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

NMP, NDP, NTP Quantification by HPLC

Ref. # WCS-Nov 1
Quantity Price*
1 sample
€ 620.00

* Pricing updated May 29th, 2015

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Download "HPLC Analysis IMPDH Inhibitors" 

Cell-based IMPDH Inhibition Assay

This 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:

  • Extraction of NMP, NDP, and NTP by SPE (Solid Phase Extraction) procedure
  • Separation by ion-paired HPLC of 13 nucleotides (Agilent 1100)
  • Quantification by ion-paired UV-HPLC (peak area at 254nm)

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.

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.

Figure 1. Nucleotide profiles of methotrexate-treated HeLa cells (10µM, 20h)
Ratio between nucleotide content in drug-treated and untreated cells are shown

Concentration of nucleotides mono- and tri-phosphate in Rbv- and DMSO-treated cells (measures as peak area, AU)
CMP 281.0 242.8
UMP 81.8 73.2
GMP 35.1 12.5
IMP 11.6 53.4
AMP 335.0 341.6
CTP 392.3 488.0
GTP 1,238.0 519.4
UTP 1,708.0 1,561.0
ATP 7,658.0 7,766.0
Figure 2. Effects of 10µM Rbv on cellular pool of nucleotide mono- and tri-phosphates

Download this document "Cell-based IMPDH inhibiton assay" 

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