Ligand-induced perturbations in Urtica dioica agglutinin

AbstractThe binding of the trisaccharide, N,N′,N″-triacetylchitotriose, to Urtica dioica agglutinin (UDA) was investigated using 1H NMR spectroscopy. UDA is a small antiviral plant lectin containing two homologous 43-amino acid domains. Carbohydrate-induced perturbations occur in one domain of UDA at trisaccharide concentrations below equimolar. Residues in the second domain are shifted at higher carbohydrate concentrations. This data confirms the presence of two binding sites of non-identical affinities per UDA monomer. Qualitative analysis of the 2D NOESY spectra indicates that UDA contains two short stretches of antiparallel β-sheet. The 1H resonance assignments for both antiparallel β-sheet sequences have been completed and there is one β-stretch per domain. A number of these β-sheet residues are perturbed in the presence of carbohydrate

Enhanced Potency of Bivalent Small Molecule gp41 Inhibitors

Low molecular weight peptidomimetic inhibitors with hydrophobic pocket binding properties and moderate fusion inhibitory activity against HIV-1 gp41-mediated cell fusion were elaborated by increasing the available surface area for interacting with the heptad repeat-1 (HR1) coiled coil on gp41. Two types of modifications were tested: 1) increasing the overall hydrophobicity of the molecules with an extension that could interact in the HR1 groove, and 2) forming symmetrical dimers with two peptidomimetic motifs that could potentially interact simultaneously in two hydrophobic pockets on the HR1 trimer. The latter approach was more successful, yielding 40–60 times improved potency against HIV fusion over the monomers. Biophysical characterization, including equilibrium binding studies by fluorescence and kinetic analysis by Surface Plasmon Resonance, revealed that inhibitor potency was better correlated to off-rates than to binding affinity. Binding and kinetic data could be fit to a model of bidentate interaction of dimers with the HR1 trimer as an explanation for the slow off-rate, albeit with minimal cooperativity due to the highly flexible ligand structures. The strong cooperativity observed in fusion inhibitory activity of the dimers implied accentuated potency due to the transient nature of the targeted intermediate. Optimization of monomer, dimer or higher order structures has the potential to lead to highly potent non-peptide fusion inhibitors by targeting multiple hydrophobic pockets

A Suite of Modular Fuorescence Assays Interrogate the Human Immunodeficiency Virus Glycoprotein-41 Coiled Coil and Assist in Determining Binding Mechanism of Low Molecular Weight Fusion Inhibitors

Several different segments of the gp41 N-heptad repeat coiled coil have been constructed using N-terminal bipyridyl modification of composite peptides and inducing trimerization by adding ferrous ions. These metallopeptides act as receptors in fluorescence-binding assays with corresponding fluorescently labeled C-peptide probes. The Fe(II) coordination complex quenches C-peptide fluorescence upon binding, and reversal of quenching by a small molecule inhibitor can be used to obtain the inhibitor-binding constant. A total of 10 peptide pairs targeting 25–46 residue segments of the coiled coil were constructed, with C-peptide probes of different lengths and binding affinities. The result is a suite of assays for exploring binding in the mM to nM range to any desired region of the coiled coil, including the hydrophobic pocket (HP), extended regions on either side of the pocket, or a region associated with T20 resistance mutations. These assays are high-throughput ready, and could be used to discover novel compounds binding along various regions of the gp41 coiled coil groove. They were used to evaluate a sub-μM low molecular weight fusion inhibitor, resulting in the finding that the molecule bound specifically to the HP and attained its potency from a low off-rate

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

[Image: see text] 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

Amphipathic Properties of HIV-1 gp41 Fusion Inhibitors

Small molecule inhibition of HIV fusion has been an elusive goal, despite years of effort by both pharmaceutical and academic laboratories. In this review, we will discuss the amphipathic properties of both peptide and small molecule inhibitors of gp41-mediated fusion. Many of the peptides and small molecules that have been developed target a large hydrophobic pocket situated within the grooves of the coiled coil, a potential hotspot for inhibiting the trimer of hairpin formation that accompanies fusion. Peptide studies reveal molecular properties required for effective inhibition, including elongated structure and lipophilic or amphiphilic nature. The characteristics of peptides that bind in this pocket provide features that should be considered in small molecule development. Additionally, a novel site for small molecule inhibition of fusion has recently been suggested, involving residues of the loop and fusion peptide. We will review the small molecule structures that have been developed, evidence pointing to their mechanism of action and strategies towards improving their affinity. The data points to the need for a strongly amphiphilic character of the inhibitors, possibly as a means to mediate the membrane - protein interaction that occurs in gp41 in addition to the protein – protein interaction that accompanies the fusion-activating conformational transition

Artificial Ion Channel Biosensor in Human Immunodeficiency Virus gp41 Drug Sensing

International audienc

The role of amphiphilicity and negative charge in glycoprotein 41 interactions in the hydrophobic pocket

The hydrophobic pocket within the coiled coil domain of HIV-1 gp41 is considered to be a hotspot suitable for small molecule intervention of fusion, although so far it has yielded only μM inhibitors. Previous peptide studies have identified specific hydrophobic interactions and a Lys-Asp salt bridge as contributing to binding affinity in the pocket. Negative charge appears to be critical for activity of small molecules. We have examined the role of charge and amphiphilic character in the interaction, by studying a series of short pocket binding peptides differing in charge, helical content and in the presence or absence of the Lys-Asp salt bridge, and a series of fatty acid salts with varying charge and hydrocarbon length. Quantitative binding analysis revealed that long range electrostatic forces and a greasy non-specific hydrophobic interaction were sufficient for μM potency. The results suggest that an extended interaction site may be necessary for higher potency. We examined a region of the coiled coil immediately C-terminal to the pocket, and found that specific salt bridge and hydrogen bond networks may reside in this region. Negatively charged groups extended towards or beyond the C-terminus of the pocket could therefore result in improved low molecular weight fusion inhibitors

Colloidal aggregate detection by rapid fluorescence measurement of liquid surface curvature changes in multiwell plates

A simple fluorescence method is reported for the detection of colloidal aggregate formation in solution, with specific applications to determination of the Critical Micelle Concentration (CMC) of surfactants and detection of small molecule promiscuous inhibitors. The method exploits the meniscus curvature changes in high density multi-well plates associated with colloidal changes in solution. The shape of the meniscus has a significant effect on fluorescence intensity when detected using a top read fluorescence plate reader, because of the effect of total internal reflection on fluorescence emission through a curved liquid surface. A dynamic range of 60% is calculated and observed, and is measured with a relative sensitivity of 2%. Facile determination of the CMC of a variety of surfactants is demonstrated, as well as a screening assay for aggregate forming properties of small drug-like compounds, a common cause of promiscuous inhibition in HTS enzyme inhibitor assays. Our preliminary results show a potential HTS assay with Z’ factor of 0.76, with good separation between aggregating and non-aggregating small molecules. The method combines the high sensitivity and universality of classic surface tension methods with simplicity and high throughput determination, enabling facile detection of molecular interactions involving a change in liquid or solid surface character