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

被引:31
作者
Faulhammer, D
Eschgfäller, B
Stark, S
Burgstaller, P
Englberger, W
Erfurth, J
Kleinjung, F
Rupp, J
Vulcu, SD
Schröder, W
Vonhoff, S
Nawrath, H
Gillen, C
Klussmann, S
机构
[1] NOXXON Pharma AG, D-10589 Berlin, Germany
[2] Johannes Gutenberg Univ Mainz, Dept Pharmacol, D-55101 Mainz, Germany
[3] Grunenthal GmbH, Mol Pharmacol, D-52078 Aachen, Germany
关键词
Spiegelmer; nociceptin; neuropeptide; biostable aptamer; RNA; pain;
D O I
10.1261/rna.5186504
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 IC50 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 GTPgammaS 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.
引用
收藏
页码:516 / 527
页数:12
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