Table 1. n-Octanoyl Ser-3 ester derivatives of ghrelin.
functionality in 9, diminish activity as well, further emphasizing the necessity of lipophilicity in the ester moiety of ghrelin. Octanoylation at Ser-2 versus Ser-3 does not significantly alter functional activity while movement to Ser-6 or Ser-18 resulted in a loss of activity. Replacement of the serine ester moiety with an amide group shows comparable activity to ghrelin as does ether or thioether replacements. The L-configuration Ser-3 is important for ghrelin activity.
The n-octanoyl side-chain has also been incorporated into peptidomimetic GHS analogs creating hybrid analogs such as 10, H, and 12. Replacement of the benzyloxy side chain of 3 with the octanoyl ester moiety results in compound 10 which has functional potency similar to ghrelin. Modifications of the aminoisobutyric moiety of 3 to mimic ghrelin, such as found in H, also results in potent ghrelin agonist (47).
Peptide Minimization - Truncation of ghrelin peptide sequence has resulted in the identification of analogs with weaker affinity to the receptor than ghrelin but similar functional activity (45, 46). As shown in Table 2, systematic minimization of the peptide backbone indicates that only the first five residues (13) are required for full activation of the receptor. Further minimization to tetrapeptide 14 results in diminished activity while the tripeptide is inactive. Other studies have shown that 13 and 14 retain some functional activity in vitro while lacking receptor affinity and in vivo efficacy for GH release (48). The tetrapeptide 14 has been evaluated by 1H-NMR analysis and compared to 2 (49). While some structural similarities were found when superimposing these two peptides, the analysis suggests that both the binding affinities and the requirement for the n-octanoyl group cannot be readily rationalized in their model.
12 GSSFLSPEHQRVQQRNH2 96 17
13 GSSFLNH2 55 11
14 GSSFNH2 889 72
Pharmacological Effects of Ghrelin Agonists on Food Intake, and Energy Balance -While extensive efforts have been directed towards optimizing molecules that stimulate pituitary GH release (4), the effects of GHSs on appetite regulation and energy homeostasis have remained considerably less characterized, particularly with respect to orally active ghrelin mimetics. In fact, only peptide ligands such as ghrelin, 2, 3, GHRP-2 (15), and ipamorelin (16). which are all distinguished by poor oral activity, have been prospectively evaluated for their adipogenic effects (2).
The earliest report of the orexigenic effects of GHSs predates the discovery of ghrelin by over 4 years (50). In this study, increases in food intake are observed with 2 after intracerebroventricular administration to rats. Similar effects have been shown for other GHSs including 15 (and analogs) and 16 (51-54). We have recently shown that GHRP-2 increases fat mass in mice lacking NPY (40). Compound 15 also increases 6-hour food intake in NPY knockout mice while not affecting 24 hour food intake. The adipogenic effects of 2 and 16 have been studied recently (55). Two weeks of treatment of 16 to GH-intact mice causes an increase in food consumption as well as fat mass, outcomes that appear to be independent of the compounds' GH releasing properties. Other work supports the hypothesis that the feeding effects observed with ghrelin and GHSs are likely independent of GH (40).
Capromorelin (17) has been shown to increase body weight in rats after extended treatment (28 days) without concomitant changes in body fat or lean mass (4, 56). The orexigenic effects of 3 have not been reported although increases in growth velocity have been observed in rats and humans (57). In humans 3 increases energy expenditure and fat free mass in obese male patients after two months of treatment (58). Total body fat was not affected in this study. The effects of SM-130686 (18) on food intake and body weight have been recently reported (59, 60). The pharmacological profile for this compound differs from previous GHS analogs in that it is a partial agonist at the GHS-R1a receptor. Unlike 15 and 16, treatment of rats for 9 days with 18 causes an increase in fat free mass. The increase in body weight observed with 18 is not accompanied with an increase in food intake.
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