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Master’s in Proteomics and Bioinformatics

2007

Synthesis and improvement of peptides isolated from the venom of cone snails

presented by

Anne Zufferey

Master’s thesis director Prof. Keith Rose

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ACKNOWLEDGEMENTS First of all, I would like to thank very much Prof. Keith Rose for giving me the opportunity to carry out my master’s internship in his research group in the department of Structural Biology and Bioinformatics. I am very grateful for his exceptional availability and his precious advice concerning both my practical work and the continuation of my scientific formation. Thank you to Dr. Jean Vizzavona who directed me during this practical training and taught me a lot about peptide chemistry and the general lab work. I am also grateful to him for his help during the writing of this report. Special thanks go to Priscille Giron, Brigitte Dufour, Nikolett Mihala and Oscar Vadas for their precious help, their support and especially their warm welcome. I am also thankful to Dr. Ron Hogg, from Prof. Bertrand’s group in the department of Basic Neurosciences for always finding time to answer my quesations and for showing me how to perform bioassays.

Finally, I would like to thank my family, my partner and my friends for their precious support.

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TABLE OF CONTENTS

ABSTRACT________________________________________________________________1

I. INTRODUCTION ______________________________________________________ 2

A. THE PROJECT ___________________________________________________________ 2

B. MULTIVALENCY_________________________________________________________ 3

C. VENOMICS AND ITS APPLICATIONS ______________________________________ 5 Conotoxin __________________________________________________________________________ 7

D. NICOTINIC ACETHYLCHOLINE RECEPTOR ______________________________ 12

II. MATERIALS AND METHODS ________________________________________ 16

III. RESULTS AND DISCUSSION_________________________________________ 19 α-conotoxin MII synthesis_____________________________________________________________ 19 Refolding of the reduced toxin _________________________________________________________ 21 Aminooxyacetylation of the peptide _____________________________________________________ 23 Linker synthesis_____________________________________________________________________ 25 Oximation _________________________________________________________________________ 28 Bioassays __________________________________________________________________________ 32

IV. CONCLUSION AND PERSPECTIVES ___________________________________ 35

V. REFERENCES _______________________________________________________ 36

VI. ABBREVIATIONS ____________________________________________________ 38

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ABSTRACT In order to develop new molecular tools and a new drug generation, venoms are largely studied as a fantastic source of highly specific “starting compounds”. In that context, a European project, named CONCO, has been settled. This project aims to identify peptide toxins of potential therapeutic interest and involves about 20 European and US labs work in consortium. Here, two different dimers of a particular toxin, α-conotoxin MII, were produced. This small toxin comes from cone snail Conus magus venom and specifically inhibits the human α3β2 nicotinic acetylcholine receptor (nAChR) subtype. The dimers are composed of an artificially synthesised conotoxin linked by a chemoselective oxime bond to a manually made polyamide spacer (or linker). There are two different kinds of dimer according to the length of the linker : a small one of 50 Ǻ and a big one of 108 Ǻ. Two controls are also produced : the native refolded conotoxin (the positive control) and a control compound, a “monomer” constituted of only one toxin linked to the spacer. All these compounds are tested on nAChR expressed in Xenopus oocytes, in collaboration with Dr. R. Hogg, from Prof. Bertrand’s group in the department of Basic Neurosciences.

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I. INTRODUCTION Knowledge in the field of molecular biology and biochemistry concerning biological and physiological processes has never been so precise and so rich. This knowledge leads to new questions and necessitates new tools to go always deeper in experiments, localisation, mechanism and inhibition of biological reactions. It is thus possible to make very fine discoveries in diseases (metabolic or/and genetic) and to define not even new drug targets, but also to design new drugs by molecular experiments or bioinformatic Docking. Chemical, biological and bioinformatic technologies allow us to improve molecules, via new functional groups or small structural modifications. In this context, the most difficult task is to find a starting molecule or small protein. Nature offers a large variety of starting molecules, often highly specialized. Some of these molecules have always been used by human beings for their medicinal or toxic properties, such as sage leaves against sore throat used in our countries or coca leaves chewed by Incas to relax after daily efforts. More recently, penicillin has been isolated from a fungus and other antibiotics correspond to improvements of this starting molecule. Now, for the first time, we are able to screen natural sources of bioactive compounds in a higher-throughput manner, to discover new drugs and to build new molecular tools aiming to answer new questions. These resources are almost inexhaustible and open new ways for treatment and diagnosis. Among these natural sources of molecules, venoms correspond to one of the most complex and specialized mixtures and we are just starting their study.

A. THE PROJECT Venoms contain a very complex mixture of polypeptides which have evolved in Nature to have a rapid and powerful biological effect. Some of their components are used today as therapeutics (for example Prialt® against severe chronic pain). For this reason, projects have started to discover more of these potential therapeutics. CONCO* is the name of a large project supported by the European Union under Framework Program 6. In this project, 20 scientific partners have formed a consortium to work principally on a cone snail species Conus consors. The project aims to better understand the action of its venom and characterize as many as possible of the bioactive compounds using genomic, transcriptomic and proteomic approaches. It is sometimes possible to improve Nature by introducing various modifications or through dimerization. CONCO aims to select, develop and optimize drug candidates discovered by genomic, transcriptomic, proteomic and functional screening approaches. Firstly, a “natural library” will be built based on this analytical information. In order to obtain sufficient material for biological testing, the identified components will be synthesized to construct a “synthetic library” of peptides by chemical or recombinant synthesis. A large scale protein-protein interaction study will also be made as well as 3D structure and in vitro, ex vivo and in vivo screening assays on medical relevant models. Candidates of interest will be optimized including by computer-assisted molecular design or structural and functional studies, and driven to the first phase of clinical study at least. Finally, a public database will be made available cost-free on the web. In this context, Prof. K. Rose’s group and Prof. D. Bertrand’s group contribute to the large scale chemical synthesis of bioactive compounds and to the functional characterization of isolated peptides on selected ion channel properties.

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My project involves attempts to improve the activity of a known venom peptide isolated from a Conus snail. It consists in synthesis of dimers linked by a spacer (also named linker) of two different sizes. The dimerized peptide is the α-conotoxin MII, a small peptide isolated from venom of Conus magus and which inhibits a certain nicotinic acethylcholine receptor (nAChR) subtype, α2β3 (see below). The peptide is produced by solid phase synthesis using Boc chemistry. The spacers are polyamide linkers manually synthesized. The smaller one is around 50 Ǻ long and the bigger about 108 Ǻ long. Finally the ligation between linker and toxin is made by a chemoselective reaction, the Oximation. Two control molecules are also prepared : the native conotoxin and a “monomer” corresponding to one toxin linked to a spacer. All these compounds are tested by Prof. Bertrand’s group on nAChR expressed in Xenopus laevis oocytes. *CONCO : Applied venomics of the cone snail species Conus consors for the accelerated, cheaper, safer and more ethical production of innovative biomedical drugs

B. MULTIVALENCY Valency is the number of independent and local interactions between two microscopic entities, from atoms to cells. Indeed a multivalent structure is able to connect to another through multiple separate connexions and to form a tight binding based on an amount of weak protein-ligand interactions. [1] Multivalency is often used by Nature to reinforce interactions or make them more specific. [2] In the case of proteins, it permits to increase the avidity of a ligand for its target, as if the ensemble of weak connexions was able to cooperate, for instance avidity of IgG which presents only two binding sites can be weaker than in the case of IgM, which interacts using ten sites. A thermodynamic model based on free energy has been build by P.I Kitov et al. to measure avidity in multivalent binding ∆Gavidity, in the absence of ligand aggregation. [1] Actually ∆Gavidity corresponds to a measure of the total entropy of both the system and the external medium, indicating if the interaction between the receptor and the ligand is favourable or not. Regarding multivalent sites with identical binding properties, avidity binding energy is due to three major elements :

1. Intrinsic free binding energy of initial biomolecular reaction of anchoring to a receptor by a first single interaction site : the first local interaction between the receptor and the ligand can be more or less favourable.

2. Intrinsic free binding energy for intramolecular binding of ligand sites to the remaining binding sites on the receptor surface : once the first local interaction is made, the others have an anchoring point which promotes their interaction.

3. A combinatorial factor reflecting the probability of association and dissociation of individual sites : each interaction depends on particular chemical and physical features and thus have their own probability of association and dissociation.

Binding mechanism is considered by the third element; actually it statistically influences avidity, increasing nonlinearly with multivalency degree. This model takes also into account an entropic driving force which acts on this kind of interaction without any constraint of the loss of conformational entropy due to the use of multiple binding sites, each interaction inducing a local molecular rearrangement. This is a measure of the disorder in the distribution of all the distinct complexes and it favours multivalent interactions between receptor and ligand (logarithmically) depending on the valency degree. So ∆Gavidity represents all

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thermodynamic enthalpic and entropic parameters of a multivalent interaction, even if there is a dependence of avidity on the density and arrangement of binding sites on the molecule surface and it allows the prediction of activity of this kind of protein on a receptor. These elementary parameters provide a basis for understanding the cooperation between the interacting sites. In the case of an interaction using 2 binding sites between receptor and ligand, the small avidity gain derives from the intramolecular free energy which corresponds to the free energy of all the interactions except the energy from the single initial binding event. By contrast, an interaction implying 10 binding sites can make significant gain of avidity entropy, even when individual interactions contribute very little. This knowledge allows us to build high avidity molecules using multivalent binding. For instance, peptabody is a pentameric recombinant protein made up of three different parts [3]: a ligand selected by screening of a large phage library on a given target, a semi-rigid hinge region and a monomer which comes from cartilage oligomeric matrix protein (COMP). Phage display allows to obtain large sequence diversity and to produce a minimal binding domain, where primary structure information is sufficient for recognition. The hinge region favours the cooperative binding of peptide “heads” (binding units). Multivalency comes from COMP part which is spontaneously associated in homopentamers, forming compact five-stranded α-helical bundle. This third part is also responsible of a remarkable solubility in free-salt water and thermostability, which are important features of bioassay tool. Here, the authors have produced Pab-S, a specific peptabody for the surface Ig idiotype of the B cell lymphoma in mouse (BCL). They built it using a fusion gene including the phage display ligand, a hinge region from camel IgG and the assembly domain from COMP and tranfected in E. Coli. Equilibrium binding studies showed that Pab-S has an avidity for IgM of BCL cells 2.105 higher than free peptide, which is the result of cooperating multiple simultaneous low affinity interactions.

Similar results have been obtained by R.H. Kramer et al. [4]. They synthesized polymer-linked ligand dimers (PLD) constituted by two cyclic GMP (cGMP) moieties linked by a PEG of different lengths. Then they tested them on a protein allosterically activated by cGMP, cyclic-nucleotide-gated channels (CNG channels) in vertabrate photoreceptor and olfactory neurons. This protein contains 4 cGMP binding sites. Their results obtained on patched rat olfactory or bovine photoreceptor CNG channels show the essential importance of the linker length on the avidity of the dimer. When the average PLD length matches the distance between binding sites on the target the avidity increases dramatically. Actually once one cGMP is bound, the diffusion of the other decreases and is constrained in a smaller distance which is equal to the length of PLD. Being closer to another site, its binding probability increases and the effective concentration decreases. This enhances the overall avidity.

Figure 1 Space-filling (Left) and ribbon (Right) representations of a model of the three-dimensional structure of Pab-S. Binding peptides are in red.Five shaded circles (radius of 40 Å) under the ribbon struc-ture schematically denote receptor mole-cules. From Terskikh et al.

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If the linker is “too long”, the increase is less marked (one “dilutes” the second binder). For a linker which is “too short” to span the binding sites, the avidity is less favorable (fig. 2)

PLD probably simultaneously occupies 2 binding sites on the channel. This is supported by the measure of dissociation constant. Indeed the channel remains open for minutes, even in conditions of continuous superfusion with agonist-free solution. Actually when one of the other binding moieties dissociates, it is held in proximity by the four (in the case of the peptabody) remaining binding units and so has a high probability of rebinding before its partners all dissociate, mostly in the medium linker case which corresponds to the ideal PEG length. This is also supported by the fact that the addition of free ligand increases the dissociation of bound PLD. So it is possible to screen the best linker length according to the multivalent target by testing a variety of PEG sizes. Note that even if this increase is progressive and begins before the optimal length because of elasticity of PLD (PEG) and possibly of elasticity of the target, this process provides also information on target binding site and can be used to build very effective antagonists, given the appropriate ligand and linker size.

Considering these examples, it is possible to improve the avidity, and so the activity, of a given ligand for its receptor provided that the receptor has several accessible binding sites. Based on this observation, the application of these properties in my project is to increase the avidity of a venom toxin for its receptor by dimerization.

C. VENOMICS AND ITS APPLICATIONS

In Nature, many organisms, from bacteria to mammals or plants, are able to produce venoms. Venom toxins present a biological action, such as pain, inability to move, insensitiveness or even death and can be either peptides either non-peptides. Venom serves to kill a prey, to digest it or to defend against a predator. In the animal kingdom, venom consists in a particular amount of more than a hundred natural bioactive substances produced mostly by a venom-producing gland and injected in prey or in aggressor through a system of injection-like hollow teeth, stings, harpoons or nematocyst tubule. These organs form the envenomation apparatus (fig 3). Venom contains also protease inhibitors and stabilizing agents. It presents various pharmacologies according to its composition. There is a relatively small number of toxin frameworks responsible for potency and stability. Their stability against chemical degradation in solution, at ambient temperature or by enzymatic process is ensured by post-translational modifications (PTM) such as carboxy-terminal amidation, glycosylation or hydroxylation of certain residues and conserved di-sulfide bonds. Actually cysteine patterns are highly conserved among classes of toxins, but there is a hypervariability between cysteine residues, which is involved in the specificity of the toxin for its target. These features make them very effective. So they are very promising

Figure 2 Diagrams of PLDs binding to a channel with four ligand-binding sites: PLDs are shown with polymers whose average length is too short (left), just right (centre), or longer (right) than necessary, to allow the ligands to span two binding sites on the channel. From Kramer et al.

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leads for new drugs and biological tools; indeed they present main characteristics for biomedical use : structure stability, target specificity and small size. [5]

Around ten million people in US suffer chronic pain which started by an injury, spinal disk problem or disease like diabetes. Then the suffering persists for weeks, months or even years. Sometimes a patient presents a partial response to traditional drugs which usually produce a large range of side effects, like sedation, nausea and cognitive changes. It is therefore necessary to find and develop new medicinal candidates, for instance based on toxins. Another useful aspect of venom for medicine is that peptide toxins are usually able to escape the immune system (including after their degradation in amino-acids) and so to limit side reactions, they cannot cross epithelial layers, including blood-brain barrier and their bioavailability is generally poor because of their hydrophilic nature and they are too big for spreading through tissues. Thus they have to be locally administered, for instance by intravenous, intramuscular, subcutaneous or epidural injection. In the case of a target both centrally and peripherally, this local administration way decreases risk of side effects. [6] There are three approaches for identification and characterization of peptides of interest. First, an assay-assisted fractionation can be made based on a given biological effect on crude venom, allowing some improvements for a high-throughput application like better separation and finally sequencing techniques. However, it identifies only the major activities. More recently, transcripomics and proteomics with identification by mass spectrometry allows to avoid assay bias. Finally, chemical synthesis confirms bioactivity and permits further characterizations across multiple targets, both in vivo and in vitro. [6] In all cases, therapeutic use of identified peptides implies as mentioned previously a certain level of stability and selectivity, and moreover its formulation, the mechanism of action knowledge and a low (or at least profitable) cost of production. These parameters make venom use in the medical field more complex. Thus projects concerning venoms aim to give 4 levels of information. Firstly, characterization of conserved cysteine patterns makes easier identification of all proteins or genome domains with the same structural signature. Because toxins seem to use structural signature to recognize their physiological targets, it is possible to find several candidates with a given recognition function which correspond to homologs of potential therapeutic value in different genomes. Secondly, these folds are shared by non-toxic proteins or peptides. These structural similarities give insights on an evolutionary relationship. Thirdly, discrimination between genes involved in cell processes and in venom production, such as PTM production, may

Figure 3 Cone snail, sea anemone, snake, Gila monster, spider and scorpion produce venom peptides with therapeutic potential. All groups are widely distributed in tropical to temperate regions, except the Gila monster which is found from south-western United States to central America. The envenomation apparatus are high-lighted (harpoon, nematocysys, teeth and sting). From Lewis et al.

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serve for development of artificial machinery to build highly PTM modified peptides or proteins. Finally, these studies give information on the animal itself and its physiology. Venom knowledge allows also to develop improved protection against envenomation. Actually, genomic and proteomic studies of toxins help to generate new concepts, like identification of widespread generic structural patterns, useful for design of new immunogens for antivenom production. Agents responsible of resistance of venomous animals against their own toxins, such as inhibiting or neutralizing substances, can also be isolated. In research, several receptors do not have specific agonist or antagonist needed for their study. Venom opens a field of potential specific ligands for these targets. For instance, mecanosensitive channels had non-specific known blockers only until the discovery by Otrow et al. of a toxin isolated from the venom of a tarantula, Gammastola spatulata.[7] This toxin, named GsMTx4, specifically inhibits the mecanosensitive channels of astrocytes and kidney cells, constituting a new tool for biochemical and electrophysiological studies of this medically important channel. The identification of an unknown sensory activator has been made by Siemens et al. [8] using a screening of twenty-two spider and scorpion venoms which produce pain. This assay-assisted fractionation of crude venoms is based on activation of capsaicin receptor (TRPV1), an excitatory channel expressed by sensory neurons of the pain pathway. A robust and reproducible signal has been observed on the TRPV1-expressing HEK-293 cell with the crude venom of a tarantula native to the West Indies, Psalmopeus cambridgei. After HPLC purification and Edman sequencing, three related peptides have been characterized and named Vanillotoxin 1, 2 and 3 (VaTx-1, 2 and 3). These toxins are members of ICK (inhibitor cysteine knot) family of spider and cone venom peptides, which are blockers of cationic channels. This family presents a conserved fold of three di-sulfide bonds and a hypervariable β-hairpin turn which confers its target specificity. The three Vanillotoxins are the first specific agonists for TRPV1 ion-channels and represent a useful new tool for understanding the mechanisms of TRP1-channels gating. The most successful venomic project is the development of Prialt®. This analgesic is a synthetic version of a ω-conotoxin (MVIIA) isolated from the cone specie Conus magus. This artificial toxin, named ziconotide or SNX-111, specifically inhibits a particular human voltage-gated channel, the N-type Ca2+ channel. This channel is highly involved in the pain pathway, inducing nocimodulators and pronociceptive neurotransmitters release by sensory neurons in the spinal cord. This drug is used in case of chronic and acute severe pain, such as respectively postsurgical setting or cancer. It is efficient in patients who have no response to opioid drugs. The only listed side effect is hypotension, probably due to Prialt® block of calcium channels on neurons which regulate blood pressure. Some of the side effects result also from the necessity of spinal injection of the treatment : it allows a spread limitation of the drug. [9] Prialt® is one of the first conotoxins derived medical treatment on the market, but some other artificial peptides, which are even more specific, are being tested at this moment. They offer a new therapeutic tool relieve suffering patients. Conotoxin All around the world, there are more than 500 cone species. These marine molluscs are carnivorous and use venom to kill their prey, from fish to marine worms. Cones inject toxins during hunting using a venom apparatus. It is constituted of a gland linked to a hollow tooth (Fig. 4) which looks like a small harpoon.

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Venoms of cone snails are among the most complex mixtures of toxins in the animal kingdom. Each venom consists in a large number of functionally different toxins (Fig. 5) and other biologically active compounds, such as stabilizing agents. Among more than 50 000 different conopeptides, only less than 0.1% is pharmacologically characterized. [10]

In general, conotoxins are small peptides of 10 to 35 residues, often disulfide bonds rich and presenting a highly specific effect on various receptors involved in pain pathway and voltage or ligand-gated ion channels crutial for hormone secretion, nerve transmission or cellular energy generation. [11] Toxins are made from a precursor sequence. Even if sequences between cysteine residues are highly variable, the signal sequence in the toxin precursor is particularly conserved across the phyla. Actually, these molluscs present a particular mechanism for introducing rapid sequence changes specifically between the cysteins, based on switching loops at the gene level (Fig. 6). This mechanism guarantees to keep the same disulfide bond pattern, while increasing variability into a given family. [12]

Figure 4 The tip of the harpoon-like tooth of Conus obscurus. The barbed, hollow tooth is used for injecting venom into the fish prey. Adapted from Olivera et al.

Figure 5 An HPLC analysis of a peptide fraction from Conus magus venom. A peptide fraction from crude C. magus venom was obtained after size fractionation on Sephadex G-25 and reverse phase HPLC. Each peak was assayed by intracranial injection of 0.5 to 2 nmol into mice. Symptoms obtained are indicated above each peak. N.A. indicates no biological activity observed. Adapted from Olivera et al.

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These features constitute so many advantages for the use of marine natural products as starting points for drug development and make them very useful tools to identify receptors or ion channels or to map the functional surface of these receptors, mainly in the nervous system. In this field, genus Conus represents an exceptionally rich source of highly evolved pharmacologically active compounds. These peptides begin even to be listed in (commercial) libraries. [13] There are two major groups of conopeptides. [11] Firstly, the disulfide rich peptides, which are the conotoxins, are encoded by gene super-families and share significant sequence similarities. The conotoxins that belong to the same family present a conserved cysteine pattern and a general pharmacological targeting specificity. Secondly, peptides with only one or no disulfide bonds will not be considered here. As previously mentioned, conotoxins are classified in families according to their conserved cysteine pattern and their targets. Here are some examples of conotoxins families and their main features.

o ω-conotoxin family : With three disulfide bonds, the ω-conotoxins present a high affinity to certain calcium channel subtypes. [14] This ion channel is a heteromultimeric protein constituted of three subunits which determine the type of channels and are the specific target of toxins. For instance the ω-conotoxins GVIA and MVIIA (the Prialt® or zicotinide previously mentioned) from respectively C. geographus and C. magus specifically act on the N-type channel. It is thus possible to discriminate between different subtypes of calcium channels using ω-conotoxins. Moreover, for instance the toxins which act on N-type channels present a therapeutic potential in pain or ischemic injury. [15]

o α-conotoxin family : α-conotoxins family includes small peptides of 10 to 25 residues and presents usually two (rarely three) disulfide bonds. Particularly stable to enzymes and in acidic conditions, these toxins are very good drug candidates. They are distributed in three subclasses : α3/5, α4/7, α4/3, according to the number of residues respectively between the second and the third cysteine and between the third and the fourth one. [10] Except for the cysteine pattern, only a proline between the second and the third cysteine is highly conserved. These toxins antagonize nicotinic acetylcholine receptor (nAChR), in muscle or in the nervous system according to their sequence and usually causes paralysis of the prey.

Figure 6 Evolution of new conotoxins. cDNA cloning has indicated that although the N-terminal end of the conotoxin precursors is highly conserved, the cone snails have a genetic mechanism for introducing rapid sequence changes specifically in loops (represented as black bars in the original peptide) between cysteine residues . The arrow represents the site of proteolytic cleavage to release the mature Cys-rich conotoxin from a prepropeptide precursor. By switching loops between cysteine residues at the gene level (perhaps by a cassette switching mechanism), three new peptides could be generated. Adapted from Olivera et al.

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Note that conotoxins from every subclass act on both kinds of target. We can assume that it is not the cysteine spacing which is responsible for selectivity. [16] The α-conotoxin MII is a sixteen residues peptide isolated from Conus magus by Cartier et al. [16]. It is a potent and highly selective competitive antagonist of a neuronal subtype of nAChR, α3β2 with an IC50 of 0.5 nM (less potent toward other subunits combinations of nAChR). In aqueous solution, a helix is formed over residues 6 to 11. (Fig. 7) It is essential for the toxin’s effect because it presents at the surface all side chains apparently needed for interaction with the receptor (i.e. Pro6, Val7, Leu10, Glu11, and Asn14). MII interaction site is between the subunits α and β and both subunit contribute to specificity. [10] Relatively stable under biological conditions, it does not cross the blood - brain barrier or intestinal mucosal membrane. In order to improve its bioavailability, Blanchfield et al. have increased its lipid solubility via incorporating a C12 2-amino-D,L-dodecanoic acid (Laa) at the N-terminal part without affecting its 3D structure. They found that this manipulation has no effect on the potency of peptide inhibition and increases its apparent permeability. [10]

A B MII is a very useful tool which acts on neuronal nAChR, but in Nature, due to its low spread potential after injection, it is possible that it targets ganglionic or adrenal nAChRs in fish to lessen the sympathetically mediated fight or escape behaviour. [10]

o δ-conotoxin family : The ten subtypes of mammalian voltage-dependant sodium channels play a fundamental role in cell membrane excitability in various localisations. The δ-conotoxin family constitutes a new probe for certain subtypes of sodium channels. For instance, δ-conotoxin EVIA from C. ermineus specifically inhibits channels in amphibian and mammalian neuronal membranes without affecting rat skeletal muscle or human cardiac muscle channels. Thus it allows the discrimination between these subtypes and opens a new field for the drug design against disease with defective nerve conduction, increasing the duration of action potential and the nerve terminal excitability and synaptic efficacy at the neuromuscular junction without any effect on the muscle itself. Its cysteine pattern consists of three disulfide bonds and so 4 loops. In Nature, this toxin immobilizes very quickly moving prey. [17]

o µ-conotoxin family : Contrary to the δ-conotoxin family, the µ-conotoxin family specifically inhibits the sodium channels of muscle membranes. It does not act on the nerve membrane channels. It possesses three disulfide bonds. [14]

o J-conotoxin family : The toxin p114a, isolated from C. palorbis, is an example of the new J family of conotoxins. It inhibits a subtype of voltage-gated potassium channel (Kv1.6) and some nicotinic acetylcholine receptor (nAChR) in the mammalian nervous system.

Figure 7 PDB views of the α-conotoxin MII Ribbon structure (A) Ribbon structure (B) with the disulfide bonds in yellow

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Presenting an amidation on the C-terminal, this small peptide contains only four cysteins and thus is stabilised by two disulfide bonds. This pattern is similar to the α-conotoxins’ one, which is supported by the effect on nAChR. The specific effects on Kv1.6 may come from a particular 3D structure. This toxin is the first which acts on two different targets, a voltage-gated channel and a ligand-gated channel, even if the first effect seems to be the major one in vivo. [11]

o κ-conotoxin family : The κ-conotoxins very specifically interact with the voltage-gated potassium channels. For instance, the κ-conotoxin PVIIA isolated from C. purpuascens only interacts with a subtype of this family of ion channel. Containing three disulfide bonds, this four-loop peptide blocks the potassium conductance in oocytes expressing the Drosophila melanogaster cloned K+ channels, but has no affinity to other subtype, even to the vertebrate one. [14]

o New conotoxin family : Venoms are not fully known and toxins which do not belong to a family are sometimes found. For instance, a new family member has been found by Kauferstein et al from Conus virgo. This new toxin, named ViTx, specifically inhibits 2 different subtypes of voltage-gated vertebrate potassium channels (Kv1.1 and Kv1.3) via an unknown mechanism. Present at a low concentration in the venom, it presents four disulfide bonds and thus does not belong to the κ-conotoxins family. [14]

Because of their potential biomedical applications, it is necessary to be able to produce refolded conotoxins at a larger scale (greater number, not amounts). Brust et al. have tried to improve methods for disulfide conopeptides formation and production of synthetic analogues. [13] Their strategy was a solid-phase synthesis in 96-well format using Fmoc chemistry using a safety catch amide linker (SCAL) on the carboxyl-terminal amide. (Fig 8) This group is stable in TFA and allows the removal of the protecting groups of the side chains on solid phase, before activation of SCAL and release of the peptide in TFA solution. Then, the oxidation of cysteine (the key step in artificial synthesis of conotoxins) is directly made in the cleavage mixture.

The only final HPLC purification step produces a high-purity peptide at a high-throughput level. With the exception of tryptophan containing peptides, this method is applicable to all disulfide peptides and could be helpful for large scale production of drugs based on toxins in Fmoc chemistry. Structures involving more than one disulfide bond are not mentioned here. Differential cysteine protection would be necessary.

Figure 8 Developed sequential side-chain cleavage, SCAL cleavage and oxidation for high-throughput production of venom peptides. From Brust et al.

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Figure 9 Ribbon diagrams of the whole muscle receptor, as viewed (A) from the synaptic cleft and (B) parallel with the membrane plane. For clarity, only the ligand-binding domain is highlighted in (A) and only the front two subunits are highlighted in (B) (α, red; β, green; γ, blue; δ, light blue). The dotted lines on the right denote the three main zones of subunit–subunit contacts. Adapted from Unwin.

D. NICOTINIC ACETHYLCHOLINE RECEPTOR Acethylcholine (ACh) is the oldest signalling molecule in terms of evolution. This molecule was even present before the nervous system, allowing cell-to-cell communication. In more evolved organisms, ACh is one of the most fundamental neurotransmitter, synthesized, stored and released by cholinergic muscarinic and nicotinic neurones. It acts on important processes, such as transmitter release, cell excitability, neuronal integration and is thus crucial for network operations and physiological functions, from sleep, fatigue, food intake, anxiety, to central processing of pain, addiction and some cognitive functions (learning, memory) in a autocrinal, justacrinal and paracrinal manner. [18] Nicotinic-type of acetylcholine receptor is a member of the pentameric “Cys-loop” superfamily of transmitter-gated ion channel, which include GABAA receptor and glycine receptor. This large glycoprotein of 290 kDa [19] is permeable mainly to calcium ions. It involves different subtypes, whose each has a particular pharmacology, physiology and distribution in the organism. The subtype of receptor is provided by its combination of subunits which determines cations permeability, activation and desensitisation kinetics, ligand pharmacology and all the functional properties. [18] It can be both hetero- or homomeric. nAChR is pentameric, but its structure, as well as its subunit composition plasticity, is not completely elucidated. The receptor consists of three different parts : a large extracellular ligand binding domain, a membrane spanning pore and a smaller intracellular domain, presenting some phosphorylation sites. Its total length is around 160 Ǻ, perpendicularly to the plasmic membrane. The ligand binding domain has a central vestibule of 20 Ǻ diameter. Muscle nAChR presents two sites for ACh (at the α-γ and α-δ interfaces) and constitutes a narrow water-filled pass across the membrane. (Fig. 9) [19] The subunits in the ligand binding domain are organised around two sets of β-sheets packed in a curled β-sandwich and joined by disulfide bonds forming the cys-loop. The membrane-spanning domain is made of four α-helical segments (M1-M4) arranged symmetrically and forming an inner ring which shapes the pore. To close the pore, the ring comes near the middle and makes a constricting hydrophobic girdle. [19]

The twelve known subunits have a high percentage of sequence identities, especially in the ACh binding site region. This makes it difficult to obtain selective probes, particularly with an interaction on the ACh binding site. In this context, venoms from snakes or cones provide a rich array of highly selective pharmacological tools toward a particular nAChR subtype through interactions with residues outside conserved regions. [20] Both hetero- and homopentamers are organised around the central channel. (Fig. 10)

A B

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Binding sites have a principal and a complementary component. In heteropentamers, the principal component is carried by α2-4 or α6, whereas the complementary one is presented by the subunit β2 or 4. In homopentamers, each subunit contributes to both components of the site. There are three different conformational states : active (or open), resting and desensitised.

o Active or open : This is the open and conducting state. In this case, an agonist (ACh or a nicotinic substance) traps the receptor in its open conformation. There are no big visible changes around the ACh binding site between the active and the desensitised states.

o Resting : This closed non-conductive state is due to an antagonist ligand, such a toxin, which stabilises a closed conformation. [20]

o Desensitised : Desensitisation is a refractory time during which the channel reminds closed and non-conductive. This state is reached after a prolonged exposure to agonist and mainly due to conformational re-arrangements in the extracell domain and phosphorylation of some sites in the cytoplasmic part of the receptor. [20] It is interesting to know that, even if the pore is closed in both resting and desensitised states, the structure of the pore is different. [20]

The energy level needed to reach a given state depends on receptor subtype. For instance, α7 desensitises faster than α3β2. ACh binding initiates two interconnected events in the binding domain. First, some local disturbance happens in the ACh-binding region. Then large-scale conformational changes involve a rotational movement in both α-helices, which induces rotation of the β-sheets and, via a movement communication through inner helices, the gate apart is broken and the alternative open-channel form is stabilized. [19] Stabilization of a given state or spontaneous transition between different conformations are also possible in an allosteric manner, for instance by certain toxins or drugs able to block the pore. There is a high number of agonists, mainly nicotinic ligands. They present a therapeutic interest and are often produced by pharmacological labs. Some antagonists are also produced,

Figure 10 (A–C) Organisation and structure of nAChRs. (A) Schematic representation of the putative transmembrane topology of nAChR subunits. The model shows the extracellular amino terminal portion, followed by three hydrophobic transmembrane domains (M1–M3), a large intracellular loop, and then a fourth hydrophobic transmembrane domain (M4). (B) Pentameric arrangement of nAChR subunits in an assembled receptor. (C) Subunit arrangement in the homomeric a7 and heteromeric a4b2 subtypes, and localisation of the ACh binding site. Adapted from Gotti et al.

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but they are less studied. The best known antagonists are peptide modulators such as venom toxins. Allosteric modulators also exist. They are divided in potentiators and inhibitors. Inhibition can be made by stabilisation of the resting or desensitised state or by increase of the desensitisation rate. nAChR family is highly permeable to calcium divalent cations (Ca2+), without required any plasmic membrane depolarisation to promote calcium influx. This property gives to nAChR a role in calcium homeostasis and signalling (calcium which can enter in the cell through this receptor is a second messenger) and also induces depolarisation of the plasmic membrane via potential activation of the voltage operated Ca2+ channel and increase of intracellular calcium through mobilisation of intern stores. Subunit combinations (and additional subunits in certain cases), which determine the nAChRs subtypes, are involved in absolute quantity and localisation (microdomains) of calcium in the cell and are thus relevant for regulation of calcium-mediated events, such as transmitters release, cell excitability, gene expression, cell differentiation and apoptosis. [1] There are different ways to classify nAChRs depending on various criterions. According to their localisation, there are two main groups : [21]

o In the nervous system, there are nine different α class subunits (from α2 to α10) and three different β (from 2 to β4). Both kinds contribute to the pharmacological specificity of each subtype and the localisation differs according to the kind of subunits. Unfortunately, these receptors are less studied. Actually, because of their heterogeneity, specific tools are necessary to distinguish between subtypes. Note that neuronal nAChRs are also found in non-neuronal tissues.

o In muscle where it is the most studied, the receptor is made of five different subunits : two α, one β, one γ (or ε), and one δ. At a high concentration at nerve-muscle synapse, it mediates fast transmission of electrical signal in response to ACh releases from the nerve terminal into the synapse.

According to binding studies, it is also possible to distribute receptors into two groups : [21] o α-Bungarotoxin sensitive nAChRs which can be homomeric or heteromeric. o α-Bungarotoxin non-sensitive nAChRs are heteromeric receptors only.

α-Bungarotoxin (α-Bgtx) is a long chain toxin isolated from snake Bungarus multicinctus. Historically, it was the first toxin used to characterize nAChR. α-Bgtx is still used as purification, affinity labeling and radioligand tool. [20] Due to their fundamental cellular effects previously mentioned, nAChRs are largely distributed in organism. Their neuronal distribution is mainly made into the brain cholinergic system. Some subtypes or subunits are more abundant in the central nervous system, for instance more than 90% of nAChRs in neural system contains α4 and β2 and the rest is mainly constituted of α7 and the most abundant in peripheral nervous system contains α3 and β3 subunits. [18] nAChRs are also present in many non-neuronal tissues, according to their subtypes. For instance, in adult muscles, α4, 5 and 7 and β2 and 4 have been found whereas α7 which is highly expressed during development and the prenatal period decrease afterwards. α3β2 is abundant in presynaptic innervating fibres and its activation facilitates ACh release. This receptor is also present in T and B lymphocytes, epidermal keratocytes, bronchial or alveolar epithelial cells, vascular endothelial cells and certain part of vascular smooth muscle where it seems involved in cell proliferation, differentiation and apoptosis. Expressed in astrocytes, nAChRs might play a role in astrocyte life or in astrocyte-neuron relationships.[18] Due to its fundamental cellular effects and its large distribution, nAChR is or seems to be involved in various pathologies where the positive effect of nicotine administration suggests the nAChR may play a role in symptoms manifestation.

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These diseases can be age-dependant, such as Tourette’s syndrome, a chronic, familial neuropsychiatric disorder involving persistent extrapyramidal movements, inappropriate vocalisations and cacolalia. nAChR seems also involved in autism, a severe developmental disorder which becomes apparent in early childhood. This disease is characterised by severely impaired social relationships and communication, planning and attention, with restrictive, odd and stereotyped behaviour. This receptor seems also involved in attention-deficit hyperactivity disorder (ADHD), which is an inheritable multigenic psychiatric disorder implicating difficulties in attention and hyperactivity. These last are positively influenced by nicotine like in schizophrenia nAChR is also involved in age-independent neurological pathologies such as epilepsy and febrile convulsions, where α4, the most common subunit in the brain, seems mutated and more ACh sensible, or depression and anxiety. Age-related neurodegenerative diseases, mainly Alzheimer’s disease (AD) and Parkinson’s disease (PD), affect the cholinergic pathway. Actually, nAChR seems to be an entry point in the cell for β-amyloid proteins, which is mainly responsible for AD. The receptor is also very important for the dopaminergic release. In PD patients, those nAChRs decrease and this fact can be considered as a possible cause of disease. nAChR is also involved in pathologies due to smoking, like number of lung diseases characterised by cell proliferation, such as carcinoma. Smoking increases also the risk of vascular complications after graft or angioplasty surgery through its action on smooth muscle cells proliferation induced by nAChR. The blood circulation is among others under cholinergic control; actually nAChR is present in key areas regulating blood pressure and thus seems involved in blood pressure and hypertension. The receptor may also play a role in inflammatory bowel diseases. This is supported by the close negative association between smoking and ulcerative colitis. Finally the most promising therapeutic application involving nAChR is the pain pathway. Even if this receptor is target of analgesic for long time, biomedical research aim to develop new drugs more subtype-specific, for instance acting on α4β2, which is the most represented subtype in the brain and seems involved in antinociceptive activity. There are several ways to study nAChRs and distinguish some subtypes. An in situ hybridization with selective subunits-specific probes allows to see the distribution of subunit mRNA. Unfortunately, methods to study protein distribution are less selective. It can be made by labelled nicotinic agents in homogenate of discrete brain areas or histological brain sections. This method is very sensitive but not subunit-specific because of the lack of subtype-specific ligand. For instance, 3H-α-conotoxin MII binds both α3 and α6 containing receptors. Immunoprecipitation or immunopurification from selected brain areas using subunit-specific antibodies present a good sensitivity and specificity, but specific antibodies are not always available or well characterised for subtype selectivity. Moreover, they sometimes do not work in bioassays. For native study, the use of polyclonal antibodies in combination with labelled nicotinic agents can be more adjusted. In vivo studies can be achieved by positron emission tomography and specific prepared nicotinic ligands. Non-invasive, this method can be used in human but necessitates some improvements in terms of ligand specificity and time and space resolution. [18]

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II. MATERIALS AND METHODS Chemicals Peptide-synthesis-grade DMF and DIEA were purchased from Biosolve. DCM, NMM, diethyl ether, dimethylsulphoxide (DMSO), carbonyldiimidazole (CDI) and trifluoromethanesulphonic acid (TFMSA) were purchased from Fluka, Switzerland. TFA was from Halocarbon, New Jersey. Acetonitrile CHROMASOLV gradient grade for high-pressure liquid chromatography (HPLC) was from Sigma–Aldrich. HBTU was from Iris Biotech GmbH Germany and BOP was from GL GLSynthesis Inc (USA). HOBt was from NovaBiochem (Switzerland) and the amino acids were from Orpegen, Germany or from NovaBiochem. Analytical High-Pressure Liquid Chromatography Analytical reverse-phase high-pressure liquid chromatography (RP-HPLC) was performed at 0.6 ml/min on Waters equipment using a Nucleosil C8 or C18 column (4 × 250 mm, 300 Ǻ 5µm particle size). Solvent A was 0.1% TFA in HPLC grade water. Solvent B was 90% acetonitrile with 0.1% TFA. Elution was done with a linear gradient of 2%/min. Preparative High-Pressure Liquid Chromatography Preparative RP-HPLC was performed at 15 ml/min on Waters equipment using a Vydac C8 column (22 × 250 mm 300 Ǻ 10–15 µm particle size). Elution with previously described solvents A and B of the peptides was done with an appropriate linear gradient, usually 1%/min. UV monitoring was at 214 nm. The peaks were collected manually and the product was recovered by lyophilization. Mass Spectrometry MALDI-TOF mass spectrometry was performed in the linear mode using sinapinic acid or α-cyano-4-hydroxycinnamic acid as matrix, on a Voyager-DE STR (Applied Biosystems) instrument equipped with delayed extraction. External calibration was performed using porcine insulin (Novo Nordisk). Solid Phase Peptide Synthesis Solid phase peptide synthesis was performed on a modified ABI 433A machine using Boc chemistry and in situ neutralization. Peptides were prepared on a 0.2 mmol scale on MBHA cross linked with 1% DVB resin (0.9 mmol/g, Senn Chemicals, Switzerland). Boc-amino acids were protected by the following groups: Arg(Tos), Asn(Xan), Asp(OcHx), Cys (4MeBzl), Glu(OcHx), His(Dnp), Lys(Z(2Cl)), Ser(Bzl), Thr(Bzl), Trp(For), Tyr(Z(2Br)). Certain protecting groups were removed prior to acid cleavage (Dnp with 20% 2-mercaptoethanol and 10% DIEA; formyl with 20% piperidine; Boc with neat TFA) with HF containing 5% p-cresol for 60 min at 0 °C. The peptides were precipitated and then washed with cold diethyl ether. The crude peptide was exposed to high vacuum overnight and purified by preparative RP-HPLC, and the purified product was lyophilized. α-conotoxin MII sequences after resin cleavage were GCCSNPVCHLEHSNLC-amide for the N-terminally modified peptide. A Boc-aminooxyacetyl group (Boc-AoA) was manually coupled on the N-

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terminal part as its N-hydroxysuccinimide ester (Boc-AoA-OSu) using 50 equivalents excess in phosphate buffer 100mM acetonitrile 50/50 (pH 8.2). Disulfide Bond Formation of α-conotoxin MII Seventy milligrams of the peptide was dissolved in 700 ml ammonium bicarbonate solution 100 mM and the pH adjusted to 7.8. After six hours at room temperature, the first analytical HPLC and MALDI have been performed. Then, after acidification with acetic acid, the solution was directly injected into preparative RP-HPLC for purification. Synthesis of a PEG–Polyamide Linker PEG-like dialdehyde linkers of various lengths were synthesized as described in Rose et al. 1999 [22]. Briefly, 0.3 mmol of Sasrin resin (1.02 mmol/g, 200–400 mesh, Bachem Switzerland) was acylated with succinic anhydride, 4 mmol in 8 ml of DMF containing 0.5 M of DMAP to which 0.4 ml of DIEA was added. Free carboxyl groups were activated with CDI for 30 min. The activated carboxyl group was then aminolyzed with 4,7,10-trioxa-1,13-tridecanediamine (commercial monodisperse PEG diamine from Fluka) in the presence of HOBt for 60 min in DMF. The growing molecule was again acylated with succinic anhydride, 4 mmol in 8 ml of DMF containing 0.5 M HOBt to which 0.4 ml of DIEA was added. The polyamide chain was further grown by successive activation, aminolysis and acylation steps. Each ‘acylation-activation-aminolysis’ cycle will add a ‘PEG-succ’ unit (–NHCH2(CH2CH2O)3CH2CH2CH2NH-COCH2CH2CO–) to the resin. After addition of the desired number of units, Boc-Ser(Bzl)-OH was coupled to the free amine. For this, 1.2 mmol (4 equivalents) of Boc-L-Ser(Bzl) was activated with 1.2 mmol (4 equivalents) HBTU dissolved in DMF. 2.4 millimols (8 equivalents) of DIEA was added, and the solution reacted for 40 min. The Kaiser ninhydrin test (Fluka) was performed to verify that the acylation coupling had succeeded. If the test was positive, the coupling was repeated for another 40 min. The products were cleaved from the resin with 1% TFA in DCM and neutralized in pyridine/methanol (9/1). HPLC preparative purification was employed. The linker was then coupled to the commercial PEG diamine or ethylene diamine (commercial EDA from Flucka) according to the desired final linker length : 2 equivalents of linker with 2 equivalents of BOP and 4 equivalents DIEA were preactivated for 5 min before reaction with 0.8 equivalent of the commercially available diamine. After incubation over night, the product was isolated by RP-HPLC. The symmetrical linker was thus obtained. The Boc and Bzl protecting groups were removed by dissolving 10 mg of the linker in 300 µl TFA for 4 min followed by the addition of 30 µl of TFMSA for 25 min. TFA was evaporated with air and the product precipitated and washed with cold ether, and dried in a dessicator under high vacuum. Serine Oxidation to a Glyoxylyl Function The N-terminal serine is oxidized with periodate to a glyoxylyl function as described in Gaertner et al. [23]. The molecule is dissolved in a 50 mM Imidazole buffer pH 7 at a final Ser concentration of 100 µM. In the presence of 50 M excess of methionine, each serine is oxidized with 4 equivalents of sodium periodate. After 5 min, 1000 equivalents (over periodate) of ethylene glycol is added and the solution is brought to pH 4–5 with acetic acid. The solution is then injected into RP-HPLC for purification.

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Oximation of the Dialdehyde Linker with Aminooxy α-conotoxin MII derivative Based on the publication of Rose [24], one equivalent of aldehyde solution (HPLC fraction concentrated) was quickly added to 1.5 excess over each aldehyde group of a 20 mM solution of aminooxypeptide (dissolved in 10 mM acetate buffer, pH 4.6 with 50% acetonitrile). The reaction mixture was stirred for 24 h at room temperature and purified by RP-HPLC, as previously described. In order to make the control asymmetric compound (monotoxin-linker), 2 equivalents of dialdehyde linker were added to 1 equivalent of aminooxy-conotoxin. After 2 hours and a HPLC and MALDI intermediary verification, 2 equivalents of methyloxylamine (from Flucka) were added. However, the incomplete reaction after 24 hours necessitated the addition of 4 equivalents of methyloxylamine to consume all the free aldehyde on the monotoxin-linker-aldehyde.

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III. RESULTS AND DISCUSSION The α-conotoxin MII is a peptide of 16 residues, with a refolded molecular mass of 1710.637 Da (it corresponds to the monoisotopic mass and is calculated with Isotopident as for every compound). Its elemental formula (after refolding) is C67H102N22O23S4. It presents two disulfide bonds, between Cys 2 and 8 and between Cys 3 and 16 :

GCCSNPVCHLEHSNLC-NH2 The C-terminal amide is generated by the resin used during the synthesis and is present in the natural material. α-conotoxin MII synthesis The α-conotoxin MII is obtained by solid phase peptide synthesis in Boc chemistry (Fig. 11). This automated method allows synthesis of small peptides, here, on MBHA resin (p-methylbenzhydrylamine) by coupling each residue on the growing peptide, according to the steps in the sequence below :

Thus, after removal protecting groups on the Histidine, Cysteine and Boc groups and cleavage from the resin, the reduced peptide is obtained and ready for HPLC purification steps and MS characterisation in linear (LM) or reflector (RM) mode. As previously mentioned, the peptide presents a C-terminal amide formed after MBHA resin cleavage.

Figure 11 Synthesiser ABI 433A

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Three synthesis of toxin are necessary to produce enough material for all dimers and control compounds. They all give the same kind of results. Here is the analytical chromatogram of the first synthesis crude material (Fig. 12) :

Figure 12 Analytical chromatogram of the first synthesis crude material of conotoxin. The reduced peptide is

indicated by an arrow. After lyophylisation, the reduced conotoxin is purified by preparative HPLC. Analytical HPLC of each collected fraction allows to pool the purer fractions in the aim of reconciling both quantity and sufficient purity before the refolding step. Here is the chromatogram of the pool (Fig. 13) and the corresponding spectrum (Fig. 14) :

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Figure 13 Analytical chromatogram of the fraction pool post purification of the crude toxin. The purity level

reaches about 90%. Note that the second peak is not characterised and disappear after refolding.

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Figure 14 Spectrum (LM) of the major peak of the purified reduced toxin (calculated mass : 1714.637 Da).

Yields of purification of each synthesis crude are of 44 %, 39.7 % and 24.7 %. These variations are due to some synthesiser difficulties during the synthesis but ample material was produced. Refolding of the reduced toxin A first refolding protocol using a redox couple L-Glutathione reduced/ L-Glutathione oxidized (5 mM/0.5 mM) is tested at small scale in phosphate buffer (pH 7.8). After one day, we stopped the folding reaction and the oxidized peptide is isolated by HPLC purification and characterized by mass spectrometry. This protocol is then applied on a larger scale to 40 mg. Unfortunately, after purification, each fraction contains an intermediary of α-conotoxin MII linked to a GSH (Fig. 15), even after a second purification by semi-preparative HPLC (Fig. 16).

Figure 15 MS spectrum (LM) of the pool descended from a pool of semi-preparative HPLC fraction (calculated

mass : 1710.637 Da).

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Figure 16 Analytical chromatogram of the semi-preparative HPLC pooled fractions after lyophylisation : it

remains a little impurity. Another strategy thus allows to avoid this GSH adduct : a refolding by air oxidation and at low peptide concentration (0.1 mg/ml) in bicarbonate buffer 100 mM (pH 7.8). After one day refolding, purification by preparative HPLC is made. It allows to pool certain fractions and to increase purity comparing to the previous refolding method (Fig. 17).

Figure 17 Chormatogram of the refolding mixture after one day. The major peak presents the correct mass.

2.2 mg of highly pure native conotoxin are lyophilized (Fig 18 p. 23) and given to Prof. Bertrand’s group for in order to have a positive control.

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Figure 18 Final chromatogram (A) and calibrated spectrum (RM) (B) of the native toxin (calculated mass :

1710.637 Da). Aminooxyacetylation of the peptide In order to assure specific attachment between the toxin and the linker, a derivatisation of the peptide is made by aminooxyacetylation of the N-terminal part. It is possible to attach the aminooxyacetyl group either on the refolded peptide or on the reduced peptide linked to the resin. This last coupling is made after removal of the Boc protecting groups with TFA. The hydrosuccinimide ester of Boc-aminooxyacetyl group (Boc-AoA-OSu) is manually linked with 1.2 equivalents and 1.2 equivalent of NMM in DMSO (pH 8.0-9.0). The process is followed by the same steps as previously mentioned : removing of the protecting groups and cleavage from the resin by HF. Unfortunately, an adduct corresponding to a mass of AoA-conotoxin + 163 Da appears (Fig. 19).

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Figure 19 Spetrum (LM) of the purifed AoA-conotoxin after coupling on the resin : an adduct of + 163 Da is

present (calculated mass : 1784.661 Da). This adduct was impossible to remove and decreased the yield of purified derivatised peptide to about 11 %. Even after refolding at small scale, the adduct is still present. The solution of this problem comes from the test of another MALDI-TOF matrix. Indeed, sinapinic acid is commonly used in this project, but when α-cyano-4-hydroxycinnamic acid is used as matrix, adducts change (Fig 20).

Figure 20 Spetrum (LM) of the purifed reduced AoA-conotoxin after coupling on the resin with α-cyano-4-

hydroxycinnamic acid as matrix : adducts change (calculated mass : 1784.661 Da). Thus these adducts are probably due to side reaction between the highly reactive aminooxy group and the matrix. Besides, a degradation of the aminooxy group seems occur (loss of -NH2 to -OH). [25] So, this method is abandoned. Aminooyxyacetylation can also be made on free refolded peptides (Fig. 21).

Figure 21 Aminooxyacetylation of the free peptide.

Two different methods of coupling on free peptide are tested at small scale (100 µg of conotoxin). The first one consists in a coupling of 50 equivalent of Boc-AoA-OSu in

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acetonitrile/bicarbonate buffer (CH3CN/H2O/NaHCO3 (47.5:47.5:5)) (pH 8-8.5). The second one is made in phosphate buffer 100 mM (pH 8.2), using the same Boc-AoA-OSu proportion. The day after, both methods are compared by HPLC (Fig. 22).

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Figure 22 Chromatogram of the AoA group coupling in bicarbonate buffer (A) and in phosphate buffer (B).

The right compound has a retention time of 32 min (confirmed by MS). Similar results are obtained after Boc protecting group removal with TFA. Thus phosphate buffer protocol is chosen for the large scale and peptide ready for linking to the spacer is produced with a yield of 73.7 %. Linker synthesis The spacer is a manually produced polyamide linker according to Rose et al. publication. These water-soluble and flexible polymer chains are very useful in biomedical field to connect peptides or increase their half-life. Essentially non-immunogenic, they also are unsusceptible to proteases. [22] [23] It is thus possible to produce linker of a precise desired size, which represents an important biological feature. Actually, as previously mentioned (see chapter concerning venomics), the length of the linker can highly influence the avidity and the bioactivity of the compound. Linkers are produced on resin beads by alternate coupling of two different units corresponding to building blocks : succinic anhydride (“suc”) and a trioxatridecanediamine (“peg”). (Fig. 23 and 24) In this project, the resin used is the Sasrin resin. Sasrin means super acid-sensitive resin, which implies that only 0.5 to 1 % of TFA is enough to cleave the linker from the solid phase, without affecting acid sensitive protecting groups.

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Figure 23 Schema of the building steps of the linker on the sasrin resin, until the cleavage of the half linker from

the solid phase.

Figure 24 Chemical view of a resin carrying a “suc”, a “peg” and a “suc” unit.

The final aim is to produce a symmetric spacer and it is thus easier to build a half of the linker on the resin and then to put two cleaved halves together (Fig. 25) using a diamine whose size depends on the final length.

Figure 25 Dimerisation steps of the linker : it is ready for deprotecting serine. (EDA = ethylene diamine,

NMP = N-methyl-2-pyrrolidone, BOP = Benzotreazole-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate)

In this project, two different linkers are produced : a small one of 50 Ǻ and a big one of 108 Ǻ. Each of them corresponds to a given number of “peg-suc” units : 50 Ǻ � -Peg-Suc-EDA-Suc-Peg- 100 Ǻ � -(Peg-Suc)2-Peg-(Suc-Peg)2- This method allows to obtain pure linker with a yield before oxidation step of 34.2 % for the small linker and of 6.7 % for the big one. This loss of yield is due to a needed second purification by semi-preparative HPLC due to a contamination (other compound stuck on the colomn). Then a HPLC purification and a lyophylisation are performed. The next step of the spacer synthesis consists in the formation of an aldehyde group, which guarantees a chemoselective ligation to the toxin aminooxyacetyl group, by oximation. This functional group is ensured by formation of glyoxylyl groups via oxidation of serines. These

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last are thus deprotected by removal of the protecting groups with TFA and TFMSA (Fig. 26) and then oxidised (Fig. 27).

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Figure 26 Chromatogram of the pool after the deprotection reaction purification of the small (A) and the big (B)

linker.

Figure 27 Oxidation of the serines makes the linker ready for oximation, by producing glyoxylyl groups. In order to obtain 1 mg of final oxidised linker and to be able to store the rest without side reactions, only 1.4 mg of the deprotected big linker and 4.3 mg of the small one are oxidised (Fig 28 and 29). [26] The rest is lyophylised.

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Figure 28 Chromatogram of the oxidated small (A) and of the big linker (B).

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Figure 29 Spectrum (RM) of the oxidised big (A) and small (B) linker (calculated mass : big linker = 1540.895

Da , small linker = 776.417 Da). Oximation In order to assure a chemoselective ligation reaction between the toxin and the linker, an oxime bond is formed from the aminooxyacetyl group and the aldehyde (Fig. 30). This specific reaction allows to avoid any cross reaction for instance with residue side chain. [24] This ligation allows more versatile assembling peptides without affecting their immunogenicity or bioactivity. [27]

Figure 30 Reaction of oximation between the spacer and two toxins. The dimer is formed.

Three compounds are build by oximation : “Small” dimer : Toxin-Small Spacer-Toxin (calculated mass : 4308.706 Da)

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“Long” dimer : Toxin-Long Spacer-Toxin (calculated mass : 5074.188 Da) Control compound : Toxin-Small Spacer (calculated mass : 2571.82 Da) They are all produced first at small scale, to test the method, and then at larger scale, to obtain around 1.5 mg of final product. The small dimer is produced by oximation of 3.8 mg of conotoxin with 0.55 mg of small linker. It is then purified by semi-preparative HPLC (Fig 31).

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Figure 31 Analytical chromatogram of the oximation reaction of the small dimer. The peak no 1 corresponds to

AoA-toxin non-consumed and the peak no 2 to the dimer. Finally, 1.6 mg of pure small dimer are produced (Fig. 32) whose 1.2 mg are given to Prof. Bertrand’s group for bioassays.

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Figure 32 Final chromatogram (LM) (A) and calibrated spectrum (B) of the small dimer. On the spectrum, the masses of 1770 and 2543 Da correspond to the cleavage of an oxim bond from the dimer because of the energy

of the laser (calculated mass : 4307.703 Da). The big dimer is made using 3.6 mg of toxin and 1 mg of long linker, before purification by semi-preparative HPLC as previously (Fig. 33).

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Figure 33 Analytical chromatogram of the oximation of the big dimer. The peak no1 corresponds to the toxin

alone and the peak no 2 corresponds to the expected dimer. Thus 2.1 mg of pure big dimer are produced (Fig. 34) whose 1.1 mg are given to Prof. Bertrand‘s group for testing.

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Figure 34 Final chromatogram (RM) (A) and calibrated spectrum (B) of the big dimer. On the spectrum, the

masses of 1768 and 3308 Da correspond to the cleavage of an oxime bond from the dimer because of the energy of the laser. The 2538 mass corresponds to the double charged dimer (calculated mass : 5072.181 Da).

The last compound to produce is the “monomer” control one. It is an asymmetric molecule made using one equivalent of the toxin and two equivalents of the linker in acetate buffer (pH 4.5). Two equivalents of methyloxylamine hydrochloride then is added after complete reaction of the toxin with the linker (verification by HPLC). The reaction is over after two days (Fig. 35) and a purification is performed.

Figure 35 Analytical chromatogram of the oximation of the control compound. The peak no 1 corresponds to

double oximation of the linker with methyloxylamine hydrochloride, the peak no 2 corresponds to the intermediary compounds, i.e. the mono-oximated linker (Ctx-linker-Ald), the peak no 3 corresponds to the

control compounds and the last one corresponds to the small dimer. Finally, 0.9 mg of control are produced (Fig. 36) and given to Prof. Bertrand’s group for biotesting.

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Figure 36 Final chromatogram (RM) (A) and calibrated spectrum (B) of the control compound (calculated

mass : 2570.078 Da). Here is a summarizing of the toxin compound produced and their corresponding masses :

Toxin compound

Isotopident mass : monoisotopic

Isotopident mass : most likely isotope combination

Experimental mass (monoisotopic and

protonated) Native toxin 1710.637 Da 1710.637 Da (100 %)* 1711.1606 Da Control compound

2570.078 Da 2571.082 Da (23.8 %)* 2572.8589 Da

Small dimer 4307.703 Da 4308.706 Da (12.8 %)* 4309.64 Da Big dimer 5072.181 Da 5074.188 Da (11.4 %)* 5074.8330 Da * Probability of the most likely isotope combination. Bioassays Bioassays are performed in collaboration with Dr. R. Hogg, from Prof. Bertrand’s group, by electrophysiological recording from human α3β2 nAChRs using the same method as in Hogg et al. publication [28]. Xenopus laevis oocytes are injected intranuclearly with cDNA (2 ng) of this receptor.

Acetylcholine (ACh) and toxin compounds are dissolved in OR2 medium (82.5 mM NaCl, 2.5 mM KCl, 5 mM CaCl2, 2 mM HEPES, adjusted to pH 7.4 with NaOH) just prior testing and recording carried out at -100 mV.

A

B

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Peak amplitude is plotted as a function of the toxin concentration on a semilog scale. Then dose-response of toxin inhibition is obtained using the formula : y = 1/1 ([toxin]/(IC50)

n) where y is the normalized response, [toxin] is the concentration of toxin and n is the Hill coefficient. nAChR response to growing concentrations of toxin, from the control to 1µM. Control effect is measured first, by exposure to ACh without toxin exposure (Fig. 37). Then 2 min toxin incubation is procssessed before ACh growing concentrations are tested.

Results show that the native conotoxin has a high inhibiting effect on the nAChR. Dimers (and control compound) do not increase receptor inhibition to ACh (Fig. 38 and 39).

This loss of bioactivity might be due to the presence of the linker. Thus the toxin concentration needed for a given level of response is higher for the compounds containing a linker. In other words, inhibition of the receptor after exposure to a toxin compound at a given concentration is lower in the case of control compound or dimer (Fig. 40).

Figure 38 Measure of the current before (green) and after (red) adding the toxin compound. The native toxin (MII) blocks current in Xenopus oocyte, but the other compounds, the control (MII linker), the small dimer (MII linker S) and the big linker (MII linker L) do not present a higher inhibition of the Ach-activated current. Their activity decreases.

Figure 39 Dose-response analysis of the native conotoxin and of the control compound. The presence of the linker decreases the activity of the toxin on the receptor.

Figure 37 Inhibition of the ACh-activated current at growing concentrations of toxin, from the control, with no toxin, to 1µM of toxin compound. The toxin inhibits the current in a concentration-dependant manner.

1 µM

Control (without toxin)

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This loss of activity can be due to several causes involving the spacer. It can hamper the interaction between the toxin and its receptor binding site. However, according to the literature, the N-terminal part does not seem important for the interaction. It is more probable that the amphiphilic properties of the spacer and/or the rigidity of the oxime bound hamper the optimal interaction. Actually, the only bioassay made with a modified α-conotoxin MII is made with a lipidic group (C12). [10] It is thus possible that a hydrophilic part prevent optimal interaction. Other experiments have thus to be performed in order to find the cause of this loss of activity.

Figure 40 Relative response of the nAChR to ACh after exposure to the native toxin (MII), the control (MII linker), the small dimer (MII linker S) and the big linker (MII linker L). All are at a concentration of 1 µM. Even if all these compounds present an inhibition of the ACh-activating current, there is an important loss of activity in the case of the dimers and the control. Note: control current amplitude without toxin is normalised to 1.0.

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IV. CONCLUSION AND PERSPECTIVES New discoveries in the biomedical field necessitate new experimental tools and lead to refined drugs. In order to have a wide source of “starting material”, the scientific community is very interested in venoms. Actually, venoms contain a high variety of small peptides, which are highly selected and specific to a target. Used for hunting by the animal, they mainly affect the nervous system and the muscular physiology of their prey, and so are relevant to several important human diseases. The aim of my project, which constitutes a preliminary work in the CONCO project, was to produce a particular toxin, the conotoxin MII, from the α family and secreted in the Conus magus snail venom, and to try to improve its avidity and bioactivity for its target by dimerisation using a linker. The target of the α-conotoxin MII is the nicotinic acetylcholine receptor (nAChR), which is involved in many fundamental physiological processes, such as pain, apprenticeship, sleeping and dependency. MII is a specific inhibiting toxin of the α3β2 nAChR subtype, which is expressed at the neuro-muscular junction. There are two dimers to produce, a small one containing a linker of 50 Ǻ and a big one containing a linker of 108 Ǻ. These sizes of linker allow to test the importance of the linker length on the bioactivity. The control compounds are a “monomer”, which corresponds to only one toxin linked to the small spacer, and the native toxin, which is the positive control. Toxin is produced by automated solid phase peptide synthesis in Boc chemistry and derivatised by coupling of an aminooxyacetyl group, which assures a chemoselective ligation with the spacer. The linkers are polyamide spacers manually made from “peg-suc” (trioxatridecanediamine and succinic anhydride) units. It is derivatised by oxidation of a serine at its both extremities in order to form an aldehyde which is necessary for the ligation to the peptide. The ligation is made by Oximation, a chemoselective reaction between an aldehyde (on the spacer) and an aminooxy group (on the peptide). It allows to avoid any side reaction. The both dimers and control compounds have been produced in an amount between 0.9 and 2.2 mg. The bioassays have been performed by Dr. R. Hogg, of the Prof. Bertrand’s group. Unfortunately, even if the positive control gives the expected results, none of the dimers presents an increase of inhibition of the nAChR. This is in spite of the success of multivalency in other areas (Introduction). Actually, the linker seems to hamper the interaction between the receptor binding site and the toxin. This effect may be due either to steric obstruction (it is possible that the N-terminal part is more important for interaction than mentioned in the literature), either to linker amphiphilicity. Another possibility is that the linker is too long. As previously mentioned (Introduction), the length of the spacer is crucial for its action. If the right spacer is not found, this can not lead to increase avidity, but the dimer is “diluted”, because the spacer hampers the second interaction, once the first is made. Thus, it is decided now to produce new dimers. In a first time, the new dimers aim to test the importance of the N-terminal part, making oximation with the linker on C-terminal extremity. They corresponds to : MII-Cter-mediumPegSuc-Cter-MII, for the small one, and MII-Cter-longPegSuc-Cter-MII, for the big one. As previously, a control compound will also be made : MII-Cter-mediumPegSuc-CH3. According to the results, new generation of toxin dimers can be envisaged and designed using computer-assisted methods and peptoid-mimetic approaches.

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V. REFERENCES 1. Kitov, P.I. and D.R. Bundle, On the nature of the multivalency effect: a

thermodynamic model. J Am Chem Soc, 2003. 125(52): p. 16271-84. 2. Michael, F.T., Adventures in multivalency, the Harry S. Fischer memorial lecture

CMR 2005; Evian, France. Contrast Media & Molecular Imaging, 2006. 1(1): p. 2-9. 3. Terskikh, A.V., et al., "Peptabody": A new type of high avidity binding protein.

PNAS, 1997. 94(5): p. 1663-1668. 4. Kramer, R.H. and J.W. Karpen, Spanning binding sites on allosteric proteins with

polymer-linked ligand dimers. Nature, 1998. 395(6703): p. 710-713. 5. Menez, A., R. Stocklin, and D. Mebs, `Venomics' or: The venomous systems genome

project. Toxicon, 2006. 47(3): p. 255-259. 6. Lewis, R.J. and M.L. Garcia, THERAPEUTIC POTENTIAL OF VENOM PEPTIDES.

Nature Reviews Drug Discovery, 2003. 2(10): p. 790-802. 7. Ostrow, K.L., et al., cDNA sequence and in vitro folding of GsMTx4, a specific peptide

inhibitor of mechanosensitive channels. Toxicon, 2003. 42(3): p. 263-274. 8. Siemens, J., et al., Spider toxins activate the capsaicin receptor to produce

inflammatory pain. Nature, 2006. 444(7116): p. 208-212. 9. McGivern, J.G., Targeting N-type and T-type calcium channels for the treatment of

pain. Drug Discov Today, 2006. 11(5-6): p. 245-53. 10. Blanchfield, J.T., et al., Synthesis, Structure Elucidation, in Vitro Biological Activity,

Toxicity, and Caco-2 Cell Permeability of Lipophilic Analogues of α-Conotoxin MII. J. Med. Chem., 2003. 46(7): p. 1266-1272.

11. Imperial, J.S., et al., A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins. Biochemistry, 2006. 45(27): p. 8331-40.

12. Olivera, B.M., et al., Conotoxins. J. Biol. Chem., 1991. 266(33): p. 22067-22070. 13. Brust, A. and A.E. Tickle, High-throughput synthesis of conopeptides: a safety-catch

linker approach enabling disulfide formation in 96-well format. J Pept Sci, 2007. 13(2): p. 133-41.

14. Kauferstein, S., et al., A novel conotoxin inhibiting vertebrate voltage-sensitive potassium channels. Toxicon, 2003. 42(1): p. 43-52.

15. Favreau, P., et al., A new omega-conotoxin that targets N-type voltage-sensitive calcium channels with unusual specificity. Biochemistry, 2001. 40(48): p. 14567-75.

16. Cartier, G.E., et al., A New alpha-Conotoxin Which Targets alpha3beta2 Nicotinic Acetylcholine Receptors. J. Biol. Chem., 1996. 271(13): p. 7522-7528.

17. Barbier, J., et al., A delta-conotoxin from Conus ermineus venom inhibits inactivation in vertebrate neuronal Na+ channels but not in skeletal and cardiac muscles. J Biol Chem, 2004. 279(6): p. 4680-5.

18. Gotti, C. and F. Clementi, Neuronal nicotinic receptors: from structure to pathology. Progress in Neurobiology, 2004. 74(6): p. 363-396.

19. Unwin, N., Refined Structure of the Nicotinic Acetylcholine Receptor at 4 A Resolution. Journal of Molecular Biology, 2005. 346(4): p. 967-989.

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22. Rose, K. and J. Vizzavona, Stepwise Solid-Phase Synthesis of Polyamides as Linkers. J. Am. Chem. Soc., 1999. 121(30): p. 7034-7038.

23. Gaertner, H.F. and R.E. Offord, Site-specific attachment of functionalized poly(ethylene glycol) to the amino terminus of proteins. Bioconjug Chem, 1996. 7(1): p. 38-44.

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VI. ABBREVIATIONS ACh Acetylcholine AD Alzheimer’s disease ADHD Attention-deficit hyperactivity disorder BCL B cell lymphoma (in mouse) Boc tert-Butoxycarbonyl group Boc-AoA Boc-aminooxyacetyl group Boc-AoA-OSu Boc-aminooxyacetyl-N-hydroxysuccinimide ester BOP Benzotreazole-1-yl-oxy-tris(dimethylamino)-

phosphoniumhexafluorophosphate cGMP Cyclic guanosine monophosphate CNG channel Cyclic-nucleotide gated channel COMP Cartilage oligomeric matrix protein Ctx Conotoxin DCM Dichloromethane DIEA N,N-Diisopropylethylamine DMF Dimethylformamide DMSO Dimethylsulfoxide EDA Ethylene diamine Fmoc Fluorenylmethoxycarbonyl group GAGA

receptor Gamma-aminobutyric acid receptor

GSH L-Glutathione reduced form GSSG L-Glutathione oxidized form HBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HEK-293 cell Human embryonic kidney cell HF Hydrogen fluoride HOBt Hydroxybenzotriazole HPLC High-performance liquid chromatography IC50 half maximal inhibitory concentration ICK Inhibitor cysteine knot Ig Immunoglobulin IgM/G Immunoglobulin M/G Kv Voltage-gated potassium channel Laa 2-amino-D,L-dodecanoic acid (C12) LM Linear mode MALDI Matrix-assisted laser desorption/ionization MBHA p-Methylbenzhydrylamine MS Mass spectrometry nAChR Nicotinic Acetylcholine Receptor NMM N-methyl-morpholine NMP N-methyl-pyrrolidone PD Parkinson’s disease PEG Polyethylene glycol PLD Polymer ligand dimer

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PTM Post-translational modification RM Reflector mode RP-HPLC Reverse-phase high-performance liquid chromatography SASRIN resin Super acid-sensitive resin SCAL Safety catch amide linker Suc Succinic anhydride TFA Trifluoroacetic acid TFMSA Trifluoromethanesulphonic acid TOF Time-of-flight TRP(V1) Caspain receptor ∆Gavidity Avidity free energy

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