Kinetics of phosphorothioate oligonucleotide metabolism in biological fluids

Gilár, Martin; Belenky, Alexei; Smisek, David L.; Bourque, André J.; Cohen, Aharon S.

doi:10.1093/nar/25.18.3615

Cited by 55 publications

(36 citation statements)

References 8 publications

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“…Capillary gel electrophoresis (CGE) [7], strong anionexchange (SAX) chromatography [8], 31 P NMR spectroscopy [8,9], matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) [10,11], liquid chromatography coupled to electrospray ionization (ESI) mass spectrometry [12,13], and tandem mass spectrometry (MS/MS) [13][14][15] are among the analytical methods that have been used to determine the quality of oligonucleotides and characterize their impurities. Ion pair-reversed phase (IP-RP) HPLC coupled with a UV detector and electrospray ionization has been identified as one of the most suitable methods because of its ability to provide increased chromatographic resolution and reliable qualitative and quantitative information [6].…”

Section: Introductionmentioning

confidence: 99%

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

Roussis

2015

J. Am. Soc. Mass Spectrom.

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Abstract.A new method is presented for determining relationships between components in complex analytical systems. The method uses the mass differences between peaks in high resolution electrospray ionization (ESI) mass spectra. It relates peaks that share common mass differences. The method is based on the fundamental assumption that peaks in the spectra having the same exact mass difference are related by the same chemical moiety/substructure. Moreover, the presence (or absence/loss) of the same chemical moiety from a series of molecules may reflect similarities in the mechanisms of formation of each molecule. The determined mass differences in the spectra are used to automatically differentiate the types of components in the samples. Contour plots and summary plots of the summed total ion signal as a function of the mass difference are generated, which form powerful tools for the rapid and automated determination of the components in the samples and for comparisons with other samples. For the first time, in this work a unique profile contour plot has been developed that permits the interactive interrogation of the mass range by mass difference data matrix to obtain valuable information about components that share a common mechanism of formation, and all possible mechanisms of formation linked to a selected precursor molecule. The method can be used as an additional and complementary method to the existing analytical methods to determine relationships between components in complex chemical systems.

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Section: Introductionmentioning

confidence: 99%

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

Roussis

2015

J. Am. Soc. Mass Spectrom.

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“…Synthetic PS-oligos have been developed as antisense probes for genomic research and medicinal applications (1,2). These oligonucleotides are promising therapeutic molecules because they are much more stable against nucleolytic degradation in blood and various cellular systems than their natural, unmodified counterparts (3)(4)(5). Their hydrolysis in plasma, kidney, and liver proceeds mainly from the 3Ј end, resulting in the appearance of the mononucleoside 5Ј-phosphorothioates identified in urine from PS-oligo-injected animals (6,7).…”

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confidence: 99%

Histidine Triad Nucleotide-binding Protein 1 (HINT-1) Phosphoramidase Transforms Nucleoside 5′-O-Phosphorothioates to Nucleoside 5′-O-Phosphates

Ozga

Dolot

Janicka

et al. 2010

Journal of Biological Chemistry

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Nucleoside 5-O-phosphorothioates are formed in vivo as primary products of hydrolysis of oligo(nucleoside phosphorothioate)s (PS-oligos) that are applied as antisense therapeutic molecules. The biodistribution of PS-oligos and their pharmacokinetics have been widely reported, but little is known about their subsequent decay inside the organism. We suggest that the enzyme responsible for nucleoside 5-O-monophosphorothioate ((d)NMPS) metabolism could be histidine triad nucleotide-binding protein 1 (Hint-1), a phosphoramidase belonging to the histidine triad (HIT) superfamily that is present in all forms of life. An additional, but usually ignored, activity of Hint-1 is its ability to catalyze the conversion of adenosine 5-O-monophosphorothioate (AMPS) to 5-O-monophosphate (AMP). By mutagenetic and biochemical studies, we defined the active site of Hint-1 and the kinetic parameters of the desulfuration reaction (P-S bond cleavage). Additionally, crystallographic analysis (resolution from 1.08 to 1.37 Å ) of three engineered cysteine mutants showed the high similarity of their structures, which were not very different from the structure of WT Hint-1. Moreover, we found that not only AMPS but also other ribonucleoside and 2-deoxyribonucleoside phosphorothioates are desulfurated by Hint-1 at the following relative rates: GMPS > AMPS > dGMPS > CMPS > UMPS > dAMPS Ͼ Ͼ dCMPS > TMPS, and during the reaction, hydrogen sulfide, which is thought to be the third gaseous mediator, was released. (Fig. 1). 5Ј-O-Phosphorothioates of ribonucleosides (NMPSNMPS and dNMPS (together denoted (d)NMPS)) are formed during the enzymatic hydrolysis of oligo(nucleoside phosphorothioate) (PS-oligos) that contain a sulfur atom attached in non-bridging positions to the phosphorus atom at each or selected internucleotide bond(s). Synthetic PS-oligos have been developed as antisense probes for genomic research and medicinal applications (1, 2). These oligonucleotides are promising therapeutic molecules because they are much more stable against nucleolytic degradation in blood and various cellular systems than their natural, unmodified counterparts (3-5). Their hydrolysis in plasma, kidney, and liver proceeds mainly from the 3Ј end, resulting in the appearance of the mononucleoside 5Ј-phosphorothioates identified in urine from PS-oligo-injected animals (6, 7). (d)NMPS may exert cytotoxic effects affecting cell proliferation, DNA or RNA synthesis, and other unknown processes (8, 9). Recently, the phosphorothioate DNA segments have been identified in bacterial DNA (10), which makes investigations into PS-oligo metabolism even more important.Although several reports have been published on the biodistribution of PS-oligos, little is known about the metabolism of the products of their degradation in vivo. It has been suggested that extracellular dNMPS and dNMP can be converted to the corresponding nucleoside by 5Ј-nucleotidase (ecto-5Ј-NT) (9). This membrane-bound enzyme preferably releases adenosine from extracellular AMP, but other purine and pyrimid...

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“…It is interesting to note that unlike the in vivo inhibitory effect, the effect of ON in vitro did not require the liposomal delivery agent. However, such an effect required a prolonged incubation of the cells with ON, i.e., 12 h before LPS stimulation, in serum-free medium to minimize degradation (15). A short-term incubation with ON, i.e., Ͻ2 h, did not result in any significant reduction in the cellular TNF-␣ response (results not shown).…”

Section: Discussionmentioning

confidence: 95%

Inhibition of TNF-α gene expression and bioactivity by site-specific transcription factor-binding oligonucleotides

Wang

Zhang

et al. 2003

American Journal of Physiology-Lung Cellular and Molecular Phys

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The present study investigated transcriptional inactivation of TNF-α gene by nuclear factor-binding oligonucleotides (ON) and their effects on pulmonary inflammatory responses in mice. PCR-based gene mutation and gel shift assays were used to identify specific cis-acting elements necessary for nuclear factor binding and transactivation of TNF-α gene by lipopolysaccharide (LPS). LPS inducibility of TNF-α was shown to require transcriptional activation by NF-κB at multiple binding sites, including the −850 (κ1), −655 (κ2), and −510 (κ3) sites, whereas the −210 (κ4) site had no effect. Maximum inducibility was associated with the activation of κ3 site. The sequence-specific, double-stranded ON targeting this site was most effective in inhibiting TNF-α activity induced by LPS. The inhibitory effect of ON on TNF-α bioactivity was also investigated using a murine lung inflammation model. Pretreatment of mice with ON, but not its mutated sequence, inhibited LPS-induced inflammatory neutrophil influx and TNF-α production by lung cells. Effective inhibition by ON in this model was shown to require a liposomal agent for efficient cellular delivery of the ON. Together, our results indicate that transcriptional inactivation of TNF-α gene can be achieved by using ON that compete for nuclear factor binding to TNF-α gene promoter. This gene inhibition approach may be used as a research tool or as potential therapeutic modality for diseases with etiology dependent on aberrant gene expression.

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Kinetics of phosphorothioate oligonucleotide metabolism in biological fluids

Cited by 55 publications

References 8 publications

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

Histidine Triad Nucleotide-binding Protein 1 (HINT-1) Phosphoramidase Transforms Nucleoside 5′-O-Phosphorothioates to Nucleoside 5′-O-Phosphates

Inhibition of TNF-α gene expression and bioactivity by site-specific transcription factor-binding oligonucleotides

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