title: Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs.
abstract: Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these "combination therapies." Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment. Unlike the widely used HIV protease inhibitors, this new class of inhibitor would target the substrate, not the protease enzyme itself. This approach offers a distinct advantage in that inhibitors of MA/CA would only need to affect a subset of the Gag molecules to disable viral replication. To discover MA/CA-specific inhibitors, we constructed a modified MA/CA fusion peptide (MA/CAΔ) that contains the HIV protease (PR) cleavage site as well as a tetracysteine motif for fluorescent labeling. The HIV PR cleavage of MA/CAΔ can then be monitored via fluorescence polarization (FP). We have adapted this FP assay for high-throughput screening and validated it according to industry standards using a 384-well plate format. We have currently tested 24,000 compounds in this assay and here detail the screening methodology and the results of this screening campaign.
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA.
authors.forename: Nancy
authors.initials: N
authors.lastname: Cheng
authors.affiliation: Department of Biochemistry and Biophysics, and the UNC Center for AIDS Research, University of North Carolina at Chapel Hill, NC, USA.
authors.forename: Sook-Kyung
authors.initials: SK
authors.lastname: Lee
authors.affiliation: LIMR Chemical Genomics Center, Inc., Lankenau Institute for Medical Research, Wynnewood, PA, USA.
authors.forename: P Scott
authors.initials: PS
authors.lastname: Donover
authors.affiliation: LIMR Chemical Genomics Center, Inc., Lankenau Institute for Medical Research, Wynnewood, PA, USA.
authors.forename: Mel
authors.initials: M
authors.lastname: Reichman
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts, Worcester, MA, USA.
authors.forename: Celia A
authors.initials: CA
authors.lastname: Schiffer
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Hull-Ryde
authors.affiliation: Department of Biochemistry and Biophysics, and the UNC Center for AIDS Research, University of North Carolina at Chapel Hill, NC, USA Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, NC, USA.
authors.forename: Ronald
authors.initials: R
authors.lastname: Swanstrom
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA Cancer Genetics Program, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC, USA bjanzen@email.unc.edu.
authors.forename: William P
authors.initials: WP
authors.lastname: Janzen
mesh_terms: D020816:Amino Acid Motifs; D019380:Anti-HIV Agents / Q000302:isolation & purification / Q000494:pharmacology*; D055808:Drug Discovery / Q000379:methods; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D009842:Oligopeptides / Q000037:antagonists & inhibitors / Q000378:metabolism; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012371:Robotics; D054852:Small Molecule Libraries; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; D005456:Fluorescent Dyes; C505205:HIV protease substrate 1; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D054301:gag Gene Products, Human Immunodeficiency Virus; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1
keywords: FlAsH; HIV protease; HTS; fluorescent polarization; orthogonally pooled chemical library