title: Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein.
abstract: Due to its highly conserved zinc fingers and its nucleic acid chaperone properties which are critical for HIV-1 replication, the nucleocapsid protein (NC) constitutes a major target in AIDS therapy. Different families of molecules targeting NC zinc fingers and/or inhibiting the binding of NC with its target nucleic acids have been developed. However, their limited specificity and their cellular toxicity prompted us to develop a screening assay to target molecules able to inhibit NC chaperone properties, and more specifically the initial NC-promoted destabilization of the nucleic acid secondary structure. Since this destabilization is critically dependent on the properly folded fingers, the developed assay is thought to be highly specific. The assay was based on the use of cTAR DNA, a stem-loop sequence complementary to the transactivation response element, doubly labelled at its 5' and 3' ends by a rhodamine 6G fluorophore and a fluorescence quencher, respectively. Addition of NC(12-55), a peptide corresponding to the zinc finger domain of NC, to this doubly-labelled cTAR, led to a partial melting of the cTAR stem, which increases the distance between the two labels and thus, restores the rhodamine 6G fluorescence. Thus, positive hits were detected through the decrease of rhodamine 6G fluorescence. An "in-house" chemical library of 4800 molecules was screened and five compounds with IC(50) values in the micromolar range have been selected. The hits were shown by mass spectrometry and fluorescence anisotropy titration to prevent binding of NC(12-55) to cTAR through direct interaction with the NC folded fingers, but without promoting zinc ejection. These non-zinc ejecting NC binders are a new series of anti-NC molecules that could be used to rationally design molecules with potential anti-viral activities.
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France.
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Shvadchak
authors.forename: Sarah
authors.initials: S
authors.lastname: Sanglier
authors.forename: Sandrine
authors.initials: S
authors.lastname: Rocle
authors.forename: Pascal
authors.initials: P
authors.lastname: Villa
authors.forename: Jacques
authors.initials: J
authors.lastname: Haiech
authors.forename: Marcel
authors.initials: M
authors.lastname: Hibert
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
authors.forename: Hugues
authors.initials: H
authors.lastname: de Rocquigny
mesh_terms: D019380:Anti-HIV Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D016325:HIV Long Terminal Repeat / Q000187:drug effects; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D020128:Inhibitory Concentration 50; D009841:Oligonucleotides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D020218:Response Elements / Q000187:drug effects; D054852:Small Molecule Libraries; D021241:Spectrometry, Mass, Electrospray Ionization; D014779:Virus Replication / Q000187:drug effects; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors*
chemical_list: D019380:Anti-HIV Agents; C071884:NCP7 protein, Human immunodeficiency virus 1; D009841:Oligonucleotides; D010455:Peptides; D054852:Small Molecule Libraries; D054301:gag Gene Products, Human Immunodeficiency Virus