Final Report Summary - PETOXSAR (Peptide-Toxins as probes for the Structure-Activity Relationship (SAR) investigation against the nicotinic acetylcholine receptors (nAChRs) subtypes)
The aim of this project was the use of several series of conotoxin analogues as probes for structure activities relationship (SAR) studies of biological important targets. Peptides are an efficient tool for SAR studies due to their benefit to easily synthesise a small library of analogues containing different characteristics. The results of such studies can lead to the design and synthesis of potentional pharmaceutical agents.
Rational design and syntheses of two different families of Conotoxins were performed during the past three years in the framework of Marie-Curie European Reintegration Grant. Analogues of a-Conotoxins were synthesised towards SAR studies of nicotinic acetylcholine receptors (nAChRs) subtypes that are important therapeutic targets for various diseases, including myasthenia gravis, Alzheimer's and Parkinson's diseases, schizophrenia, as well as for the cessation of smoking.
Furthermore, an additional objective was established during Marie Curie Reintegration Grant. Design and synthesis of novel small library of µ-Conotoxin analoques was performed in order to investigate the biological system of sodium ion channels that are targets against both chronic neuropathic and chronic inflammatory pain conditions. Drugs that block these channels may have therapeutic efficacy with doses that are far below those that impair nerve impulse propagation or cardiovascular function.
Twenty three rational designed rich disulfide bonds peptides were synthesised manually or by automated peptide synthesiser, oxidised, identified and purified. More specifically, 12 analogues of a-conotoxin that targets to nAchRs and eleven 11 analogues of µ-Conotoxin against sodium ion channels were produced chemically based on:
(i) the sequence alignment of the a- or µ- conotoxin families in combination to theirs specificity to various enzyme subtypes;
(ii) previous biological tested analogues already reported; and
(iii) computational methods.
Several techniques were studied in order to optimise the two (a-analogues) or three (µ-analogues) disulfide bond formation towards the enhanced yield and purity of the produced peptides. Specifically, random co-oxidation under highly optimised conditions were applied for µ-conotoxins analogues and disulfide bonds will be formed in one step oxidation, and selective stepwise oxidation methods were applied for the a-Conotoxins analogues. The previous experience of Dr Galanis was important for the successful production of the peptide analogues. Biological evaluation of the synthetic analogues is under progress and can shed light for the design and the synthesis of the next generation of analogues. Part of the results of Conotoxin analogues have been presented in a several conferences and the future perspective is to constitute a part of a peer-reviewed publication while all the synthetic peptides biological evaluation will be completed. Furthermore, partly results of biological studies will be a basis for collaboration with Greek research and development (R&D) company that will potentially fund the continuation of current project towards the design of new Conotoxin analogues.
Furthermore, the development of state-of-the-art methods and techniques for the synthesis of high rich disulfide bond a-neurotoxins and analogues has been attempted. The main goal of this concept was the application of the native chemical ligation reaction of peptide fragments with the disulfide bridges pre-formed. Studies of native chemical ligation of disulfides rich peptides were performed using small peptide models containing four cysteine residues. The peptide fragments were attempted to be engaged via chemical ligation after the formation of disulfide bonds of one fragment (the C-terminus). In conventional native chemical ligation reaction, the use of thiol agents is essential as catalysts and in terms of avoiding the oxidation of the - SH groups of potential Cys residues and of the - SH-group that participates onto the reaction. In our method, the use of thiol moieties were prohibited as the goal was to perform the reaction using pre-formed disulfide bonds of the two fragments. Thus, the use of elevated temperature chemical ligation of the peptide fragments in order to overcome the obstacle of the thiol moieties absence during the reaction was studied. Even after optimisation studies in terms of temperature, pH, time reaction and the use of microwave or conventional heating, poor yield results and many impurities were obtained. This part of the project was the most difficult and risky as development of new strategies was required. Hitherto, non-successful studies for chemical ligation of pre-formed disulfide bonded fragments have been reported. However, alternative techniques are under investigation in the Laboratory of Pharmacognocy and Chemistry of Natural products for the engagement of disulfide bridged peptide fragments.
Conclusively, two set of a- and µ-Conotoxins have been successfully synthesised by rational design and synthesis optimisation of disulfide bond formation strategies. These peptides will be evaluated biologically for their activity against two important biological systems as nAChRs and Na+ channels towards structure-activity studies. Furthermore, chemical ligation reaction preliminary studies of pre-formed disulfide bond peptide fragments have been performed, and further investigation is planned. These projects will lead in the near future to a couple of publications in peer reviewed journals.
Additionally, during Reintegration Grant Dr Galanis had the opportunity to complete a few last steps of previous project in collaboration with the two previous laboratories that he worked during his previous Marie Curie IEF grant, Dr Morten Grotli, University of Gothenburg, Sweden and Dr Fernando Albericio, University of Barcelona, Spain. In this frame, Dr Galanis had a short visit (one week) to Sweden for scientific discussion and experimental work. One more article was published in peer reviewed journal. Furthermore, Dr Galanis had the chance to complete another project that was in collaboration with the host institution, result in another peer reviewed article publication.
Rational design and syntheses of two different families of Conotoxins were performed during the past three years in the framework of Marie-Curie European Reintegration Grant. Analogues of a-Conotoxins were synthesised towards SAR studies of nicotinic acetylcholine receptors (nAChRs) subtypes that are important therapeutic targets for various diseases, including myasthenia gravis, Alzheimer's and Parkinson's diseases, schizophrenia, as well as for the cessation of smoking.
Furthermore, an additional objective was established during Marie Curie Reintegration Grant. Design and synthesis of novel small library of µ-Conotoxin analoques was performed in order to investigate the biological system of sodium ion channels that are targets against both chronic neuropathic and chronic inflammatory pain conditions. Drugs that block these channels may have therapeutic efficacy with doses that are far below those that impair nerve impulse propagation or cardiovascular function.
Twenty three rational designed rich disulfide bonds peptides were synthesised manually or by automated peptide synthesiser, oxidised, identified and purified. More specifically, 12 analogues of a-conotoxin that targets to nAchRs and eleven 11 analogues of µ-Conotoxin against sodium ion channels were produced chemically based on:
(i) the sequence alignment of the a- or µ- conotoxin families in combination to theirs specificity to various enzyme subtypes;
(ii) previous biological tested analogues already reported; and
(iii) computational methods.
Several techniques were studied in order to optimise the two (a-analogues) or three (µ-analogues) disulfide bond formation towards the enhanced yield and purity of the produced peptides. Specifically, random co-oxidation under highly optimised conditions were applied for µ-conotoxins analogues and disulfide bonds will be formed in one step oxidation, and selective stepwise oxidation methods were applied for the a-Conotoxins analogues. The previous experience of Dr Galanis was important for the successful production of the peptide analogues. Biological evaluation of the synthetic analogues is under progress and can shed light for the design and the synthesis of the next generation of analogues. Part of the results of Conotoxin analogues have been presented in a several conferences and the future perspective is to constitute a part of a peer-reviewed publication while all the synthetic peptides biological evaluation will be completed. Furthermore, partly results of biological studies will be a basis for collaboration with Greek research and development (R&D) company that will potentially fund the continuation of current project towards the design of new Conotoxin analogues.
Furthermore, the development of state-of-the-art methods and techniques for the synthesis of high rich disulfide bond a-neurotoxins and analogues has been attempted. The main goal of this concept was the application of the native chemical ligation reaction of peptide fragments with the disulfide bridges pre-formed. Studies of native chemical ligation of disulfides rich peptides were performed using small peptide models containing four cysteine residues. The peptide fragments were attempted to be engaged via chemical ligation after the formation of disulfide bonds of one fragment (the C-terminus). In conventional native chemical ligation reaction, the use of thiol agents is essential as catalysts and in terms of avoiding the oxidation of the - SH groups of potential Cys residues and of the - SH-group that participates onto the reaction. In our method, the use of thiol moieties were prohibited as the goal was to perform the reaction using pre-formed disulfide bonds of the two fragments. Thus, the use of elevated temperature chemical ligation of the peptide fragments in order to overcome the obstacle of the thiol moieties absence during the reaction was studied. Even after optimisation studies in terms of temperature, pH, time reaction and the use of microwave or conventional heating, poor yield results and many impurities were obtained. This part of the project was the most difficult and risky as development of new strategies was required. Hitherto, non-successful studies for chemical ligation of pre-formed disulfide bonded fragments have been reported. However, alternative techniques are under investigation in the Laboratory of Pharmacognocy and Chemistry of Natural products for the engagement of disulfide bridged peptide fragments.
Conclusively, two set of a- and µ-Conotoxins have been successfully synthesised by rational design and synthesis optimisation of disulfide bond formation strategies. These peptides will be evaluated biologically for their activity against two important biological systems as nAChRs and Na+ channels towards structure-activity studies. Furthermore, chemical ligation reaction preliminary studies of pre-formed disulfide bond peptide fragments have been performed, and further investigation is planned. These projects will lead in the near future to a couple of publications in peer reviewed journals.
Additionally, during Reintegration Grant Dr Galanis had the opportunity to complete a few last steps of previous project in collaboration with the two previous laboratories that he worked during his previous Marie Curie IEF grant, Dr Morten Grotli, University of Gothenburg, Sweden and Dr Fernando Albericio, University of Barcelona, Spain. In this frame, Dr Galanis had a short visit (one week) to Sweden for scientific discussion and experimental work. One more article was published in peer reviewed journal. Furthermore, Dr Galanis had the chance to complete another project that was in collaboration with the host institution, result in another peer reviewed article publication.