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Investigating discovery strategies and pharmacological properties of stereodefined phosphorodithioate LNA gapmers.

ABSTRACT: The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS₂) introduces second sulfur into the phosphorothioate linkage to remove the chirality of phosphorus atom. Here, we describe the application of symmetrical non-bridging PS₂ linkages in the context of stereodefined locked nucleic acids (LNAs) antisense oligonucleotides with the goal of reducing chiral complexity and, ultimately, resulting in single molecules. In addition, we propose a rather simple strategy to rapidly identify stereodefined gapmers, combining PS₂ and a preferred stereochemistry motif (RSSR), which supports RNase-H-mediated target knockdown. Pharmacological efficacy and metabolic stability are investigated systematically using ApoB as a target sequence, where in vivo data correlate well to what is observed in vitro.

SUBMITTER: Duschmale J

PROVIDER: S-EPMC9271985 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Investigating discovery strategies and pharmacological properties of stereodefined phosphorodithioate LNA gapmers.

Duschmalé Jörg J Schäublin Adrian A Funder Erik E Schmidt Steffen S Kiełpiński Łukasz J ŁJ Nymark Helle H Jensen Klaus K Koch Troels T Duschmalé Martina M Koller Erich E Møller Marianne Ravn MR Schadt Simone S Husser Christophe C Brink Andreas A Sewing Sabine S Minz Tanja T Wengel Jesper J Bleicher Konrad K Li Meiling M

Molecular therapy. Nucleic acids 20220622

The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS<sub>2</sub>) introduce ...[more]

PMID: 35860384

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Project description:Human stem cell-derived neurons are increasingly considered powerful models in drug discovery and disease modeling, despite limited characterization of their molecular properties. Here, we have conducted a detailed study of the properties of a commercial human induced Pluripotent Stem Cell (iPSC)-derived neuron line, iCell [GABA] neurons, maintained for up to 3 months in vitro. We confirmed that iCell neurons display neurite outgrowth within 24 h of plating and label for the pan-neuronal marker, βIII tubulin within the first week. Our multi-electrode array (MEA) recordings clearly showed neurons generated spontaneous, spike-like activity within 2 days of plating, which peaked at one week, and rapidly decreased over the second week to remain at low levels up to one month. Extracellularly recorded spikes were reversibly inhibited by tetrodotoxin. Patch-clamp experiments showed that iCell neurons generated spontaneous action potentials and expressed voltage-gated Na and K channels with membrane capacitances, resistances and membrane potentials that are consistent with native neurons. Our single neuron recordings revealed that reduced spiking observed in the MEA after the first week results from development of a dominant inhibitory tone from GABAergic neuron circuit maturation. GABA evoked concentration-dependent currents that were inhibited by the convulsants, bicuculline and picrotoxin, and potentiated by the positive allosteric modulators, diazepam, chlordiazepoxide, phenobarbital, allopregnanolone and mefenamic acid, consistent with native neuronal GABAA receptors. We also show that glycine evoked robust concentration-dependent currents that were inhibited by the neurotoxin, strychnine. Glutamate, AMPA, Kainate and NMDA each evoked concentration-dependent currents in iCell neurons that were blocked by their selective antagonists, consistent with the expression of ionotropic glutamate receptors. The NMDA currents required the presence of the co-agonist glycine and were blocked in a highly voltage-dependent manner by Mg2+ consistent with the properties of native neuronal NMDA receptors. Together, our data suggest that such human iPSC-derived neurons may have significant value in drug discovery and development and may eventually largely replace the need for animal tissues in human biomedical research.

Dataset Information

Investigating discovery strategies and pharmacological properties of stereodefined phosphorodithioate LNA gapmers.

Publications

Investigating discovery strategies and pharmacological properties of stereodefined phosphorodithioate LNA gapmers.

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