Solution conformation of Leu27 hGRF(1-32)NH2 and its deamidation products by 2D NMR

The solution structure and helical content of a human growth hormone releasing factor analog, Leu²⁷ hGRF(1–32)NH₂ (hGRF), and its deamidation products Asp⁸ Leu²⁷ hGRF(l-32)NH₂ and isoAsp⁸ Leu²⁷ hGRF(1-32)NH₂, were determined by CD and 2D NMR. Chemical-shift assignments of ¹H NMR resonances were made from DQFCOSY, HOHAHA and NOESY spectra, and qualitative secondary structure was determined from NOESY spectra. 2D NMR studies in aqueous MeOH showed the Asn⁸, Asp⁸ and isoAsp⁸ hGRF analogs to have significant α-helical character. However, the β-linked isoAsp⁸ analog did not retain helical structure in the N-terminal region, most likely because of disruption of the hydrogen bonding pattern upon substitution of the extra methylene into the peptide backbone. The helical content, as determined by CD, was ~ 12% in 0% MeOH for all three peptides, and 77, 72 and 69% in 80% MeOH for the Asn⁸, Asp⁸ and isoAsp⁸ hGRF analogs, respectively. However, 2D NMR solution structure data indicated a decrease in helicity in the N-terminal region for the isoAsp⁸ analog when compared with the other two analogs. In the Asn⁸ and Asp⁸ hGRF analogs, the helix began at Asp³ or Ala⁴, while the isoAsp⁸ analog helix was disrupted until Arg. The higher helicity value for the Asn⁸ peptide over the isoAsp⁸ analog may be associated with reported biological activity, where the in vitro activity decreased from 100 to 4 and < 1% for Asn⁸, Asp⁸ and isoAsp⁸ hGRF, respectively.     

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.     

Pegylated peptides V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo

In the present study, human growth hormone-releasing factor (hGRF) and analogs were successfully pegylated at the carboxy-terminus using a novel solid- and solution-phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF(1-29)-NH2, [Ala¹⁵-hGRF(1-29)-NH₂, [desNH₂ 2Tyr1, o-Ala2, Ala15-hGRF(1-29)-NH2 and [His1, Val2, Gin8, Ala15, Leu27-hGRF(1-32)-OH were each C-terminally extended using a Gly-Gly-Cys-NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl-PEG reagent. PEG moieties of 750, 2000, 5000 or 10000 molecular weight (MW) were examined to determine the effect of polymer weight on activity. Initial biological evaluations in vitro revealed that all C-terminally pegylated hGRF analogs retained high growth hormone (GH)-releasing potencies, regardless of the MW of PEG polymer employed. Two of these pegylated hGRF analogs, [desN₂Tyr, D-Ala², Ala¹⁵-hGRF(1-29)-Gly-Gly-Cys(NH₂)-S-Nle- PEG₅₀₀₀ and [His, Val², Gin⁸, Ala¹⁵, Leu²⁷-hGRF(1-32)-Gly-Cys(NH2)-S-Nle-PEG5000, were subsequently evaluated in both pig and mouse models and found to be highly potent (in vivo potency range = 12-55-fold that of native hGRF). Relative to their non-pegylated counterparts, these two pegylated hGRF analogs exhibited enhanced duration of activity. © Munksgaard 1997.     

Potent agonists of growth hormone-releasing hormone

Analogs of the 29 amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) in position 29 have been synthesized by the solid phase method, purified, and tested in vitro and in vivo. The majority of the analogs contained desaminotyrosine (Dat) in position 1, but a few of them had Tyr¹, or N-MeTyr¹. Some peptides contained one or more additional l - or d -amino acid substitutions in positions 2, 12, 15, 21, 27, and/or 28. Compared to the natural sequence of GH-RH(1-29)NH₂, [Dat′,Ala¹⁵]GH-RH(1-28)Agm (MZ-3-191) and [d -Ala²,Ala¹⁵]GH-RH(l-28)Agm (MZ-3-201) were 8.2 and 7.1 times more potent in vitro, respectively. These two peptides contained Met²⁷. Their Nle²⁷ analogs, [Dat^I,Ala¹⁵,Nle²⁷]GH-RH(1-28)Agm(MZ-2-51), prepared previously (9), and [^D-Ala²,Ala¹⁵,Nle²⁸]GH-RH(l-28)Agm(MZ-3-195) showed relative in vitro potencies of 10.5 and 2.4, respectively. These data indicate that replacement of Met²⁷ by Nle²⁷ enhanced the GH-releasing activity of the analog when the molecule contained Dat¹-Ala² residues at the N-terminus, but peptides containing Tyr¹-D-Ala² in addition to Nle²⁷ showed decreased potencies. Replacement of Ser²⁸ with Asp in multi-substituted analogs of GH-RH(l-28)Agm resulted in a decrease in in vitro potencies compared to the parent compound. Thus, the Ser²⁸-containing MZ-2-51, and [Dat¹,Ala¹⁵,d -Lys²¹,Nle²⁷]GH-RH(l-28)Agm, its Asp²⁸ homolog (MZ-3-149), possessed relative activities of 10.5 and 5.6, respectively. In vivo after the iv injection, the analogs [Dat¹,Ala¹⁵,Nle²⁷,Asp²⁸]GH-RH(l-28)Agm (MZ-3-149), [Dat¹, Ala¹⁵]GH-RH(l-28)Agm, (MZ-3-191) and [d -Ala²,Ala^,5]GH-RH(l-28)Agm (MZ-3-201) showed a potency equivalent to 7.6, 4.9 and 3.3 times that of GH-RH(1-29)NH₂, respectively, at 5 min and 20.3, 4.3 and 1.7 times higher, respectively, at 15 min. After sc administration, analogs MZ-3-149, MZ-3-191, and MZ-3-201 were shown to be 63.7, 55.2 and 56.8 times more potent than the parent hormone at 15 min and 57.6, 60.6, and 42.6 times more active, respectively, at 30 min. In addition, MZ-3-149 had prolonged GH-releasing activity as compared to the standard, and proved to be more potent than MZ-2-51, the most active member of our previous series (8, 9). Our studies indicate that very potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.     

Importance of the C‐terminal phenylalanine of gastrin for binding to the human CCK2 receptor

Abstract: The importance of the C‐terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK₂ receptor were assessed using analogs of [Leu¹⁵]G(11?17). The following peptides were synthesized, Ac[Leu¹⁵]G(11?17), Ac[Leu¹⁵]G(11?16)NH₂, [Leu¹⁵]G(11?17), [Leu¹⁵,Ala¹⁷]G(11?17), [Leu¹⁵,Abu¹⁷]G(11?17), [Leu¹⁵,Val¹⁷]G(11?17), [Leu¹⁵,Leu¹⁷]G(11?17), [Leu¹⁵,Cha¹⁷]G(11?17), [Leu¹⁵,Trp¹⁷]G(11?17), [Leu¹⁵,Tic¹⁷]G(11?17), [Leu¹⁵, d ‐Phe¹⁷]G(11?17) and [Leu¹⁵,p‐X‐Phe¹⁷]G(11?17), where X = F, Cl, Br, I, OH, CH₃, NH₂ and NO_2. Competition binding experiments with [³H]CCK‐8 were performed using human CCK₂ receptors stably expressed in CHO cells. Phe¹⁷ was shown to be important for binding. A hydrophobic side‐chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side‐chain either in the g(+) or g(–) conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d ‐Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des‐Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK₂ receptor.     

Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor

Products of the degradation of human growth hormone-releasing factor (GRF) in aqueous solutions (15–200 μM) have been isolated and fully characterized. The cleavage product, GRF(4–44)-NH₂, and the isomer-ization product, [β-Asp³]GRF(1–44)-NH₂, from the degradation of GRF(1–44)-NH₂ in acidic solution and the corresponding products, GRF(4–29)-NH₂ and [β-Asp³]GRF(1–29)-NH₂, from the degradation of GRF(1–29)-NH₂ have been isolated and characterized. The products, [β-Asp⁸]GRF(1–44)-NH₂ and [Asp⁸]GRF(1–44)-NH₂, from the deamidation of GRF(1–44)-NH₂ at pH 8.0 and the corresponding products, [β-Asp⁸]GRF(1–29)-NH₂ and [Asp⁸]GRF(1–29)-NH₂, from the deamidation of GRF(1–29)-NH₂ have been isolated and characterized. AH the degradation products of GRF(1–44)-NH₂ and GRF(1–29)-NH₂ were evaluated for biological activity and found to have much lower in vitro potencies than the parent peptides. Degradation occurs at Asp³ and Asn⁸ and the kinetics of these various transformations versus pH and temperature have been studied. GRF is most stable at pH 4–5. At pH below the pK_a of the Asp³ side-chain (pH<4), cleavage at Asp³-Ala⁴ is the major route of degradation. For pH>4, isomerization of Asp³ to β-Asp³ (iso-Asp³) predominates. The rates of cleavage and isomerization are simple first order and vary with pH, independent of buffer concentration, such that the protonated (COOH) form of Asp³ undergoes cleavage while the ionized (COO^-) form isomerizes. The more rapid deamidation of Asn⁸ to generate β-Asp⁸ and Asp⁸ in about a 4:1 ratio, presumably via a cyclic imide intermediate, occurs at pH < 5 and is general base-catalyzed. Evidence was also obtained for direct hydrolysis of protonated Asn⁸ in GRF(1–29)-NH₂ at pH<2 to give exclusively [Asp⁸]GRF(1–29)-NH₂. The deamidation of Asn⁸ in GRF(1–29)-NH₂ at pH 8.0, 22–55°C, is relatively insensitive to temperature for T>37°C, possibly due to conformational constraints. Asp²⁵ and Asn³⁵ are sterically, conformationally, or otherwise hindered with respect to these changes as no degradation at these sites was observed under the conditions employed.     

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

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

Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment

Five analogues of human parathyroid hormone (hPTH-(20-34)-NH₂, I; cyclo[Lys²⁶-Asp³⁰]-hPTH-(20-34)-NH₂, II; cyclo[Glu²²-Lys²⁶]-hPTH-(20-34)-NH₂, III; cyclo[Lys²⁷-Asp³⁰]-hPTH-(20-34)-NH₂, IV; and [Leu²⁷]-hPTH-(20-34)-NH₂, V) were tested for their ability to promote membrane-bound protein kinase C (PKC) activity in a rat osteosarcoma cell line (ROS 17/2). Analogues I, II and V stimulated PKC activity in the picomolar range, whereas analogues III and IV did not stimulate this activity at any concentration tested. The circular dichroism spectra in neutral, aqueous buffer showed an increase in α-helix in analogues II, III and V as compared to I; this increase appeared to be in the region of the cyclic lactam structure. Analogue IV did not adopt a helical structure, even in the presence of 40% trifluoroethanol, a helix-promoting solvent. The remaining analogues showed a three- to four-fold enhancement of α-helix in this solvent. Analogues II and III had increased retention times in reversed-phase chromatography, as compared to I and IV, This is consistent with a stabilization of amphiphilic helix in analogues II and III compared with I and IV, The data suggest that in the region bounded approximately by residues 24–32, an amphiphilic α-helix is important for correct functional binding to the PTH receptor.     

Serine and d-serine position-1 substituted angiotensin II analogues Synthesis using new 2,3,5,6-tetrafluorophenyl active esters and biological activities

[Ser¹], ( 32 ), [d -Ser¹]- ( 29 ), [Ser¹, Leu⁸]- ( 31 ), and [d -Ser¹, Leu⁸] angiotensin II ( 30 ) were synthesized by a repetitive method in solution using new protected amino acid 2,3,5,6-tetrafluorophenyl active esters. 32 and 29 were agonists, and 31 and 30 were specific antagonists to angiotensin II (AII) receptors determined by the rabbit aortic strip (RAS) and rat blood pressure (RBP) assays. It was found that the hydroxymethyl side chains of serine and D-serine in position-l has an important influence on the agonistic activity of the analogues. The pressor activities of 32 and 29 were 129 and 314%, respectively, as potent as AII. On the other hand, 31 and 30 were effective in antagonizing the AII-induced contraction of RAS and rise in RBP.     

Isolation and Identification of Peptide Degradation Products of Heat Stressed Pramlintide Injection Drug Product

Purpose. This report summarizes the identification of nine deamidation and four hydrolysis products from a sample of pramlintide injection final drug product that was subjected to stress at 40°C for 45 days. Methods. The pramlintide degradation products were isolated by strong cation exchange HPLC followed by reversed-phase HPLC. Subsequent to isolation, the molecular weight of each component was determined by liquid chromatography-mass spectrometry (LC/MS). Further characterization was accomplished by amino acid sequence analysis and/ or enzymatic (thermolysin) digestion followed by LC/MS and sequence analysis. Results. The isolated products were identified as [iso-Asp²¹]-pramlintide, [iso-Asp³]-pramlintide, and [iso-Asp²²]-pramlintide, the deamidation products of pramlintide with rearrangement at Asn²¹, Asn³, and Asn²², respectively. Also found were [Asp/iso-Asp¹⁴]-pramlintide, and [Asp/iso-Asp³⁵]-pramlintide, the deamidation products at Asn¹⁴, and Asn³⁵, and [Asp²¹]-pramlintide together with [Asp²²]-pramlintide. For the deamidations at the 14^th and 35^th residues, it could not be determined whether the substance corresponded to the Asp or the iso-Asp product. The [Asp²¹] and [Asp²²] products could not be separated from each other chromatographically but were both identified in a single fraction. Two minor degradation products were also identified as deamidated species. However, the sites of deamidation remain unknown. Also identified were [l-18]-pramlintide, [l-19]-pramlintide, [19-37]-pramlintide, and [20-37]-pramlintide, the products of hydrolytic peptide backbone cleavage at amino acids His¹⁸/Ser¹⁹ and Ser¹⁹/Ser²⁰, respectively. One other product was isolated and tentatively identified as a cyclic imide intermediate preceeding deamidation. Conclusions. The primary mode of thermally induced degradation for this peptide is deamidation. A second degradation mechanism is peptide backbone hydrolysis.     

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.     

β-ENDORPHIN: SYNTHESIS AND ANALGESIC ACTIVITY OF SEVERAL ANALOGS MODIFIED IN POSITIONS 2 AND 5

The solid-phase syntheses of [Sar²]-, [Ala²]-, [D-Leu²]-, [D-Lys²]-β- endorphins and [Pro⁵]-, [Leu⁵]-, [D-Leu⁵]-, [D-Ala², D-Leu⁵]-β-endorphins are described. The synthetic peptides were purified by chromatography on carboxymethylcellulose and partition chromatography on Sephadex G-50. They were characterized by partition chromatography on agarose, thin-layer chromatography, paper electrophoresis, and amino acid analyses of acid and enzymic hydrolysates. Bioassay of the synthetic analogs for analgesic activity by the tail-flick method showed the D-Leu² analog to be 48% as potent as β_h-endorphin while the Ala², D-Lys², Leu⁵, and [D-Ala², D-Leu⁵] analogs were 8 to 17% as active. The Sar², D-Leu⁵, and Pro⁵ analogs were less than 1% as potent.     

New analogs of human growth hormone-releasing hormone (1-29) with high and prolonged antagonistic activity

Based on our previous results, in conjunction with various structural considerations, 19 new analogs of the GHRH antagonist [PhAc-Tyr¹,D-Arg²,Phe(pCl)⁶,Abu¹⁵,Nle²⁷,Agm²⁹]hGHRH(1-29) (MZ-5-156) were synthesized by the solid-phase method. These compounds were designed to develop further analogs of this class with increased receptor-binding affinity. All analogs had Abu¹⁵ and Nle²⁷ modifications and were acylated with phenylacetic acid at the N-terminus. Most of the analogs had d -Arg² and Phe(pCl)⁶ substituents and Agm²⁹ or Arg²⁹-NH₂ at the C-terminus. Additional single substitutions consisted of the incorporation of d - or l -Tic¹, d -Tic², Tic⁶ or Phe(pNO₂)⁶ and Arg²⁹-NH₂. The Arg²⁹-NH₂ analog of MZ-5-156 (KT-48) was further modified by single substitutions using Pal¹; d -Tpi²; d - or l -Phe⁴; Phe(pX)⁶×= F, Cl, I; Tyr⁷; Aib⁸; Tyr(Me)¹⁰ or Phe(pCl)¹⁰. Four peptides had multiple substitutions. All the analogs were evaluated for their ability to inhibit GH release induced by hGHRH(1-29)NH₂in vitro and some were also tested in vivo. Peptides [PhAc-Tyr¹,D-Arg²,Phe(pI)⁶,Abu¹⁵,Nle²⁷]hGHRH(1-29)NH₂ (KT-30), [PhAc-Tyr¹,D-Arg²,Phe(pCl)⁶,Aib⁸,Abu¹⁵,Nle²⁷]hGHRH(1-29)NH₂ (KT-50) and [PhAc-Tyr¹,D-Arg²,Phe(pCl)⁶,Tyr(Me)¹⁰,Abu¹⁵,Nle²⁷]hGHRH(1-29)NH₂ (KT-40) with Phe(pI)⁶, Aib⁸ or Tyr(Me)¹⁰modifications, respectively, showed high and prolonged inhibitory effect in superfused rat pituitary system. Analog KT-50 also exhibited a strong and long-term inhibitory activity in vivo in rats. Most of the new analogs showed high binding affinities to rat pituitary GHRH receptors.     

Synthesis,biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp⁸-Lys¹²)-[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂, i.e. cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp⁸ to Lys¹² proceeded rapidly (～2h) using the BOP reagent. Substitution of Ala¹² with d -Ala² and/or NH₂-terminal replacement (desNH₂-Tyr¹ or N-MeTyr¹) in the cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ contains a long α-helical segment even in aqueous solution. A series of cyclo^8.12 stereoisomers containing d -Asp⁸ and/or d -Lys¹² were prepared and also found to be highly potent and to retain significant α-helical conformation. The high biological activity of cyclo^8.12[N-MeTyr¹,d -Ala²,Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ may be explained on the basis of retention of a preferred bioactive conformation.     

Synthesis and in vitro and in vivo activity of analogs of growth hormone-releasing hormone (GH-RH) with C-terminal agmatine

In the search for more active analogs of human growth hormone-releasing hormone (GH-RH), 37 new compounds were synthesized by solid phase methodology, purified, and tested biologically. Most of the analogs contained a sequence of 27 amino acids and N-terminal desaminotyrosine (Dat) and C-terminal agmatine (Agm), which are not amino acids. In addition to Dat in position 1 and Agm in position 29, the majority of the analogs had Ala¹⁵ and Nle²⁷ substitutions and one or more additional L- or D-amino acid modifications. [Dat¹, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-51) was the most active analog. Its in vitro GH-releasing potency was 10.5 times higher than that of GH-RH(1-29)NH₂ and in the i.v. in vivo assay, MZ-2-51 was 4-5 times more active than the standard. After s.c. administration to rats, MZ-2-51 showed an activity 34 times higher at 15min and 179 times greater at 30min than GH-RH(1-29)NH₂ and also displayed a prolonged activity. D-Tyr¹⁰, D-Lys¹², and D-Lys¹² homologs of MZ-2-51 also showed enhanced activities. Thus, [Dat¹, D-Tyr¹⁰, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-159), [Dat¹, D-Lys¹², Ala¹⁵, Nle²⁷]GH-RH(1-28)AGM (MZ-2-57), and [Dat¹, Ala¹⁵, D-Lys²¹, Nle²⁷]GH-RH(1-28)Agm (MZ-2-75) were 4-6 times more active in vitro than GH-RH(1-29)NH₂. In vivo, after i.v. administration, analog MZ-2-75 was equipotent and analogs MZ-2-159 and MZ-2-57 about twice as potent as the standard. After S.C. administration, the potencies of MZ-2-57 and MZ-2-75 were 10-14 times higher than the standard at 15 min and 45-89 times greater when determined at 30 min. Most of the analogs containing two or more D-amino acid substitutions were less active than GH-RH(1-29)NH₂ or inactive. Our studies indicate that very potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.     

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.     

Solid phase synthesis of human growth hormone-releasing factor analogs containing a bicyclic β-turn dipeptide

Three analogs derived from the N-terminal 29-residue fragment of human growth hormone-releasing factor (hGRF) which contained a bicyclic β-turn dipeptide (BTD) at 7-8,8-9, and 9-10 positions were synthesized by solid phase methodology to ascertain if the β-turns are important for the biological activity of hGRF and also to show the applicability of the BTD unit to solid phase synthesis. All three analogs were obtained in good yield and purity indicating that the BTD unit can be used in the usual condition of solid phase synthesis. The capacity of these analogs to release growth hormone (GH) was tested in an in vitro bioassay using rat anterior pituitary cells. All three BTD-containing analogs showed the same maximal GH secretion with parallel dose-response curves to that of hGRF(1-29)NH₂, except their relative potencies were very low.     

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.     

Preparation and opioid activities of N-methylated analogs of [D-Ala2, Leu5]enkephalin

Analogs of opioid pentapeptide [D-Ala², Leu⁵]enkephalin were prepared using two kinds of N-methylation reactions, namely quaternization and amide-methylation. Quaternization reaction with CH³I-KHCO³ in methanol was applied to the deprotected N-terminal group of the pentapeptide derivatives affording trimethylammonium group-containing analogs. [Me₃⁺ Tyr¹,D-Ala², Leu⁵]enkephalin and its amide were found to show opioid activity on guinea pig ileium assay only slightly lower than the parent unmethylated peptides. Application of amide-methylation reaction using CH₃I-Ag₂O in DMF to the protected pentapeptide yielded a pentamethyl derivative in which all of the five N atoms were methylated. Deprotection of the derivative gave pentamethyl analogs of [D-Ala², Leu⁵]enkephalin, which showed no significant activity on the guinea pig ileum assay and opiate-receptor binding assay.     

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.