title: Functional label-free assays for characterizing the in vitro mechanism of action of small molecule modulators of capsid assembly.
abstract: HIV capsid protein is an important target for antiviral drug design. High-throughput screening campaigns have identified two classes of compounds (PF74 and BI64) that directly target HIV capsid, resulting in antiviral activity against HIV-1 and HIV-2 laboratory strains. Using recombinant proteins, we developed a suite of label-free assays to mechanistically understand how these compounds modulate capsid activity. PF74 preferentially binds to the preassembled hexameric capsid form and prevents disruption of higher-order capsid structures by stabilizing capsid intersubunit interactions. BI64 binds only the monomeric capsid and locks the protein in the assembly incompetent monomeric form by disrupting capsid intersubunit interactions. We also used these assays to characterize the interaction between capsid and the host protein cleavage and polyadenylation specific factor 6 (CPSF6). Consistent with recently published results, our assays revealed CPSF6 activates capsid polymerization and preferentially binds to the preassembled hexameric capsid form similar to the small molecule compound, PF74. Furthermore, these label-free assays provide a robust method for facilitating the identification of a different class of small molecule modulators of capsid function.
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Latesh
authors.initials: L
authors.lastname: Lad
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Sheila
authors.initials: S
authors.lastname: Clancy
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: David
authors.initials: D
authors.lastname: Koditek
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Melanie H
authors.initials: MH
authors.lastname: Wong
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Debi
authors.initials: D
authors.lastname: Jin
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Anita
authors.initials: A
authors.lastname: Niedziela-Majka
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Giuseppe A
authors.initials: GA
authors.lastname: Papalia
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Magdeleine
authors.initials: M
authors.lastname: Hung
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Stephen
authors.initials: S
authors.lastname: Yant
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: John R
authors.initials: JR
authors.lastname: Somoza
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Eric
authors.initials: E
authors.lastname: Hu
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Chienhung
authors.initials: C
authors.lastname: Chou
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Winston
authors.initials: W
authors.lastname: Tse
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Randall
authors.initials: R
authors.lastname: Halcomb
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Roman
authors.initials: R
authors.lastname: Sakowicz
authors.affiliation: Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States.
authors.forename: Nikos
authors.initials: N
authors.lastname: Pagratis
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001562:Benzimidazoles / Q000494:pharmacology; D015374:Biosensing Techniques / Q000379:methods*; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015497:HIV-1; D054884:Host-Pathogen Interactions / Q000187:drug effects; D007211:Indoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D019380:Anti-HIV Agents; C000598985:BI64 compound; D001562:Benzimidazoles; D007211:Indoles; C000591221:PF-3450074; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; C450562:cleavage factor Im, human; D039221:mRNA Cleavage and Polyadenylation Factors; D010649:Phenylalanine