Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs

Campbell, Robert M.; Lee, Yung; Mowles, Thomas F.; McIntyre, Kim W.; Ahmad, Mushtaq; Félix, Arthur; Heimer, Edgar P.

doi:10.1016/0196-9781(92)90188-9

Cited by 12 publications

(4 citation statements)

References 15 publications

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“…6,7 The hGRF(1-29)-NH 2 fragment has been shown to be the shortest fragment of native hGRF [hGRF(1-44)-NH 2 ] retaining a significant GH-releasing activity. [8][9][10] The potency of the PEGylated analogues was found to be strongly dependent upon the site of PEGylation. PEGylation at the C-terminus of hGRF-(1-29)-NH 2 and a number of analogues was shown not to affect the in vitro biological activity with respect to the parent non-PEGylated compounds, whereas the duration of activity of the PEGylated derivatives was greatly enhanced in vivo because of a reduced clearance from blood and tissues.…”

Section: Introductionmentioning

confidence: 99%

“…The site-directed PEGylation strategy is nevertheless becoming very attractive also in the field of bioactive peptides, and it has been successfully employed to enhance the pharmacolgical properties of fragment 1−29 of the human growth hormone-releasing factor [hGRF(1−29)−NH 2 ] 4,5 and analogues. , The hGRF(1−29)−NH 2 fragment has been shown to be the shortest fragment of native hGRF [hGRF(1−44)−NH 2 ] retaining a significant GH-releasing activity. − The potency of the PEGylated analogues was found to be strongly dependent upon the site of PEGylation. PEGylation at the C-terminus of hGRF(1−29)−NH 2 and a number of analogues was shown not to affect the in vitro biological activity with respect to the parent non-PEGylated compounds, whereas the duration of activity of the PEGylated derivatives was greatly enhanced in vivo because of a reduced clearance from blood and tissues .…”

Section: Introductionmentioning

confidence: 99%

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NMR Structure of Two Novel Polyethylene Glycol Conjugates of the Human Growth Hormone-Releasing Factor, hGRF(1−29)−NH₂

et al. 2003

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Two novel mono-PEGylated derivatives of hGRF(1-29)-NH(2) [human growth hormone-releasing factor, fragment 1-29] have been synthesized by regio-specific conjugation of Lys(12) or Lys(21) to a monomethoxy-PEG(5000) chain (compounds Lys(12)PEG-GRF and Lys(21)PEG-GRF). The PEG moiety has been covalently linked at the amino group of a norleucine residue via a carbamate bond. The Lys(12)PEG-GRF regioisomer was found to be slightly less active in vitro than both the unmodified peptide and Lys(21)PEG-GRF. To assess whether the differences in the biological activity of the PEGylated analogues could be related to conformational rearrangements induced by the PEG moiety, the structure of these PEGylated derivatives has been worked out (TFE solution) by means of NMR spectroscopy and molecular dynamics. Secondary structure shifts, hydrogen/deuterium exchange kinetics, temperature coefficients of amide protons, and NOE-based molecular models point out that hGRF(1-29)-NH(2), Lys(21)PEG-GRF and Lys(12)PEG-GRF share a remarkably similar pattern of secondary structure. All three compounds adopt an alpha-helix conformation which spans the whole length of the molecule, and which becomes increasingly rigid on going from the N-terminus to the C-terminus. Residues Lys(12) and Lys(21) are enclosed in all the compounds considered into well-defined alpha-helical domains, indicating that PEGylation either at Lys(12) or Lys(21) does not alter the tendency of the peptide to adopt a stable alpha-helix conformation, nor does it induce appreciable conformational mobility in the proximity of the PEGylation sites. No significant variation of the amphiphilic organization of the alpha-helix is observed among the three peptides. Therefore, the different biological activities observed for the PEGylated analogues are not due to conformational effects, but are rather due to sterical hindrance effects. The relationship between the biological activitiy of the mono-PEGylated derivatives and sterical hindrance is discussed in terms of the topology of interaction between hGRF(1-29)-NH(2) and its receptor.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

NMR Structure of Two Novel Polyethylene Glycol Conjugates of the Human Growth Hormone-Releasing Factor, hGRF(1−29)−NH₂

et al. 2003

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“…Ling et al [36] established by removing AAs from the C-terminus portion of the molecule that the GRF (1-29)NH 2 was the shortest peptide with similar in vitro [37] and in vivo [38,39] biological activities when compared to the native hGRF (1-44)NH 2 . Thereafter, the next goals were to establish secondary structures allowing better bioactive conformations and resistance to degradation processes as well as higher receptor affinity.…”

Section: Active Corementioning

confidence: 99%

Growth Hormone-Releasing Factor: Structural Modification or Protection for More Potent Analogs

Dubreuil

Désévaux²

2006

CCHTS

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Growth hormone-releasing factor was discovered in 1982 by Guillemin and has been subjected to intense investigations because of its huge potential applications. The major concerns encountered with the native molecules were their short half-lives in vivo in many species including man, precluding the practical use of these peptides for medical or production purposes. Many efforts to produce analogs of shorter length, more resistant to degradation and having higher affinity to the receptors have been made during the last decades. The present paper presents a quick review of the work done to produce such analogs.

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“…We recently described11 a GRF analog, [desNH2Tyr1,D-Ala2,Ala15]-GRF(l-29)-NH2 (4), which possesses a 4-5fold higher intrinsic potency than the parent hormone in vitro11 and has a significantly longer duration of action in vivo. 12 The increased in vitro potency is primarily a result of the replacement of the native Gly16 residue by Ala which enhances the -helicity of the peptide and maximizes the amphiphilic character of the central -helix13 which may lead to improved receptor affinity.14 The longer in vivo duration of action arises from replacement of the native Tyr ^Ala2 by desNH^Tyr^D-Ala2 which confers resistance to proteolysis by dipeptidyl aminopeptidase IV (DPP IV).16 In considering various methods for large-scale synthesis of the target analog 4, recombinant DNA synthesis is not an option as 4 contains two important features that are not afforded by existing recombinant DNA methods; namely the C-terminal amide, essential for high potency,6 and the unnatural N-terminal residues, desNI^Tyr^D-Ala2. To overcome this limitation, we have devised an enzymatic semisynthetic route to 4 from the precursor, [Ala16'29]-GRF(4-29)-OH (1) (eq 3).…”

Section: Introductionmentioning

confidence: 99%

Enzymic semisynthesis of a superpotent analog of human growth hormone-releasing factor

Bongers

Lambros²,

Li³

et al. 1992

J. Med. Chem.

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A superpotent analog of human growth hormone-releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), was prepared from the precursor, [Ala15,29]-GRF(4-29)-OH (1), by a two-step enzymatic semisynthesis. The amidated C-terminus, essential for high biological potency, was obtained via a carboxypeptidase Y-catalyzed exchange of Ala29-OH for Arg29-NH2 to produce [Ala15]-GRF(4-29)-NH2 (2). The N-terminal desNH2Tyr-D-Ala moiety, which greatly increases in vivo duration of action, was then incorporated by V8 protease-catalyzed condensation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR [R = CH3CH2- (3a) or 4-NO2C6H4CH2-(3b)]. The main focus of this report was to develop conditions to use the V8 protease-catalyzed coupling while avoiding a competing cleavage of the proteolytically-sensitive Asp25-Ile26 bond in GRF. Conversion of 2 to 4 in couplings employing the alpha-ethyl ester of the acyl component 3a was limited to about 60% by competing proteolysis at Asp25-Ile26. This system was adequate for preparing, isolating, and fully characterizing the target analog 4 and identifying the side products. The 4-nitrobenzyl ester 3b proved to be a superior substrate, resulting in 90% conversion of 2 to 4 with no detectable loss to proteolysis and requiring significantly lesser amounts of catalyst. These results demonstrate that enzymatic semisynthesis of a biologically-active peptide amide which contains unnatural amino acids at the N-terminus can be achieved from a biosynthetic precursor in good yield and purity.

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Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs

Cited by 12 publications

References 15 publications

NMR Structure of Two Novel Polyethylene Glycol Conjugates of the Human Growth Hormone-Releasing Factor, hGRF(1−29)−NH₂

NMR Structure of Two Novel Polyethylene Glycol Conjugates of the Human Growth Hormone-Releasing Factor, hGRF(1−29)−NH₂

Growth Hormone-Releasing Factor: Structural Modification or Protection for More Potent Analogs

Enzymic semisynthesis of a superpotent analog of human growth hormone-releasing factor

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Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs

Cited by 12 publications

References 15 publications

NMR Structure of Two Novel Polyethylene Glycol Conjugates of the Human Growth Hormone-Releasing Factor, hGRF(1−29)−NH2

NMR Structure of Two Novel Polyethylene Glycol Conjugates of the Human Growth Hormone-Releasing Factor, hGRF(1−29)−NH2

Growth Hormone-Releasing Factor: Structural Modification or Protection for More Potent Analogs

Enzymic semisynthesis of a superpotent analog of human growth hormone-releasing factor

Contact Info

Product

Resources

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NMR Structure of Two Novel Polyethylene Glycol Conjugates of the Human Growth Hormone-Releasing Factor, hGRF(1−29)−NH₂

NMR Structure of Two Novel Polyethylene Glycol Conjugates of the Human Growth Hormone-Releasing Factor, hGRF(1−29)−NH₂