Early attempts to identify a specific receptor for the GHRPs by the Merck group and by others (64,65) made use of [3H] and [125I]-labeled ligands derived from GHRP-6. Success was limited by their relatively low specific activity and high nonspecific binding nor, in the experience of the Merck group, did the secretagogue activity of compounds correlate well with their potency in displacing labeled ligand. With discovery of MK-0677 and the demonstration of its high selectivity in respect to other receptors, a renewed effort was made to produce from it a radioligand of high specific activity. [125I] could not be used in the benzyloxy-para-position of MK-0677 since considerable loss of intrinsic secretagogue potency would ensue. Nor could the Bolton-Hunter reagent be used since conjugation of the MK-0677 amino group would lead to complete loss of bioactivity. Instead Dean et al. (66) developed a synthesis of methane [35S]-sulfonyl chloride and utilized this reagent in the preparation of [35S]-MK-0677 in greater than 99% radiochemical purity with specific activities ranging from 700 to 1100 Ci/mmol. They were thus able to achieve high specific activity in a potent and selective radioligand with relatively low lipophilicity (log P = 3.0).
With the availability of [35S]-MK-0677, Pong et al. (29)identified a saturable, high-affinity binding site in porcine and rat anterior pituitary membranes. Its KD of 161 ± 11 pM in rat pituitary membranes closely corresponded to a K of 240 pM determined by the displacement of the radioligand by unlabeled MK-0677. This K for receptor binding is slightly lower than its EC50 of 1.3 nM for GH secretion in the rat pituitary cell. Corresponding data for GHRP-6 (Ki = 6 nM) were in line with its potency in the cell culture assay (EC50 = 10 nM). The specific binding of [35S]-MK-0677 was Mg2+ dependent and inhibited by the GTPyS, which is consistent with the receptor being G-protein linked. Importantly, double reciprocal plot analysis of saturation isotherms for the [35S] MK-0677 binding demonstrated that GHRP-6 inhibition could be overcome by increasing concentrations of [35S]-MK-0677. These data suggest that the two secreta-gogues interact competitively at the same receptor and further confirm the peptidomi-metic nature of MK-0677.
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