The biological screening section of our research group evaluates the test compounds for their capability to bind to or interact with certain target proteins. Targets of particular interest for our group are transport proteins and receptors involved in neurotransmission. So far, assays for the following targets have been established by our research group:
- GABA uptake inhibition (mGAT1, mGAT2, mGAT3, mGAT4, hGAT-1) and glycine (GlyT-1)
- GABAA- und GABAB-receptors
- NMDA-receptor complex (binding sites for PCP, glycine, glutamate, ifenprodil, polyamine)
To characterize the test compounds regarding GABA uptake, their capability to inhibit the 3H-GABA-uptake in synaptosomes (from pig brain) or in cells expressing GABA uptake proteins (GAT) is investigated. We use transient or stable transfected HEK293 and COS7 cells heterologously expressing the protein targets.
Binding assays to determine the affinity to a certain binding site of a receptor (usually in form of a membrane fraction of pig brain) are based on competition experiments between a 3H--marked radioligand and the test compounds. In addition, we have developed a novel method to perform competitive binding assays that allows us to use “native” markers instead of radioactive markers. The quantification of the “native” markers is performed by mass spectrometry. For the first time, the applicability of this new method was proven for the evaluation of test compounds regarding their affinity to the dopamine D1- receptor. Further applications following this principle are being developed.
Liquid scintillation counter
Laminar air flow
Publications about the test systems:
- G. Höfner, K. T. Wanner, „Characterisation of [3H]MK-801 binding and its cooperative modulation by pig brain membranes“, J. Recept. Signal Transduct. Res., 1996, 16, 297-313.
- G. Höfner, K. T. Wanner, „Characterization of the binding of [3H]MDL 105,519, a radiolabelled antagonist for the N-methyl-D-aspartate-associated glycine site, to pig cortical brain membranes“, Neurosci. Lett., 1997, 226, 79-82.
- G. Höfner, K. T. Wanner, „[3H]ifenprodil binding to NMDA receptors in porcine hippocampal brain membranes“, Eur. J. Pharmacol., 2000, 394, 211-219.
- G. Höfner, K. T. Wanner, „Kompetitive Bindungsstudien leicht gemacht – mit nativem Marker und massenspektrometrische Quantifizierung“, Angew. Chem., 2003, 115, 5393-5395.
- G. Höfner, K. T. Wanner, „Evaluation of GABA uptake in subcellular fractions of bovine frontal cortex and brainstem“, Neurosci. Lett., 2004, 364, 53-57.
- A. Kragler, G. Höfner, K. T. Wanner, "Novel parent structures for inhibitors of the murine GABA transporters mGAT3 and mGAT4", Eur. J. Pharmacol., 2005, 519, 43-47.
- C. Zepperitz, G. Höfner, K. T. Wanner, "MS-Binding assays: kinetic, saturation and competitive experiments based on quantitation of bound marker – exemplified by the GABA transporter mGAT1", ChemMedChem, 2006, 1,208-217.