Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4

Kinetic parameters extracted from fitting the binding curves of concentration series of three different mAbs against a single preparation of immobilized CXCR4 ACMs.

[a]<p>KD for 12G5 binding to CXCR4-ACMS shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110847#pone-0110847-g002" target="_blank">Figure 2</a> was 60.2±17 nM.</p><p>Kinetic parameters extracted from fitting the binding curves of concentration series of three different mAbs against a single preparation of immobilized CXCR4 ACMs.</p

Sensorgrams of mAb 12G5 binding (100 nM) to CXCR4-ACMs immobilized at low RU (ca. 1400) via biotin/spteptavidin immobilization of the embedding polymersome matrix.

<p>The analyte was injected in triplicate, at cycle 7, 14, and 21. Intermediate cycles involved blank injections. The blue line shows the fit to the curve assuming 1∶1 binding kinetics. The inset shows the relative decrease in binding activity of the surface as measured by the binding level 4 s before the end of the injection.</p

Kinetic screening of 12G5 mAb binding to CXCR4-ACMs immobilized onto biosensor chips.

<p>A: Ab was injected at increasing concentrations (6.25–400 nM) over 100 s, followed by a buffer wash (without regeneration) between injections (immobilization level: ca. 5000 RU; biotin/streptavidin immobilization). B. Saturation binding of 125-I SDF1α to CXCR4-ACMs. A dissociation constant of 8.4 nM was determined. C. The same series of measurements as shown in Fig. 2 A, conducted using immobilized VLPS (immobilization level: 5000 RU).</p

Targeting opioid receptor heterodimers: Strategies for screening and drug development

G-protein-coupled receptors are a major target for the development of new marketable drugs. A growing number of studies have shown that these receptors could bind to their ligands, signal, and be internalized as dimers. Most of the evidence comes from in vitro studies, but recent studies using animal models support an important role for dimerization in vivo and in human pathologies. It is therefore becoming highly relevant to include dimerization in screening campaigns: the increased complexity reached by the ability to target 2 receptors should lead to the identification of more specific hits that could be developed into drugs with fewer side effects. In this review, we have summarized results from a series of studies characterizing the properties of G-protein-coupled receptor dimers using both in vitro and in vivo systems. Since opioid receptors exist as dimers and heterodimerization modulates their pharmacology, we have used them as a model system to develop strategies for the identification of compounds that will specifically bind and activate opioid receptor heterodimers: such compounds could represent the next generation of pain relievers with decreased side effects, including reduced drug abuse liability