AHelicallystabilized Ghrh Analogs

As we have seen, the accumulated evidence is very strong for almost the whole GHRH(1-29) sequence existing as an amphiphilic a-helix in its receptor-bound state. There are two ambiguous regions emanating from the computer prediction and NMR data—the areas around Gly in position 15 and the 7-10 sequence. Replacement of Gly15 with Ala or a-aminoisobutyric acid (Aib) should increase a-helicity (Ala and Aib being, in that order, the best amino acids for helix enhancement) and indeed around fivefold-increases in in vitro GH releasing potency have been reported for many Ala15- and Aib15-analogs. These results have been reviewed recently (50). A similar Ala-substitution strategy was applied to positions 8 and 9 (51). All Ala8, Ala9, and Ala8,9-analogs exhibited (51) increased in vitro potency thus finally ruling out the presence of a P-turn in this region. Also, another consequence of a helical conformation is that the Asn8 and Lys12 side-chains should be in close proximity and, indeed, these positions can be covalently linked through Asp and Lys side-chains with complete retention of potency (52).

A more extensive Ala and Aib-substitution study (52) utilizing GHRH(1-29)NH2 was aimed at probing both the functionality of each amino acid side chain and a-helical propensity along the chain and yielded much useful information. It was found that the substitution of Ala in positions 8,9,15,22, and 24 resulted in significant increases in in vitro GH-releasing potencies and that substitution in position 16, 18, 19, 24, 25, and 26 resulted in complete retention of potency. Thus, an amazing number of more complex hydrophobic or hydrophilic amino acid side chains could be replaced with the simple methyl group side chain of Ala. Aib substitution in positions 8 and 22 resulted in increased potencies and conserved potencies when present in positions 9, 16, 18, 24, 25, and 28. We found (53), quite unexpectedly, that simultaneous substitution of Ala at all those positions that had produced enhanced or conserved potency resulted in no loss of potency. In fact, [D-Ala2, Ala89,15,16 18,22,24-28]GHRH(1-29)NH2 (NC-9-45; Table 2), has 48% Ala content and most of its functional amino acids replaced with Ala and yet was still 1.9 times more potent than the parent peptide. Incorporation of Aib in favorable positions 8, 18, and 24 (53) to give [D-Ala2, Aib8,18,24, Ala9,15,16,22,25-28]GHRH(1-29)NH2(NC-9-96; Table 2) resulted in a 2.6-fold increase in GH-releasing potency.

Chou-Fasman secondary structure and hydrophobicity calculations were performed (54) on one of the new high-Ala content analogs (JF-01-40) in comparison with GHRH(1-29) itself and the results are shown in the lower panel of Fig. 3. The new analog, not surprisingly given the number of Ala residues present, is predicted to adopt an a-helical conformation with high probability throughout the whole length of the chain (lower panel, Fig. 3). The calculated hydrophobicity profile (Fig. 2, lower panel) is also interesting to compare to GHRH(1-29) itself. The three hydrophobic and two hydrophilic domains (the latter essentially constituting the pair of -Arg-Lys- sequences) within the chain are now highlighted and might very well comprise the recognition sites for GHRH binding to its receptor. It is also tempting to conclude that the pockets of high hydrophobicity variably separated by periodic basic amino acids (see highlighted residues in the sequences in Fig. 1) are responsible for the binding specificity of the various members of this series of related peptides.

The stabilized helix also permits removal of C-terminal residues with retention of high potency. With the GHRH(1-29)NH2 sequence itself, removal of two and three C-terminal amino acids results in a 10-fold and 100-fold loss of potency, respectively (Table 2).

Table 2

Structure and GH-Releasing Potencies Relative to GHRH(1-29)NH2 on Rat Pituitary Cells of High Ala/Aib-Content GHRH Analogs with Shortened Chain Lengths

Table 2

Structure and GH-Releasing Potencies Relative to GHRH(1-29)NH2 on Rat Pituitary Cells of High Ala/Aib-Content GHRH Analogs with Shortened Chain Lengths o

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