Growth Hormone-releasing Factor Analog Research Articles

Synthesis and [formula omitted] bioactivity of human growth hormone-releasing factor analogs substituted with a single D-amino acid

Fifty-four analogs of human growth hormone-releasing factor (hGRF) substituted with a single D-amino acid were synthesized by solid phase methodology. Their capacity to release growth hormone was tested on rat anterior pituitary cells in monolayer culture. Among the series of 28 analogs, which had the amino acid at each position of hGRF (1–29)NH 2, except glycine at position 15, substituted by the corresponding D-isomer, [D-Ala 2]-, [D-Asp 3]-, [D-Asn 8]-, [D-Tyr 10]-, [D-Asp 25]-, [D-Met 27]-, [D-Ser 28]-, and [D-Arg 29]hGRF(1–29)NH 2 were as potent as hGRF(1–29)NH 2, while [D-Ile 5]-, [D-Phe 6]-, [D-Thr 9]-, and [D-Val 29]hGRF(1–29)NH 2 showed quite low potencies. Effects of substitution with other D-amino acids in positions 2,3,8,9,10 and 11 were also studied. In most cases, the resulting analogs showed decreased potency, but still retained high intrinsic activity. Only [D-Arg 2]hGRF(1–29)NH 2 showed very low intrinsic activity and some antagonistic property.

ChemInform Abstract: Differential Effects of N‐Terminal Modifications on the Biological Potencies of Growth Hormone Releasing Factor Analogues with Varying Chain Lengths.

ChemInformVolume 18, Issue 24 Natural Products ChemInform Abstract: Differential Effects of N-Terminal Modifications on the Biological Potencies of Growth Hormone Releasing Factor Analogues with Varying Chain Lengths. D. H. COY, D. H. COY Inst. Org. Chem., Univ., D-7000 Stuttgart 80Search for more papers by this authorW. A. MURPHY, W. A. MURPHY Inst. Org. Chem., Univ., D-7000 Stuttgart 80Search for more papers by this authorV. A. LANCE, V. A. LANCE Inst. Org. Chem., Univ., D-7000 Stuttgart 80Search for more papers by this authorM. L. HEIMAN, M. L. HEIMAN Inst. Org. Chem., Univ., D-7000 Stuttgart 80Search for more papers by this author D. H. COY, D. H. COY Inst. Org. Chem., Univ., D-7000 Stuttgart 80Search for more papers by this authorW. A. MURPHY, W. A. MURPHY Inst. Org. Chem., Univ., D-7000 Stuttgart 80Search for more papers by this authorV. A. LANCE, V. A. LANCE Inst. Org. Chem., Univ., D-7000 Stuttgart 80Search for more papers by this authorM. L. HEIMAN, M. L. HEIMAN Inst. Org. Chem., Univ., D-7000 Stuttgart 80Search for more papers by this author First published: June 16, 1987 https://doi.org/10.1002/chin.198724299AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume18, Issue24June 16, 1987 RelatedInformation

Effect of long-term administration of an analog of growth hormone-releasing factor on the GH response in rats

The effect of the repeated or continuous administration of an analog of GH releasing factor (GH-RF), D-Tyr-1, D-Ala-2, Nle-27, GH-RF (1–29)-NH 2 (DBO-29), on the subsequent response to this peptide was investigated in pentobarbital-anesthetized male rats. A sc administration of this analog induced a greater and more prolonged GH release than doses 10 times larger of GH-RF(1–29). The GH increase after sc injection of 10 μg/kg bw of the analog was greater than that induced by iv administration of 2 μg/kg bw of GH-RF(1–44). Pretreatment with 10 μg/kg bw of the analog did not affect the pituitary response to a strong stimulus (20 μg/kg bw) of GH-RF(1–44), 24 h later. Pretreatment with the analog in doses of 10 μg/kg bw, sc twice a day, 5 days per week for 4 weeks, significantly diminished the GH release in response to a sc injection of the analog (10 μg/kg bw), as compared to vehicle-pretreated controls (P <0.01). On the other hand, a continuous sc administration of 0.4 μg/h of the analog to intact rats for 7 days did not result in a decrease in GH response to a sc injection of the analog (10 μg/kg bw). Since the rats injected repeatedly with the analog for 4 weeks still showed a marked, although somewhat reduced response, analogs of this type may be useful clinically.

Tissue and plasma distribution of exogenous growth hormone-releasing factor analogue (GRF1-29NH 2) after intravenous, subcutaneous and intraperitoneal injection in the rat

1. The administration of 125I-labelled growth hormone-releasing factor (GRF) analogue 1-29NH 2 by intravenous, subcutaneous or intraperitoneal injection to rats leads to rapid (i.v.) or slow (s.c. and i.p.) increases in plasma radioactivity followed by extensive breakdown of the peptide. 2. Tissues possessing GRF-like immunoreactivity such as gastric antrum (but not fundus), duodenum and ileum showed in vivo specific uptake of 125I-GRF probably mediated by vasoactive intestinal peptide (VIP) receptors. 3. Pituitary (the primary target organ for GRF) but neither thyroid nor parathyroid exhibited specific uptake of 125I-GRF.

Solution structure of human growth hormone releasing factor: Combined use of circular dichroism and nuclear magnetic resonance spectroscopy

The solution structures of two human growth hormone releasing factor analogues, 27Leu 45Gly-hGHRF(1–45)OH and 27Nle-hGHRF(1–29)NH 2, are investigated by means of circular dichroism and nuclear magnetic resonance spectroscopy. Using circular dichroism spectroscopy, it is shown that both peptides adopt ordered structures at low concentrations of trifluoroethanol (~30%). Quantitative analysis of the circular dichroism spectra indicates that the same number of residues, approximately 23 to 25, are in a helical state in both peptides. Using two-dimensional nuclear magnetic resonance methods all proton resonances of the 27Nle-hGHRF(1–29)NH 2 fragment are assigned and its secondary structure is determined from a qualitative interpretation of the nuclear Overhauser enhancement data. Two distinctive regions of α-helix are present extending from residues 6 to 13 and 16 to 29.

Amphiphilic growth hormone releasing factor (GRF) analogs: Peptide design and biological activity in vivo

The first twenty-nine amino acids of human Growth Hormone Releasing Factor (hGRF) possess a distinct amphiphilic character. This is seen as twisted hydrophobic and hydrophilic bands in the helical net projection. Four amidated analogs were designed by optimizing amphiphilic and helical potentials of the native sequence. These designed analogs, with up to eight-amino acid changes, were tested in sheep via intravenous injection. The growth hormone-stimulating activities of the analogs were significantly higher when compared to bovine Growth Hormone Releasing Factor (bGRF44-NH2). This suggests that the amphiphilic conformation of GRF(1-29) is important to the receptor.

Production and Characterization of Growth Hormone Releasing Factor Analogs Through Recombinant DNA and Chemical Techniques

A growth hormone releasing factor analog [Leu27,Hse44]GRF(1–44)lactone has been made by recombinant DNA techniques. The peptide was expressed from a multiple–copied GRF gene fused to lacZ. The monomeric GRF peptide analog with a C–terminal homoserine lactone was obtained upon treatment of the isolated fusion protein with cyanogen bromide. The yield of the analog increased with the number of copies fused to β–galactosidase. The C–terminal homoserine moiety was chemically converted to the primary amide, [Leu27,Hse44]GRF(1–44)NH2 and also the n–butyl and n–dodecyl amides, [Leu27, Hse44]GRF(1–44)NHC4H9 and [Leu27,Hse44]GRF(1–44)NHC12H25. The primary amide exhibited a slightly higher releasing activity than the naturally occurring hpGRF(1–44)NH2 whereas the secondary amide analogs had lower activities.

Specific labelling of high-affinity vasoactive intestinal peptide receptors in rat liver membranes by a growth hormone-releasing factor analog.

(125I-His1, D-Ala2, Nleu27)-growth hormone-releasing factor (GRF) (1-29)-NH2, initially developed as a possible radioligand for identifying GRF receptors in the anterior pituitary, was found to bind to rat hepatic membranes. The tracer was stable, bound rapidly and reversibly, and its dissociation was accelerated by GTP. Radioligand binding was enhanced by the divalent cations Mg2+, Ca2+ and Mn2+ and inhibited by the chelating agent EDTA. Vasoactive intestinal peptide (VIP), PHI, secretin, GRF(1-29)-NH2, (His1, D-Ala2, Nleu27)-GRF(1-29)-NH2, and (D-Ala2, Nleu27)-GRF(1-29)-NH2 dose-dependently inhibited tracer binding. The order of potency of the unlabelled peptides tested suggested that (125I-His1, D-Ala2, Nleu27)-GRF(1-29)-NH2 specifically identified a high-affinity subclass of VIP receptors in rat liver membranes.

Structural requirements for the activation of rat anterior pituitary adenylate cyclase by growth hormone-releasing factor (GRF): discovery of (N-Ac-Tyr1, D-Arg2)-GRF(1-29)-NH2 as a GRF antagonist on membranes.

The efficacy and potency of 14 GH-releasing factor (GRF) analogs, substituted in position 1 to 7, on adenylate cyclase activation in crude homogenates from rat anterior pituitary were related to those of human pancreatic GRF(1-29)-amide and vasoactive intestinal peptide. Among several D-amino acid substitutions, that in position 2 was the only one to yield a super-agonist [with a Kact (concentration required for half-maximal adenylate cyclase activation) 2 times lower than that of GRF(1-29)-NH2]. By contrast, D-isomer substitution in position 1 and 3 was without effect and D-isomer substitution in position 4, 6, or 7 decreased the affinity of the analog. The N-acetylated analog of GRF was as potent and active as the parent peptide, and the identity of the amino acid in position 2 of (N-Ac-Tyr1)-GRF(1-29)-NH2 proved to be determining for enzyme activation, with D-Phe2 and D-Trp2 derivatives acting as partial agonists and the (N-Ac-Tyr1,D-Arg2) analog being an efficient competitive antagonist of GRF(1-29)-NH2. With use of this antagonist, it was possible to demonstrate that GRF and vasoactive intestinal peptide receptors represent distinct entities in the rat anterior pituitary.

Administration of human pancreatic growth hormone-releasing factor (GRF) analogs enhances responsiveness of cultured rat pituitary cells to GRF

The aim of this study was to investigate whether anterior pituitary responsiveness to human pancreatic growth hormone-releasing factor containing 29 amino acids (GRF-29) can be modulated by GRF-29 itself. Male rats were injected (sc) daily for 3 days with 50 ug of GRF-29, or were treated twice daily for 14 days with 5 ug of [D-Ala-2]-GRF-29 (a potent GRF agonist). Control animals were injected with saline. After the last injection, pituitaries were removed, dispersed, cultured for 96 h and then challenged with either GRF-29 or [D-Trp-6]-LHRH (a LHRH agonist). Cultured cells from analog-treated rats were more responsive to GRF-29 stimulation than were cells obtained from controls. In contrast, neither treatment altered the response to [D-Trp-6]-LHRH. These studies indicate that periodic administration of GRF analogs can increase hypophyseal GRF responsiveness. Such control may be an important component in the physiological regulation of GH secretion and has important implications for potential therapeutic uses of GRF analogs.

Synthesis and [formula omitted] bioactivity of C-terminal deleted analogs of human growth hormone-releasing factor

A series of C-terminal deleted analogs of human growth hormone-releasing factor (hGRF) with either an amidated or a free carboxylic acid C-terminus were synthesized by solid phase methodology. Their capacity to re;ease growth hormone was tested on rat anterior pituitary cells in monolayer culture. A gradual decrease of bioactivity down to 23% relative to hGRF was noted when the C-terminal amino acids were deleted to hGRF(1–34)OH. Further deletions, however, did not decrease the bioactivity because the potencies of the fragments, hGRF(1–31)NH 2, (1–30)NH 2 and (1–29)NH 2 remained at about 50% of that of hGRF. Continual deletion of residues to hGRF(1–23)NH 2, (1–22)NH 2 and (1–21)NH 2 still yielded bioactive fragments with full intrinsic activity despite very low potency. Only with the deletion down to hGRF(1–19)NH 2 did the bioactivity completely disappear. Thus, together with the data published in a previous paper (1), the minimal biologically active core of hGRF with full intrinsic activity comprises the fragment (3–21).

Synthesis and [formula omitted] bioactivity of human growth hormone-releasing factor analogs substituted at position-1

Eight position-1 analogs of the 40-amino acid fragment and two position-1 analogs of human growth hormone-releasing factor were synthesized by solid phase methodology and their capacity to release growth hormone was determined using rat anterior pituitary cells in monolayer culture. Relative to hGRF(1–40)OH, which was arbitrarily assigned a potency value of 1, [D-Tyr 1]hGRF(1–40)OH, [Phe 1]hGRF(1–40)OH, [Trp 1]hGRF(1–40)OH, [His 1]hGRF(1–40)OH, [Ala 1]hGRF(1–40)OH, [(N-Ac)Tyr 1]hGRF(1–40)OH, Arg 0-hGRF(1–40)OH and Ala 0-hGRF(1–40)OH have potencies of 0.022, 0.038, 0.003, 0.351, 0.010, 0.032, 0.002 and 0.007 respectively. Relative to hGRF(1–44)NH 2 = 1, [(3-Me)His 1]hGRF(1–44)NH 2 and [[O-Me)Tyr 1]hGRF(1–44)NH 2 have potiencies of 0.132 and 0.001 respectively. These results demonstrate the prerequisite for an aromatic residue at position-1 for potent biological activity and also suggest that the capacity for hydrogen bond formation with the first residue is required for full receptor-ligand interaction.

Super-active analogs of growth hormone-releasing factor (1–29)-amide

Human pancreatic growth hormone releasing factor (1–29)-amide [hpGRF (1–29)-NH 2] and the following analogs: [D-Tyr-1]-hpGRF (1–29)-NH 2, [D-Ala-2]-hpGRF (1–29)-NH 2, [D-Asp-3]-hpGRF (1–29)-NH 2, and [N-Ac-Tyr-1]-hpGRF (1–29)-NH 2 were synthesized using solid phase methodology and tested for their ability to stimulate growth hormone (GH) secretion in the rat and the pig in vivo. [D-Ala-2]-hpGRF (1–29)-NH 2 was approximately 50 times more potent than the parent molecule in eliciting GH secretion in the rat. The other analogs were less active, but all were more potent than the 1–29 amide in the rat. [D-Tyr-1]-hpGRF (1–29)-NH 2 was 10 times more potent, [D-Asp-3]-hpGRF (1–29)-NH 2 7 times more potent, and the acetylated molecule approximately 12 times more potent than hpGRF (1–29)-NH 2.