Document Detail


A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid.
MedLine Citation:
PMID:  23763575     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.
Authors:
Sook-Kyung Lee; Nancy Cheng; Emily Hull-Ryde; Marc Potempa; Celia A Schiffer; William Janzen; Ronald Swanstrom
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Validation Studies     Date:  2013-07-11
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Jul 
Date Detail:
Created Date:  2013-12-05     Completed Date:  2014-02-20     Revised Date:  2014-07-31    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4929-40     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Capsid / metabolism*
Cell Line
Fluorescein / chemistry
HIV Protease / metabolism*
HIV-1 / drug effects*,  pathogenicity,  physiology
Humans
Substrate Specificity
Virus Assembly
Virus Replication
Grant Support
ID/Acronym/Agency:
P01 GM066524/GM/NIGMS NIH HHS; P01-GM066524/GM/NIGMS NIH HHS; P30 AI050410/AI/NIAID NIH HHS; P30 CA016086/CA/NCI NIH HHS; P30 CA16086/CA/NCI NIH HHS; P30-AI50410/AI/NIAID NIH HHS; R01 GM064347/GM/NIGMS NIH HHS; R01GM65347/GM/NIGMS NIH HHS; R21 NS073052/NS/NINDS NIH HHS; R21 NS073052/NS/NINDS NIH HHS; T32 AI007001/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
2321-07-5/Fluorescein; EC 3.4.23.-/HIV Protease
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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