Structure–activity relationships of arodyn,a novel acetylated kappa opioid receptor antagonist*,†

Abstract: We previously reported that the novel dynorphin A (Dyn A, Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐Arg‐Ile‐Arg‐Pro‐Lys‐Leu‐Lys‐Trp‐Asp‐Asn‐Gln) analog arodyn (Ac[Phe^1,2,3,Arg⁴,d ‐Ala⁸]Dyn A‐(1–11)NH₂, Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002) J. Med. Chem. vol. 45, pp. 5617–5619) is a κ opioid receptor‐selective peptide [K_i(κ) = 10 nm , K_i ratio (κ/μ/δ) = 1/174/583] which exhibits antagonist activity at κ opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure–activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric d ‐amino acid substitution in the N‐terminal ‘message’ sequence, NMePhe substitution individually in positions 1–3, and modifications in position 1. The results for the Ala‐substituted derivatives indicated that Arg⁶ and Arg⁷ are the most important residues for arodyn's nanomolar binding affinity for κ opioid receptors. Ala substitution of the other basic residues (Arg⁴, Arg⁹ and Lys¹¹) resulted in lower decreases in affinity for κ opioid receptors (three‐ to fivefold compared with arodyn). Of particular interest, while [Ala¹⁰]arodyn exhibits similar κ opioid receptor binding as arodyn, it displays higher κ vs. μ opioid receptor selectivity [K_i ratio (κ/μ) = 1/350] than arodyn because of a twofold loss in affinity at μ opioid receptors. Surprisingly, the Tyr¹ analog exhibits a sevenfold decrease in κ opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr¹]arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing κ opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe¹]arodyn which exhibits high affinity [K_i(κ) = 4.56 nm ] and exceptional selectivity for κ opioid receptors [K_i ratio (κ/μ/δ) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nm Dyn A‐(1–13)NH₂. Thus [NMePhe¹]arodyn is a highly κ opioid receptor‐selective antagonist that could be a useful pharmacological tool to study κ opioid receptor‐mediated activities.     

Synthesis and biological activity of dynorphin-(1 - 13) and analogs substituted in positions 8 and 10

Dynorphin-(1–13) (Dyn-(1–13)) and various analogs substituted in positions 8 and 10 were synthesized by the solid-phase technique and analyzed for their ability to inhibit the electrically evoked contraction of the guinea pig ileum (GPI) and to compete with the binding of [³H]-ethylketocyclazocine (EKC, k ligand), [³H]-[D-Ala², MePhe⁴-Gly-ol⁵]-enkephalin (DAGO, μ ligand) and [³H]-[D-Ser², Thr⁶]-Leu-enkephalin (DSLET, δ ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Introduction of Ala in position 8 decreased the activity of the peptide on the GPI by 50% but induced a 2.22-fold increase in its affinity for the k receptor ([³H]-EKC binding displacement from guinea pig cerebellum; Ki of 0.05 nM as compared with 0.11 nM for Dyn-(1–13)). On the other hand, the ability of [Ala⁸]-Dyn-(1–13) to displace the binding of [³H]-DSLET from rat brain membranes was decreased by a factor of 1.7 while its affinity for the μ receptor was not greatly affected ([³H]-DAGO displacement; Ki of 0.44nM as compared with 0.50nM for Dyn-(1–13)). Replacement of position 8 by D-Ala caused similar changes in the activity of the peptide but the increase in its affinity for the k site was somewhat smaller (Ki of 0.08 nM as compared with 0.11 nM). [D-Pro¹⁰]-Dyn-(1–13) was equipotent to [Ala⁸]-Dyn-(1–13) in the GPI but its affinity for the μ binding site was decreased by a factor of 2.7 as compared with Dyn-(1–13). The affinity of [D-Pro¹⁰]-Dyn-(1–13) for the other binding sites (k and δ) was not greatly affected. Replacement of positions 8 or 10 by Trp or D-Trp decreased the activity on the GPI by more than 50% as well as the affinity for most receptor types. These data indicate that the selectivity of Dyn(1–13) for the k opioid receptor can be increased either by increasing its affinity for the K binding site ([Ala⁸]-Dyn-(1–13)) or lowering its potency for other binding sites, [Ala⁸]-Dyn-(1–13) being less potent on δ sites and [D-Pro¹⁰]-Dyn-(1–13) less potent on μ sites.     

The 10th and 11th residues of short length N‐terminal PTH(1–12) analogue are important for its optimum potency

Abstract: The 10th and 11th residues of parathyroid hormone PTH(1–12) analogues were substituted to study the structure and function of PTH analogues. The substitution of Ala¹⁰ of [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1–12)NH₂ with Glu¹⁰ and/or the Arg¹¹ with Ile¹¹ markedly decreased cAMP generating activity. Data from circular dichroism (CD) and the nuclear magnetic resonance (NMR) structural analysis of [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1–12)NH₂ revealed tight α‐helical structures, while the Glu¹⁰ and/or Ile¹¹ substituted analogues showed unstable α‐helical structures. We conclude that 10th and 11th residues are important for stabilizing its helical conformation and that destabilization of the α‐helical structure, induced by substituting the above residues, remarkably affect its biological potency.     

Synthetic linear and cyclic glucagon antagonists

The synthesis and biological activities of seven new glucagon analogues are reported. The design of com- pounds 2-5 is based on potent antagonists recently reported from this laboratory, where we have focused on modifications in the N-terminal region. In this report we have concentrated specifically on modifications to histidine-1. In addition we have prepared two cyclic compounds 7 and 8 , related to a linear in vivo antagonist [Glu⁹]glucagon, reported by Medeld (Unson et al. (1987) Proc. Natl. Acad. Sci. USA 84 , 4083-4087). The N-terminal modifications involved substitution of His¹ by the unnatural conformationally constrained residue (S)-5,6,7,8-tetrahydro-5-oxoimidazo(1,5-c)pyrimidine-7-carboxylic acid (Toc), desaminohistidine (dHis) and 3-(4-nitrobenzyl)histidine. The structures of the new compounds are as follows. [Toc¹,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17,18,Glu²¹]glucagon ( 2 ); [Toc¹,d Phe⁴,Tyr⁵,Arg¹²,Lys^17,18,Glu²¹]glucagon amide ( 3 ); [3-(4-nitrobenzyl)His¹,d Phe⁴,Tyr⁵,Arg¹²,Lys^17,18,Glu²¹]glucagon ( 4 ); [dHis¹,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17,18,Glu²¹]glucagon ( 5 ); [dHis¹,Glu⁹]glucagon ( 6 ); (desHis¹)glucagon amide ( 7 ); (desHis¹)-glucagon amide ( 8 ). The binding potencies of the linear analogues, as expressed a percentage of glucagon binding, are 2.6 ( 2 ), 0.13 ( 3 ), 0.8 ( 4 ), 0.8 ( 5 ), 2.2 ( 6 ). Both cyclic analogues 7 and 8 show biphasic binding curves. The IC₅₀ values for 7 at the high and low finity sites are 1.5 and 167 nm , respectively (IC₅₀ of glucagon = 1.3 nm ). The IC₅₀ values for ₅₀ at the high and low affinity sites are 4.7 and 3451 nm , respectively. The cyclic analogues are characterized by fast atom bombardment mass spectrometry of endoproteinase ASP-N digests. The specificity of the enzyme used in these studies enables differentiation of isomers of the cyclic glucagon analogues which differ only in the position of cyclic amide bond. Analogues 2,3 and 5–8 are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase (AC) system. Analogue 4 is a partial agonist (5.7% compared to glucagon) of AC. Introduction of unusual amino acids which do not contain a primary α-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases.     

β-ENDORPHIN: SYNTHESIS AND MORPHINE-LIKE ACTIVITY OF ANALOGS WITH D-AMINO ACID RESIDUES IN POSITIONS 1, 2, 4 AND 5

The solid-phase syntheses of [D-Tyr¹]-, [D-Ala²]-, [D-Phe⁴]-, and [D-Met⁵]-β_c-endorphins are described. A comparison of certain methods of purification and criteria of homogeneity is made with the use of these compounds. Bioassay of these synthetic analogs both in vitro and in vivo show that [D-Ala²]-β_c-endorphin possesses significant opiate activity whereas the other analogs have minimal activity.     

Cyclic morphiceptin analogs: cyclization studies and opioid activities in vitro

Attempts were undertaken to develop cyclic β-casomorphin-5 analogs with improved opioid activity profiles by deletion of the glycine residue in position 5, leading to analogs structurally related to the opioid peptide morphiceptin (H-Tyr-Pro-Phe-Pro-NH₂). The tetrapeptide sequence Boc-Tyr(tBu)-D-Xaa-Phe-Yaa-OH (Xaa = Lys, Orn, A₂bu; Yaa = Pro in L- or D-configuration) was used to study the influence of ring size and chirality on the yield of cyclization between the ω-amino group of Xaa and the C-terminal carboxyl group. In all cases the cyclization reaction was performed under identical experimental conditions to allow a direct comparison with regard to yield and homogeneity. The reaction products were purified by crystallization and liquid chromatography, and were characterized by HPLC, TLC, electrospray mass spectrometry and ¹H-NMR spectroscopy. In none of the reactions performed with the cyclization precursors containing proline in the L-configuration could a cyclic monomer be detected, and the cyclodimer ( 7–9 ) was the exclusive product in each case. Cyclodimerization was also the favored reaction in the attempted formation of the 11-membered ring of the cyclic [D-A₂bu², D-Pro⁴]-morphiceptin analog 12 , since only traces of the monomer were found. In the case of both the [D-Lys², D-Pro⁴]-analog 10 and the [D-Orn², D-Pro⁴]- analog 11 , the cyclomonomer/cyclodimer ratio was about 80: 20. The cyclic monomers 10 and 11 showed high opioid activity in the μ-receptor-representative guinea pig ileum assay (IC₅₀= 2–5 nM) and in the δ-receptor representative mouse vas deferens assay (IC₅₀= 50–60 nM), whereas the potency of the cyclodimers was 2–3 orders of magnitude lower in both in vitro bioassays.     

Influence of 1‐aminocyclohexane‐1‐carboxylic acid in position 2 or 3 of AVP and its analogues on their pharmacological properties

Abstract: In this study we describe the synthesis and some pharmacological properties of seven new analogues of arginine vasopressin (AVP) substituted in position 2 or 3 with 1‐aminocyclohexane‐1‐carboxylic acid (Acc). All peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities. The Acc³ modifications of AVP, dAVP, [d ‐Arg⁸]VP and [Cpa¹]AVP have been found to be deleterious for interaction with all three neurohypophyseal hormone receptors, as judged from the several orders of magnitude decreased biological activities, whereas Acc² substitution selectively altered the interaction with the receptors. Two of the new analogues, [Acc²]AVP and [Acc², d ‐Arg⁸]AVP, are potent antidiuretic agonists. [Acc²]AVP exhibits moderate pressor agonistic activity and weak antiuterotonic properties. [Acc², d ‐Arg⁸]AVP has been found to be a weak antagonist in the pressor and uterotonic tests. Another analogue – [Cpa¹, Acc³]AVP – turned out to be a highly selective V₂ agonist. This is an unexpected effect, as its parent peptide, [Cpa¹]AVP is a very potent V_1a receptor antagonist. This is the first Cpa¹ modification to have resulted in V₂ agonism enhancement. Besides providing useful information about structure–activity relationships, our results could open up new possibilities in the design of highly potent and selective V₂ agonists.     

Synthetic glucagon antagonists and partial agonists

This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N-terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C-terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys^17.18,Glu²¹]glucagon,¹ a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [d -Phe⁴,Tyr⁵,Arg¹²]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N-terminal modifications involved substitution of His¹ by the unnatural conformationally constrained residue, 4,5,6,7-tetrahydro-1H-imidazo[c]pyridine-6-carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe⁶ residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip¹, Lys^17.18,Glu²¹]glucagon (2); [Tip¹,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17.18,Glu²¹ glucagon (3); [dHis¹,d -Phe⁴,Tyr⁵,Arg¹², Lys^17.18,Glu²¹ glucagon (4); [dHis¹,Asp³,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17.18,Glu²¹]glucagon (5); des-Phe⁶-[Tip¹,D-Phe⁴,Tyr⁵Arg¹²,Glu²¹ glucagon (6); des-Phe⁶-[Asp³,d -Phe⁴,Tyr⁵,Arg¹²,Glu²¹]glucagon (7) The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3-7 are antagonists unable to activate the AC system even at concentrations as high as 10^?5m . In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve. Hence these compounds are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase system.     

N-Methylated Cyclic Enkephalin Analogues Retain High Opioid Receptor Binding Affinity

In an effort to improve the bioavailability of the non-selective, cyclic enkephalin analogues H-Dmt-c[d -Cys-Gly-Phe-d (or L )-Cys]NH₂ (Dmt = 2′,6′-dimethyltyrosine), analogues N-methylated at the Phe⁴ and/or Cys⁵ residue were synthesized. In comparison with the non-methylated parent peptides, all mono- and N-di-methylated analogues in general retained high binding affinities at all three opioid receptors and high opioid agonist potencies in functional opioid activity assays. The results indicate that the progressive conformational restriction in these compounds upon mono- and di-N-methylation did not significantly affect the in vitro opioid activity profile. A low-energy conformer identified for the conformationally most restricted analogue of the series, H-Dmt-c[D -Cys-Gly-Phe(NMe)-L -Cys(NMe)]NH₂ (6), showed good spatial overlap of the essential pharmacophoric moieties with those in the proposed μ receptor-bound conformation of the μ-selective opioid peptide JOM-6 [H-Tyr-c(S-Et-S)[D -Cys-Phe-D -Pen]NH₂] (Pen = penicillamine) [Mosberg M.I. and Fowler C.B. (2002) J Peptide Res; 60:329–335], in agreement with the moderate μ selectivity determined for this compound. An analogue of 6 containing (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Dmt¹ was an opioid antagonist with quite high opioid receptor binding affinities and can be expected to show improved bioavailability because of its further increased lipophilicity and reduced hydrogen-bonding capacity.     

CONFORMATIONAL ENERGY CALCULATIONS ON ENKEPHALINS AND ENKEPHALIN ANALOGS:

Conformational energy calculations were carried out on the peptide enkephalins (ENK) and selected analogs to find those conformers of low energy. The analogs studied include [D-Ala²]Enk-NH₂, [D-Ala²]Enk, [D-Met², Pro⁵]Enk-NH₂, [D-Ala², D-Phe⁵]Enk, [D-Ala², D-Leu⁵]Enk, [D-Ala², (N-Me)Phe⁴, Met⁵] Enk-NH₂ and [D-Ala², (N-Me)Met⁵]Enk-NH₂. When the low-energy conformers for all the analogs are compared, different allowed backbone conformations are found which orient the functional side-chains such that three classifications of structures appear. Each classification shows a unique configuration of side- chain positions in space even though different backbone conformations are found within each classification.     

Cyclic Opioid Peptide Agonists and Antagonists Obtained Via Ring‐Closing Metathesis

The opioid peptide H‐Tyr‐c[D‐Cys‐Phe‐Phe‐Cys]NH₂ cyclized via a methylene dithiother is a potent and selective μ opioid agonist (Przydial M.J. et al., J Peptide Res, 66, 2005, 255). Dicarba analogues of this peptide with Tyr, 2′,6′‐dimethyltyrosine (Dmt), 3‐[2,6‐dimethyl‐4‐hydroxyphenyl)propanoic acid (Dhp) or (2S)‐2‐methyl‐3‐(2,6‐dimethyl‐4‐hydroxyphenyl)propanoic acid [(2S)‐Mdp] in the 1‐position were prepared. The peptides were synthesized on solid‐phase by substituting d ‐allylglycine and (2S)‐2‐amino‐5‐hexenoic acid in position 2 and 5, respectively, followed by ring‐closing metathesis. Mixtures of cis and trans isomers of the resulting olefinic peptides were obtained, and catalytic hydrogenation yielded the saturated –CH₂–CH₂– bridged peptides. All six Tyr¹‐ and Dmt¹‐dicarba analogues retained high μ and δ opioid agonist potency and showed only slight or no preference for μ over δ receptors. As expected, the six Dhp¹‐ and (2S)‐Mdp¹‐dicarba analogues turned out to be μ opioid antagonists but, surprisingly, displayed a range of different efficacies (agonism, partial agonism or antagonism) at the δ receptor. The obtained results indicate that the μ versus δ receptor selectivity and the efficacy at the δ receptor of these cyclic peptides depend on distinct conformational characteristics of the 15‐membered peptide ring structure, which may affect the spatial positioning of the exocyclic residue and of the Phe³ and Phe⁴ side chains.     

Structure-activity relationships of dermorphin analogues containing N-substituted amino acids in the 2-position of the peptide sequence

A series of dermorphin analogues containing an N-alkylated amino-acid residue Xaa in the 2-position of the peptide sequence was synthesized (Xaa =N-methylalanine, proline, pipecolic acid, N-methylphenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [Tic]). These peptides have the potential of assuming a cis Tyr^l-Xaa² peptide bond. Their in vitro opioid activity profiles were determined in μ and δ-receptor-representative binding assays and bioassays. Aside from [D-Pro²]dermorphin, all analogues showed high affinity for μ and/or δ-opioid receptors. Whereas most compounds were found to be full μ-agonists in the guinea pig ileum (GPI) assay, [Tic²]dermorphin (compound 7) was a partial μ-agonist. Replacement of Gly⁴in 7 with Phe resulted in an analogue (8) with weak μ-antagonist activity. Furthermore, analogues 7 and 8 both were potent § antagonists (K_c= 3–40 nM) against the §-agonists Leuenkephalin, DPDPE and deltorphin I in the mouse vas deferens (MVD) assay. Compound 3, containing l -Pro in the 2-position, turned out to be one of the most μ receptor-selective linear dermorphin analogues reported to date. Low-temperature HPLC experiments using micropellicular octadecyl silica as stationary phase revealed conformational heterogeneity of the dermorphin analogues which was ascribed to cis-trans isomerization around the Tyr^l-Xaa²-and Tyr⁵-Pro⁶ peptide bonds. In the case of analogue 7 four separate peaks corresponding to the four possible isomers were apparent at -5°C. Since opioid peptide analogues with a non-N-akylated l -amino acid residue in the 2-position are nearly inactive and cannot assume a cis peptide bond at the 1–2 position, these results support the hypothesis that the bioactive conformation of opioid peptides containing an N-alkylated l -amino acid residue in position 2 is characterized by a cis Tyr^l-Xaa² peptide bond.     

Comparative biological activities of potent active-site analogues of α-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several α-melanotropin (α-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ and Ac-[Tyr⁴]-α-MSH_4–11-NH₂ were compared with α-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ was found to be less active than Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr⁴]-α-MSH_4–11-NH₂ was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ over Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on frog melanocytes may be related to the fact that the shorter 4–10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of α-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle⁴]-α-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity. Similarly, incorporation of D-Phe-7 into Tyr-4 containing melanotropin fragments produced analogues Ac-[Tyr⁴, D-Phe⁷]-αMSH_4–10-NH₂ and Ac-[Tyr⁴, D-Phe⁷]-α-MSH_4–11-NH₂, which also exhibited greatly increased biological activity in all three assay systems. Both of these analogues were also found to have prolonged activity in the frog skin bioassay but little or no prolonged activity in the lizard skin bioassay. These two analogues turned out to be full agonists in the mouse melanoma adenylate cyclase bioassay and were equipotent to α-MSH. These results demonstrate that substitution of tyrosine for methionine at position-4 dramatically affects the potency and prolonged activity of these melanotropin analogues and the melanotropic activities observed as a result of such substitutions are themselves affected by concomitant substitutions at the 7(Phe) and 11 (Lys) positions of the analogues.     

Differentiation of the structural features of melanotropins important for biological potency and prolonged activity in vitro

Several α-melanotropin (α-MSH) analogues have been synthesized and tested for their melanotropin activities in order to determine the functional importance of certain amino acids near the primary active sequence of α-MSH, H-(Glu)-His-Phe-Arg-Trp-Gly-OH, on the biological activities of the hormone. In particular, we have examined the importance of the 4 and 11 positions in conjunction with the substitution of l -Phe in position 7 by d -Phe on potency and prolonged activity of the hormone. In the frog (Rana pipiens) skin system the relative potencies were found to be: [Nle⁴, d -Phe⁷]-α-MSH (60) > α-MSH (1.0) > Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–11-NH₂ (0.16) > Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–10-NH₂ (0.02) · Ac-[d -Phe⁷]-α-MSH_5–11-NH₂ (0.01) > Ac-[Nle⁴]-α-MSH_4–10-NH₂ (0.002) = Ac-[Nle⁴]-α-MSH_4–11-NH₂ > Ac-α-MSH_4–10-NH₂ (0.0003) · Ac-α-MSH_5–11-NH₂ (0.0002). On the other hand the relative potencies on the lizard (Anolis carolinensis) skin system were found to be: Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–10-NH₂ (10) · Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–11-NH₂ (8.0) · Ac-[Nle⁴, d -Phe⁷]-α-MSH (5.0) > α-MSH (1.0) = Ac-[Nle⁴]-α-MSH_4–11-NH₂ = Ac-[d -Phe⁷]-α-MSH_5–11-NH₂ > Ac-[Nle⁴]-α-MSH_4–10-NH₂ (0.06) > Ac-α-MSH_5–11-NH₂ (0.01) > Ac-α-MSH_4–10-NH₂ (0.004). Detailed analyses of these data suggest species-dependent differences in the stereostructural relationships of the residues in the 4, 7, and 11 positions for melanotropic potency in vitro. Particularly noteworthy is the observation that the 4–11 fragment analogue Ac-[Nle⁴]-α-MSH_4–11-NH₂ is equipotent to α-MSH in the lizard assay system, suggesting that the 1–3, 12, and 13 residues of α-MSH are not involved in the binding or transduction in this system. Examination of the ability of these α-melanotropin analogues to effect sustained biological activity (prolongation) following removal of exogenous peptide from the bioassay medium showed striking differences in the two systems. On the lizard skin assay, only [Nle⁴, d -Phe⁷]-α-MSH, Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–11-NH₂ and Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–10-NH₂ effect marked prolonged melanotropic activity as compared to α-MSH. In contrast, on the frog skin assay, only [Nle⁴, d -Phe⁷]-α-MSH, Ac-[Nle⁴, d -Phe⁷]-α-MSH_4–11-NH₂, Ac-α-MSH_5–11-NH₂, and Ac-[Nle⁴]-α-MSH_4–10-NH₂ exhibited significant prolonged activity. These results demonstrate that relative potency and prolongation of melanotropic activity are not directly related, but rather are the manifestation of different, species-dependent structural and topographical requirements for peptide-receptor interactions related to binding and signal transduction.     

Radioiodinated [D-Ala2, Met 5] enkephalin

¹²⁵I[D-Ala², Met⁵] enkephalin with high specific activity (122–185 Ci/mmol) was prepared and purified by Sep-Pak C₁₈ reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved ¹²⁵I[D-Ala², Met⁵] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same R_f values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24d?. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing ¹²⁵I[D-Ala², Met⁵] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala², Met⁵] enkephalin and [D-Ala², Leu⁵] enkephalin for 50% inhibition of ¹²⁵I[D-Ala², Met⁵] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the ¹²⁵I[D-Ala², Met⁵] enkephalin binding to any significant extent. The ¹²⁵I[d-Ala², Met⁵] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

Para-Substituted phenylalanine-4 analogues of [l-Ala3]. DPDPE: highly selective δ opioid receptor ligands

Several para-substituted Phe⁴ analogues of the δ₁-selective antagonist [l -Ala³]. DPDPE (DPADPE) were prepared and evaluated for their brain-binding and in vitro pharmacological effects. Unlike the p-haloPhe⁴ analogues of DPDPE and the deltorphins, similar analogues of DPADPE with electron-withdrawing groups substituted at the para-position of the Phe⁴ aromatic ring did not all have increased potency and selectivity for δ opioid receptors, but all retained high potency and selectivity for δ opioid receptors greater than DPDPE. © Munksgaard 1997.     

Comparative biological activities of potent analogues of α-melanotropin

Several α-melanotropin (α-MSH) analogues with para substituted aromatic and nonaromatic amino acids in the 7-position of the hormone were prepared and their melanotropic activities determined in the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. D and L-Phe(p-NO₂), D- and L-Tyr, D- and L-Ala, and Gly were substituted in the 7-position. The use of substituted D or L-aromatic amino acids in the 7th position of the central Ac-[Nle⁴] -α-MSH_4–11 - NH₂ fragment resulted in a loss in potency relative to the corresponding phenylalanine-containing analogue. The loss in potency cannot be due entirely to steric hindrance at the melanophore receptor, since nonaromatic amino acids substituted in the 7th position of this octapeptide fragment also generally led to a loss in biological activity. We reported previously that replacement of phenylalanine-7 by its D enantiomer led to a marked increase in potency in each fragment analogue tested. Analogues containing other D amino acids in the 7th position also were more potent than their L amino acid-containing analogues with one exception: Ac-[Nle⁴, Ala⁷]-α-MSH_4–11-NH₂ was more potent than Ac-[Nle⁴, D-Ala⁷]-α-MSH_4–11-NH₂ in the frog skin bioassay. Replacement of phenylalanine-7 by glycine resulted in a large decrease in potency in both bioassays, illustrating the importance of the side chain group, in this position of α-MSH, to biological potency of the hormone.     

Analogues of arginine vasopressin modified in the N‐terminal part of the molecule with enantiomers of N‐methylphenylalanine

Abstract: Four new analogues of arginine vasopressin (AVP) substituted in positions 2 and 3 with all possible combinations of enantiomers of N‐methylphenylalanine were synthesized and studied to assess the influence of N‐methylation of the peptide bonds between the first three amino acids on the pharmacological properties of the resulting peptides. The next three analogues were designed to learn how the shortening of the peptide chain, by removal of one of the N‐methylphenylalanine residues, would affect pharmacological properties of the resulting compounds. The activity of the analogues was tested in the in vitro uterotonic, pressor and antidiuretic tests. None of the prepared analogues displayed significant biological activity with the exception of [Me‐d ‐Phe², Me‐Phe³]AVP and [Me‐d ‐Phe^2,3]AVP, which showed low antiuterotonic activity (pA₂ = 6.6 and pA₂ = 6.4, respectively). Our results, while not impressive in terms of biological activity, may be helpful for designing potent and selective oxytocin antagonists.     

Spectrum of the μ-, δ- and κ-binding sites in homogenates of rat brain

1 In homogenates of rat brain, the binding characteristics of tritiated opiates and opioid peptides were examined and the relative capacities of μ-, δ- and κ-binding sites of the opiate receptor determined by saturation analysis.

2 In competition experiments, binding of the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin at the μ-site was displaced by [D-Ala²,D-Leu⁵]enkephalin with rather low affinity (K_I = 12.6 nM) and more readily by the ketazocine-like compounds (-)-ethylketazocine (K_I = 3.1 nM) and (-)-bremazocine (K_I = 0.32 nM), which also displaced the binding of [³H]-[D-Ala²,D-Leu⁵]enkephalin from the δ-site. In contrast, the binding to the κ-site was easily displaced by ethylketazocine (1.0 nM) and bremazocine (0.37 nM) but not by the μ-ligand [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin (K_I = 2000-3000 nM) or the δ-ligand [D-Ala²,D-Leu⁵]enkephalin (K_I > 20,000 nM).

3 The dissociation equilibrium constant (K_D) and the binding capacity (pmol/g) of the μ-binding site were determined with the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin. For the δ-site, [³H]-[D-Ala²,D-Leu⁵]enkephalin was used in the presence of unlabelled [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin in order to suppress cross-reactivity to the μ-binding site. For the estimation of κ-binding, [³H]-(±)-ethylketazocine or [³H]-(-)-bremazocine were used in the presence of unlabelled μ- and δ-ligands for the suppression of cross-reactivities to the μ- and δ-binding sites.

4 In rat brain the capacity of the μ-binding site was 7.3 pmol/g brain, that of the δ-binding site 6.7 pmol/g brain and that of the κ-binding site 2.0 pmol/g brain. Thus, the κ-binding site had the lowest value whereas in the guinea-pig brain the capacity of the μ-binding site was lower than that of the δ- or κ-binding site.

     

Structure-activity relationships of CCK26-33-related analogues modified in position 33

In the sequence of the C-terminal octapeptide of cholecystokinin, the phenylalanine amide residue in position 33 is of primary importance for the biological activity. Indeed, removal of Phe³³-NH₂ is a modification known to induce antagonist properties. The influence of the chemical nature of the Phe³³-NH₂ side chain on the biological activity of CCK₈ was investigated through replacement of this residue by several amino acids with different lipophilic properties in the sequence of Boc(Nle²⁸, Nle³¹)CCK_27-33, an equipotent analogue of CCK₈. The binding properties of these new CCK-related analogues: Boc(Nle²⁸,Nle³¹,X³³)CCK_27-33 were measured on both mouse brain and guinea pig pancreatic membranes, and their peripheral activities on amylase secretion and contractions of guinea pig ileum. Among the various peptides modified in position 33, Boc(Nle²⁸,Nle³¹,Naa³³-NH₂)CCK_27-33(Naa = naphthylalanine) and Boc(Nle²⁸,Nle³¹,Cha³³-NH₂)CCK_27-33(Cha = cyclohexylalanine) displayed high affinities for central and peripheral CCK-receptors and proved to be full agonists of CCK₈ in the peripheral tests while Boc(Nle²⁸,Nle³¹,Ala³³-NH₂)CCK_27-33 was completely inactive. This suggests that, at the level of the Phe³³-NH₂ subsite, the critical factor for optimal interaction with CCK-receptors is not the aromatic nature of the side chain but its size and hydrophobicity.