Identification of drug binding sites in GABA-A receptors

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the central nervous system. Most of the actions of GABA are mediated by GABA A receptors. These are chloride channels that can be opened by GABA and are composed of five protein subunits. GABA A receptors are the site of action of a variety of pharmacologically and clinically important drugs such as anxiolytics, anaesthetics, anticonvulsants, muscle relaxants, hypnotics and drugs that influence vigilance, learning and memory. Depending on their subunit composition GABA A receptors exhibit distinct pharmacological and electrophysiological properties. GABA A receptor subtypes present in the brain exhibit a distinct regional and cellular distribution and mediate different effects of drugs. Currently available drugs interact with many different subtypes and exhibit a broad spectrum of action. Drugs that selectively interact with specific GABA A receptor subtypes thus will exhibit very selective clinical effects and consequently less side effects. Although many compounds have been developed that can modulate the GABA induced chloride current of these receptors, for most of them no binding site has been identified so far. Recently structural models were developed by the applicant that suggest potential compound binding pockets in multiple places of the receptor. In the present project a combination of computational, electrophysiological and biochemical methods will be employed to investigate some of these so far unexplored putative drug binding pockets and to identify compounds using them as binding sites. Based on computational models site directed mutagenesis will be employed to introduce cysteine residues in selected positions. The resulting receptors will then be modified with cysteine reactive reagents that in turn will serve as steric hindrance to compound binding. A selective loss of compound action is a first hint for a binding interaction and will be followed up by more specific experiments. In addition, the regions around the introduced cysteines will be structurally mapped and the data integrated into structural models. All compounds that can be assigned to a specific binding site, regardless of their pharmacological profile, can be used for further screening and are lead compounds for the development of drugs interacting with this specific binding site. Results will facilitate the development of novel subtype selective modulators of this important drug target and pave the way for a structure guided drug design.

The scientific goal of this study was the identification of binding sites on GABA-A receptors for new therapeutic principles. Many widely used medications, such as narcotic, hypnotic, anxiolytic and antiepileptic agents, act by binding to the most important inhibitory receptor type of the central nervous system, the GABA-A receptor (gamma amino butyric acid receptor type A). Many subtypes of these receptors exist. Innovative drug research is generally aimed at addressing these subtypes selectively as unselectively acting agents show side effects such as drowsiness in antiepileptics. Owing to the complex structure of these receptors, not all binding sites at which compounds can influence their function are known. For rational drug design, information about binding sites is important. Thus, the main goal of this project was the localization and investigation of uncharacterized binding sites in GABA-A receptors by using molecular biological and pharmacological methods, as well as the identification of compounds acting via these sites that would offer new therapeutic principles in the treatment of sleep- and anxiety disorders or epilepsy. The main result of the study was the identification of a previously unknown binding site. This site has strong similarity with the already well known and therapeutically heavily used binding site for benzodiazepines. Therefore, structural models of this site were also developed in the course of the project that will enable future structural studies of the new site, for which very little data exists. It was shown for the newly identified site that is formed by two subunits, that recognition of receptor subtypes via both participating subunits is possible. Thus, the development of selective compounds will be feasible. In summary, the foundation for new therapeutic principles utilizing this important receptor was laid in this project. Our current work aims to identify more chemotypes that might act selectively at this group of binding sites so that a broad range of leads can be created as basis for a further drug development.