Molecular Mechanisms of Ligand-Receptor Interaction
Molecular Mechanisms Determining the Specificity of Interaction of Three-Finger Neurotoxins with Acetylcholine Receptors
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- MED-DRG/Drug Discovery/Medicine
3 Approved without Funding
Institute of Bioorganic Chemistry, Russia, Moscow
- Freie Universität Berlin, Germany, Berlin\nBarrow Neurological Institute, USA, AZ, Phoenix
Project summaryThe project goal is to study the molecular mechanisms underlying regulation of ligand-gated ion channels on the example of nicotinic acetylcholine receptor (nAChR) and snake neurotoxins. Special attention will be paid to the determination of structural elements participating in ligand-receptor interactions.
The complex investigation will involve experimental methods of gene and protein engineering and NMR spectroscopy, as well as theoretical methods of molecular modeling and molecular dynamics. The main objects of the proposed study are snake neurotoxins of three different classes selective for different subtypes of nAChRs: short chain neurotoxin II from Naja oxiana venom (effective inhibitor of muscle type nAChR), recombinant analogs of long chain alpha-neurotoxins from Naja oxiana (effective inhibitor of both muscle and neuronal alpha7 nAChRs) and weak toxin from Naja kaouthia (“weak” inhibitor of both muscle and neuronal alpha7 nAChRs).
High-level expression systems for production of recombinant neurotoxins and extracellular domain of nAChR (developed by the authors) permit to produce isotope-labeled analogues of these proteins for subsequent NMR investigation. Modern NMR techniques will allow us to study the spatial structures of neurotoxins and extracellular domain of nAChR, as well as to delineate the intermolecular interactions at atomic level. In the project course the amino acid residues mediating ligand-receptor interactions will be revealed. This knowledge should give clues to understanding the mechanisms of interaction between the pharmacologically different subtypes of nAChRs and other members of ‘cys-loop’ membrane receptors family (5-HT3 serotonin, GABA-A and glycine receptors) and their antagonists.
The proposed project includes the following milestones:
- optimization of bacterial expression systems for producing of “weak” neurotoxin, isotope-labeled analogues of extracellular domain of nAChR, neurotoxins and their mutants;
- identification and comparison of structural determinants of various types of neurotoxins (long and short chain alpha-neurotoxins, “weak” neurotoxin), important for interaction with different types of nAChR in order to establish relationships between the types of observed activities and the toxins structures;
- identification of the changes in the spatial structure of neurotoxins and extracellular nAChR domain caused by ligand-receptor interaction;
- modeling the interaction between neurotoxins and nAChRs and revealing the mechanisms underlying regulation of ligand-gated ion channels.
The results of the project will be important not only as a fundamental knowledge, but, in perspective, will be useful for elaboration of a new-type biopharmaceuticals. Based on the obtained data we will suggest the algorithm of creation of synthetic selective inhibitors for different nAChR types. These synthetic inhibitors of nAChRs could be used as drug prototypes for the treatment of a number of nervous diseases, including Parkinsonism, Alzheimer disease and nicotinic addiction.
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