Inhibition of ghrelin action in vitro and in vivo by an RNA-Spiegelmer

Abstract

Employing in vitro selection techniques, we have generated biostable RNA-based compounds, so-called Spiegelmers, that specifically bind n-octanoyl ghrelin, the recently discovered endogenous ligand for the type 1a growth hormone secretagogue (GHS) receptor. Ghrelin is a potent stimulant of growth hormone release, food intake, and adiposity. We demonstrate that our lead compound, L-NOX-B11, binds ghrelin with low-nanomolar affinity and inhibits ghrelin-mediated GHS-receptor activation in cell culture with an IC(50) of 5 nM. l-NOX-B11 is highly specific for the bioactive, n-octanoylated form of ghrelin. Like the GHS receptor, it does not recognize the inactive unmodified peptide and requires only the N-terminal five amino acids for the interaction. The i.v. administration of polyethylene glycol modified l-NOX-B11 efficiently suppresses ghrelin-induced growth hormone release in rats. These results demonstrate that the neutralization of circulating bioactive ghrelin leads to inhibition of ghrelin's secretory effects in the CNS.

Fig. 4.

Ghrelin-mediated GH release is suppressed by prior i.v. administration of Spiegelmer l-NOX-B11. (A) Animals were injected i.v. with either PBS (curve a) or the indicated doses of Spiegelmer (curves b–d) 15 min before stimulation with 3 nmol of ghrelin. Plasma GH levels were determined at the indicated time points. (B) The ghrelin-induced stimulation of GH release is not inhibited by a random Spiegelmer control sequence.

Fig. 1.

The central 47 nucleotides of RNA B11 are sufficient for binding to d-ghrelin. (A) Secondary structure predictions for full-length (Left) and truncated (Right) RNA B11. (B) On the basis of the predicted secondary structure the 81-mer B11 was truncated to 47 nt and the K_d for ghrelin of the resulting RNA NOX-B11 was determined. No loss in affinity was associated with the truncation.

Fig. 2.

Ghrelin-mediated activation of GHS-R1a is inhibited in the presence of Spiegelmer l-NOX-B11. GHS-R1a-expressing CHO cells were stimulated with ghrelin at 5 nM in the presence of the indicated concentrations of Spiegelmer l-NOX-B11, and the resulting Ca²⁺-associated fluorescence was measured. The response to ghrelin binding to the receptor is suppressed by l-NOX-B11 in a dose-dependent manner, whereas a nonspecific l-RNA sequence does not affect receptor activation.

Fig. 3.

Spiegelmer l-NOX-B11 binds the n-octanoylated N terminus of ghrelin. l-NOX-B11 requires the octanoylated N terminus of ghrelin for binding and inhibition. GHS-R1a-expressing CHO cells were stimulated with 10 nM ghrelin (A) or ghrelin-(1–5) (B) in the presence of 30 nM l-NOX-B11, 300 nM desoctanoyl ghrelin (d.-o. Ghr.), or both. Ca²⁺-associated fluorescence triggered by ghrelin's binding to the receptor was measured. (C) The sequence of rat ghrelin, showing the minimal binding motif for l-NOX-B11 (boxed) and the differing amino acids in the human sequence (shaded). See Results for details.