Η χ-κωνοτοξίνη MrIA, που απομονώθηκε από το δηλητήριο του θαλάσσιου σαλιγκαριού-κώνου Conus Marmoreus, είναι ένα 13-αμινοξέων πεπτίδιο (Asn1-Gly2-Val3-Cys4-Cys5-Gly6-Tyr7-Lys8–Leu9-Cys10-His11-HyP12-Cys13-OH) με δύο δισουλφιδικούς δεσμούς μοτίβου Cys1-Cys4 και Cys2-Cys3. Η κωνοτοξίνη αυτή στοχεύει εκλεκτικά τον μεταφορέα την νορεπινεφρίνης (ΝΕΤ) και αποτελεί πιθανό φάρμακο ως αναλγητικό. Η φαρμακοφόρος ομάδα αποτελείται από τη γ-στροφή που σχηματίζεται από το βρόγχο μεταξύ των Cys στις θέσεις 5 και 10 και από τα ενδιάμεσα αμινοξέα αυτών. Το αμιδιωμένο C-τελικό παράγωγο στο οποίο έχει πραγματοποιηθεί αντικατάσταση της Asn1 με pGlu είναι σήμερα υποψήφιο φάρμακο κατά του νευροπαθητικού πόνου, με ονομασία Xen2174. Οι μελέτες που έχουν ασχοληθεί με τη Ν-τελική περιοχή του χ-MrIA έχουν δείξει πως η Val3, σε αντίθεση με τα δύο πρώτα αμινοξέα της αλληλουχίας, (Asn1, Gly2), παίζει σημαντικό δομικό ρόλο. Σκοπός της διατριβής αυτής είναι η σύγκριση συνθετικών μεθόδων παραλαβής του φυσικού πεπτιδίου χ-MrIA-ΝΗ2 καθώς και ο σχεδιασμός και η σύνθεση αναλόγων του με τροποποιήσεις στην Ν-τελική αλληλουχία. Επίσης συντέθηκε ένα ανάλογο με Ala στη θέση 6 και ένα ανάλογο στο οποίο οι Cys4 και Cys13 έχουν αντικατασταθεί από Glu4 και Lys13 αντίστοιχα Η σύνθεση των αναλόγων πραγματοποιήθηκε με την Fmoc/tBu μεθοδολογία επί στερεάς φάσεως, χρησιμοποιώντας ως στερεό υπόστρωμα την Sieber Amide ρητίνη ή την Rink Amide MBHA ρητίνη. Σαν αντιδραστήρια σύζευξης χρησιμοποιήθηκαν α) DIC/HOBt, β) TBTU/DIEA/HOBt, γ) ΗBTU/DIEA/HOBt, δ) HATU/DIEA. Η δημιουργία των δισουλφιδικών δεσμών έγινε σε δύο στάδια με οξείδωση με DMSO ή / και με οξείδωση με I2/AcOH Τα πειραματικά αποτελέσματα έδειξαν πως καλύτερες αποδόσεις και καθαρότερα προϊόντα επιτυγχάνονται όταν η επιμήκυνση της πεπτιδικής αλυσίδας γίνεται σε υπόστρωμα Sieber Amide, οι συζεύξεις των αμινοξέων με τη χρήση ουρονικών παραγώγων και η οξείδωση με τη μέθοδο I2/AcOH. Η δομή επιλεκτικών αναλόγων μελετήθηκε με πειράματα πυρηνικού μαγνητικού συντονισμού (NMR). / The χ-conopeptide MrIA isolated from the venom of the cone snail Conus Marmoreus, is a 13-residue peptide with two disulfide bonds forming pairs Cys1-Cys4 and Cys2-Cys3. This conotoxin targets selectively the norepinephrine transporter (NET) and is a potent analgesic. The pharmacophore group consists of an inverse γ-turn formed by the first loop Cys2-Cys3 and the residues located between them. The amidated C-terminal derivative bearing the replacement of Asn1 by pGlu led to the potent drug against neuropathetic pain, Xen2174. However, not much is known about the N-terminal region of χ-MrIA. Studies have shown that Val3 has an important structural role, as opposed to the first two amino acids of the peptide sequence. The aim of this research is the design and synthesis of analogs of χ-MrIA-NH2 with substitutions at residues Asn1, Gly2, Val3, Gly6 as well as Cys4 and Cys13 of the peptide sequence and the comparison of these synthetic methods. These analogues were synthesized by solid peptide synthesis, using the Fmoc/tBu methodology. Sieber Amide resin and Rink Amide MBHA resin were used as solid support and a) DIC/HOBt, b) TBTU/DIEA/HOBt, c) TBTU/DIEA/HOBt, d) HATU/DIEA as coupling reagents. The formation of disulfide bonds was achieved in two steps by oxidation with DMSO and / or oxidation by iodine. We concluded that the preferred method for this peptide synthesis is Sieber Amide as solid support and uronic derivatives as coupling reagents, while for the formation of disulfide bonds, the oxidation with iodine. Some of these analogs are studied by NMR spectroscopy for structure determination.

Οι Κωνοτοξίνες είναι μικρά πεπτίδια πλούσια σε δισουλφιδικούς δεσμούς, τα οποία προέρχονται από τα δηλητήρια των θαλάσσιων σαλιγκαριών. Οι κωνοτοξίνες αυτές στοχεύουν διαφορετικούς υποδοχείς στο νευρικό σύστημα με υψηλή εκλεκτικότητα και ισχύ, αποτελώντας χρήσιμους φαρμακευτικούς δείκτες ή και φάρμακα. Μια ομάδα κωνοτοξινών είναι οι μ-κωνοτοξίνες, οι οποίες έχουν παρουσιάσει αναλγητική δράση. Οι μ-κωνοτοξίνες έχουν συγκεκριμένα δομικά χαρακτηριστικά πολύ σημαντικά για την βιολογική τους δραστικότητα. Περιέχουν τρείς δισουλφιδικούς δεσμούς μεταξύ των Cys1-Cys4, Cys2-Cys5 και Cys3-Cys6, σχηματίζοντας τρείς θηλιές (loop) στον σκελετό του πεπτιδίου. Οι μ-κωνοτοξίνες ερευνήθηκαν σαν πιθανά φαρμακευτικά εργαλεία εξαιτίας της ικανότητας τους να αναστέλλουν διαφορετικούς νευρικούς υποτύπους διαύλων Νατρίου και συνεπώς να στοχεύουν όχι μόνο κατά του χρόνιου πόνου αλλά και κατά άλλων ασθενειών. Στόχος της παρούσας διατριβής είναι η σύνθεση νέων μ-κωνοπεπτιδίων με πιθανή αναλγητική δράση, τα οποία στοχεύουν κυρίως σε πρωτεΐνες –κανάλια ιόντων Νατρίου, προκαλώντας αναστολή της μετάδοσης σήματος στα νευρικά κύτταρα. Η σύνθεση των αναλόγων πραγματοποιήθηκε με την Fmoc/But μεθοδολογία επί στερεάς φάσεως, χρησιμοποιώντας ως στερεό υπόστρωμα την Sieber Amide ρητίνη. Ο σχηματισμός των τριών δισουλφιδικών γεφυρών επιτεύχθηκε σε ένα στάδιο στην υγρή φάση με την χρήση ρυθμιστικών αναγωγικών μέσων. Στην παρούσα φάση, τα συντιθέμενα πεπτίδια βρίσκονται σε στάδιο βιολογικής αξιολόγησης μέσω ηλεκτροφυσιολογικών ανταγωνιστικών πειραμάτων. / Conotoxins are small disulfide rich peptides derived from the venom of Conus snails. They target different receptors in the nervous system with high selectivity and potency making them valuable as drug leads or drug themselves. One group of conotoxins, μ-conotoxins have been shown to have potential as analgesic treatment. μ-conotoxins have concrete conformational features, which are very crucial to their biological activity. They contain three disulfide bonds among Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6, forming three loops in their backbone. μ-conotoxins studied as potent pharmacological tools due to their ability to inhibit different neuronal subtypes of sodium channels and likewise conotoxins target not only the chronic pain treatment but also target against other disease’s treatment. The goal of this present thesis is to synthesize new μ-conopeptides, with a potent analgesic activity, which target mostly proteins-sodium channels, inhibiting the nerve impulse transmission at the neuromuscular injection. These analogues were synthesized using the Fmoc/But methodology by SPPS, using Sieber Amide resin as solid support. The formation of three disulfide bonds was accomplished in one step in the solution phase using the redox buffer strategy. At present, the synthesized cono-peptides are under biological evaluation through electrophysiological competitive experiments.

Major constituents of the venom of various animals are peptidogenic in nature. Marine snails belonging to the species Conus are venomous predators that use small, structurally constrained peptides present in their venom for prey capture and defense. It is known that ~500 Conus species are present in nature and the venom of each of these Conus species is a complex mixture of nearly 100 peptides accounting for > 50,000 peptides with little overlap among the different species. The peptides isolated from the venom of Conus species are commonly known as conotoxins or conopeptides. Some of the common targets of these peptides include the different ion channels like Na+, K+, and Ca2+, and receptor subtypes such as nicotinic acetylcholine and NMDA receptors. The ion channels and receptor subtypes were targeted by conopeptides with high degree of specificity and selectivity. The structural information on the peptides from cone snails can prove to be a valuable starting tool for the understanding of the function of different ion channels and hence in the design of neuropharmacologically active drugs. Conotoxins are disulfide-rich peptides and the number of disulfide generally ranges from two to five. Based on the arrangement of cysteines in their primary sequence, they are classified into different superfamilies. The signal sequences of the precursors belonging to a particular superfamily are highly conserved and hence the members within the same family have, in common, the unique disulfide arrangement and pharmacological activity. Conotoxins are classified into eleven superfamilies till date. In order to understand the underlying the principles involved in the action of these peptides on different ion channels, one needs to know the three-dimensional structures which, in potential, will help in the identification of the pharmacophores responsible for the observed pharmacological activity. With the aim of studying the structure-activity relationships found among the conotoxins, we have initiated a study on the peptides isolated from the marine snails found in the Indian coastal waters. This thesis is focused in the structural studies of the peptide toxins from marine cone snails and a terrestrial scorpion. The tool used for the structural studies of these peptide toxins is Nuclear Magnetic Resonance Spectroscopy. Chapter 1 provides an overview of the peptide toxins found among various animal species with more emphasis on conotoxins and scorpion toxins. In addition, the rationale behind the present study has also been explained. Chapter 2 describes the structure determination of two conopeptides isolated from Conus amadis, δ-Am2766 and Am2735, which are active on mammalian sodium channels. The structural aspects and comparison with other known conopeptides belonging to the same superfamily as that of these two peptides have also been described. Solution NMR studies of Ar1446 and Ar1248, two conopeptides isolated from the species Conus araneosus have also been studied using Homonuclear NMR methods. Ar1446 is a three disulfide-bonded peptide. Our studies have revealed that this peptide has a novel disulfide connectivity not previously observed in the M superfamily or any other superfamily of conotoxins. The structural features of Ar1446 will be described along with the NMR studies on two-disulfide bonded peptide, Ar1248, belonging to the A-superfamily of conotoxins. The main problem faced in the kind of study of peptides isolated from natural sources is the amount that can be isolated and purified to homogeneity. In order to obtain large quantities of peptides, we have successfully used Cytochrome b5 as fusion host to clone, over express and purify these peptides using recombinant methods. The use of recombinant methods has aided in the preparation of isotopically enriched peptides. The use of cyt b5 as fusion host for the large scale production of some of the peptides from Indian marine snails is described in Chapter 4. A novel pharmacologically active linear peptide, Mo1659 isolated from Conus monile, have been studied using Heteronuclear NMR methods. This peptide was cloned, over expressed and purified using Cytochrome b5 as a fusion host. Another linear peptide, Mo1692 (also from Conus monile), has been prepared using the same method and was studied using Homonuclear NMR methods. Both these peptides were liberated from the fusion host using cyanogen bromide cleavage and were subsequently purified using RP-HPLC. The results of the biosynthetic preparation and NMR studies of these two peptides have been described in Chapter 5. Chapter 6 describes the solution structure determination of a novel scorpion toxin characterized in the venom of the Indian red scorpion Buthus tamulus. The cloning, over expression, folding and purification of BTK-2 is described here. The structure and the function of this recombinantly produced BTK-2 will also be described.

Venomous animals have evolved a vast array of peptide toxins for prey capture and defense. Nature has evolved the venoms into a huge library of active molecules with high selectivity and affinity, which could be explored as therapeutics or serve as a template for drug design. The individual components of venom i.e. toxins are used in ion channel and receptor studies, drug discovery, and formulation of insecticides. ‘Venominformatics is a systematic bioinformatics approach in which classified, consolidated and cleaned venom data are stored into repositories and integrated with advanced bioinformatics tools and computational biology for the analysis of structure and function of toxins.’ Conus peptides (conopeptides), the main components of Conus venom, represent a unique arsenal of neuropharmacologically active molecules that have been evolutionarily tailored to afford unprecedented and exquisite selectivity for a wide variety of ion-channel subtypes and neuronal receptors. Ziconotide (ω-conotoxin MVIIa from Conus magus (Magician's cone snail)), is proven as an intrathecally administered N-type calcium channel antagonist for the treatment of chronic pain (U.S. Food and Drug Administration. Center for Drug Evaluation and Research) attesting to the pharmaceutical importance of Conus peptides. From the point of view of protein sequence and structure analysis, conopeptides can serve as attractive systems for the studies in sequence comparison, pattern extraction, structure–function correlations, protein–protein interactions and evolutionary analysis. Despite their importance and extensive experimental investigations on them, they have been hardly explored through in silico methods. The present thesis is perhaps the first attempt at deploying a multi-pronged bioinformatics approaches for studies in the burgeoning field of conopeptides. In the process of sequence-structure-function studies of conopeptides, we have created several sequence patterns of different conopeptide families and these have been accepted for inclusion in international databases such as PROSITE, the first pattern database to have been developed (http://www.expasy.org/prosite) and INTERPRO (http://www.ebi.ac.uk/interpro). More importantly, we have carried out extensive literature survey on the peptides for which we have defined the patterns to create PROSITE compatible documentation files (PDOC6004, PDOC60025 and PDOC60027). We have also created a series of sequence patterns and associated documentation filesof pharmaceutically promising peptides from plants and venomous animals (including O-conotoxin and P-conotoxin superfamily members) with knottin scaffold. Knottins provide appealing scaffolds for protein engineering and drug design due to their small size, high structural stability, strong sequence tolerance and easy access to chemical synthesis. The sequence patterns and associated documentation files created by us should be useful in protein family classification and functional annotation. Even though patterns might be useful at the family level, they may not always be adequate at the superfamily level due to hypervariability of mature toxins. In order to overcome this problem, we have demonstrated the applicationos of multi-class support vector machines (MC-SVMs) for the successful in silico classification of the mature conotoxins into their superfamilies. TheI- and J-conotoxin-superfamily members were analyzed in greater detail. On the basis of in silico analysis, we have divided the 28 entries previously grouped as I-conotoxin superfamily in UniProtKB/Swiss-Prot (release 49.0) into I1 and I2 superfamilies inview of their having two different types of signal peptides and exhibiting distinct functions. A comparative study of the theoretically modeled structure of ViTx from Conus virgo, a typical member of I2-conotoxin superfamily, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels. Putative complexes created by us of very recently characterized J-superfamily conotoxin p11-4a with Kv1.6 suggest that the peptide interacts with negatively charged extracellular loops and pore-mouth of the potassium channel and blocks the channel by covering the pore as a lid, akin to previously proposed blocking mechanism of kM-conotoxin RIIIK from Conus radiatus to Tsha1 potassium channel. This finding provides a pointer to experimental work to validate the observations made here. Based on differences in the number and distribution of the positively charged residues in other conopeptides from the J-superfamily, we hypothesize different selectivity profile against subtypes of the potassium channels for these conopeptides. Furthermore, the present thesis reports the application of order-statistic filters and hydrophobicity profiles for predicting the location of membrane-spanning helices. The Proposed method is in particular effective for the class of helical membrane proteins, namely the therapeutically important voltage-gated ion channels, which are natural targets of several conotoxins. Our suggested ab initio approach is comparatively better than other spatial filters, confirming to the efficacy of including the concept of order or ranking information for prediction of TM helicdes. Such approaches should be of value for improved prediction performance including in large-scale applications. In addition, anlaysis has been carried out of the role of context in the relationship between form and function for the true PDB hits of some nonCys-rich PROSITE patterns. We have found specific examples of true hits of some PROSITE patterns displaying structural plasticity by assuming significantly different local conformation, depending upon the context. The work was carried out as a part of the research interest in our group in studying structural and other features of protein sequence patterns. The Contributions of the candidate to venominormatics include, creation of protein sequence patterns and information highlighting the importance of the patterns as gleaned from the lteratures for family classification: profile HMM and MC-SVMs for conotoxin superfamily classification; in silico characterization of I1 and I2 conotoxin superfamilies; studies of interaction with Kv1 channels of typical members of I2 and 3 conotoxin superfamilies and development of improved methods for detecting membrane-spanning helices. Chapter I starts with a brief account of venominformatics; bioinformatics for venoms and toxins. Chapter 2 presents a regular expression based classification of Conus peptides. Chapter 3 revisits the 28 entries previously grouped as I-conotoxin superfamily in UniProt Swiss-Prot knowledgebase (release 49.0) having four disulfide bonds with Cys arrangement C-C-CC-CC-C-C and they inhibit or modify ion channels of nerve cells. Chapter 4 describes pseudo-amino acid composition and MC-SVMs approach for conotoxin superfamily classification. Chapter 5 describes in silico detection of binding mode with Kv1.6 channel of J-superfamily conotoxin p114a from bermivorouos cone snail, Conus planorbis. Chapter 6 presents a comparative sequence-structure-function analysis of naturally occurring Cys-rich peptides having the Knottin or inhibitor cystine knot(ICK) scaffold, from different plants and venomous animals based on information available in the knottin database(http://knottin.cbs.cnrs.fr/). Chapter 7 describes the application of order-statistic filters and hydrophobicity profiles for detecting membrane-spanning helices. Chapter 8 describes the role of context in the relationship between form and function for the true PDB hits of some non Cys-rich PROSITE patterns. Chapter 9 summaries the important findings of the present studies on naturally occurring bioactive Cys-rich peptides with emphasis on Conus peptides and their interactions with respective target such as voltage-gated ion channels.