NMR structure of two novel polyethylene glycol conjugates of the human growth hormone-releasing factor, hGRF(1-29)-NH₂

Two novel mono-PEGylated derivatives of hGRF(1-29)-NH₂ [human growth hormone-releasing factor, fragment 1-29] have been synthesized by regio-specific conjugation of Lys¹² or Lys²¹ to a monomethoxy-PEG₅₀₀₀ chain (compounds Lys¹²pEG-GRF and Lys²¹PEG-GRF). The PEG moiety has been covalently linked at the amino group of a norleucine residue via a carbamate bond. The Lys¹²PEG-GRF regioisomer was found to be slightly less active in vitro than both the unmodified peptide and Lys²¹PEG-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)-NH₂, Lys²¹PEG-GRF and Lys¹²PEG-GRF share a remarkably similar pattern of secondary structure. All three compounds adopt an α-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 Lys¹² and Lys²¹ are enclosed in all the compounds considered into well-defined α-helical domains, indicating that PEGylation either at Lys¹² or Lys²¹ does not alter the tendency of the peptide to adopt a stable α-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 α-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)-NH₂ and its receptor.