Sandbox Reserved 712: Difference between revisions
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These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PR<sub>DRV5</sub> which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the K<sub>i</sub> value for all the samples in comparison to the wild-type virus. PR<sub>DRV5</sub> - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in k<sub>i</sub> value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ K<sub>i</sub> values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref> | These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PR<sub>DRV5</sub> which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the K<sub>i</sub> value for all the samples in comparison to the wild-type virus. PR<sub>DRV5</sub> - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in k<sub>i</sub> value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ K<sub>i</sub> values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref> | ||
[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig. | [[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.4 Relative vitality values. <ref name="Molecular"/>]] | ||
The relative vitality values are defined as v = (K<sub>i</sub>k<sub>cat</sub>/K<sub>m</sub>)<sub>MUT</sub>/(K<sub>i</sub>k<sub>cat</sub>/K<sub>m</sub>)<sub>WT</sub>. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more does the mutated PR support the viral replication. <ref name="Kinetic"> PMID:7626598 </ref> | The relative vitality values are defined as v = (K<sub>i</sub>k<sub>cat</sub>/K<sub>m</sub>)<sub>MUT</sub>/(K<sub>i</sub>k<sub>cat</sub>/K<sub>m</sub>)<sub>WT</sub>. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more does the mutated PR support the viral replication. <ref name="Kinetic"> PMID:7626598 </ref> | ||
The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PR<sub>DRV4</sub> > PR<sub>DRV1</sub> > PR<sub>DRV2</sub> > PR<sub>DRV6</sub>). Due to the fact that PR<sub>DRV5</sub> does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.( | The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PR<sub>DRV4</sub> > PR<sub>DRV1</sub> > PR<sub>DRV2</sub> > PR<sub>DRV6</sub>). Due to the fact that PR<sub>DRV5</sub> does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig.4) <ref name="Molecular"> PMID:19535439 </ref> | ||
Despite the many mutations the k<sub>cat</sub> values were still between 30% and 50% of the wild-type value. In contrast the K<sub>m</sub> values of the mutants were (mostly) four- to eightfold higher than the wild-type PR. | Despite the many mutations the k<sub>cat</sub> values were still between 30% and 50% of the wild-type value. In contrast the K<sub>m</sub> values of the mutants were (mostly) four- to eightfold higher than the wild-type PR. | ||