A Sensitive Assay Using a Native Protein Substrate for Screening HIV-1 Maturation Inhibitors Targeting the Protease Cleavage Site between the Matrix and Capsid

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• 作者：Sook-Kyung Lee ; Nancy Cheng ; Emily Hull-Ryde ; Marc Potempa ; Celia A. Schiffer ; William Janzen ; Ronald Swanstrom
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：July 23, 2013
• 年：2013
• 卷：52
• 期：29
• 页码：4929-4940
• 全文大小：460K
• 年卷期：v.52,no.29(July 23, 2013)
• ISSN：1520-4995

文摘

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鈥?value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC¹²⁸⁰ 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.