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Selective ligands of nicotinic acetylcholine receptor subtypes designed on the basis of X-ray structures of complexes of acetylcholine-binding proteins with agonists and antagonists

Ziel

The major objective of this project is to obtain an accurate insight into the organization of ligand-binding sites in pharmacologically distinct nAChR subtypes and on this basis design novel compounds with increased affinity and selectivity for identification and precise quantification of different nAChRs at neurodegenerative and psychiatric diseases. Comprehensive testing of biological activity of the designed and synthesized compounds will also provide the framework for developing new agonists and antagonists of nAChRs for medical applications. The project stems from our results on the X-ray structures of acetylcholine-binding proteins (AChBP), which are unique high-resolution models of ligand-binding domains of nAChRs, both in free state and in the complexes with agonists and antagonists.

The latter include a-conotoxins, and the X-ray structure of an AChBP complex with a-conotoxin is a corner stone of the project. This AChBP structure, and recent results on AChBP from other laboratories e.g. on the Torpedo nAChR, will serve as a basis for modelling the extra-cellular domains of distinct neuronal nAChRs. With the aid of docking and molecular dynamics simulations novel a-conotoxins will be designed with the expected higher selectivity and potency. The series of such analogues, directed against homooligomeric (a7) or heteromeric (a3ß2,a4ß2 ) nAChRs will be prepared by solid-phase peptide synthesis, and their activity will be tested both against AChBPs and diverse nAChRs. This will be performed on the isolated proteins and respective cell lines using radioligand analysis, isothermal titration calorimetry and surface plasmon resonance. The most crucial for the assessment of potency and selectivity of the obtained compounds will be their analysis using electrophysiology experiments on a panel of neuronal nAChRs heterologously expressed in the Xenopus oocytes.

Experimental data on the alpha-conotoxin interaction with different nAChRs, naturally occurring and mutated AChBPs will be used in subsequent new rounds of design and synthesis of more potent and selective analogues. New synthetic a-conotoxins which will show the desired potency and selectivity will be also crystallized with AChBPs to extend our knowledge on the a-conotoxin-receptor recognition and, additionally, to verify our designing approach. For a-conotoxin analogues, satisfying the criteria of high affinity and selectivity, the respective labelled forms (radioactive, fluorescent, biotinylated etc.) will be obtained for subsequent studies. The project, which combines the approaches of protein and peptide chemistry, structural biology and modern electrophysiology involves a continuation of the already established successful collaboration of the four groups with the respective expertise reflected in recent publications.

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Letzte Aktualisierung: 16 März 2017

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Permalink: https://cordis.europa.eu/project/id/INTAS2005-1000008-7919/de

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