Bioactive Spiegelmers


Williams et al. showed in 1997 that "selection-reflection" is a viable means of producing bioactive and nuclease-resistant ligands for bioactive target molecules (Williams et al., 1997). Selecting for the all-d form of vasopressin - a cyclic non-apeptide - they finally generated a 55-nucleotide l-DNA that binds to the natural vasopressin with equal (low micromolar) affinity. This DNA Spiegelmer was active in an assay using cultured renal cells which expressed the V2 receptor, antagonizing vasopressin-stimulated receptor activation with a calculated IC50 in the lower micromolar range. An l-DNA control sequence with identical base composition and the same putative secondary structure was virtually inactive in the assay.

Gonadotropin-releasing hormone

Another peptide hormone that had been addressed by a "selection-reflection" approach is gonadotropin-releasing hormone I (GnRH; synonyms: gonadoliberin, luteinizing hormone-releasing hormone, LHRH) (Leva et al., 2002). GnRH is a key peptide hormone in the regulation of mammalian reproduction. It is a deca-peptide that is released from hypothalamic neurons in a pulsatile manner, binding to receptors on gonadotrophic cells on the pituitary. Here, GnRH stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in turn trigger the production of sexual steroids. Therefore, GnRH and its receptor have been identified as therapeutic targets for sex steroid-dependent conditions such as prostate and breast cancer, endometriosis, as well as in assisted-reproduction techniques.

For the first time, RNA as well as DNA Spiegelmers were identified for the same target molecule. The oligonucleotides exhibit dissociation constants between 50 and 100 nmol/L in equilibrium dialysis assays. As demonstrated with surface plasmon resonance experiments, both Spiegelmers showed very high specificity for GnRH, since even the exchange of a single amino acid, as in chicken GnRH (Arg8pGln), resulted in a dramatic loss of binding affinity. The unrelated peptide hormones vasopressin or oxytocin, which are of similar molecular weight, were not recognized by the Spiegelmers at all. Cell culture experiments with transfected Chinese hamster ovary (CHO) cells that stably expressed the human GnRH receptor revealed that the Spiegelmers inhibit binding of l-GnRH to its receptor with an IC50 of 200 and 50nmol/L for the 48-nt RNA and the 60-nt DNA Spiegelmer, respectively

Calcitonin gene-related peptide

An RNA Spiegelmer directed to the migraine-associated molecule calcitonin gene-related peptide 1 (CGRP) resulted from a mirror-image selection against rat d-CGRP (Vater et al., 2003). This selection was performed using the so-called "Tailored-SELEX" process which relies on in vitro selection of a primerless RNA library and custom-designed primers/adapters that are added by ligation before and removed within the amplification processes. The calculated affinity (Kd = 2.5 nmol/L at 37 °C) of the best Spiegelmer almost reaches the binding of the neuropeptide to its receptor (EC50 = 1 nmol/L). The potential of this molecule to act as a CGRP antagonist in vivo and its feasibility in the treatment of migraine is currently under investigation (Karl Messlinger, personal communcation).


Another pain-related target is the endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ), which plays a prominent role in the regulation of several biological functions such as pain and stress. High-affinity RNA Spiegelmers were shown to antagonize the interaction of N/OFQ with its receptor ORL1 in a binding competition assay exhibiting an IC50 of 110 nmol/L (Faulhammer et al., 2004). Furthermore, the identified Spiegelmers inhibited N/OFQ-induced GTPgS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor and showed an antagonistic effect to the N/OFQ-ORL1 receptor system that was functionally coupled with G protein-regulated inwardly rectifying K+ channels in Xenopus laevis oocytes.

Substance P

The mirror image of the neuropeptide l-substance P served as a target to demonstrate the robustness of an automated in vitro selection protocol (Eulberg et al., 2005). Substance P is an 11-amino-acid peptide that belongs to the tachykinin family and has a wide variety of biological activities, such as peripheral vasodilation, smooth muscle contraction, pain transmission, and neurogenic inflammation. The identified 44-nucleotide Spiegelmer ligand binds substance P with high affinity (Kd = 40 nmol/L at 37 °C) and also inhibits substance P action in a bio-activity assay using substance P-sensitive human AR42J pancreatic cells (IC50 z 45 nmol/L).

18.3 Mirror-Image Evolutionary Techniques: Selection-Reflection | 431 Spiegelmer Activity in Vivo

Gonadotropin-releasing hormone

The functional activity of mirror-image oligonucleotides in an animal model was demonstrated first with a GnRH-inhibiting DNA Spiegelmer (Wlotzka et al., 2002). In an effort to maximize the pharmacological response of the active Spiegelmer molecule by slowing down its renal clearance from the bloodstream, the l-DNA-oligonucleotide was modified with a 40kDa branched polyethylene glycol (PEG) moiety to its 5' end. PEGylation of the Spiegelmer had no effect on its binding behavior towards GnRH, as both the PEGylated and the non-PEGylated versions showed the same activity in cell culture assays with an IC50 of approximately 20nmol/L. The PEGylated Spiegelmer was further studied in a widely used rat model of GnRH regulation in which the serum LH concentration in orchidectomized (castrated) animals represented the efficacy parameter: due to orchidectomization, the lack of testosterone production led to an increase in the frequency of GnRH pulses resulting in high LH plasma levels. Neutralization of the GnRH activity in turn leads to a normalization of the LH plasma concentration to the level of intact untreated animals.

The DNA Spiegelmer was administered 8 days after castration, when the animals displayed stably high LH levels. The subcutaneously administered non-PEGylated Spiegelmer (100mg/kg) showed maximal GnRH antagonism 1.5 h after administration, which levelled off during the following hours. The effect of the PEGylated Spiegelmer, given intravenously at 150mg/kg, lasted longer and was comparable to that of the current gold-standard Cetrorelix™, a GnRH receptor antagonist, administered at a dose of 100 mg/kg to control groups.


Ghrelin was discovered in stomach extract by Kangawa and co-workers (Kojima et al., 1999). The 28-amino-acid peptide is the only endogenous ligand for the growth hormone secretagogue receptor 1 (GHS-R1a) known to date. Receptor binding and activation requires the presence of a post-translationally added octa-noic, decanoic, or decenoic acid modification at Ser3. This modification is essential for bioactivity, whereas more than 20 amino acid residues may be deleted from the C-terminus without compromising receptor binding in vitro. RNA Spiegelmers were generated that bind with extremely high specificity to the octanoy-lated form of ghrelin (Helmling et al., 2004). The best binding candidate NOX-B11 displays a binding affinity in the low nanomolar range (Kd z 35nmol/L). NOX-B11 also inhibits ghrelin-mediated stimulation of cells expressing GHS-R1a (IC50 z 5nmol/L). Furthermore, the Spiegelmer clearly differentiates between the octanoylated and desoctanoylated forms of ghrelin and requires only the N-terminal five amino acids of bioactive ghrelin for binding, as demonstrated in competition experiments.

The in vivo activity of the Spiegelmer was studied in several animal models. Since ghrelin is known to induce growth hormone (GH) release as well as acute food intake, these parameters were analyzed in the first instance. After stimulating rats with 3 nmol of (octanoylated) ghrelin, an increase of GH in the serum could be detected. This effect was dose-dependently alleviated by NOX-B11, which was administered 15min before the ghrelin stimulus. A 5- to 10fold molar excess of NOX-B11 over ghrelin blocked GH release completely.

Two similar surrogate studies in rats were carried out to monitor the effects of NOX-B11 on the parameter acute food intake as well as neuronal activation in the arcuate nucleus (Kobelt et al., 2005). Spiegelmer NOX-B11 was able to reduce ghrelin-induced (3 nmol per animal) food intake dose-dependently. Whereas at a dose of lnmol per animal no effect was observed, 30 nmol of NOX-B11 per rat totally abolished effects ofexogenous ghrelin. A comparable result was obtained by monitoring ghrelin-induced c-Fos-like immunoreactivity in the arcuate nucleus. NOX-B11 specifically reduced neuronal activation as compared to a nonfunctional control Spiegelmer.

These data confirm the potential therapeutic utility of Spiegelmers in the living organism. They further indicate that treatment with a ghrelin-binding Spiegelmer may provide an approach to the treatment of obesity and obesity-related diseases through blocking of peripheral, endogenous ghrelin. First results indicate that ghrelin blockade by chronic infusion of NOX-B11 leads to weight loss in diet-induced obese mice (L. P. Shearman, personal communication).

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