Sandbox Reserved 712
From Proteopedia
(→Enzymatic analysis of recombinant PRs) |
(→Enzymatic analysis of recombinant PRs) |
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{{Zitat|Vitality describes the relative ability of a given PR species to hydrolyze its substrate in the presence of an inhibitor. The higer the vitality value the greater the advantage given to that PR species.}} | {{Zitat|Vitality describes the relative ability of a given PR species to hydrolyze its substrate in the presence of an inhibitor. The higer the vitality value the greater the advantage given to that PR species.}} | ||
| - | === Enzymatic analysis of recombinant PRs === | ||
| - | 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. | ||
| - | |||
| - | Despite there were many mutations the k<sub>cat</sub> values still were 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. <ref name="Molecular" /> | ||
| - | |||
| - | (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in this study]) | ||
| - | |||
| - | Inhibition constants were determined by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor. | ||
| - | PR<sub>drv5</sub> - which only had a specific activity of 5% of the wild-type value - also shows a smaller difference in k<sub>i</sub> value for darunavir, even if it contains 20 mutations.(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ K<sub>i</sub> values for the inhibitors of PR mutants]) Among those 20, some are responsible for cross-resistance to other PIs (L10I, L33F, M46L, I54V, A71V, V82T, I84V, L89V and L90M). <ref name="Molecular" /> | ||
=== X-ray strucure analysis of PRdrv1 and PRdrv5 === | === X-ray strucure analysis of PRdrv1 and PRdrv5 === | ||
Revision as of 14:49, 28 December 2012
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| 3ggu, resolution 1.80Å () | |||||||||
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| Ligands: | |||||||||
| Gene: | Gag-Pol, pol (Viruses) | ||||||||
| Activity: | HIV-1 retropepsin, with EC number 3.4.23.16 | ||||||||
| Related: | 3ggt | ||||||||
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| Resources: | FirstGlance, OCA, PDBsum, RCSB | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
Contents |
Description
3ggu is a drug resistant HIV protease. Shown is a patient's variant in complex with darunavir.
HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes. HIV proteases contain a highly conserved region Asp - Thr - Gly (Asp25, Thr26 and Gly27), with the aspartic residue beeing the active site in the aspartyl protease.[1]
Because of its importance for the life-cycle of the retrovirus, HIV-PR are the major target for anti-HIV treatment. HIV protease inhibitors are the most potent agens used in anti-HIV treatment. However it occurs that HIV-PR develop a resistance to the inhibitor. [2]
3ggu (also PRDRV5) 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. [3]
Activity
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Darunavir as a PI
Prevalence of daruanvir resistance mutations in a large clinical database
Enzymatic analysis of recombinant PRs
Samples PRdrv1 to PRdrv6 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.
Despite there were many mutations the kcat values still were between 30 and 50% of the wild-type value. In contrast the Km values of the mutants were (mostly) four- to eightfold higher than the wild-type PR. [3]
(Complete Table: Enzyme characteristics of PR variants analyzed in this study)
Inhibition constants were determined by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor. PRdrv5 - which only had a specific activity of 5% of the wild-type value - also shows a smaller difference in ki value for darunavir, even if it contains 20 mutations.(Complete Table: Ki values for the inhibitors of PR mutants) Among those 20, some are responsible for cross-resistance to other PIs (L10I, L33F, M46L, I54V, A71V, V82T, I84V, L89V and L90M). The three amprenavir- and darunavir-associated mutations (V32I, I47V and I54L/M) don't exist in PRdrv5 (but in PRdrv1 and PRdrv6. [3]
Vitality describes the relative ability of a given PR species to hydrolyze its substrate in the presence of an inhibitor. The higer the vitality value the greater the advantage given to that PR species.
X-ray strucure analysis of PRdrv1 and PRdrv5
Structure
Applications
External Resources
References
- ↑ Kohl NE, Emini EA, Schleif WA, Davis LJ, Heimbach JC, Dixon RA, Scolnick EM, Sigal IS. Active human immunodeficiency virus protease is required for viral infectivity. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4686-90. PMID:3290901
- ↑ Watkins T, Resch W, Irlbeck D, Swanstrom R. Selection of high-level resistance to human immunodeficiency virus type 1 protease inhibitors. Antimicrob Agents Chemother. 2003 Feb;47(2):759-69. PMID:12543689
- ↑ 3.0 3.1 3.2 Saskova KG, Kozisek M, Rezacova P, Brynda J, Yashina T, Kagan RM, Konvalinka J. Molecular characterization of clinical isolates of human immunodeficiency virus resistant to the protease inhibitor darunavir. J Virol. 2009 Sep;83(17):8810-8. Epub 2009 Jun 17. PMID:19535439 doi:10.1128/JVI.00451-09
Contributors
Julia Baaske, Angelika Wackerl
