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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

Ganciclovir (GCV) phosphorylation catalysed by the wild type and UL97 mutant proteins (designated by the amino acid change present). Insect cells were infected with the wild type or mutant UL97 expressing baculoviruses at an MOI 10. The medium was supplemented with tritiated GCV at 48 hours. The nucleotides were harvested at 72 hours and bound to DE81 filter paper. The phosphorylated GCV is plotted as percentage phosphorylation compared to the wild type UL97 (set at 100%). Data are the mean +/- one standard deviation of three experiments.
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Figure 3: Ganciclovir (GCV) phosphorylation catalysed by the wild type and UL97 mutant proteins (designated by the amino acid change present). Insect cells were infected with the wild type or mutant UL97 expressing baculoviruses at an MOI 10. The medium was supplemented with tritiated GCV at 48 hours. The nucleotides were harvested at 72 hours and bound to DE81 filter paper. The phosphorylated GCV is plotted as percentage phosphorylation compared to the wild type UL97 (set at 100%). Data are the mean +/- one standard deviation of three experiments.

Mentions: Insect cells were infected with the wild type or mutant UL97-expressing baculoviruses at high multiplicity and after 48 hours, the medium was supplemented with tritiated GCV. The phosphorylation of GCV catalysed by each of the mutant UL97 proteins was compared to wild type GCV phosphorylation (normalised to 100%). The data summarised in Figure 3 show that GCV phosphorylation by the genotypic GCV-resistant UL97 mutants (M460I, H520Q, A594T and L595F) was reduced to between 10% and 20% of the levels of GCV phosphorylation catalysed by the wild type UL97 protein. As expected, mutation at the invariant lysine (K355Q) produced a protein that was unable to phosphorylate GCV. Although mutation of M460 showed a substantial loss of GCV phosphorylation, mutation of the nearby 456 codon had no effect on GCV phosphorylation. Mutations around the His-X-aromatic-hydrophobic motif at codons H662L and V665I showed a reduction in GCV phosphorylation by 12% and 72% of wild type levels respectively. Interestingly, the maribavir-resistant UL97 mutant (L397R) exhibited a significant impairment in GCV phosphorylation (approximately 10% of wild type).


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

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

Ganciclovir (GCV) phosphorylation catalysed by the wild type and UL97 mutant proteins (designated by the amino acid change present). Insect cells were infected with the wild type or mutant UL97 expressing baculoviruses at an MOI 10. The medium was supplemented with tritiated GCV at 48 hours. The nucleotides were harvested at 72 hours and bound to DE81 filter paper. The phosphorylated GCV is plotted as percentage phosphorylation compared to the wild type UL97 (set at 100%). Data are the mean +/- one standard deviation of three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Ganciclovir (GCV) phosphorylation catalysed by the wild type and UL97 mutant proteins (designated by the amino acid change present). Insect cells were infected with the wild type or mutant UL97 expressing baculoviruses at an MOI 10. The medium was supplemented with tritiated GCV at 48 hours. The nucleotides were harvested at 72 hours and bound to DE81 filter paper. The phosphorylated GCV is plotted as percentage phosphorylation compared to the wild type UL97 (set at 100%). Data are the mean +/- one standard deviation of three experiments.
Mentions: Insect cells were infected with the wild type or mutant UL97-expressing baculoviruses at high multiplicity and after 48 hours, the medium was supplemented with tritiated GCV. The phosphorylation of GCV catalysed by each of the mutant UL97 proteins was compared to wild type GCV phosphorylation (normalised to 100%). The data summarised in Figure 3 show that GCV phosphorylation by the genotypic GCV-resistant UL97 mutants (M460I, H520Q, A594T and L595F) was reduced to between 10% and 20% of the levels of GCV phosphorylation catalysed by the wild type UL97 protein. As expected, mutation at the invariant lysine (K355Q) produced a protein that was unable to phosphorylate GCV. Although mutation of M460 showed a substantial loss of GCV phosphorylation, mutation of the nearby 456 codon had no effect on GCV phosphorylation. Mutations around the His-X-aromatic-hydrophobic motif at codons H662L and V665I showed a reduction in GCV phosphorylation by 12% and 72% of wild type levels respectively. Interestingly, the maribavir-resistant UL97 mutant (L397R) exhibited a significant impairment in GCV phosphorylation (approximately 10% of wild type).

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