WO2001027158A2 - Sequences de recepteurs olfactifs - Google Patents

Sequences de recepteurs olfactifs Download PDF

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Publication number
WO2001027158A2
WO2001027158A2 PCT/US2000/027582 US0027582W WO0127158A2 WO 2001027158 A2 WO2001027158 A2 WO 2001027158A2 US 0027582 W US0027582 W US 0027582W WO 0127158 A2 WO0127158 A2 WO 0127158A2
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WO
WIPO (PCT)
Prior art keywords
seq
olfactory
polypeptides
receptor
scent
Prior art date
Application number
PCT/US2000/027582
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English (en)
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WO2001027158A3 (fr
Inventor
Joel Bellenson
Dexster Smith
Doron Lancet
Gustavo Glusman
Tania Fuchs
Itai Yanai
Original Assignee
Digiscents
Yeda Research And Development Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Digiscents, Yeda Research And Development Co., Ltd. filed Critical Digiscents
Priority to AU29037/01A priority Critical patent/AU2903701A/en
Publication of WO2001027158A2 publication Critical patent/WO2001027158A2/fr
Publication of WO2001027158A3 publication Critical patent/WO2001027158A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L9/00—Disinfection, sterilisation or deodorisation of air
    • A61L9/015—Disinfection, sterilisation or deodorisation of air using gaseous or vaporous substances, e.g. ozone
    • A61L9/02—Disinfection, sterilisation or deodorisation of air using gaseous or vaporous substances, e.g. ozone using substances evaporated in the air by heating or combustion
    • A61L9/03—Apparatus therefor
    • A61L9/035—Apparatus therefor emanating multiple odours
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L9/00—Disinfection, sterilisation or deodorisation of air
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L9/00—Disinfection, sterilisation or deodorisation of air
    • A61L9/015—Disinfection, sterilisation or deodorisation of air using gaseous or vaporous substances, e.g. ozone
    • A61L9/04—Disinfection, sterilisation or deodorisation of air using gaseous or vaporous substances, e.g. ozone using substances evaporated in the air without heating
    • A61L9/12—Apparatus, e.g. holders, therefor
    • A61L9/125—Apparatus, e.g. holders, therefor emanating multiple odours

Definitions

  • the olfactory system contains millions of olfactory sensory neurons (OSNs) located in the olfactory epithelium of the nasal cavity. In humans, the olfactory epithelium occupies an area of approximately 5 cm 2 .
  • the OSNs are bipolar with one end extending through the supporting cell into the mucosal layer, terminating in hairlike cilia. These cilia are the site of the olfactory receptors (OR) where the odorant ligands are thought to bind (Mombaerts Curr. Opin. Genet. Dev. 1999 9, 315-320, Hildebrand et al., Annu. Rev. Neurosci., 1997, 20, 595- 631).
  • Scents can be captured, analyzed and recorded by a sensory device using various methods. Scent capture can be initiated by the user or by an automatic sensing system.
  • a scent can be analyzed in terms of its interaction with olfactory neurons of a mammalian, preferably human, olfactory system, or by the expression of individual receptors under appropriate conditions and appropriate assay conditions in multiwell plates or in terms of its perception by a panel of mammalian, preferably human, subjects.
  • the interaction with olfactory neurons can be determined experimentally, in vitro, by determining the interaction of an odorant with olfactory receptors of a given type.
  • the interaction with olfactory receptor can be determined using a computer simulation which provides information regarding the interaction of an odorant with the olfactory receptors.
  • a panel of subjects can be used to represent odors in terms of their perception.
  • the data so generated can be used to represent a scent in a manner which can be recorded in digital or other format, stored in media such as computer memory, disks, or printed format, and transmitted over a data network.
  • the representation of the scent can be used to re-create the scent at a local or remote site using an emitter module.
  • the representation of the scent allows for scent editing, where desirable aspects of an odor are enhanced or added and undesirable aspects are attenuated or eliminated.
  • the invention also embraces a method to determine single nucleotide polymorphisms in olfactory receptors, comprising the steps of uniquely amplifying olfactory receptor sequences from DNA obtained from one or more individuals, based on primers designed according to the first 25 bases and the last 25 bases of any combination of, or each of, SEQ ID NO: 153 through SEQ ID NO: 1084, and determining the similarities and differences between said amplified DNA and the corresponding receptor from SEQ ID NO: 153 through SEQ ID NO: 1084.
  • Figure 2 depicts the isolated polynucleotide sequences, which encode polypeptides involved in olfactory sensation, corresponding to SEQ ID NOs: 111 - 152.
  • the present invention provides isolated polynucleotides comprising sequences that encode polypeptides which are involved in olfactory sensation and which can be used to screen odorant ligands, e.g., odorant receptor agonists and antagonists.
  • odorant ligand refers to a molecule that has the potential to bind to an olfactory receptor. Equivalent terms employed herein include “odorant”, “odorant molecule” and “odorant compound”.
  • binding or “interaction” as used herein with respect to odorant ligands refers to the interaction of ligands with the receptor polypeptide where the ligands may serve as either agonists and/or antagonists of a given receptor or receptor function. An odorant ligand may thus directly cause a perception of odor (an agonist), or may block the perception of odor (an antagonist).
  • any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form. Not all linkages in a polynucleotide need be identical.
  • heterologous means derived from (i.e., obtained from) a genotypically distinct entity from the rest of the entity to which it is being compared.
  • a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, thus becoming a heterologous polynucleotide.
  • a promoter which is linked to a coding sequence with which it is not naturally linked is a heterologous promoter.
  • Enrichment can be measured on an absolute basis, such as weight per volume of solution, by specific activity or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more preferred. Thus, for example, a 2-fold enrichment is preferred, 10-fold enrichment is more preferred, 100- fold enrichment is more preferred, 1000-fold enrichment is even more preferred.
  • a substance can also be provided in an isolated state by processes such as chemical synthesis or recombinant expression.
  • a "reagent" polynucleotide, polypeptide, or antibody is a substance provided for a reaction, the substance having some known and desirable function in the reaction.
  • polynucleotides involved in olfactory sensation can be obtained using chemical synthesis, recombinant methods, or PCR.
  • Vectors pDBV52 and pDBV53 are suitable for expression.
  • Another example of an expression vector/host cell system is the baculovirus (e.g., nuclear polyhedrosis virus)/insect cell (e.g., sf9 cells) system.
  • Human olfactory receptor polypeptides are expressed from olfactory receptor cDNA by methods well-known to those of skill in the art.
  • a cDNA or portion thereof is inserted in an expression vector using standard molecular cloning techniques. Coupled in vitro transcription and translation of such a vector results in expression of the OR protein encoded by the cDNA.
  • In vivo expression of a OR polypeptide is accomplished by inserting an OR cDNA into a eucaryotic or procaryotic expression vector, of which many are known in the art, to genereate an OR expression construct.
  • mammalian viral vectors including, but not limited to, retroviruses, lentiviruses, Semliki Forest viruses, vaccinia viruses, adenoviruses and adeno-associated viruses.
  • Vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, direct injection, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is an infectious agent, such as a virus).
  • the choice of means of introducing vectors or polynucleotides encoding polypeptides involved in olfactory sensation will often depend on the host cell, as will be well known to those of skill in the art.
  • Host cells transformed with polynucleotides encoding polypeptides involved in olfactory sensation are host cells transformed with (i.e., comprising) polynucleotides encoding polypeptides involved in olfactory sensation, and/or vectors having polynucleotide(s) sequences encoding polypeptides involved in olfactory sensation, as described above.
  • Both prokaryotic and eukaryotic host cells may be used.
  • Prokaryotic hosts include bacterial cells, for example E. coli, B. subtilis, and mycobacteria.
  • eukaryotic hosts are yeast, insect, avian, plant and mammalian cells. Host systems are known in the art and need not be described in detail herein.
  • the host cells of this invention can be used, «ter alia, as repositories of polynucleotides encoding polypeptides involved in olfactory sensation, and/or vehicles for production of polynucleotides encoding polypeptides involved in olfactory sensation, and/or polypeptides involved in olfactory sensation . They may also be used as vehicles for in vivo delivery of polypeptides involved in olfactory sensation .
  • Polypeptides can be purified by, for example, phase partitioning, affinity methods, gel filtration and ion exchange, as well as additional methods known by those skilled in the art. Further characterization of the expressed polypeptide can be achieved by purification of the polypeptide using techniques known in the art.
  • Amino acid residues which can be conservatively substituted for one another include but are not limited to: glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine; lysine/arginine; and phenylalanine/tyrosine.
  • conservative substitutions are known in the art, and preferably, the amino acid substitutions would be such that the substituted amino acid would possess similar chemical properties as that of the original amino acid.
  • polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post- translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation.
  • Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region.
  • Other methods of modification include using coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution and chelation. Modified polypeptides involved in olfactory sensation are made using established procedures in the art.
  • the invention also encompasses fusion proteins comprising one or more polypeptides involved in olfactory sensation.
  • an fusion protein contains one or more polypeptides involved in olfactory sensation and another amino acid sequence to which it is not attached in the native molecule, for example, a heterologous sequence or a homologous sequence from another region.
  • Useful heterologous sequences include, but are not limited to, sequences that provide for secretion from a host cell, intracellular trafficking, and stability/degradation. Other useful heterologous sequences are ones which facilitate purification. Examples of such sequences are known in the art and include those encoding epitopes such as Myc, HA (derived from influenza virus hemagglutinin), His-6, or FLAG.
  • Other heterologous sequences that facilitate purification are derived from proteins such as glutathione S-transferase (GST), maltose-binding protein (MBP), or the Fc portion of immunoglobulin.
  • polypeptides capable the construction of expression vectors encoding intact (i.e., native) polypeptide, functional equivalents and functional fragments thereof, modified forms or recombinant forms.
  • a polynucleotide encoding the desired polypeptide, or a fusion protein can be ligated into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems can be used.
  • the polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification or isolation of the polypeptides expressed in host systems can be accomplished by any method known in the art (e.g.
  • prokaryotic host cells include, for example, E. coli and B. subtilis.
  • eukaryotic host cells include yeast, avian, insect, plant, and animal cells such as COS7, HeLa, CHO, HEK-293 and other mammalian cells.
  • the polypeptides are at least partially purified from other cellular constituents.
  • the polypeptides are at least 70%, more preferably at least 80%, even more preferably at least 90% or most preferably at least 95% pure.
  • purity can be calculated as a weight percent of the total protein content of the preparation. More highly purified polypeptides may also be obtained and are encompassed by the present invention. Methods of protein purification are known in the art and are not described in detail herein. For membrane-bound proteins, the lipid content of the preparation, which is required to maintain the structure and function of the protein, is excluded from the purity calculation.
  • polypeptides of this invention have a variety of uses. They can be used, for example, to screen odorant ligands in order to determine the scent representations, scent profiles or scent fingerprints of particular odorant molecules and further to characterize the effect of functional groups and chemical characteristics on perceived smell.
  • Methods for screening odorant compounds using odorant receptors in neuronal cells are known in the art (Firestein et al., WO
  • Analysis of the scent can be performed in a number of ways. Various embodiments of the scent analysis system are presented. Examples of how these embodiments might operate are also presented, although it should be emphasized that the invention is not limited by any particular theory of olfactory perception or scent analysis.
  • the sensory subsystem comprises a series of olfactory receptors, which selectively bind with the chemical component(s) making up the scent.
  • the scent can be characterized in terms of which of the approximately 1 ,000 olfactory receptors the scent component(s) bind to, and the strength of the interaction of the component(s) with those receptors.
  • Each olfactory receptor can be considered an orthogonal basis vector; the entire set of olfactory receptors can be considered a set of basis vectors spanning "olfactory space.” This is analogous to vectors pointing along the x, y, and z directions in three-dimensional space, where any point in space can be represented by a combination of the x, y, and z basis vectors (with each of the x, y, and z vectors multiplied by the appropriate scalar quantity).
  • the intensity of interaction of a scent with an olfactory receptor determines the magnitude of the vector along that particular "axis" in olfactory space. Thus, every scent can be uniquely described by a vector representation in olfactory space.
  • a representation of a scent in such a manner that the scent can later be re-created is defined as scent profiling.
  • the aforementioned vector representation is one example of a scent profile.
  • a receptor primary scent component is defined as a chemical that interacts with one and only one scent receptor.
  • a receptor complex scent component is defined as a chemical that interacts with more than one scent receptor; the receptor complex scent component can interact with each of the scent receptors to different degrees, to equal degrees, or can interact with some receptors to the same degree and others to different degrees.
  • Olfactory receptors are proteins which fall in the class of seven transmembrane domain G protein-coupled receptors, and are found in olfactory neurons in vivo. Binding of an odorant to an olfactory receptor causes second messenger systems to become activated or inhibited in the cell, leading to increased cellular production of second messenger molecules such as cyclic AMP. These second messenger systems in turn lead to the depolarization of the olfactory neuron, or other changes in the state of the neuron, which provides the signal to the nervous system that the odorant has been detected. With a complete set of receptor primary scent components, any scent can be re-created with the knowledge to the degree to which it interacts with each olfactory receptor.
  • the instant invention encompasses such complete sets of receptor primary scent components.
  • Other embodiments of the invention encompass sets of receptor primary scent component chemicals which provide the ability to re-create a particularly desired subset of scents, but not necessarily all possible scents.
  • Still more embodiments encompass sets of receptor primary scent component chemicals which provide the ability to approximate particular scents, while not necessarily exactly re-creating the interaction profile of the particular scents.
  • a receptor complex scent will be an acceptable approximation to a receptor primary scent. That is, if a given receptor complex scent interacts with a first scent receptor strongly, but interacts with other scent receptors less strongly, it can be considered an approximation to a receptor primary scent component for the first receptor.
  • receptor primary or quasi-primary scent component chemicals provides the most conceptually straightforward method of re-creating scents.
  • another embodiment of the invention encompasses the use of receptor complex scent components for re-creating scents.
  • An example of such an embodiment would be recreation of a scent that activates olfactory receptors designated OR1, OR2, OR3, OR4, OR5 and OR6 (for the sake of illustration, it is assumed that the olfactory receptors are stimulated to an equal extent).
  • Perceptive primary scents are defined as scents that give a single scent perception, for example, the scent "lemon” as perceived by a human.
  • a perceptive primary scent can be composed of one or more receptor primary scent components, one or more receptor complex scent components, or a mixture of one or more receptor primary scent components and one or more receptor complex scent components. Since perceptive primary scents are to some extent subjective, identification of perceptive primary scents can be performed by using a panel of subjects who evaluate and describe scents.
  • the approximate binding constant of the composition to the olfactory receptors is determined.
  • the degree of activation of the olfactory receptor by the composition is determined. For receptor antagonists, binding will occur, but no activation will occur; the invention embraces the identification of such antagonists.
  • scent profiling is a representation of a scent relative to a mammalian olfactory system in such a manner as to provide useful information about the interaction of the scent with that olfactory system, such as sufficient information to enable re-creation of the scent from receptor primary scent components.
  • scent fingerprinting can, but does not necessarily, provide such information.
  • scent finge ⁇ rinting Another use of scent finge ⁇ rinting is quality control of a manufacturing process. Many food items, such as freshly-baked bread and pastries, sauces, and cheeses, have distinct odors.
  • a manufacturer can record a scent finge ⁇ rint for a given food item, e.g. spaghetti sauce for packaging in jars. The quality of the product can then be monitored at various stages in manufacture and storage, and deviations from the established scent finge ⁇ rint can be used to alert the manufacturer to problems in manufacture or storage.
  • Quality control scent finge ⁇ rints are not limited to food items, but can be used in any circumstance where a volatile component of an item of manufacture can be used as a quality control indicator, e.g., perfume, deodorants, solvent mixtures, etc.
  • the TM2 primer set contained 32 oligonucleotides, representing all possible nucleotide sequences capable of encoding the TM2 amino acid sequence motif P-M-Y-F/L-F/Y-F/L, and designed to be non-degenerate at their 3' ends. Sequences of the TM2 primers are as follows:

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Abstract

La présente invention concerne des séquences polynucléotidiques codant pour des polypeptides qui interviennent dans la sensation olfactive. L'invention concerne également les polypeptides codés par ces séquences polynucléotidiques, des vecteurs renfermant les séquences polynucléotidiques et des cellules hôtes transfectées avec lesdites séquences. De plus, cette invention concerne des variants fonctionnels et des homologues des ces séquences polynucléotidiques et des polypeptides codés par ces polynucléotides. Sont également décrits des banques de polypeptides. Par ailleurs, l'invention concerne l'utilisation de ces polypeptides et des banques de polypeptides pour le criblage de molécules odorantes, le but étant de déterminer des correspondances (représentation, empreinte ou profil des odeurs) entre des récepteurs d'odeurs individuels (les polypeptides) et des molécules odorantes particulières. Enfin, l'invention porte sur l'utilisation de représentations d'empreintes et de profils d'odeurs pour recréer et mettre au point des odeurs.
PCT/US2000/027582 1999-10-08 2000-10-06 Sequences de recepteurs olfactifs WO2001027158A2 (fr)

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AU29037/01A AU2903701A (en) 1999-10-08 2000-10-06 Olfactory receptor sequences

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US15861599P 1999-10-08 1999-10-08
US60/158,615 1999-10-08
US18480900P 2000-02-24 2000-02-24
US60/184,809 2000-02-24

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