Spiegelmer Inhibition of MCP-1/CCR2--Potential as an Adjunct Immunosuppressive Therapy in Transplantation.

The rejection process remains the key unsolved issue after transplantation of disparate tissue. The CC chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) has been reported to be involved in the process of alloimmune interaction. Spiegelmers are l-oligonucleotides that can be designed to bind to pharmacologically relevant target molecules. Here, we tested a high-affinity Spiegelmer-based MCP-1 inhibitor (mNOX-E36) in an allogeneic heart transplant model. Fully vascularized allogeneic heterotopic heart transplantations from BALB/c to C57BL/6 mice were performed. Mice were either treated with the anti-MCP-1-Spiegelmer (mNOX-E36) in monotherapy or in combination with subtherapeutic doses of cyclosporine A (CsA) (10 mg/kgBW/day) for 10 days. Controls received equivalent doses of a non-functional Spiegelmer (revmNOX-E36). Graft survival of allogeneic heart transplants was slightly but significantly prolonged under mNOX-E36 monotherapy (median graft survival 10 day ± 0.7) compared to revmNOX-E36 (median graft survival 7 day ± 0.3; P = 0.001). A synergistic beneficial effect could be seen when mNOX-E36 was administered in combination with subtherapeutic doses of CsA (18 day ± 2.8 versus 7 day ± 0.3; P < 0.0001). Levels of inflammatory cytokines and 'alarmins' were significantly reduced, and the number of F4/80(+) cells was lower under combination therapy (1.8% ± 1.3%; versus 14.6% ± 4.4%; P = 0.0002). This novel inhibitor of the MCP-1/CCR2 axis (mNOX-E36), which has already proven efficacy and tolerability in early clinical trials, alleviates acute rejection processes in allogeneic transplantation especially when combined with subtherapeutic doses of CsA. Thus, mNOX-E36 may have potential as an adjunct immunomodulatory agent.

A novel CGRP-neutralizing Spiegelmer attenuates neurogenic plasma protein extravasation.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) plays an important role in the pathology of migraine, and recent clinical trials suggest the inhibition of CGRP-mediated processes as a new therapeutic option in migraine. In this study, we describe the generation of NOX-L41, a CGRP-neutralizing mirror-image (L-)aptamer (Spiegelmer) and investigate its in vitro and in vivo function. EXPERIMENTAL APPROACH: A CGRP-binding Spiegelmer was identified by in vitro selection. Binding studies were performed using surface plasmon resonance (SPR), and the inhibitory activity was determined in cell-based assays. The pharmacokinetic profile comparing i.v. and s.c. dosing was analysed in rats. Intravital two-photon microscopy was employed to follow extravasation from meningeal vessels. Finally, in vivo efficacy was tested in a model of electrically evoked meningeal plasma protein extravasation (PPE) in rats. KEY RESULTS: We identified NOX-L41, a novel CGRP-neutralizing Spiegelmer. SPR studies showed that NOX-L41 binds to human and rat/mouse CGRP with sub-nanomolar affinities and is highly selective against related peptides such as amylin. In vitro, NOX-L41 effectively inhibited CGRP-induced cAMP formation in SK-N-MC cells. In rats, NOX-L41 had a plasma half-life of 8 h. Pharmacodynamic studies showed that NOX-L41 extravasates from blood vessels in the dura mater and inhibits neurogenic meningeal PPE for at least 18 h after single dosing. CONCLUSIONS AND IMPLICATIONS: This is the first description of the CGRP-neutralizing Spiegelmer NOX-L41. Preclinical studies confirmed a role for CGRP in neurogenic PPE and provided proof-of-concept for the potential use of this new drug candidate for the treatment or prevention of migraine.

Hematopoietic stem and progenitor cell mobilization in mice and humans by a first-in-class mirror-image oligonucleotide inhibitor of CXCL12.

NOX-A12 is a PEGylated mirror-image oligonucleotide (a so-called Spiegelmer) that binds to CXCL12 (stromal cell-derived factor-1, SDF-1) with high affinity thereby inhibiting CXCL12 signaling on both its receptors, CXCR4 and CXCR7. In animals, NOX-A12 mobilized white blood cells (WBCs) and hematopoietic stem and progenitor cells (HSCs) into peripheral blood (PB). In healthy volunteers, single doses of NOX-A12 had a benign safety profile and also dose-dependently mobilized WBCs and HSCs into PB. HSC peak mobilization reached a plateau at five times the baseline level at an i.v. dose of 5.4 mg/kg. In accordance with the plasma half-life of 38 h, the duration of the WBC and HSC mobilization was long lasting and increased dose-dependently to more than 4 days at the highest dose (10.8 mg/kg). In conclusion, NOX-A12 may be appropriate for therapeutic use in and beyond mobilization of HSCs, e.g., in long-lasting mobilization and chemosensitization of hematological cancer cells.

Podocytes produce homeostatic chemokine stromal cell-derived factor-1/CXCL12, which contributes to glomerulosclerosis, podocyte loss and albuminuria in a mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: Chemokine (C-X-C motif) ligand 12 (CXCL12) (also known as stromal cell-derived factor-1 [SDF-1]-alpha) is a homeostatic chemokine with multiple roles in cell homing, tumour metastasis, angiogenesis and tissue regeneration after acute injuries. However, its role in chronic diseases remains poorly defined, e.g. in chronic glomerular diseases like diabetic glomerulosclerosis. We hypothesised that CXCL12 may have a functional role during the evolution of diabetic glomerulosclerosis, either by assisting glomerular repair or by supporting the maladaptive tissue remodelling in response to hyperglycaemia and glomerular hyperfiltration. METHODS: To define the functional role of CXCL12 in the progression of glomerular disease, we used the CXCL12-specific inhibitor NOX-A12, an L: -enantiomeric RNA oligonucleotide (Spiegelmer). A mouse model of type 2 diabetes (db/db mice) was used. Male db/db mice, uni-nephrectomised at 6 weeks of age, received subcutaneous injections with a PEGylated form of NOX-A12, non-functional control Spiegelmer or vehicle on alternate days from 4 to 6 months of age. RESULTS: Immunostaining localised renal CXCL12 production to glomerular podocytes in db/db mice with early or advanced diabetic nephropathy. CXCL12 inhibition significantly reduced the degree of glomerulosclerosis, increased the number of podocytes, prevented the onset of albuminuria and maintained the peritubular vasculature without affecting blood glucose levels, body weight or glomerular macrophage infiltration. CONCLUSIONS/INTERPRETATION: We conclude that podocytes produce CXCL12, which contributes to proteinuria and glomerulosclerosis in our mouse model of type 2 diabetes. This novel pathomechanism provides the first evidence that CXCL12 could be a therapeutic target in (diabetic) glomerulosclerosis.

The anti-ghrelin Spiegelmer NOX-B11-3 blocks ghrelin- but not fasting-induced neuronal activation in the hypothalamic arcuate nucleus.

The hypothalamic arcuate nucleus (Arc) is the presumed target site for the orexigenic hormone ghrelin, which is secreted from the stomach during fasting. Ghrelin directly activates Arc neurones. Similar to exogenous ghrelin, overnight food deprivation also induces c-Fos expression in the Arc of mice. In the present study, we tested the role of endogenous ghrelin in the fasting-induced c-Fos expression in the Arc of mice. We used NOX-B11-3, the latest generation of the recently developed ghrelin-binding compounds, so-called RNA Spiegelmers (SPM) to block endogenous ghrelin action during food deprivation. The specificity and potency of this compound was also tested in electrophysiological and immunohistological experiments. In electrophysiological in vitro single cell recordings, NOX-B11-3 effectively blocked the excitatory effect of ghrelin in the medial Arc (ArcM) of rats whereas the biologically inactive control SPM had no effect. Furthermore, NOX-B11-3 (15 mg/kg i.p.) potently suppressed ghrelin-induced (25 microg/kg s.c., 12 h after SPM injection) c-Fos expression in the Arc. However, when injected at the beginning of a 14-h fasting period, the same dose of NOX-B11-3 had no effect on the c-Fos expression in the Arc of mice. These results demonstrate that NOX-B11-3 is a long-acting compound, which effectively blocks the effect of exogenous ghrelin on neuronal activity in the Arc under in vitro and in vivo conditions. Furthermore, increased ghrelin signalling does not appear to be a necessary factor for the activation of Arc neurones during food deprivation or other fasting-related signals might have masked or compensated for the loss of the ghrelin effect.

Anti-ghrelin Spiegelmer NOX-B11 inhibits neurostimulatory and orexigenic effects of peripheral ghrelin in rats.

BACKGROUND AND AIMS: Ghrelin, the natural ligand of the growth hormone secretagogue receptor 1a, is the most powerful peripherally active orexigenic agent known. In rodents, ghrelin administration stimulates growth hormone release, food intake, and adiposity. Because of these effects, blocking of ghrelin has been widely discussed as a potential treatment for obesity. Spiegelmer NOX-B11 is a synthetic l-oligonucleotide, which was previously shown to bind ghrelin. We examined the effects of NOX-B11 on ghrelin induced neuronal activation and food intake in non-fasted rats. METHODS: Animals received various doses of NOX-B11, inactive control Spiegelmer, or vehicle intravenously. Ghrelin or vehicle was administered intraperitoneally 12 hours later and food intake was measured over four hours. Neuronal activation was assessed as c-Fos-like immunoreactivity in the arcuate nucleus. RESULTS: Treatment with NOX-B11 30 nmol suppressed ghrelin induced c-Fos-like immunoreactivity in the arcuate nucleus and blocked the ghrelin induced increase in food intake within the first half hour after ghrelin injection (mean 1.13 (SEM 0.59) g/kg body weight; 4.94 (0.63) g/kg body weight versus 0.58 (0.58) g/kg body weight; p<0.0001). Treatment with NOX-B11 1 nmol or control Spiegelmer had no effect whereas treatment with NOX-B11 10 nmol showed an intermediate effect on ghrelin induced food intake. CONCLUSIONS: Spiegelmer NOX-B11 suppresses ghrelin induced food intake and c-Fos induction in the arcuate nucleus in rats. The use of an anti-ghrelin Spiegelmer could be an innovative new approach to inhibit the biological action of circulating ghrelin. This may be of particular relevance to conditions associated with elevated plasma ghrelin, such as the Prader-Willi syndrome.

Control of neuronal branching by the death receptor CD95 (Fas/Apo-1).

The CD95 (Apo-1/Fas)/CD95 ligand (CD95L) system is best characterized as a trigger of apoptosis. Nevertheless, despite broad expression of CD95L and CD95 in the developing brain, absence of functional CD95 (lpr mice) or CD95L (gld mice) does not alter neuronal numbers. Here, we report that in embryonic hippocampal and cortical neurons in vivo and in vitro CD95L does not induce apoptosis. Triggering of CD95 in cultured immature neurons substantially increases neurite branches by promoting their formation. The branching increase occurs in a caspase-independent and death domain-dependent manner and is paralleled by an increase in the nonphosphorylated form of Tau. Most importantly, lpr and gld mutants exhibit a reduced number of dendritic branches in vivo at the time when synapse formation takes place. These data reveal a novel function for the CD95 system and add to the picture of guidance molecules in the developing brain.

First look at RNA in L-configuration.

Nucleic acid molecules in the mirror image or L-configuration are unknown in nature and are extraordinarily resistant to biological degradation. The identification of functional L-oligonucleotides called Spiegelmers offers a novel approach for drug discovery based on RNA. The sequence r(CUGGGCGG).r(CCGCCUGG) was chosen as a model system for structural analysis of helices in the L-configuration as the structure of the D-form of this sequence has previously been determined in structural studies of 5S RNA domains, in particular domain E of the Thermus flavus 5S rRNA [Perbandt et al. (2001), Acta Cryst. D57, 219-224]. Unexpectedly, the results of crystallization trials showed little similarity between the D- and the L-forms of the duplex in either the crystallization hits or the diffraction performance. The crystal structure of this L-RNA duplex has been determined at 1.9 A resolution with R(work) and R(free) of 23.8 and 28.6%, respectively. The crystals belong to space group R32, with unit-cell parameters a = 45.7, c = 264.6 A. Although there are two molecules in the asymmetric unit rather than one, the structure of the L-form arranges helical pairs in a head-to-tail fashion to form pseudo-continuous infinite helices in the crystal as in the D-form. On the other hand, the wobble-like G.C(+) base pair seen in the D-RNA analogue does not appear in the L-RNA duplex, which forms a regular double-helical structure with typical Watson-Crick base pairing.

A new class of Spiegelmers containing 2'-fluoro-nucleotides.

Synthesis of 2'-fluoro-nucleosides from L-arabinose in order to perform the synthesis of 2'-fluoro-Spiegelmers binding to a neuropeptide.

Mirror-image RNA that binds D-adenosine.

A 58-mer L-RNA ligand that binds to naturally occurring D-adenosine with a dissociation constant of 1.7 microM in solution was identified from a combinatorial library employing mirror-design. The corresponding D-RNA ligand shows identical binding affinity to L-adenosine. Reciprocal chiral specificity was also evident from ligand discrimination; the binding affinity of the L-RNA ligand for D-adenosine was 9000-fold greater than its affinity for L-adenosine and vice versa. While the D-RNA ligand was rapidly degraded in human serum, the L-RNA ligand displayed an extraordinary stability. This indicates the potential application of specifically designed L-RNA ligands as stable monoclonal antibody analogues and the development of highly stable L-ribozymes.

Mirror-design of L-oligonucleotide ligands binding to L-arginine.

The high affinity and selectivity of nucleic acid ligands have clearly demonstrated that RNA can be targeted to a variety of molecules. In practice, however, the use of unmodified aptamers is impeded by the low stability of RNA in biological fluids. Here we describe the mirror-design of a stable 38-mer L-oligoribonucleotide ligand that binds to L-arginine. This L-RNA ligand was also able to bind to a short peptide containing the basic region of the human immunodeficiency virus type-1 Tat-protein. The L-RNA ligand displayed the expected stability in human serum. These findings may contribute to the identification of novel diagnostics and pharmaceuticals.

Detection of multiple conformations of the E-domain of 5S rRNA from Escherichia coli in solution and in crystals by NMR spectroscopy.

NMR spectroscopy of the E-domain fragment of Escherichia coli 5S rRNA indicates that this molecule exists in solution as either a stem-loop or as a duplex with two U-U base pairs in the bulge region. At temperatures below 27 degrees C, interconversion between the monomeric and dimeric forms in solution occurs on a time scale of weeks and allows the preparation of samples on which NMR structure determinations can be carried out on predominantly monomeric or dimeric species. The NMR results obtained provide comparison data for the distinction between A- and B-form E.coli 5S rRNA and for the possible kinetics of conversion between these forms. NMR evidence is presented that the duplex form also exists in crystals and suggestions are made for means to obtain stem-loop conformations of E-domain and other small RNA stem-loop sequences in crystals.

Initial analysis of 750 MHz NMR spectra of selective 15N-G,U labelled E. coli 5S rRNA.

The overall folding of an RNA molecule is reflected in its base pairing pattern. The identification of that pattern provides a first step towards the determination of the structure of an RNA molecule. We show that the application of heteronuclear NMR methods at 750 MHz to E. coli 5S rRNA (120 nucleotides) selectively labelled with 15N in guanine and uridine allows observation of base pairing patterns for a larger RNA molecule. We also present evidence that the fold of the E-domain of the 5S rRNA (nt 79-97) as a contiguous part of the 5S rRNA and as an isolated molecule is virtually the same.

N-terminal modification and amino-acid sequence of the ribosomal protein HmaS7 from Haloarcula marismortui and homology studies to other ribosomal proteins.

The ribosomal protein HmaS7 from the 30S subunit of the extreme halophilic archaeum Haloarcula marismortui was isolated by semi-preparative RP-HPLC. The complete amino-acid sequence of this protein was determined by automated microsequence analysis of appropriate peptide fragments from several proteinase digests. The entire protein consists of 205 amino acids with a corresponding molecular mass of 22580 Da. The modification at the amino-terminal amino acid was deblocked so that the N-terminal amino acids could be sequenced and the type of the modification was identified as an acetyl group by electrospray mass spectrometry of suitable peptides. Homology studies of HmaS7 showed similarities to ribosomal proteins derived from organisms of all three urkingdoms, such as to EcoS7, HmoS7, MvaS7, SacS7 and RatS7; due to the strong sequence homologies found within the archaebacterial ribosomal proteins we conclude that the protein sequence which was determined for S7 from Methanococcus vannielii by nucleotide sequencing of the gene should be about 20 or 30 amino acids longer than previously published (Lechner, K., Heller, G. & Böck, A. (1989) J. Mol. Evol. 29, 20-27).