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.
journal_name
Proc Natl Acad Sci U S Aauthors
Al-Mawsawi LQ,Fikkert V,Dayam R,Witvrouw M,Burke TR Jr,Borchers CH,Neamati Ndoi
10.1073/pnas.0511254103subject
Has Abstractpub_date
2006-06-27 00:00:00pages
10080-5issue
26eissn
0027-8424issn
1091-6490pii
0511254103journal_volume
103pub_type
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