Sandbox Reserved 712
From Proteopedia
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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
Darunavir (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large penal of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to darunavir. [4] One reason for the wide spectrum of darunavir suspectibilty is the strong binding of the PI to the main-chains of the protease active-site amino acids. [5] Darunavir shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. [6] Darunavir resistance-associated mutations are V11I, V32I, L33F, I47V, I50V, I54L, I54M, G73S, L76V, I84V and L89V. Those mutations occured in patientes who have a high number of PI resistance-associated mutations. [7]
Prevalence of daruanvir resistance mutations in a large clinical database
Phenotypic susceptibility to PIs
PRDRV5 (3ggu) shows twenty mutations of amino acids. The PR mutations that are associated to the darunavir resistance are L33F, I84V and L89V.
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 strutural related PI of darunavir, it only shows a 24-fold suscepetibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L, I54M. Those were not found PRDRV5 which explains the smaller phenotypic changes in the suscepetibility to darunavir.(table4) Nevertheless we can observe an increase of the Ki value for all the samples in comparison to the wild-type virus. (table6) [3]
The relative vitality values are defined as v = (Kikcat/Km)MUT/(Kikcat/Km)WT. 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. [8]
The relative vitality is related to the phenotypic changes in the suspectibilty to darunavir. Due to the fact that PRDRV5 does not have the key mutations, it has in comparison to the other samples a low vitality value for darunavir and the structural related amprenavir. (Fig2) [3]
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. [9]
