Award
Dr Larissa Balakireva, CEO & Founder of NovoCIB, was awarded with the Trophy of
"Femmes en Or 2011, Femme de l'Innovation"
in September 2011
.

Nucleoside Kinase Enzymes

NOVOCIB produces human recombinant deoxycytidine kinase, UMP-CMP kinase (CMK), adenosine kinase and cN-II phosphotransferase. These enzymes are available in a lyophilized form.

ADK enzyme dCK enzyme CMK enzyme cN-II enzyme
Natural substrates Adenosine
Inosine
Deoxycytidine
Cytidine
Deoxyadenosine
Deoxyguanosine
dCMP
CMP
UMP
Deoxyinosine
Inosine
Nucleoside analogues substrates Ribavirin
Tubercidin
Mizoribin
Cladribine
Fludarabine
Gemcitabine (dFdC)
Lamivudine
Aracytidine (araC)
Fluorodeoxyuridine
dFdCMP
(Gemcitabine monophosphate)
3TCMP
araCMP
(Aracytidine monophosphate)
Adefovir (PMEA)
Dideoxyinosine
Ribavirin
Acyclovir
Download our brochure "NovoCIB's Nucleoside kinases" 


Nucleoside kinases: rate-limiting step of nucleoside analogues activation

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 the phosphorylation of cytidine analogues. These kinases provide the first two steps of the activation of highly effective anti-cancer and anti-viral drugs, such as 1-β-D-arabinofuranosylcytosine (araC, aracytidine), 2’,2’difluorodeoxycytidine (dFdC, gemcitabine), and 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, 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 which 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 (ADK). Recently it has been demonstrated that cytosolic 5’-nucleotidase II (cN-II) can also phosphorylate ribavirin. This could contribute to the development of ribavirin-induced haemolytic anemia in vivo(2). NOVOCIB has developed both human recombinant adenosine kinase (ADK) and cytosolic nucleotidase II (cN-II) nucleoside phosphorylation assays to evaluate the properties of new ribonucleoside analogues in comparison with those of ribavirin.

References
1. R. C. Willis et al. (1978): Adenosine Kinase Initiates the Major Route of Ribavirin Activation in a Cultured Human Cell Line Proc. Natl. Acad. Sci. USA 75(7), 3042-3044
2. J. Z. Wu et al. (2005): Phosphorylation of ribavirin and viramidine by adenosine kinase and cytosolic 5'-nucleotidase II: Implications for ribavirin metabolism in erythrocytes Antimicrob. Agents Chemother. 49(6), 2164-2171

Download this document  "NovoCIB's Nucleoside kinases" 


ADK - Adenosine Kinase (EC 2.7.1.20)

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Synonyms: ADK, Adenosine 5'-phosphotransferase

Adenosine kinase (ADK) is a ubiquitous enzyme that catalyzes the transfer of gamma-phosphate from ATP to 5’-hydroxyl of adenosine, generating AMP and ADP. Adenosine (AR) is an important modulator of central nervous system (CNS) functions with a half-life of seconds. Facilitated diffusion of adenosine across the cell membrane closely couples adenosine concentrations in the intracellular and extracellular compartments. Inhibition of adenosine kinase results in selective increase of local adenosine concentrations and reduced seizure susceptibility and nociception in vivo.
Adenosine kinase is an attractive and experimentally validated target for the development of new analgesic and anti-inflammatory agents. ADK has recently emerged as a novel target to predict and to prevent epileptogenesis.
The X-ray crystallographic structure of human ADK has been described and provides structural basis for rational design and optimisation of new ADK inhibitors.
In addition, this enzyme is responsible for the phosphorylation and consequent clinical activity of several therapeutically useful nucleosides analogues, including the antiviral drug ribavirin, immunosuppressive drug mizoribine, and anticancer C-nucleoside, tiazofurin.
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dCK - Deoxycytidine Kinase (EC 2.7.1.74)

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Human deoxycytidine kinase (dCK) plays a key role in the salvage pathway of deoxynucleotides synthesis, providing resting cells with deoxynucleotides for DNA repair and mitochondrial DNA synthesis. The enzyme has a broad substrate specificity and provides the phosphorylation of both purine and pyrimidine deoxynucleosides (e.g. deoxyadenosine (dA), deoxyguanosine (dG)), deoxycytidine (dC), and pyrimidine ribonucleoside, cytidine (C). The enzyme can utilize both ATP and UTP as phosphate donor with UTP being the preferred substrate.
Deoxycytidine kinase is responsible for the phosphorylation and activation of numerous nucleoside analogs used to treat cancer (e.g. cytarabine, gemcitabine, cladribine and fludarabine) including nucleoside analogs of non-physiological L-chirality (e.g. 3TC, lamivudine, anti-HIV, and anti-hepatitis B agent). Three-dimensional structures of dCK in complex with various pyrimidine and purine D- and L-nucleosides have been solved providing structural basis for activation of L- and D-nucleoside analogs.
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CMK - UMP-CMP Kinase (EC 2.7.4.14)

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Synonyms: cytidylate kinase, deoxycytidylate kinase, cytidine monophosphate kinase (CMK, CMPK), uridine monophosphate kinase (UMK, UMPK), uridine monophosphate / cytidine monophosphate kinase (UCK), UMP/CMP kinase (UMP/CMPK)

UMP-CMP kinase (CMK) plays a critical role in supplying cells with nucleotides by catalysing the phosphorylation of CMP, UMP and dCMP to their respective diphosphate form. CMK plays also an important role in the activation of cytidine analogues, aracytidine and gemcitabine, a mainstay of leukaemia and lymphoma therapy. CMK has a remarkable ability to phosphorylate L-nucleotides from their monophosphate to diphosphate forms as shown for beta-L-2’3’-dideoxy-3’thiacytidine (L-SSdC, 3-TC or lamividune), an anti-HIV and anti-hepatitis B drug.
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cN-II - cytosolic 5'-nucleotidase II (EC 3.1.3.5)

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Synonyms: cytosolic 5'-nucleotidase/phosphotransferase, High Km 5’-nucleotidase (hkm-NT), cytosolic purine 5’-nucleotidase (purine 5'-NT), IMP/GMP-specific 5’-nucleotidase (IMP/GMP-specific 5'-NT)

Cytosolic 5'-nucleotidase II (cN-II) is one of the seven known mammalian nucleotidases that specifically catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5’-monophosphates (IMP, dIMP, GMP, dGMP) and regulates cellular pool of IMP and GMP. The enzyme also acts as a phosphotransferase catalyzing the transfer of a phosphate from a nucleoside monophosphate to a nucleoside acceptor – preferentially inosine and deoxyinosine. Unlike the other 5’-nucleotidases, cN-II is allosterically regulated by adenine/guanine nucleotides and 2,3-biphosphoglycerate.
Moreover, cytosolic 5'-nucleotidase II phosphorylates anti-viral and anti-tumour nucleoside analogues such as 2’3’-dideoxyinosine, carbovir, acyclovir and ribavirin.
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Related Links
Nucleoside Kinase Assay Kits
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