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The effects of maribavir on the autophosphorylation of ganciclovir resistant mutants of the cytomegalovirus UL97 protein.

Shannon-Lowe CD, Emery VC - Herpesviridae (2010)

Bottom Line: Wild type and site directed mutant forms of the human cytomegalovirus UL97 gene product were expressed using recombinant baculoviruses and the purified products used to assess the effects of maribavir on the ganciclovir (GCV) kinase and protein kinase (PK) activities.Enzyme kinetic experiments demonstrated that maribavir was a competitive inhibitor of ATP with a Ki of 10 nM.Maribavir is a potent competitive inhibitor of the UL97 protein kinase function and shows increased activity against the M460I GCV-resistant mutant which may impact on the management of GCV drug resistance in patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infection, Centre for Virology, UCL (Royal Free Campus Campus), Rowland Hill Street, Hampstead, London NW3 2QG, UK. v.emery@medsch.ucl.ac.uk.

ABSTRACT

Background: The UL97 protein kinase of human cytomegalovirus phosphorylates the antiviral drug ganciclovir and is the target of maribavir action. A detailed enzyme kinetic analysis of maribavir on the various enzymatic functions of wild type and ganciclovir resistant forms of UL97 is required.

Methods: Wild type and site directed mutant forms of the human cytomegalovirus UL97 gene product were expressed using recombinant baculoviruses and the purified products used to assess the effects of maribavir on the ganciclovir (GCV) kinase and protein kinase (PK) activities.

Results: Maribavir was a potent inhibitor of the autophosporylation of the wild type and all the major GCV resistant UL97 mutants analysed (M460I, H520Q, A594V and L595F) with a mean IC50 of 35 nM. The M460I mutation resulted in hypersensitivity to maribavir with an IC50 of 4.8 nM. A maribavir resistant mutant of UL97 (L397R) was functionally compromised as both a GCV kinase and a protein kinase (~ 10% of wild type levels). Enzyme kinetic experiments demonstrated that maribavir was a competitive inhibitor of ATP with a Ki of 10 nM.

Discussion: Maribavir is a potent competitive inhibitor of the UL97 protein kinase function and shows increased activity against the M460I GCV-resistant mutant which may impact on the management of GCV drug resistance in patients.

No MeSH data available.


Related in: MedlinePlus

IC50 of maribavir for the wild type and mutant UL97 Proteins. The wild type and mutant UL97 proteins were subjected to protein kinase assays with varying concentrations of maribavir (0.01-1.0 μM). The autoradiographs were analysed using the BioRad Multianalyst software and UL97 phosphorylation plotted as a percentage of the total phosphorylation in the absence of maribavir. An exponential decay curve was fitted and the IC50 of maribavir was determined for each of the proteins. The alteration of x-axis scale of the M460I graph should be noted. The IC50 for each species is shown.
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Figure 6: IC50 of maribavir for the wild type and mutant UL97 Proteins. The wild type and mutant UL97 proteins were subjected to protein kinase assays with varying concentrations of maribavir (0.01-1.0 μM). The autoradiographs were analysed using the BioRad Multianalyst software and UL97 phosphorylation plotted as a percentage of the total phosphorylation in the absence of maribavir. An exponential decay curve was fitted and the IC50 of maribavir was determined for each of the proteins. The alteration of x-axis scale of the M460I graph should be noted. The IC50 for each species is shown.

Mentions: Quantitative image analysis of the autoradiographs generated from the protein kinase assays using increasing concentrations of maribavir allowed calculation of the IC50 for each UL97 protein under investigation (Figure 6). The IC50 for wild type UL97 was 34 nM, and for the mutants H520Q, A594T, L595F, the D456A, H662L and V665I the IC50 was 33 nM, 31 nM, 28 nM 34 nM, 40 nM and 30 nM respectively. There was no autophosphorylation of the K355Q and N461G mutants hence these proteins were not examined in the presence of maribavir. The maribavir-resistant mutant (L397R) did not exhibit a reduction in autophosphorylation even when drug concentrations were increased to 100 μM. In contrast to the comparable IC50 values observed between wild type UL97 and the GCV resistant mutants at amino acids 520, 594 and 595, the M460I mutant was hypersensitive to maribavir with an IC50 of 4.8 nM (Figure 6).


The effects of maribavir on the autophosphorylation of ganciclovir resistant mutants of the cytomegalovirus UL97 protein.

Shannon-Lowe CD, Emery VC - Herpesviridae (2010)

IC50 of maribavir for the wild type and mutant UL97 Proteins. The wild type and mutant UL97 proteins were subjected to protein kinase assays with varying concentrations of maribavir (0.01-1.0 μM). The autoradiographs were analysed using the BioRad Multianalyst software and UL97 phosphorylation plotted as a percentage of the total phosphorylation in the absence of maribavir. An exponential decay curve was fitted and the IC50 of maribavir was determined for each of the proteins. The alteration of x-axis scale of the M460I graph should be noted. The IC50 for each species is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3050433&req=5

Figure 6: IC50 of maribavir for the wild type and mutant UL97 Proteins. The wild type and mutant UL97 proteins were subjected to protein kinase assays with varying concentrations of maribavir (0.01-1.0 μM). The autoradiographs were analysed using the BioRad Multianalyst software and UL97 phosphorylation plotted as a percentage of the total phosphorylation in the absence of maribavir. An exponential decay curve was fitted and the IC50 of maribavir was determined for each of the proteins. The alteration of x-axis scale of the M460I graph should be noted. The IC50 for each species is shown.
Mentions: Quantitative image analysis of the autoradiographs generated from the protein kinase assays using increasing concentrations of maribavir allowed calculation of the IC50 for each UL97 protein under investigation (Figure 6). The IC50 for wild type UL97 was 34 nM, and for the mutants H520Q, A594T, L595F, the D456A, H662L and V665I the IC50 was 33 nM, 31 nM, 28 nM 34 nM, 40 nM and 30 nM respectively. There was no autophosphorylation of the K355Q and N461G mutants hence these proteins were not examined in the presence of maribavir. The maribavir-resistant mutant (L397R) did not exhibit a reduction in autophosphorylation even when drug concentrations were increased to 100 μM. In contrast to the comparable IC50 values observed between wild type UL97 and the GCV resistant mutants at amino acids 520, 594 and 595, the M460I mutant was hypersensitive to maribavir with an IC50 of 4.8 nM (Figure 6).

Bottom Line: Wild type and site directed mutant forms of the human cytomegalovirus UL97 gene product were expressed using recombinant baculoviruses and the purified products used to assess the effects of maribavir on the ganciclovir (GCV) kinase and protein kinase (PK) activities.Enzyme kinetic experiments demonstrated that maribavir was a competitive inhibitor of ATP with a Ki of 10 nM.Maribavir is a potent competitive inhibitor of the UL97 protein kinase function and shows increased activity against the M460I GCV-resistant mutant which may impact on the management of GCV drug resistance in patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infection, Centre for Virology, UCL (Royal Free Campus Campus), Rowland Hill Street, Hampstead, London NW3 2QG, UK. v.emery@medsch.ucl.ac.uk.

ABSTRACT

Background: The UL97 protein kinase of human cytomegalovirus phosphorylates the antiviral drug ganciclovir and is the target of maribavir action. A detailed enzyme kinetic analysis of maribavir on the various enzymatic functions of wild type and ganciclovir resistant forms of UL97 is required.

Methods: Wild type and site directed mutant forms of the human cytomegalovirus UL97 gene product were expressed using recombinant baculoviruses and the purified products used to assess the effects of maribavir on the ganciclovir (GCV) kinase and protein kinase (PK) activities.

Results: Maribavir was a potent inhibitor of the autophosporylation of the wild type and all the major GCV resistant UL97 mutants analysed (M460I, H520Q, A594V and L595F) with a mean IC50 of 35 nM. The M460I mutation resulted in hypersensitivity to maribavir with an IC50 of 4.8 nM. A maribavir resistant mutant of UL97 (L397R) was functionally compromised as both a GCV kinase and a protein kinase (~ 10% of wild type levels). Enzyme kinetic experiments demonstrated that maribavir was a competitive inhibitor of ATP with a Ki of 10 nM.

Discussion: Maribavir is a potent competitive inhibitor of the UL97 protein kinase function and shows increased activity against the M460I GCV-resistant mutant which may impact on the management of GCV drug resistance in patients.

No MeSH data available.


Related in: MedlinePlus