title: A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid.
abstract: 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.affiliation: Department of Biochemistry and Biophysics and UNC Center for AIDS Research, ‡Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, §Department of Microbiology and Immunology, and ∥Cancer Genetics Program, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Sook-Kyung
authors.initials: SK
authors.lastname: Lee
authors.forename: Nancy
authors.initials: N
authors.lastname: Cheng
authors.forename: Emily
authors.initials: E
authors.lastname: Hull-Ryde
authors.forename: Marc
authors.initials: M
authors.lastname: Potempa
authors.forename: Celia A
authors.initials: CA
authors.lastname: Schiffer
authors.forename: William
authors.initials: W
authors.lastname: Janzen
authors.forename: Ronald
authors.initials: R
authors.lastname: Swanstrom
mesh_terms: D002213:Capsid / Q000378:metabolism*; D002460:Cell Line; D019793:Fluorescein / Q000737:chemistry; D016333:HIV Protease / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects* / Q000472:pathogenicity / Q000502:physiology; D006801:Humans; D013379:Substrate Specificity; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D016333:HIV Protease; D019793:Fluorescein