Development of a Cell Membrane Microarray as a new high throughput screening tool for drug discovery

Gabriel Barreda-Gómez1, Egoitz Astigarraga1, Mª Teresa Giralt2, Rafael Rodríguez-Puertas2. 1IMG Pharma Biotech S.L., R & D; 48940, Spain, 2Universidad del País Vasco, Farmacología; 48940, Spain

DNA, protein or tissue microarrays can be considered one of the most miniaturized high throughput screening (HTS) systems used in research to date. These microarrays are time and cost-saving, but they have important limitations when they are applied to drug discovery, as they are not appropriate for drug screening. In contrast, we have developed cell membrane microarrays for both the pharmacological characterization of the primary and secondary targets of a compound, and the prediction of its toxicity to obtain a safety profile.

The present work demonstrates the technical advantages of the use of cell membrane microarrays applied to different drug discovery processes, such as radioligand binding, [35S]GTPgammaS functional coupling and immunochemistry. The microarrays were composed of membrane homogenates isolated from different rat tissues and from cells that over-express a specific G protein-coupled receptor (GPCR). They were incubated with specific radioligands to label binding sites for cannabinoids, muscarinic and GABAergic compounds: [3H]-SR141716A; [3H]-NMS; [3H]-CGP54626. The specificity of the binding was tested using unlabelled drugs (WIN55,212-2, carbachol or baclophen, respectively). The [35S]GTPgammaS assay was used to calculate the activity of the compounds activating G proteins. Another set of cell membrane microarrays was used to test the specificity of several antibodies.

The radioligand binding profiles obtained with cell membrane microarrays matched with those obtained using conventional techniques. Moreover, a positive correlation was observed between the density of [3H]-CGP54626 bindings sites and the functional coupling induced by baclophen, as it also was for the cholinergic muscarinic compounds. Furthermore, the density of [3H]-NMS binding sites correlated with that quantified using the anti-M1 antibody for the M1 muscarinic receptor subtype.

Thus, cell membrane microarrays not only allow the identification of the target receptors and the organs or tissues in which they are located, but also indicate effects that the analyzed molecule could induce on specific tissues or cells. In summary, cell membrane microarrays are useful for studying the selectivity and affinity of compounds independently of their chemical structure, and also to determine the drug safety profile in a single miniaturized assay (WO2007/012688).

Acknowledgments: Biscay County Council (Beaz; 3/12/01/2010/00002), Basque Government (Spri; Ekintzaile) and Basque Government Research group in Neurochemistry and Neurodegeneration (IT440-10).