Photolabeling identifies position 172 of the human AT(1) receptor as a ligand contact point: receptor-bound angiotensin II adopts an extended structure.

An angiotensin II (AngII) peptidic analogue in which the third residue (valine) was substituted with the photoreactive p-benzoyl-L-phenylalanine (Bpa) was used to identify ligand-binding sites of the human AT(1) receptor. High-affinity binding of the analogue, (125)I-[Bpa(3)]AngII, to the AT(1) receptor heterologously expressed in COS-7 cells enabled us to efficiently photolabel the receptor. Chemical and enzymatic digestions of the (125)I-[Bpa(3)]AngII-AT(1) complex were performed, and receptor fragments were analyzed in order to define the region of the receptor with which the ligand interacts. Results show that CNBr hydrolysis of the photolabeled receptor gave a glycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143-243 of the AT(1) receptor. Digestion of the receptor-ligand complex with Endo Lys-C or trypsin followed by PNGase-F treatment yielded fragments of 7 and 4 kDa, defining the labeling site of (125)I-[Bpa(3)]AngII within residues 168-199 of the AT(1) receptor. Photolabeling of three mutant receptors in which selected residues adjacent to residue 168 were replaced by methionine within the 168-199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage revealed that the bound photoligand (125)I-[Bpa(3)]AngII forms a covalent bond with the side chain of Met(172) of the second extracellular loop of the AT(1) receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended beta-strand conformation when bound to the receptor and would orient itself within the binding domain by having its N-terminal portion interacting with the second extracellular loop and its C-terminus interacting with residues of the seventh transmembrane domain.

Subtleties of structure-agonist versus antagonist relationships of opioid peptides and peptidomimetics.

The development of novel delta opioid antagonists and delta opioid agonists structurally derived from the prototype delta antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH), is reviewed. Both delta antagonists and delta agonists with extraordinary potency and unprecedented delta receptor selectivity were discovered. Some of them are already widely used as pharmacological tools and are also of interest as potential therapeutic agents for use in analgesia. The results of the performed structure-activity studies revealed that the delta antagonist versus delta agonist behavior of this class of compounds depended on very subtle structural differences in diverse locations of the molecule. These observations can be best explained with a receptor model involving a number of different inactive and active receptor conformations.

The TIPP opioid peptide family: development of delta antagonists, delta agonists, and mixed mu agonist/delta antagonists.

The discovery of the prototype delta opioid antagonists TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) in 1992 was followed by extensive structure-activity relationship studies, leading to the development of analogues that are of interest as pharmacological tools or as potential therapeutic agents. Stable TIPP-derived delta opioid antagonists with subnanomolar delta receptor binding affinity and extraordinary delta receptor selectivity include TIPP[Psi] (H-Tyr-TicPsi[CH(2)NH]Phe-Phe-OH] and TICP[Psi] (H-Tyr-TicPsi[CH(2)NH]Cha-Phe-OH); Cha: cyclohexylalanine), which are widely used in opioid research. Theoretical conformational analyses in conjunction with the pharmacological characterization of conformationally constrained TIPP analogues led to a definitive model of the receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related delta opioid antagonists, which is characterized by all-trans peptide bonds. Further structure-activity studies revealed that the delta antagonist vs delta agonist behavior of TIP(P)-derived compounds depended on very subtle structural differences in diverse locations of the molecule and suggested a delta receptor model involving a number of different inactive receptor conformations. A further outcome of these studies was the identification of a new class of potent and very selective dipeptide delta agonists of the general formula H-Tyr-Tic-NH-X (X = arylalkyl), which are of interest for drug development because of their low molecular weight and lipophilic character. Most interestingly, TIPP analogues containing a C-terminal carboxamide group displayed a mixed mu agonist/delta antagonist profile, and thus were expected to be analgesics with a low propensity to produce tolerance and physical dependence. This turned out to be the case with the TIPP-derived mu agonist/delta antagonist DIPP-NH(2)[Psi] (H-Dmt-TicPsi[CH(2)NH]Phe-Phe-NH(2)); Dmt: 2',6'- dimethyltyrosine).

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

Two different models for the receptor-bound conformation of delta-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 Tyr1 and Tic2 aromatic rings and N-terminal amino group with the corresponding aromatic rings and nitrogen atom of the nonpeptide delta-antagonist naltrindole. However, in one model the peptide bond between the Tyr1 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-Tyr(psi)[CH2NH]Tic-Phe-Phe-OH and H-Tyr(psi)[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 delta-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 ("omega1") of the reduced peptide bond between Tyr and Tic (or MeTic) had a value of 180 degrees (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 "omega1" had a value of 0 degrees (cis conformation). Conformers with that same bond in the gauche conformation ("omega1" = -60 degrees) 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 delta-peptide antagonists containing an N-terminal H-Tyr-Tic-dipeptide segment must have all trans peptide bonds.

The octapeptide angiotensin II adopts a well-defined structure in a phospholipid environment.

The conformational properties adopted by angiotensin II in a phospholipid micelle solution were studied by NMR spectroscopy and molecular modelling. The octapeptide was found to assume a well-defined hairpin structure with its C- and N-termini approaching to within 0.76 nm of each other. Three of the residues had fixed side chain configurations; Tyr4 (g+), His6 (g-) and Val3 (g-). Consequently, the His6 and Tyr4 aromatic rings were consistently close together. Conformers containing a cis His6-Pro7 peptide bond were observed for the peptide in a purely aqueous sample but completely disappeared when lipid vesicles were added to the sample. This result is explained by the existence of a very stable hydrogen bond between the Phe8 NH and the His6 carbonyl group of the lipid-solvated trans isomer, resulting in the formation of an inverse gamma turn centered on Pro7. 1H-NMR selective line broadening was apparent for several of the angiotensin II protons upon titration of an aqueous sample with less than stoichiometric amounts of 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer vesicles. The data obtained were consistent with the structure derived for micelle-bound angiotensin II, indicating that conformations adopted by the peptide hormone in the presence of micelles and lipid-bilayer vesicles are similar.

Crucial role of the residue R280 at the F'-G' loop of the human granulocyte/macrophage colony-stimulating factor receptor alpha chain for ligand recognition.

The receptor for granulocyte/macrophage colony-stimulating factor (GM-CSF) is composed of two chains, alpha and betac. Both chains belong to the superfamily of cytokine receptors characterized by a common structural feature, i.e., the presence of at least two fibronectin-like folds in the extracellular domain, which was first identified in the growth hormone receptor. The GM-CSF receptor (GMR)-alpha chain confers low affinity binding only (5-10 nM), whereas the other chain, betac, does not bind GM-CSF by itself but confers high affinity binding when associated with GMR-alpha (25-100 pM). The present study was designed to define the assembly of the GMR complex at the molecular level through site-directed mutagenesis guided by homology modeling with the growth hormone receptor complex. In our three-dimensional model, R280 of GMR-alpha, located in the F'-G' loop and close to the WSSWS motif, is in the vicinity of the ligand Asp112, suggesting the possibility of electrostatic interaction between these two residues. Through site directed mutagenesis, we provide several lines of evidence indicating the importance of electrostatic interaction in ligand-receptor recognition. First, mutagenesis of GMR-alphaR280 strikingly ablated ligand binding in the absence of beta common (betac); ligand binding was restored in the presence of betac with, nonetheless, a significant shift from high (26 pM) toward low affinity (from 2 to 13 nM). The rank order of the dissociation constant for the different GMR-alphaR280 mutations where Lys > Gln > Met > Asp, suggesting the importance of the charge at this position. Second, a mutant GM-CSF with charge reversal mutation at position Asp112 exhibited a 1,000-fold decrease in affinity in receptor binding, whereas charge ablation or conservative mutations were the least affected (10-20-fold). Third, removal of the charge at position R280 of GMR-alpha introduced a 10-fold decrease in the association rate constant and only a 2-fold change in the dissociation rate constant, suggesting that R280 is implicated in ligand recognition, possibly through interaction with Asp112 of GM-CSF. For all R280 mutants, the half-efficient concentrations of GM-CSF required for membrane (receptor binding) to nuclear events (c-fos promoter activation) and cell proliferation (thymidine incorporation) were in the same range, indicating that the threshold for biologic activity is governed mainly by the affinity of ligand-receptor interaction. Furthermore, mutation of other residues in the immediate vicinity of R280 was less drastic. Sequence alignment and modeling of interleukin (IL)-3R and IL-5R identified an arginine residue at the tip of a beta turn in a highly divergent context at the F'-G' loop, close to a conserved structural element, the WSXWS motif, suggesting the possibility of a ligand association mechanism similar to the one described herein for GMR.

Hydrophobic forces are responsible for the folding of a highly potent natriuretic peptide analogue at a membrane mimetic surface: an NMR study.

A conformational study by nmr spectroscopy was performed with the highly active 28 residue hybrid natriuretic peptide analogue pBNP1 [M. Mimeault, A. De Léan, M. Lafleur, D. Bonenfant, and A. Fournier (1995) Biochemistry, Vol. 34, pp. 955-964], which consists of the cyclic peptide core of pBNP32 and the N- and C-terminal exocyclic segments of rANP (99-126). In purely aqueous solution pBNP1 exhibits random coil behavior as evidenced by the almost complete absence of structurally significant nmr observables. By contrast, elements of secondary structure emerged upon the addition of dodecylphosphocholine micelles to the aqueous sample. Nuclear Overhauser effect distance-restrained molecular dynamics simulations in conjunction with torsional angle determinations permitted the generation of reasonable model of the lipid-bound conformation of pBNP1. According to this model, pBNP1 adopts turn-like features in the cyclic and C-terminal regions of the peptide, but remains quite flexible in the N-terminal segment. Two hydrophobic cores separated by a hydrophilic cleft were also evident in the generated structure. A mechanism is proposed whereby the hydrophobic interactions necessary to stabilize a folded structure of pBNP1 are facilitated by the presence of the membrane-like polar/apolar interface provided by the phospholipid micelles.

Effect of aromatic amino acid substitutions in the 3-position of cyclic beta-casomorphin analogues on mu-opioid agonist/delta-opioid antagonist properties.

The beta-casomorphin-5 analog H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-] (2-Nal = 2-naphthylalanine) was the first reported cyclic opioid peptide with mixed mu agonist/delta antagonist properties [R. Schmidt et al. (1994) J. Med. Chem. 37, 1136-1144]. The 2-Nal3 residue in this peptide was replaced with benzothienylalanine (Bta) (3), His(Bzl) (4), Tyr(Bzl) (5), 4'-benzoylphenylalanine (Bpa) (6), 4'-benzylphenylalanine (Bzp) (7), thyronine (Thy) (8), thyroxine (Thx) (9), 4'-biphenylalanine (Bip) (10), 4'-biphenylglycine (Bpg) (12) and 3,3-diphenylalanine (Dip) (14), and the in vitro opioid activity profiles of the resulting compounds were determined in mu and delta receptor-representative binding assays and bioassays. Analogues 3, 12 and 14 were full agonists in the mu receptor-representative guinea-pig ileum (GPI) assay and also were agonists in the delta receptor-representative mouse vas deferens (MVD) assay. The agonist effects of the latter compounds in the MVD assay were antagonized by the highly selective delta antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP), indicating that they were triggered by delta receptor activation. The Bzp3- and Bip3- containing peptides 7 and 10 turned out to be mu antagonists against the mu selective agonist H-Tyr-D-Ala-Phe-Phe-NH2 in the GPI assay. The other analogues were weak partial mu agonists which displayed remarkably decreased mu receptor affinity as compared to parent peptide 1. Compounds 4-10 were found to be delta antagonists in the MVD assay. Analogues 4 and 9 exhibited delta antagonist potency similar to that of parent peptide 1, while compounds 5-8 and 10 showed 3-12-fold higher delta antagonist potency against DPDPE and deltorphin I and, in most cases, increased delta receptor affinity. These results indicate that the delta receptor tolerates bulky aromatic side chains in the 3-position of cyclic beta-casomorphin analogs with either delta agonist or delta antagonist properties. However, these compounds displayed drastically reduced mu receptor affinity in nearly all cases.

Role of hydrophobic substituents in the interaction of opioid Tyr-Tic dipeptide analogs with dodecylphosphocholine micelles. Molecular partitioning in model membrane systems.

The conformational properties of three Tyr-Tic-NH-R dipeptide analogs [where R = (CH2)2-Ph, (CH2)3-Ph or (CH2)2-cHx; Ph = phenyl; cHx = cyclohexyl and Tic = tetrahydroisoquinoline-3-carboxylic acid] have been investigated in purely aqueous solution and in the presence of fully deuterated dodecylphosphocholine micelles. H-Tyr-Tic-NH-(CH2)2-Ph is an opioid delta-agonist, whereas H-Tyr-Tic-NH-(CH2)3-Ph is a fairly potent delta-antagonist. H-Tyr-Tic-NH-(CH2)2-cHx is a less potent delta-antagonist. 1H-NMR spectra revealed that conformers containing cis and trans configurations of the Tyr-Tic peptide bond were present in all compounds in H2O and the H2O/lipid solvent. Analyses of the NMR data for the compounds in H2O indicate that in all three dipeptides the C-terminal substituent is flexible and the Tyr-side-chain adopts a trans orientation in most of the conformations. This promotes a compact Tyr-Tic structure. NOE patterns observed for the compounds in the micelle solution indicate that Tyr has an even greater tendency to assume a trans side chain configuration in the biphasic-solvent system. This feature was more pronounced in the trans conformers than in the cis conformers. Specific lipid-peptide interactions were indicated by NOESY spectra acquired for micelle samples incorporating 20% (by mass) protonated lipid. According to the obtained NOE data, Tyr and Tic form an aromatic cluster which preferentially inserts into the lipid interior of the micelle for the trans conformers of all three dipeptides and for the cis conformer of H-Tyr-Tic-NH-(CH2)2-Ph. For the cis isomers, partitioning of the C-terminal substituents into the lipid phase exhibited more diverse behaviour. The cis conformers of H-Tyr-Tic-NH-(CH2)3-Ph and H-Tyr-Tic-NH-(CH2)2-cHx preferentially anchor to the micelle via their C-terminal substituent, while the corresponding region in H-Tyr-Tic-NH-(CH2)2-Ph remains flexible and immersed in the aqueous phase. The inconsistent mode of peptide-micelle interaction observed for cis conformers of the three compounds studied is explained in terms of differences in their dipeptide-substituent hydrophobicities. The more apolar the substituent, the greater its tendency to preferentially insert into the lipid core of the micelle. Amide-proton temperature coefficients measured for the three peptides revealed differences amongst the cis and trans isomers. The amide proton in the trans conformer of each compound is highly exposed to the aqueous phase in both solvent systems studied, whereas the cis NH proton of each peptide is only partially exposed. These results demonstrate that a subtle structural modification of an active peptide analog can result in dramatic changes of its biological activity and its mode of partitioning at a membrane surface.

Distinct conformational preferences of three cyclic beta-casomorphin-5 analogs determined using NMR spectroscopy and theoretical analysis.

The conformational properties of three cyclic beta-casomorphin analogs based on the general formula H-Tyr-c[-D-Orn-2-Nal-D-Pro-Xaa-] (2-Nal = 2-naphthylalanine; Xaa = D-Ala, Sar or NMe-Ala) in DMSO solution were investigated using NMR spectroscopy in conjunction with molecular modeling techniques. The D-Ala5- and Sar5-analogs (compounds 1 and 2 respectively) are potent mixed mu-agonist/delta-antagonists with high mu- and delta-opioid receptor affinities, whereas the NMe-Ala5-analog (compound 3) is a potent mu-agonist and a weak partial delta-agonist. Distinct conformational differences emerged for the three compounds studied. Flexibility in the bare ring structures was found to increase in the order 3 < 2 < 1. The increased structural rigidity of 3 may be responsible for its low delta-receptor affinity as compared to the two other analogs. A low fractional population of conformers containing two cis peptide bonds was found for compound 2 but not for analog 1 or 3. Initial evidence for this observation was obtained from NMR differential line-broadening experiments and later confirmed by molecular mechanics simulations. Comparison of the temperature dependence of amide proton chemical shifts acquired for the three cyclic analogs indicate a large degree of intramolecular hydrogen bonding for 1 but not for the other two peptides.

Influence of sample pH on the conformational backbone dynamics of a pseudotripeptide (H-Tyr-Tic psi [CH2-NH]Phe-OH) incorporating a reduced peptide bond: an NMR investigation.

In the present paper we investigate the influence of sample pH on the conformational and dynamical properties of the pseudotripeptide H-Tyr-Tic psi [CH2-NH]Phe-OH (TIP[psi]; Tic: 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid) using various one- and two-dimensional nmr techniques in conjunction with molecular modeling. Studies were conducted at three different pH levels corresponding to the zwitterionic peptide containing a formal positive charge (pH 3.1), the deprotonated molecule (pH 9.1), and a situation at neutral pH (pH 7.2) involving both protonated and deprotonated states of the reduced peptide bond. Analysis of the one-dimensional1H-nmr spectra reveals that in solution TIP[psi] is in slow dynamic exchange between conformations containing cis and trans configurations of the Tyr-Tic bond. An nmr pH dependence study of the cis:trans ratio indicated that the exchange process was governed by the protonation state of the reduced bone amine. From the nmr data, reduced peptide bond pK alpha values of 6.5 and 7.5 were determined for the cis and trans conformers, respectively. It was concluded that conformations containing a trans Tyr-Tic bond are stabilized at low pH by an intramolecular hydrogen bond between the Tyr carbonyl and the reduced peptide bond protonated amine. This observation was corroborated by molecular mechanics investigations that revealed low energy trans structures compatible with nmr structural data, and furthermore, were consistently characterized by the existence of a strong N+ H ... O = C interaction closing a seven-membered cycle. The dynamics of cis-trans isomerization about the Tyr-Tic peptide bond were probed by nmr exchange experiments. The selective presaturation of exchanging resonances carried out at several temperatures between 50 and 70 degrees C allowed the determination of isomerization rate constants as well as thermodynamic activation parameters. delta G not equal to values were in close agreement with the cis-->trans energy barrier found in X-Pro peptide fragments (approximately 83 kJ/mol). A large entropic barrier determined for the trans-->cis conversion of TIP[psi] (5.7 JK-1 mol-1 at pH 3.1;6.5 J K-1 mol-1 at pH 9.1) is discussed in terms of decreased solvent molecular ordering around the conformers possessing a trans Tyr-Tic bond. Evidence that the neutral form of the reduced peptide bond gains rigidity upon protonation was obtained from relaxation measurements in the rotating frame. T1 rho measurements of several protons in the vicinity of the reduced peptide bond were made as a function of spin-lock field.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative analysis of various proposed models of the receptor-bound conformation of H-Tyr-Tic-Phe-OH related delta-opioid antagonists.

A molecular mechanics study (grid search and energy minimization) was performed with six delta opioid peptide antagonists containing a tetrahydroisoquinoline-3-carboxylic acid (Tic) residue in the 2-position of the peptide sequence. Compounds examined were the highly potent and selective TIP(P) peptides H-Tyr-Tic-Phe-OH (TIP), H-Tyr-Tic psi[CH2-NH]Phe-OH (TIP[psi]), H-Tyr-Tic-Phe-Phe-OH (TIPP), and H-Tyr-Tic psi[CH2-NH]Phe-Phe-OH (TIPP[psi]), and the weakly active analogues H-Tyr-Tic-NH2 and H-Tyr-Tic-Ala-OH. Low energy conformers of the peptides were examined for their compatibility with three proposed models of the delta receptor-bound conformation. Model 1, based on spatial overlap of the Tyr1 and Phe3 aromatic rings and N-terminal amino group of the peptides with the corresponding aromatic rings and nitrogen atom of the nonpeptide delta-antagonist naltrindole, was ruled out because of the demonstrated importance of the Tic2 aromatic ring for delta antagonism and because of the somewhat elevated energies of the conformers consistent with this model. Models of the receptor-bound conformation based on superimposition of the Tyr1 and Tic2 aromatic rings and N-terminal amino group of the peptides with the corresponding moieties in naltrindole included an all-trans peptide bond conformer [model 2, proposed by B.C. Wilkes and P.W. Schiller (1994) Biopolymers, Vol. 34, pp. 1213-1219] and a conformer with a cis peptide bond between the Tyr1 and Tic2 residues (model 3, originally proposed by P.A. Temussi et al. [(1994) Biochemical and Biophysical Research Communications, Vol. 198, pp. 933-939]. For all six peptides low energy conformers consistent with both model 2 and model 3 were identified; however, peptide conformers corresponding to model 2 showed better coplanarity of the Tyr1 aromatic ring and the phenol ring in naltrindole than peptide conformers corresponding to model 3. Both models remain plausible candidate structures for the receptor-bound conformation of delta antagonists of the TIP(P) class. TIP(P) analogues containing additional conformational constraints need to be developed in order to arrive at a unique model.

An all D-amino acid opioid peptide with central analgesic activity from a combinatorial library.

A synthetic combinatorial library containing 52,128,400 D-amino acid hexapeptides was used to identify a ligand for the mu opioid receptor. The peptide, Ac-rfwink-NH2, bears no resemblance to any known opioid peptide. Simulations using molecular dynamics, however, showed that three amino acid moieties have the same spatial orientation as the corresponding pharmacophoric groups of the opioid peptide PLO17. Ac-rfwink-NH2 was shown to be a potent agonist at the mu receptor and induced long-lasting analgesia in mice. Analgesia produced by intraperitoneally administered Ac-rfwink-NH2 was blocked by intracerebroventricular administration of naloxone, demonstrating that this peptide may cross the blood-brain barrier.

Theoretical conformational analysis of the opioid delta antagonist H-Tyr-Tic-Phe-OH and the mu agonist H-Tyr-D-Tic-Phe-NH2.

A molecular mechanics study (grid search and energy minimization) of the highly delta receptor-selective delta opioid antagonist H-Tyr-Tic-Phe-OH (TIP; Tic: tetrahydroisoquinoline-3-carboxylic acid) resulted in four low energy conformers with energies within 2 kcal/mol of that of the lowest energy structure. These four conformers contain trans peptide bonds only and represent compact structures showing various patterns of aromatic ring stacking. The centrally located Tic residue imposes several conformational constraints on the N-terminal dipeptide segment; however, the results of molecular dynamics simulations indicated that this tripeptide still shows some structural flexibility, particularly at the Phe3 residue. Analogous studies performed with the structurally related mu receptor-selective mu agonist H-Tyr-D-Tic-Phe-NH2 resulted in low energy structures that were also compact but showed patterns of ring stacking different from those obtained with TIP. Superimposition of low energy conformers of TIP and H-Tyr-D-Tic-Phe-NH2 revealed that the Phe3 residues of the L-Tic- and the D-Tic peptide were always located on opposite sides of the plane defined by the Tic residue, thus providing an explanation for the distinct activity profiles of the two compounds in structural terms. Attempts to demonstrate spatial overlap between the pharmacophoric moieties of low energy conformers of TIP and the nonpeptide delta antagonist naltrindole were made by superimposing either the Tyr1 and Tic2 aromatic rings and the N-terminal amino group or the Tyr1 and Phe3 aromatic rings and the N-terminal amino group of the peptide with the corresponding aromatic rings and nitrogen atom in the alkaloid structure.(ABSTRACT TRUNCATED AT 250 WORDS)

TIPP[psi]: a highly potent and stable pseudopeptide delta opioid receptor antagonist with extraordinary delta selectivity.

Pseudopeptide analogues of the delta opioid antagonists H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-Tic-Phe-OH (TIP) containing a reduced peptide bond between the Tic2 and Phe3 residues were synthesized. The two compounds, H-Tyr-Tic psi [CH2NH]Phe-Phe-OH (TIPP [psi]) and H-Tyr-Tic psi-[CH2NH]Phe-OH (TIP [psi]), were tested in mu-, delta-, and kappa-receptor-selective binding assays and in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays. In comparison with their respective parent peptides, both pseudopeptide analogues showed increased delta antagonist potency in the MVD assay, higher delta receptor affinity and further improved delta receptor selectivity. The more potent compound, TIPP [psi], displayed subnanomolar delta receptor affinity and in direct comparisons with other selective delta ligands was shown to have unprecedented delta specificity (Ki mu/Ki delta = 10,500). Furthermore, this compound turned out to be highly stable against enzymatic degradation and, unlike other delta antagonists, showed no mu or kappa antagonist properties. TIPP [psi] is likely to find wide use as a pharmacological tool in opioid research.

Differential stereochemical requirements of mu vs. delta opioid receptors for ligand binding and signal transduction: development of a class of potent and highly delta-selective peptide antagonists.

Opioid peptide analogs consisting entirely of aromatic amino acid residues and containing conformationally restricted phenylalanine derivatives in position 2 of the peptide sequence were synthesized and pharmacologically characterized in vitro. Both diastereoisomers of H-Tyr-(D or L)-NMePhe-Phe-Phe-NH2 (NMePhe is N alpha-methylphenylalanine) were mu-receptor-selective, were full agonists in the mu-receptor-representative guinea pig ileum assay, and were partial agonists in the mouse vas deferens assay, with the L-NMePhe2 analog displaying somewhat higher intrinsic activity than the D-NMePhe2 analog. Further conformational restriction at position 2 in the sequence, as achieved through substitution of D- or L-tetrahydro-3-isoquinoline carboxylic acid (Tic), produced a configuration-dependent differential effect on receptor selectivity and intrinsic activity, leading to a potent mu-selective mu agonist (the D-Tic2 analog) with increased intrinsic activity in the mouse vas deferens assay and to a potent delta-selective delta antagonist (the L-Tic2 analog). These results demonstrate that imposition of conformational constraints in a peptide not only may alter receptor selectivity but also may decrease, totally abolish, or even enhance intrinsic activity. The tetrapeptide H-Tyr-Tic-Phe-Phe-NH2 was a moderately potent full agonist in the guinea pig ileum assay and, thus, represents a compound with mixed mu-agonist/delta-antagonist properties. The corresponding peptide with a free C-terminal carboxyl group H-Tyr-Tic-Phe-Phe-OH showed high delta-receptor affinity (Ki delta = 1.2 nM), unprecedented delta selectivity (Ki mu/Ki delta = 1410), high potency as delta antagonist (Ke = 3-8 nM against various delta agonists in the mouse vas deferens assay) and, unlike other delta antagonists, had no mu-antagonist properties. The tripeptides H-Tyr-Tic-Phe-OH and H-Tyr-Tic-Phe-NH2 were also delta antagonists.

Conformationally restricted deltorphin analogues.

Conformationally restricted deltorphin analogues were synthesized either through incorporation of cyclic phenylalanine analogues in position 2 or 3 of the peptide sequence or through various side chain-to-side chain cyclizations. Compounds were tested in mu-, delta-, and kappa-receptor selective binding assays and in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays. Replacement of Phe3 in [D-Ala2]deltorphin I with 2-aminoindan-2-carboxylic acid (Aic) or L- or D-2-aminotetralin-2-carboxylic acid (Atc) resulted in agonist compounds which retained the high delta receptor selectivity of the parent peptide. Substitution of a tetrahydroisoquinoline-3-carboxylic acid (Tic) residue in the 2-position of [D-Ala2]deltorphin I and of [Phe4,Nle6]deltorphin produced a partial delta agonist, H-Tyr-Tic-Phe-Asp-Val-Val-Gly-NH2, and a pure delta antagonist, H-Tyr-Tic-Phe-Phe-Leu-Nle-Asp-NH2, respectively. The latter antagonist displayed high delta selectivity (Ki mu/Ki delta = 502) and was a potent antagonist against selective delta agonists in the MVD assay (Ke congruent to 10 nM). Various [D-Ala2]-deltorphin I analogues cyclized between the side chains of Orn (or Lys) and Asp (or Glu) residues substituted in positions 2 and 4, 4 and 7, and 2 and 7 were essentially nonselective. Comparison with corresponding N-terminal tetrapeptide analogues revealed that the C-terminal tripeptide segment in the deltorphin heptapeptides made a crucial contribution to delta affinity and delta selectivity in the case of the agonist peptides but not in the case of the antagonist.

Molecular dynamics simulations of opioid peptide analogs containing multiple conformational restrictions.

Molecular dynamics simulations were performed on the potent and slightly mu-receptor selective cyclic dermorphin analog H-Tyr-D-Orn-Phe-Glu-NH2 as well as on analogs containing a conformationally restricted phenylalanine derivative in place of Phe in the 3 position of the peptide sequence. Peptides studied included the potent and highly mu-selective analogs H-Tyr-D-Orn-Aic-Glu-NH2 (Aic = 2-aminoindan-2-carboxylic acid), H-Tyr-D-Orn-Atc-Glu-NH2 (Atc = 2-aminotetralin-2-carboxylic acid) and H-Tyr-D-Orn-D-Atc-Glu-NH2, and the weakly active analog H-Tyr-D-Orn-Tic-Glu-NH2 (Tic = tetrahydroisoquinoline-3-carboxylic acid). Four different starting conformations were chosen for each peptide, and after equilibration each simulation was allowed to proceed for 100 picoseconds at 600 degrees K. The 14-membered ring structures in the Phe-, Aic-, L- and D-Atc-containing analogs showed moderate structural flexibility, while the peptide ring in the Tic-containing analog was more rigid. As theoretically predicted, the phi 3 and psi 3 angles of the Aic-, L- and D-Atc-containing analogs were limited to values of either about +50 degrees or -50 degrees during almost the entire period of the simulations. In the Tic-containing analog the phi 3 and psi 3 angles were 0 degrees and 90 degrees, respectively, and did not change for the entire duration of the simulation. The side chains of the constrained amino acids showed limited movement, but transitions between the allowed conformations did occur on the time scale of the simulations.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly efficient photoaffinity labeling of the hormone binding domain of atrial natriuretic factor receptor.

A high-efficiency photoaffinity derivative of atrial natriuretic factor (ANF) was developed for studying the peptide binding domain of the receptor protein and for better characterization of this receptor in tissues with a low density of binding sites. The position of the photosensitive residue was chosen on the basis of a molecular conformational model and on structure-activity relationship studies which both indicate that the carboxy-terminal end of the peptide is part of a hydrophobic pole likely to interact deeply within the ANF binding pocket of the receptor. Selection of the photoreactive residue p-benzoylphenylalanine (BPA) as a substitute for arginine in position 125 of the peptide sequence led to a photoaffinity derivative with a high (63%) efficiency of covalent incorporation to the receptor protein. This derivative (BPA-ANF) has a 10-fold lower affinity when compared with ANF, but it is a full agonist in stimulating cGMP production and inhibiting aldosterone secretion in bovine adrenal zona glomerulosa. Photoaffinity labeling with BPA-ANF specifically identifies ANF-R1 and ANF-R2 receptor proteins with a 10-fold higher efficiency than with azido derivatives of ANF or with cross-linking agents. This new ANF derivative therefore appears to be useful for studying ANF receptors in tissues with low levels of expression, for locating receptor following cellular internalization, and for tagging proteolytic fragments of the receptor amenable to amino acid microsequencing.