With about 70% of inhibiting neurons γ-amino butyric acid (GABA) is the the most important inibiting neuro transmitter in the central nervous system (CNS). Several neurological diseases, including epilepsy, Huntington's Chorea, and Morbus Parkinson are thought to be caused by pathologically reduced GABA neuro-transmission.
The concentration of extra-cellular GABA in the brain is regulated by specific transport proteins, so-called GABA transport proteins (GAT). To date, four different subtypes, mGAT1 - mGAT4 (nomenclature for mouse) have been identified.
The concentration of the subtypes varies in different regions of the brain, as do the cell types on which they can be located predominantly and their sub-cellular distribution. Therefore, the different GABA transport subtypes have presumably different functions as well.
As mGAT1 and mGAT4 can be found in all areas of the synaptic cleft, it is assumed that they are directly involved in the synaptic signal transmission and that their most important task is to terminate GABA-ergic signals by reabsorbing the spilled GABA. mGAT2 and mGAT3 are not present in the synaptic cleft but can be located at some distance. Additionally, these transporters can be found on a number of special cell structures. It is therefore assumed that the task of these transporters is to control the "crosstalk" between synapses, to prevent a "spillover" of the neurotransmitter and to regulate the general "GABA status" in the brain.
The goal of our research is to develop new, subtype selective inhibitors of the GABA transporters that would allow a deeper insight into the physiological functions of each GAT protein, to study the therapeutic potential of each GAT subtype, and, in the long run, possibly develop new drugs.
Our research group is also testing enantiomerically pure compounds, nitrogen heterocycles, and amino acid derivatives for their potential as inhibitors of GABA transport proteins. We therefore work intensively on the development of generally applicable, asymmetric synthesis methods to produce these groups of compounds as well. The biological evaluation is conducted with radioligand binding assays.
Additionally, MS binding studies are used. MS binding studies are a new procedure developed by us that works along principles very similar to those of radioligand binding assays. The great advantage of the MS binding assay is that no radioactive substances are necessary. Instead of using radio ligands, MS binding assays employ native markers which are quantified through mass spectrometry.
The results of the biological evaluation are used to create structure activity relationships and 3D binding models as basis for further optimization (Molecular Modeling).
Characterizing the affinity of test compounds for defined targets is one of the fundamental challenges in the area of drug development. Traditionally, binding assays employing radioactive or fluorescent ligands are used to assess the affinity of a test compound. The principle of marking ligands has a number of serious drawbacks. The additional synthetic step required for labelling means higher expenses and a limited availability of marked ligands. Additional limitations are caused by the reduction of the affinity that is very often observed after the introduction of large fluorescent groups, or the safety precautions that have to be observed when handling radioligands.
All these disadvantages can be avoided by replacing the marked ligand with a native (i.e. unmarked) ligand that is analysed by mass spectrometry. We have termed this type of binding assay, in analogy of radioligand binding assays, 'MS Binding Assay' (see scheme below). MS binding assays are universally applicable and, in principle, can be used in all common types of radioligand binding assays like saturation, competition or kinetic experiments. In our research group we have already established MS binding assays for dopamine D1 and D2 receptors as well as for the GAT1 subtype of the GABA transporters. We are working on additional test systems.
Scheme: Competitive MS binding assay with quantification of the target-bound marker. After the incubation of the target with marker and test substance the target-marker complex is separated by filtration. Subsequently the bound marker is liberated from the target-marker complex and the marker quantified using LC-ESI-MS-MS.