Sandbox Reserved 712: Difference between revisions
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=== X-ray structure analysis of 3ggu === | === X-ray structure analysis of 3ggu === | ||
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig. | [[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.1 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]] | ||
[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig. | [[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.2 Structural changes in PR<sub>DRV5</sub> mutant relative to wild-type PR. <ref name="Molecular" />]] | ||
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig. | [[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.3 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]] | ||
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations. | The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations. | ||
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps. | Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps. | ||
PR<sub>DRV5</sub> contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and | PR<sub>DRV5</sub> contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and | ||
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig. | <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.1, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites). | ||
The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>). | The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>). | ||
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<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>, | <scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>, | ||
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge. | <scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge. | ||
The pictures on the right (Fig. | The pictures on the right (Fig.2 and Fig.3) show the regions (indicated in blue) that undergo structural changes caused by the mutations. | ||
To see the full images, with changes in PR<sub>DRV1</sub> and comparative structure of wild-type, PR<sub>DRV1</sub> and PR<sub>DRV5</sub> follow the links: | To see the full images, with changes in PR<sub>DRV1</sub> and comparative structure of wild-type, PR<sub>DRV1</sub> and PR<sub>DRV5</sub> follow the links: | ||
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PR<sub>DRV</sub> mutants relative to wild-type PR] and | [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PR<sub>DRV</sub> mutants relative to wild-type PR] and | ||
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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''' == | == '''Phenotypic susceptibility and enzymatic analysis''' == | ||
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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. | ||
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HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today. | HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today. | ||
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance. The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future. | The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance.<ref name="Molecular" /> The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future. | ||
== '''External Resources''' == | == '''External Resources''' == | ||