Synthesis and characterization of a new labeled gastrin ligand, 125I-BH-[Leu15]-gastrin-(5-17), on binding to canine fundic mucosal cells and Jurkat cells

In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we have synthesized and characterized a new labeled gastrin ligand, ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) [(3-[¹²⁵I]iodo-4-hydroxyphenyl)-propionyl-[Leu¹⁵]-gastrin-(5-17)]. Binding of ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) to isolated canine fundic mucosal cells was specific, saturable and of high affinity. ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) and ¹²⁵I-BH-CCK-8 [(3-[¹²⁵I]iodo-4-hydroxyphenyl)-propionyl-CCK-8] interact with isolated canine fundic mucosal cells with small differences in maximal binding capacities and affinities, 3800 ± 900 binding sites/cell (Kd = 0.52 ± 0.23 nM) and 6200 ± 1100 binding sites/cell (K_d= 0.31 ± 0.18 nM), respectively. The relative order of potencies for gastrin and CCK analogs in displacing ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) binding correlated well with those obtained using ¹²⁵I-BH-CCK-8. Selective CCK/gastrin antagonists L-364,718 (MK-329) and L-365,260 also inhibited ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) binding. These results indicate that ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) binds to gastrin receptors in isolated canine fundic mucosal cells. We have also characterized ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) binding to the human Jurkat lymphoblastic cell line (Jurkat cells) known to express the CCK-B/gastrin receptor. Saturation experiments have shown that both ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) and ¹²⁵I-BH-CCK-8 interact with a single class of high-affinity binding sites in the Jurkat cell line. Binding characteristics of ¹²⁵I-BH-[Leu¹⁵]-Gastrin-(5-17) and ¹²⁵I-BH-CCK-8 were estimated to be about 2500 ± 400 binding sites/cell (Kd= 0.19 ± 0.09 nM) and 2400 ± 350 binding sites/cell (Kd= 0.06 ± 0.02 nM), respectively. Furthermore, the apparent affinities of CCK analogs and selective antagonists MK-329 and L-365,260 for the sites labeled by both probes were identical. The biological activity of cold ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) and [Leu¹⁵]-gastrin-(5-17) was demonstrated by their ability to increase intracellular calcium concentration in Jurkat cells. All these experiments showed that ¹²⁵I-BH-[Leu¹⁵]-gastrin-(5-17) provides a convenient ligand for gastrin receptor binding studies. © Munksgaard 1994.     

The agonist SR 146131 and the antagonist SR 27897 occupy different sites on the human CCK1 receptor

1-[2-(4-(2-Chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid (SR 27897) is an effective CCK₁ receptor antagonist, while the structurally related molecule 2-[4-(4-chloro-2,5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl]-5,7-dimethyl-indol-1-yl-1-acetic acid (SR 146131) is a highly potent and specific agonist for the same receptor. To discover how the two molecules interact with the human cholecystokinin (CCK) CCK₁ receptor, we have carried out binding and activity studies with 33-point mutated receptors. Only six mutants showed altered [³H]SR 27897 binding properties, Lys¹¹⁵, Lys¹⁸⁷, Phe¹⁹⁸, Trp²⁰⁹, Leu²¹⁴ and Asn³³³. In contrast, numerous mutations throughout the receptor either reduced SR 146131 agonist potency, Phe⁹⁷, Gly¹²², Phe¹⁹⁸, Trp²⁰⁹, Ile²²⁹, Asn³³³, Arg³³⁶ and Leu³⁵⁶ or increased it, Tyr⁴⁸, Cys⁹⁴, Asn⁹⁸, Leu²¹⁷ and Ser³⁵⁹. Only mutations of Phe¹⁹⁸, Trp²⁰⁹ and Asn³³³ affected both SR 27897 and SR 146131 binding or activity. The collated information was used to construct molecular models of SR 27897 and SR 146131 bound to the human CCK₁ receptor. The clear difference in the binding sites of SR 27897 and SR 146131 offers a molecular explanation for their contrasting pharmacological characteristics.     

Dermorphin‐based potential affinity labels for µ‐opioid receptors*

Abstract: Dermorphin and [Lys⁷]dermorphin, selective µ‐opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the ‘message’ or ‘address’ sequences as potential peptide‐based affinity labels for µ‐receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe³ and Phe⁵ was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine‐containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. In dermorphin, introduction of the electrophilic groups in the ‘message’ domain lowered the binding affinity by > 1000‐fold; only [Phe(p‐NH₂)³]dermorphin retained nanomolar affinity for µ‐receptors. Modifications in the ‘address’ region of both dermorphin and [Lys⁷]dermorphin were relatively well tolerated. In particular, [Phe(p‐NH₂)⁵,Lys⁷]dermorphin showed similar affinity to dermorphin, with almost 2‐fold higher selectivity for µ‐receptors. [Phe(p‐NHCOCH₂Br)⁵]‐ and [Phe(p‐NHCOCH₂Br)⁵,Lys⁷]dermorphin exhibited relatively high affinity (IC₅₀ = 27.7 and 15.1 nm , respectively) for µ‐receptors. However, neither of these peptides inhibited [³H]DAMGO binding in a wash‐resistant manner.     

An affinity label for δ‐opioid receptors derived from [d‐Ala2]deltorphin I*

Abstract: A series of potential affinity label derivatives of the amphibian opioid peptide [d ‐Ala²]deltorphin I were prepared by incorporation at the para position of Phe³ (in the ‘message’ sequence) or Phe⁵ (in the ‘address’ sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the δ‐opioid receptor affinity, selected analogs retained nanomolar affinity for δ receptors. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited moderate affinity (IC₅₀ = 83 nm ) and high selectivity for δ receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in δ‐receptor affinity (80‐ and >1200‐fold, respectively, compared with [d ‐Ala²]deltorphin I). In the ‘address’ sequence, the Phe(p‐NH₂)⁵ derivative showed the highest δ‐receptor affinity (IC₅₀ = 32 nm ), while the Phe(p‐NHCOCH₂Br)⁵ and Phe(p‐NCS)⁵ peptides displayed four‐ and tenfold lower δ‐receptor affinities, respectively. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited wash‐resistant inhibition of [³H][d ‐Pen²,D‐Pen⁵]enkephalin (DPDPE) binding to δ receptors at a concentration of 80 nm . [d ‐Ala², Phe(p‐NCS)³]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the ‘message’ sequence of the peptide.     

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.     

Synthesis and evaluation of potential affinity labels derived from endomorphin‐2

Abstract: In an attempt to identify potential peptide‐based affinity labels for opioid receptors, endomorphin‐2 (Tyr‐Pro‐Phe‐PheNH₂), a potent and selective endogenous ligand for µ‐opioid receptors, was chosen as the parent peptide for modification. The tetrapeptide analogs were prepared using standard Fmoc‐solid phase peptide synthesis in conjunction with incorporation of Fmoc‐Phe(p‐NHAlloc) and modification of the p‐amino group. The electrophilic groups isothiocyanate and bromoacetamide were introduced into the para position on either Phe³ or Phe⁴; the corresponding free amine‐containing peptides were also prepared for comparison. The peptides bearing an affinity label group and their free amine analogs were evaluated in a radioligand‐binding assay using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. Modification on Phe⁴ was better tolerated than on Phe³ for µ‐receptor binding. Among the analogs tested, [Phe(p‐NH₂)⁴]endomorphin‐2 showed the highest affinity (IC₅₀ = 37 nm ) for µ‐receptors. The Phe(p‐NHCOCH₂Br)⁴ analog displayed the highest µ‐receptor affinity (IC₅₀ = 158 nm ) among the peptides containing an affinity label group. Most of the compounds exhibited negligible binding affinity for δ‐receptors, similar to the parent peptide.     

Replacement of Tyr-SO3H by a p-carboxymethyl-phenylalanine in a CCK8-derivative preserves its high affinity for CCK-B receptor

The sulfated tyrosine present in the sequence of CCK₈ Asp²⁶-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-PheNH₂, seems to play a critical role in the recognition of CCK-A binding sites. In this work, we have investigated whether the presence of an anionic charge on the tyrosine moiety is strictly necessary and whether the sulfate moiety interacts with a divalent cation in the receptor subsite. For this purpose, the novel amino acids (l,d ) Phe(p-CH₂CO₂H) and (l,d ) Phe(p-CH₂CONHOH), as well as their l -resolved forms were introduced into the sequence of Ac[X²⁷, Nle²⁸, Nle³¹]-CCK_27-33 by solid phase method. The biological activities of these new derivatives were compared to two almost equiactive analogues of CCK₈, Ac[Phe(p-CH2SO₃H)²⁷, Nle²⁸, Nle³¹]-CCK_27-33 and Boc[Nle²⁸, Nle³¹]-CCK₂₇-3₃ (BDNL) and to the nonsulfated analogue of the latter peptide (BDNL NS). All these new CCK-related analogues behave as agonists in stimulating pancreatic amylase release and display high affinity for brain binding sites (K₁～ 3-11 nm ) but the only peptides which retain affinity for CCK-A receptors (K₁～ 20 nm ) are those containing a p-caiboxymethyl phenylalanine. Thus, introduction of this amino acid under an esterified form on the side chain, into specific and potent CCK-B agonists could allow compounds endowed with good bioavailabilities to be obtained.     

Binding of endomorphin‐2 to μ‐opioid receptors in experimental mouse mammary adenocarcinoma

Abstract: Endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) binds with high affinity and selectivity to the μ‐opioid receptor. In the present study, [¹²⁵I]endomorphin‐2 has been used to characterize μ‐opioid‐binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [¹²⁵I]endomorphin‐2 (1 nm ) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K_d = 18.79 ± 1.13 nm , B_max = 635 ± 24 fmol/mg protein) and the other shows low affinity and higher capacity (K_d = 7.67 ± 0.81 μm , B_max = 157 ± 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [¹²⁵I]endomorphin‐2 binding site was [d ‐1‐Nal³]morphiceptin > endomorphin‐2 ? [d ‐Phe³]morphiceptin > morphiceptin > [d ‐1‐Nal³]endomorphin‐2, indicating binding of these peptides to μ‐opioid receptors. The uptake of ¹³¹I‐labeled peptides administered intraperitoneally to tumor‐bearing mice was also investigated. The highest accumulation in the tumor was observed for [d ‐1‐Nal³]morphiceptin, which reached the value of 8.19 ± 1.14% dose/g tissue.     

Synthesis and biological activity of ΔPhe4-enkephalins*

The ΔPhe⁴-enkephalins have been synthesized and examined in an in vitro receptor binding assay and an in vivo tail flick analgesia test. The ΔPhe⁴ residue was derived from Boc-Gly-Phe(β-OH)-OH by spontaneous dehydration and azlactonization. The dipeptide azlactone was coupled directly with H-Leu-OBzl to yield a tripeptide which was converted into the pentapeptides after stepwise coupling with two amino acids using the water soluble EDC-HOBt method. Dehydroenkephalins were liberated with hydrogen fluoride in the presence of anisole. In the radioligand binding assay which did not contain an enzyme inhibitor [d -Ala², ΔPhe⁴, Leu⁵] enkephalin was almost twice as active as saturated [d -Ala², d -Leu⁵] -enkephalin. The ΔPhe⁴-enkephalins exhibited a considerably diminished activity as compared with the saturated peptide in the in vivo analgesic assay. These results are discussed with regard to the enzyme stability and receptor preference of dehydroenkephalins.     

Structure-activity studies of hydrophobic amino acid replacements at positions 9, 11 and 16 of glucagon

We have designed and synthesized eight compounds 2-9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis¹,Va1⁹,11e^11,16] glucagon amide, (3) [desHis¹,Val^9,11,16]glucagon amide, (4) [desHis¹,Va1⁹,Leu^11,16]glucagon amide, (5) [desHis¹,Nle⁹,11e^11,16]glucagon amide, (6) [desHis¹,Nle⁹,Val^11,16]glucagon amide, (7) [desHis¹,Nle⁹,Leu^11,16]glucagon amide, (8) [desHis¹,Val⁹,Leu^11,16,Lys^17,18,Glu²¹]glucagon amide and (9) [desHis¹,Nle⁹,Leu^11,16,Lys^17,18,Glu²¹]glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC₅₀= 1.5 nM), analogues 2-9 were found to have IC₅₀ values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with PA₂, values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively. © Munksgaard 1997.     

Syntheses,opioid binding affinities,and potencies of dynorphin A analogues substituted in positions 1, 6, 7, 8 and 10

Structural, stereochemical, stereoelectronic and conformational requirements for biological activity of dynorphin A_1–11-NH₂ analogues at opioid receptors were explored by substitution of Tyr¹, Arg⁶, Arg⁷, Ile⁸ and Pro¹⁰ with other amino acid residues. Interestingly, substitution of Tyr¹ with N^α-Ac-Tyr^l, D-Tyr¹, Phe¹ or p-BrPhe¹ led to analogues that were quite potent at κ opioid receptors, and additional substitution of Ile⁸with D-Ala⁸ and/or Pro¹⁰ with D-Pro¹⁰ retained high potency in brain binding assay: [N^α-Ac-Tyr¹]- (1), [D-Tyr¹]- (2) [Phe¹]- (3), [Phe¹. D-Ala⁸]- (5), [p-BrPhe¹, D-Ala^s]- (6), [Phe¹, D-Pro¹⁰]- (7) and [Phe¹, D-Ala⁸, D-Pro¹⁰]-Dyn A_1–11-NH₂ (8) had IC₅₀(nM) binding affinities of 13.2, 18.6, 1.64, 1.26, 1.84, 2.44 and 1.62 nM, respectively. The D-Phe¹ analogue 4, however, was only weakly active (610 nM). All of the analogues except 4 were modestly selective for κ vs. μ guinea pig brain opioid receptor (11- to 88–fold) and quite selective for κ vs. δ receptors (65–576). However, all of the analogues appeared to have very low or essentially no activity in the guinea pig ileum and mouse vas deference functional bioassays, and one analogue, 5, appeared to have weak antagonist activities. On the other hand, if constrained amino acids such as β-methylphenylalanine or 1,2,3,4-tetrahydroisoquinoline carboxylic acid, and hydroxyproline were placed in the 1 position, inactive analogues or analogues with greatly reduced potency and biological activity were obtained (compounds 12–14). It had previously been suggested that the Arg⁶ and Arg⁷ residues were critical for biological activity. However, when we replace either one of these residues, [Nle⁶]Dyn A_1–11 (9) and [Nle⁷]Dyn A_1–11-NH₂ (10) were both highly potent binders in κ receptor binding studies (IC₅₀= 0.95 and 0.43 nM, respectively), and interestingly also were potent in μ and δ binding studies. Furthermore, both of the analogues were modestly potent in the GPI and MVD assays (94, 65 nM; 31, 81 nM, respectively). These results demonstrate that basic residues at positions 6 and 7 in dynorphin are not very important for binding to κ opioid receptors. Finally, many of the compounds reported here showed high selectivity for central vs. peripheral κ opioid receptors, with compound 4 being the most selective (63 000-fold).     

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.     

Effect of secondary structure on the rate of deamidation of several growth hormone releasing factor analogs

The objective of this study was to determine whether the rates of deamidation of Asn⁸ in selected growth hormone releasing factor (GRF) analogs were related to the peptide's secondary structures in solution. Bovine or human [Leu²⁷]GRF(1–32)NH₂ (both having Gly at position 15), [Ala¹⁵ Leu²⁷]bGRF(1–32)NH₂ and [Pro¹⁵ Leu²⁷]bGRF(1–32)NH₂ were used as model peptides. The peptide helical content (assessed by CD) increased with the increasing methanol concentration and was as follows: 7, 12 and 18% in 0% MeOH; 24, 48 and 52% in 40% MeOH; and 41, 77 and 81% in 80% MeOH for Pro¹⁵ Leu²⁷ bGRF(1–32)NH₂, [Leu²⁷]hGRF(1–32)NH₂, and Ala¹⁵ Leu²⁷ bGRF(1–32)NH₂, respectively. 2D NMR studies done in the presence of 40% CD₃OH indicated more helical structure for the Ala¹⁵ analog as compared to [Len²⁷]hGRF(1–32)NH₂. In both these peptides Asn⁸ was included in the helical region. In contrast, the lack of conformational information for the Pro¹⁵ analog indicated little helical structure around Asn⁸. The peptides’ deamidation rates decreased and their half-lives increased with increasing MeOH concentrations. At 40% MeOH, the least helical Pro¹⁵ bGRF analog (t_1/2= 10.78 h) deamidated 1.5 and 2 times faster than its Gly¹⁵ (t_1/2= 15.74 h) and Ala¹⁵ (t_1/2= 21.53 h) counterparts, respectively. This study indicates that helical environment around Asn⁸ in GRF makes this residue less prone to deamidation.     

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.     

Analogs of Substance P modified at the C‐terminus which are both agonist and antagonist of the NK‐1 receptor depending on the second messenger pathway

Abstract: The initial goal of this study was to analyze, using photolabeling, the interactions between Substance P and its tachykinin NK‐1 receptor. Therefore, the photoreactive amino acid para‐benzoyl‐phenylalanine (pBzl)Phe was incorporated into the Substance P sequence from position 4 to 11 leading to Bapa⁰[(pBzl)Phe^x]SP analogs. Biotinyl sulfone‐5‐aminopentanoic acid (Bapa) was introduced in order to purify the covalent complex. These photoreactive SP analogs were first assayed for their affinity for the two binding sites associated with the NK‐1 receptor, as well as for their potency in activating the phospholipase C and adenylate cyclase pathways. All analogs photoreactive from position 4 to 11 have moderate to high affinity for the two NK‐1 receptor‐binding sites, except for the analog modified at position 7. This affinity could be correlated to their potency to activate the phospholipase C and adenylate cyclase pathways, except for the analog photoreactive at position 11. Bapa⁰[(pBzl)Phe¹¹]SP was found to be an agonist in the phospholipase C pathway and an antagonist in the adenylate cyclase pathway, other analogs modified at position 11 were therefore analyzed. Among these, Bapa⁰[Pro⁹, (pBzl)Hcy(O₂)¹¹]SP is a partial agonist, whereas Bapa⁰[Hcy(ethylaminodansyl)¹¹]SP is a full agonist in the phospholipase C pathway, the two analogs being antagonist in the adenylate cyclase pathway. These results show that analogs of SP can be simultaneously agonist at one binding site and antagonist at the other binding site associated with the NK‐1 receptor.     

Synthesis and receptor binding affinity of both E-and Z-dehydro-phenylaline4 enkephalins*

The dehydrophenylalanine⁴-enkephalin having the E-configuration (Δ^EPhe; phenyl and C = O, cis) was prepared by photoisomerization of the Z-isomer with 3100 Å light, followed by reversed-phase HPLC separation of the resulting mixture of the Z- and E-isomers. In the radioligand receptor binding assays, the E-isomer of [D-Ala², ΔPhe⁴, Leu⁵]enkephalin exhibited an extremely diminished affinity as compared with the Z-isomer, namely 150–260-fold loss of affinity for the delta and mu opiate receptors. The results indicate that the interrelationship of the Tyr¹ and Phe⁴ residues in the enkephalin molecule seems to be of great importance in receptor recognition.     

Characterization of a novel binding site for 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]bradykinin on epithelial membranes of guinea pig ileum

We have recently shown that (a) [^125]I-Tyr8]bradykinin (BK) recognized bradykinin binding sites in guinea pig epithelium membranes with a K_d value of 1.6 nM and a B_max of 156 fmol/mg protein, and (b) B₂ agonists and some B₂ antagonists, such as D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK, inhibited this specific binding with a K_i value of 32 nM. In the present study, we have radioiodinated the B₂ antagonists Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK and have performed a full characterization of the binding properties of this tracer in the same membrane preparation. Equilibrium experiments performed in the absence or presence of an excess of BK (10⁻⁵ M) showed that ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK specifically labelled two different sites. One of these is the same as the site labelled by [¹²⁵I-Tyr⁸]BK, and this indicates that ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK interacts specifically with kinin B₂ receptors. Equilibrium experiment performed in the presence of an excess of BK (10⁻⁵ M) indicated that specific binding of ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK to the second site is also saturable and Scatchard analysis showed that the site is of high affinity with a K_d of 16.8 nM and a B_max of 2.08 pmol/mg protein. Surprisingly, unlabelled B₂ agonists such as bradykinin, [Tyr⁸]BK,[Leu⁸]BK,[Hyp³,Tyr⁸(OMe)]BK,D-Arg-[Hyp³]BK and kallidin were found to be inactive on this second site. A series of B₂ receptor antagonists, Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK, D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK, D-Arg-[Hyp³,Leu^5.8,D-Phe⁷]BK, D-Arg-[Hyp³,Gly⁶,D-Phe⁷,Leu⁸]BK and D-Arg-[Hyp³,Thi^5.8,D-Phe⁷]BK inhibited ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu Leu⁸]BK binding with K_i values of 25.0, 20.9, 15.8, 64.6 and 6606.9 nM respectively. On the other hand, [Thi^5.8,D-Phe⁷]BK did not interfere with ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK but was found to be a potent inhibitor of [¹²⁵I-Tyr⁸]BK binding (K_i = 53.7 nM). As expected, B₁ receptor agonists, antagonists and peptides non-related to BK such as substance P, neurokinin A, neurokinin B, angiotensin II, bombesin, vasopressin and the calcitonin gene related peptide were unable to compete with ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK. The results show that ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK is interacting with two distinct binding sites in the guinea pig epithelium: one is the well known bradykinin B₂ receptor and the other is a new, non-characterized binding site that interacts exclusively with some bradykinin receptor antagonists.     

Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal d-Trp or d-Tpi

In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the d forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin(6-14) and Leu¹³Ψ(CH₂NH)Phe¹⁴ -bombesin (6-14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14-27)-induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of ¹²⁵I-Tyr⁴ -bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H-345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14-27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14-27)-induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H-345 cells and suppressed the GRP(14-27)-induced increase of [³H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist d -Tpi⁶,Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin (6-14)(RC-3095) was slightly more potent in these assays than d -Trp⁶,Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin (6-14)(RC-3125). Nevertheless, d -Trp⁶ Leu¹³Ψ (CH₂NH)Phe¹⁴-bombesin (6-14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14-27)-induced amylase secretion. This antagonist RC-3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC₅₀ value less than 1 nm . Our work indicated that the substitution of d -Trp and d -Tpi at position 6 of the pseudononapeptide bombesin analogs (Ψ^13-14), in which the Met¹⁴ residue is replaced by Leu or Phe, results in potent bombesin/GRP antagonists with improved in vivo activity.     

Potent pseudopeptide bombesin-like agonists and antagonists

Bombesin-like pseudopeptides have been synthesized, and certain physicochemical properties and biological activities have been examined. Bombesin and the related peptide litorin were modified at positions 13–14 and 8–9, respectively, with ψ[CH₂S] and ψ[CH₂N(CH₃)]. [Phe¹³ψ[CH₂S]Leu¹⁴]bombesin and [Phe⁸ψ[CH₂S]-Leu⁹]litorin bound to the murine pancreatic bombesin gastrin releasing peptide receptor with similar dissociation constants (K_d= 3.9 and 3.4 nM. respectively). Increased potency was achieved by oxidation of the thiomethylene ether to two diastereomeric sulfoxides (isomer I, K_d= 1.6 nM and isomer II, K_d= 0.89nM. Further oxidation to the sulfone decreased potency ([Phe⁸ψ[CH₂SO₂]Leu⁹]litoin, K_d= 9.9nM). All five analogs were receptor antagonists as determined by phosphatidylinositol turnover in murine pancreas. In contrast to these peptide backbone substitutions, a ψCH₂N(CH₃)] at the 8–9 amide bond position resulted in an agonist. The analogs were compared with those of litorin (K_d= 0.1 nM) and [Leu⁹]litorin (K_d= 0.17 nM) by CD and fluorescence spectroscopy. The CD spectra demonstrated ordered conformation for all the peptides in TFE. Different conformations corresponding to agonist and antagonist peptides were suggested by CD. Based on the pH-dependence of the fluorescence spectra of the peptides in a zwitterionic detergent, two titratable groups were identified (pK_a= 6.3 and 8.5). The lower pK_a is found in the agonist analogs but not in the ψ[CH₂S]-containing antagonist.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs

Abstract: Ascidiacyclamide (ASC), cyclo(‐Ile¹‐Oxz²‐d ‐Val³‐Thz⁴‐)₂ (Oxz=oxazoline and Thz=thiazole) has a C₂‐symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers 49 , 459–469]. In this study, the modifications were extended. The Ile¹ residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d ‐Ile, and the d ‐Val³ residue was replaced by Val. The structures of these analogs were analyzed by X‐ray diffraction, ¹H NMR and CD techniques. X‐Ray diffraction analyses revealed that the [Ala¹], [Aib¹] and [Phe¹]ASC analogs are folded, whereas [Val¹]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly¹] and [Phe¹]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib¹], [d ‐Ile¹] and [Val³]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d ‐Ile¹] and [Val³]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly¹]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val³]ASC. In the cytotoxicity test using P388 cells, [Val¹]ASC exhibited the strongest activity, whereas the epimers of ASC ([d ‐Ile¹] and [Val³]ASCs), showed fairly moderate activities.