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S. aureus vs. Human IMPDH As bacterial resistance to antibiotics has become a serious worldwide health problem, there is a continuing need to develop newer and more potent antibiotics that represent a new class of compounds not previously used to treat bacterial infection. One attractive strategy for developing new antibiotics is to inhibit bacterial IMPDH, a bacterial enzyme necessary for the de novo synthesis of purine nucleotides, and therefore, necessary for bacterial cell growth and division. Mammalian and bacterial IMPDHs are known to have significantly different kinetic properties and inhibitor sensitivities. NOVOCIB's PRECICE® IMPDH Screening Assay Kit allows the screening of specific bacterial IMPDH inhibitors with no inhibitory effect on Human IMPDH activity, needed for lymphocyte proliferation. |
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PRECICE® IMPDH Screening Assay Kit
PRECICE® IMPDH Screening Assay Kit uses two IMPDH enzymes in parallel for the screening of specific inhibitors. Bacterial recombinant IMPDH from Staphylococcus aureus and Human recombinant IMPDH, type II, are used to screen IMPDH inhibitors and assesss inhibitor's specificity.
Validation
Mammalian and bacterial IMPDHs are known to have significantly different kinetic properties and inhibitor sensitivities(1,2). The experiments done with previously cloned human IMPDH2 and bacterial IMPDH of Staphylococcus aureus, are illustrated below. In agreement with published data, mycophenolic acid (MPA) inhibits human IMPDH type II >20-times more efficiently than bacterial IMPDH with IC50 values of 100nM and 2.6µM, respectively (A). In contrast, mizoribine monophosphate displays the opposite selectivity (B). It is a more potent inhibitor of bacterial IMPDH with respective IC50 values of 12nM and 185nM for bacterial and human enzymes.
PRECICE® IMPDH Screening Assay Kit is a useful tool for the selection of species-specific IMPDH inhibitors.
Validation
Mammalian and bacterial IMPDHs are known to have significantly different kinetic properties and inhibitor sensitivities(1,2). The experiments done with previously cloned human IMPDH2 and bacterial IMPDH of Staphylococcus aureus, are illustrated below. In agreement with published data, mycophenolic acid (MPA) inhibits human IMPDH type II >20-times more efficiently than bacterial IMPDH with IC50 values of 100nM and 2.6µM, respectively (A). In contrast, mizoribine monophosphate displays the opposite selectivity (B). It is a more potent inhibitor of bacterial IMPDH with respective IC50 values of 12nM and 185nM for bacterial and human enzymes.
PRECICE® IMPDH Screening Assay Kit is a useful tool for the selection of species-specific IMPDH inhibitors.
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IMPDH inhibition: Effect of mizoribine monophosphate (A) and MPA (B) on human recombinant IMPDH II (red curve) and bacterial recombinant IMPDH of Staphylococcus aureus (blue curve). Enzymatic assays are performed in duplicate at 37°C in 0.1M KH2PO4 buffer pH 8.0 in the presence of 1mM DTT, 200µM NAD, 200µM IMP, 60nM Human IMPDH, Type 2 or 95nM IMPDH S.aureus. Reaction is followed in an iEMS Reader MF (Labsystems) microtiter plate reader at 340nm.
Monophosphorylated mizoribine is produced enzymatically by phosphorylation of mizoribine (MP Biochemicals) using NOVOCIB's adenosine kinase (AK). |
| Kit Content: | • Bacterial recombinant IMPDH (S. aureus)
• Human recombinant IMPDH, type II |
• NAD
• IMP |
• Reaction Buffer (10x) |