Sandbox Reserved 712: Difference between revisions
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HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref> | HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref> | ||
3ggu (also PR<sub>DRV5</sub>) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. Darunavir is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref> | |||
== '''Darunavir: HIV-Protease Inhibitor''' == | == '''Darunavir: HIV-Protease Inhibitor''' == | ||
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Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and | Darunavir resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and | ||
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref> | <scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref> | ||
== '''Structure''' == | == '''Structure''' == | ||
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It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" /> | It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" /> | ||
== '''Phenotypic susceptibility and enzymatic analysis''' == | |||
Samples PR<sub>DRV1</sub> to PR<sub>DRV6</sub> have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs. | |||
PR<sub>DRV5</sub> (3ggu) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and | |||
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>. | |||
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 ampenavir, 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.1 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 supports the mutated PR 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.(Fig1) <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. | |||
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) <ref name="Molecular" /> | |||