Differential effects of N-terminal modifications on the biological potencies of growth hormone releasing factor analogues with varying chain lengths.

The excellent retention of biological potencies observed with human growth hormone releasing factor analogues with chains 29-44 amino acid residues long is suddenly lost when further amino acid residues are removed from the C-terminus. For instance, 1-27 and 1-24 exhibited little biological activity (greater than 1%) in vivo and in vitro in the rat. Studies were made to determine whether this was due to conformational changes rather than simply the loss of amino acids needed for direct receptor interactions. These involved the introduction of mild conformational restraint in the N-terminal region by the introduction of D-amino acid residues previously shown to increase the potency of the 1-29 peptide. D-Ala in position 2, responsible for a 40- to 50-fold increase in activity in the 1-29 species, resulted in little increase in the potency of 1-27 or 1-24 sequences. However, N-terminal acetylation, responsible for a 12-fold increase in 1-29 in vivo potency, caused greater than 50-fold increase in 1-27 potency but had little effect on 1-24 potency. Likewise, D-Asn in position 8 was far more effective in increasing the potency of the 1-27 sequence compared to the 1-29 ([D-Asn8]-GRF(1-29)NH2, 220% vs. [D-Asn8, Leu27]-GRF(1-27)NH2, 53%; in vivo]. This differential effect was even more clear in vitro. The highest in vivo potency in the 1-27 series was achieved with [D-Asp3,D-Asn8,Leu27]-GRF(1-27)NH2 (200%); however, this analogue was still far less potent than its 1-29 counterpart (3800%). None of the D-amino acid substitution strategies were effective in increasing 1-22 peptide potencies to detectable levels. The results indicate that the effect of N-terminal substitutions and resulting potencies of the GRFs is very much dependent on chain length, perhaps suggesting that C-terminal amino acids promote conformational effects at the N-terminus and/or vice versa.