ABSTRACT
Roy G. Smith Merck Research Laboratories, Merck and Co., Inc., Rahway, New Jersey
I. INTRODUCTION
Pulsatile GH release from the pituitary gland is thought to be regulated by episodic changes in two hypothalamic hormones, growth hormone-releasing hormone (GHRH) and the inhibitory hormone, somatostatin. The discovery of synthetic growth hormone-releasing peptides (GHRPs) by Bowers and co-workers (1-5) that also stimulate GH release was an important fundamental observation. It demanded continued evaluation of whether the physiology of GH release was governed by more than just GHRH and somatostatin and whether GHRPs were mimicking an unknown natural hormone that also played a role. With this in mind, in 1987, the Merck group focused on this issue for two reasons. First, understanding this pathway would likely lead to new strategies that could be exploited for the treatment of GH deficiencies. Second, because GHRPs were small molecules, they might be appropriate templates for the design of nonpeptide analogs. Peptidomimetics have an important advantage over peptides because their structures are more readily modified to allow optimization of oral bioavailability and pharmacokinetic properties. Extensive investigations of the biological properties of GHRPs and peptidomimetics lead one to conclude that these molecules mimic a natural hormone. Indeed, most recently, the cloning and sequencing of the receptor for these novel GH secretagogues (GHSs) showed the receptor to be a highly conserved G-protein-coupled receptor that apparently does not belong to any of the known families of G-protein-coupled receptors. Because of its apparent pivotal role in modulating
38 Smith
II. MECHANISM OF ACTION IN SOMATOTROPHS
The early studies of Cheng et a!. demonstrated that GHRP-6, in contrast to GHRH, appeared to signal through the phospholipase C (PLC) pathway (6). They showed that the effects of GHRP-6 were mimicked, at least partially, by activators of protein kinase C (PKC) and that the effects of GHRP6 were antagonized by a protein kinase C inhibitor (6). Moreover, prolonged exposure of rat anterior pituitary cells to an activating phorbol ester almost completely desensitized the cells to GHRP-6, without affecting their response to GHRH. These data were consistent with suggestions that the receptors for GHRH and GHRP-6 were distinct and that the GHRP-6 receptor was G-protein-coupled and transduced its signal through phospholipase C. From these conclusions and from a consideration of the structural features, the Merck group searched for a pharmacophore that would mimic either GHRP-6 or GHRH by screening a library of nonpeptidyl "privileged structures" that were known to bind to G-protein-coupled receptors (7). With this approach, the first potent nonpeptide GH secretagogue, L692,429, that increased GH release from rat pituitary cells was discovered (8). As shown in Figure 1 the (S)-enantiomer of L-692,429, L-692,428,
~ 50
0 ~--~--~--~--~~--~--~--~--~--~ 0.0 0.5 1.0
Cone (~M) 1.5 2.0
Figure 1 Dose-dependent increase in GH secretion from rat pituitary cells in response to L-692,429 but not L-692,428 demonstrating the stereoselectivity of the response. (From Ref. 8.)
Mechanism of a Non-GHRH GHS's Action 39
The conclusions of the earlier mechanistic studies of Cheng et a!. suggesting that the GHRP-6 signal was transduced through phospholipase C (6) were supported by more direct evidence. Herrington and Hille (1 0) and Bresson-Bepoldin and Dufy-Barbone (11) reported that GHRP-6 stimulated Ca2 + release from intracellular stores. Adams et a!. (12) showed that both GHRP-6 and the nonpeptide mimetic L-692,429 (8) caused increased in inositol triphosphate (IP3) and Mau et a!. demonstrated translocation of protein kinase C (13). These observations strengthened the notion that GHRP-6 and L-692,429 interacted with a G-protein-coupled receptor that
H 0 CH3 CH3 II "\ / ~N-C-CH2-C-NH2
L-692,429
L-692,585
L-692,428
MK-0677
Figure 2 Structural variants of mimetics of GHRP-6 that with the exception of L692,428, the S = enantiomer of L-692,429, are potent GH secretagogues.
