Potent Opioid Peptide Agonists Containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylalanine (Bcp) in Place of Tyr

Analogues of the opioid peptides H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ (non‐selective), H‐Tyr‐d ‐Arg‐Phe‐Lys‐NH₂ (μ‐selective) and dynorphin A(1‐11)‐NH₂ (κ‐selective) containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylanine (Bcp) in place of Tyr¹ were synthesized. All three Bcp¹‐opioid peptides retained high μ opioid receptor binding affinity, but showed very significant differences in the opioid receptor selectivity profiles as compared with the corresponding Tyr¹‐containing parent peptides. The cyclic peptide H‐Bcp‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ turned out to be an extraordinarily potent, μ‐selective opioid agonist, whereas the Bcp¹‐analogue of dynorphin A(1‐11)‐NH₂ displayed partial agonism at the μ receptor. The obtained results suggest that the large biphenylethyl substituent contained in these compounds may engage in a hydrophobic interaction with a receptor subsite and thereby may play a role in the ligand’s ability to induce a specific receptor conformation or to bind to a distinct receptor conformation in a situation of conformational receptor heterogeneity.     

Effect of sulfate position on rnyotropic activity of the gastrin/CCK-like insect leucosulfakinins

Leucosulfakinin and leucosulfakinin-II are sulfated insect neuropeptides with homology to human gastrin II and cholecystokinin. Biological evaluation of the analog [Ser(SO₃H)², Tyr⁵]leucosulfakinin-II, two cholecystokinin analogs containing key leucosulfakinin amino acid replacements, and a Tyr(SO₃H) position-analog series has demonstrated that while the presence of a sulfate group is critical to leucosulfakinin myotropic activity, its position is not. Most strikingly, the analog [Tyr (SO₃H)⁵,Phe⁶,Nle⁹]leucosulfakinin, in which the sulfate moiety is shifted one position towards the N-terminus relative to the parent peptide, retains a very significant 38% of the parent's threshold activity. A shift in the location of the sulfate group by two to five positions toward the N-terminus led to reduction of potency to a significant but low plateau of activity, whereas a shift by more than one position towards the C-terminus led to a complete loss of activity. The introduction of a single residue (Arg) in the 7 position of CCK-8 transforms this inactive mammalian hormone into a myotropically-active leucosulfakinin analog on the isolated cockroach hindgut.     

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.     

Functional preference of the constituent amino acid residues in a phage‐library‐based nonphosphorylated inhibitor of the Grb2‐SH2 domain

Abstract: A nonphosphorylated disulfide‐bridged peptide, cyclo(Cys‐Glu¹‐Leu‐Tyr‐Glu‐Asn‐Val‐Gly‐Met‐Tyr⁹‐Cys)‐amide (termed G1) has been identified, by phage library, that binds to the Grb2‐SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2‐SH2 domain association (IC₅₀ of 15.5 µm ) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2‐SH2 domain in a pTyr‐independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side‐chains of amino acids Glu¹, Tyr³, Glu⁴, Asn⁵, and Met⁸. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2‐SH2 domain in a β‐turn‐like conformation. Replacement of Tyr³ or Asn⁵ with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y‐X‐N consensus sequence similar to that found in natural pTyr‐containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu¹, i.e. the amino acid N‐terminal to Y³, remarkably reduces the binding affinity. The position of the Glu¹ side‐chain is confirmed to provide a complementary role for pTyr³, as demonstrated by the low micromolar inhibitory activity (IC₅₀ = 1.02 µm ) of the nonphosphorylated peptide 11 , G1(Gla¹), in which Glu¹ was replaced by γ‐carboxy‐glutamic acid (Gla).     

Inhibition of binding of phospholipase Cγ1 SH2 domains to phosphorylated epidermal growth factor receptor by phosphorylated peptides

A series of tyrosine-containing peptides 1–12: Asp-Ala-Asp-Glu-Tyr⁹⁹²(PO₃H₂)-Leu-Ile-Pro-Gln-Gln-Gly-OH (1) Asp-Ala-Asp-Glu-Tyr⁹⁹² -Leu-Ile-Pro-Gln-Gln-Gly-OH (2) Phe-Leu-Pro-Val-Pro-Glu-Tyr¹⁰⁶⁸(PO₃H₂)-Ile-Asn-Gln-Ser-Val-OH (3) Phe-Leu-Pro-Val-Pro-Glu-Tyr¹⁰⁶⁸ -Ile-Asn-Gln-Ser-Val-OH (4) Asp-Asn-Pro-Asp-Tyr¹¹⁴⁸JR(PO₃H₂)-Gln-Gln-Asp-Phe-Phe-OH (5) Asp-Asn-Pro-Asp-Tyr¹¹⁴⁸ -Gln-Gln-Asp-Phe-Phe-OH (6) Ala-Glu-Tyr¹¹⁷³(PO₃H₂)-Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (7) Ala-Glu-Tyr¹¹⁷³ -Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (8) Ala-Glu-Tyr¹¹⁷³(PO₃H₂)-Leu-Arg-Val-Ala-OH (9) Ala-Glu-Tyr¹¹⁷³ -Leu-Arg-Val-Ala-OH (10) Tyrl¹¹⁷³(PO₃H₂)-Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (11) Tyr¹¹⁷³ -Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (12) (six pairs with and without the tyrosine phosphorylated) has been synthesized. The peptides were derived from tyrosine autophosphorylation sites in the epidermal growth factor receptor (EGFR): Tyr 992, 1068, 1148 and 1173. Peptide 1, derived from the Tyr 992 site, inhibited binding of a ³⁵S-labelled fusion protein containing both of the SH2 domains from PLCγ1 to the phosphorylated EGFR with an IC₅₀ of 8 μM. All of the phosphorylated peptides except 11 (1, 3, 5, 7 and 9) inhibited this binding to some degree (20–55%) at 10 p μM. The nonphosphorylated peptides were inactive in this assay. The nonphosphorylated peptides 2, 4, 6, 8, 10 and 12 were obtained by standard solid-phase synthetic methodologies using both Boc/benzyl and Fmoc/tert-butyl strategies. The phosphorylated peptides 1, 3, 5, 7, 9 and 11 were similarly obtained using a Fmoc/tert-butyl strategy incorporating unprotected N^x-Fmoc-Tyr, followed by phosphitylation and oxidation of the tyrosine in the resin-bound peptide. In addition, Asp-Ala-Asp-Glu-Phe⁹⁹²(4-CH₂PO₃H₂)-Leu-Ile-Pro-Gln-Gln-Gly-OH (15), an analog of 1 incorporating an enzymatically stable phosphotyrosine mimic, 4-phosphonomethyl-l -phenylalanine, was synthesized and found to be inactive.     

The receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related δ-opioid antagonists contains all trans peptide bonds

Two different models for the receptor-bound conformation of δ-opioid peptide antagonists containing the N-terminal dipeptide segment H-Tyr-Tic (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) have been proposed. Both models are based on spatial overlap of the Tyr and Tic² aromatic rings and N-terminal amino group with the corresponding aromatic rings and nitrogen atom of the nonpeptide δ-antagonist naltrindole. However, in one model the peptide bond between the Tyr and Tic² residues assumes the trans conformation, whereas in the other it is in the cis conformation. To distinguish between these two models, we prepared the two peptides H-Tyrψ[CH₂NH]. Tic-Phe-Phe-OH and H-Tyrψ[CH₂NH]. MeTic-Phe-Phe-OH (MeTic = 3-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in which a cis peptide bond between the Tyr and Tic (or MeTic) residues is sterically forbidden. Both compounds turned out to be moderately potent δ-opioid antagonists in the mouse vas deferens assay. A molecular mechanics study performed with both peptides resulted in low-energy conformations in which the torsional angle (“ω₁”) of the reduced peptide bond between Tyr and Tic (or MeTic) had a value of 180°(trans conformation) and which were in good agreement with the proposed model with all trans peptide bonds. Furthermore, this study confirmed that neither of these two peptides could assume low-energy conformations in which “ω₁” had a value of 0°(cis conformation). Conformers with that same bond in the gauche- conformation (“ω₁”= -60“) were also identified, but were higher in energy and showed no spatial overlap with naltrindole. On the basis of these results it is concluded that the receptor-bound conformation of δ-peptide antagonists containing an N-terminal H-Tyr-Tic-dipeptide segment must have all trans peptide bonds. © Munksgaard 1998.     

THERMAL PERTURBATION SPECTROPHOTOMETRY OF LULIBERIN AND MODEL HIS-TRP PEPTIDES

Thermal perturbation (TP) spectra of luliberin were measured at pH 5–8, and compared with the model chromophores N-Ac-Tyr-NH₂, N-Ac-Trp-NH₂, t-Boc-His-Trp-NH₂, H-His-Trp-OH, Ac-His-Trp-OH, tryptophan and cyclo- [His-Trp] (all l -isomers). Between pH 5 and neutrality, the major TP extremum of the Trp³ residue of luliberin increases by about 50%. A similar effect is seen for luliberin acetylated on Tyr⁵. The effect with luliberin is attributed to the protonation of the His² residue. One proposed explanation is that the protonated imidazole orients water around the nearby indole in a different way than does unprotonated imidazole. The Tyr⁵ residue of luliberin behaves like N-Ac-Tyr-NH₂, and is considered to be well exposed to solvent. The TP spectra of N-Ac-Trp-NH₂, t-Boc-His-Trp-NH₂, Ac-His-Trp-OH, and cyclo-[His-Trp] are pH-independent from pH 5 to 8. The TP spectrum of H-His-Trp-NH₂ has a bell-shaped pH dependence, rising from normal at pH 3.5 to above normal at pH 6, and returning to normal at pH 8. Luliberin and model peptides show that fluorescence and TP spectra of His-Trp sequences can respond differently to pH.     

Calorimetric study of tryptophan synthase α-subunit and two mutant proteins

Heat-denaturation of tryptophan synthase α-subunit from E. coli and two mutant proteins (Glu 49 ± Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein. The partial specific heat capacity in the native state at 20°, C_p²⁰, has been found to be (0.43 ± 0.02) cal ± K^-1 ± g^-1, the unfolding heat capacity change, Δ_dC_p, (0.10 ± 0.01) cal ± K^-1 ± g^-1, and the unfolding enthalpy value extrapolated to 110°, Δ_dh¹¹⁰, (9.3 ± 0.5) cal ± g^-1 for the three proteins. The value of C_p²⁰ was larger than those found for fully compact protein and that of Δ_dh¹¹⁰ was smaller. Unfolding Gibbs energy, Δ_dG at 25° for Wild-type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal ± mol^-1 at pH 9.3, respectively. Unfolding enthalpy, Δ_dH, of the three proteins seemed to be the same and equal to (23.2 ± 1.2) kcal ± mol^-1 at 25°. As a consequence of the same value of Δ_dH and the different value in Δ_dG, substantial differences in unfolding entropy, Δ_dS, were found for the three proteins. The values of Δ_dG for the three proteins at 25° coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.     

Linear and cyclic N-terminal galanin fragments and analogs as ligands at the hypothalamic galanin receptor

The neuropeptide galanin (1–29) binds with high affinity to hypothalamic receptors (K_D～ 0.9 nM) and regulates feeding behavior. The N-terminal fragments (1–16), (1–16)NH₂ are high affinity (K_D～ 6nM) full agonists in vivo and in vitro.l -Ala substitutions show that amino acid residues Gly¹, Trp², Asn⁵, Tyr⁹, and Gly¹² are important for the high affinity binding of galanin (1–16). Shortening the fragment (1–16) to galanin (1–7) causes a gradual drop of affinity: galanin (1–15), (1–14), and (1–13) have submicromolar K_D values and galanin (1–12) has K_D～ 3 μm . Cyclic analogs of galanin (1–12) of different ring size were synthesized by condensing Gly¹ and Gly¹² without or with spacer groups. These analogs, independent of ring size, had a lower affinity than the linear galanin (1–12). Derivatization of the N-terminus of galanin (1–29), (1–16), and (1–12) all resulted in a large drop of affinity for the receptors, suggesting again the importance of the free N-terminal Gly.     

Functional and structural consequences of aromatic residue substitutions within the kringle-2 domain of tissue-type plasminogen activator

Abstract: Aromatic amino acid residues within kringle domains play important roles in the structural stability and ligand-binding properties of these protein modules. In previous investigations, it has been demonstrated that the rigidly conserved Trp²⁵ is primarily involved in stabilizing the conformation of the kringle-2 domain of tissue-type plasminogen activator (K2_tPA), whereas Trp⁶³, Trp⁷⁴, and Tyr⁷⁶ function in ω-amino acid ligand binding, and, to varying extents, in stabilizing the native folding of this kringle module. In the current study, the remaining aromatic residues of K2_tPA, viz., Tyr², Phe³, Tyr⁹, Tyr³⁵, Tyr⁵², have been subjected to structure–function analysis via site-directed mutagenesis studies. Ligand binding was not significantly influenced by conservative amino acid mutations at these residues, but a radical mutation at Tyr³⁵ destabilized the interaction of the ligand with the variant kringle. In addition, as reflected in the values of the melting tempe0ratures, changes at Tyr⁹ and Tyr⁵² generally destabilized the native structure of K2_tPA to a greater extent than changes at Tyr², Phe³, and Tyr³⁵. Taken together, results to date show that, in concert with predictions from the crystal structure of K2_tPA, ligand binding appears to rely most on the integrity of Trp⁶³ and Trp⁷⁴, and aromaticity at Tyr⁷⁶. With regard to aromatic amino acids, kringle folding is most dependent on Tyr⁹, Trp²⁵, Tyr⁵², Trp⁶³, and Tyr⁷⁶. As yet, no obvious major roles have been uncovered for Tyr², Phe³, or Tyr³⁵ in K2_tPA.     

Cyclic enkephalin analogs containing various para‐substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists*

Abstract: The cyclic enkephalin analog H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ is a highly potent opioid agonist with IC₅₀s of 35 pm and 19 pm in the guinea‐pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe¹‐analog of this peptide showed 370‐fold and 6790‐fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr¹ hydroxyl‐group in the interaction with μ and δ opioid receptors. In the present study, the effect of various substituents (‐NH₂, ‐NO₂, ‐CN, ‐CH₃, ‐COOH, ‐COCH₃, ‐CONH₂) introduced in the para‐position of the Phe¹‐residue of H‐Phe‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ on the in vitro opioid activity profile was examined. Most analogs showed enhanced μ and δ agonist potencies in the two bioassays, except for the Phe(pCOOH)¹‐analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH₃)¹‐ and the Phe(pCONH₂)¹‐analogs. The latter compound showed subnanomolar μ and δ agonist potencies and represents the most potent enkephalin analog lacking the Tyr¹ hydroxyl‐group reported to date. Taken together, these results indicate that various substituents introduced in the para‐position of Phe¹ enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure‐activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.     

Differential contribution of the conserved tyrosine residues to activity and structural stability of Ophiophagus hannahα‐neurotoxins

Abstract: Two α‐neurotoxins, Oh‐4 and Oh‐7, from king cobra (Ophiophagus hannah) venom were subjected to Tyr modification with tetranitromethane. Selective nitration of Tyr⁴ in Oh‐4 caused a slight decrease in lethal toxicity of 11% and a decrease in nicotinic acetylcholine receptor (nAchR)‐binding activity of 28%, whereas nitration of Tyr⁴ in Oh‐7 resulted in an ≈ 60% decrease in lethality and nAchR‐binding activity. When the Tyr²³ in Oh‐4 or Tyr²² in Oh‐7 appears to be ‘buried’ in the toxin following further modification, the toxins lost their biological activity and conformational change concurrently. Nevertheless, the dinitrated Oh‐4 retained a β‐sheet structure as revealed by CD spectra and exhibited a precipitin reaction with anti‐Oh‐4 sera. These results indicate that both Tyr⁴ and Tyr²² play a crucial role in the neurotoxicity of Oh‐7, whereas intact Tyr²³ is involved in the manifestation of the toxicity of Oh‐4 to a greater extent. In contrast to Oh‐4, the conformational stability of Oh‐7 depends heavily upon the integrity of Tyr²².     

Mechanisms of antibacterial formaldehyde delivery from noxythiolin and other ‘masked-formaldehyde’ compounds

Formaldehyde release in aqueous solutions of noxythiolin (N-methyl-N'-hydroxymethyl thiourea) has been monitored by nuclear magnetic resonance (n.m.r.) spectroscopy. The results suggest that antibacterial activity in such solutions resides mainly in the free formaldehyde. N.m.r. spectroscopy also demonstrated slow C-N bond rotation in noxythiolin and N-methylthiourea, with ΔG? of ca 15 kcal mol^?1 (63 kJ mol^?1)-N-Hydroxymethyl imidazole is marginally more effective than corresponding hydrated formaldehyde solutions, an effect which is attributed to more rapid turnover of unhydrated formaldehyde as detected by saturation transfer n.m.r. spectroscopy. These observations are combined with the known delivery of lethal iminium ions, R₂N⁺=CH₂, by compounds of the form R₂NCH₂X (X = OH, NR₂; R is alkyl) to suggest a single consistent explanation of the antibacterial properties of a wide range of masked formaldehyde compounds.     

s-cis,s-trans Isomerism about the Ala-Pro peptide bond in Nα-phospho- and Nα-phosphono-l-alanyl-l-prolines by 31P n.m.r.

Nα-(Phenethylphosphono)-l -alanyl-l -proline 1, a potent inhibitor of angiotensin converting enzyme, exhibits two ³¹P n.m.r. resonances (intensity ratio one to one), which exchange with a constant (k₁) of about 1 s^?1 and a free energy of activation ΔG*=～ 20kcal/mol at 23° in deuterated dimethylsulfoxide. Two resonances in exchange are also observed in deuterium oxide at pH 7.5. Thus the exchanging ³¹P resonances report the s-cis, s-trans conformational equilibrium about the alanyl-proline peptide bond. Similar results were observed with Nα-[(O-phenyl)-phenethylphosphono]-l -alanyl-l -proline 2. Nα -(O-phenylphospho)-l -alanyl-l -proline, 3 Nα-(O, O′-diphenylphospho)-l -alanyl-l -proline 4, and Nα-[2-(2-oxo-1, 3, 2-dioxaphosphiranyl)]-l -alanyl-l -proline 5 in deuterated dimethyl sulfoxide, deuterium oxide, and deuterochloroform. A ¹³C n.m.r. spectrum of 5 confirmed the presence of s-cis and s-trans resonances for the proline carbons in the same intensity ratio observed by ³¹P n.m.r.     

Conformational analysis of endomorphin‐1 by molecular dynamics methods

Abstract: Endomorphin‐1 (EM1, H‐Tyr‐Pro‐Trp‐Phe‐NH₂) is a highly potent and selective agonist for the μ‐opioid receptor. A conformational analysis of this tetrapeptide was carried out by simulated annealing and molecular dynamics methods. EM1 was modeled in the neutral (NH₂‐) and cationic (NH‐) forms of the N‐terminal amino group. The results of NMR measurements were utilized to perform simulations with restrained cis and trans Tyr¹‐Pro² peptide bonds. Preferred conformational regions in the Φ₂–Ψ₂, Φ₃–Ψ₃ and Φ₄–Ψ₄ Ramachandran plots were identified. The g(+), g(?) and trans rotamer populations of the side‐chains of the Tyr¹, Trp³ and Phe⁴ residues were determined in χ₁ space. The distances between the N‐terminal N atom and the other backbone N and O atoms, and the distances between the centers of the aromatic side‐chain rings and the Pro² ring were measured. The preferred secondary structures were determined as different types of β‐turns and γ‐turns. In the conformers of trans‐EM1, an inverse γ‐turn can be formed in the N‐terminal region, but in the conformers of cis‐EM1 the N‐terminal inverse γ‐turn is absent. Regular and inverse γ‐turns were observed in the C‐terminal region in both isomers. These β‐ and γ‐turns were stabilized by intramolecular H‐bonds and bifurcated H‐bonds.     

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.     

Conformational analysis of μ-selective [D-Ala2,MePhe4]enkephalins

The conformational space of the potent μ-selective opioids [D-Ala²,MePhe⁴,Gly-o1⁵]enkephalin (DAGO) and [D-Ala²,MePhe⁴,Gly-o1⁵]enkephalin (FK 33-824) has been analyzed by ¹H-NMR spectroscopy and theoretical calculations involving systematic conformational searching and energy minimizations. A cis-trans equilibrium of the Gly³-MePhe⁴ amide bond is induced by the N-methyl group, and the more energetically favoured trans isomer is proposed as the biologically relevant form. A compact interaction between the side chains of Tyr¹ and D-Ala² was demonstrated by NOE and ROE effects in both peptides in D₂O and DMSO-d₆, further supported by shielding of the D-Ala² methyl protons in both solvents. Analysis of coupling constants, NOE and ROE data indicated significant restriction of the conformational freedom of the MePhe⁴ side-chain for both peptides in the two solvents. The NMR results and theoretical calculations point towards folded low energy conformations characterized by a β₁₁-type turn around Gly³-MePhe⁴. For the trans isomer, a Tyr¹-MePhe⁴ phenyl ring separation between 8.5 and 12.5 Å was accompanied by proximity between the D-Ala² side chain and the C-terminal in low energy conformations The results are in good agreement with available data on related active enkephalins. The conformational effects induced by simultaneous incorporation of D-Ala² and MePhe⁴ in enkephalins is discussed in the light of the enhanced μ-opioid receptor selectivity and activity of these peptides.     

Folded conformations of the δ-selective opioid dermenkephalin with head-to-tail interactions. A simulated annealing study through NMR restraints

Despite similar tripeptide N-termini, dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) and dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH₂), naturally occuring opioid peptides from frog skin, exhibit high affinity but contrasting selectivity for the μ- and δ-opioid receptors, respectively. Structure-activity relationship studies have shown that the N-terminal tripeptide, Tyr-D-Xaa-Phe (where Xaa is either Ala or Met), is necessary for binding with both the μ- and δ-receptors while the nature and/or the conformation of the C-terminus His-Leu-Met-Asp-NH₂ of dermenkephalin are responsible for addressing the peptide to the δ-receptor. In order to examine the conformational characteristics that are related to the selectivity of dermenkephalin towards the δ-receptor, 50 NOE restraints (10 between nonadjacent residues), and 7 dihedral angles, derived from a two-dimensional ¹H-NMR study of dermenkephalin in dimethyl sulfoxide, were used in simulated annealing and energy minimization procedures. Twenty-four resulting conformers (60% of the generated structures) with no severe distance restraint violation were pooled into seven groups and three related families. These 24 conformers show close proximity between the two methionine residues, S-shaped structures, mean planes of N-terminal and C-terminal moieties almost at right angles to each other, a C-terminus region above the plane of the N-terminal region and g^? as preferential orientation in the side chain of the. Aside these similarities, families of conformers differ by the preferential orientation in the side chain of Tyr (t or g^?) and proximity between Tyr and Asp, or Tyr and the C-terminus. In contrast to previous models, practically no β-turn structures exist for dermenkephalin, most of the NH hydrogen bonds participating to γ-turns. The possible relationship between the conformational characteristics of dermenkephalin and the δ-opioid receptor selectivity is discussed. © Munksgaard 1996.     

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.     

Synthesis and Neuropeptide Y Y1 Receptor Antagonistic Activity of N,N-Disubstituted ω-Guanidino- and ω-Aminoalkanoic Acid Amides

Potent arpromidine-type histamine H₂ receptor agonists such as BU-E-76 (He 90481) were among the first non-peptides reported to display weak neuropeptide Y (NPY) Y₁ receptor antagonist activity. In search of new chemical leads for the development of more potent NPY antagonists, a series of N,N-disubstituted ω-guanidino and ω-aminoalkanoic acid amides were synthesized on the basis of structure-activity relationships and molecular modeling studies of arpromidine and related imidazolylpropylguanidines. In one group of compounds the imidazole ring was retained whereas in the second group it was replaced with a phenol group representing a putative mimic of Tyr³⁶ in NPY. Although the substitution patterns have not yet been optimized, the title compounds are NPY Y₁ antagonists in human erythroleukemia (HEL) cells (Ca²⁺ assay) achieving pKB values in the range of 6.3–6.6. For representative new substances tested in the isolated guinea pig right atrium histamine H₂ receptor agonism could not be found. In the N-(diphenylalkyl)amide series, compounds with a trimethylene chain were more active Y₁ antagonists than the ethylene homologs. Concerning the spacer in the ω-amino or ω-guanidinoalkanoyl portion, the best activity was found in compounds with a four- or five-membered alkyl chain or a 1,4-cyclohexylene group. Surprisingly, in contrast to the phenol series, in the imidazole series the compounds with a side chain amino group turned out to be considerably more potent than the corresponding strongly basic guanidines. Thus, the structure-activity relationships appear to be different for the diphenylalkylamide NPY antagonists with one or two basic groups.