Characterization of the diffusion of epidermal growth factor receptor clusters by single particle tracking.

A number of studies have shown that receptors of the epidermal growth factor receptor family (ErbBs) exist as higher-order oligomers (clusters) in cell membranes in addition to their monomeric and dimeric forms. Characterizing the lateral diffusion of such clusters may provide insights into their dynamics and help elucidate their functional relevance. To that end, we used single particle tracking to study the diffusion of clusters of the epidermal growth factor (EGF) receptor (EGFR; ErbB1) containing bound fluorescently-labeled ligand, EGF. EGFR clusters had a median diffusivity of 6.8×10(-11)cm(2)/s and were found to exhibit different modes of transport (immobile, simple, confined, and directed) similar to that previously reported for single EGFR molecules. Disruption of actin filaments increased the median diffusivity of EGFR clusters to 10.3×10(-11)cm(2)/s, while preserving the different modes of diffusion. Interestingly, disruption of microtubules rendered EGFR clusters nearly immobile. Our data suggests that microtubules may play an important role in the diffusion of EGFR clusters either directly or perhaps indirectly via other mechanisms. To our knowledge, this is the first report probing the effect of the cytoskeleton on the diffusion of EGFR clusters in the membranes of live cells.

Design of monodisperse and well-defined polypeptide-based polyvalent inhibitors of anthrax toxin.

The design of polyvalent molecules, presenting multiple copies of a specific ligand, represents a promising strategy to inhibit pathogens and toxins. The ability to control independently the valency and the spacing between ligands would be valuable for elucidating structure-activity relationships and for designing potent polyvalent molecules. To that end, we designed monodisperse polypeptide-based polyvalent inhibitors of anthrax toxin in which multiple copies of an inhibitory toxin-binding peptide were separated by flexible peptide linkers. By tuning the valency and linker length, we designed polyvalent inhibitors that were over four orders of magnitude more potent than the corresponding monovalent ligands. This strategy for the rational design of monodisperse polyvalent molecules may not only be broadly applicable for the inhibition of toxins and pathogens, but also for controlling the nanoscale organization of cellular receptors to regulate signaling and the fate of stem cells.

Insight into NSAID-induced membrane alterations, pathogenesis and therapeutics: characterization of interaction of NSAIDs with phosphatidylcholine.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most widely consumed pharmaceuticals, yet both the mechanisms involved in their therapeutic actions and side-effects, notably gastrointestinal (GI) ulceration/bleeding, have not been clearly defined. In this study, we have used a number of biochemical, structural, computational and biological systems including; Fourier Transform InfraRed (FTIR). Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) spectroscopy, and cell culture using a specific fluorescent membrane probe, to demonstrate that NSAIDs have a strong affinity to form ionic and hydrophobic associations with zwitterionic phospholipids, and specifically phosphatidylcholine (PC), that are reversible and non-covalent in nature. We propose that the pH-dependent partition of these potent anti-inflammatory drugs into the phospholipid bilayer, and possibly extracellular mono/multilayers present on the luminal interface of the mucus gel layer, may result in profound changes in the hydrophobicity, fluidity, permeability, biomechanical properties and stability of these membranes and barriers. These changes may not only provide an explanation of how NSAIDs induce surface injury to the GI mucosa as a component in the pathogenic mechanism leading to peptic ulceration and bleeding, but potentially an explanation for a number of (COX-independent) biological actions of this family of pharmaceuticals. This insight also has proven useful in the design and development of a novel class of PC-associated NSAIDs that have reduced GI toxicity while maintaining their essential therapeutic efficacy to inhibit pain and inflammation.

Structural association of nonsteroidal anti-inflammatory drugs with lipid membranes.

The location and distribution of ibuprofen, a model nonsteroidal anti-inflammatory drug, in a phospholipid bilayer was examined in molecular detail by a combination of neutron diffraction and computer simulations. In addition to their use as antipyretic, analgesic, and anti-inflammatory drugs, such nonsteroidal anti-inflammatory drugs are used in the treatment of a number of diseases including cancer and Alzheimer's. As a side effect, they have been known to cause gastrointestinal toxicity, although the molecular mechanism of their action is poorly understood. In this study, we have used contrast variation-based neutron diffraction to determine the position of the drug in a 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine lipid bilayer and explore changes to the bilayer structure upon drug incorporation. In its charged state, the drug was found to locate in the polar headgroup region of the phospholipid bilayer, to induce bilayer thinning, and to increase the number of water molecules closely associated with the bilayer. These structural insights are consistent with molecular dynamics simulations and earlier macroscopic experiments of vesicle structure and dynamics. Using MD simulations, the neutral ibuprofen, typically observed at low pH and inaccessible to the diffraction studies, was found to locate deeper within the bilayer than the charged form.

Structure-based design of a heptavalent anthrax toxin inhibitor.

The design of polyvalent molecules, consisting of multiple copies of a biospecific ligand attached to a suitable scaffold, represents a promising approach to inhibit pathogens and oligomeric microbial toxins. Despite the increasing interest in structure-based drug design, few polyvalent inhibitors based on this approach have shown efficacy in vivo. Here we demonstrate the structure-based design of potent biospecific heptavalent inhibitors of anthrax lethal toxin. Specifically, we illustrate the ability to design potent polyvalent ligands by matching the pattern of binding sites on the biological target. We used a combination of experimental studies based on mutagenesis and computational docking studies to identify the binding site for an inhibitory peptide on the heptameric subunit of anthrax toxin. We developed an approach based on copper-catalyzed azide-alkyne cycloaddition (click-chemistry) to facilitate the attachment of seven copies of the inhibitory peptide to a ß-cyclodextrin core via a polyethylene glycol linker of an appropriate length. The resulting heptavalent inhibitors neutralized anthrax lethal toxin both in vitro and in vivo and showed appreciable stability in serum. Given the inherent biocompatibility of cyclodextrin and polyethylene glycol, these potent well-defined heptavalent inhibitors show considerable promise as anthrax antitoxins.

Partitioning of nonsteroidal antiinflammatory drugs in lipid membranes: a molecular dynamics simulation study.

Using the potential of mean constrained force method, molecular dynamics simulations with atomistic details were performed to examine the partitioning and nature of interactions of two nonsteroidal antiinflammatory drugs, namely aspirin and ibuprofen, in bilayers of dipalmitoylphosphatidylcholine. Two charge states (neutral and anionic) of the drugs were simulated to understand the effect of protonation or pH on drug partitioning. Both drugs, irrespective of their charge state, were found to have high partition coefficients in the lipid bilayer from water. However, the values and trends of the free energy change and the location of the minima in the bilayer are seen to be influenced by the drug structure and charge state. In the context of the transport of the drugs through the bilayer, the charged forms were found to permeate fully hydrated in contrast to the neutral forms that permeate unhydrated.

Small-angle neutron scattering studies of phospholipid-NSAID adducts.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to have strong interactions with lipid membranes. Using small-angle neutron scattering, the effect of ibuprofen, a prominent NSAID, on the radius of small unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and their bilayer structure was studied systematically as a function of pH (ranging from 2 to 8) and drug-to-lipid mole ratio (from 0/1 to 0.62/1 mol/mol). Ibuprofen with a pK(a) of approximately 4.6 was found to significantly affect the bilayer structure at all pH values, irrespective of the charge state of the drug. At low pH values, the drug reduces the bilayer thickness, induces fluid-like behavior, and changes headgroup hydration. The incorporation of the drug in the lipid bilayer while affecting the local bilayer structure and hydration of the lipid does not affect the overall stability of the vesicle dispersions over the pH range studied.

Effect of pH and ibuprofen on the phospholipid bilayer bending modulus.

The lipid bilayer bending modulus, characterized by thermal undulations, is often affected by the presence of membrane active molecules. However, complex interplay between headgroup charges, hydration, and bilayer structural parameters such as bilayer thickness make it difficult to understand the changes in bending modulus. Using neutron spin-echo measurements, the effect of ibuprofen, a model nonsteroidal anti-inflammatory drug, on the bending modulus of phospholipid membranes is studied as a function of pH and temperature. Ibuprofen was found to lower the bending modulus at all pH values. We present molecular insights into the observed effect on membrane dynamics based on molecular dynamics simulations and small-angle neutron scattering based structural perturbations as well as changes in zwitterionic headgroup electrostatics due to pH and addition of ibuprofen.