Identification of fragments targeting an alternative pocket on HIV-1 gp41 by NMR screening and similarity searching

Abstract

The HIV-1 envelope glycoprotein gp41 fusion intermediate is a promising drug target for inhibiting viral entry. However, drug development has been impeded by challenges inherent in mediating the underlying protein-protein interaction. Here we report on the identification of fragments that bind to a C-terminal sub-pocket adjacent to the well-known hydrophobic pocket on the NHR coiled coil. Using a specifically designed assay and ligand-based NMR screening of a fragment library, we identified a thioenylaminopyrazole compound with a dissociation constant of ∼500μM. Interaction with the C-terminal sub-pocket was confirmed by paramagnetic relaxation enhancement NMR experiments, which also yielded the binding mode. Shape-based similarity searching detected additional phenylpyrazole and phenyltriazole fragments within the library, enriching the hit rate over random screening, and revealing molecular features required for activity. Discovery of the novel scaffolds and binding mechanism suggests avenues for extending the interaction surface and improving the potency of a hydrophobic pocket binding inhibitor.

Druggability analysis of membrane proteins by DoGSiteScorer

“…Pocket_0 is located in the loop region that connecting NHR and CHR helixes. Pocket_1 and Pocket_2 are located on the surface of the helix surface and are in good agreements with the experimental data, confirming the binding pockets of gp41 determined by NMR spectroscopy (S. D. Chu & Gochin, 2013;. These two binding pockets provide very valuable targets for developing small molecule inhibitors that can inhibit the HIV virus entering into the host cells.…”

“…Several gp41 have been prepared for drug discovery (S. D. Chu et al, 2015;. Chu et al have discovered another binding site on gp41 by using a novel fragment library screening combined with chemoinformatics (S. D. Chu & Gochin, 2013). Phospholamban is a membrane protein (MacLennan & Kranias, 2003).…”

Systematic Targeting of Protein–Protein Interactions

“…Fragment-based lead discovery and determination of structure-activity relationships by NMR have now become established methods for the development of PPI inhibitors[29, 30, 72-76]. Other methods including X-ray crystallography, differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence spectroscopy assays have also been adapted to address the challenge of finding lead fragments[64, 65, 77-79].…”

Swapped-Domain Constructs of the Glycoprotein-41 Ectodomain Are Potent Inhibitors of HIV Infection

“…29, 30 N40-containing constructs contained 12 fewer residues at the C-terminus, including NHR residues A582-A591. We 37, 38 and others 39 have determined that this 10 residue segment C-terminal to the pocket mediates important inter-domain interactions, and we therefore included it in our longer constructs. We additionally made C28(L4)N45, to probe this C-terminal segment while reducing fraying of the NHR helix at the hydrophobic pocket (unpublished results).…”