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.

Fig. 1.

Structures of earlier antiviral hydroxycoumarin compound 1 and the unmodified compound 2, with the benzophenone-linked coumarins (compounds 3–5) and positive control compound 6.

Fig. 2.

Compound 3 binds to IN at a 1:1 ratio. (A) MALDI-TOF of an HPLC fraction containing the IN core complexed to compound 3 along with the IN core domain alone (control). (B) Two ESI-MS spectra showing the IN core domain alone, and complexed with compound 3. (C) ESI-MS spectrum showing the tryptic peptide T7 bound to one molecule of compound 3.

Fig. 3.

Resistance to coumarins observed in nonconservative W132 mutants and C130S. Comparison of the IC₅₀ values for IN inhibitory compounds 3 (A), 4 (B), 5 (C), and 6 (D) in different mutants. 3′-processing values are indicated by open bars, whereas strand transfer values are indicated by filled bars.

Fig. 4.

Coumarins bind to a region that may disrupt formation of active IN multimers. Shown are symmetric dimers with a tetrameric arrangement for a catalytically functional IN core domain. Compound 3 is shown in white ball and stick, the side chains of the ¹²⁸AACWWAGIK¹³⁶ residues are shown as green stick, the magenta and yellow ribbon shows two IN monomers, whereas red sticks and brown spheres represent the conserved DDE motif and Mg²⁺ ion, respectively.

Fig. 5.

Coumarins are less potent against Ca²⁺-induced preassembled IN-DNA complexes. Shown are the inhibitory effects of compound 3 on preassembled WT IN complex. Concentration of compound 3 decreases successively by a fraction of two-thirds from 100 μM to 1.2 μM.