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Spiegelmers are high-affinity L-enantiomeric oligonucleotide ligands that display high resistance to enzymatic degradation compared with D-oligonucleotides. The target binding properties of Spiegelmers can be designed by an in vitro-selection process starting from a random pool of oligonucleotides. Applying this method, a Spiegelmer with high affinity (K D ‫؍‬ 20 nM) for the peptide hormone, gonadotropin-releasing hormone (GnRH) was isolated. The Spiegelmer acts as an antagonist to GnRH in Chinese hamster ovary cells stably expressing the human GnRH receptor, and its activity is unchanged by linking to 40-kDa polyethylene glycol. In a castrated rat model the Spiegelmer further demonstrated strong GnRH antagonist activity, which is more pronounced and persists longer with the polyethylene glycol-linked derivative. Furthermore, in rabbits the anti-GnRH Spiegelmer was shown to have a very low, possibly negligible immunogenic potential. These studies suggest that Spiegelmers could be of substantial interest in the development of new pharmaceutical approaches against GnRH and other targets.in vitro selection ͉ mirror-image oligonucleotide ͉ animal model ͉ aptamer ͉ immunogenicity S piegelmers are mirror-image, high-affinity oligonucleotide ligands composed of L-ribose or L-2Ј-deoxyribose units. The chiral inversion results in high stability in plasma compared with natural D-oligonucleotide ligands, aptamers, suggesting that Spiegelmers may display favorable in vivo behavior and present future potential for therapeutic and diagnostic applications (1). Spiegelmers thus offer a promising alternative to aptamers, the limited in vivo stability of which continues to be a major obstacle to clinical development despite extensive efforts to improve the structure of the oligonucleotide backbone (2, 3).Spiegelmers can fold into distinct three-dimensional structures generating high-affinity ligands that can be selected against defined pharmacological targets. High-affinity Spiegelmers with the desired target-binding properties can be identified by using an adaptation of the SELEX (systematic evolution of ligands by exponential enrichment) procedure (4). Because L nucleic acids are not compatible with SELEX because of the enantio specificity of the enzymes used for amplification, a ''mirror-image'' SELEX approach is used. The first step is to select an aptamer against the enantiomeric form of the natural target. After trimming to the minimal binding motif, the equivalent L form of the aptamer, the Spiegelmer, then is synthesized, and because of the reciprocal chirality, this Spiegelmer binds with high affinity to the natural target. The basic concept of combining molecular evolution with chiral inversion stemmed from the identification of a D-peptide ligand for the SH3 domain of c-Src by using a phage display approach (5). Mirror-image RNA ligands to adenosine and arginine as well as an enantiomeric DNA specific for vasopressin were identified by mirror-image SELEX and have been described previously (1, 6, 7).Gonadotropin-releasi...

These findings provide new therapeutic perspectives for the treatment of muscle injuries, sarcopenia, and cachectic disease, but also imply that such a substance could be abused for doping purposes.

The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (G q , G s , and G i ) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic ␣T3-1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca 2؉ mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the G i -coupled m2 muscarinic receptor interacted with a chimeric G s protein (G s i5) containing the C-terminal five amino acids of G␣ i2 , the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of ␣T3-1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive elementdriven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH-elicited rises in [Ca 2؉ ] i . In ␣T3-1 and CHO#3 cell membranes, addition of [␣-32 P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of G␣ q/11 but not of G␣ i , G␣ s or G␣ 12/13 proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the G q/11 family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor.

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