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Resistance of Nucleoside Analogues to PNP-catalyzed phosphorolysis
Because of its cleaving activity, PNP can represent a threat for therapeutic efficacy of Nucleoside Analogues. In vivo, it can be responsible for the cleavage and the subsequent deactivation of Nucleoside Analogues, then unable to be phosphorylated by nucleoside kinases. The resistance to PNP-catalyzed cleavage may be worth being investigated to increase the therapeutic efficacy of Nucleoside Analogues.
Because of its cleaving activity, PNP can represent a threat for therapeutic efficacy of Nucleoside Analogues. In vivo, it can be responsible for the cleavage and the subsequent deactivation of Nucleoside Analogues, then unable to be phosphorylated by nucleoside kinases. The resistance to PNP-catalyzed cleavage may be worth being investigated to increase the therapeutic efficacy of Nucleoside Analogues.
Purine Nucleoside Phosphorylase Cleaving Activity (in vitro)
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Aim: To evaluate the resistance of Nucleoside Analogues to phosphorolysis catalyzed by human Purine Nucleoside Phosphorylase (PNP).
Nucleoside Analogues deactivation by PNP: In vivo, phosphorolysis is highly favoured over purine nucleoside synthesis and is coupled with two additional enzymatic reactions: oxidation of the liberated purine base by xanthine oxidase (XO) and its phosphoribosylation by hypoxanthine-guanine phosphoribosyltransferase (HGPRT)(1). Thus, PNP plays a key role in the salvage pathway of the purine metabolism, enabling the cell to utilize purine bases recovered from metabolized purine ribo- and deoxyribonucleosides to synthesize purine nucleotides.
This phosphorolysis reaction of purine nucleosides catalyzed by PNP has a direct impact on the therapeutic efficacy of Nucleoside Analogues. Antitumour or antiviral nucleoside analogues are likely to be cleaved by PNP before being phosphorylated by the cell nucleoside kinases and converted to the active nucleotide form. For instance, 2',3'-dideoxyguanosine (ddG)(2), 9-β-D-arabinofuranosyl guanine (AraG)(3)3 as well as one of its produg, Nelarabine (Arranon®, GSK)(4), which is intracellularly converted to AraG by Adenosine deaminase (ADA), are PNP resistant nucleoside analogues, whereas 2',3'-dideoxyinosine (ddI)(5) is easily cleaved in vivo by PNP.
Since acyclonucleoside analogues are particularly resistant to cleavage by PNP though phosphorylated by viral thymidine kinases (TK), they are generally considered as excellent candidates as antiviral agents (e.g. aciclovir, ganciclovir)(6).
Note that Ganciclovir is not only PNP resistant, but is also a PNP inhibitor.
PNP enzyme: The enzyme used in each of this assay is a human recombinant PNP, cloned by NOVOCIB from human cells, expressed in E. coli, and produced and purified by NOVOCIB (click here for details). PNP purity and activity are controlled before running every assay.
Description of the Nucleoside Resistance assay: The assays are performed at 25°C or 37°C in 200µl of reaction buffer (lower volumes are available if compound saving is a constraint) on 96-well microplate. Pipetting is done by a Multiprobe® II Robotic Liquid Handling System (Packard BioScience). Inosine is used as a positive control and adenosine as a negative control.
Note that if inosine is a natural substrate of PNP and is consequently actively cleaved by a wide range of PNPs, adenosine resists to Human PNP phosphorolytic activity but is easily cleaved by other PNPs, such as E.coli PNP for instance. This is why evaluating the cleavage resistance of Nucleoside Analogue using Human PNP may represent a decisive advantage.
HPLC analysis: The products derived from the phosphorolysis of a nucleoside analogue by Human PNP are identified and quantified by HPLC, in comparison with positive (using inosine) and negative (using adenosine) controls.
Nucleoside Analogues deactivation by PNP: In vivo, phosphorolysis is highly favoured over purine nucleoside synthesis and is coupled with two additional enzymatic reactions: oxidation of the liberated purine base by xanthine oxidase (XO) and its phosphoribosylation by hypoxanthine-guanine phosphoribosyltransferase (HGPRT)(1). Thus, PNP plays a key role in the salvage pathway of the purine metabolism, enabling the cell to utilize purine bases recovered from metabolized purine ribo- and deoxyribonucleosides to synthesize purine nucleotides.
This phosphorolysis reaction of purine nucleosides catalyzed by PNP has a direct impact on the therapeutic efficacy of Nucleoside Analogues. Antitumour or antiviral nucleoside analogues are likely to be cleaved by PNP before being phosphorylated by the cell nucleoside kinases and converted to the active nucleotide form. For instance, 2',3'-dideoxyguanosine (ddG)(2), 9-β-D-arabinofuranosyl guanine (AraG)(3)3 as well as one of its produg, Nelarabine (Arranon®, GSK)(4), which is intracellularly converted to AraG by Adenosine deaminase (ADA), are PNP resistant nucleoside analogues, whereas 2',3'-dideoxyinosine (ddI)(5) is easily cleaved in vivo by PNP.
Since acyclonucleoside analogues are particularly resistant to cleavage by PNP though phosphorylated by viral thymidine kinases (TK), they are generally considered as excellent candidates as antiviral agents (e.g. aciclovir, ganciclovir)(6).
Note that Ganciclovir is not only PNP resistant, but is also a PNP inhibitor.
PNP enzyme: The enzyme used in each of this assay is a human recombinant PNP, cloned by NOVOCIB from human cells, expressed in E. coli, and produced and purified by NOVOCIB (click here for details). PNP purity and activity are controlled before running every assay.
Description of the Nucleoside Resistance assay: The assays are performed at 25°C or 37°C in 200µl of reaction buffer (lower volumes are available if compound saving is a constraint) on 96-well microplate. Pipetting is done by a Multiprobe® II Robotic Liquid Handling System (Packard BioScience). Inosine is used as a positive control and adenosine as a negative control.
Note that if inosine is a natural substrate of PNP and is consequently actively cleaved by a wide range of PNPs, adenosine resists to Human PNP phosphorolytic activity but is easily cleaved by other PNPs, such as E.coli PNP for instance. This is why evaluating the cleavage resistance of Nucleoside Analogue using Human PNP may represent a decisive advantage.
HPLC analysis: The products derived from the phosphorolysis of a nucleoside analogue by Human PNP are identified and quantified by HPLC, in comparison with positive (using inosine) and negative (using adenosine) controls.
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 Cladribine |
| Superposed HPLC spectra of two Nucleoside Resistance assays:30 mn incubation at 25°C
Left: Inosine cleavage by Human PNP Right: Cladribine (deoxyadenosine analogue) resistance to cleavage by Human PNP. Blue: without Human PNP Red: with Human PNP. |
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