MXPA00005010A - Histamine and serotonin binding molecules - Google Patents

Abstract

The present invention relates to histamine and serotonin binding molecules that possess a binding site with the precise molecular configuration that is necessary to confer on the molecule a high affinity for histamine. The invention includes proteins, peptides and chemical compounds that possess this molecular configuration and that are thus able to bind to histamine with high affinity. These molecules may be used in the regulation of the action of histamine or serotonin, the detection and quantification or histamine or serotonin and in the treatment of various diseases and allergies. The molecules may also be used as components of vaccines directed against blood-sucking ectoparasites.

Description

MOLECULES OF UNION TO HISTA INA AND SEROTONINA The present invention relates to histamine and serotonin binding molecules. More particularly, the present invention relates to molecules that possess a binding site with the precise molecular configuration that is necessary to confer in the molecule a high affinity for histamine. Included as the embodiments of the present invention are those "proteins, peptides" and chemical compounds which possess this molecular configuration and which are thus capable of binding to histamine with high affinity.The molecules of the present invention can be used in the regulation of the action of histamine or serotonin and thus are useful in the detection and quantification of histamine or serotonin and in the treatment of various diseases and allergies.The vasoactive amines such as histamine and serotonin are mediators of inflammation and regulators of certain physiological processes in animals, including humans.Histamine is present in secretory granules of mast cells and basophils and is formed by the REF .: 120459 decarboxylation of histidine. Also present in cornice and plants and can be synthesized synthetically from histidine or citric acid. The main actions of histamine in humans are the stimulation of gastric secretion, contraction of most smooth muscle tissue, cardiac stimulation, vasodilatation and increased vascular permeability. In addition to its regulatory role in immune reactions and inflammatory processes, histamine also modulates the production of many cytokines in the body (including those that regulate inflammation) and may interfere with the expression of cytokine receptors. In addition, histamine promotes wound healing. The main pathophysiological roles of histamine are as a stimulant of gastric acid secretion and as a mediator of type I hypersensitivity reactions such as urticaria and hay fever. Histamine and its receptors also have pathological aspects to their functions. They play dominant roles in allergies such as asthma, allergic rhinitis, atopic dermatitis and allergies to food and drugs, which affects a large number of people and are a major cause of disease and mortality. Histamine or its receptors may also be comprised either directly or indirectly or in autoimmune disease, for example, arthritis, and in tumor growth (Falus, 1994). The hormone serotonin (also known as 5-hydroxytryptamine) is also a vasoconstrictor and a neurotransmitter. It can also increase vascular permeability, induce dilatation of capillaries and cause contraction of non-vascular smooth muscle. Serotonin is present in the brain and intestinal tissues and is produced by the pineal gland and by blood platelets. The pathological aspects related to serotonin include abnormal blood pressure, migraine, physiological disorders, respiratory disease and coronary heart disease. Serotonin agonists and antagonists are used to treat some of these disorders, but again they often have undesirable side effects. Anti- hislamin drugs are used extensively, especially for the treatment of allergies, most of these drugs are compounds that are structurally related to histamine, and bind to their receptor (s), thus obstructing the interaction of histamine with its receptor (s). These drugs since they are currently available frequently have undesirable side effects (for example numbness) and are not always effective. Histamine produces its actions by an effect on specific histamine receptors that are of three main types, Hi, H2 and H3, distinguished by means of antagonist drugs and selective agonists. Hi and H2 histamine antagonists have clinical uses, but at present histamine H3 antagonists are used mainly as research tools. Intracellular histamine appears to be involved in cell growth (promotion of tumor growth) and tissue repair. Intracellular histamine receptors, currently undefined, are thought to be involved in these processes (Falus, 1994). The histamine receptors have been the subject of concentrated research for several years. However, little information is available regarding the structure of the active site of these molecules, in reality the H3 receptor has not yet been cloned. The lack of any direct structural information for these proteins presumably is due to the fact that the histamine receptor proteins are membrane proteins that are denatured in the absence of the lipid and consequently are very difficult to crystallize. Based on the fact that Hi and H2 type receptors correspond to the broad class of seven transmembrane G protein coupled receptors, it can be assumed that they are mainly alpha-helical. Several sequence-directed mutagenesis studies have been conducted on many receptors, which have indicated that certain residues are important for binding to histamine. In the H2 receptor, it is believed that Asp98, Asp186, and Thr190 contribute to the histamine binding receptacle (Gantz et al., 1992). Hx-receptor antagonists, conventional as anti- his- tamines, are widely used to treat allergic reactions including allergic rhinitis (hay fever), urticaria, insect bites and drug hypersensitivities. Drugs lacking receptor agonists, sedatives or muscarinics are preferred. Hi receptor antagonists are also used as anti-emetics for the prevention of motor or other nausea causes including severe morning sickness. The actions of the receptor antagonists, muscarinic of some anti- histamines probably contribute to the efficacy, but they can also cause side effects. Some antagonists of the Hi receptor are completely strong sedatives and can be used for this action. However, there are numerous undesirable effects of commonly used Hi receptor antagonists. When used for purely anti-histamine actions, all effects on the CNS are undesirable. When used for its sedative or anti-emetic actions, some of the CNS effects such as dizziness, ear ringing and fatigue are undesirable. Excessive doses can cause excitement and can cause seizures in children. The peripheral anti-muscaric actions are always undesirable. The most common of this is the dryness of the mouth, very blurred vision, constipation and retention of urine may also occur. In the undesirable effects not related to the pharmaceutical action of the drug are also seen. In this way, gastrointestinal disturbances are completely common, while allergic dermatitis can follow the topical application of these drugs. H2 antagonists are frequently used as inhibitors of gastric acid secretion. They are used as the drugs of choice in the treatment of peptic ulcer, as second-line drugs in the treatment of Zollinger-Ellison syndrome and for treating reflux esophagitis. Unwanted effects have been reported, including diarrhea, dizziness, muscle aches, momentary rashes, and hyper-gastrinemia. Some H2 receptor antagonists can cause gynecomastia in men and confusion in the elderly. In addition to these unwanted effects, some histamine antagonists are problematic if taken with alcohol or drugs. For example, Seldane antibacterial used in combination with antibiotics and antifungals can cause life-threatening side effects.Therefore, it can be seen that the drugs used to control the actions of histamine are not always effective. The reason why they may have limited efficacy may be related to the specificity of these drugs for only a sub-class of histamine receptors, particularly when a certain class of conditions requires interference with a larger class of receptors. Molecules that currently bind to histamine by themselves will compete for histamine binding "with all receptors and thus may be more suitable for the treatment of certain conditions.Thus, there is a great need for effective antagonists of histamine and serotonin that do not generate the side effects that impair their api icabi lity to the treatment of human and animal disorders There is also a great need for the quantification of histamine, for example, in food products, various body fluids (for example, plasma or urine) or cell culture supernatant to monitor the effects of certain allergens, for example, or to indicate a potential antagonistic therapy, specific to an allergic reaction. The commonly used systems (radioimmunoassays and ELISA) use antibodies against histamine or against histamine derivatives. However, histamine is not very immunogenic, making it difficult to formulate high affinity antibodies against it, and most of the quantification systems that are currently used are not very sensitive or require the modification of the histamine that is measured (e.g. by methylation or acylation). The disorder of "" 'molecules that bind to histamine in its natural form that will replace the antibodies in the assay like these will provide a highly sensitive system for the measurement of unmodified histamine. Similarly, molecules that bind to serotonin could be used for the quantification of this molecule. Molecules capable of binding to histamine have previously been identified in ectoparasites that feed on blood. For example, a hemoprotein carrying nitric, salivary, or troforin oxide from the triatoma insect Rh odn i u s prol i xus has been found to bind to histamine (Ribeiro and alker, 1994). The isolation of four tick-binding amine-binding proteins (VABPs) from ticks is described in Co-pending International Patent Application No. PCT / GB97 / 01372 which is owned by the Applicant of the present invention. The contents of PCT / GB97 / 01372 are incorporated in the present application in its entirety. These proteins bind to histamine and are closely related to each other. They are named MS-HBP1, FS-HBP1, FS-HBP2 and D.RET6. Some of these molecules also tend to be serotonin (for example, FS-HBP2). In other cases, such as in the case of D.RET6 for example, the binding of serotonin is thought to alter the affinity of the molecules for histamine. The DNA sequences encoding these proteins are currently being used to isolate other related proteins from the same family from the same and different species. These molecules appear to differ markedly from histamine binding proteins from any of the Hx, H2 or H3 families and appear to bind to histamine in a different way. The elucidation of the structure of the histamine binding site that is in molecules will markedly accelerate the rational design of effective histamine antagonists that will not likely suffer from the side effects that are associated with conventional anti-his t amine agents.

BRIEF DESCRIPTION OF THE INVENTION According to a first aspect of the present invention, there is provided a histamine or serotonin binding compound capable of binding to histamine or serotonin with a constant dissociation of less than 10"7M and having a binding site comprising the residues of amino acid phenylalanine, isoleucine or leucine in the I position, tryptophan in. position I and aspartate or glutamate in positions III and IV, where residues I or IV are placed substantially the same as residues 108, 42, 39 and 82, respectively in any of SEQ ID Nos. 1 or 2, with the residues 107, 41, 38 and 78 in SEQ ID NO: 3 or in residues 122, 54, 50 and 95 in SEQ ID NO: 4 and the functional equivalents thereof. Subsequently, this binding site will refer to it as the "first binding site". The proteins identified in SEQ ID Nos. 1 or 4 are known as FS-HBP1, FS-HBP2, MS-HBP and D.RETβ, respectively. According to a second aspect of the present invention, there is provided a histamine or serotonin binding compound capable of binding to histamine or serotonin with a dissociation constant of less than 10 ~ 7M and having the binding site comprising the residues of amino acids phenylalanine or isoleucine in residue I, tryptophan in residue II and aspartate or glutamate in residues III and IV, where residues I or IV are placed substantially identical as residues 98, 137, 24 and 120, respectively in any of SEQ ID Nos. 1 or 2, or residues 95, 138, 23 and 120 in SEQ ID NO: 3 or residues 112, 149, 35 and 135 in SEQ ID NO: 4, and functional equivalents thereof. Subsequently, this binding site will be referred to as the "second binding site". According to a third aspect of the invention, there is provided a histamine or serotonin binding compound capable of binding to histamine or serotonin with a constant dissociation of less than 10"7M and comprising both the first and second binding sites described previously, and the functional equivalents thereof For some histamine binding compounds containing both the first and second binding sites (such as D.RET6), the binding of serotonin to the compound is thought to alter the affinity of the compound for histamine Other chemical compounds with an action related to serotonin may also influence the binding of histamine to a histamine-binding compound that contains both the first and the second binding sites. of cysteinyl (such as leukotriene D4 and leukotriene E), the activation factor of platelets and thromboxanes. As an aspect of the present invention, there is provided a histamine or serotonin binding compound capable of binding to histamine or serotonin with a constant dissociation of less than 10 ~ 7M and comprising the sequence D.RET6 (SEQ ID NO: 4) or a fragment thereof and comprising a first and a second binding site as defined above, or functional equivalents thereof, wherein the binding of serotonin to the compound increases the affinity of the compound for histamine.

By "binding site" is meant the specific region of the compound that contributes directly to the binding of a molecule of histamine or serotonin. As such, the binding to this site will comprise the molecular recognition events between the binding site and the histamine or serotonin molecule, regulated by functional complementarities of shape, size, charges, H bonds, hydrophobic interactions and pi and van forces. der aal. The interactions can also comprise covalent chemical bonds. By the term "functional equivalent" is meant compounds that possess the desired binding site and include any macromolecule or molecular entity that binds to histamine or serotonin with a constant dissociation of 10 ~ 7M or less and which possess an equivalent complementarity of form that possessed by the binding sites of the histamine or serotonin binding molecules identified in any of SEQ ID Nos. 1 to 4. A functionally equivalent complementarity of forms can be provided by any hydrogen, oxygen, phosphorus and nitrogen atom which are substantially placed as identified in the structures described herein. Certain methods of generating compounds with affinity for a molecule of interest have until recently been relatively primitive. The notion of combination chemistry and the generation of combination libraries has developed, however, at high speed and facilitates the rational design and improvement of molecules with desired properties. These techniques could be used to generate molecules possessing identical binding sites and similar to those of the histamine or serotonin binding sites identified herein. These compounds can be generated by rational design, using for example normal synthesis techniques in combination with molecular modeling and computer visualization programs. Under these techniques, the "day" compound with a framework similar to the histamine or serotonin binding site is optimized by combining a variety of components and substituents. Alternatively, or as a step in the structure-guided design of a molecular entity, combination chemistry can be used to generate or refine the structure of the compounds that emit the histamine or serotonin binding site of the binding compounds. to histamine or serotonin by producing congenial combination arrangements around a frame stage. These steps may include normal synthesis of peptide or organic molecules with a split-phase and solid-phase recombination process or unitary parallel combination synthesis using either solid phase or solution techniques (see, for example, Hogan, 1997 and references cited in the same ) . Alternatively, or as a portion of a histamine or serotonin binding molecule of the present invention, functional equivalents may comprise fragments or variants of the proteins identified in Figures 1 to 4 or closely related proteins that exhibit significant homology to the sequence. By "fragment" is meant any portion of the complete protein sequence that retains the ability to bind to vasoactive amines with a dissociation constant of 10 ~ 7M or less. Accordingly, fragments containing single or multiple amino acid deletions of any term of the protein or internal stretches of the primary amino acid sequence form an aspect of the present invention. Variants may include, for example, mutants containing substitutions, insertions or deletions of amino acids from the wild-type sequence of Figures 1 to 4. The man skilled in the art will understand that the residues that contribute to the binding of the amines vasoactive in the four proteins explicitly identified herein remain in the relevant position for binding to histamine or serotonin through the framework structure of the protein. In this way, the framework residues of the proteins are responsible for the exact placement of the binding amino acids. Accordingly, it is contemplated that any molecular framework capable of retaining these side chains of amino acids at the positions necessary for binding to histamine or serotonin will be suitable for use in accordance with the present invention. Of particular suitability will be the cyclic peptides maintained in a precise framework by their linking groups and bonds.

The amino acid side chains can be maintained in a position substantially identical to their position at the histamine or serotonin binding site of the native histamine or serotonin binding compounds. Preferably, the cyclic peptides comprise between 6 and 30 amino acids, preferably between 8 and 20 amino acids. Of particular suitability is the cyclic octapeptide Ala-Glu-Ala-Phe-Ala-Glu-Ala-Trp. The biologically active peptides are histamine or serotonin binding sites according to the present invention can be generated using phage libraries.The nucleic acids are encoded for the amino acid residues identified as participants in the binding of histamine or serotonin, together with the nucleic acid encoding the surrounding residues of the framework can be fused to give an idiogenic polypeptide unit of between 10 and 1000 residues, preferably between 25 and 100 residues, by fusion of this nucleic acid fragment with what it encodes for a phage protein, for example pIII of the bacteriophage fd, the fusion molecule can be displayed on the phage surface.The detection of the phage library with histamine or serotonin will then identify these clones of interest.These clones can then be subjected to rounds Interactive mutagenesis and detect to improve the affinity of the molecules generated for histamine os erotonin Residues with physical properties analogous to those comprising the histamine or serotonin binding site may also be part of a molecule according to the present invention. For example, with respect to the FS-HBP2 protein, any of the charged residues glutamate or aspartate can occupy position 39 and 82 in the sequence. At position 108 in the sequence, it is contemplated that any hydrophobic amino acid residue may occupy this site, provided that the spherical interests are satisfied with respect to the molecular configuration of the binding site. Phenylalanine, isoleucine and leucine are preferred residues in this position. In position 42, tryptophan is preferred. Additionally, at position 100 in the sequence of the histamine binding compounds, it is preferred that a tyrosine residue be present. This molecule is thought to contribute to the stability of histamine at the binding site. Any molecular structure that retains this side chain of amino acids or an equivalent in this position forms an aspect of the present invention. Due to the variations in length and sequence of the four proteins explicitly described herein, the method for reducing residues differs between proteins. However, it will be "" evident from the alignment based on Figure 22 that the residues correspond to the residues numbered according to the sequence of FS-HBP2 It is contemplated that the proteins according to the present invention can be stabilized Because of the presence of disulphide bridges in the structure, for example, the cysteines found at positions 48, 169, 119 and 148 of FS-HBP2 are conserved in four amine histima proteins identified hitherto. FS-HBP2, one between cysteines 48 and 169, the other between 148 and 119. Accordingly, any protein fragment designed to limit the structure of the lateral binding site of the histamine or serotonin binding compound, these cysteine residues may are present in the sequence, so that one or both disulfide bridges are formed within the protein structure.It is preferred that in addition to the high affinity with which the compounds of the present invention When they bind to histamine or serotonin, this binding phenomenon is also specific for histamine or serotonin. The advantages of this specificity that confer to the compounds will be obvious to one skilled in the art. For example, for use as a pharmaceutical product or in the quantification of the histamine content of a solution, it is of primary importance that compounds other than histamine are not bound by the compounds of the present invention. In the case of a pharmaceutical product, the lack of specificity can lead to unwanted side effects; used in the quantification of histamine, non-specificity will lead to erroneous guidance and inaccurate results. According to a fifth aspect of the invention, there is provided a protein comprising the amino acid sequence given in SEQ ID NO: 5. This sequence encodes a salivary protein of Rhipicephalus appe? Diculatus named Ra-Res. Its main sequence was inferred from a cDNA obtained by detecting a cDNA library of the salivary gland of R. appendiculatus with the antiserum of a guinea pig that has developed resistance against ticks of R. appendiculatus. According to a sixth aspect of the invention, a protein comprising the amino acid sequence given in SEQ ID NO: 6 is provided. This sequence encodes a protein called Av-HBP from Amblyomma variegatum ticks. The preliminary results of the histamine binding studies suggest a constant equilibrium dissociation (Kd) of 7.3 nM. According to a seventh aspect of the invention, a protein comprising the amino acid sequence given in either SEQ ID NO: 7 or SEQ ID NO: 8 is provided. These sequences encode proteins called Ib / Bm-HBPl and Ih / Bm-HBP2, respectively, and were isolated from a mixed cDNA expression library of Boophilus microplus / Ixodes hexagonus. The library was detected with probes constructed from RT-PCR products obtained from the RNA of the salivary gland of Boophilus microplus / Ixodes hexagonus, using degenerate primers of which the design is based on conserved domains with the HBPs of R. appendiculatus ( FS-HBP1, FS-HBP2, MS-HBP1 and D. RET6). According to an eighth aspect of the invention, there is provided a protein comprising the amino acid sequence given in SEQ ID NO: 9, the amino acid sequence given in SEQ ID NO: 10, the amino acid sequence given in SEQ ID NO: 11. These sequences code for the proteins named Ib / Bm-HPB3, Ih / Bm-HBP4 and Ib / Bm-HBP5, respectively. These sequences were also isolated from the cDNA expression library, mixed Boophilus microplus / Ixodes hexagonus, mentioned above. These sequences show a convincing sequence similarity with the HBPs of R. appendiculatus (FS-HBP1, FS-HBP2, MS-HBP1 and D.RET6), but also contain extensive domains that are not present in the traditional sequences (see Figure 22). ). As will be clear to the person skilled in the art, the invention includes functionally equivalent derivatives and fragments of the protein sequences given in SEQ ID Nos. 6 to 11. "Functionally equivalent" is used in this context to indicate derivatives and fragments that require the ability to bind to vasoactive amines or containing epitopes that can be used in the development of antibody vaccines that target any of the Ra-Res, Av-HBP, Ih / Bm-HBPl, Ih / Bm-HBP2, Ih proteins / Bm-HBP3, Ih / Bm-HBP4, or Ih / Bm-HBP5. Derivatives can be derived from "the wild-type sequences of these proteins by substitutions, additions, insertions and / or single or multiple deletions of amino acids or by chemical modification of one or more of the amino acids, for example by deglycosides and of the glycosylated forms The invention also includes proteins in the same family as Ra-Res, Av-HBP, Ih / Bm-HBPl, Ih / Bm-HBP2, Ih / Bm-HBP3, Ih / Bm-HBP4 or Ih / Bm-HBP5 A protein is considered as corresponding to the same family as any of these proteins if 40% or more of the amino acids in the sequence are conserved.For example, the proteins can be compared in this way using the order GCG pileup (Program manual for the Wisconsin package, 1994, separation creation permeability = 2.50, separation extension penalty = 0.05) The proteins of these aspects of the invention include natural biological variants or geographical variations raficas- within the species from which the proteins are derived. For example, a derivative may include an additional protein or polypeptide fused to one of these proteins at its amino or carboxy terminus, or added internally in the sequence "" The purpose of the additional polypeptide may be to aid in the detection, expression of the Separation or purification of the protein or can be to grant additional properties to the protein as desired Synthetic molecules designed to mimic the tertiary structure or the active site of the proteins Ra-Res, Av-HBP, Ih / Bm-HBPl, Ih / Bm-HBP2, Ih / Bm-HBP3, Ih / Bm-HBP4, or Ih / Bm-HBP5 constitute a further aspect of the invention For many applications, the compounds according to the present invention can be fused to a molecule effector or indicator such as a tag, toxin or bioactive molecule These molecules may comprise an additional protein or polypeptide fused to the histamine or serotonin binding compound at its amino or carboxy or additional terminus nar internally. The purpose of the additional polypeptide may be to aid in the detection, expression, separation or purification of the histamine or serotonin binding compound or may be to confer additional properties to the compound as desired. Particularly suitable candidates for fusion will be indicator molecules such as luciferase, green fluorescent protein, or horseradish peroxidase. The reaction labels can be radiolabels or molecules that are detectable in a spectroscopic manner, for example fluorescent or phosphorescent chemical groups. Binding molecules such as streptavidin or biotin can also be used. Additionally, other peptides or polypeptides may be fused to a histamine or serotonin binding compound. Suitable peptides can be, for example, β-galactosidase, glutathione-Stransferase, luciferase, poly ihis tidine tags, secretion signal peptides, the Fe region of an antibody, the FLAG peptide, the cellulose binding domain , calmodulin and the maltose binding protein. Antibodies or peptides used to target histamine or serotonin binding compounds more effectively towards a site of action (e.g., antibodies against mast cell membrane proteins), may also be fused to histamine binding compounds or serotonin These fusion molecules can be fused chemically, using methods such as chemical crosslinking. "Suitable methods will be well known to those skilled in the art and can comprise for example, thiol group crosslinking of the cysteine residues, or crosslinking using In most cases, chemical cross-linking will be used to fuse non-protein compounds, such as cyclic peptides and labels, and when it is desired to fuse two protein molecules, the method of choice will often be to fuse the molecules genetically. In order to generate a recombinant fusion protein, the genes or gene portions coding for proteins or protein fragments of interest are engineered to form a contiguous gene arranged so that the codons of the two sequences are transcribed in the frame. The compounds of the present invention may also comprise These bind to histamine or serotonin bound to a support that can be used to remove, isolate, or extract histamine or serotonin from body tissues, blood or food products. The support can comprise any suitable inert material and includes gels, magnificent or other gels, microspheres, binding columns and resins. If it is proteinaceous, the histamine or serotonin binding compound can be derived from any organism possessing a protein in the same family as the histamine or serotonin binding compounds identified to date. By "protein family" is meant a group of polypeptides that share a common function and exhibit a sequence homology common among the portions present in the appropriate polypeptide sequences. By sequence homology it is meant that the appropriate polypept sequences are related by divergence from a common ancestor.

Preferably, proteins or protein fragments are derived from ectoparasites that feed on blood, spiders, scorpions or vipers or other poisonous animals. More preferably, proteins or protein fragments are derived from ticks, more preferably Ixodid ticks such as, for example, Rhipicepha lus appendiculatus. More preferably, the proteinaceous compounds according to the present invention are derived from any of the proteins FS-HBP1, FS-HBP2, MS-HBP1, D.RET6, Ra-Res, Av-HBP, Ih / Bm -HBPl, Ih / Bm-HBP2, Ih / Bm-HBP3, Ih / Bm-HBP4 or Ih / Bm-HBP5. The protein or peptide compounds according to the invention will preferably be expressed in recombinant form by the expression of coding DNA in an expression vector in a host cell. These expression methods are well known to those skilled in the art and many are described in detail in DNA cloning; a practical approach, Volume II: Expression Systems, edited by D.M. Glover (IRL Press, 1995) or in DNA cloning: a practical approach, Volume IV; Mamma 1 ian systems, edited by D.M. Glover (IRL Press, 1995). Protein compounds can also be prepared using known techniques of genetic engineering such as random or sequence directed mutagenesis as described, for example, in Molecular Cloning: a Laboratory Manual; 2nd edition (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press) or Protein Engineering: A Practical approach (edited by A. R. Rees et al., IRL Press 1993). Suitable expression vectors can be chosen for the host of choice. The vector may contain a recombinant DNA molecule encoding the compounds of the present invention operably linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector contains this recombinant DNA molecule under the control of a promoter recognized by the transcription machinery of the host. Suitable hosts include commonly used prokaryotic species, such as E. coli, or eukaryotic yeasts which can be made to express high levels of recombinant proteins and which can be easily cultured in large quantities. Mammalian cell lines cultured in vitro are also suitable, particularly when using virus-filed expression systems such as baculovirus expression systems comprising the use of insect cells as hosts. The compounds can also be expressed in vivo, for example in insect larvae or in mammalian tissues. According to a ninth aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of a histamine or serotonin according to the first or second aspect of the invention or a protein according to the third, fourth, fifth, sixth , seventh or eighth aspects of the invention, in conjunction with a pharmaceutically acceptable excipient. Suitable excipients are well known to those skilled in the art and may comprise, for example, phosphate-buffered saline (0.01M phosphate sacs, 0.138M NaCl, 0.00271-: KCl, pH7.4). The pharmaceutical compositions may also contain additional preservatives to ensure a shelf life prolonged in storage.

According to a still further aspect, the present invention provides for the use of the compounds of a histamine or serotonin of the first, second, third or fourth aspects of the invention, of a protein according to the fifth, sixth, septic or eighth aspects of the invention or of the pharmaceutical compositions of the ninth aspect of the invention in therapy. The histamine or serotonin binding compounds, proteins or compositions can be used as anti-inflammatory agents that can be used to bind histamine or serotonin in mammals, to arrange in this way its action and to control its pathological effects. This causes sequestration and thus decreases the effective concentration of histamine or serstonin in the body. This results in a physiological response warmed or even completely abolished, depending on the dose used. The histamine or serotonin binding compounds of the present invention can also be used as anti-inflammatory agents or agents to counteract the effects of allergic reactions on the body. According to this aspect of the invention, the histamine or serotonin binding compounds, proteins or compositions can be used in conjunction with serotonin in order to alter the affinity of the compounds for histamine. For example, for the compound D.RET6 it is shown here that serotonin significantly increases the affinity of the compound for histamine. Compounds related in action to serotonin can also be used, such as cysteinyl leukotrienes (such as leukotriene D4 or leukotriene E4), platelet activation factor, or thromboxanes. The histamine or serotonin binding compound according to the first, second, third or fourth aspect of the invention or protein according to the fifth, sixth, seventh or eighth aspects of the invention may constitute the active, unique component of the composition or it can be part of a therapeutic package, such as a component of creams for topical administration to insect bites, vipers or scorpions, or to skin affected by dermatitis. The proteins can also be used as carrier molecules for histamine or serotonin and compounds related to histamine or serotonin, in creams, oils, powders, or pills, to provide slow release of the bound histamine or serotonin. The invention also comprises the use of the compounds of the present invention as histamine or serotonin binding components in equipment for the detection or quantification of histamine or serotonin levels (e.g., in blood, nasal lavage fluid, tissues or products). food). This equipment will resemble radio-immunoassay equipment and will comprise a histamine-binding compound or "serotonin" according to the present invention and a detection means that allows the exact quantification of the amount of histamine or serotonin in the fluid. The amount of radiolabelled histamine or serotonin, for example, titrated histamine or serotonin titrated, is added to the sample being measured.Histamine or serotonin in the sample will then compete with labeled histamine or serotonin for binding to the limited number of sites of binding possessed by the histamine or serotonin binding compounds also present in the sample, in this way, the amount of histamine or serotonin present in the sample can be accurately assessed.

One aspect of the present invention comprises kits that incorporate the histamine or serotonin binding compounds of the present invention. The histamine or serotonin binding compounds can be attached to magnetic beads, agarose beads or can be fixed to the bottom of a multi-well plate. This will allow the removal of marked, unbound histamine or serotonin from the sample after incubation. Alternatively, the protein can be attached to SPA beads (Proximity Assay for Scintillation), in which case there is no need to remove the unbound ligand. Using a set of unlabeled histamine or serotonin standards, the results obtained with this standard can be compared with the results obtained with the sample to be measured. The histamine or serotonin binding compounds of the first, second, third or fourth aspects of the invention, or proteins according to the fifth, sixth, seventh or eighth aspects of the invention can also be used for the detection of histamine or serotonin. Any technique common in the art, such as a detection method, can be used and can comprise the use of transfer techniques (Towbin et al., 1979), binding columns, gel delay, chromatography, or any of the other methods suitable that are widely used in the art. In another embodiment, the histamine or serotonin binding compound can be fused either genetically or synthetically to another protein such as alkaline phosphatase, luciferase or peroxidase in order to facilitate its detection. It may be preferred to include serotonin or a related compound in the kits according to this aspect of the invention, in order to alter the affinity of the histamine binding compound for histamine. These related compounds include cysteinyl leukotrienes (such as leukotriene D4 or leukotriene E4), platelet activation factor, and thromboxanes. These will be particularly preferred with the compound D.RET6 or a functional equivalent thereof is used as the active histamine binding compound of the kit. The invention also comprises the use of the histamine or serotonin binding compound of the first, second, third or fourth aspect of the invention, or proteins according to the fifth, sixth, seventh or eighth aspects of the invention as histamine binding entities. or serotonin., attached to a support that can be used to remove, isolate or extract histamine or serotonin (from body tissues, blood or food products). The support can comprise any suitable material and includes gels, beads, microspheres, binding columns and resins. The histamine or serotonin binding compound can be linked, for example chemically or enzymatically, to the reactive groups on these supports. The present invention also includes the use of a histamine or serotonin binding compound of a first, second, third or fourth aspects of the invention, by a protein according to the fifth, sixth, seventh or eighth aspects of the invention as tools in the study of inflammation, processes related to inflammation or other physiological aspects of vasoactive amines such as the role of histamine in the formation of gastric ulcers. For example, the histamine and serotonin binding compound can be used for the depletion of histamine or serotonin in cell cultures or in inflamed animal tissues, in order to study the importance of histamine or serotonin in these systems. The histamine or serotonin compounds can be pre-incubated with serotonin, or a related compound to implement the affinity of the compounds for histamine. The present invention also provides the use of the histamine or serotonin binding compounds of the first, second, third or fourth aspects of the invention, and of a protein according to the fifth, sixth, seventh or eighth aspects of the invention, such as immunogens for use as vaccines of metazoan parasites and in particular as protective immunogens in the control of diseases caused by arthropods and other metazoan parasites Suitable candidates for vaccination include domestic animals such as cattle., goats, sheep, dogs, cats and other animals that require protection against metazoan parasites, especially ticks. The vaccine may include adjuvants of the type that are known in the art. Nucleic acid molecules comprising a nucleotide sequence encoding a histamine or serotonin binding compound of the first, second, third or fourth aspects of the invention, purify for a protein according to the fifth, sixth, seventh or eighth aspects of the invention form additional aspects of the invention. These molecules include DNA, cDNA and RNA, as well as synthetic species of nucleic acid. The complementary DNAs that code for the histamine or serotonin molecules, according to the proteins FS-HBP1, FS-HBP2, MS-HBP1, D.RET6, Ra-Res, Av-HBP, Ih / Bm-HBPl, Ih / Bm-HBP2, Ih / Bm-HBP3, Ih / Bm-HBP4 or Ih / Bm-HBP5, are described herein in Figures 1 to 11 (nucleotides and amino acids are given in their normal one-letter abbreviations). The preferred nucleic acid molecule, according to the invention, comprises a nucleotide fragment identical to or complementary to any portion of any of the nucleotide sequences shown in Figures 1 to 11 which codes for a histamine or serotonin binding compound, or a sequence that degenerates or is substantially homologous with it, or that hybridizes with this sequence. By "substantially homologous" is meant to mean the sequence exhibiting at least 60% sequence homology. "Hybridization sequences" included within the scope of the invention are those junctions under non-severe normal conditions (6 X SSC / 50% formamide at room temperature) and washed under conditions of low stringency (2 x SSC, room temperature, or 2 x SSC, 42 ° C) or preferably under normal conditions of high severity, for example, 0.1 x SSC, 65 ° C (where SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2). The nucleic acid sequences according to the invention may be single or double stranded DNA, cDNA or RNA. "Preferably, the nucleic acid sequences comprise DNA The invention also includes cloning and expression vectors containing the DNA of the invention These cloning vectors will incorporate appropriate transcriptional and transductional control sequences, for example, enhancer elements, promoter-operator regions, termination stop sequences, mRNA stability sequences, start and stop codons or In addition, in the absence of a signal peptide naturally effective in the protein sequence, it may be desirable to cause the recombinant protein to segregate from certain hosts. Therefore, additional components of these vectors may include sequencing nucleic acid encodes encoding the secretion processing signaling sequences. The vectors according to the invention include plasmids and viruses (including both bacteriophage and eukaryotic viruses). Many of these vectors and expression systems are well known and documented in the art. Particularly suitable viral vectors include vectors based on baculovirus, adenovirus and vaccinia virus. The expression of heterologous polypeptides and polypeptide fragments in prokaryotic cells such as E. coli is well established in the art; see, for example Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press or DNA cloning: a practical aproach, Volume II: Expression systems, edited by D.M. Glover (IRL Press, 1995). Expression in eukaryotic cells in culture is also an option available to those skilled in the art for the production of heterologous proteins; for example O'Reilly et al., (1994) Baculovirus expression, vectors - a laboratory manual (Oxford University Press) or DNA cloning: a practical approach, Volume IV: Mammalian systems, edited by D.M. Glover (IRL Press, 1995). Suitable vectors can be chosen or constructed for the expression of histamine or serotonin binding proteins, which contain the appropriate regulatory-as sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. The vectors can be plasmids, virals for example bacterifagos or fagemid, as appropriate. For additional details see Molecular Cloning; a Laboratory Manual. Many known techniques and protocols for the manipulation of nucleic acid, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of AD? in cells and gene expression, and protein analysis, are described in detail in Short Protocols i si Molecular Biology, Second Edition, Ausubel et al., eds., (John Wiley &Sons, 1992) or Pro tein Engineering: A Pra cti ca l approa ch (edited by AR Rees et al., IRL Press 1993). For example, in eukaryotic cells, the vectors of choice are virus-based. A further aspect of the present invention provides a host cell containing a nucleic acid encoding a histamine or serotonin binding compound of the first, second, third or fourth aspects of the invention, or coding for a protein according to the fifth, sixth, seventh or eighth aspects of the invention. A still further aspect provides a method comprising introducing the nucleic acid into a host cell or organism. The introduction of nucleic acid can employ any available technique. Eukaryotic cells, in suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection or transduction using retroviruses and other viruses, such as vaccinia or for insect cells, baculovirus. In bacterial cells, suitable techniques may include transformation with calcium chloride, electroporation or transfection using bacteriophage. The introduction of nucleic acid can be followed by making or allowing expression of the nucleic acid, for example by culturing host cells under conditions for gene expression. In one embodiment, the nucleic acid of the invention and integrates into the genome (eg, chromosomes) of the host cell. The integration can be "" promoted by inclusion of sequences that promote recombination with the genome, according to normal techniques. Transgenic animals transformed to express or overexpress r in the germ line one or more histamine or serotonin binding compounds as described herein form a still further aspect of the invention, together with methods for their production. There are now many techniques for introducing transgenes from the junctions or germ lines of an organism, as illustrated for example in Watson et al., (1994) Recombinant DNA (2nd edition), Scientific American Books. A variety of techniques for introducing the vectors according to the present invention are known and can be used prokaryotic or eukaryotic enzymes. The proper techniques of transformation or transfection are well described in the literature Mol ecul a r on on g a a ry Labora tory Man u al: 2nd edition, (Sambrook et al., 1989, Cold spring Harbor Laboratory Press). In eukaryotic cells, the expression systems can be either transient (eg, episomal) or permanent (chromosomal integration) according to the needs of the system. See, for example, Short Pro t ocol s in Mol e cul a r Bi ol ogy, Second Edition Ausubel et al., Eds., (John Wiley & amp;; Sons, 1992). All documents mentioned in the text are incorporated herein by reference. The various aspects and embodiments of the present invention will now be described in more detail by way of example, with particular reference to the histamine or serotonin binding compounds, prot einacea, isolated from ticks. It will be appreciated that modification of the detail can be made without departing from the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is the sequence of FS-HBP1 (SEQ ID NO: 1), which shows the sequencing primers and the sequencing strategy. Figure 2 is the sequence of FS-HBP2 (SEQ ID NO: 2), which shows the sequencing primers and the sequencing strategy. Figure 3 is the sequence of MS-HBP1 (SEQ ID NO: 3), which shows the sequencing primers and the sequencing strategy. Figure 4 is the sequence of D.RET6 (SEQ ID NO: 4), which shows the sequencing primers and the sequencing strategy. Figure 5 is the sequence of ra-RES (SEQ ID NO: 5). The asparagine that are part of the putative glycosylation recognition sites are underlined and shown in italics. The wavy line denotes a possible nesting site I; the double line indicates the putative polyadenylation signal and the poly A-extremity is shown in bold typeface. Figure 6 is the sequence of Av-HBP (SEQ ID NO: 6). The sequence of Av-HBP cDNA and its inferred primary structure. The cDNA has a remarkably long non-coding region, a 5 'direction of the stop codon. Figure 7 is the sequence of Ih / Bm-HBPl (SEQ ID NO: 7). Figure 8 is the sequence of Ih / Bm-HBP2 (SEQ ID NO: 8). Figure 9 is the sequence of Ih / Bm-HBP3 (SEQ ID NO: 9). Figure 10 is the sequence of Ih / Bm-HBP4 (SEQ ID NO: 10). Figure 11"is the sequence of Ih / Bm-HBP5 (SEQ ID NO: 11) Figure 12 is a 12% SDS-polyacrylamide gel obtained with silver showing the fractions obtained from a resin column of nickel used to purify recombinant D.RET6 expressed in insect cells, A through flow fraction, B, first wash fraction (10 volumes of phosphate buffer pH 6.5), C, second wash fraction (with imidazole), mM); D, third wash fraction (with 10 mM imidazole); E fourth wash fraction (with 15 mM imidazole); F, fifth wash fraction (with 20 mM imidazole); G, 300 mM imidazole elution fraction; H, D.RET 6 purified after ion exchange chromatography, Figure 13 is a 12% SDS polyacrylamide gene obtained with silver from D.RET6 overexpressed in insect cells. D.RET6 purified as a monomer (25 kDa), dimero (50 kDa) and trimer (85 kDa) and lanes C which is after the deglycosylation. The cleaved oligosaccharides were retained in the stacking gel and can be seen at the top of lanes C. The excess of PNGase-F appears as a band of approximately 30 kDa. Paths D show the purified monomeric form of D.RET6. Figure 14 shows the detection by Western transference of the histamine protein, native in the extract of the salivary gland of Derma cen t o r re t i cu l a t us. Path A, non-reduced form; path B, reduced form; path C, deglycosylated form. Figure 15: Determination of the IC50 of the three tagging competitors by example, histamine, 1-met i lhis tamine and 3-me t i lhis tamine by generating a competitive binding curve of recombinant D.RET6. The graphically derived IC 50 of unlabeled ligands above the radioactive histamine shift of the binding site by 50% are 133 nM, 750 μM and 365 μM, respectively. Figure 16 shows the curves "saturation of histamine binding in the absence of (solid line) or presence of (discontinuous lines) serotonin. * No serotonin, B 2. 4 μM and? 23.8 μM serotonin, respectively. Figure 17: Radioactive histamine binding assay of recombinant D.RET6 in the absence (A) and presence of serotonin at 2.4 μM (B) and 23.8 μM (C). Figure 18: Representation of unit histamine in the histamine binding site binding pocket of a cyclic peptide. Figure 19: Three-dimensional representation of the bound histamine in the histamine binding site binding pocket of the cyclic peptide. Figure 20: (a) diagram and tape showing the arrangement of molecules A and B in the asymmetric unit of FS-HBP2. Sheet 13 is labeled A-I and a-helices 1-3. The histamine ligands are shown as a representation of ball and stick, together with TyrlOO. The arrow indicates the direction of the view in (b) and the region in the table shows the increased portion of (c). (b) This view shows the propeller and the extended turn that occludes the barrel entrance. (c) Stereoscopic view of the individual hydrogen bond contacts between the residues in molecules A and B. (d) Traces of Ca superimposed on molecules A and B [performed using SHP, (Stuart et al-, 1979)] . Figure 21: Stereoscopic views of the H (a), L (b) and apo-L (c) histamine binding sites, respectively. In each case. Map 2 | Fo | - | Faith | is exhibited "around the ligand." The figure shows the core structure of the ligand.; that makes contact with the waste and the elements of the. secondary structures are marked. In (c) the joined structure L (transparent trace) has been superimposed on the coordinates apo. Figure 22: An alignment of the amino acid sequence inferred by cDNA of the various HBPs, created using the pileup commands of the Genetics Computer Group. (1994). Program Manual for the Wisconsin Package, Version 8. (575 Science Drive, Madison, Wisconsin, USA 53711).

EXAMPLES Ticks Ticks were created according to Jones et al., (1988), as described in detail in the co-pending International Patent Application PCT / GB97 / 01372, the contents of which are incorporated herein in their entirety. The identification of the proteins FS-HBP1, FS-HBP2 and D-RET6, and the cloning of the coding genes is also described in PCT / GB97 / 01372 (see Examples 1, 2 and 3). -dO (Rhipicephalus appendiculatus) with mRNA from the salivary glands of non-fed R. appendiculatus ticks (127 males, 12 females) The library (Amblyomma variegatum) was constructed with mRNA isolated from salivary glands of ticks A. variegatum adult, partially fed (50 males, fed for 6-7 days, and 50 females, fed for 4 to 5 days) The Ih / Bm library (a mixed library of Ixodes hexagonus and Boophylus microplus) was constructed with the mRNA mixed in the salivary glands of Boophilus microplus ticks partially fed (30 females) and Ixodes hexagonusk fed for three days (20 males / 20 females).

Example 1: Cloning and sequencing of the libraries of bnblyomma variegatum, lxod.es hexagonus and Boophilus microplus Using the pure, total cRNA extraction kit (Bioline Ltd., UK), total RNA was isolated from the salivary glands of the mblyomma va riega tum, Ixodes hexagonus and Boophilus miroplus ticks partially fed. The RNA samples were subjected to reverse transcriptase polymerase chain reactions (RT-PCT), using the One-Tube Titan RT-PCR system (Boehringer Mannheim) and the degenerate primers: 5 '-AAYGGNGARCAYCARGAYGCNTGGAA; and 5 '-KTRTMRTCNGTNRYCCANARYTCRTA, the design of which was based on the conserved domains in the HBP of Rhipicephalus appendiculatus. Cyclization conditions were in accordance with the manufacturer's suggestion, but a fixation temperature of 48 ° C was used. The RT-PCR products were ligated into the pGEM-T vector (Promega) and partially sequenced, using the SP6 and T7 primer sites flanking the cloning site. The inserts from which the inferred amino acid sequences showed similarity to the original HBpi ceph HBPs were labeled with digoxigenin, using the High Prime DNA labeling equipment (Boehringer Mannheim), and used as probes to detect the libraries . An anti-digoxigenin antibody conjugated with alkaline phosphatase was used to visualize probe hybridization. The Ra-dO library was detected (according to Mierendrof et al., 1987) with guinea pig serum that has acquired resistance against R ticks. appendí cu l a t us after repeated infestations. The pBluescript SK (-) phagemids of positive clones were excised in vivo, using an auxiliary phage R408, as described by Shrot et al. , (1988).

Example 2: Expression of recombinant protein In Example 3 of PC / B97 / 01372 the expression of purification of FS-HBP1, FS-HBP2, MS-HBP1 is described in detail. 1) Construction of clones for D.RET6 Using an expression system based on E. coli, the DNA sequence coding for D was subcloned. RET 6 (from Glu 29 to Leu 109) as a fragment of Bcll / Xhol in pET-23a (+) digested with BamHI / XhoI in the same reading frame as the 6x His mark using the PCR technique with the following primers 5 '-TATATGATCAGAAAACCCGCTCTGGG-3' and 5 'TATACTCGAGCCA "GGGTTCGCCGT-3' (enzyme recognition sites are underlined.) The recombinant plasmid was transformed into the host strain AD494 (DE3) pLysS, which uses the T7 system, and The use of the sequencing procedure was confirmed.The D.RET6 was also expressed in the bacteria.The bacterial transformant was cultured at 37 ° C in the Lur ia-Bert ani medium containing ampicillin and chloramphenicol.The culture was induced in its path of exponential procedure (OD60o approximately 0.5) using isopropyl-β-Dt iogalactopyranes gone 0.5-1 mM (IPTG) and continued further for at least 4 hours before collection by centrifugation (4500 xg, 4 ° C, 5 minutes). The slurry was re-dispersed in the lysis buffer containing 6M, 20 mM Tris pH 8 and 500 mM NaCl, sonically briefly treated on ice, a few drops of Triton-acquisition were isolated, and mixed by stirring 4 ° C for 5 minutes. The supernatant was collected by centrifugation at 7500xg 4 ° C for 30 minutes. The D.RET 6 was also expressed in the baculovirus expression system. The DNA fragment containing the complete coding sequence of D.RET6 was amplified using the 5 'oligonucleotides -TATGAAGATGCAGGTAGTGC-3' and 5'-ATATGATCAGCCAGGGTTCGCCGT-3 '. The resulting PCR product was made at the diamond end, digested with Bell, and ligated with the transfer vector pAcCL 29-1 -6xHis (Li vings tone, 1989), which was prepared to have a lozenge end at a Sac site. I and an adhesive end at a BamHI site. The ligation product was then transferred to E. coli (XL1-zul) and the transformant was cultured to produce the plasmid. The plasmid was verified for the absence of any unwanted mutation by complete re-sequencing. The Sf9 cells were co-transfected at different ratios with the recombinant transfer vector and DNA of the polyhedrosis virus, A ut ograp "ha ca l orn i ca cut with Bsu36I (BacPAKd) in the presence of 8 μg of Lipofectin (Gibco BRL) 24 μl reaction For expression, recombinant baculoviruses were identified as gal-act negative plaques when plated on marine plaque agar The putative recombinant clones were plaque purified once more. of a lysate in infected Sf21 cells which gave a positive result in a Western blot using enough serum of D.RET6 expressed antibacterial, polyclonal was amplified and used a production of the recombinant protein.For each subsequent production of the fusion protein, detected Sf9 cells with a baculovirus at MOI (multiplicity of infection) of about 5 were cultured for 2-3 days before harvesting the supernatant po r centrifugation at lOOOxg for 5 minutes. After a precipitation of ammonium sulfate to 60% supernatant, the sediment was discarded. The sediment obtained in the precipitation of 100% ammonium sulfate was re-dissolved in buffer A (50 mM sodium phosphate, 300 mM NaCl and 10 pH glycerol). 2) Protein purification production of antiserum The purification steps were the same for both bacterial and baculovirus expression systems. Briefly, the Ni2 + -nitrile-tri-acetic acid resin, previously equilibrated in buffer A, was added to the overhead or the solution. After incubation on a roller (2 hours 4 ° C), the resin was transferred to a column and washed with 20 volumes in phosphate buffer (50 mM sodium phosphate, 300 mM NaCl and 10% glycerol, pH 6.5 ). The protein was diluted with 5 volumes of 300 mM imidazole, pH 8, concentrated and the solvent was replaced with phosphate buffered saline using a Centricon-10 (Amicon) filtration linkage. Recombinant D.RET6 was further purified on a HiTrap SP column (Pharmacia) using a gradient of 0.1-0.6 M NaCl in 50 mM MES. PH 6.2. To determine the location and solubility of the protein expressed in E. coli, the culture of the bacterial transformant was sampled before and 4 hours after induction by IPTG and processed according to the protocol described in the Qiaexpressionis t (QIAGEN) booklet. . Briefly, the sample was centrifuged, lysed by a freeze-thaw cycle, and briefly treated with sound, then centrifuged to separate the insoluble proteins and the soluble cytosolic protein fraction. In another culture shown, cells were harvested and osmotic shocked first by re-dispersing in 30 mM Tris solution containing "20% sucrose (pH 8) and incubated at room temperature with agitation in the presence of EDTA 1 mM The cells were harvested and re-dispersed in ice-cooled solution of low osmotic concentration (5 mM MgSO4) .The proteins released in the solution were collected by centrifugation.All fractions were analyzed together with the fraction pre-induced by SDS. 12% -PAGE The preparation of fusion proteins for the production of antibodies and the technique for immunizing with small amounts of antigens are described by Sambrook et al., 1989. Briefly, the anti-D.RET6 antiserum was prepared. of guinea pig by repeated intraperitoneal immunization (x3 of guinea pigs with 10% SDS-PAGE gel portions, homogenized containing amounts in micrograms and purified D.RET6 expressed in a manner bacterial in saline buffered with phosphate. 3) Electrophoresis and Western transfer The SDS-PAGE gels in the Western blot demonstrate the expression of FS-HBP1, FS-HB02, MS-HBP1 is shown in PCT / GB / 97/01372 (see Figures 6 and 7). Salivary glands (and other tissues) were excised from ticks at different time points of the feeding period, and homogenized in PBS. The homogenates were centrifuged at 10,000 g for 5 minutes and the supernatants were subjected to dodecyl-sulfat or de-sodium-polyacrylamide electrophoresis (SDS-PAGE; Laemmli, 1970). Figures 8 and 9 show the expression of D.RET6 in E. coli and insect cells, respectively. For Western blotting, the proteins were transferred to no t cells (Gelman Sciences) by semi-dry electrotransfer (Kyhse-Anderson, 1984) using an MS-HBP1 AE-6675 Hroizblot from the first day p.a. until the end of the feeding period. Figure 14 shows a Western blot showing the expression of the protein D.RET6. 4) . Expression and purification of the Av-HBP protein The "av-HBP cDNA purification region was amplified by PCR (95 ° C for 30 '50 ° C for 30 °°, 72 ° C for 30' 18 cycles) using a start primer designed to add a Sac site. I in the 5 'direction of the start codon (5' -TATGAGCTCATGAACTCTGCCTTGTGG; the Sacl site is underlined) and an inverted primer (5'-TATGGATCCGGGGTGGCCTCACCG) containing a BamHI site (subraday) .The product was ligated between the sites Sac I and BamHl of the transfer vector pAcC129.1 (Livingstone and Jones, 1989) that has been modified by the insertion of six histidine codons and a stop codon, in the 3 'direction of the BamH1 recognition site (see original patent). This resulted in the addition of the lie- (His) 6 sequence to the carboxy terminus of Av-HBP. The co-transfection of Sf21 cells of Spodoptera with the transfer vectors and baculovirus (BacPakß), and amplification of the recombinant virus, but according to Kitts and Posee (1993). The cells of Tri chopl usi n i (High Five-, Invitrogen) and the TC100 medium containing 10 -% fetal bovine serum (Gibco BRL) were used for protein expression. After an incubation of 60 hours at 28 ° C, the 0.0025 cm (0.001 inch segregated) was precipitated from the medium with ammonium sulfate (in the 45-80% saturation fraction) and re-dissolved in a phosphate buffer of 50 mM (pH 8.0), which contains 300 mM NaCl and 10% glycerol (buffer A).

(Clontech) were added to capture proteins labeled with oligohistidine incubation of 1 hour at 4 ° C). The beads were applied to a 10 ml column and washed with buffer A (20 volumes), then with 50 mM sodium phosphate buffer (pJ 7.0), containing 300 mM NaCl and 10% glycerol (20 volumes), and finally with 50 mM sodium phosphate buffer (pJ 7.0, 20 volumes). The protein was eluted using 200 mM imidazole in sodium phosphate buffer (100 mM, pH adjusted to 0) Example 3: Characterization of proteins 1) Histamine binding assays The purified recombinant proteins were subjected to histamine binding assays as set forth in Warlo and Bernard (1987). This method used protein precipitation to separate the. bound free ligand (radiolabelled histamine) by the addition of polyethylene glycol (molecular weight 8000) and centrifugation. In all experiments, thin layer chromatographies were run in an acetate-ammonia solvent system after a four-hour incubation period to ensure that the metabolism of histamine has not occurred. The saturable binding of 3H-histamine was obtained with FS-HBP1, FS-HBP2, MS-HBP1 rHBP (see Figure 20 of PCT / GB97 / 01372). For AV-HBP, the semi-purified recombinant protein was incubated with varying amounts of titled histamine, and the amount of unit radiolabel was determined by liquid scintillation counting. The separation of the free ligand from the bound was obtained following the method described by Warlow and Bernard (1987), which uses polyethylene glycol (PEG 800) to precipitate the protein. The results (not shown) suggest that the constant equilibrium dissociation (Kd) for histamine is approximately 7.3 nM. 2) Radioligand binding assays for D.RET6 Recombinant D.RET6 was diluted with 1.5% γ-globulin (Sigma) and used in a set of experiments. Fifty microliters of the protein solution were incubated with 50 μl of a 1: 2500 dilution of [2, 5-3H] -his tamma-diHCl (1 μCi / μl) solution (Amersham) at room temperature for at least 3 hours with or without increasing concentrations of unlabeled histamine. All the tests were carried out in a total volume of 200 μl. Incubations were determined by adding 125 μl of PEG 800 (36% w / v in PBS) and centrifuged in a microcentrifuge at maximum speed for 12 minutes to collect the bound protein. The tubes were centrifuged once more to remove all the supernatant without disturbing the cements. Subsequently, the cements were redissolved in PBS. Three milliliters of the liquid scintillation cocktail (Beckman) were added and the radioactivity was measured using a liquid scintillation counter (Wallac, 1217 Rackbeta). In a second set of experiments, a competitive binding assay was used to compare three competing, unlabeled ligands (histamine, 1-met ilhis tamine and 3-methylhis tamine). To study "the effect of serotonin on histamine binding activity, they were added μl of PBS (as a control experiment) or 10 μl of serotonin (50 μM or 500 μM) to each binding assay of 200 μl. For the analysis of the data, the ligand affinity constant was estimated from the Scatchard plots as previously described by Hulme, 1992, Recep t or-Li gand In t era ct ons, IRL Press, Oxford). Non-linear regression was used to fit the data (Motulsky, 1987, FASEB J, 1: 365-374) and two straight asymptotic lines were made as described elsewhere (Feldman, 1972, Analytical Biochem 48: 317). From the plotted curve line, two straight, asymptotic lines were drawn according to Feldman (1972) consisting of two histamine binding sites of approximately Kd 6 x 10 ~ 8 M and 2 x 10 ~ 6 M. The comparison of The ability of histamine and its methododers to displace radioactive histamine indicated that binding by D.RET6 was specific for histamine (Figure 15). In a surprising way, the saturation curves and the corresponding Scatchard plots for histamine binding in the presence of serotonin revealed a marked synergistic effect (Figures 15 and 16). At a final concentration of 2.38 μM of serotonin, the Kd for the two binding sites for histamine was 1.1 x 10 ~ 9 M and 1 x 10"M, and with 23.8 μM of serotonin, 1.3 x 10" M and 1 x 10, - '6 M, respectively. Thus, in the presence of serotonin, the binding affinity of D.RET6 for its ligand, histamine, was found to be increased by six fold. Recently, it has been reported that external stimuli (including serotonin) regulate the activity of the mammalian Hl receptor, which causes increased affinity to ligand (Bloemers, S.M. M. Verheule, S., Peppelenbosch, M.E.

P., Smit, M.J., Tertoolen, L.G.J., and De Laat, S. (1998) The Journal of Biology, Chemi s try 273 (4), 2249-2255). Although the molecular details remain undefined, a conformational change in the • Hl receptor, induced by serotonin, has been proposed to explain the increase in affinity for histamine (Bloemers, 1998). Thus, it seems possible that the synergistic effect of serotonin on the histamine binding protein of the tick has been developed to counteract the enhanced binding affinity induced by serotonin of Hl receptors. This mechanism can therefore enable the tick protein to compete with the serotonin-sensitized histamine receptors of the host at the feeding site. Since the adult D. retee feeds on a variety of domestic and wild mammals, including dogs, horses, cattle, sheep, deer, foxes, hares, and porcupines, the synergistic effect of serotohine can provide flexibility in the performance of the histamine binding protein of the tick under a range of host-specific hemostatic responses.

This finding has important implications for the design of molecules with histamine binding activity and gives important understanding of the mechanism of action of these tick proteins, along with molecules designed to mimic their action. For example, in order to alter the affinity of these molecules for histamine, serotonin can be distributed simultaneously in an appropriate amount.

Example 4: Crystallization of proteins Purified FS-HBP2 was dialyzed against 10 mM histamine in water (the pH of the histamine solution was adjusted to 6.8 using NaOH), and concentrated using Centricon 10, centrifugation units (Amicon) at a final protein concentration of 20 μg / μl. A hanging droplet of 3 μl of the protenine / his-tamine solution combined with 2 μl of mother liquor was allowed to equilibrate at room temperature with 1 ml of mother liquor [buffer MES 0.1 M (pH 6.5), containing Cobalt hexahydrate 0.01 M and 1.8 M ammonium sulfate (Hampton Research)]. Native data at resolution at 2.24 A and data derived at 3 Á were collected at room temperature using Mar-research imaging plate detectors at home attached to Rigaku rotating anode generators. The frozen native and semi-apo data were collected (at a resolution of 1.24 Á and 1.35 Á, respectively) at the Brookhaven National Laboratory's synchrontron using a Brandis individual module CCD detector (see Table 1). The semi-apo crystal was prepared by immersion in several changes of the histamine-free mother liquor during the week prior to data collection. The mother liquor containing 30% glycerol was used as a freezing solution. The crystals were maintained at 100 K using a cryocurrent (Oxford Cryosystems). The freezing resulted in a different change in the dimensions of the unit cell, with the smaller axes b and c and the axis a slightly larger than at room temperature. The diffraction data were processed with DENZO and SCALEPACK (Otwinowski and Minor, 1997).

Put in phase and construction of the model The structure was resolved by MIR using cis-platinum derivatives and trimethoprimethyl acetate (Holden and Rayment, 1991) (Table 1). Initially, 10-15 ° of data was collected for each new derivative. Data collection was continued only for crystals that showed appreciable isomorphic differences, otherwise they were transferred back to the mother liquor for the next soaking experiment. In Patterson's difference maps they allowed two binding sites to be located, by inspection, for "each heavy metal." Additional sites and the common origin for the two derivatives were found by different Fourier techniques. degree of non-isomorphism (see Table 1)., scaling and calculation based on the isomorphic difference data was carried out with the 3DSCALE and DIFFER home programs (Stuart et al., 1979). The MIR-phases were calculated with MLPHARE (Otwmowski, 1991), and were improved by solvent flattening with the GAP program (Grimes and Stuart, unpublished). The initial maps revealed the presence of two molecules in the asymmetric unit. The non-crystallographic symmetry operator that refers to these molecules was determined and refined with GAP starting from the coordinates of the heavy metal binding sites and the chosen marker positions of the electron density map. The average of the non-crystallographic symmetry resulted in a high quality electronic density map, which can be easily interpreted. The interpretation of the electronic density map used FRODO (Jones, 1985) and 0 (Jones et al., 1991).

"TABLE 1 (a) determination of structure and refinement (b) Refining Statistics Refinement and analysis The initial map immediately revealed that FS-HBP2 has lipocalin topology. The coordinates of the retinoic acid binding protein (IRBP) were used as an initial framework for the construction of the model. The initial model was defined against the data set at room temperature, (native 1, table 1) using XPLOR (Brtinger, 1992) (rigid body, positional refinement and B-factor). The resulting model was refined against the higher resolution data (native 2). The refinement of the rigid body produced improved NSC operators that allowed the positional B-factor and positional refinement at a resolution of 1.24Á. Additional refinement included a complete refinement of the anisotropic B-factor and mock binding. The water and histamine molecules were then included along with a volumetric correction of solvent. During the course of the refinement, the stereochemical restrictions were modified so that the electrostatic and van der Waals terms of the obj ective function were eventually omitted. PROCHECK (Laskowski et al., 1993) was used for the validation of the structure. The assignments of the secondary structure used DSSP (Kabsch and Sander, 1983). Structural overlays used SHP (Stuart et al., 1979). The atomic coordinates used for the comparisons were obtained from the Protein Data Bank, Brookhaven National Laboratory.

Determination of the structure of rFS-HBP2 The complex of FS-HBP2 - this tamine crystallizes in a group of orthohombic space (P2? 2? 2?) With 2 molecules per asymmetric unit. The dimensions of the unit cell vary, specifically in the freezing (table I). The structure was solved by the use of two heavy-atom derivatives and was refined using XPLOR (Brunger, 1992) to produce a final model with reasonable stereochemistry (RMS junction 0.013Á) and R-factor of 18.4%, for all data in the resolution range 20-21.4 Á (table I). 91.1% of the waste is within the most favorable region of the Ramachandran chart, no waste is in the region not allowed. The two crystallographically distinct molecules (A and BJ are very similar (deviations of rms for the atoms C ~ 0.6 Á) .The significant differences are in the turns in lys points of crystalline contrast (figure 20) (the conformation of the side chain and the Val21 residue also differs between molecules A and B, but this is unlikely to affect biological function.) These molecules contain two histamines, linked at the sites denoted H and L. The error in coordinates for most hydrogen atoms is less than 0.2 Á (Luzzatti, 1952) The molecules A and B in the glass have an extensive contact, the hydrogen bonds of the main chain link a short strand that follows the a3-helix of molecule A (residues A143 to A146) at the end of strand B of molecule B (residues B54 to B57) (Figure 20a, c). Other hydrogen bonds link A150 ND2 to B25 0 and A167 OG to B59 OD 1. These molecules are not related by a two-fold axis. The various residues on the surface of the protein have multiple conformations in the crystal, however, these do not have a role in the structural and functional interactions discussed later. In the line with the excellent diffraction, the crystallographic B-factors are rather low (table 1), indicating that the molecule has a core of considerable rigidity. The refined model for molecule A consists of the Al-A 171 residues of the native protein together with the Ile- (His) 6 carboxy-terminal tradename, engineered. Although the course of the polypeptide chain could be plotted in this region, the positions of the three His side chains were not clear and thus were molded as Ala. "No density was observed for the (His) 6 mark on the molecule B (residue Bl also sae omitted due to the disorder).

Complete structure Volume of the molecule (approximate dimensions 33Á x 38Á x 45Á) is an antiparallel ß-barrel of 8 strands, whose topology places FS-HBP2 within the lipocalin superfamily (Figure 13 &Flower, 1996). Lipocalins are typically extracellular proteins that carry small hydrophobic ligands with varying degrees of specificity. The β-barrel of HBP houses two molecules of histamine and the protein is terminated by amino-terminal and carboxy-terminal extensions, partially helical to the β-barrel (Figure 13a). From the high level of sequence similarities, it is expected that the complete structures of the three Ra-HBPs are very similar (deviation of RMS between Ca atoms ~ 1Á).

Unique Characteristics The various characteristics of the functional significance set FS-HBP2 are based on other lipocalinas. The unusually extended amino terminus of FS-HBP2 runs initially along the barrel, forming hydrogen bonding interactions with the β-strands F and G before forming the a and a2 helix. The substantial a2 helix is placed over the "mouth" of the barrel, interacting with the waste on the shore to form a significant barrier between the junction cavities and the outside (Figure 20a, b). One side of the helix is held in place by hydrogen bonds to the B and C strands joining the handle. The other side of the hydrogen bonds of the helix to the F and G strands (the F-G loop is particularly prominent in HBP, in line with a functional role). As with other lipocalins, there is an a3-helix, which joins butt at the midpoint of the barrel. The FS-HBP2, the helix is immobilized to the barrel at its extremities by two disulfide bridges (Cysll9 to Cysl48 and Cys48 to Cysl68). Three structurally conserved regions (SCR), which are in close proximity to the structure, have been identified as determinants of the lipocalin fold (Flower, 1996). The SCRI includes a 3/10 helix followed by a Gly-x-Trp sequence in strand A. In FS-HBP2, an a-helix replaces the helix "3/10 and Asn-Val-Tyr (residues 27-29) replaces the signature sequence In several lipocalins SCR2 contains a Thr-Asp-Try portion While FS-HBP2 does not have this signature sequence, there are conformational similarities in this region (such as a characteristic twist in the F-strand). The final conserved region between strand H and a3 contains a residue of Arg or Lys, close to the conserved Trp residue of SCRI In HBP2, this loop contains the tripeptide Thr-Asp-Tyr (139-141), which usually is present in SCR2 Surprisingly, HBP2 contains several different elements of the sequence that are also reminiscent of misplaced lipocalin fragments (although the sequence Glu-Lys-Val-Thr-Ala in strand D is a perfect match to the equivalent portion of the retonic acid binding protein, epi ridimal; Newcomer, 1993). Perhaps the single most notable feature of the HBP structure is the presence of two ligand binding sites with the barrel core. One of the histamine molecules binds across the width of the binding cavity, with the G and H strands remaining to be stripped from the rest of the sheet, distorting the canonical hydrogen bonding pattern.

Two binding sites differing in affinity for histamine To fully study the relative affinities of the two histamine binding sites, and to probe the conformational implications of histamine binding, we attempted to remove the histamine from the preformed crystals. A rigorous regime of crystal immersion was followed in the mother liquor (minus histamine) followed by data collection at 100K. The diffraction data revealed that the histamine was lost from one of the binding sites in both the crystallographically independent molecules while the second site remained fully occupied. It is inferred from this that, in the crystal, one site has a much slower speed than the other. The similar behavior of the two molecules in different environments within the crystal strongly suggests that one site, called H, has a higher intrinsic affinity than the other, called L. The observed ligand-protein interactions support this assignment. The "union" sites are located, outside the center, at the opposite ends of the barrel (Figure 20a). Unusually for a lipocalin (but according to the hydrophilic nature of the ligand) both binding cavities sequester a number of charged residues inside the barrel. These residues are critical components of the histamine binding sites (Figure 21a, b). The difference by RMS in the length of stabilization hydrogen bonds between molecules A and B is 0.08Á; therefore no distinction was made between the two molecules in this respect. The side chain of the TyrlOO residue and a bridge water molecule form a wall that separates the H and L sites. However, a network of hydrogen bonds links the two sites, the components of which are almost completely conserved between the different sites. HBP. In site H, the histamine ligand is perpendicular along the axis of the barrel, leading to distortions in the structure of HBP, in comparison to other lipocalinas. The aromatic side chains of Trp42 and Phel08 are arranged, parallel but slightly off center, to form strong 11-11 stacking interactions (McGaughey et al., 1998) with the imidazole of the histamine molecule. The phenol ring of TrylOO is perpendicular to the plane of the imidazole ring of histamine, which contributes to additional il-Il interactions. There is an extensive network of hydrogen bonds between the nitrogen atoms of the histamine and the carboxylates of the Glu (82 and 135) and Asp (39 and 110) residues, both directly and via the bridging water molecules. From this environment it is expected that the H-site histamine binds in the di-cationic form. In site L, the histamine molecule is parallel to the barrel with the imidazole portion pointing to the center of the barrel. The types of interactions at site L are similar to site H, but less favorable. The imidazole ring of histamine has base stacking interactions with the phenolic ring of the side chain Phe98, which are almost perpendicular. There are also van der Waals interactions with the phenolic ring of Try29, while Ser20, Asp24 OEI, TyrlOO OH and Aspl20 0E2 form hydrogen bonds with the nitrogen atoms of histamine. Therefore, TrylOO has a role in binding both the histamine molecules, although using different modes of interaction. The non-ordered water molecules are present at the L site and the volumetric calculations suggest that the histamine is slightly less tightly packed at this site.The analysis by the B-factor confirms, however, that both histamine ligands bind rigidly (Table I) The poorer setting of histamine at site L raises the question of whether a preferred compound of histamine could be the natural ligand for this cavity.

Structure of FS-HBP2 with a Unified Histamine The structure of FS-HBP2 without histamine at the L site was determined on both the A molecule and the B molecule, although on the B molecule, a remnant of histamine was seen (approximately 10 % of acupation). The model for this 'semi-apo' structure has an R-factor of 18.7% (table 1) The structural details are essentially the same for molecule A and B. For four water molecules they have replaced the histamine at the L site, one of which takes the position of the Cß atom of histamine (Figure 21c), while the others buffer stabilization by occupying positions equivalent to the hydrogen atoms of histamine. The removal of histamine from site L causes only a slight change (of the order of a few tenths of an Angstrom, the total change per RMS at the Cot positions is only 0.1 Á) and the conformations of the side chain remain essentially unchanged , (figure 21c). Histamine remains attached to a full occupancy at site H, consistent with a much higher affinity.

Implications of the Sequence Variation Within the HBP Family The variation of the sequence between the three HBPs is still present for residues directly included in the histamine binding, especially those that contribute to the L cavity. The complete FS-HBP2 resembles the FS-HBPl more closely than the FS-HBPl, however, the L-cavity of FS-HBP2 is actually more similar to that of MS-HBP1 than FS-HBPl, despite the fact that, completely, the FS-HBP2 resembles FS-HBPl more strongly. In FS-HBPl, the histidine and tyrosine residues replace Ser20 and AsP120, respectively. These changes must modify both the shape and load characteristics of the joint cavity. "In contrast to the numerous changes at the L site, only the substitution of Phe 108 for a Leu has an effect on the H site of FS-HBPl The Kd for FS-HBP2 and MS-HBP1 are similar, and approximately 10 times lower than that for FS-HBPl, suggesting that modifications in the L site modulate the binding affinities observed.The modeling of the FS-HBPI structure indicates that the loss of aromatic residues may open an internal cavity, presumably changing the specificity. -HBP3, the specific, male protein, has more substantial changes compared to FS-HBP2, including insertions and deletions in the regions of the loop.The MS-HBP1 contains an additional Cys (150), which is likely to be in the If the surface of the molecule is responsible for the formation of the covalent dimers discussed above, the putative glycosylation site, Asndl, is also likely to be exposed to the solvent (Figure 13).

Example 5: Design of a cyclic, synthetic peptide with histamine binding activity There are two chains in the histamine binding proteins identified in Figures 17 and 18, A and B. Each chain binds to two histamines, one in amounts of μmol and one in pmol amounts. The geometric investigation of the four binding sites is summarized in Tables 2 and 3 below. These data indicate that in the pmol junction cavities, the positively charged quaternary nitrogen limb of histamine binds to two negatively charged residues (Asp 110 and Glu 135). Similarly, the positively charged imidazole ring binds to two negatively charged residues (Glu 82 and Asp 39), through the two ring nitrogens. There are also two aromatic rings for the positively charged interactions between the nitrogen of the imidazole ring and Trp 42 and Phe 108. 1) Peptide design The analysis of active sites suggested that in order to mimic histamine binding sites, two separate types of interaction should be considered. The first type of interaction is that between positively charged centers (nitrogen tip and imidazole ring) and negatively charged residues. The second type of interaction is between the positively charged imidazole ring and the aromatic ring. This leads to the design of several cyclic peptide systems that incorporate negatively charged waste, for example, Glu, and aromatic residues, for example, Phe. Initial modeling studies indicated that the cyclic hexapeptides would not be sufficiently flexible to allow histamine recognition. The modeling of cyclic octotic systems indicates that they will potentially allow histamine binding. The sequence for the cyclic octapeptide suggested is: Ala-Glu-Ala-Phe-Ala-Glu-Ala-Trp Figures 17 and 18 show a minimized conformation of the cyclic octapeptide / histamine system. These figures reveal the suggested mode of binding between histamine and cyclic peptide. At either 'end' of the cyclic peptide are two negatively charged Glu residues to interact with the positively charged nitrogen extremity and the imidazole ring. The Phe and Trp residues on opposite sides of the imidazole ring allow the interaction of positive-aromatic charge. The interaction distances between the different centers (positive charge to negative charge and positive charge to aromatic group) are shown in table 2.

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It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following.

CLAIMS 1. A histamine or serotonin binding compound, characterized in that it is capable of binding to histamine or serotonin with a dissociation constant of less than 10 ~ 7M and having a binding site comprising the amino acid residues phenylalanine, isoleucine or leucine in the position I, tryptophan in position II and aspartate or glutamate in positions III and IV where residues I or IV are "placed substantially the same as residues 108, 42, 39 and 82, respectively in any of figures 1 or 2 , or residues 107, 41, 38 and 78 in Figure 3 or residues 122, 54, 50 and 95 in Figure 4, and functional equivalents thereof, wherein the numbering of the amino acid residues refers to the sequence of the mature protein that lacks the guiding sequence. 2. A histamine or serotonin binding compound, characterized in that it is capable of binding to histamine or serotonin with a dissociation constant of less than 10 ~ 7M and having a binding site comprising the amino acid residues phenylalanine or isoleucine in residue I , tryptophan in the residues II and aspartate or glutamate in the residues III and IV where the residues I to IV are placed substantially the same as residues 98, 137, 24 and 120, respectively, in any of figures 1 or 2, or residues 95, 138, 23 and 120 in Figure 3 or residues 112, 149, 35 or 135 in Figure 4, and functional equivalents thereof, wherein the numbering of the amino acid residues refers to the sequence of the mature protein that lacks the guiding sequence. 3. A histamine binding compound, characterized in that it is capable of binding to histamine or serotonin with a dissociation constant of less than 10"7M, and having two binding sites, the first binding site comprising the amino acid residues phenylalanine, isoleucine or leucine in position I, tryptophan in position II and aspartate or glutamate in positions III and IV where residues I to IV are placed substantially the same as residues 108, 42, 39 and 82, respectively in either of Figures 1 or 2, or residues 107, 41, 38 and 78 in Figure 3 or residues 122, 54, 50 and 95 in Figure 4, and the second binding site comprising the amino acid residues phenylalanine or isoleucine in residue I, tryptophan in residue II and aspartate or glutamate in residues I to IV are placed substantially the same as residues 98, 137, 24 and 120, respectively in either of figures 1 or 2, or residues 95 , 138, 23 and 120 in figure 3 ol Residues 112, 149, 35 and 135 in Figure 4, functional equivalents thereof, wherein the numbering of the amino acid residues refers to the sequence of the mature protein lacking the leader sequence. 4. A histamine binding or serotonin binding compound according to claim 1 or 3, characterized in that it additionally comprises in the residue V, a tyrosine residue, wherein the residue V is placed substantially the same as the residue 100 in the sequence of any of Figures 1 or 2, residue 97 in Figure 3 or residue 114 in Figure 4, and functional equivalents thereof, wherein the amino acid residue numbering refers to the sequence of the mature protein that lacks the guide sequence . A histamine or serotonin binding compound according to claim 2 or 3, characterized in that it additionally comprises in residue V, a tyrosine residue, wherein residue V is placed substantially the same as residue 29 in the protein sequence of either of Figures 1 to 2, residue 28 in Figure 3 or residue 40 in Figure 4, and functional equivalents thereof, wherein the numbering of the amino acid residues refers to the sequence of the mature protein that is lacking of the guide sequence. 6. A histamine or serotonin binding compound according to any preceding claim, characterized in that the compound is stabilized by either or both of the disulfide bridges formed between cysteines 48 and 169 and cysteines 148 and 119 in the protein sequence of any of the Figures 1 or 2, cysteines 47 and 175 and cysteines 151 and 119 of Figure 3 or cysteines 162 or 134 of Figure 4, wherein the amino acid residue numbering refers to the sequence of the mature protein lacking the guide sequence. 7. A histamine or serotonin binding compound according to any of the preceding claims, characterized in that it comprises a peptide, or a fragment of any of the proteins whose amino acid sequences are presented in Figures 1-4. 8. The histamine or serotonin binding compound according to claim 7, characterized in that it comprises a cyclic peptide. 9. The histamine or serotonin binding compound according to claim 8, characterized in that the cyclic peptide comprises the sequence Ala-Glu-Ala-Phe-Ala-Glu-Ala-Trp.

. The histamine or serotonin binding compound according to any of claims 1 to 9, characterized in that it comprises a synthetic compound. 11. A protein characterized in that it comprises the Ra-Res amino acid sequence given in Figure 5 or functionally equivalent derivative or functionally equivalent fragment thereof. 12. A protein characterized in that it comprises the aV-HBP amino acid sequence given in Figure 6 or functionally equivalent derivative or functionally equivalent fragment thereof. 13. A protein characterized in that it comprises the amino acid sequence Ih / Bm-HBPl given in Figure 7 or functionally equivalent derivative or functionally equivalent fragment thereof. 14. A protein characterized in that it comprises the amino acid sequence Ih / Bm-HBP2 given in Figure 8 or functionally equivalent derivative or functionally equivalent fragment thereof.

. A protein characterized in that it comprises the amino acid sequence Ih / Bm-HBP3 given in Figure 9 or functionally equivalent derivative or functionally equivalent fragment thereof. 16. A protein characterized in that it comprises the amino acid sequence Ih / Bm-HBP4 given in Figure 10 or functionally equivalent derivative or functionally equivalent fragment thereof. 17. A protein characterized in that it comprises the amino acid sequence Ih / Bm-HBP5 given in Figure 11 or functionally equivalent derivative or functionally equivalent fragment thereof. 18. The histamine or serotonin binding compound according to any of claims 1 to 10 or protein according to any of claims 11 to 17 characterized in that it is produced by recombinant DNA technology. 19. A histamine or serotonin binding compound or protein according to any of the preceding claims, characterized in that it binds specifically to histamine.

. The histamine or serotonin binding compound or protein according to any of the preceding claims, characterized in that it has an effector molecule or indicator attached thereto. 21. The histamine or serotonin binding compound or protein according to any preceding claim, characterized in that it is derived from ectoparasites that feed on blood, spiders, scorpions or vipers and poisonous animals. 22. The histamine or serotonin binding compound or protein according to claim 21, characterized in that it is derived from ticks. 23. The histamine or serotonin binding compound or protein according to claim 22, characterized in that it is derived from Ixodid ticks. 24. The histamine or serotonin binding compound or protein according to claim 23, characterized in that it is derived from Rhipicephalus appandiculatus, D. Reticulatus, Amblyomma variagatu, Boophilus microplus or Ixodes hexa gen us.

. The histamine or serotonin binding compound or protein according to any of the preceding claims, characterized in that it is associated with one or more carbohydrate moieties. 26. The histamine or serotonin binding compound or protein according to any of the preceding claims, characterized in that it is associated with one or more peptides or polypeptides. 27. The histamine or serotonin binding compound or protein according to claim 26, characterized in that it is fused genetically or chemically to one or more peptides or polypeptides. 28. The histamine or serotonin binding compound or protein according to any of the preceding claims, characterized in that it is bound to a brand or toxin. 29. The histamine or serotonin binding compound or protein according to any of the preceding claims, characterized in that "" is attached to a support, such as a resin.

. A therapeutic or diagnostic composition, characterized in that it comprises a histamine or serotonin binding compound or protein according to any of the preceding claims. 31. A therapeutic or diagnostic composition, according to claim 30, characterized in that it additionally comprises serotonin.

A therapeutic diagnostic composition, in accordance with rei indication 31, characterized in that it additionally comprises a cysteinyl leukotriene, platelet activation factor, or a thromboxane. 33. A vaccine characterized in that it comprises a histamine or serotonin binding compound according to any of claims 1-10 or protein according to any of claims 11-17. 34. The histamine or serotonin binding compound or protein according to any one of claims 1 to 29, or composition according to any of claims 30 to 32 for use in therapy.

. The histamine or serotonin binding compound or protein according to any of claims 1 to 29, for use as a pharmaceutical product. 36. The use of the histamine or serotonin binding protein compound according to any of claims 1 to 29, as a pharmaceutical product. 37. The histamine or serotonin binding compound or protein according to any of claims 1 to 29, for use in a vaccine. 38. The use of the histamine or serotonin binding protein compound according to any of the rei indications 1 to 29, in a vaccine. 39. The histamine or serotonin binding compound or protein according to any of claims 1 to 29, for use in the detection or quantitation of histamine in human, animal or plant and food material. 40. The histamine or serotonin binding compound according to any of claims 1 to 29, for use in the depletion or removal of histamine from food products, cell cultures or human, animal or plant and food material. 41. The histamine or serotonin binding compound according to claims 1 to 29, for use in the binding or detection of histamine in humans or animals. 42. The histamine or serotonin binding compound or protein according to any one of claims 1 to 29, for use as an anti-hisminic agent, an anti-inflammatory drug or in the treatment of allergy. 43. The histamine or serotonin binding compound or protein according to any of claims 1 to 29, for use as a tool in scientific research regarding the role of histamine in biological processes. 44. The use of a histamine or serotonin binding compound according to any one of claims 1 to 29, in conjunction with a pharmaceutically acceptable carrier in the manufacture of a medicament for the treatment or prevention of inflammation or allergic reaction in humans or animals. 45. A nucleic acid compound, characterized in that it encodes a histamine or serotonin binding protein or molecule according to any one of claims 1 to 29, or that it hybridizes to the nucleic acid molecule under normal hybridization conditions. 46. The nucleic acid molecule according to claim 45, characterized in that it comprises DNA, "cDNA or RNA. 47. An expression cloning vector, characterized in that it comprises a nucleic acid molecule of. according to any of claims 45 or 46. 48. The vector according to the indication rei 47, characterized in that it is based on vi rus. 49. A host cell characterized in that it is transformed or transfected with the vector of any of claims 47 or 48.

Claims (1)

1. 50. A transgenic animal, characterized in that it has been transformed by a nucleic acid molecule according to any of claims 45 or 56 or vector according to any of claims 47 or 48. MOLECULES OF UNION TO HISTAMINE AND SEROTONINE SUMMARY OF THE INVENTION The present invention relates to histamine or serotonin binding molecules that possess a binding site with the precise molecular configuration that is necessary to confer in the molecule a high affinity for histamine. The invention includes proteins, peptides and chemical compounds which possess this molecular configuration and which are thus capable of binding to histamine with high affinity. These molecules can be used in the regulation of the action of histamine or serotonin, the detection and quantification of histamine or serotonin and in the treatment of various diseases and allergies. The molecules can also be used as components of vaccines directed against ectoparasites that suck blood.