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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

Effects of maribavir on the autophosphorylation of wild type UL97 and the various mutant proteins. Autoradiographs showing the Inhibition of autophosphorylation of wild type and mutant UL97 proteins by maribavir. The presence or absence of maribavir (0.5 μM) is indicated by + or-above each lane. UI = uninfected control insect cells.
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Figure 5: Effects of maribavir on the autophosphorylation of wild type UL97 and the various mutant proteins. Autoradiographs showing the Inhibition of autophosphorylation of wild type and mutant UL97 proteins by maribavir. The presence or absence of maribavir (0.5 μM) is indicated by + or-above each lane. UI = uninfected control insect cells.

Mentions: In the absence of maribavir, the GCV resistant UL97 mutants H520Q, A594T and L595F exhibited autophosphorylation levels equivalent to the wild type UL97 (Figure 5). However, the M460I mutant consistently exhibited approximately 85% of the autophosphorylation levels of the wild type UL97 (p = 0.01). Mutations of the invariant lysine (K355Q) completely negated autophosphorylation, as did mutation of the arginine at amino acid 461. Interestingly, the mutation that confers maribavir resistance to UL97 (L397R) resulted in a reduction in autophosphorylation to approximately 8% of the wild type levels. The remaining mutations within the UL97 gene (D456A, H662L and V665I) did not result in any change in autophosphorylation compared to that catalysed by the wild type UL97.


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

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

Effects of maribavir on the autophosphorylation of wild type UL97 and the various mutant proteins. Autoradiographs showing the Inhibition of autophosphorylation of wild type and mutant UL97 proteins by maribavir. The presence or absence of maribavir (0.5 μM) is indicated by + or-above each lane. UI = uninfected control insect cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effects of maribavir on the autophosphorylation of wild type UL97 and the various mutant proteins. Autoradiographs showing the Inhibition of autophosphorylation of wild type and mutant UL97 proteins by maribavir. The presence or absence of maribavir (0.5 μM) is indicated by + or-above each lane. UI = uninfected control insect cells.
Mentions: In the absence of maribavir, the GCV resistant UL97 mutants H520Q, A594T and L595F exhibited autophosphorylation levels equivalent to the wild type UL97 (Figure 5). However, the M460I mutant consistently exhibited approximately 85% of the autophosphorylation levels of the wild type UL97 (p = 0.01). Mutations of the invariant lysine (K355Q) completely negated autophosphorylation, as did mutation of the arginine at amino acid 461. Interestingly, the mutation that confers maribavir resistance to UL97 (L397R) resulted in a reduction in autophosphorylation to approximately 8% of the wild type levels. The remaining mutations within the UL97 gene (D456A, H662L and V665I) did not result in any change in autophosphorylation compared to that catalysed by the wild type UL97.

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