly active (2% of the residual activity) when tested in vitro for their ability to interact with their receptor channel and in vivo for their neurotoxic activity in mice. This suggests that disulfide bridge Cys¹²-Cys²⁸ is essential for the folding process. In contrast, the lack of disulfide bridge Cys³-Cys²¹ does not affect the folding and the maintenance of bioactive conformation of Leiurotoxin I.     

β-Endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays

Biological activity of synthetic β-endorphin (β-EP) analogs containing dermorphin or dynorphin-A-(1 – 13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH₂-terminal 1–7 segment of camel β-EP [β_c-EP-(1–7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1–7)] -β_c-EP was 120 times more potent than β_c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH₂-terminal 1–13 segment of human β-EP [β_h-EP-(1–13)] with dynorphin-A-(1–13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1–13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that β_c-EP-(8–31) facilitates the dermorphin moiety to act on opiate μ and δ receptors but not on the ± receptor, while β_h-(14–31) reduces the action of dynorphin on μ, δ and k receptors.     

β-Endorphin

A double-headed analog of human β-endorphin (β_h-EP), N, N'-bis (β-endorphinyl)-cystine (II), has been synthesized by the solid-phase method, along with β_h-EP-Cys(CH₂CONH₂)-OH (I) and (Tyr³¹]-β_h-EP (III). Their relative potencies in a radioreceptor-binding assay were: B_h-EP, 100; II, 235; I, 170; and III, 204. In the tail-flick test for analgesic activity their relative potencies were: β_hEP, 100; II, 86; I, 93; and III, 116.     

Enkephalin analogs: synthesis and properties of analogs with lipophilic or extended carboxyl-terminus. Quantitative structure-activity relationship of analogs modified in residue position 5

Four analogs of enkephalin (EK) have been synthesized by the solid-phase method and their biological activities have also been investigated. All four analogs were less active than Met-enkephalin (Met-EK) as shown by relative potencies in the guinea pig ileum (GPI) assay: Met-EK, 100; [Phe⁵]-EK-NH₂, 59; [Trp⁵]-EK-NH₂, 11; Met-EK-Cys(Cam)-OH, 37; and N, N'-bis(Met-EK)-cystine, 34. Two of the analogs were more potent than Met-EK as shown by relative potencies in the mouse tail-flick assay for analgesia: Met-EK, 100; [Phe⁵]-EK-NH₂, 1340; [Trp⁵]-EK-NH₂, 1640. Quantitative structure-activity relationship calculations were carried out for GPI potencies of analogs substituted in position 5. The calculation indicated that, in this position, the bulkiness had the main influence.     

1H n.m.r. investigation of conformational features of cyclic enkephalinamide analogs

The conformational basis for the differing opioid receptor selectivities of the cyclic cystine-containing analogs, [D-Cys², D(or L)-Cys⁵] enkephalinamide and the related penicillamine-containing analogs, [D-Pen², D(or L)-Cys⁵] enkephalinamide (penicillamine =β,β dimethylcysteine) was investigated by ¹H n.m.r. in aqueous solution. Comparison of chemical shift, temperature dependence of amide proton chemical shift, and coupling constant data suggests similar overall conformations for corresponding penicillamine- and cystine-containing analogs. Differences in conformation and flexibility do appear in the carboxamide terminal region of the corresponding analogs, which may account for their selectivities for different classes of opioid receptors.     

ß-endorphin: synthesis and properties of analogs modified at position 6

Two analogs of ß-endorphin (ß-EP) were synthesized by the solid-phase method: [D-Ser⁶]-ß_c-EP (I) and [Pro⁶]-ß_c-EP (II)(ß_c-EP = camel ß-EP). Radioreceptor binding assay with use of tritiated human ß-EP (ß_h-EP) as primary ligand gave relative potencies as follows: ß_c-EP, 100; I, 32; II, 28. Relative potencies in an analgesia assay were: ß_c-EP, 100; I, 115; II, 31.