title: Discovery and structural characterization of a new inhibitor series of HIV-1 nucleocapsid function: NMR solution structure determination of a ternary complex involving a 2:1 inhibitor/NC stoichiometry.
abstract: The nucleocapsid (NC) protein is an essential factor with multiple functions within the human immunodeficiency virus type 1 (HIV-1) replication cycle. In this study, we describe the discovery of a novel series of inhibitors that targets HIV-1 NC protein by blocking its interaction with nucleic acids. This series was identified using a previously described capsid (CA) assembly assay, employing a recombinant HIV-1 CA-NC protein and immobilized TG-rich deoxyoligonucleotides. Using visible absorption spectroscopy, we were able to demonstrate that this new inhibitor series binds specifically and reversibly to the NC with a peculiar 2:1 stoichiometry. A fluorescence-polarization-based binding assay was also developed in order to monitor the inhibitory activities of this series of inhibitors. To better characterize the structural aspect of inhibitor binding onto NC, we performed NMR studies using unlabeled and (13)C,(15)N-double-labeled NC(1-55) protein constructs. This allowed the determination of the solution structure of a ternary complex characterized by two inhibitor molecules binding to the two zinc knuckles of the NC protein. To the best of our knowledge, this represents the first report of a high-resolution structure of a small-molecule inhibitor bound to NC, demonstrating sub-micromolar potency and moderate antiviral potency with one analogue of the series. This structure was compared with available NC/oligonucleotide complex structures and further underlined the high flexibility of the NC protein, allowing it to adopt many conformations in order to bind its different oligonucleotide/nucleomimetic targets. In addition, analysis of the interaction details between the inhibitor molecules and NC demonstrated how this novel inhibitor series is mimicking the guanosine nucleobases found in many reported complex structures.
authors.affiliation: Department of Chemistry, Boehringer Ingelheim (Canada) Ltd., Research & Development, 2100 Cunard Street, Laval, QC, Canada H7S 2G5. Electronic address: RESGeneral.LAV@boehringer-ingelheim.com.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Goudreau
authors.affiliation: Department of Chemistry, Boehringer Ingelheim (Canada) Ltd., Research & Development, 2100 Cunard Street, Laval, QC, Canada H7S 2G5. Electronic address: oliver.hucke@boehringer-ingelheim.com.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hucke
authors.affiliation: Department of Chemistry, Boehringer Ingelheim (Canada) Ltd., Research & Development, 2100 Cunard Street, Laval, QC, Canada H7S 2G5.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Faucher
authors.affiliation: Department of Chemistry, Boehringer Ingelheim (Canada) Ltd., Research & Development, 2100 Cunard Street, Laval, QC, Canada H7S 2G5.
authors.forename: Chantal
authors.initials: C
authors.lastname: Grand-Maître
authors.affiliation: Department of Chemistry, Boehringer Ingelheim (Canada) Ltd., Research & Development, 2100 Cunard Street, Laval, QC, Canada H7S 2G5.
authors.forename: Olivier
authors.initials: O
authors.lastname: Lepage
authors.affiliation: Department of Chemistry, Boehringer Ingelheim (Canada) Ltd., Research & Development, 2100 Cunard Street, Laval, QC, Canada H7S 2G5.
authors.forename: Pierre R
authors.initials: PR
authors.lastname: Bonneau
authors.affiliation: Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., Research & Development, 2100 Cunard Street, Laval, QC, Canada H7S 2G5.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Mason
authors.affiliation: Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., Research & Development, 2100 Cunard Street, Laval, QC, Canada H7S 2G5.
authors.forename: Steve
authors.initials: S
authors.lastname: Titolo
mesh_terms: D019380:Anti-HIV Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D019380:Anti-HIV Agents; C071884:NCP7 protein, Human immunodeficiency virus 1; D054301:gag Gene Products, Human Immunodeficiency Virus