June 15, 2012 (Vol. 32, No. 12)

As Focus Shifts to Membrane-Bound Proteins, Tools for Identification of Drug Targets Are Needed

SPR (surface plasmon resonance) biosensors are used in drug discovery as tools to provide rich content surrounding the interaction of biomolecules. This includes label-free binding data such as kinetic and equilibrium constants and concentration determination. Such information is essential when developing novel drugs that are required to have high affinities for their targets.

Researchers are using biosensors earlier in drug discovery to more quickly weed out molecules that are destined to fail. The ProteOn™ XPR36 protein interaction array system has been widely accepted by label-free drug discovery researchers. It uses a novel 6 x 6 multiplexed array to provide 36 interaction data points simultaneously.

There has been a recent shift in drug targets from soluble proteins to membrane-associated and trans-membrane proteins and receptors. Measuring the interaction of lipid assemblies—such as vesicles, liposomes, and lipoparticles—with drugs is also becoming more common as researchers unearth critical answers about drug delivery, virology, and signal transduction.

Bio-Rad Laboratories launched two new kits for use with the ProteOn system to address these specific issues: The ProteOn Liposome Capturing Kit and ProteOn GLC Lipid Kit. Both kits come with easy-to-follow protocols and produce high-quality data to study interactions involving lipid assemblies and trans-membrane proteins with other proteins, peptides, and small molecules.

Studying Drug-Membrane Interactions

The ProteOn Liposome Capturing Kit provides a novel hydrophilic surface chemistry that is used to capture intact lipoparticles, vesicles, and lipid assemblies with low non-specific binding, easy surface regeneration, and the ability to capture multiple layers of lipid assemblies for increased sensitivity.

The ProteOn LCP sensor chip surface is saturated with single-stranded biotinylated DNA molecules from the Liposome Capturing Kit, which enables liposomes tagged with cholesterol-labeled double-stranded DNA molecules to be captured to the surface through DNA hybridization.

This kit has been used to measure the binding of small molecule drugs to a variety of lipid membranes captured to the ProteOn LCP sensor chip surface (Figure 1). This type of analysis is important in drug discovery and development where drugs with ideal efficacy should be both soluble in an aqueous environment and able to pass through lipophilic barriers such as cell membranes. This balance between hydrophilicity and lipophilicity is an essential aspect to consider in drug discovery.

Many other in vitro methods have been developed, but only a few accurately predict drug transport across biological membranes. Researchers have been analyzing drug partitioning using liposomes, which mimic the lipid bilayer geometry and ionic characteristics of a biological membrane.

Figure 1. Interaction analysis achieved using the ProteOn Liposome Capturing Kit. The experiment screened 14 small molecule drugs against 5 types of liposomes: plain POPC, plain DSPC/CHOL 67:33 mol/mol, plain DSPC/CHOL 55:45 mol/mol, plain DSPC/CHOL/mPEG2000-DSPE 50:45:5 mol/mol, and DSPC/CHOL 55:45 mol/mol with 350 mM (NH4)2SO4 (ammonium gradient). POPC–1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; DSPC– 1,2-distearoyl-sn-glycero-3-phosphocholine; CHOL–cholesterol. Normalized maximum signals of the interactions between the drugs and the liposomes. Peak signals were normalized by dividing by the compound’s molecular weight. Strong signals were observed when using plain POPC liposomes and ammonium sulfate gradient DSPC/CHOL (NH4)2SO4 liposomes, while other liposomes showed no binding. Most of the weakly basic drugs that were tested showed binding interaction with ammonium sulfate gradient liposomes (6, 7, 8, 11, 12, 13, 14), which was expected since the acidic pH inside these liposomes protonates the weak bases, making them less lipophilic, leading eventually to their accumulation inside the liposomes [Barenholz, 2006].1 Strong binders identified during screening were subject to full kinetic analysis (data not shown).

Figure 1

Toxicological and Viral Research

Understanding the interaction of peptide or protein toxins with cell membranes is of increasing interest in academic fields such as virology and also in the pharmaceutical industry for drug discovery. The fusion activity of melittin, the active substance in honey bee venom, which manifests its toxicity by increasing the permeability of cellular membranes, was studied.

Using the ProteOn Liposome Capturing Kit, melittin was shown to bind to lipid bilayer membranes of plain POPC liposomes. As the concentration of melittin increases, the melittin molecules bound to the membrane start to reorient to a transmembrane configuration and form oligomeric pores (Figure 2A). Eventually at even higher concentration melittin ruptures the liposomes to produce micelle structures (Figure 2B).

Figure 2A. Interaction analysis of plain POPC liposomes and melittin achieved by the ProteOn Liposome Capturing Kit.

Drug Target Characterization

As many essential biological functions involve the participation of membrane proteins such as GPCRs, ion channels, and membrane-bound enzymes, they constitute a significant fraction of top drug targets. SPR platforms have been used with limited success to characterize the interactions of drug compounds against membrane protein targets. In utilizing the new GLC Lipid Kit from Bio-Rad Laboratories, improved success and an increase in throughput for this important analysis was observed.

A model system of CXCR4-embedded lipoparticles and associated α CXCR4-antibody were analyzed using the ProteOn GLC Lipid Kit. The GLC Lipid Kit is based on the traditional approach of capturing liposomes using a lipophilic surface chemistry.

Reagents are used to control surface lipophilicity of the sensor chip to enable lipid assembly capture. The surface can be effectively regenerated using detergents. CXCR4 is a typical GPCR protein, and lipoparticles, virus-like particles with lipid bilayer membranes, have been increasingly used in membrane protein research for this reason. Kinetic analysis of the interaction can be measured as shown in Figure 3.

Figure 2B. Sensorgram of the interaction analysis. The analyte melittin was injected in a twofold dilution series from 92 µM to 3 µM. At the highest melittin concentration, after an increase in response of ~2,500 RU (indicated by 92 µM curve), there is a decrease in response due to formation of melittin/lipid micelles that are detached from the surface. Running buffer: 10 mM Tris-HCl at pH 7.5 with 100 mM NaCl; Referencing used: blank channel reference.

Figure 3. (A) Interaction analysis of CXCR4 lipoparticles and anti-CXCR4 antibody achieved by the ProteOn GLC Lipid Kit. This interaction is a model system for membrane protein-protein interaction analysis. Lipoparticles were produced by Integral Molecular. (B) Sensorgrams from two channels showing an increase in response upon an increase of analyte (anti-CXCR4 antibody was injected at concentrations 33, 11, 1.2, 0.41 nM). Running buffer: PBS; Referencing used: channel reference (null lipoparticle surface).

Figure 3


Membrane-bound proteins are the next lucrative source of therapeutic drug targets. Since they have a wide range of functions, including immune response and cell signaling, these drugs may be used to treat a wide range of diseases. SPR biosensors provide real-time, label-free kinetic data making them valuable tools for the identification of drug targets.

The ProteOn XPR36 bridges the need for a sensitive and high-throughput drug target approach with the ability to effectively capture and regenerate the target of interest. The two new lipid capture solutions from Bio-Rad aid therapeutic development by facilitating the interrogation of drug targets by biologics and small molecules. The three case studies described in this article illustrate how the power of the ProteOn combined with the flexibility of the two new innovative kits from Bio-Rad will accelerate your research.

Laura Moriarty, Ph.D. ([email protected]), is a product manager in the protein function division of Bio-Rad Laboratories.


1. Barenholz, Y. Amphipathic weak base loading into preformed liposomes having a transmembrane ammonium ion gradient: from the bench to approved Doxil. Liposome Technology, Volume II (3rd Edition) 2: 1-25 (2006).

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