title: Discovery of a small-molecule HIV-1 integrase inhibitor-binding site.
abstract: Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photo-crosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide (128)AACWWAGIK(136) was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling/MS analysis with site-directed mutagenesis/molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Laith Q
authors.initials: LQ
authors.lastname: Al-Mawsawi
authors.forename: Valery
authors.initials: V
authors.lastname: Fikkert
authors.forename: Raveendra
authors.initials: R
authors.lastname: Dayam
authors.forename: Myriam
authors.initials: M
authors.lastname: Witvrouw
authors.forename: Terrence R
authors.initials: TR
authors.lastname: Burke
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
authors.forename: Nouri
authors.initials: N
authors.lastname: Neamati
mesh_terms: D015110:4-Hydroxycoumarins / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001577:Benzophenones / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003374:Coumarins / Q000737:chemistry* / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D015195:Drug Design; D004351:Drug Resistance / Q000235:genetics; D019427:HIV Integrase / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics; D019428:HIV Integrase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D017434:Protein Structure, Tertiary / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
chemical_list: C512589:4-(bis(4-hydroxy-2-oxo-2H-benzopyran-3-yl)methyl)benzophenone; D015110:4-Hydroxycoumarins; D001577:Benzophenones; D003374:Coumarins; D019428:HIV Integrase Inhibitors; D014364:Tryptophan; D019427:HIV Integrase; D003545:Cysteine