•
Phosphorylation of Nucleoside Analogues (In vitro)
NOVOCIB offers a range of Contract Research Services for in vitro phosphorylation of nucleoside analogues with recombinant nucleoside kinases.
NOVOCIB offers a range of Contract Research Services for in vitro phosphorylation of nucleoside analogues with recombinant nucleoside kinases.
| Adenosine Kinase (AK) | Deoxycytidine Kinase (dCK) | UMP-CMP Kinase (CMK) |
dCK-CMK (Coupled Assay) | 5'-nucleotidase (cN-II) | |
| Natural substrates | Adenosine
Inosine |
Deoxycytidine
Cytidine Deoxyadenosine Deoxyguanosine |
dCMP
CMP UMP |
Deoxycytidine
Cytidine |
Deoxyinosine
Inosine |
| Nucleoside analogues substrates | Ribavirine
Tubercidin Mizoribine |
Cladribine
Fludarabine Gemcitabine (dFdC) Lamivudine Aracytidine (araC) Fluorodeoxyuridine |
dFdCMP
(Gemcitabine monophosphate) 3TCMP araCMP (Aracytidine monophosphate) Adefovir (PMEA) |
Gemcitabine (dFdC)
Lamivudine Aracytidine (araC) Fluorodeoxyuridine |
Dideoxyinosine
Ribavirine Acyclovir |
See also our Nucleoside Kinase - IMPDH coupled assay
Nucleoside kinases: rate-limiting step of nucleoside analogues activation
(^Top)
Nucleoside analogues have proven to be a highly successful class of anti-cancer and anti-viral drugs. The therapeutic efficacy of nucleoside analogues is dependent of their intracellular phosphorylation. Two cellular nucleoside kinases, deoxycytidine kinase (dCK) and UMP-CMP kinase (CMK) are critical for phosphorylation of cytidine analogues. These kinases provide the first two steps of activation of highly effective anti-cancer and anti-viral drugs, such as 1-β-D-arabinofuranosylcytosine (araC, aracytidine), 2’,2’difluorodeoxycytidine (dFdC, gemcitabine), β-D-2’3’-dideoxycytidine (ddC). Both kinases phosphorylate unnatural L-nucleosides (e.g., β-L-2’3’-dideoxy-3’thiacytidine, L-SSdC, 3-TC or lamividune). Kinetic constants of araC, dFdC and 3TC phosphorylation by recombinant dCK and UMP-CMPK have been published. The comparison of phosphorylation properties of new nucleoside analogues with those of known drugs provides the rational basis for selection of analogues of better therapeutic potential.
To characterize the phosphorylation properties of new nucleoside analogues, NOVOCIB has developed human recombinant dCK and human recombinant CMK nucleoside phosphorylation assays. As shown in Table 1 (here below), CMK assay must be performed with monophosphate forms of nucleoside analogues and requires preliminary phosphorylation of nucleoside analogues and their purification. To circumvent this time-consuming step, NOVOCIB has developed a coupled dCK-CMK nucleoside phosphorylation assay that delivers in one step the critical information on both dCK and CMK substrate properties of nucleoside analogue.
Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a purine nucleoside analogue with a broad-spectrum antiviral activity. Since the 1970's(1), it is known that the initial step of ribavirin (as well as viramidine) phosphorylation is provided by adenosine kinase (AK). Recently it has been demonstrated that cytosolic 5’-nucleotidase II (cN-II) can also phosphorylate ribavirin, that could contribute to the development of ribavirin-induced haemolytic anemia in vivo(2). NOVOCIB has developed both human recombinant adenosine kinase (AK) and cytosolic nucleotidase II (cN-II) nucleoside phosphorylation assays to evaluate the properties of new ribonucleoside analogues in comparison with those of ribavirin.
To characterize the phosphorylation properties of new nucleoside analogues, NOVOCIB has developed human recombinant dCK and human recombinant CMK nucleoside phosphorylation assays. As shown in Table 1 (here below), CMK assay must be performed with monophosphate forms of nucleoside analogues and requires preliminary phosphorylation of nucleoside analogues and their purification. To circumvent this time-consuming step, NOVOCIB has developed a coupled dCK-CMK nucleoside phosphorylation assay that delivers in one step the critical information on both dCK and CMK substrate properties of nucleoside analogue.
Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a purine nucleoside analogue with a broad-spectrum antiviral activity. Since the 1970's(1), it is known that the initial step of ribavirin (as well as viramidine) phosphorylation is provided by adenosine kinase (AK). Recently it has been demonstrated that cytosolic 5’-nucleotidase II (cN-II) can also phosphorylate ribavirin, that could contribute to the development of ribavirin-induced haemolytic anemia in vivo(2). NOVOCIB has developed both human recombinant adenosine kinase (AK) and cytosolic nucleotidase II (cN-II) nucleoside phosphorylation assays to evaluate the properties of new ribonucleoside analogues in comparison with those of ribavirin.
Download this document "NovoCIB's Nucleoside kinases" 
Phosphorylation by Adenosine Kinase (in vitro) (^Top)
Aim: Characterization of substrate properties (Km and Vmax) of new nucleoside analogues for human adenosine kinase in comparison with properties of known nucleoside analogues (e.g., ribavirine, tubercidine or mizoribine).
Phosphorylation by Deoxycytidine Kinase Assay (in vitro)
(^Top)
Aim: Characterization of substrate properties (Km and Vmax) of new nucleoside analogues for human deoxycytidine kinase in comparison with the properties of known nucleoside analogues (e.g., aracytidine, gemcitabine, cladribine and lamivudine).
Phosphorylation by UMP-CMP Kinase (CMK, in vitro)
(^Top)
Aim: Characterization of substrate properties (Km and Vmax) of monophosphate forms of new nucleoside analogues for human CMK in comparison with monophosphate forms of natural nucleosides or of reference nucleoside analogues (AraC-MP, dFdC-MP).
Phosphorylation by Deoxycytidine Kinase and UMP-CMP Kinase
(Coupled Assay, in vitro)
(^Top)
Aim: Coupled dCK-CMK nucleoside phosphorylation assay is a cost-effective rapid assay that delivers in one step the critical information on both dCK and CMK substrate properties of a nucleoside analogue.
Transphosphorylation by 5'-nucleotidase (cN-II, in vitro)
(^Top)
Aim: Characterization of substrate properties of new nucleoside analogues for human cN-II phosphotransferase activity in comparison with ribavirine as reference nucleoside analogue.
Please, contact us for further details.