Tripeptide Analogs and A Pharmacophore Model

The evidence summarized above strongly supports the designation of MK-0677 as a mimetic of GHRP-6 because they are functionally equivalent, they bind competitively to the same receptor, and this binding responds similarly to E124^Q124 mutation. Furthermore, an analog of GHRP-6 with aminoisobutyric acid substituted for its N-terminal histidine is a highly active secretagogue (70). Even if one assumes binding correspondence near Glu124, the overlap, if any, of their other pharmacophore groups is not certain. Ambiguities in defining the active conformation of GHRP-6 also limit definitive comparisons with MK-0677. However, the Merck group (27) hypothesized in general agreement with Momany et al. (9) and Momany (71) that GHRP-6 has a bent conformation when bound to the receptor. Using the published conformation of Schoen et al. (27) and superimposing the N-termini of GHRP-6, and MK-0677, we hypothesized that the spiroindoline of MK-0677 and the indole group of Trp4 in GHRP-6 may share the same binding site on the receptor. To test this hypothesis, some of the compounds in Tables 7 and 8 were synthesized by Yang et al. (72). Reasonable

Table 7 Spiropiperidine Replacements h r

Compound_R_EC50 (nM)'

Compound_R_EC50 (nM)'

h aData from ref. 72.

h aData from ref. 72.

potency was observed with the P-napthylmethylamide 51 (EC50 = 85 nM) and the indolylethylamide 52 (EC50 = 57 nM). However, most strikingly, the D-amino acid derivatives 54 (EC50 = 3 nM) and 56 (EC50 = 6 nM) are more active in our assay than GHRP-6 (EC50 = 10 nM). In contrast, the hydrophobic, aliphatic amino acid 57 was poorly active (EC50 = 5 5 5 nM) and the absence of an amino acid side-chain in 58 led to even less activity (EC50 = 1060 nM). This SAR study achieved for us a reduction of the GHRP-6 structure to the tripeptide level with retention of greater activity than GHRP-6 in the (D)-isomers of compounds 54 and 56. A similar achievement based on conformationally restricted analogs and molecular modeling studies also brought McDowell et al. (25) to the conclusion that the minimum pharmacophore required for the expression of GHRP type activity is only a basic amino group and two aromatic amino acids. The fact that we were able to convert MK-0677 to a highly active peptide of the same size strongly supports a peptide-peptidomimetic relationship at this tripeptide level.

Table 8

Tripeptide Growth Hormone Secretagogues

CKH^"

Compound R Stereochemistry EC50 (nM)a

Compound R Stereochemistry EC50 (nM)a

C02Et

"Data from ref. 72.

C02Et

"Data from ref. 72.

Peptide and peptidomimetic agonist ligands need not bind to receptors in the same way (73). However, if the tripeptides and MK-0677 do bind with correspondence of their amino and aromatic residues, then, in this instance, the privileged structure seems to be simply a conformationally rigid moiety which is able to share the binding site of an aromatic amino acid of the tripeptide. Ariens' concept of an accessory binding site as applied to biogenic amine antagonists would then not be necessary nor apply to these peptidomimetic secretagogue agonists.

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