Methods for L-ribooligonucleotide sequence determination using LCMS

The ability to verify the sequence of a nucleic acid-based therapeutic is an essential step in the drug development process. The challenge associated with sequence identification increases with the length and nuclease resistance of the nucleic acid molecule, the latter being an important attribute of therapeutic oligonucleotides. We describe methods for the sequence determination of Spiegelmers, which are enantiomers of naturally occurring RNA with high resistance to enzymatic degradation. Spiegelmer sequencing is effected by affixing a label or hapten to the 5′-end of the oligonucleotide and chemically degrading the molecule in a controlled fashion to generate fragments that are then resolved and identified using liquid chromatography-mass spectrometry. The Spiegelmer sequence is then derived from these fragments. Examples are shown for two different Spiegelmers (NOX-E36 and NOX-A12), and the specificity of the method is shown using a NOX-E36 mismatch control

Synthesis of conformationally restricted melatonin analogues

The pineal hormone melatonin plays a major role in the regulation of seasonal cycles and the control of circadian rhythms in mammals, reptiles and birds. The emerging potential of melatonin in the therapy of human rhythm disorders (i.e. SAD and shift-work syndrome) and its application in agriculture (control of reproductive cycles in farm-animals such as sheep and horses) has caused a dramatic upsurge of interest in synthesising melatonin agonists and antagonists. As part of a programme to examine the three-dimensional structure of the melatonin receptor the synthesis and biological activity of several structural melatonin analogues with conformationally restricted N-acyl-3-ethanamine side-chain was investigated. The tricyclic analogue N-butanoyl-4-aminomethyl-6-methoxy-9-methyl-1,2,3,4-tetra- hydrocarbazole showed the highest affinity in the 2-[125I]-iodomelatonin radioligand binding assay in chick brain membranes (Ki=378 pM, melatonin Ki=580 pM). Displacement of the 6-methoxy moiety effected a change from agonistic to antagonistic properties in the pigment aggregation response test involving melanophores obtained from the neural crest of Xenopus laevis embryos, [beta]-Alkylated melatonin analogues were prepared by Bischler methodology and showed, as well as 4-bromo-melatonin, a retention in binding potency. Several synthetic routes towards N-acyl-nortryptamines, 2-substituted cyclopent[b]- and 9-substituted cyclohept[b]-indoles were investigated. Biological results were evaluated by correlating the binding affinity of the melatonin analogues with the conformation of the side chain, the nature of the N-acylating group, and the spatial distance between the methoxy and amide pharmacophores

Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers are l-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared to d-oligonucleotides. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC(50) values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPγS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes from Xenopus laevis, NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K(+) channels

SDF-1 Inhibition Targets the Bone Marrow Niche for Cancer Therapy

Summary: Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l-oligonucleotide (olaptesed-pegol). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells. : Roccaro et al. show that stromal-cell-derived factor-1 (SDF-1) is highly expressed in active multiple myeloma (MM), as well as in bone marrow (BM) sites of tumor metastasis, and report on a high-affinity PEGylated mirror-image l-oligonucleotide (olaptesed pegol) that specifically binds and neutralizes SDF-1 in vitro and in vivo. Using in vivo murine and xenograft mouse models, the authors document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces clonal plasma cell homing and growth, thereby inhibiting MM disease progression