Cell-based IMPDH Inhibition AssayThis assay has been specially tailored to validate IMPDH inhibitor in cultured cells. After incubation of cultured cells with the inhibitor, this high performance liquid chromatography analysis of nucleotides consists in:
Cell-based IMPDH inhibition assay was validated with mycophenolic acid (MPA), ribavirin and mizoribin, recognized inhibitors of IMPDH. When applied for the study of nucleoside analogues, this assay can also reveal the formation of their mono-, di-, and triphosphate forms, indicating that nucleoside analogues enter the cells and are readily phosphorylated by cellular kinases. |
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Example: Ribavirine (Rbv)
Numerous nucleoside analogues (NA) are currently used to treat viral infections. They are usually designed to inhibit one viral target. This remains in contrast with the observation that ribavirin, a purine nucleoside analogue currently used as a part of bi-therapy against hepatitis C infection, has multiple modes of action:
- depletion of intracellular GTP pools by inhibition of the cellular IMPDH,
- inhibition of viral polymerase activity,
- induction of error catastrophe as a result of accumulation of mutations in the viral genome.
Even if direct relationship between ribavirin antiviral action and IMPDH inhibition has not been demonstrated, the depletion of cellular GTP should result in an increased frequency of ribavirin triphosphate incorporation by viral polymerase due to a lower intracellular concentration of its natural competitor.
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| Figure 1. Nucleotide profiles of methotrexate-treated HeLa cells (10µM, 20h) Ratio between nucleotide content in drug-treated and untreated cells are shown |
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Concentration of nucleotides mono- and tri-phosphate in Rbv- and DMSO-treated cells (measures as peak area, AU)
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| Figure 2. Effects of 10µM Rbv on cellular pool of nucleotide mono- and tri-phosphates |
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