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Nucleoside Kinase - IMPDH Coupled Assay
Contract Research Service for the Screening of Nucleoside Analogues as IMPDH inhibitors
This Contract Research Service is specially designed to screen the potential inhibitory effect of Nucleoside Analogues on IMPDH enzyme. These two-step assay consists in phosphorylating a nucleoside analogue with a recombinant nucleoside kinase and in evaluating IMPDH inhibition by the produced monophosphorylated form of NA.
Contract Research Service for the Screening of Nucleoside Analogues as IMPDH inhibitors
This Contract Research Service is specially designed to screen the potential inhibitory effect of Nucleoside Analogues on IMPDH enzyme. These two-step assay consists in phosphorylating a nucleoside analogue with a recombinant nucleoside kinase and in evaluating IMPDH inhibition by the produced monophosphorylated form of NA.
Aim: Rapid evaluation of monophosphate forms of nucleoside analogues as IMPDH inhibitors
IMP Dehydrogenase (IMPDH, E.C. 1.1.1.205) catalyzes the pivotal step in guanine nucleotide biosynthesis. By converting inosine monophosphate (IMP) to xanthosine monophosphate (XMP), IMPDH controls the guanine nucleotide pool. A number of nucleoside analogues (e.g. ribavirin, mizoribine) are known to inhibit IMPDH after being monophosphorylated. The therapeutic consequences of IMPDH inhibition vary from different analogues - mizoribine is an immunosuppressor and ribavirin is a broad spectrum antiviral. Even if direct relationship between ribavirin antiviral action and IMPDH inhibition by ribavirin monophosphate has not been demonstrated, the depletion of cellular GTP might result in an increased frequency of ribavirin triphosphate incorporation by viral polymerase due to a lower intracellular concentration of its natural competitor.
Enzymes: Nucleoside Kinases (AK or cN-II) and IMPDH Type II (eventually Bacterial (Staphylococcus aureus) IMPDH) used in the assays are recombinant, expressed in E. coli, and produced and purified by NOVOCIB (see Enzymes & Kits > Nucleoside Kinases and Enzymes & Kits > Purine Metabolism Enzymes). The purity of each enzyme is controlled by SDS-PAGE. Protein concentration is measured by Bradford method (Bio-Rad). Enzymatic activities are systematically controlled before performing any phosphorylation-inhibition coupled assay.
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IMP Dehydrogenase (IMPDH, E.C. 1.1.1.205) catalyzes the pivotal step in guanine nucleotide biosynthesis. By converting inosine monophosphate (IMP) to xanthosine monophosphate (XMP), IMPDH controls the guanine nucleotide pool. A number of nucleoside analogues (e.g. ribavirin, mizoribine) are known to inhibit IMPDH after being monophosphorylated. The therapeutic consequences of IMPDH inhibition vary from different analogues - mizoribine is an immunosuppressor and ribavirin is a broad spectrum antiviral. Even if direct relationship between ribavirin antiviral action and IMPDH inhibition by ribavirin monophosphate has not been demonstrated, the depletion of cellular GTP might result in an increased frequency of ribavirin triphosphate incorporation by viral polymerase due to a lower intracellular concentration of its natural competitor.
| Adenosine Kinase (AK) | cytosolic 5'-nucleotidase (cN-II) | |
| Natural substrates | Adenosine
Inosine |
Deoxyinosine
Inosine |
| Nucleoside analogues substrates | Ribavirine
Tubercidin Mizoribine |
Dideoxyinosine
Ribavirine Acyclovir |
Enzymes: Nucleoside Kinases (AK or cN-II) and IMPDH Type II (eventually Bacterial (Staphylococcus aureus) IMPDH) used in the assays are recombinant, expressed in E. coli, and produced and purified by NOVOCIB (see Enzymes & Kits > Nucleoside Kinases and Enzymes & Kits > Purine Metabolism Enzymes). The purity of each enzyme is controlled by SDS-PAGE. Protein concentration is measured by Bradford method (Bio-Rad). Enzymatic activities are systematically controlled before performing any phosphorylation-inhibition coupled assay.
 Phosphorylation of nucleoside analogue is provided by specific nucleoside kinase and confirmed by spectrophotometric quantification of formed ADP in LDH-PK coupled reaction. A confirmation by HPLC analysis of the formation of monophosphorylated forms is available upon request. |
 IMPDH inhibition: Effect of monophosphorylated nucleoside analogues on human recombinant IMPDH II. Enzymatic assays performed in duplicate are carried out at 37°C in 0.1M KH2PO4 buffer pH 8.0 in the presence of 2mMDTT, 200µM NAD, 200µM IMP and 0.2 µM IMPDH II and increasing concentration of monophosphorylated nucleoside. Reaction is followed in an iEMS Reader MF (Labsystems) microtiter plate reader at 340nm. |
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