Probing the mechanism of drug/lipid membrane interactions using Biacore

Assay conditions were established to screen a panel of drugs for binding to liposome surfaces using a surface plasmon resonance (SPR) biosensor. Drugs were found to bind negligibly or reversibly or were retained on the liposome surface. Cationic amphiphilic drugs fell into the last class and correlated with drugs that induce phospholipidosis in vivo. To a first approximation, a single-site model yielded apparent binding affinities that adequately described a drug's dose-dependent binding to liposome surfaces. Affinities ranged at least 1000-fold within the drug panel. A liposome's drug-binding capacity and affinity depended on both the lipid headgroup and the drug's structure. Although a drug's charge state generally dominated whether or not it remained bound to the liposome, subtle structural differences between members of certain drug families led to them having widely differing binding affinities. A comparison between the dissociation of drugs from liposome surfaces by Biacore and the lipid retention measurements determined by a parallel artificial membrane permeability assay was drawn. The results from this study demonstrate the potential of using SPR-based assays to characterize drug/liposome-binding interactions.     

Elucidating the Signal Responses of Multi-Parametric Surface Plasmon Resonance Living Cell Sensing: A Comparison between Optical Modeling and Drug–MDCKII Cell Interaction Measurements

In vitro cell-based assays are widely used during the drug discovery and development process to test the biological activity of new drugs. Most of the commonly used cell-based assays, however, lack the ability to measure in real-time or under dynamic conditions (e.g. constant flow). In this study a multi-parameter surface plasmon resonance approach in combination with living cell sensing has been utilized for monitoring drug-cell interactions in real-time, under constant flow and without labels. The multi-parameter surface plasmon resonance approach, i.e. surface plasmon resonance angle versus intensity plots, provided fully specific signal patterns for various cell behaviors when stimulating cells with drugs that use para- and transcellular absorption routes. Simulated full surface plasmon resonance angular spectra of cell monolayers were compared with actual surface plasmon resonance measurements performed with MDCKII cell monolayers in order to better understand the origin of the surface plasmon resonance signal responses during drug stimulation of cells. The comparison of the simulated and measured surface plasmon resonance responses allowed to better understand and provide plausible explanations for the type of cellular changes, e.g. morphological or mass redistribution in cells, that were induced in the MDCKII cell monolayers during drug stimulation, and consequently to differentiate between the type and modes of drug actions. The multi-parameter surface plasmon resonance approach presented in this study lays the foundation for developing new types of cell-based tools for life science research, which should contribute to an improved mechanistic understanding of the type and contribution of different drug transport routes on drug absorption.     

Kinetic screening of antibodies from crude hybridoma samples using Biacore

Experimental and data analysis protocols were developed to screen antibodies from hybridoma culture supernatants using Biacore surface plasmon resonance biosensor platforms. The screening methods involved capturing antibodies from crude supernatants using Fc-specific antibody surfaces and monitoring antigen binding at a single concentration. After normalizing the antigen responses for the amount of antibody present, a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants (k(a) and k(d)) and affinity (K(D)) could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of antigen were performed to validate the reliability of single-concentration measurements. The screening protocols can be used to characterize antigen binding kinetics to approximately 200 antibody supernatants per day using automated Biacore 2000 and 3000 instruments.     

A synthetic membrane protein in tethered lipid bilayers for immunosensing in whole blood

Tethered lipid bilayers, containing a transmembrane synthetic ligand-gated ion channel (SLIC), have been formed on gold surfaces. The SLIC was designed as a highly selective receptor and reporter protein to detect antibodies in whole blood, which are of importance in malaria diagnosis. The specific binding of the antibody to the sensor surface was monitored on-line with label-free surface-sensitive techniques either optically by surface plasmon resonance in whole blood or electrically by measuring the channel activity of SLIC in blood serum. We demonstrate the feasibility of a highly sensitive and easily applicable whole blood biosensor on the basis of simple commercially available components. The sensor might find applications in the field of infectious diseases such as point-of-care diagnostics of malaria, high content quality control of blood samples of donors, or monitoring the efficacy of vaccination.     

Lipid-induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: determination and biological specificity

While antimicrobial and cytolytic peptides exert their effects on cells largely by interacting with the lipid bilayers of their membranes, the influence of the cell membrane lipid composition on the specificity of these peptides towards a given organism is not yet understood. The lack of experimental model systems that mimic the complexity of natural cell membranes has hampered efforts to establish a direct correlation between the induced conformation of these peptides upon binding to cell membranes and their biological specificities. Nevertheless, studies using model membranes reconstituted from lipids and a few membrane-associated proteins, combined with spectroscopic techniques (i.e. circular dichroism, fluorescence spectroscopy, Fourier transform infra red spectroscopy, etc.), have provided information on specific structure-function relationships of peptide-membrane interactions at the molecular level. Reversed phase-high performance chromatography (RP-HPLC) and surface plasmon resonance (SPR) are emerging techniques for the study of the dynamics of the interactions between cytolytic and antimicrobial peptides and lipid surfaces. Thus, the immobilization of lipid moieties onto RP-HPLC sorbent now allows the investigation of peptide conformational transition upon interaction with membrane surfaces, while SPR allows the observation of the time course of peptide binding to membrane surfaces. Such studies have clearly demonstrated the complexity of peptide-membrane interactions in terms of the mutual changes in peptide binding, conformation, orientation, and lipid organization, and have, to a certain extent, allowed correlations to be drawn between peptide conformational properties and lytic activity.     

Binding of vancomycin group antibiotics to D-alanine and D-lactate presenting self-assembled monolayers

Peptides terminating in -Lys-D-Ala-D-Ala, -Lys-D-Ala-L-Ala and -Lys-D-Ala-D-Lactate were covalently coupled via an N-terminal aminohexanoic acid linker to a self-assembled monolayer of HS(CH2)15CO2H on a thin gold film. Binding of the glycopeptide antibiotics vancomycin and chloroeremomycin to these surfaces was then measured using a surface plasmon resonance biosensor. Both antibiotics bound with micromolar affinity to the D-Ala-terminating surface and with millimolar affinity to the D-Lactate-terminating surface. Increasing density of these covalently attached peptides on the surface had no effect on the resultant affinities of either antibiotic for the surface. In contrast, when the lipid-anchored peptide N-alpha-docosanoyl-epsilon-acetyl-Lys-D-Ala-D-Ala was inserted into a supported lipid monolayer, the affinity of the strongly dimerizing antibiotic chloroeremomycin for the surface showed a dependence on ligand density. This was not the case with the weakly dimerizing antibiotic vancomycin. The lipid monolayer surface, which is a more realistic model of the surface of a bacterium, was thus better suited for the study of the cooperative binding interactions that occur between dimeric glycopeptide antibiotics and surface-bound ligands.     

Comparative study of horseradish peroxidase immobilization on modification of a gold surface using a surface plasmon resonance method

Horseradish peroxidase is immobilized by a periodate method on the gold surfaces previously modified with 16-mercapto-hexadecanoic acid or with hydrogen disulfide and soybean trypsin inhibitor. The effect of gold surface modification conditions on the immobilization of the enzyme as well as on the properties of the immobilized glycoprotein are studied using surface plasmon resonance technique. Restoration of the ability to bind specific antibodies is demonstrated for the immobilized enzyme. The low level of non-specific antibody binding to the immobilized glycoprotein is also shown.     

A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane

A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (β-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer’s disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer’s disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.     

Binding and selective detection of the secretory N-terminal domain of the alzheimer amyloid precursor protein on cell surfaces.

The secretory N-terminal domain of the Alzheimer amyloid precursor protein (sAPP) evokes specific responses in cells on binding to their surfaces. Because APP is expressed in a large variety of cell types, the localization of sAPP binding requires detection techniques that selectively recognize sAPP as a ligand. For this purpose, we prepared antibodies against recombinant sAPP695 (sAPPrec) previously expressed in E. coli. Such antibodies were found to distinguish between sAPPrec and cellular APP or sAPP, as shown by immunocytochemistry and by immunoblot. In addition, they allowed the selective localization of bound sAPPrec on cell surfaces without any signal from cellular APP or sAPP. Saturation of sAPPrec binding to cell surfaces, as determined radiometrically, was reached at 10 nM [125I]-sAPPrec. Binding was specific because it was almost completely inhibited by a 100-fold excess of unlabeled sAPPrec. This specificity of binding was confirmed by surface plasmon resonance spectroscopy. Binding of sAPPrec to cell surfaces occurred in patches and was dependent on the state of cell differentiation. The sAPPrec used in this study contains heparin binding sites, but enzymatic removal of cell surface associated heparin did not affect sAPPrec binding. Aldehyde fixation of cells strongly inhibited their ability to bind sAPPrec. The data point to a fixation-sensitive sAPPrec binding protein which is detectable in the form of patches and therefore is part of assembled cell surface microdomains.     

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes

Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.     

Interaction of antiinflammatory drugs with EPC liposomes: calorimetric study in a broad concentration range

Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/partition phenomenon was quantified in terms of the partition coefficient (K(p)), and/or the equilibrium constant (K(b)). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio >100:1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, DeltaH, and indirectly, DeltaG and DeltaS. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quantification of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction-possibly the consequence of a modification of the membrane's physical properties as it becomes saturated with the drug.     

Computational and experimental investigation of DNA repair protein photolyase interactions with low molecular weight drugs

This paper reports the previously unknown interactions between eight low molecular weight commercially available drugs (130–800 Da) and DNA repair protein photolyase using computational docking simulations and surface plasmon resonance (SPR) experiments. Theoretical dissociation constants, K_d, obtained from molecular docking simulations were compared with the values found from SPR experiments. Among the eight drugs analyzed, computational and experimental values showed similar binding affinities between selected drug and protein pairs. We found no significant differences in binding interactions between pure and commercial forms of the drug lornoxicam and DNA photolyase. Among the eight drugs studied, prednisone, desloratadine, and azelastine exhibited the highest binding affinity (K_d = 1.65, 2.05, and 8.47 μM, respectively) toward DNA photolyase. Results obtained in this study are promising for use in the prediction of unknown interactions of common drugs with specific proteins such as human clock protein cryptochrome. Copyright © 2013 John Wiley & Sons, Ltd.     

Drug–drug plasma protein binding interactions of ivacaftor

Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR‐protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co‐administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co‐administered CF drugs for human serum albumin (HSA) and α₁‐acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site‐selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug–drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug–drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug–drug interactions of ivacaftor with co‐administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. Copyright © 2015 John Wiley & Sons, Ltd.     

Binding of imipramine to phospholipid bilayers using radioligand binding assay

Binding of the tricyclic antidepressant imipramine (IMI) to neutral and negatively charged lipid membranes was investigated using a radioligand binding assay combined with centrifugation or filtration. Lipid bilayers were composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS). IMI binding isotherms were measured up to IMI concentration of 0.5 mmol/l. Due to electrostatic attraction, binding between the positively charged IMI and the negatively charged surfaces of PS membranes was augmented compared to binding to neutral PC membranes. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherms were described both by surface partition coefficients and by binding parameters (association constants and binding capacities). It was confirmed that binding of IMI to model membranes is strongly affected by negatively charged phospholipids and that the binding is heterogeneous; in fact, weak surface adsorption and incorporation of the drug into the hydrophobic core of lipid bilayer can be seen and characterized. These results support the hypothesis suggesting that the lipid part of biological membranes plays a role in the mechanism of antidepressant action.     

Partitioning of local anesthetics into membranes: surface charge effects monitored by the phospholipid head-group

The binding of the charged form of two local anesthetics, dibucaine and etidocaine, to bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was measured simultaneously with ultraviolet spectroscopy and deuterium magnetic resonance. Because of their amphiphilic molecular structure, both drugs intercalate between the lipid molecules, increasing the surface area and imparting a positive electric charge onto the membrane. The ultraviolet (UV) binding isotherms were therefore analyzed in terms of a model which specifically took into account the bilayer expansion as well as the charge-induced concentration variations near the membrane surface. By formulating a quantitative expression for the change in surface area upon drug intercalation and combining it with the Gouy-Chapman theory, the binding of charged dibucaine and etidocaine to the lipid membrane was best described by a partition equilibrium, with surface partition coefficients of 660 +/- 80 M-1 and 11 +/- 2 M-1 for dibucaine and etidocaine, respectively (pH 5.5, 0.1 M NaCl/50 mM buffer). Deuterium magnetic resonance demonstrated further that the binding of drug changed the head-group conformation of the lipid molecules. Invoking the intercalation model, a linear variation of the deuterium quadrupole splittings of the choline segments with the surface charge density was observed, suggesting that the phosphocholine head-group may act as a 'molecular electrometer' with respect to surface charges.     

Aminoacyl-tRNA synthetase-interacting multi-functional protein, p43, is imported to endothelial cells via lipid rafts

An aminoacyl-tRNA synthetase subunit, p43, was previously demonstrated to be released from mammalian cells, and to function as an extracellular regulator of both angiogenesis and inflammatory responses (Ko et al., [2001] J Biol Chem, 276; 23028; Park et al.[2002], J Biol Chem 277; 45243). Here, we report that p43 is internalized to the endothelial cells via lipid rafts. Exogenous p43 was co-localized on bovine aorta endothelial cells with cholera toxin B (CTB), which binds to cholesterol-enriched lipid rafts. The p43 was rapidly internalized to the cells, as early as 5 min after binding to the surfaces of the cells. p43 bound to the isolated lipid rafts, and its interaction with the lipid rafts, was prevented by high salt content, but not by detergent. This suggests that ionic bonds are involved in the molecular association of p43 with the lipid rafts. Taken together, we conclude that p43 binds to the endothelial cell surface via lipid rafts.     

Real-time monitoring of binding events on a thermostabilized human A2A receptor embedded in a lipid bilayer by surface plasmon resonance

Membrane proteins (MPs) are prevalent drug discovery targets involved in many cell processes. Despite their high potential as drug targets, the study of MPs has been hindered by limitations in expression, purification and stabilization in order to acquire thermodynamic and kinetic parameters of small molecules binding. These bottlenecks are grounded on the mandatory use of detergents to isolate and extract MPs from the cell plasma membrane and the coexistence of multiple conformations, which reflects biochemical versatility and intrinsic instability of MPs. In this work ,we set out to define a new strategy to enable surface plasmon resonance (SPR) measurements on a thermostabilized and truncated version of the human adenosine (A_2A) G-protein-coupled receptor (GPCR) inserted in a lipid bilayer nanodisc in a label- and detergent-free manner by using a combination of affinity tags and GFP-based fluorescence techniques. We were able to detect and characterize small molecules binding kinetics on a GPCR fully embedded in a lipid environment. By providing a comparison between different binding assays in membranes, nanodiscs and detergent micelles, we show that nanodiscs can be used for small molecule binding studies by SPR to enhance the MP stability and to trigger a more native-like behaviour when compared to kinetics on A_2A receptors isolated in detergent. This work provides thus a new methodology in drug discovery to characterize the binding kinetics of small molecule ligands for MPs targets in a lipid environment.     

Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip

The dextran matrix of a surface plasmon resonance (SPR) sensor chip modified with hydrophobic residues (BIAcore sensor chip L1) provides an ideal substrate for liposome adsorption. Liposomes of different lipid compositions are captured on the sensor chips by inserting these residues into the liposome membrane, thereby generating stable lipid surfaces. To gain a more detailed understanding of these surfaces, and to prove whether the liposomes stay on the matrix as single particles or form a continuous lipid layer by liposome fusion, we have investigated these materials, using atomic force microscopy (AFM) and fluorescence microscopy. Force measurements with AFM probes functionalized with bovine serum albumin (BSA) were employed to recognize liposome adsorption. Analysis of the maximal adhesive force and adhesion energy reveals a stronger interaction between BSA and the dextran matrix compared to the lipid-covered surfaces. Images generated using BSA-coated AFM tips indicated a complete and homogeneous coverage of the surface by phospholipid. Single liposomes could not be detected even at lower lipid concentrations, indicating that the liposomes fuse and form a lipid bilayer on the dextran matrix. Experiments with fluorescently labeled liposomes concurred with the AFM studies. Surfaces incubated with liposomes loaded with TRITC-labeled dextran showed no fluorescence, indicating a complete release of the encapsulated dye. In contrast, surfaces incubated with liposomes containing a fluorescently labeled lipid showed fluorescence.