USOO8637259B1

(12) United States Patent (10) Patent No.: US 8,637,259 B1 Chatelain et al. (45) Date of Patent: Jan. 28, 2014

(54) METHODS OF DENTIFYING MODULATORS (56) References Cited OF OLFACTORY RECEPTORS INVOLVED IN THE PERCEPTION OF SWEAT U.S. PATENT DOCUMENTS CARBOXYLIC ACDS 2008, 0299586 A1 12/2008 Han et al. (75) Inventors: Pierre Chatelain, Brussels (BE); Alex FOREIGN PATENT DOCUMENTS Veithen, Genappe (BE) WO 2012/O29922 3, 2012 (73) Assignee: ChemCom S.A., Brussels (BE) OTHER PUBLICATIONS Laska M, et al. Chem. Senses 29:101-109, 2004.* (*) Notice: Subject to any disclaimer, the term of this Krautwurst B, Chemistry & Biodiversity. 5:842-852, 2008.* patent is extended or adjusted under 35 Idan Menashe, et al., Genetic Elucidation of Human Hyperosmia to U.S.C. 154(b) by 0 days. Isovaleric Acid, PloS Biology, Nov. 2007, vol. 5, Issue 11, p. 2462. (21) Appl. No.: 13/024.325 * cited by examiner Primary Examiner — Robert Landsman (22) Filed: Feb. 9, 2011 (74) Attorney, Agent, or Firm — Edwards Wildman Palmer LLP. Ralph A. Loren; Daniel W. Clarke (51) Int. C. (57) ABSTRACT GOIN33/53 (2006.01) The invention relates to the identification of carboxylic acids, GOIN33/567 (2006.01) present in human Sweat, as natural ligands of a specific Sub (52) U.S. C. group of seven olfactory receptor (OR) belonging to class 1 USPC ...... 435/7.1:435/7.2:435/7.21: 435/810; within the OR classification. The invention encompasses the 435/975 use of the interaction of OR polypeptides and carboxylic (58) Field of Classification Search acids as the basis of Screening assays for agents that specifi None cally modulate the activity of the seven ORS of the invention. See application file for complete search history. 33 Claims, 13 Drawing Sheets

U.S. Patent Jan. 28, 2014 Sheet 4 of 13 US 8,637,259 B1

Figure 2A: concentration dependent activation of OR52L1, measured with luciferase assay

O S g O C logEC50 as -3.05 CD D Of O O v O Q CD O c CD O (f) C S C -

Log Pentanoic acid (M) U.S. Patent Jan. 28, 2014 Sheet 5 of 13 US 8,637,259 B1

Figure2B: concentration dependent activation of OR52E8, measured with luciferase assay

a. O h - O O logEC50 = -3,98 CD 2 f O O H O SS CD CD CD CD f CD C S - -3 -1 Log 3-hydroxy-3-methylhexanoic acid (M) U.S. Patent Jan. 28, 2014 Sheet 6 of 13 US 8,637,259 B1

Figure 2C: concentration dependent activation of OR52E1, measured with luciferase assay

s O 2 O c O C CD log EC50 e-4.43 2 co O O w O SS CD c c CD C cf. CD . 8 s -

Log butanoic acid (M) U.S. Patent Jan. 28, 2014 Sheet 7 of 13 US 8,637,259 B1

Figure 2D: concentration dependent activation of OR52A5, measured with luciferase assay

2 O C o CD logEC50 as -4,52 e f C C - O SS Y CD C c c f c 1 O 8 -

Log4-ethyloctanoic acid) (M) U.S. Patent Jan. 28, 2014 Sheet 8 of 13 US 8,637,259 B1

Figure2E: Concentration dependent activation of OR512, measured with luciferase assay

90 logEC50s -3.61 - logEC50s -4.17

Log butanoic acid(M) Logisovaleric acid) (M)

s O . O iogEC50 - -3.73 C logEC50=-3.41 d 2 O O O s d () d O f E -

Log 2-methylhexanoic acid) (M)

og EC50 = -4.42 logEC50s -4,33

Log trans-3-methyl-2hexenoic ac) (M) Logbenzoic acid) (M) U.S. Patent Jan. 28, 2014 Sheet 9 of 13 US 8,637,259 B1

Figure 2F: concentration dependent activation of OR5112, measured with luciferase assay

120 logEC50= 4.07 100

80

60

40

20

-9 -7 -5 -3 -1 Log (pentanoic acid) (M)

90 80 iogEC50 E-3.08 70 60 50 40 30 20 10

-9 -7 -5 -3 -1 Log (3-methylhexanoic ac) (M) U.S. Patent Jan. 28, 2014 Sheet 10 of 13 US 8,637,259 B1

Figure 2G: concentration dependent activation of OR52B2, measured with luciferase assay

12 --~~~~~~~~~~~~~~~~~~~~~~~~~~~: log EC50 = -3.83. 10 logEC50 as

I -9 -7 -5 -3 -1 -7 -5 -3 Log hexanoic acid (M) Log heptanoic acid (M) is 18 lic s 25 cc 16 logEC50 = -4.08 8 20 iogEC50s -4.28 S 14 9 i 12 C 10 8. o C S$ 8 S. 3 6 9. 9 4 9 f 2 8 5 O I 5 -9 -7 -5 -3 -1 -9 -7 -5 -3 -1 Lognonanoic acid (M) Log decanoic acid (M) U.S. Patent Jan. 28, 2014 Sheet 11 of 13 US 8,637,259 B1

Figure2H: concentration dependent activation of OR52B2, measured with luciferase assay

logEC50 = -3.83

-3

-- 9 C O iog ECS{} : -4.47 d 2 O O O V O SSO c) C d c) O d S E - Log undecanoic acid) (M) U.S. Patent Jan. 28, 2014 Sheet 12 of 13 US 8,637,259 B1

Figure 2: concentration dependent activation of OR56A5, measured with luciferase assay

3. 8 12 logEC50 as 2.83 g 8 logEC50 = -3.24 g 10 9. 20 8 8. g w Yrg 6 8 a 10 g 4. ) C 2 E .

- -9 -7 -5 -3 -

Log hexanoic acid) (M)

logEC50= -4.42 og EC50 = -4.4 t

Lognonanoic acid (M) Log decanoic acid (M)

50 Fog EC50 -3.2

Log 2-methylheptanoic acid (M) U.S. Patent Jan. 28, 2014 Sheet 13 of 13 US 8,637,259 B1

Figure 2J: concentration dependent activation of OR56A5, measured with luciferase assay

7O ayaayaaaaayaaaayaaaaaaa.

60 log EC50s -4.1 - 50 40 30 20 O

Logoctanoic acid) (M)

50 YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY o logEC50= -3.97 8 40

30

1 O

Log undecanoic acid (M) US 8,637,259 B1 1. 2 METHODS OF DENTIFYING MODULATORS humans. Class 2 ORs probably represent an adaptation to the OF OLFACTORY RECEPTORS INVOLVED IN terrestrial life where the detection of airborne odorants is THE PERCEPTION OF SWEAT required. CARBOXYLCACDS Mechanisms of Odor Perception Each OR is able to interact with different molecules, and FIELD OF THE INVENTION each odorant molecule can activate more than one OR. Thus, odor perception does not rely on the simple activation of a The present invention relates to the characterization of single OR, but rather on multiple activations of several ORs. olfactory receptors. In particular, the present invention relates An odor (which can be a single molecule or a mixture) is 10 paired with a unique set of activated ORs that are sufficient for to seven class 1 olfactory receptors and the identification of its discrimination and characterization. Odorant concentra their natural ligands corresponding to carboxylic acids tion can dramatically affect the profile of an odor as some present in human Sweat. The present invention provides additional ORS may be recruited (high concentration) or not assays and methods of screening for compounds, particularly activated (low concentration). Therefore, the set of activated antagonists or blockers, modulating the interaction between ORs will differ for different odor concentrations, leading to olfactory receptors and their respective natural ligands. The varying odor perceptions. With a pool of 380 ORs, the num present invention further provides compositions and methods ber of possible combinations is almost infinite, thus explain comprising the above-mentioned compounds to counteract ing the outstanding discrimination properties of the olfactory Sweat malodors. system. Odorant receptors are expressed in specialized olfac tory sensory neurons (OSNs) located at the top of the nasal BACKGROUND OF THE INVENTION cavity in a small area that constitutes the olfactory epithelium. Filiform extensions at one end of these cells contain the ORS Olfactory Receptors on their surface and float in the nasal mucus where the odor The genes coding for olfactory receptors (ORS) represent ants are dissolved. At the opposite end, the OSN extends its the largest family of genes (3% of the whole genome) in the 25 axon across the ethmoid bone at the base of the cranium to human body dedicated to a single physiological function. connect to the olfactory bulb a small region of the brain These ORs belong to the superfamily of G protein coupled dedicated to the integration of the olfactory stimuli. An out receptors (GPCRs). GPCRs are membrane receptors usually standing feature of the tens of millions of OSNs scattered located at the surface of many different cell types. The com throughout the olfactory epithelium is that each one expresses mon features of these receptors consist of seven transmem 30 only one of the about 400 OR genes available in the human brane spans that form a barrel within the cell membrane and genome. The OSNs expressing the matching gene connect in their capacity to interact with heterotrimeric GTPase and their axons to the same subregion of the olfactory bulb form thereby transducing a signal upon binding of their activators. ing a structure called a glomerulus. It is from this organization of OSNs that the coding of an odor by a specific set of In the human genome, about 900 sequences containing activated ORS is translated geographically in the bulb by a characteristic signatures of olfactory receptors have been corresponding pattern of activated glomeruli. This informa found. However, 60% of these appear to encode non-func tion is further transmitted to the olfactory area of the cortex tional pseudogenes, thereby leaving humans with about 380 where it is decoded and analyzed. In OSNs, triggering of the different OR proteins. ORs are characterized by 6 conserved OR promotes the activation of an olfactory-specific G protein amino acid motifs in their sequence. The first is the FILLG 40 (Galpha-olf) that stimulates a type III adenylate cyclase to motif (SEQID No. 17) located in the extracellular N-terminal produce cyclic AMP; this plays the role of a second messen end of the receptor. It corresponds to a highly conserved ger. Upon binding to a cAMP-gated cation channel, this mes phenylalanine and glycine separated by 3 variable but mostly senger induces the entry of calcium into the cell. Calcium hydrophobic amino acids. The other motifs include causes the opening of another channel that promotes the exit LHTPMY (SEQ ID No. 18) in intracellular loop 1, MAY 45 of chloride ions, and hence triggers an action potential of the DRYVAIC (SEQ ID No. 19) at the end of transmembrane neuron leading to a signal to the respective brain area. domain 3 and the beginning of intracellular loop 2, SY (SEQ Characterisation of Odorant Molecules with ORS ID No. 20) at the end of transmembrane domain5, FSTCSSH Cultured cell lines have been widely used to characterize (SEQID No. 21) in the beginning of transmembrane domain and study receptors of interest in both academic and industrial 6, and PMLNPF (SEQID No. 22) in transmembrane domain 50 contexts. This approach involves introduction of the corre 7. sponding gene into the cells, and Subsequent promotion of its The mammalian ORs are usually subdivided in two distinct stable or transient overexpression. The activity of the receptor classes. Class 1 ORs, also called fish-like receptors, form a can be monitored using a functional assay. The use of easy homogenous group that is more closely related to ORS found to-culture cell lines along with easy-to perform functional in fish and are therefore assumed to represent a conserved 55 assays facilitates several thousand measurements per day. relic maintained throughout the evolution of the vertebrates. Typically, in the pharmaceutical industry, it is common to test The persistence of this group of ancestral ORS Suggests that libraries of 1,000,000 compounds per day on non-olfactory they play an important role in mammalian chemical percep receptors. In the aftermath of OR discovery, several attempts tion. In humans, class 1 ORs encompass 68 non-pseudogenic were made to express ORs in the cell lines suitable for the sequences that correspond to potential functional proteins. 60 expression of non-olfactory receptors, but they remained These receptors share several characteristic domains in their largely unsuccessful. The reason for Such a setback can be sequence that allows their classification as “class 1 ORs. It is found, not in the failure of the cell to produce the receptor, but also to be noted that some amino acids located in the trans rather in its inability to send the receptor to the surface of the membrane domains are highly conserved within the members cell. A technique aimed at improving the functional expres of the fish-like ORs. In contrast to the fish-like ORs, class 2 65 sion of ORS requires engineering a conventional cell line to ORs first appeared in tetrapode vertebrates and expanded to make it suitable for OR expression. In fact, it had long been form the majority of the OR repertoire presently known in Suspected that correct expression and targeting of the OR at US 8,637,259 B1 3 4 the cell surface requires an OSN-specific intracellular major molecule responsible for the characteristic cheesy odor machinery that is absent in a non-olfactory cell line. Thor released by Sweating feet (Ara et al. 2006. Can. J. Microbiol. ough analysis of the expression in OSNs revealed two mem Vol. 52 pp. 357-364). Acids are not directly produced by the bers of a new family of proteins that are specific to this apocrine glands. They appear under the form of glutamine sensory cell. When co-introduced into a conventional cell line conjugates and are released under the action of skin bacteria along with a model OR, the so-called receptor transport pro enzymes. The abundance of several malodorants may there teins 1 and 2 (RTP1 and RTP2) enhanced both the cell surface fore vary from one individual to another, depending on the expression and the response of the receptor to its cognate composition of his bacterial flora. odorants. The production of cAMP arising in the cell upon activation of the OR by its odorant molecules may be detected 10 by an indirect approach that consists of the use of a reporter TABLE 1 gene, as described in (Saito et al., 2004 Cell Vol. 119, 679 Carboxylic acids contributing to Sweat malodor 691). This gene is placed under the control of a cAMP induc ible promoter and is expressed only upon induction by cAMP. Molecule References Odor descriptor Different genes can be used for this purpose, but one of the 15 acetic acid 1, 2, 3 sharp pungent sour vinegar most popular ones encodes the light-producing protein propanoic acid 2, 3 pungent acidic cheesy vinegar butanoic acid 1, 2, 3 sharp acetic cheese butter fruit luciferase. While cleaving its substrate, luciferin, this enzyme ISOValeric acid 2, 3 Sour stinky feet Sweaty cheese releases light that is readily detected and quantified. The tropical intensity of light emitted reflects the amount of luciferase pentanoic acid 3 sickening putrid acidic Sweaty produced, which is proportional to the cAMP increase and rancid therefore directly related to the activity of the receptor. One of hexanoic acid 2, 3, 4, 5 sour fatty sweat cheese 2-methylhexanoic acid 4 acid, animalic, honey, civet, Sweet the advantages of reporter gene assays is dependent upon the 3-methylhexanoic acid 4 Sweaty, butyric signal amplification between receptor activation and reporter (E)-3-methyl-2- 4 acid, Sweaty, fruity, fatty, production. This makes the assay particularly sensitive to hexenoic acid labdanum, hay, Soupy 3-hydroxy-3- 6 pungent Sweaty weak responses that can hardly be detected by other func 25 methylhexanoic acid tional assays. Heptanoic acid rancid sour cheesy Sweat Other functional assays have also been used to demonstrate 2-methylheptanoic acid 4 Sour-fruity, Sweet, slightly the activation of an OR by its odorant ligand. One of these fatty-oily Octanoic acid fatty waxy rancid oily vegetable assays consists in monitoring the increase in cytosolic cal cheesy cium that occurs upon activation of the receptor intracellular 30 4-ethyloctanoic acid 4 costus, fatty, greasy calcium increase (Krautwurtz D. etal. 1998. Cell 95,917-26). nonanoic acid 4 waxy dirty cheese cultured dairy So far, the identification of odorant activators has only been decanoic acid 3 acid, hot iron, metallic, waxy, reported for few mouse odorant ORs. Example of mouse OR Soapy, metal, candle Undecanoic acid 5 waxy creamy cheese fatty coconut deorphanization are given in Malnic et al., 1999, Cell 96, benzoic acid 2 Sweet; benzoin, powdery 713-23; Saito H. et al. 2009. Sci. Signal. 2, 1-14). 35 phenylacetic acid 6 Sweet, animal-honey The identification of human OR activators has also been 1. Yamazaki et al. (2010) Anti-Aging Medicine. 7(6): 60-652. reported. Examples of deorphanized human ORS are e.g. 2. Gallagher et al. (2008) Br. J. Dermatol. 159(4): 780-7913. given in FujitaYetal. 2007. J. Recept. Signal. Transduct. Res. 3. Ara et al. (2006) Can. J. Microbiol. 52: 357-3644. 27, 323-34; Keller A. et al. 2007. Nature 449, 468-72; Mat 4. Zeng et al. (1991) J. Chem. Ecol. 17(7): 1469-14925. 40 5. Labows et al. (1999) Antiperspirants and Deodorants, 2nd Editioned K. Laden, Cosmetic arazzo V. etal. 2005. Chem. Senses 30, 195-207: Saito H. etal. Science and Technology Series Vol. 20 Marcel Dekker Inc, New York, 59-826. 2009. Sci. Signal. 2, 1-14; Sanz, G. et al. 2005. Chem. Senses 6. Natsch et al (2006) Chem. & Biodiv, 3:1-20 30, 69-80; Schmiedeberg K. et al. 2007. J Struct. Biol. 159, 400-12.; Shirokova E. et al. 2004. J. Biol. Chem. 280, 11807 Different strategies have been developed to counteract 15. Spehr M. et al. 2003. Science 299, 2054-58. Wetzel, K. Sweat malodors. The most conventional ones consist in over et al. 1999. J. Neurosci. 19, 7426-33; Sallmann et al. PCT No. 45 powering the malodor with a pleasant fragrance. In a more WO2006/094704. Sophisticated approach, the fragrance is designed to harmo For several of the receptors, more than one ligand has been nize well with the malodor and to shift the perception to a identified. Odorants activating the same OR can belong to more pleasant character. In this case, the fragrance does not different odorant families such as alcohol, aldehyde, esters, need to be a strong odorant by itself. etc (Sanz, G. et al. 2005. Chem. Senses 30, 69-80; Saito H. et 50 An alternative way for reducing malodor consists of lim al. 2009. Sci. Signal. 2, 1-14). iting the production of odorant molecules. This can be Body Malodors achieved either by limiting the skin bacteria population with In our modern Society, odors released by the human body, bacteriostatic agents or by blocking the enzymes responsible and more precisely in the Sweat, are often considered as for the malodorant release. The development of antagonists unpleasant or even offensive. Significant efforts have been 55 and/or blockers that would specifically block the receptors for made by the cosmetic industry to counteract the perception of a malodor molecule can also be considered. An ideal blocker these odors. Amongst the various categories of molecule would have no odor perse, would not affect the bouquet and present in human Sweat, short chain carboxylic acids are of therefore would give a full creative freedom to perfumers. particular importance. Indeed, more than 50 different acids In the present invention it has Surprisingly been discovered have been identified in Sweat. A series of acids is assumed to 60 that seven olfactory receptors belonging to class 1 of ORS are participate in or to be important for the genesis of malodour activated by carboxylic acids present in human Sweat. This (Table 1). For example, 3-hydroxy-3-methylhexanoic acid or unexpected discovery allows the identification of com (E)-3-methyl-2-hexenoic possess a pungent odorand both are pounds, which is of interest for the perfumer and flavorist known to be important contributors to axillary malodor (Zeng companies. Indeed, the identified natural ligands of these et al. 1991, J. Chem. Ecolog. Vol. 17 pp 1469-1492; Gautschi 65 seven olfactory receptors are known to be important constitu et al., 2007, Chimia, Vol. 61 pp 27–32). Isovaleric acid, ents of sweat malodor. The identification and the use of block another short chain carboxylic acid, has been identified as the ers or antagonists of these olfactory receptors in a fragrance US 8,637,259 B1 5 6 composition in order to modify the perception of Sweat mal nonanoic acid, decanoic acid, or undecanoic acid to OR52B2, odor represents an original concept that can open a new or for influencing the activation of OR52B2 by hexanoic acid, possibility for deodorant development. heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, or undecanoic acid. SUMMARY OF THE INVENTION In another embodiment said agent is tested for influencing the binding ofbutanoic acid, isovaleric acid, pentanoic acid, The present invention relates to the identification of seven hexanoic acid, 2-methylhexanoic acid, 3-methylhexanoic Olfactory Receptors (ORS) belonging to class 1 of ORs, acid, (E)-3-methyl-2-hexanoic acid, or benzoic acid to namely, OR52L1, OR52E8, OR52B2, OR51I2, OR52E1, OR51I2, or for influencing the activation of OR5112 by OR52A5 and OR56A5 (the ORs of the invention), as natural 10 butanoic acid, isovaleric acid, pentanoic acid, hexanoic acid, receptors for carboxylic acids present in human Sweat. The 2-methylhexanoic acid, 3-methylhexanoic acid, (E)-3-me invention encompasses the use of the interaction of these OR thyl-2-hexanoic acid, or benzoic acid polypeptides and carboxylic acids as the basis of Screening In a further embodiment said agent is tested for influencing assays for agents that modulate the activity of the ORs of the the binding ofbutanoic acid to OR52E1, or for influencing the invention. 15 activation of OR52E1 by butanoic acid. The invention also encompasses kits for performing In yet another embodiment said agent is tested for influ screening methods based upon the interaction of the 7 ORS encing the binding of 4-ethyloctanoic acid to OR52A5, or for invention with carboxylic acids. influencing the activation of OR52A5 by 4-ethyloctanoic The invention encompasses a method of identifying an acid. agent that modulates the activity of one or more of the ORs of In another embodiment, said agent is tested for influencing the invention, said method comprising: a) contacting an OR the binding of hexanoic acid, heptanoic acid, 2-methylhep polypeptide with a carboxylic acid in the presence and in the tanoic acid, octanoic acid, nonanoic acid, decanoic acid, or absence of a candidate modulator under conditions permit undecanoic acid to OR56A5, or for influencing the activation ting the binding of said carboxylic acid to said OR polypep of OR56A5 by hexanoic acid, heptanoic acid, 2-methylhep tide; and b) measuring the binding of said OR polypeptide to 25 tanoic acid, octanoic acid, nonanoic acid, decanoic acid, or said carboxylic acid, wherein a decrease in binding in the undecanoic acid. presence of said candidate modulator, relative to the binding Preferably, said one or more OR polypeptides is defined by in the absence of the candidate modulator, identifies the can the amino acid sequence of SEQID NOS. 2, 4, 6, 8, 10, 12 or didate modulator as an agent that modulates the activity of 14. The invention further encompasses a method of detecting OR of the invention. 30 in a sample the presence of an agent that modulates the The invention thus provides for a method for identifying an activity of the OR of the invention in a sample, said method agent that modulates the function of one or more Olfactory comprising a) contacting an OR polypeptide with carboxylic Receptors (ORS) selected from the group consisting of acid in the presence and in the absence of said sample under OR52L1, OR52E8, OR52B2, OR51I2, OR52E1, OR52A5 conditions permitting the binding of said carboxylic acid to and OR56A5 comprising the steps of: 35 said OR polypeptide; and b) measuring the binding of said a) contacting said one or more ORS with one or more OR polypeptide to said carboxylic acid, wherein a decrease in carboxylic acid(s) selected from the group of consisting of binding in the presence of the sample, relative to the binding butanoic acid, isovaleric acid, pentanoic acid, hexanoic acid, in the absence of the candidate modulator, indicates the pres 2-methylhexanoic acid, 3-methylhexanoic acid, (E)-3- ence, in the sample of an agent that modulates the activity of methly-2-hexenoic acid, 3-hydroxy-3-methylhexanoic acid, 40 an OR in said sample. heptanoic acid, 2-methylheptanoic acid, octanoic acid, The invention further encompasses a method of identifying 4-ethyloctanoic acid, nonanoic acid, decanoic acid, unde an agent that modulates the function of the OR of the inven canoic acid and benzoic acid, in the presence and in the tion said method comprising: a) contacting an OR polypep absence of said agent under conditions permitting the binding tide with a carboxylic acid in the presence and in the absence of said carboxylic acid(s) to said ORS or permitting the acti 45 of a candidate modulator, under conditions permitting acti vation of said ORs by said carboxylic acid(s), vation of said OR polypeptide by carboxylic acid; and b) b) comparing the binding of said one or more ORS to said measuring a signaling activity of said OR polypeptide, one or more carboxylic acid(s), or the activity of said one or wherein a change in the activity in the presence of said can more ORs, in the presence and in the absence of said agent, didate modulator relative to the activity in the absence of said wherein a difference in binding or activity in the presence of 50 candidate modulator identifies said candidate modulator as an said agent, relative to the binding or activity in the absence of agent that modulates the function of said OR. the agent, identifies the agent as an agent that modulates the The invention further encompasses a method of identifying function of said one or more ORS in response to said one or an agent that modulates the function of the OR of the inven more carboxylic acid(s). tion, said method comprising: a) contacting an OR polypep In a preferred embodiment, said agent is tested for influ 55 tide with a candidate modulator; b) measuring a signaling encing the binding of said carboxylic acids to all 7 ORs listed activity of said OR polypeptide in the presence of said can therein, or for influencing the activation of all 7 ORs listed didate modulator; and c) comparing the activity measured in therein by said carboxylic acids. the presence of said candidate modulator to said activity In a further embodiment, said agent is tested for influenc measured in a sample in which said OR polypeptide is con ing the binding of pentanoic acid to OR52L1, or for influenc 60 tacted with carboxylic acid at its ECso, wherein said candi ing the activation of OR52L1 by pentanoic acid. date modulator is identified as an agent that modulates the In another embodiment said agent is tested for influencing function of the OR when the amount of the activity measured the binding of 3-hydroxy-3-methylhexanoic acid to OR52E8. in the presence of the candidate modulator is at least 10% of or for influencing the activation of OR52E8 by 3-hydroxy-3- the amount induced by said carboxylic acid present at its methylhexanoic acid. 65 ECso. In a next embodiment, said agent is tested for influencing The invention further encompasses a method of detecting the binding of hexanoic acid, heptanoic acid, octanoic acid, in a sample the presence of an agent that modulates the US 8,637,259 B1 7 8 function of an OR of the invention, said method comprising: MAP kinase activity, tyrosine kinase activity, melanophore a) contacting an OR polypeptide with carboxylic acid in the assay, receptor initialization assay, or reporter gene expres presence and in the absence of said sample; b) measuring a Sion. When the G-protein binding/coupling or exchange is signaling activity of said OR polypeptide; and c) comparing measured, of all Ga subunits possible preferably the behav the amount of said activity measured in a reaction containing 5 iours of GTP-binding protein G protein alpha-olf subunit OR and carboxylic acid without said sample to the amount of (olfactory), also G-olf, is studied. The sequence of the human said activity measured in a reaction containing OR, carboxy G-olf subunit has been deposited previously at the Genebank lic acid and said sample, wherein a change in said activity in under accession number L10665. However, G-olf subunits of the presence of said sample relative to the activity in the other species may be used and studied. absence of said sample indicates the presence of an agent that 10 In a preferred embodiment, the measuring of the signaling modulates the function of OR in said sample. The invention further encompasses a method of detecting activity comprises using a fluorescence or luminescence in a sample the presence of an agent that modulates the assay. Fluorescence and luminescence assays may comprise function of an OR of the invention, said method comprising: the use of Ca" sensitive fluorophores including fluo3, Fluo4 a) contacting an OR polypeptide with said sample; b) mea 15 or Fura, (Molecular probes); Ca3-kit family (Molecular Suring a signaling activity of said OR polypeptide in the Device) and aequorin. Furthermore, said assays may apply an presence of said sample; and c) comparing said activity mea automated fluorometric or luminescent reader such as FDSS Sured in the presence of said sample to said activity measured (Hammamatsu) or FLIPR (Molecular Device). in a reaction in which said OR polypeptide is contacted with The invention further encompasses a method of modulat carboxylic acid present at its ECso, wherein an agent that ing the activity of an OR of the invention in a cell, said method modulates the function of the OR is detected if the amount of comprising the step of delivering to said cell, a carboxylic the activity measured in the presence of said sample is at least acid or agent that modulates the activity of an OR polypep 10% of the amount induced by the carboxylic acid present at tide, such that the activity of the OR is modulated. its ECso. In another embodiment of any one of the preceding meth According to the present invention, when using binding 25 ods, the method is a high throughput screening method. methods the carboxylic acid may be detectably labeled. In In another embodiment of any one of the preceding meth said methods, the carboxylic acid may be detectably labeled ods, the agent is part of a chemical library or animal organ with a moiety selected from the group consisting of a radio eXtractS. isotope, a fluorophore, and a quencher of fluorescence. According to the present invention, the agent identified or In one embodiment of any one of the preceding methods, 30 detected by any of the preceeding methods, or the composi the contacting is performed in or on a cell expressing said OR tion comprising said agent, may be used to counteract Sweat polypeptide. According to the present invention, said cell may malodour. Alternatively, these may be used for the prepara be, but is not limited to, Human embryonic kidney cells tion of odorant blockers or odorant antagonists. For instance (Hek293), Chinese hamster cells (CHO), Monkey cells an OR blocker or antagonist may be used as a deodorant. An (COS), primary olfactory cells, Xenopus cells, insect cells, 35 OR blocker or antagonist may be added to a fragrance or yeast or bacteria. perfume formulation already used as a deodorant to reinforce In another embodiment of any one of the preceding meth its efficacy. ods, the contacting is performed in or on synthetic liposomes The present invention also encompasses a composition (see Tajib et al., 2000, Nature Biotechnology 18: 649-654, comprising an isolated OR polypeptide and a carboxylic acid. which is incorporated herein by reference) or virus-induced 40 In a preferred embodiment, said composition encompasses budding membranes containing an OR polypeptide (see all 7 ORs identified herein, or any combination thereof of 2,3, WO0102551, 2001, incorporated herein by reference). 4, 5, or 6 receptors. In another embodiment of any one of the preceding meth The present invention further relates to the use of carboxy ods, the method is performed using a membrane fraction from lic acids for the production of a kit for Screening agents that cells expressing said OR polypeptide. 45 modulate the signaling of OR of the invention, or in combi In a preferred embodiment of either one of the preceding nation with OR of the invention for the production of a kit to methods, the method is performed on a protein chip. screen odorant blockers or odorant antagonists. In another preferred embodiment of either one of the pre In addition, the present invention encompasses the use of a ceding methods, the measuring is performed using a method carboxylic acid present in mammalian Sweat as a ligand for selected from label displacement, Surface plasmon reso 50 OR of the invention. nance, fluorescence resonance energy transfer, fluorescence The present invention also relates to an antibody recogniz quenching, and fluorescence polarization. ing the carboxylic acid/OR of the invention complex or frag In another embodiment of either one of the preceding ments thereof. methods, the agent is selected from the group consisting of a The invention further encompasses a kit comprising an peptide, a polypeptide, an antibody or antigen-binding frag 55 isolated OR polypeptide or several isolated OR polypeptides, ment thereof, a lipid, a carbohydrate, a nucleic acid, and a a carboxylic acid and packaging materials therefore; an iso Small organic molecule. lated polynucleotide encoding an OR polypeptide or several According to the present invention, when a functional isolated polynucleotides encoding an OR polypeptide, a car assay is used, the step of measuring a signaling activity of the boxylic acid, and packaging materials therefore; a kit com OR of the invention may comprise detecting a change in the 60 prising a cell expressing an OR polypeptide or membranes level of a second messenger. thereof or several cells expressing an OR polypeptide or In another embodiment, the step of measuring a signaling membranes thereof, a carboxylic acid and packaging materi activity comprises measurement of guanine nucleotide bind als therefore. Said cell may be transformed with a polynucle ing/coupling or exchange, adenylate cyclase activity, cAMP otide encoding said OR. In a preferred embodiment, said kit Protein Kinase C activity, Protein Kinase A activity phos 65 encompasses all 7 ORs identified herein, or any combination phatidylinosotol breakdown, diacylglycerol, inositol triphos thereof of 2, 3, 4, 5, or 6 receptors and their respective car phate, intracellular calcium, calcium flux, arachidonic acid, boxylic acids. US 8,637,259 B1 9 10 BRIEF DESCRIPTION OF THE FIGURES activity assays described herein. The measurable activity can be measured directly, as in, for example, measurement of FIG. 1A-FIG. 1C show the DNA and corresponding cAMP or diacylglycerol levels. Alternatively, the measurable polypeptide sequences encoding the seven Olfactory Recep activity can be measured indirectly, as in, for example, a tors (ORs) of the invention. reporter gene assay. For most of these assays, kits are com FIGS. 2A-FIG. 2J correspond to a concentration-response mercially available. analysis of the seven receptors of the invention with their Carboxylic acids according to the invention are carboxylic different activators that are all carboxylic acids found in acids present in mammalian Sweat, preferably human Sweat, Sweat. These analyses have been performed according to the which participate in or are important for the genesis of Sweat procedure described in “Experimental procedure” 10 malodour (cf. e.g. Table 1). A “blocker' or “blocking compound according to the DETAILED DESCRIPTION OF THE INVENTION invention is a molecule that attenuates or abolishes the per ception of an odorelicited by one or more odorant molecules. Definitions A blocker may act by interacting with an OR that transduces 15 the said odor or by interacting with the natural ligand for the As used herein, the term “Olfactory Receptor polypeptides receptor. A "blocking compound” of the invention can (ORs) in general refers to polypeptides from the G protein decrease the intracellular response induced by an agonist, for coupled receptor family generated in olfactory neurons. ORS example a carboxylic acid present in human Sweat, by at least may have the ability to interact with odorant molecules and to 10%, preferably 15-25%, more preferably 25-50% and most transduce the odorant signal. The terms “Olfactory Receptors preferably, 50-100%. A “blocker can also refer to a nucle (ORs) according to the invention' or “Olfactory Receptor otide sequence encoding a blocker of the invention. A polypeptides according to the invention” refer to the group of blocker, useful according to the present invention, includes, 7 olfactory receptors that have been shown in the present but is not limited to an antibody, Small molecule, aptamer, invention to be able to selectively detect carboxylic acids. photoaptamer, modified natural ligand, etc. which specifi Examples of olfactory receptors according to the invention 25 cally binds to at least a portion of an OR which is required for include, but are not limited to polypeptides having at least signal transduction through carboxylic acids (such as the 80% amino acid identity, and preferably 90%. 95%, 96%, ligand binding site), or which is capable of blocking or reduc 97%, 98%, 99% or higher, including 100% amino acid iden ing (e.g., by at least 10%) the signal transduction pathway tity, to the sequence represented in FIG. 1A-FIG.1C (SEQID which is coupled to the OR. Preferably, the blocking agent is NOs 2, 4, 6, 8, 10, 12 and 14). Said homology may relate to the 30 preferably volatile, or can be made volatile in combination whole polypeptide or only part of the polypeptide Such as with appropriate solvents or additives. CDR domain (ligand-binding domain of the receptor). As used herein, an “antagonist’ is a ligand which binds to According to Pilpel and Lancet (Protein Science 8:969-977, a receptor and inhibits the intracellular response induced by a 1999) the CDR domain of a GPCR may be defined following ligand oran agonist, for example a carboxylic acid present in the indications published: TM3-#4, TM3-#8, TM3-#11, 35 human sweat, by at least 10%, preferably 15-25%, more TM3-#12, TM3-#15, TM4-#11, TM4-#15, TM4-#19, TM4 preferably 25-50% and most preferably, 50-100%, as com #22, TM4-#23, TM4-#26, TM5-#3, TM5-#6, TM5-#7, TM5 pared to the intracellular response in the presence of an ago #10, TM5-#11 and TM5-il 13, wherein TMX indicates the nist and in the absence of an antagonist. The antagonist may transmembrane region of said receptor, and # indicates the be competitive i.e. it binds at the same site as the agonist or amino acid position whithin said region. 40 ligand, but does not activate an intracellular response initiated As used herein, the term “OR polynucleotide' refers to a by an active form of the receptor and therefore avoids the polynucleotide that encodes the OR polypeptides as defined activation by said ligand or agonist. Alternatively, the antago herein. Preferably, said polynucleotide has an identity of at nist may be non-competitive, i.e. it binds to a site other than least 80% or more, preferably 90%, 95%, 96%, 97%, 98%, the agonist or ligand binding site and blocks the receptor in an 99% or higher, including 100% nucleic acid identity, to the 45 inactive conformation and therefore avoids the transduction sequence represented in FIG. 1A-FIG. 1C (SEQID NOs 1,3, of the olfactory signal by the agonist. 5, 7, 9, 11 and 13). As used herein, “natural ligand” refers to a naturally occur As used herein, the term “OR binding refers to specific ring ligand which binds to a receptor in a manner that is at binding of an odorant molecule by an OR polypeptide. least equivalent to a carboxylic acid present in human Sweat, Examples of odorant molecules include, but are not limited to 50 Such as the carboxylic acids as exemplified herein. A “natural carboxylic acids, esters, alcohols and amines. ligand does not refer to an engineered ligand that is not found As used herein, the term “OR signaling activity” refers to in nature and that is engineered to bind to a receptor, where it the initiation or propagation of signaling by an OR polypep did not formerly do so in a manner different, either in degree tide. OR signaling activity is monitored by measuring a or kind, from that which it was engineered to do. Such an detectable step in a signaling cascade by assaying one or more 55 engineered ligand is no longer naturally-occurring but is of the following: stimulation of GDP for GTP exchange on a “non-natural” and is derived from a naturally occurring mol G protein and most particularly G-olf; alteration of adenylate ecule. cyclase activity; protein kinase C modulation; protein kinase As used herein, a “modulator” refers to a compound that A modulation; phosphatidylinositol breakdown (generating increases or decreases the cell Surface expression of a recep second messengers diacylglycerol, and inositol triphos 60 tor of the invention, increases or decreases the binding of a phate); intracellular calcium flux; activation of MAP kinases: ligand to ORS of the invention, or any compound that modulation of tyrosine kinases; internalization assay; modu increases or decreases the intracellular response initiated by lation of gene or reporter gene activity; or melanophore assay. an active form of the ORs of the invention, either in the A detectable step in a signaling cascade is considered initi presence or absence of a ligand for the receptor, for example ated or mediated if the measurable activity is altered by 10% 65 a carboxylic acid present in human Sweat. A modulator or more above or below a baseline established in the substan includes an antagonist, or blocker, as defined herein. A modu tial absence of a carboxylic acid relative to any of the OR lator can be for example, a small molecule, a polypeptide, a US 8,637,259 B1 11 12 peptide, an antibody or antigen-binding fragment thereof, a term “membrane associated’ refers to those cellular constitu lipid, a carbohydrate, a nucleic acid, an aptamer, a photo ents that are either integrated into a lipid membrane or are aptamer, or a small chemical compound or Small organic physically associated with a component that is integrated into molecule. Candidate modulators can be natural or synthetic a lipid membrane. compounds, including, for example, synthetic Small mol- 5 As used herein, the term 'second messenger assay pref ecules, compounds contained in extracts of animal, plant, erably comprises the measurement of guanine nucleotide bacterial or fungal cells, as well as conditioned medium from binding or exchange, adenylate cyclase, intra-cellular cAMP Such cells. intracellular inositol phosphate, intra-cellular diacylglycerol As used herein, the terms “increase' and “decrease' refer concentration, arachidonic acid concentration, MAP to a change in amount of ligand binding to the ORS of the 10 kinase(s) or tyrosine kinase(s), protein kinase C activity, or invention and/or cell signalling through ORS of the invention reporter gene expression or an aequorin-based assay accord of at least 10%. An “increase' or “decrease' in binding or signalling is preferably measured in response to contacting ing to methods known in the art and defined herein. ORs of the invention with a ligand in the presence of a can As used herein, the term 'second messenger” refers to a didate modulator, wherein the change in binding or signalling 15 molecule, generated or caused to vary in concentration by the is relative to the binding or signalling in the absence of the activation of a G-Protein Coupled Receptor that participates candidate modulator. in the transduction of a signal from that GPCR. Non-limiting As used herein, the term “small molecule' refers to a examples of second messengers include cAMP, diacylglyc compound having a molecular mass of less than 3000 Dal erol, inositol triphosphate, arachidonic acid release, inositol tons, preferably less than 2000 or 1500, still more preferably 20 triphosphate and intracellular calcium. The term “change in less than 1000, and most preferably less than 600 Daltons. A the level of a second messenger refers to an increase or “Small organic molecule' is a Small molecule that comprises decrease of at least 10% in the detected level of a given second carbon. messenger relative to the amount detected in an assay per As used herein, the terms “change”, “difference'. formed in the absence of a candidate modulator. “decrease', or “increase' as applied to e.g., binding or sig- 25 As used herein, the term “aequorin-based assay” refers to nalling activity or amount of a Substance refer to an at least an assay for GPCR activity that measures intracellular cal 10% increase or decrease in binding, signalling activity, or for cium flux induced by activated GPCRs, wherein intracellular example, level of mRNA, polypeptide or ligand relative to a calcium flux is measured by the luminescence of aequorin standard in a given assay. expressed in the cell. As used herein, the term “conditions permitting the binding 30 As used herein, the term “binding refers to the physical of carboxylic acid to an OR of the invention” refers to con association of a molecule (e.g., a ligand Such as a carboxylic ditions of, for example, temperature, salt concentration, pH acid or an antibody) with a receptor (e.g., OR of this inven and protein concentration under which the OR binds a car tion). As the term is used herein, binding is “specific’ if it boxylic acid. Exact binding conditions will vary depending occurs with an ECso or a Kd of 1 mM less, generally in the upon the nature of the assay, for example, whether the assay 35 range of 1 mM to 10 nM For example, binding is specific if the uses viable cells or only a membrane fraction of cells. How ECs or Kd is 1 mM, 500 uM, 100 uM, 10uM,9.5uM, 9 uM, ever, because the ORs of the invention are cell surface pro 8.5uM, 8 uM, 7.5uM,7uM, 6.5uM, 6 uM, 5.5uM,5uM, 4.5 teins, favored conditions will generally include physiological uM, 4 uM, 3.5uM, 3 M, 2.5uM, 2 uM, 1.5uM, 1 uM, 750 salt (90 mM) and pH (about 7.0 to 8.0). Temperatures for nM, 500 nM, 250 nM or 100 nM or less. binding can vary from 15° C. to 37°C., but will preferably be 40 As used herein, the term “ECs” refers to that concentra between room temperature and about 30°C. The concentra tion of a compound at which a given activity, including bind tion of carboxylic acid in a binding reaction will also vary ing of a carboxylic acid or other ligand and a functional from about 0.5 to 2 uM, but will preferably be about 1 uM. activity of a OR, is 50% of the maximum for that OR activity As used herein, the term “sample” refers to the source of measurable using the same assay in the absence of compound. molecules being tested for the presence of an agent or modu- 45 Stated differently, the “ECs” is the concentration of com lator compound that modulates binding to or signalling activ pound that gives 50% activation, when 100% activation is set ity of an OR of the invention. A sample can be an environ at the amount of activity that does not increase with the mental sample, a natural extract of animal, plant, yeast or addition of more agonist. bacterial cells or tissues, a clinical sample, a synthetic sample, As used herein, the term “saturation” refers to the concen or a conditioned medium from recombinant cells or from a 50 tration of a carboxylic acid present in human Sweat or other fermentation process. ligand at which further increases in ligand concentration fail As used herein, a “tissue' is an aggregate of cells that to increase the binding of ligand or OR-specific signalling perform a particular function in an organism. The term “tis activity. sue' as used herein refers to cellular material from aparticular As used herein, the term “ICs is the concentration of an physiological region. The cells in a particular tissue can com- 55 antagonist or blocker that reduces the maximal activation of prise several different cell types. A non-limiting example of an OR of the invention by 50%. this would be brain tissue that further comprises neurons and As used herein, the term “decrease in binding” refers to a glial cells, as well as capillary endothelial cells and blood decrease of at least 10% in the amount of ligand binding cells, all contained in a given tissue section or sample. In detected in a given assay with a known or Suspected modu addition to solid tissues, the term “tissue' is also intended to 60 lator of OR of the invention relative to binding detected in an encompass non-solid tissues, such as blood. assay lacking that known or Suspected modulator. As used herein, the term “membrane fraction” refers to a As used herein, the term "G-Protein coupled receptor,” or preparation of cellular lipid membranes containing an OR of “GPCR refers to a membrane-associated polypeptide with 7 the invention. As the term is used herein, a “membrane frac alpha helical transmembrane domains. Functional GPCRs tion' is distinct from a cellular homogenate, in that at least a 65 associate with a ligand or agonist and also associate with and portion (i.e., at least 10%, and preferably more) of non-mem activate G-proteins. OR polypeptides of the invention are brane-associated cellular constituents has been removed. The GPCRS. US 8,637,259 B1 13 14 As used herein, the term “antibody' is the conventional A. OR Polypeptides. immunoglobulin molecule, as well as fragments thereof Assays using the interaction of OR polypeptides and car which are also specifically reactive with one of the subject boxylic acids require a source of OR polypeptides. The poly polypeptides. Antibodies can be fragmented using conven nucleotide and polypeptide sequence of human ORS are pre tional techniques and the fragments screened for utility in the sented herein in FIG. 1A-FIG. 1C. The human OR52L1, same manner as described herein below for whole antibodies. OR52E8, OR52B2, OR51I2, OR52E1, OR52A5 and For example, F(ab')2 fragments can be generated by treating OR56A5 polynucleotide sequences are also available at Gen antibody with pepsin. The resulting F(ab')2 fragment can be Bank Accession No.s NM 001005173 (SEQ ID NO.1), treated to reduce disulfide bridges to produce Fab fragments. NM 001005168 (SEQ ID NO3), NM 001004052 (SEQ The antibody of the present invention is further intended to 10 ID NO.5), NM 001004754 (SEQ ID NO.7), NG 033197 include bispecific, single-chain, and chimeric and humanised (SEQ ID NO:9), NM 001005160 (SEQ ID NO.11), molecules having affinity for a polypeptide conferred by at NM 001146033 (SEQ ID NO.13), respectively. The least one CDR region of the antibody. In preferred embodi polypeptide sequences are also recorded at accession Nos. ments, the antibody further comprises a label attached thereto 15 Q8NGH7 (SEQ ID NO:2), Q61 FG1 (SEQ ID NO.4), and able to be detected, (e.g., the label can be a radioisotope, Q96RD2 (SEQID NO.6), Q9H344 (SEQID NO.8), Q8NGJ3 fluorescent compound, chemiluminescent compound, (SEQ ID NO.10), Q9H2C5 (SEQ ID NO.12), and P0C7T3 enzyme, or enzyme co-factor). The antibodies, monoclonal or (SEQID NO.14) respectively in the Uniprot database. polyclonal and their hypervariable portion thereof (FAB, One skilled in the art can readily amplify an OR sequence FAB", etc.) as well as the hybridoma cell producing the anti from a sample containing mRNA encoding the protein bodies are a further aspect of the present invention which find through basic PCR and molecular cloning techniques using a specific industrial application in the field of diagnostics and primers or probes designed from the known sequences. Also, monitoring of specific diseases, preferably the ones hereafter since OR genes are intron-less genes, a person skilled in the described. Inhibitors according to the invention include but art can amplify an OR sequence from genomic DNA. are not limited to labeled monoclonal or polyclonal antibod 25 The expression of recombinant polypeptides is well known ies or hypervariable portions of the antibodies. in the art. Those skilled in the art can readily select vectors As used herein, the term “OR constitutive activity” refers and expression control sequences for the expression of OR to a measurable activity of an olfactory receptor expressed polypeptides according to the invention in eukaryotic or into a cell that occurs spontaneously without addition of a prokaryotic cells. OR polypeptides are preferably associated ligand for the said olfactory receptor. 30 with the cell membrane or synthetic liposomes in order to As used herein, the term “inverse agonist” refers to a mol have binding or signaling function. Methods for the prepara ecule that binds to and decreases or suppresses the constitu tion of cellular membrane fractions are well known in the art, tive activity of an OR. e.g., the method reported by Hubbard & Cohn, 1975, J. Cell The invention relates to the discovery that carboxylic acids Biol. 64: 461–479, which is incorporated herein by reference. present in human Sweat are natural ligands for a specific 35 In order to produce membranes comprising OR polypeptides, groups of olfactory receptors, the OR polypeptides as defined one can e.g. apply such membrane isolation techniques to herein. The OR/carboxylic acids interaction is useful for cells endogenously or recombinantly expressing one of the screening assays for agents that modulate such an interaction OR polypeptides of the invention. Alternatively, OR polypep and thus the function of the OR. This OR/carboxylic acid tides can be integrated into membrane preparations by dilu interaction also provides for the identification of modulators 40 tion of detergent Solution of the polypeptide (see, e.g., Sala which could be of interest in industry. mon et al., 1996, Biophys. J. 71:283-294, which is Assays for the Identification of Agents that Modulate the incorporated herein by reference). Activity of ORS B. Carboxylic Acids Present in Sweat. Agents that modulate the activity of ORS can be identified The structure of such carboxylic acids are well known by a in a number of ways that take advantage of the interaction of 45 skilled person. In addition, the person skilled in the art may said receptors with carboxylic acids. For example, the ability easily derive equivalent acids from said structure and may to reconstitute OR/carboxylic acid binding either in vitro, on easily test if said equivalents are able to bind and/or modulate cultured cells or in vivo provides a target for identification of the OR polypeptides. Carboxylic acids may be isolated from agents that disrupt that binding. Assays based on disruption of natural samples, or chemically synthesized. binding can identify agents, such as Small organic molecules, 50 Methods which can be used to quantify said acids may be, from libraries or collections of such molecules. Alternatively, but are not limited to, a) for extraction and purification: Sol Such assays can identify agents in samples or extracts from vent extraction, oil extraction, Vapour extraction, CO2 Super natural Sources, including plant, fungal, or bacterial extracts critical extraction, liquid chromatography, distillation, gas or even human tissue samples. Modulators of OR/carboxylic chromatography; b) for quantifying: gas chromatography, acid binding can then be screened using a binding assay or a 55 liquid chromatography and mass spectrometry. Said methods functional assay that measures downstream signaling through are well known in the art. the said receptor. Both binding assays and functional assays Carboxylic acids may be used in purified form or used as are validated using carboxylic acids. compositions. The amounts of the acid necessary in a given Another approach that uses the OR/carboxylic acid inter binding or functional assay according to the invention will action more directly to identify agents that modulate OR 60 vary depending upon the assay, but will generally use 1 LM to function measures changes in OR downstream signaling 1000 uMoflabeled and 10 uM to 10 mMofunlabeled acid per induced by candidate agents or candidate modulators. These assay. If necessary for a given assay, a carboxylic acid can be functional assays can be performed in isolated cell membrane labeled by incorporation or addition of radioactive labels as fractions or on cells expressing the receptor on their Surfaces. pointed out above. The following description provides methods for both bind 65 C. Assays to Identify Modulators of ORS Activity ing and functional assays based upon the interaction of ORS The discovery that carboxylic acids are ligands of seven and carboxylic acids. ORs belonging to the class 1 olfactory receptor family per US 8,637,259 B1 15 16 mits the development of Screening assays to identify modu et al., 1993, Nature 365:649; Parma et al., 1998, J. Pharmacol. lators of ORs activity. The screening assays will have two Exp. Ther. 286:85; and Parent et al., 1996, J. Biol. Chem. general approaches. 271:7949. 1) Ligand binding assays, in which cells expressing one or Ligand Binding and Displacement Assays: more ORS according to the invention, membrane extracts 5 One can use OR polypeptides expressed in a cell, or iso from Such cells, or immobilized lipid membranes comprising lated membranes containing receptor polypeptides, along one or more ORS according to the invention are exposed to a with a carboxylic acid in order to screen for compounds that labeled carboxylic acid known to bind said one or more ORS inhibit the binding of carboxylic acids to OR polypeptides. and a candidate compound. Following incubation, the reac When identified in an assay that measures binding or car tion mixture is measured for specific binding of the labeled 10 boxylic acid displacement alone, compounds will have to be carboxylic acid to said ORs. Compounds that interfere with or subjected to functional testing to determine whether they act displace labeled carboxylic acid from the ORS can be identi as agonists, antagonists or inverse agonists. fied as modulators, preferably blockers or antagonists of OR For displacement experiments, cells expressing an OR activities. Functional analysis can be performed on positive polypeptide (generally 25,000 cells per assay or 1 to 100 g of compounds to determine in which of these categories they fit. 15 membrane extracts) are incubated in binding buffer (e.g., 50 Binding of a compound may be classified into 3 main mM Hepes pH 7.4; 1 mM CaCl2: 0.5% Bovine Serum Albu categories: competitive binding, non-competitive binding min (BSA) Fatty Acid-Free; and 0.5 mM MgCl2) for 1.5 hrs and uncompetitive binding. A competitive binding compound (at, for example, 27°C.) with labeled carboxylic acid in the resembles a second (reference) compound and binds to the presence or in the absence of increasing concentrations of a same binding pocket of a target molecule (here receptor). candidate modulator. To validate and calibrate the assay, con Upon addition, the competitive binding compound displaces trol competition reactions using increasing concentrations of said second compound from said target. A non-competitive unlabeled carboxylic acid can be performed. After incuba binding compound does not bind to the same binding pocket tion, cells are washed extensively, and bound, labeled car of the target molecule as a second (reference) compound but boxylic acid is measured as appropriate for the given label may interact with the effect of said second compound on said 25 (e.g., Scintillation counting, enzyme assay, fluorescence, target molecule. The second compound is not displaced upon etc.). A decrease of at least 10% in the amount of labeled addition of the non-competitive binding compound. An carboxylic acid bound in the presence of the candidate modu uncompetitive-binding compound binds to the target mol lator indicates displacement of binding by the candidate ecule when a second compound is already bound. Coopera modulator. Candidate modulators are considered to bind spe tive binding means that a compound facilitates the binding of 30 cifically in this or other assays described herein if they dis another compound which may be a reference compound. The place 50% of the labeled carboxylic acid. cooperative effect is thus seen in the analysis of the Kd of said Alternatively, binding or displacement of binding can be other compound. monitored by surface plasmon resonance (SPR). Surface 2) Functional assays, in which a signaling activity of ORS plasmon resonance assays can be used as a quantitative is measured. 35 method to measure binding between two molecules by the a) For agonist screening, cells expressing ORS or mem change in mass near an immobilized sensor caused by the branes prepared from them are incubated with a candidate binding or loss of binding of carboxylic acid from the aqueous compound, and a signaling activity of ORS is measured. The phase to a OR polypeptide immobilized in a membrane on the assays are validated using a carboxylic acid as agonist, and sensor. This change in mass is measured as resonance units the activity induced by compounds that modulate receptor 40 versus time after injection or removal of the carboxylic acidor activity is compared to that induced by the carboxylic acid. candidate modulator and is measured using a Biacore Bio An agonist or partial agonist will have a maximal biological sensor (Biacore AB). OR polypeptides can be immobilized on activity corresponding to at least 10% of the maximal activity a sensor chip (for example, research grade CM5 chip; Biacore of the carboxylic acid when the agonist or partial agonist is AB) in a thin film lipid membrane according to methods present at 100 uMorless, and preferably will have 50%, 75%, 45 described by Salamon et al. (Salamon et al., 1996, Biophys.J. 100% or more, including 2-fold, 5-fold, 10-fold or more 71: 283-294; Salamon et al., 2001, Biophys. J. 80: 1557 activity than the carboxylic acid. 1567; Salamon et al., 1999, Trends Biochem. Sci. 24: 213 b) For antagonist Screening, cells expressing ORS or mem 219, each of which is incorporated herein by reference.). branes isolated from them are assayed for signaling activity in Sarrio et al. demonstrated that SPR can be used to detect the presence of a carboxylic acid with or without a candidate 50 ligand binding to the GPCRA(1) adenosine receptor immo compound. Antagonists will reduce the level of carboxylic bilized in a lipid layer on the chip (Sarrio et al., 2000, Mol. acid-stimulated receptor activity by at least 10%, relative to Cell. Biol. 20:5164-5174, incorporated herein by reference). reactions lacking the antagonist. Conditions for carboxylic acid binding to an OR of the inven c) For inverse agonist screening, cells expressing constitu tion in an SPR assay can be fine-tuned by one skilled in the art tive OR activity or membranes isolated from them are used in 55 using the conditions reported by Sarrio et al. as a starting a functional assay that measures an activity of the receptor in point. the absence of carboxylic acid ligands. Inverse agonists are SPR can assay for modulators of binding in at least two those compounds that reduce the constitutive activity of the ways. First, a carboxylic acid can be pre-bound to immobi OR by at least 10%. Overexpression of OR may lead to lized OR polypeptide, followed by injection of the candidate constitutive activation. OR can be overexpressed by placing it 60 modulator at approximately 10 ul/min flow rate and a con under the control of a strong constitutive promoter, e.g., the centration ranging from 1 nM to 1000 uM, preferably about CMV early promoter. Alternatively, certain mutations of con 100 uM. Displacement of the bound carboxylic acid can be served GPCR amino acids oramino acid domains tend to lead quantified, permitting detection of modulator binding. Alter to constitutive activity. See for example: Kjelsberg et al., natively, the membrane-bound carboxylic polypeptide can be 1992, J. Biol. Chem. 267:1430; McWhinney et al., 2000. J. 65 pre-incubated with a candidate modulator and challenged Biol. Chem. 275:2087: Ren et al., 1993, J. Biol. Chem. 268: with a carboxylic acid. A difference in carboxylic acid bind 16483; Samama et al., 1993, J. Biol. Chem. 268:4625; Parma ing to the OR exposed to the modulator relative to that on a US 8,637,259 B1 17 18 chip not pre-exposed to the modulator will demonstrate bind rescence polarization, relative to a mixture without the can ing. In either assay, a decrease of 10% or more in the amount didate inhibitor, if the candidate inhibitor disrupts or inhibits of carboxylic acid bound is in the presence of candidate the interaction of the OR with the carboxylic acid. Fluores modulator, relative to the amount of carboxylic acid bound in cence polarization is well suited for the identification of small the absence of candidate modulator indicates that the candi molecules that disrupt the formation of polypeptide or protein date modulator inhibits the interaction of the OR and the complexes. A decrease of 10% or more in fluorescence polar carboxylic acid. A Biacore system can be plugged to a system ization in samples containing a candidate modulator, relative identifying candidate modulator Such as mass spectrometry, to fluorescence polarization in a sample lacking the candidate or gas chromatography. modulator, indicates that the candidate modulator inhibits the Another method of measuring inhibition of binding of 10 OR/carboxylic acid interaction. carboxylic acid to OR uses fluorescence resonance energy Another alternative for monitoring OR/carboxylic acid transfer (FRET). FRET is a quantum mechanical phenom interactions uses a biosensor assay. ICS biosensors have been enon that occurs between a fluorescence donor (D) and a described by AMBRI (Australian Membrane Biotechnology fluorescence acceptor (A) in close proximity to each other Research Institute: http//www.ambri.com.au/). In this tech (usually <100 A of separation) if the emission spectrum of D 15 nology, the association of molecules such as an OR and a overlaps with the excitation spectrum of A. The molecules to carboxylic acid, is coupled to the closing of gramacidin be tested, e.g., a carboxylic acid and an OR polypeptide, are facilitated ion channels in Suspended membrane bilayers and labeled with a complementary pair of donor and acceptor thus to a measurable change in the admittance (similar to fluorophores. While close to each other due to the OR/car impedence) of the biosensor. This approach is linear over six boxylic acid interaction, fluorescence emitted upon excita orders of magnitude of admittance change and is ideally tion of the donor fluorophore will have a different wavelength Suited for large scale, high throughput screening of Small from that emitted in response to the excitation wavelength molecule combinatorial libraries. A 10% or greater change when the molecules are not bound, thus allowing quantifica (increase or decrease) in admittance in a sample containing a tion of bound versus unbound polypeptides by measurement candidate modulator, relative to the admittance of a sample of emission intensity at each wavelength. Donor/acceptor 25 lacking the candidate modulator, indicates that the candidate pairs of fluorophores with which to label the target molecules modulator inhibits the interaction of OR and carboxylic acid. are well known in the art. It is important to note that in assays of acid-protein inter A variation on FRET uses fluorescence quenching to moni action, it is possible that a modulator of the interaction need tor molecular interactions. One molecule in the interacting not necessarily interact directly with the domain(s) of the pair can be labeled with a fluorophore, and the other with a 30 proteins that physically interact. It is also possible that a molecule that quenches the fluorescence of the fluorophore modulator will interact at a location removed from the site of when brought into close apposition with it. A change in fluo acid-protein interaction and cause, for example, a conforma rescence upon excitation is indicative of a change in the tional change in the OR polypeptides. Modulators (inhibitors association of the molecules tagged with the fluorophore: or agonists) that act in this manner are nonetheless of interest quencher pair. Generally, an increase in fluorescence of the 35 as agents to modulate the activity of ORs. labeled OR polypeptide is indicative that carboxylic acid Any of the binding assays described can be used to deter bearing the quencher has been displaced. For quenching mine the presence of an agent in a sample, e.g., a tissue assays, a 10% or greater increase in the intensity of fluores sample, that binds to OR molecule, or that affects the binding cent emission in samples containing a candidate modulator, of carboxylic acid to ORs. To do so, OR polypeptides are relative to samples without the candidate modulator, indicates 40 reacted with carboxylic acid or another ligand in the presence that the candidate modulator inhibits OR/carboxylic acid or in the absence of the sample, and carboxylic acid or ligand interaction. binding is measured as appropriate for the binding assay Bioluminescence Resonance Energy Transfer (BRET) is a being used. A decrease of 10% or more in the binding of system for monitoring intermolecular interactions in vivo. carboxylic acid or other ligand indicates that the sample con The assay is based on non-radiative energy transfer between 45 tains an agent that modulates carboxylic acid or ligand bind fusion proteins containing Renilla luciferase (Rluc) and e.g. ing to OR polypeptides. Yellow Fluorescent Protein (YPF) or Green Fluorescent Pro Proteins Chips tein (GFP). The BRET signal is generated by the oxidation of The methods of the present invention may be applied on a coelenterazine derivative substrate. Said system may apply protein chips. Said protein chip may be, but is not limited to, a cell-permeable and non-toxic coelenterazine derivative Sub 50 a glass slide or a nitrocellulose membrane. Array-based meth strate Deep BleuCTM (DBC) and a mutant of the Green Fluo ods for protein chips are well known in the art. The protein rescent Protein (GFP) as acceptor. When the donorand accep arrays preferably comprise one or more OR polypeptides tor are in close proximity the energy resulting from the according to the invention or fragments thereof that are catalytic degradation of the DBC is transferred from Rluc to responsible for the binding with carboxylic acids. The protein GFP which will then emit fluorescence at its characteristic 55 chip preferably comprises all 70R polypeptides according to wavelength. This method allows higher distance between the the invention, or fragments thereofthat are responsible for the two tested molecules and is fluorophore-angle independent. binding with carboxylic acids. In addition to the surface plasmon resonance, FRET and Functional Assays of Receptor Activity BRET methods, fluorescence polarization measurement is i. GTPase/GTP Binding Assays: useful for quantification of carboxylic acid-receptor binding. 60 For GPCRs such as OR polypeptides, a measure of recep The fluorescence polarization value for a fluorescently tor activity is the binding of GTP by cell membranes contain tagged molecule depends on the rotational correlation time or ing receptors. In the method described by Traynor and tumbling rate. Protein complexes, such as those formed by an Nahorski, 1995, Mol. Pharmacol. 47: 848-854, incorporated OR associating with a fluorescently labeled carboxylic acid, herein by reference, one essentially measures G-protein cou have higher polarization values than uncomplexed, labeled 65 pling to membranes by measuring the binding of labeled GTP carboxylic acid. The inclusion of a candidate inhibitor of the to the membrane. For GTP binding assays, membranes iso OR/carboxylic acid interaction results in a decrease in fluo lated from cells expressing the receptor are incubated in a US 8,637,259 B1 19 20 buffer containing 20 mMHEPES, pH 7.4, 100 mMNaCl, and expressing the OR polypeptide (mock-transfected cells) but 10 mMMgCl2, 80 uM35S-GTPy5 and 3 uMGDP. The assay treated with the candidate modulator. mixture is incubated for 60 minutes at 30° C., after which When performed in the absence of a carboxylic acid, the unbound labeled GTP is removed by filtration onto GF/B assay can be used to identify an agonist or inverse agonist of filters. Bound, labeled GTP is measured by liquid scintillation 5 an OR activity. When the assay is performed in the presence counting. In order to assay for modulation of carboxylic of a carboxylic acid, it can be used to assay for an antagonist. acid-induced OR activity, membranes prepared from cells 1) a Fluo3, 4, Fura2, and Calcium3 (Molecular Device) expressing an OR polypeptide are mixed with a carboxylic Based-Assay. acid, and the GTP binding assay is performed in the presence Fluorescence-based assays take advantage of calcium and in the absence of a candidate modulator of OR activity. A 10 fluxes triggered by receptor activation: either calcium decrease of 10% or more in labeled GTP binding as measured entrance through CNG for instance or calcium release from by Scintillation counting in an assay of this kind containing endoplasmic reticulum. Some fluorophores including but not the candidate modulator, relative to an assay without the limited to Fluo3, Fluo4 and Fura2 (Molecular Probes) and modulator, indicates that the candidate modulator inhibits OR Calcum3 kit series (Molecular Device) are known to bind activity. 15 calcium. Such fluorophore-calcium complexes emit fluores A similar GTP-binding assay can be performed without the cence at specific wavelengths. Thereby, upon activation of a carboxylic acid to identify compounds that act as agonists. In G-protein coupled receptor, calcium released from endoplas this case, the carboxylic acid-stimulated GTP binding is used mic reticulum or entered through CNG binds to fluorophore as a standard. A compound is considered an agonist if it leading to specific fluorescence emission. OR-overexpress induces at least 50% of the level of GTP binding induced by ing cells are incubated for 30 to 60 minutes with a solution of the carboxylic acid when the compound is present at 1 mM or 1 to 8 uM fluorophore at 37°C. After thorough washing with less, and preferably will induce a level the same as or higher saline buffer, 50 ul of the same buffer is poored into each well than that induced by the carboxylic acid. containing cells (6 to 1536). Tested agonists are then injected GTPase activity is measured by incubating the membranes 25 into such loaded cells and activation of an OR is followed by containing an OR polypeptide with gamma-32P-GTP. Active fluorescence measurement. GTPase will release the label as inorganic phosphate, which is Intracellular calcium levels are “changed if fluorescence detected by separation of free inorganic phosphate in a 5% intensity increases or decreases by 10% or more in a sample suspension of activated charcoal in 20 mM HPO, followed of cells, expressing an OR polypeptide and treated with a by Scintillation counting. Controls include assays using mem 30 candidate modulator, relative to a sample of cells expressing branes isolated from cells not expressing OR (mock-trans an OR polypeptide but not treated with the candidate modu fected), in order to exclude possible non-specific effects of the lator or relative to a sample of cells not expressing an OR candidate compound. polypeptide (mock-transfected cells) but treated with the can In order to assay for the effect of a candidate modulator on didate modulator. OR-regulated GTPase activity, membrane samples are incu 35 bated with carboxylic acid, with and without the modulator, 2) Depolarization/Hyperpolarization Membrane Assay followed by the GTPase assay. A change (increase or (DiBac Fluorophore for Instance). decrease) of 10% or more in the level of GTP binding or The principle of this assay is to follow depolarization of the GTPase activity relative to samples without modulator is cell membrane. The anionic probe DiBAC4(3) partitions indicative of carboxylic modulation by a candidate modula 40 between intra- and extracellular compartments in a mem tOr. brane potential-dependent manner. With increasing mem ii. Downstream Pathway Activation Assays: brane potential (depolarization), the probe further partitions a. Calcium Flux—the Aequorin-Based Assay. into the cell resulting in an increase of fluorescence. Con The aequorin assay takes advantage of the responsiveness versely, hyperpolarization leads to a decrease of fluorescence of mitochondrial or cytoplasmic apoaequorin to intracellular 45 due to dye extrusion. calcium release or calcium flux (entrance) induced by the The DiBAC4(3) probe is excited with a wavelength of 488 activation of GPCRs (Stables et al., 1997, Anal. Biochem. nm, and emits at a wavelength of 540 nm. 252: 115-126; Detheux et al., 2000, J. Exp. Med., 192 1501 On the day of the experiment, add the glucose to the assay 1508; both of which are incorporated herein by reference). buffer (saline buffer) to a final concentration of 10 mM and Briefly, OR-expressing clones are transfected to coexpress 50 the DiBAC4(3) probe to a final concentration of 5uM. Main mitochondrial or cytoplasmic apoaequorin and G-alpha-16 or tain the assay buffer at 37°C. Remove the cell culture medium G-olf. Cells are incubated with 5 uM Coelenterazine H or and rinse twice each well containing OR-overexpressing cells derivates (Molecular Probes) for 4 hours at room temperature, with 200 ul of pre-heated assay buffer. Place 180 ul of Assay washed in DMEM-F 12 culture medium and resuspended at a buffer containing DiBAC4(3) and incubate the cells for 30 concentration of 0.5x106 cells/ml. Cells are then mixed with 55 min at the appropriate temperature. Cell plates will be ready test agonist peptides and light emission by the aequorin is for assay after these 30 mins. incubation. Collect baseline for recorded with aluminometer for 30 sec. Results are expressed 2 mins. prior any addition. Add 20 Jul of candidate modulators as Relative Light Units (RLU). Controls include assays using to the appropriate well and collect the data for an additional membranes isolated from cells not expressing C356 (mock 25 mins. transfected), in order to exclude possible non-specific effects 60 Membrane polarization is “changed if fluorescence inten of the candidate compound. sity increases or decreases by 10% or more in a sample of Aequorin activity or intracellular calcium levels are cells, expressing an OR polypeptide and treated with a can “changed if light intensity increases or decreases by 10% or didate modulator, relative to a sample of cells expressing an more in a sample of cells, expressing an OR polypeptide and OR polypeptide but not treated with the candidate modulator treated with a candidate modulator, relative to a sample of 65 or relative to a sample of cells not expressing an OR polypep cells expressing the OR polypeptide but not treated with the tide (mock-transfected cells) but treated with the candidate candidate modulator or relative to a sample of cells not modulator. US 8,637,259 B1 21 22 3) Melanophore Assay. tions should be performed using extracts of mock-transfected The melanophore assay is a color-based assay. Basically cells to exclude possible non-specific effects of some candi cells used for this assay are derived from skin of the frog date modulators. Xenopus Laevis. These immortalized cells contain melano Assays should be performed using cells or extracts of cells Somes, which are organelles containing dark pigment. Acti expressing an OR polypeptide, treated or not treated with a Vation of endogenous or recombinant GPCR that triggeracti carboxylic acid with or without a candidate modulator. Con Vation of adenylate cyclase or phospholipase C lead to trol reactions should be performed using mock-transfected melanosome dispersion and therefore cell darkening. Alter cells, or extracts from them in order to exclude possible natively, a GPCR that inhibits adenylate cyclase orphospho non-specific effects of some candidate modulators lipase C leads to cell lightening. Thereby, instead of measur 10 ing concentrations of second messenger, one can easily The level of cAMP is “changed” if the level of cAMP pinpoint hit observing cell coloration change. This color detected in cells, expressing an OR polypeptide and treated change can easily be quantified on a microplate reader mea with a candidate modulator of OR activity (or in extracts of suring absorbance at 650 nM or by examination on a video such cells), using the RIA-based assay of Horton & Baxen imaging System. 15 dale, 1995, Supra, increases or decreases by at least 10% b. Adenylate Cyclase Assay: relative to the cAMP level in similar cells not treated with the Assays for adenylate cyclase activity are described by Ken candidate modulator. imer & Nirenberg, 1981, Mol. Pharmacol. 20: 585-591, d. Phospholipid Breakdown, DAG Production and Inositol incorporated herein by reference. That assay is a modification Triphosphate Levels: of the assay taught by Solomon et al., 1974, Anal. Biochem. Receptors that activate the breakdown of phospholipids 58: 541-548, also incorporated herein by reference. Briefly, can be monitored for changes due to the activity of known or 100 ul reactions contain 50 mM Tris-Hcl (pH 7.5), 5 mM Suspected modulators of an OR by monitoring phospholipid MgCl2, 20 mM creatine phosphate (disodium salt), 10 units breakdown, and the resulting production of second messen (71 ug of protein) of creatine phosphokinase, 1 mM C-32P gers DAG and/or inositol triphosphate (IP3). Methods of ATP (tetrasodium salt, 2 uCi), 0.5 mM cyclic AMP, G-3H 25 measuring each of these are described in Phospholipid Sig labeled cyclic AMP (approximately 10,000 cpm), 0.5 mM naling Protocols, edited by Ian M. Bird. Totowa, N.J., Ro20-1724, 0.25% ethanol, and 50-200 ug of protein homo Humana Press, 1998, which is incorporated herein by refer genate to be tested (i.e., homogenate from cells expressing or ence. See also Rudolph et al., 1999, J. Biol. Chem. 274: not expressing an OR polypeptide, treated or not treated with 11824-11831, incorporated herein by reference, which also carboxylic acid with or without a candidate modulator). 30 describes an assay for phosphatidylinositol breakdown. Reaction mixtures are generally incubated at 37° C. for 6 Assays should be performed using cells or extracts of cells minutes. Following incubation, reaction mixtures are depro expressing an OR, treated or not treated with carboxylic acid teinized by the addition of 0.9 ml of cold 6% trichloroacetic with or without a candidate modulator. Control reactions acid. Tubes are centrifuged at 1800xg for 20 minutes and each should be performed using mock-transfected cells, or extracts supernatant solution is added to a Dowex AG50W-X4 col 35 from them in order to exclude possible non-specific effects of umn. The cAMP fraction from the column is eluted with 4 ml Some candidate modulators. of 0.1 mM imidazole-HCl (pH 7.5) into a counting vial. According to the invention, phosphatidylinositol break Assays should be performed in triplicate. Control reactions down, and diacylglycerol and/or inositol triphosphate levels should also be performed using protein homogenate from are “changed if they increase or decrease by at least 10% in cells that do not express an OR polypeptide. 40 a sample from cells expressing an OR polypeptide and treated Assays should be performed using cells or extracts of cells with a candidate modulator in the presence or in the absence expressing an OR, treated or not treated with a carboxylic acid of carboxylic acid, relative to the level observed in a sample with or without a candidate modulator. Control reactions from cells expressing a carboxylic polypeptide that is not should be performed using mock-transfected cells, or extracts treated with the candidate modulator. from them in order to exclude possible non-specific effects of 45 e. PKC Activation Assays: Some candidate modulators Growth factor receptor tyrosine kinases tend to signal via a According to the invention, adenylate cyclase activity is pathway involving activation of Protein Kinase C(PKC). “changed if it increases or decreases by 10% or more in a which is a family of phospholipid- and calcium-activated sample taken from cells treated with a candidate modulator of protein kinases. PKC activation ultimately results in the tran OR activity, relative to a similar sample of cells not treated 50 Scription of an array of proto-oncogene transcription factor with the candidate modulator or relative to a sample of cells encoding genes, including c-fos, c-myc and c-jun, proteases, not expressing an OR polypeptide (mock-transfected cells) protease inhibitors, including collagenase type I and plasmi but treated with the candidate modulator. Alternatively, a nogen activator inhibitor, and adhesion molecules, including decrease of activity by 10% or more by the candidate modu intracellular adhesion molecule I (ICAMI). Assays designed lator of OR polypeptides in a sample treated with a reference 55 to detect increases in gene products induced by PKC can be compound may be tested. used to monitor PKC activation and thereby receptor activity. c. cAMP Assay: In addition, activity of receptors that signal via PKC can be Intracellular cAMP is measured using a cAMP radioim monitored through the use of reporter gene constructs driven munoassay (RIA) or cAMP binding protein according to by the control sequences of genes activated by PKC activa methods widely known in the art. For example, Horton & 60 tion. This type of reporter gene-based assay is discussed in Baxendale, 1995, Methods Mol. Biol. 41: 91-105, which is more detail below. incorporated herein by reference, describes an RIA for For a more direct measure of PKC activity, the method of cAMP. Kikkawa et al., 1982, J. Biol. Chem. 257: 13341, incorporated A number of kits for the measurement of cAMP are com herein by reference, can be used. This assay measures phos mercially available, such as the High Efficiency Fluorescence 65 phorylation of a PKC substrate peptide, which is subse Polarization-based homogeneous assay marketed by LJL quently separated by binding to phosphocellulose paper. This Biosystems and NEN Life Science Products. Control reac PKC assay system can be used to measure activity of purified US 8,637,259 B1 23 24 kinase, or the activity in crude cellular extracts. Protein kinase should be performed using mock-transfected cells, or extracts Csample can be diluted in 20 mM HEPES/2 mMDTT imme from them in order to exclude possible non-specific effects of diately prior to assay. Some candidate modulators The substrate for the assay is the peptide Ac-FKKSFKL PKA activity activity is “changed if the level of activity is NH2 (SEQID NO: 15), derived from the myristoylated ala increased or decreased by 10% or more in a sample from cells, nine-rich protein kinase C substrate protein (MARCKS). The expressing an OR polypeptide, treated with a candidate Km of the enzyme for this peptide is approximately 50 uM. modulator relative to PKA kinase activity in a sample from Other basic, protein kinase C-selective peptides known in the similar cells not treated with the candidate modulator. art can also be used, at a concentration of at least 2-3 times g. Kinase Assays: their Km. Cofactors required for the assay include calcium, 10 MAP kinase activity can be assayed using any of several magnesium, ATP phosphatidylserine and diacylglycerol. kits available commercially, for example, the p38 MAP Depending upon the intent of the user, the assay can be Kinase assay kit sold by New England Biolabs (Cat #9820) or performed to determine the amount of PKC present (activat the FlashPlateTM MAP Kinase assays sold by Perkin-Elmer ing conditions) or the amount of active PCK present (non Life Sciences. activating conditions). For most purposes according to the 15 Assays should be performed using cells or extracts of cells invention, non-activating conditions will be used. Such that expressing an OR, treated or not treated with a carboxylic acid the PKC that is active in the sample when it is isolated is with or without a candidate modulator. Control reactions measured, rather than measuring the PKC that can be acti should be performed using mock-transfected cells, or extracts vated. For non-activating conditions, calcium is omitted in the from them in order to exclude possible non-specific effects of assay in favor of EGTA. Some candidate modulators The assay is performed in a mixture containing 20 mM MAP Kinase activity is “changed if the level of activity is HEPES, pH 7.4, 1-2mMDTT, 5 mMMgCl2, 100 uMATP-1 increased or decreased by 10% or more in a sample from cells, uCi Y-32P-ATP, 100 ug/ml peptide substrate (~100 uM), 140 expressing an OR polypeptide, treated with a candidate uM/3.8 LM phosphatidylserine/diacylglycerol membranes, modulator relative to MAP kinase activity in a sample from and 100 uM calcium (or most preferably 500 uMEGTA). 48 25 similar cells not treated with the candidate modulator. ul of sample, diluted in 20 mM HEPES, pH 7.4, 2 mM DTT Direct assays for tyrosine kinase activity using known syn is used in a final reaction volume of 80 ul. Reactions are thetic or natural tyrosine kinase Substrates and labeled phos performed at 30°C. for 5-10 minutes, followed by addition of phate are well known, as are similar assays for other types of 25 ul of a solution containing 100 mM ATP and 100 mM kinases (e.g., Ser/Thr kinases). Kinase assays can be per EDTA with a pH value of 8.0, which stops the reactions. 30 formed with both purified kinases and crude extracts prepared After the reaction is stopped, a portion (85 ul) of each from cells expressing an OR polypeptide, treated with or reaction is spotted onto a Whatman P81 cellulose phosphate without a carboxylic acid, with or without a candidate modu filter, followed by washes: four times 500 ml of 0.4% phos lator. Control reactions should be performed using mock phoric acid, (5-10 min. per wash); and a final wash in 500 ml transfected cells, or extracts from them in order to exclude 95% EtOH, for 2-5 min. Bound radioactivity is measured by 35 possible non-specific effects of some candidate modulators. Scintillation counting. Specific activity (cpm/nmol) of the Substrates can be either full length protein or synthetic pep labeled ATP is determined by spotting a sample of the reac tides representing the substrate. Pinna & Ruzzene (1996, tion onto P81 paper and counting without washing. Units of Biochem. Biophys. Acta 1314: 191-225, incorporated herein PKC activity, defined as nmol phosphate transferred permin, by reference) list a number of phosphorylation substrate sites are calculated as follows: 40 useful for measuring kinase activities. A number of kinase substrate peptides are commercially available. One that is The activity, in UNITS (nmol/min) is: = (cpm on particularly useful is the “Src-related peptide.” paper)x (105ul total 85ul spotted) (assay time, (RRLIEDAEYAARG (SEQ ID NO: 16); available from min) (specific activity of ATP cpm/nmol). Sigma i A7433), which is a substrate for many receptor and An alternative assay can be performed using a Protein 45 nonreceptor tyrosine kinases. Because the assay described Kinase C Assay Kit sold by PanVera (Cat. # P2747). below requires binding of peptide substrates to filters, the Assays are performed on extracts from cells expressing an peptide Substrates should have a net positive charge to facili OR polypeptide, treated or not treated with a carboxylic acid tate binding. Generally, peptide Substrates should have at with or without a candidate modulator. Control reactions least 2 basic residues and a free amino terminus. Reactions should be performed using mock-transfected cells, or extracts 50 generally use a peptide concentration of 0.7-1.5 mM. from them in order to exclude possible non-specific effects of Assays are generally carried out in a 25 Jul Volume com Some candidate modulators. prising 5 ul of 5x kinase buffer (5 mg/mL BSA, 150 mM According to the invention, PKC activity is “changed by a Tris-Cl (pH 7.5), 100 mM MgC12; depending upon the exact candidate modulator when the units of PKC measured by kinase assayed for, MnCl2 can be used in place of or in either assay described above increase or decrease by at least 55 addition to the MgCl), 5 ul of 1.0 mM ATP (0.2 mM final 10%, in extracts from cells expressing an OR and treated with concentration), gamma-32P-ATP (100-500 cpm/umol), 3 ul a candidate modulator, relative to a reaction performed on a of 10 mM peptide substrate (1.2 mM final concentration), cell similar sample from cells not treated with a candidate modu extract containing kinase to be tested (cell extracts used for lator. kinase assays should contain a phosphatase inhibitor (e.g. f. PKA Activation Assays 60 0.1-1 mM sodium orthovanadate)), and HO to 25 ul. Reac PKA activity can be assayed using any of several kits tions are performed at 30°C., and are initiated by the addition available commercially, for example from molecular device of the cell extract. IMAP PKA assay kit, or from promega ProFluor PKA assay Kinase reactions are performed for 30 seconds to about 30 kit. minutes, followed by the addition of 45 ul of ice-cold 10% Assays should be performed using cells or extracts of cells 65 trichloroacetic acid (TCA). Samples are spun for 2 minutes in expressing an OR, treated or not treated with a carboxylic acid a microcentrifuge, and 35ul of the Supernatant is spotted onto with or without a candidate modulator. Control reactions Whatman P81 cellulose phosphate filter circles. The filters US 8,637,259 B1 25 26 are washed three times with 500 ml cold 0.5% phosphoric In order to assay OR activity with carboxylic acid-respon acid, followed by one wash with 200 ml of acetone at room sive transcriptional reporter construct, cells that stably temperature for 5 minutes. Filters are dried and incorporated express an OR polypeptide are stably transfected with the P is measured by scintillation counting. The specific activ reporter construct. To Screen for agonists, untreated cells are ity of ATP in the kinase reaction (e.g., in cpm/pmol) is deter exposed to candidate modulators, or exposed to a carboxylic mined by spotting a small sample (2-5 ul) of the reaction onto a P81 filter circle and counting directly, without washing. acid, and expression of the reporter is measured. The car Counts per minute obtained in the kinase reaction (minus boxylic acid-treated cultures serve as a standard for the level blank) are then divided by the specific activity to determine of transcription induced by a known agonist. An increase of at the moles of phosphate transferred in the reaction. 10 least 10% in reporter expression in the presence of a candidate Assays should be performed using cells or extracts of cells modulator compared to reporter expression in the absence of expressing an OR, treated or not treated with a carboxylic acid any modulator indicates that the candidate is a modulator of with or without a candidate modulator. Control reactions OR activity. An agonist will induce at least as much, and should be performed using mock-transfected cells, or extracts 15 preferably the same amount or more reporter expression than from them in order to exclude possible non-specific effects of the carboxylic acid. Partial agonists may activate the receptor Some candidate modulators. less compared to the carboxylic acid. This approach can also Tyrosine kinase activity is “changed if the level of kinase be used to screen for inverse agonists where cells express an activity is increased or decreased by 10% or more in a sample OR polypeptide at levels such that there is an elevated basal from cells, expressing an OR polypeptide, treated with a activity of the reporter in the absence of carboxylic acid or candidate modulator relative to kinase activity in a sample other agonists. A decrease in reporter activity of 10% or more from similar cells not treated with the candidate modulator. in the presence of a candidate modulator, relative to its h. Transcriptional Reporters for Downstream Pathway absence, indicates that the compound is an inverse agonist. Activation: 25 To screen for antagonists, the cells expressing an OR and The intracellular signal initiated by binding of a modulator carrying the reporter construct are exposed to a carboxylic to a receptor, e.g., an OR polypeptide of the invention, sets in acid (or another agonist) in the presence and absence of a motion a cascade of intracellular events, the ultimate conse candidate modulator. A decrease of 10% or more in reporter quence of which is a rapid and detectable change in the 30 expression in the presence of candidate modulator, relative to transcription and/or translation of one or more genes. The the absence of the candidate modulator, indicates that the activity of the receptor can therefore be monitored by mea candidate is an antagonist of OR activity. Suring the expression of a reporter gene driven by control Controls for transcription assays include cells not express sequences responsive to OR activation. ing an OR of the invention but carrying the reporter construct, 35 as well as cells with a promoter less reporter construct. Com As used herein “promoter” refers to the transcriptional pounds that are identified as modulators of OR-regulated control elements necessary for receptor-mediated regulation transcription should also be analyzed to determine whether of gene expression, including not only the basal promoter, but they affect transcription driven by other regulatory sequences also any enhancers or transcription-factor binding sites nec and by other receptors, in order to determine the specificity essary for receptor-regulated expression. By selecting pro 40 and spectrum of their activity. moters that are responsive to the intracellular signals resulting The transcriptional reporter assay, and most cell-based from agonist binding, and operatively linking the selected assays, are well Suited for Screening chemical libraries of promoters to reporter genes whose transcription, translation chemical compounds for those that modulate OR activity. The or ultimate activity is readily detectable and measurable, the 45 libraries can be, for example, libraries from natural Sources, transcription based reporter assay provides a rapid indication e.g., plants, animals, bacteria, etc. of whether a given receptor is activated. Candidate Modulators Useful According to the Invention Reporter genes Such as luciferase, Chloramphenicol Candidate modulators can be screened from large libraries Acetyl Transferase (CAT), Green Fluorescent Protein (GFP), of synthetic or natural compounds. Numerous means are beta-lactamase or beta-galactosidase are well known in the 50 currently used for random and directed synthesis of various art, as are assays for the detection of their products. kinds of compounds. Synthetic compound libraries are com Genes particularly well Suited for monitoring receptor mercially available from a number of companies including, activity are the “immediate early’ genes, which are rapidly for example, Maybridge Chemical Co. (Trevillet, Cornwall, induced, generally within minutes of contact between the 55 UK), Comgenex (Princeton, N.J.), Brandon Associates (Mer receptor and the effector protein or ligand. The induction of rimack, N.H.), and Microsource (New Milford, Conn.). A immediate early gene transcription does not require the Syn rare chemical library is available from Aldrich (Milwaukee, thesis of new regulatory proteins. In addition to rapid respon Wis.). Combinatorial libraries of small organic molecules are siveness to ligand binding, characteristics of preferred genes available and can be prepared. Alternatively, libraries of natu useful to make reporter constructs include: low or undetect 60 ral compounds in the form of bacterial, fungal, plant and able expression in quiescent cells; induction that is transient animal extracts are available from e.g., Pan Laboratories and independent of new protein synthesis; Subsequent shut (Bothell, Wash.) or MycoSearch (NC), or are readily pro off of transcription requires new protein synthesis; and duceable by methods well known in the art. Additionally, mRNAs transcribed from these genes have a short half-life. It 65 natural and synthetically produced libraries and compounds is preferred, but not necessary that a transcriptional control are readily modified through conventional chemical, physi element have all of these properties for it to be useful. cal, and biochemical means. US 8,637,259 B1 27 28 EXAMPLES carboxylic acids has been boxed in bold. The seven tested class 1 receptors have all responded specifically and exclu The invention is further illustrated by the following non sively to carboxylic acids. limiting examples. Each molecule was tested at 3 different concentrations (1 mM,316 uM, 100 uM). The different molecules of the tested Experimental Procedure libraries were disposed at the same concentration into 96 well plates (1 well/molecule) containing cells expressing the Cell Culture and Cell Line Generation receptor of interest. The activity of the tested molecules was Cells were maintained in minimal essential medium measured using the lucifierase activity as explained above. (EMEM, Lonza) containing 10% fetal bovine serum (M10). 10 The median luciferase activity induced by the tested mol HEK293T-RTP1A1/RTP2 cells were generated by transfect ecules and the associated Standard deviation were deter ing HEK293T with an expression vector containing the mined. Putatively active molecules (hits) were defined as sequences of the chaperone proteins RTP1A1 and RTP2 and molecules inducing a luciferase activity higher or equal to the a resistance gene to puromycin, using Lipofectamine 2000. A median--2 standard deviations. recombinant cell population was selected by adding 10 ug/ml 15 of puromycin into the culture medium. Monoclonal popula Table 2 summarizes the results of this deorphanization. tions were further obtained by limit dilution procedure. Each OR-activating molecule couple is indicated by a black Briefly, a cell suspension was diluted to contain 1 cell per ml square at the intersection of the column corresponding to the and this dilution was dispatched in poly-D-lysine-coated 96 receptor and the row corresponding to the molecule. The wells plates (200 ul of dilution per well). After 5 days of results clearly show that the seven ORs of the invention are culture, the presence and number of cell colonies per well was activated by carboxylic acids which are present in human checked under a phase contrast microscopic. After 5 addi SWeat. tional days of culture, wells containing a single colony were The 7 ORs of the invention were further included in a large harvested and each collected population was amplified inde screening campaign aiming to test different molecule librar pendently. 25 ies that do not contain carboxylic acids. These screenings Odorant Molecule Dilution were performed as described above. A total of 823 molecules Odorant molecules were diluted at a concentration of 1 were tested on OR52L1, OR52E8, OR51I2, OR52A5 and mole/liter (M) into dimethyl sulfoxide (DMSO) to generate OR56A6. A total of 592 molecules were tested on OR52B2 stock solutions. and a total of 777 were tested on OR52E1. The complete list For screening experiments, stock solutions of odorant mol 30 of the tested molecules is given in Table 3. None of the ecules were diluted in EMEM disposed in 96-well plates. molecules gave a hit on any of the 7 ORs of the invention. This Plates containing the tested molecules (1 molecule? well) at a result confirms the very high selectivity of the 7 ORs of the concentration of 2 mM, at a concentration of 632 uMandata concentration of 200 uM were prepared. invention to carboxylic acid ligands. For concentration-response analysis, serial dilutions of the 35 The 7 tested ORs from class 1 (corresponding to the ORs tested molecules were prepared from Stock solutions in of the invention, namely: OR52L1, OR52E8, OR52B2, EMEM plated into 96-well plates. OR51I2, OR52E1, OR52A5 and OR56A5) were therefore Luciferase Assay. found to respond specifically and exclusively to carboxylic For the initial deorphanisation screening and dose-re acids. sponse analysis, a Luciferase-based gene reporter assay 40 (Promega, Leiden, The Nederlands) was used as described in Example 2 Saito et al. (2004). Briefly, cells were platted on poly-D- lysine-coated 96-well plates (BDBioscience, Erembodegem Dose-Response Analysis of Ligand-OR Interaction Dorp, Belgium) and transfected with a plasmid containing the CRE-luciferase and a plasmid containing the olfactory recep 45 The hits were validated by concentration-response analy tor. Sixteen hours after transfection, the culture medium was sis. Semi-logarithmic serial dilutions of hit molecules, from 1 replaced by serum-free EMEM containing the tested ligandat mM to 316 nM, were tested on the responding ORS using the a determined concentration. After four hours of incubation at luciferase assay as described above. 37° C. degree, cells were lysed and processed for lumines Results are given in Table 2. Full results are given in FIG. cence measurement according to the manufacturer's proto 50 2A-FIG. 2.J. cols. Luminescene emission was recorded on a Spectra Max We observed that each of the 7 ORS tested respond to at M5 reader (Molecular Devices, Sunnyvale, Calif.). Results least one molecule containing a carboxylic function. A care were expressed as percentage of the response induced by 10 ful comparison of these activators with the known carboxylic uM of the adenylate cyclase activator Forskolin. acids occurring in human Sweat revealed that each of the 55 receptors responds to at least one carboxylic acid released in Example 1 Sweat. Some of these acids, such as hexanoic acid, 3-methyl hexanoic acid, (E)-3-methyl-2-hexenoic acid, 3-hydroxy-3- Screening of Odorant Molecule Libraries methylhexanoic acid, heptanoic acid, octanoic acid, 4-ethy loctanoic acid, (Zeng et al. 1991; J. Chem. Ecolog. Vol. 17 pp Odorant molecule libraries containing carboxylic acids 60 1469-1492; Natsch et al. 2006 Chem. & Biodiv. Vol. 3 pp and other types of molecules were used to identify activators 1-20) are known to be important promoters of human Sweat of the seven ORs of the invention. The deorphanisation cam malodor. paign was performed on the seven olfactory receptors with a These 7 ORs of the invention are therefore involved in the series of 148 odorant molecules. Sixteen carboxylic acids perception of Sweat malodorelicited by carboxylic acids and present in Sweat were included within the 148 tested odorants. 65 constitute valuable candidate receptors for the identification Black Squares correspond to a response of a receptor to one of antagonists and/or blockers that would block the percep odorant molecule. The part of the table corresponding to tion of malodor. US 8,637,259 B1 29 30 TABLE 2

Activation of ORS according to the invention by carboxylic acids originating from Sweat.

butanoic acid Isovaleric acid pentanoic acid hexanoic acid 2-methylhexanoic acid 3-methylhexanoic acid

(E)-3-methyl-2-hexenoic acid

3-hydroxy-3-methylhexanoic acid heptanoic acid 2-methylheptanoic acid octanoic acid 4-ethyloctanoic acid nonanoic acid

decanoic acid

undecanoic acid

benzoic acid (R)-(+)-Citronellal (R)-(+)-Pulegone (S)-(-)-Citronellol 1,4-Butanedithiol 1-amino-2-phenylethane 1-Butaneethiol 1-cyclohexylethanol 1-Furfurylpyrrole 1-hepten-3-ol 1-Propanol 2,6-Dimethylthiophenol 2-Butanone 2-cyclohexylethanol 2-Methylbutyl acetate 2-Methylpyridine 2-Nonanone 3,4-Dimethoxyphenyl acetone 3-Octanone 4-(4-Methoxyphenyl)-2-butanone 4-(methylthio)butanol -Hydroxy-3-methoxyphenylacetone 4-Hydroxybenzaldehyde 4-Propylphenol 5-Hexen-1-ol Acetophenone a-ionone Allyl cyclohexylpropionate Allyl mercaptain Allyl sulfide alpha-Methylbenzyl alcohol AMYL BENZOATE Amyl Salicylate Androstanolone Anisyl acetate US 8,637,259 B1 31 32 TABLE 2-continued

Activation of ORS according to the invention by carboxylic acids originating from Sweat.

a-pinene a-terpineol Benzophenone Benzyl acetate Benzymercaptain Butyraldehyde Carvacrol Caryophyllene Cinnamyl acetate | | | | | | | cis-6-Nonenal CITRAL DIMETHYLACETAL Coumarin EMICyclopentadecanone H D-Carvone DECALACTONE DELTA DECYLACETATE dihexyl fumarate Dihydroanethole Dihydroeugenol Dimethylsulfide DIONE Ethyl 2-mercaptopropionate Ethyl p-anisate Ethyl phenylacetate Ethylvanillin Eucalyptol oridile Frutonile Furfuryl butyrate FurFuryl methylsulfide Gammajasmolactone gamma-undcalactone Geranyl acetate Guaiacol Heptaldehyde

Hexana Hexyl octanoate Isoamyl laurate Isobornylcyclohexanol Jasmacyclene Jasmatone JASMOLACTONE Jessate Lauryl alcohol L-Nicotine Lyral Menthalactone Menthol Methyl 3-nonenoate Methyl anthranilate METHYL HEPTENONE PURE Methyl Laitone methyl salicylate Nectaryl NONADIENOL-2,6 US 8,637,259 B1 33 34 TABLE 2-continued Activation of ORS according to the invention by carboxylic acids originating from Sweat.

Nonanal

Nonyl Alcohol n-Valeraldehyde Ocimene o-cresol Octanal Octyl propionate OXYOCTALINE FORMATE Para-methoxyacetophenone p-cresyl methyl ether p-Dimethoxybenzene Phenethyl 2-furoate Phenyl acetate Phenylethanol Piperonyl acetate Piperonyl isobutyrate p-Mentha-8-thiol-3-one PRENYL BENZOATE Propenylguaethol PROPYLIDENEPHTHALIDE Pyrrole Raspberry ketone

etrahydrogeraniol etrahydromyrcenol Thymol tridecenenitrile undecanal

Undecavertol Undecene-2-nitrile Vanilin Verdyl propionate Vetiveryl Acetate

TABLE 3 TABLE 3-continued Complete list of odorant molecules tested on the 7 ORs of the invention. Complete list of odorant molecules tested on the 7 ORs of the invention. gamma Dodecalactone (natural) 45 Isobutyl phenylacetate Hexyl isobutyrate Benzyl phenylacetate 3-Acetyl-2,5-dimethylthiophene Anisyl phenylacetate Acetaldehyde ethyl phenylethyl acetal Triacetin Methyl isoeugenol 2-Methyl-4-phenyl-2-butanol 4-Isopropylcyclohexanol Methyl cinnamate Ethyl maltol 50 Benzyl isobutyrate Prenyl benzoate Ethyl cinnamate 2-Methyl-3-(p-methoxyphenyl)propanal Benzyl butyrate (+)-Camphene Benzyl cinnamate Ethyl acetoacetate ethylene glycol ketal Phenethyl acetate Acetanisole Benzyl ether 4,5-Dihydro-3(2H)thiophenone 55 Phenethyl cinnamate Styrene Cinnamyl acetate Benzyl alcohol 2-Phenoxyethyl isobutyrate Benzaldehyde 1-Bromo-2-phenylethylene Benzyl mercaptain 2,2-Dimethyl-3-(3-methylphenyl)propanol 2-Ethylpyridine 2-Methyl-3-(p-isopropylphenyl)propionaldehyde alpha,alpha-Dimethylphenethyl alcohol p-Tolylacetaldehyde Dimethylbenzyl carbinyl butyrate 60 1-Phenyl-3-methyl-3-pentanol alpha-Methylcinnamaldehyde Anisyl acetate Methyl phenylacetate p-Propyl anisole Phenylacetaldehyde dimethyl acetal gamma-Octalactone Diphenyl ether Cinnamic alcohol alpha-Amylcinnamyl alcohol Cinnamaldehyde alpha-Hexylcinnamaldehyde 6 5 gamma-Nonalactone p-Tolyl phenylacetate 2-Cyclohexyliden-2-phenylacetonitrile US 8,637,259 B1 35 36 TABLE 3-continued TABLE 3-continued Complete list of odorant molecules tested on the 7 ORs of the invention. Complete list of odorant molecules tested on the 7 ORs of the invention. Phenethyl formate Benzyl salicylate gamma-Undecalactone 5 Maltol (-)-C-Terpineol 2,6-Dimethylthiophenol 4-Methylanisole 4-Butanedithiol Ethyl 6-acetoxyhexanoate Prenyl acetate Anisyl alcohol 8-Octanedithiol 3-Decen-2-one 2-Furyl methyl ketone gamma-Heptalactone 10 Methyl salicylate Ethyl propionate 2-Acetyl-5-methylfuran Diethyl malonate Fenchone Ethylbutyrate Benzophenone Acetal Styrallyl propionate Geranyl acetate sobutylbenzoate Ethylene brassylate Benzyl benzoate omega-Pentadecalactone 15 Heliotropin Butyl laurate indole 3,7-Dimethyl-1-octanol Ethylvanillin Citronellol Vanillin (+)-Citronellal Ammonium sulfide Geraniol Ethyl 3-phenylglycidate Nerol Triethylamine Isoamylbutyrate Methylp-anisate Ethylheptanoate 4-Methylacetophenone Ethyl octanoate Cuminaldehyde p-Cresol ,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopentag-2- Dimethyl Succinate benzopyran Solution 2,6-Dimethyl-5-heptenal 25 alpha-Amylcinnamaldehyde 1-Propanethiol Benzylpropionate Hydroxycitronellal Cinnamyl cinnamate Isopentylamine 3-Phenylpropyl acetate Ethyl isovalerate Phenylacetaldehyde Benzenethiol 3-Phenyl-1-propanol 3-Methylpyridine 30 Phenoxyethanol 2-Methylpyridine 4-Ethylphenol 2-Methylpyrazine 4-Hydroxybenzaldehyde Octyl isobutyrate p-Anisaldehyde 16-Hexadecalactone 4-Heptanone Butyl 10-undecenoate Ethylnonanoate Butylamine 35 2,5-Dimethylpyrazine -Butanethiol Myrcene 3-Propanedithiol Propionaldehyde Pyrrole Soamyl alcohol Sopropyl myristate Ethylhexanoate Diethyl sebacate Allylhexanoate Methyl decanoate 2-Heptanone 40 Butyl acetate Soamyl formate Soamyl acetate n-Valeraldehyde Ethyl myristate Pyridine Sobornyl acetate Piperidine alpha-Ionone 6-Methyl-5-hepten-2-one 3 methyl 4 (2,6,6 trimethyl 2 cyclohex-1-yl-3 buten 2 One) alpha Methyl 2-octynoate 45 isomethyl ionone Hexylamine Butylated hydroxytoluene Hexyl alcohol 9-Decen-1-ol Ethyl octadecanoate 2-Hexylcyclopentanone Heptaldehyde 2,4-Dimethyl-2-(1,1,4,4-tetramethyltetralinyl)-1,3-dioxolan Hexyl octanoate 2,6-Dimethyl-2-heptanol Methyl 2-nonenoate 50 Menthalactone Methyl 2-nonynoate Amyl 2-furoate Methyl 10-undecenoate Methyl anthranilate Methyl laurate Guaicwood acetate Myrcenyl acetate 3-Carene 2-Undecanone Methyl 3-nonenoate Octyl acetate 3,5-Dimethyl-1,2-cyclopentadione Decyl acetate 55 Syringaldehyde 2-Acetylpyridine Methyl 3-(methylthio)propionate Decanal 6-Isopropylquinoline 10-Undecen-1-ol Furfuryl 3-methylbutanoate Undecanal 2-n-Heptylcyclopentanone 10-Undecena Cuminyl nitrile Dodecyl aldehyde 60 cis-4-(1-Methylethyl)cyclohexanemethanol Methyl stearate 1,3,3-Trimethyl-2-norbornanyl acetate Methyl linoleate 3,7-Dimethyl-1,3,6-octatriene Dihydrolasmone dI-Limonene Linallyl acetate 2-t-Butylcyclohexyloxy-2-butanol Linallyl formate Benzyl acetate (2,6,6-Trimethylcyclohexa-1,3-dienyl)methanal 65 Phenethyl isovalerate 2(2,4-Dimethylcyclohex-3-en-1-yl)-5-methyl-5-(1-methylpropy-) p-Methylphenyl acetate

US 8,637,259 B1 39 40 TABLE 3-continued TABLE 3-continued Complete list of odorant molecules tested on the 7 ORs of the invention. Complete list of odorant molecules tested on the 7 ORs of the invention. Octyl 2-furoate Ethyl 2-ethyl-6,6-dimethyl-2-cyclohexenecarboxylate alpha-Furfuryl octanoate 5 Ethyl tiglate Ethyl 2-methylpentanoate 3,5,5-Trimethylhexyl acetate Furfurylheptanoate Formaldehyde cyclododecyl ethyl acetal 9-Decenal Terpinolene 3,7-Dimethyl octanenitrile D-(+)-Xylose trans-Nerolidol Isobutyl 2-butenoate 2,5-Dimethyl-4-methoxy-3 (2H)furanone 10 3-Heptanol Acetaldehyde ethyl linallyl acetal Menthanyl acetate (Z)-3-Hexenyl isobutyrate Acetoxymethyl-isolongifolene Octahydro-7-methyl-1,4-methanonaphthalen-6-2H)-one 2,3-Dimethylpyrazine Anetho Ethyl 2-methyl-6-pentyl-4-oxocyclohex-2-enecarboxylate 3,7-Dimethyl-7-methoxyoctan-2-ol Butyl formate Diisopropyl disulfide 15 Allyl sulfide p-Menthane-3,8-diol Tetrahydrogeranial Diacety d-Limonene 2-Ethylhexanal ethylene glycol acetal 2,3-Pentanedione Cinnamyl nitrile Tributyrin 2-Phenyl-2-butenal Phenethyl alcohol Octahydrocoumarin 2-Pentylbutyrate 2,2'-(Dithiodimethylene)difuran 2O Methyl 2-furoate 3,4-Hexanedione cis-3-Hexenyl cis-3-hexenoate Farneso L-Menthyl lactate NOOTKATONE Sobutyl tiglate -Fenchone Ethyl pyruvate ALPHA. CEDRENE ((-)-a-Cedrene;1S,2R,5S)-2,6,6,8- 3,7-Dimethyl-2,6-nonadienenitrile Tetramethyltricyclo[5.3.1.01.5undec-8-ene) 25 Dihydrocarveol 4-Cineole Cyclohexyl acetate Methylatrarate Furfuryl acetate Eucalyptol Furfuryl butyrate 2-Pentylcyclopentanone Methylbutyrate cis-Jasmone Ethyl trans-2-butenoate Acetovanillone 30 2-Methylbutyl acetate Dihydro-alpha-terpineol 6-Methylheptan-3-one Cyclopentadecanone Ethyl methylsulfide 4-Dithiane Dimethyl disulfide Borneol 3-Penten-2-one n-Amyl phenylacetate Hexyl salicylate Acetoin 35 Diethyl malate 3,7-Dimethyl-2,6-Octadienenitrile Propylhexanoate -Citronellyl nitrile Ethyl undecanoate 3-(Methylthio)-1-hexanol Ethyl palmitate 2,4,6-Trimethyl-4-phenyl-1,3-dioxane Butyl 2-methylvalerate Ethyl (+)-2-hydroxycaproate Soamyl laurate Methyl 3,3-dimethylbicyclo[2.2.1]heptan-3-carboxylate 2-Isobutyl-4-hydroxy-4-methyltetrahydropyran Phenylethyl n-butyl ether 40 Sopropylbutyrate trans-2-Undecenal Amyl octanoate 4-Isopropylbenzyl alcohol Phenethylamine 3-Ethylpyridine Propyl isobutyrate Methylbenzyl ether 4-Propylphenol Ethyl valerate L-Carvone Amylhexanoate 45 5-(2,2,3-Trimethylcyclopent-3-en-1-yl)-3-methylpentan-2-ol Sobutyl propionate trans-4-Decenal Ethyl 3-hydroxybutyrate 2.5,9,10-Tetramethyl-5,6-dehydro-1-decallyl formate Tricyclodecenyl acetate Maltol isobutyrate 3-Methyl-1-cyclopentadecanone 2,2,5-Trimethyl-5-pentylcyclopentanone Hexyl phenylacetate 4-Methyl-5-thiazoleethanol acetate 2-Acetyl-3,5(6)-dimethylpyrazine 50 Isoamyl isovalerate 3,5,5-Trimethylhexanal Methyl tiglate Octahydro-2,3,8,8-tetramethyl-2-acetonaphthone Hexanal 2-Butyl-4,4,6-trimethyl-1,3-dioxane 4-Allyl-2,6-dimethoxyphenol Thujone sopropyl 2-methylbutyrate 4-(4-Hydroxyphenyl)-2-butanone p-lsobutyl-alpha-methylhydrocinnamaldehyde Ethylene dodecanedioate 55 trans-2-Hexen-1-al 3-Methyl-5-phenyl-1-pentanol 4-Heptenal 3-Methyl-5-phenyl-1-pentanal 3,5,5-Trimethylhexyl formate 3-Butylidenephthalide 3,3,5-Trimethylcyclohexylethylether 2-Columaranone Sopropyl alcohol Methyl isovalerate cis-3-Hexenyl methyl carbonate 3-Methyl-2-buten-1-ol Allyl amylglycolate 2,6-Nonadienal (trans, cis) 60 para-Ethyl-alpha,alpha-dimethyldihydrocinnamaldehyde Phenylethyl isoamyl ether Hydratropaldehyde propylene glycol acetal 4-Carvomenthenol Phenethyl pivalate Caryophylene acetate ,1-Dimethoxy-2,2,5-trimethyl-4-hexene 2-Methyl-3-tetrahydrofuranthiol Methylsulfoxide delta-Damascone Ethyl-2-t-butylcyclohexylcarbonate 2,6-Xylenol 65 7-Formyl-5-isopropyl-2-methylbicyclo[2.2.2]oct-2-ene 1-Phenyl-1,2-propanedione Amylcyclohexyl acetate (mixed isomers) US 8,637,259 B1 41 42 TABLE 3-continued TABLE 3-continued Complete list of odorant molecules tested on the 7 ORs of the invention. Complete list of odorant molecules tested on the 7 ORs of the invention. cis-3-Hexenyl tiglate 2-Nonanone Hexyl benzoate 5 2,4-Dimethyl-4-phenyltetrahydrofuran (-)-Ambroxide Skatole Isolongifolene epoxide N-Methyl-N-phenyl-2-methylbutyramide Phenylethyl isopropyl ether Diethyl phthalate 4-Methyl-4-phenyl-2-pentyl acetate 3-Methyldodecanonitrile Grisalva Isobornyl isobutyrate 2-Ethoxy-9-methylene-2,6,6-trimethylbicyclo[3.3.1..nonane 10 2-Methoxybiphenyl Methyl 1-methyl-4-isopropylbicyclo[2.2.2]oct-5-enecarboxylate Methyl 2-methylbutyrate Methyl sorbate Allyl mercaptain Cyclomugual Isoamyl Salicylate 2-Methyldecanonitrile Caryophyllene 6- or 7-Ethylideneoctahydro-5,8-methano.2H-1-benzopyran-2-one p-Methoxybenzonitrile 5-Ethyl-3-hydroxy-4-methyl-2(5H) furanone 15 Tetrahydrolinalool delta-Decalactone Diethyl L-tartrate gamma-Decalactone o-t-Butylcyclohexyl acetate 1-(2,2,6-Trimethylcyclohexyl)-3-hexanol 2-Isopropylphenol 2-methoxyphenol reaction products with hydrogenated 2,2- Isopulegyl acetate dimethyl-3-methylenebicyclo[2.2.1]heptane (-)-Isopulegol delta-Undecalactone Menthone 1-p-Menthene-8-thiol 2O Thymol 1-Propanol Salicylaldehyde delta-Dodecalactone 1-Methylnaphthalene Phenethyl 2-furoate Trichloromethyl phenyl carbinyl acetate 2.5,5-Trimethyloctahydro-2-naphthol 2-Methylpentyl 2-methylpentanoate 2-Methyl-4-(2,6,6-trimethyl-2-cyclohexenyl)butanal 2-Phenylpropionaldehyde dimethyl acetal Methyl trans-2-octenoate 25 Naphthalene 2.2,6-Trimethyl-6-vinyltetrahydropyran 2-Naphthalenethiol 2,2-Dimethyl-5-(1-methylpropen-1-yl)tetrahydrofuran Coumarin Ethyl 2-methylbutyrate trans-Isoeugenylbenzyl ether Citronellyloxyacetaldehyde trans-2-Hexen-1-ol Butylbutyryllactate cis-3-Hexen-1-ol Acetaldehyde phenethylpropyl acetal 30 Methyl beta-naphthyl ketone Allyl phenoxyacetate 4-Allyl-1,2-dimethoxybenzene Ethylamine beta-Naphthyl ethyl ether Acetaldehyde 2-Phenylpropionaldehyde Ethanethiol Methylbenzoate 1,5-Dimethyl bicyclo(3.2.1)octan-8-one oxime Methyl nicotinate Dimethylsulfide 35 Ethylbenzoate Citral dimethyl acetal alpha-Methylbenzyl acetate (+)-Camphor -Ethylhexyl tiglate Cedarwood oil alcohols Ethylp-anisate Cedryl acetate Soamylbenzoate Terpinyl isobutyrate Geranyl benzoate 3-Methyl-3-pentanol Piperine (O-6-Hexadecenlactone 40 Propenylguaethol Soamyl cinnamate 2-Hexyl-2-cyclopenten-1-one Sobutyl acetoacetate o-Cresol sobutylbenzyl carbinol 2,5-Xylenol Ethyl 3-methyl-3-phenylglycidate 2-(2-(4-Methyl-3-cyclohexen-1-yl)propyl)cyclopentanone (1S)-(-)-C-Pinene 2,3-Heptanedione L-Fenchone 45 Eugenol Triethylcitrate Isoeugenol Linallyl isobutyrate Ethyl isobutyrate Linallyl cinnamate Isobutyl isobutyrate Linalool Citronellyl isobutyrate Sobutylamine Tetrahydrofurfuryl alcohol sobutyl alcohol 50 alpha-Terpineol sobutyraldehyde alpha-Methylbenzyl alcohol 2-Butanol alpha-Phellandrene 2-Butanone gamma-Terpinene Pyruvaldehyde alpha-Terpinene Methyl acetate p-Cymene alpha-Irone 55 Composé Inconnu Allyl alpha-ionone Forskolin Geranium bourbon Sodium Sulfide hydrate 1,3-Dimethylbut-3-enyl isobutyrate Sodium methanethiolate Cognac oil, green 3-mercapto-1-pentanol alpha-Terpinyl acetate 3-mercapto-2-methyl-1-butanol p-tert-Butyl-alpha-methyldihydrocinnamic aldehyde 3-Mercapto-3-methyl-1-hexanol alpha-Pinene 60 trimethylamine HCL Ethyl octahydro-4,7-methano.3aHindene-3a-carboxylate putrescine Musk ketone cadaverine Musk xylol morpholine 4-Methyl-3-decen-5-ol 4-methylmorpholine 2.4.4,7-Tetramethyl-6,8-nonadiene-3-one oxime Urea Acetyl diisoamylene 65 Androstadienol 3,5,6,6-Tetramethyl-4-methylene-2-heptanol geovertol

US 8,637,259 B1 45 46 TABLE 3-continued TABLE 3-continued Complete list of odorant molecules tested on the 7 ORs of the invention. Complete list of odorant molecules tested on the 7 ORs of the invention. 2,4-dimethyl-2H4H.4aH,5H.9bH-indeno1,2-d1.3dioxine (3aR.5aS,9aS,9bS)-3a,6,6,9a-tetramethyl-2,4,5,5a,7,8,9,9b-octahydro 1-1-(3,3-dimethylcyclohexyl)ethoxy-2-methylpropan-2-ylpropanoate 5 1H-benzoebenzofuran 2-1-(3,3-dimethylcyclohexyl)ethoxy-2-methylpropyl propanoate

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 22

<21 Os SEQ ID NO 1 &211s LENGTH: 990 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

<4 OOs SEQUENCE: 1 atgactittgg tttcttttitt ct ctitt.cct c to caa.gc.cat tdataatgct cottagcaat 60 tdaagctgga ggctatc.cca gcctt Cttitt ct cotggtag ggattic Cagg tittagaggaa 12O agc.ca.gcact ggattgcact gcc.cctgggc atcc titt acc to cittgctitt agtgggcaat 18O

gttaccatt c tott catcat ctdgatggac ccatccttgc accalat citat gtacct ctitc 24 O ctgtc catgc tagctgc cat cqacctggitt ctdgcct cot coactgcacc caaag.ccctt 3 OO gcagtgctcc tigttcatgc cc acgagatt ggg tacatcg totgcctgat coagatgttc 360 ttcatcCatg cattctic ct c catggagitta gggg tacttg tdgc catggc tictggattgc 42O

tatgtagcca tttgtcaccc cttgcac cat tccacaatcc togcatcCagg ggtcat aggg 48O

cgcatcggala tigtggtgct ggtgagggga ttact actcc titat coccitt CCCC attittg 54 O ttgggaacac titatic ttctg. ccaagccacc at cataggcc atgcct attg tdaacatatg 6 OO gctgttgttga aacttgcct g ct cagaalacc acagt caatic gagct tatgg gctgactatg 660

gccttgcttg tattgggct ggatgttctg gc cattggtg titt cctatgc cc acat cotc 72O Caggcagtgc tigaaggt acc agggagtgag gC cc.gacitta aggcgtttag cacatgtggc 78O

tot catattt gtgtcatcct gg.tcttctat gtcc ctdgaa ttitt ct cott cotcact cac 84 O cgctittggtc at catgtacc ccatcatgtc. catgttcttic toggccacacg gtat ct cotc 9 OO

atgccacctg. c9ct caatcc tdttgtctat ggagtgaaga ct cagcagat CCC cagcga 96.O

gtgct cagag titttacaca aaaggattaa 990

<21 Os SEQ ID NO 2 &211s LENGTH: 329 212s. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs SEQUENCE: 2 Met Thr Lieu Val Ser Phe Phe Ser Phe Leu Ser Llys Pro Lieu. Ile Met 1. 5 1O 15

Lieu Lleu Ser Asn. Ser Ser Trp Arg Lieu. Ser Glin Pro Ser Phe Lieu. Lieu. 2O 25 3 O

Val Gly Ile Pro Gly Lieu. Glu Glu Ser Gln His Trp Ile Ala Leu Pro 35 4 O 45

Lieu. Gly Ile Lieu. Tyr Lieu. Lieu Ala Lieu Val Gly Asn Val Thir Ile Lieu. SO 55 60

Phe Ile Ile Trp Met Asp Pro Ser Lieu. His Glin Ser Met Tyr Lieu Phe 65 70 7s 8O

Lieu. Ser Met Lieu Ala Ala Ile Asp Lieu Val Lieu Ala Ser Ser Thr Ala US 8,637,259 B1 47 48 - Continued

85 90 95

Pro Ala Lieu Ala Wall Lieu. Lieu. Wall His Ala His Glu Ile Gly Tyr 1OO 105 11 O

Ile Wall Cys Lieu. Ile Gln Met Phe Phe Ile His Ala Phe Ser Ser Met 115 12 O 125

Glu Luell Gly Wall Lieu. Wall Ala Met Ala Luell Asp Cys Wall Ala Ile 13 O 135 14 O

Cys His Pro Lieu. His His Ser Thr Ile Luell His Pro Gly Wall Ile Gly 145 150 155 160

Arg Ile Gly Met Wall Wall Lieu Wall Arg Gly Luell Lell Lell Luell Ile Pro 1.65 17O 17s

Phe Pro Ile Lieu. Luell Gly Thr Lieu. Ile Phe Glin Ala Thir Ile Ile 18O 185 19 O

Gly His Ala Glu. His Met Ala Wall Wall Lell Ala Cys Ser 195 2OO

Glu Thir Thir Wall Asn Arg Ala Tyr Gly Luell Thir Met Ala Luell Lieu Wall 21 O 215

Ile Gly Luell Asp Val Lieu Ala Ile Gly Wall Ser Ala His Ile Lieu. 225 23 O 235 24 O

Glin Ala Wall Lieu Lys Val Pro Gly Ser Glu Ala Arg Lell Ala Phe 245 250 255

Ser Thir Gly Ser His Ile Cys Wall Ile Luell Wall Phe Tyr Wall Pro 26 O 265 27 O

Gly Ile Phe Ser Phe Lieu. Thir His Arg Phe Gly His His Wall Pro His 27s 28O 285

His Wall His Wall Lieu. Lieu Ala Thr Arg Luell Lell Met Pro Pro Ala 29 O 295 3 OO

Lell Asn Pro Lieu Wall Tyr Gly Val Thir Glin Glin Ile Arg Glin Arg 3. OS 310 315 32O

Wall Luell Arg Wall Phe Thr Gln Lys Asp 3.25

<210s, SEQ ID NO 3 &211s LENGTH: 954 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 3 atggcaggaa gaatgtctac gtctaatcac acc cagttcc at cottct to att cotactg 6 O

Ctgggt at CC Cagggctaga agatgtgcac atttggattg gtgtc.cc titt tittctttgttg 12 O tat cittgttg cacticcitggg aaacactgct citc.ttgtttg tgatccagac tgagcagagt 18O citccatgagc ctato tacta citt cotggcc atgttggatt c cattgacct gggcttgtct 24 O acagccacca tcc ccaaaat gttgggcatc ttctggttca ataccaaaga aat at Cttitt 3OO ggaggctgcc titt ct cacat gttct tcatc catttgttca Ctgctatgga gag cattgttg 360 ttggtggc.ca tggcctittga cc.gctacatt gcc atttgca aac ct ctitcg gtacaccatg atcCtcacca gcaaaatcat cagcct catt gCagg cattg Ctgtc.ctgag gag cctgtac atggttgttc cactggtgtt tot cottctg aggctg.ccct tctgtgggca tcqtat catc 54 O c ct catact t attgtgagca catgggcatt catcaaagtic aacattaggit ttggcc ttgg caa.catat ct citc.ttgttac tggatgttat cott attatt 660 citct cotatg t caggat.cct g tatgctgtc ttctgcc togc Cct Cotggga agct cqactic 72 O aaagct ct ca acacctgtgg ttct catatt ggtgttatct tagcc tttitt tacaccagoa US 8,637,259 B1 49 - Continued tttittitt cat t cittgacaca totgttittggc cataatat co cacagtatat acat attata 84 O ttagccaa.cc td tatgtggit tdtcc cacca gcc ct caatic ctd taatcta toggagt cagg 9 OO acaaag caga titcgagagag agtgctgagg atttitt Ctca agaccalatca ctaa 954

<210s, SEQ ID NO 4 &211s LENGTH: 317 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 4 Met Ala Gly Arg Met Ser Thr Ser Asn His Thr Glin Phe His Pro Ser 1. 5 1O 15 Ser Phe Lieu. Lieu. Lieu. Gly Ile Pro Gly Lieu. Glu Asp Wal His Ile Trp 2O 25 3O Ile Gly Val Pro Phe Phe Phe Val Tyr Lieu Val Ala Lieu. Leu Gly Asn 35 4 O 45

Thir Ala Lieu Lleu Phe Wall Ile Glin Thr Glu Glin Ser Lieu. His Glu Pro SO 55 6 O Met Tyr Tyr Phe Lieu Ala Met Lieu. Asp Ser Ile Asp Lieu. Gly Lieu. Ser 65 70 7s 8O Thr Ala Thr Ile Pro Llys Met Leu Gly Ile Phe Trp Phe Asn. Thir Lys 85 90 95 Glu Ile Ser Phe Gly Gly Cys Lieu Ser His Met Phe Phe Ile His Phe 1OO 105 11 O Phe Thr Ala Met Glu Ser Ile Val Lieu Val Ala Met Ala Phe Asp Arg 115 12 O 125 Tyr Ile Ala Ile Cys Llys Pro Leu Arg Tyr Thr Met Ile Lieu. Thir Ser 13 O 135 14 O Lys Ile Ile Ser Lieu. Ile Ala Gly Ile Ala Val Lieu. Arg Ser Lieu. Tyr 145 150 155 160 Met Val Val Pro Leu Val Phe Leu Lleu Lieu. Arg Lieu Pro Phe Cys Gly 1.65 17O 17s His Arg Ile Ile Pro His Thr Tyr Cys Glu. His Met Gly Ile Ala Arg 18O 185 19 O Lieu Ala Cys Ala Ser Ile Llys Val Asn. Ile Arg Phe Gly Lieu. Gly Asn 195 2OO 2O5 Ile Ser Lieu. Lieu Lleu Lieu. Asp Val Ile Lieu. Ile Ile Lieu. Ser Tyr Val 21 O 215 22O Arg Ile Lieu. Tyr Ala Val Phe Cys Lieu Pro Ser Trp Glu Ala Arg Lieu 225 23 O 235 24 O Lys Ala Lieu. Asn. Thir Cys Gly Ser His Ile Gly Val Ile Lieu Ala Phe 245 250 255 Phe Thr Pro Ala Phe Phe Ser Phe Lieu. Thr His Arg Phe Gly His Asn 26 O 265 27 O Ile Pro Glin Tyr Ile His Ile Ile Leu Ala Asn Lieu. Tyr Val Val Val 27s 28O 285 Pro Pro Ala Lieu. Asn Pro Val Ile Tyr Gly Val Arg Thr Lys Glin Ile 29 O 295 3 OO Arg Glu Arg Val Lieu. Arg Ile Phe Lieu Lys Thr Asn His 3. OS 310 315

<210s, SEQ ID NO 5 &211s LENGTH: 972 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens US 8,637,259 B1 51 52 - Continued

<4 OOs, SEQUENCE: 5 atgagt caca ccaatgttac cat cit to cat cctgcagttt ttgtcct tcc tggcatc cct 6 O gggttggagg CttatcaCat ttggctgtca attacct ctitt gcct cattta cat cactgca 12 O gtcCtgggaa acagoat cct gatagtggitt attgt catgg aacgtaacct t catgtgcc c 18O atgt atttct t cottctgaat gctggc.cgt.c atgga catcc tgctgtctac caccactgtg 24 O

Ccca aggc cc tagc.cat citt ttggcttcaa gcacatalaca ttgcttittga tgcctgtgtc. 3OO acccaaggct totttgtc.ca tatgatgttt gtgggggagt cagctat cot gttagc.catg 360 gcctittgatc gctttgttggc catttgttgcc c cact gagat atacaiacagt gctaa catgg

Cctgttgttgg ggaggattgc tctggc.cgtc atcaccc.gaa gctitctgcat Catct tcc.ca gtcatatt ct tgctgaag.cg gctg.ccct tc tgcct aacca acattgttcc to act CCtac 54 O tgtgagcata ttggagtggc tcqtttagcc tgtgctgaca t cactgttaa catttggitat ggct tcticag tgcc cattgt catggtcatc ttggatgtta t cct catcgc tgttgtc.ttac 660 t cactgat co tcc.gagcagt gtttcqtttg c cct c cc agg atgct cqgca caaggcc ct c 72 O agcacttgttg gct cocacct ctgtgtcatc cittatgttitt atgttccatc citt citt tacic ttattgaccc at cattttgg gcqtaatatt cct caacatg to catat citt gctggccaat 84 O

Ctttatgtgg cagtgccacc aatgctgaac cc cattgtct atggtgtgaa gactalagcag 9 OO atacgtgagg gtgtagcc.ca ccggttctitt gacat Calaga Cttggtgctg tacct cocct 96.O

Ctgggct cat a.a. 972

<210s, SEQ ID NO 6 &211s LENGTH: 323 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6

Met Ser His Thir ASn Wall Thir Ile Phe His Pro Ala Wall Phe Wall Lieu 1. 5 1O 15

Pro Gly Ile Pro Gly Lieu. Glu Ala Tyr His Ile Trp Lell Ser Ile Pro 25

Lell Cys Luell Ile Tyr Ile Thr Ala Val Lieu. Gly Asn Ser Ile Lieu. Ile 35 4 O 45

Wall Wall Ile Val Met Glu Arg Asn Lieu. His Wall Pro Met Phe Phe SO 55 6 O

Lell Ser Met Lieu Ala Val Met Asp Ile Lieu. Lieu. Ser Thir Thir Thir Wall 65 70 8O

Pro Ala Lieu Ala Ile Phe Trp Lieu. Glin Ala His Asn Ile Ala Phe 85 90 95

Asp Ala Val Thr Glin Gly Phe Phe Wal His Met Met Phe Val Gly 105 11 O

Glu Ser Ala Ile Lieu. Lieu. Ala Met Ala Phe Asp Arg Phe Wall Ala Ile 115 12 O 125

Ala Pro Lieu. Arg Tyr Thr Thr Wall Lieu. Thir Trp Pro Wall Val Gly 13 O 135 14 O

Arg Ile Ala Lieu Ala Wall Ile Thr Arg Ser Phe Ile Ile Phe Pro 145 150 155 160

Wall Ile Phe Lieu Lleu Lys Arg Lieu Pro Phe Cys Lell Thir Asn Ile Wall 1.65 17O 17s

Pro His Ser Tyr Cys Glu. His Ile Gly Val Ala Arg Lell Ala Cys Ala 18O 185 19 O US 8,637,259 B1 53 54 - Continued

Asp Ile Thr Val Asn Ile Trp Tyr Gly Phe Ser Wall Pro Ile Wal Met 195 2O5

Wall Ile Lieu. Asp Val Ile Lieu. Ile Ala Wal Ser Tyr Ser Luell Ile Lieu. 21 O 215 22O

Arg Ala Val Phe Arg Lieu Pro Ser Glin Asp Ala Arg His Ala Lieu 225 23 O 235 24 O

Ser Thr Cys Gly Ser His Lieu. Cys Wall Ile Lieu. Met Phe Wall Pro 245 250 255

Ser Phe Phe Thir Lieu Lleu. Thir His His Phe Gly Arg Asn Ile Pro Glin 26 O 265 27 O

His Wal His Ile Lieu. Lieu. Ala Asn Leu Tyr Val Ala Wall Pro Pro Met 27s 285

Lieu. ASn Pro Ile Val Tyr Gly Val Llys Thir Lys Glin Ile Arg Glu Gly 29 O 295 3 OO

Wall Ala His Arg Phe Phe Asp Ile Lys. Thir Trp Thir Ser Pro 3. OS 310 315 32O Lieu. Gly Ser

<210s, SEQ I D NO 7 &211s LENGT H: 939 212. TYPE : DNA <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 7 atggggttgt t caatgtcac t caccctgca ttct tcc toc tgactggitat CCCtggtctg 6 O gaga.gctict c act cotggct gtcagggc cc Ctctg.cgtga tgtatgctgt ggCCCttggg 12 O ggaaatacag tgatcc tigca ggctgttgcga gtggagcc.ca gcc to catga gcc catgitac 18O tact tcctgt c catgttgtc citt cagtgat gtggc catat c catggccac actg.cccact 24 O gtact.ccgaa cct tctgcct caatgcc.cgc aaCat Cactt ttgatgcctg tctaatt cag 3OO atgttt citta tt cact tott ctic catgatg gaat Caggta ttctgctggc catgagttitt 360 gaccgctatg tggc.catttg tgacc ccttg cgctatgcag ctgtgct cac cactgaagtic attgctgcaa tgggitt tagg tgcagctgct cgaagct tca t caccottt t coctott coc tt tott atta agaggctgcc tat ctdcaga tccaatgttc titt cit cactic c tact gcctg 54 O cacccagaca tgatgaggct tgcctgtgct gatat cagta t caacagcat ctatogactic tttgttcttg tat coacct it tgg catggac citgttttitta tott cotctic ctatotgctic 660 attctg.cgitt Ctgtcatggc cactgct tcc cgtgaggaac gcct caaagc tot Calacaca 72 O tgtgttgtcac at atcc tiggc tgtacttgca titt tatgtgc Caatgattgg ggit ct coaca gtgcaccgct ttgggaagca tgtcc catgc tacatacatg t cct catgtc aaatgtgtac 84 O ctatttgtgc ctic ctdtgct Calacc Ctectic atttatagog c caagacaaa ggaaatc.cgc 9 OO cgagccattt tcc.gcatgtt to accacatc. aaaatatga 939

<210s, SEQ I D NO 8 &211s LENGT H: 312 212. TYPE : PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 8 Met Gly Lieu Phe Asn Val Thr His Pro Ala Phe Phe Leu Lieu. Thr Gly 1. 5 15 Ile Pro Gly Lieu. Glu Ser Ser His Ser Trp Leu Ser Gly Pro Leu. Cys 25 US 8,637,259 B1 55 56 - Continued

Wall Met Tyr Ala Val Ala Lieu. Gly Gly Asn Thir Wall Ile Luell Glin Ala 35 4 O 45

Wall Arg Wall Glu Pro Ser Lieu. His Glu Pro Met Tyr Phe Luell Ser SO 55 6 O

Met Luell Ser Phe Ser Asp Wall Ala Ile Ser Met Ala Thir Luell Pro Thir 65 70

Wall Luell Arg Thr Phe Cys Lieu. Asn Ala Arg ASn Ile Thir Phe Asp Ala 85 90 95

Luell Ile Gln Met Phe Lieu. Ile His Phe Phe Ser Met Met Glu Ser 105 11 O

Gly Ile Luell Lieu Ala Met Ser Phe Asp Arg Wall Ala Ile 115 12 O 125

Pro Luell Arg Tyr Ala Ala Val Lieu Th Thr Glu Wall Ile Ala Ala Met 13 O 135 14 O

Gly Luell Gly Ala Ala Ala Arg Ser Phe Ile Thir Lell Phe Pro Leul Pro 145 150 155 160

Phe Luell Ile Lys Arg Lieu Pro Ile Ser Asn Wall Luell Ser His 1.65 17O 17s

Ser Lieu. His Pro Asp Met Met Arg Luell Ala Ala Asp Ile 185 19 O

Ser Ile Asn Ser Ile Tyr Gly Lieu Phe Wall Luell Wall Ser Thir Phe Gly 195

Met Asp Luell Phe Phe Ile Phe Lieu. Ser Tyr Wall Lell Ile Luell Arg Ser 21 O 215 22O

Wall Met Ala Thir Ala Ser Arg Glu Glu Arg Luell Ala Luell Asn. Thir 225 23 O 235 24 O

Wall Ser His Ile Lieu Ala Val Lieu Ala Phe Wall Pro Met Ile 245 250 255

Gly Wall Ser Thr Val His Arg Phe Gly Lys His Wall Pro Cys Tyr Ile 26 O 265 27 O

His Wall Luell Met Ser Asn Val Tyr Lieu. Phe Wall Pro Pro Wall Luell Asn 285

Pro Luell Ile Tyr Ser Ala Lys Thr Lys Glu Ile Arg Arg Ala Ile Phe 29 O 295 3 OO

Arg Met Phe His His Ile Lys Ile 3. OS 310

<210s, SEQ ID NO 9 &211s LENGTH: 927 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 9 atgaatacca citctatttica t cott act ct ttcottct to tgggaattic C tgggctggala 6 O agtatgcatc tctgggttgg ttt toott to tittgctgtgt tcc tigacagc tgtcCttggg 12 O aatlatcacca toctittttgt gattcagact gacagtagt c to CatcatCC catgttctac 18O titcc toggc.ca ttctgtcatc tattgaccc.g ggcctgtcta Cat CCaC cat c cctaaaatg 24 O cittggcacct tctggtttac Cctgagagaa atctoctittg aaggatgcct tacccagatg 3OO t tott catcc. acctgtgcac tgg catggaa t cagctgtgc ttgttggc cat ggcct atgat 360 tgctatgtgg c catctgtga ccctcitttgc tacacgttgg tgctgacaaa Caaggtggtg t cagtt atgg cactggcc at Ctttctgaga c cct tagt ct ttgtcat acc Ctttgttcta 48O tittatcCtaa ggct tccatt ttgttggacac Calaattatto Ctcat actta cggtgagcac 54 O US 8,637,259 B1 57 58 - Continued atgggcattg ccc.gc.ctgtc. ttgttgc.ca.gc at Cagggitta a catcatCta tggct tatgt gccatctota tcc tiggtc.tt togacat cata gcaattgtca titt cotatgt acagatccitt 660 tgtgctgt at ttctact citc titcacatgat gcacgactica aggcatt cag Cacctgtggc 72 O t ct catgtgt gtgtcatgtt gactittctat atgcctgcat tgttct catt catgacc cat aggtttggtc ggaatatacct cactittatc cacattct to tggctaattit Ctgtgtagt c 84 O attic cacctg citct caactic totaattitat ggtgtcagaa c caaacagat tagagcacala 9 OO gtgctgaaaa tgtttittcaa taaataa 927

SEQ ID NO 10 LENGTH: TYPE : PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 10

Met Asn. Thir Thr Lell Phe His Pro Tyr Ser Phe Lell Lell Luell Gly Ile 1. 5 1O 15

Pro Gly Luell Glu Ser Met His Lieu. Trp Val Gly Phe Pro Phe Phe Ala 25

Wall Phe Luell Thir Ala Val Lieu. Gly Asn. Ile Thr Ile Lell Phe Wall Ile 35 4 O 45

Glin Thir Asp Ser Ser Lieu. His His Pro Met Phe Tyr Phe Luell Ala Ile SO 55 6 O

Lell Ser Ser Ile Asp Pro Gly Lieu. Ser Thir Ser Thir Ile Pro Lys Met 65 70 7s 8O

Lell Gly Thir Phe Trp Phe Thir Lieu. Arg Glu Ile Ser Phe Glu Gly Cys 85 90 95

Lell Thir Glin Met Phe Phe Ile His Lieu. Cys Thr Gly Met Glu Ser Ala 105 11 O

Wall Luell Wall Ala Met Ala Tyr Asp Cys Tyr Val Ala Ile Asp Pro 115 12 O 125

Lell Cys Thir Lell Wall Lieu. Thir Asn Llys Val Wall Ser Wall Met Ala 13 O 135 14 O

Lell Ala Ile Phe Lell Arg Pro Lieu. Wall Phe Wall Ile Pro Phe Wall Lieu 145 150 155 160

Phe Ile Luell Arg Lell Pro Phe Cys Gly His Glin Ile Ile Pro His Thr 1.65 17O 17s

Gly Glu His Met Gly Ile Ala Arg Lieu. Ser Ala Ser Ile Arg 18O 185 19 O

Wall Asn Ile Ile Tyr Gly Lieu. Cys Ala Ile Ser Ile Lell Wall Phe Asp 195

Ile Ile Ala Ile Wall Ile Ser Tyr Wall Glin Ile Lell Ala Wall Phe 21 O 215

Lell Luell Ser Ser His Asp Ala Arg Lieu Lys Ala Phe Ser Thir 225 23 O 235 24 O

Ser His Wall Wall Met Lieu. Thir Phe Tyr Met Pro Ala Luell Phe Ser 245 250 255

Phe Met Thir His Arg Phe Gly Arg Asn. Ile Pro His Phe Ile His Ile 26 O 265 27 O

Lell Luell Ala Asn Phe Cys Val Val Ile Pro Pro Ala Lell Asn Ser Wall 28O 285

Ile Tyr Gly Wall Arg Thr Lys Glin Ile Arg Ala Glin Wall Luell Lys Met 29 O 295 3 OO US 8,637,259 B1 59 - Continued Phe Phe Asn Lys 3. OS

SEQ ID NO 11 LENGTH: 945 TYPE: DNA ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 11 atgc.cgacat t caatggctic agt citt catg c cct citgcgt. ttatactaat tgggatt CCt 6 O ggtctggagt Cagtgcagtg ttggattggg att cott tot ctgccatgta t cittattggit 12 O gtgattggaa att CCCtaat tittagittata atcaaatatg aaaacagcct CCatataccC 18O atgtacattt ttittggcc at gttggcagcc acaga cattg CaCttalacac ctgcatt citt 24 O cc caaaatgt taggcatctt ctggttt cat ttgc.ca.gaga tttcttittga 3OO tittcaaatgt ggct tatt ca ct cattccag gcaattgaat cgggt at CCt tctggcaatg 360 gccCtggatc gctatgtggc catctgtatic c ccttgagac atgccaccat cittitt cocag cagttcttaa ct catattgg acttggggtg acact caggg ctgcc attct tataatacct t cct tagggc t catcaaatg ctgtctgaaa cactatogaa ctacagt cat citct cact ct 54 O tact.gtgagc acatggcc at cgtgaagctg gct actgaag at atc.cgagt caacaagata tatggccitat ttgttgccitt tgcaatcc ta gggtttgaca taatlattitat aac Cttgtc.c 660 tatgtc.caaa titt titat cac tgtctitt cag ctgc.cccaga aggaggcacg attcaaggcc 72 O tittaataCat gcattgcc.ca catttgttgtc titcct acagt to tacct tot tgcctitcttic tott tott ca. cacacaggitt tggttcacac attaccaccat a tatt Catat cct cittgtca 84 O aatctttacC tgttagt ccc acct t t t c to aac cc tattg tctatggagt gaagaccalag 9 OO caaatt cqtg accatattgt gaaagttgttt ttcttcaaaa. agtaa 945

<210s, SEQ ID NO 12 &211s LENGTH: 314 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 12

Met Pro Thr Phe Asn Gly Ser Val Phe Met Pro Ser Ala Phe Ile Lieu. 1. 5 1O 15

Ile Gly Ile Pro Gly Lieu. Glu Ser Val Glin Cys Trp Ile Gly Ile Pro 25 3O

Phe Ser Ala Met Tyr Lieu. Ile Gly Val Ile Gly Asn Ser Luell Ile Lieu. 35 4 O 45

Wall Ile Ile Llys Tyr Glu Asn. Ser Lieu. His Ile Pro Met Ile Phe SO 55 6 O

Lell Ala Met Lieu Ala Ala Thr Asp Ile Ala Lieu. Asn Thir Ile Lieu. 65 70 7s 8O

Pro Met Leu Gly Ile Phe Trp Phe His Lieu. Pro Glu Ile Ser Phe 85 90 95

Asp Ala Leu Phe Gln Met Trp Lieu. Ile His Ser Phe Glin Ala Ile 105 11 O

Glu Ser Gly Ile Lieu. Lieu. Ala Met Ala Lieu. Asp Arg Tyr Wall Ala Ile 115 12 O 125

Ile Pro Lieu. Arg His Ala Thr Ile Phe Ser Glin Glin Phe Lieu. Thir 13 O 135 14 O

His Ile Gly Lieu. Gly Val Thr Lieu Arg Ala Ala Ile Lell Ile Ile Pro 145 150 155 160 US 8,637,259 B1 61 62 - Continued

Ser Luell Gly Lieu. Ile Llys Cys Cys Lieu Lys His Tyr Arg Thir Thir Wall 1.65 17O 17s

Ile Ser His Ser Tyr Cys Glu. His Met Ala Ile Wall Lys Luell Ala Thr 18O 185 19 O

Glu Asp Ile Arg Val Asn Lys Ile Tyr Gly Lieu. Phe Wall Ala Phe Ala 195

Ile Luell Gly Phe Asp Ile Ile Phe Ile Thir Lieu. Ser Wall Glin Ile 21 O 215 22O

Phe Ile Thir Wall Phe Glin Leu Pro Gln Lys Glu Ala Arg Phe Lys Ala 225 23 O 235 24 O

Phe Asn Thir Cys Ile Ala His Ile Cys Val Phe Lell Glin Phe Tyr Lieu. 245 250 255

Lel Ala Phe Phe Ser Phe Phe Thr His Arg Phe Gly Ser His Ile Pro 26 O 265 27 O

Ile His Ile Lieu Lleu Ser Asn Lieu. Tyr Lell Lell Wall Pro Pro 28O 285

Phe Luell Asn Pro Ile Val Tyr Gly Val Lys Thr Lys Glin Ile Arg Asp 29 O 295 3 OO

His Ile Wall Llys Val Phe Phe Phe 3. OS 310

<210s, SEQ ID NO 13 &211s LENGTH: 942 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 13 atgacattac cca.gcaacaa citc. cact tcc. c cagt ctittg aat tott cott catttgttt c 6 O cc.cagttt co agagctggca gCactggctg totctg.cccc t cago: ct cot citt cotcctg 12 O gccatggggg c caatgccac c ctitctgatc acCatctato tggaa.gc.ctic tctgcaccag 18O cc cctd tact acctgcticag c ct cost ct co Ctgctggaca tcqtact citg cct caccgt.c 24 O atc.cccaagg tcc tiggcc at cittctggttt gacct cagat caatcagctt c cctdcctgc 3OO titcc titcaga tgttcatcat gaacagttitt Ctgactatgg agt cctdcac att catgat c 360 atggcc tatg accoctatgt ggc catctgc aagcc cctac agtact catc CatCatcact gatcaatttg ttgctagggc tgc catctitt gttgttggc.ca ggaatggcct tct tact atg

CCtatic Coca tact t t cit to tcqacticaga tactgtgcag gacacat cat Caagaactgc 54 O atctgtacta acgtgtctgt gtctaaactic tcttgttgatg a catcaccitt gaatcagagc taccagtttg ttataggttg gaccctgctg ggctctgacc t cat cott at tgttctotct 660 tact t t t t ta. t cittgaaaac tgtgctaagg attalagggtg agggagatat ggccaaagct 72 O c tagg tactt gtggttcc.ca citt catcc to at cost cit tot t caccacagt Cctgctggitt ctgg to atca cta acctggc Caggaagaga att cotc.cgg atgtc.cc cat cctgct caac 84 O atcc tdcacc acct tatt co cc.cagctctg aac cc cattg tittatggtgt gagaac Caag 9 OO gagatcaa.gc agggaatc.ca gaacctgctg aagaggttgt a.a. 942

<210s, SEQ ID NO 14 &211s LENGTH: 313 212. TYPE : PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 14

Met Thir Lieu. Pro Ser Asn. Asn. Ser Thir Ser Pro Wall Phe Glu Phe Phe US 8,637,259 B1 63 - Continued

1. 5 1O 15 Lieu. Ile Cys Phe Pro Ser Phe Glin Ser Trp Gln His Trp Leu Ser Lieu. 2O 25 3O Pro Lieu. Ser Lieu Lleu Phe Lieu. Lieu Ala Met Gly Ala Asn Ala Thr Lieu. 35 4 O 45 Lieu. Ile Thr Ile Tyr Lieu. Glu Ala Ser Lieu. His Gln Pro Leu Tyr Tyr SO 55 6 O Lieu. Lieu. Ser Lieu Lleu Ser Lieu. Lieu. Asp Ile Val Lieu. Cys Lieu. Thr Val 65 70 7s 8O Ile Pro Llys Val Lieu Ala Ile Phe Trp Phe Asp Lieu. Arg Ser Ile Ser 85 90 95 Phe Pro Ala Cys Phe Leu Gln Met Phe Ile Met Asn Ser Phe Lieu. Thr 1OO 105 11 O Met Glu Ser Cys Thr Phe Met Ile Met Ala Tyr Asp Arg Tyr Val Ala 115 12 O 125 Ile Cys Llys Pro Leu Gln Tyr Ser Ser Ile Ile Thr Asp Glin Phe Val 13 O 135 14 O Ala Arg Ala Ala Ile Phe Val Val Ala Arg Asn Gly Lieu. Lieu. Thir Met 145 150 155 160 Pro Ile Pro Ile Lieu. Ser Ser Arg Lieu. Arg Tyr Cys Ala Gly His Ile 1.65 17O 17s Ile Lys Asn. Cys Ile Cys Thr Asn Val Ser Val Ser Llys Lieu. Ser Cys 18O 185 19 O Asp Asp Ile Thr Lieu. Asn Glin Ser Tyr Glin Phe Val Ile Gly Trp Thr 195 2OO 2O5 Lieu. Lieu. Gly Ser Asp Lieu. Ile Lieu. Ile Val Lieu. Ser Tyr Phe Phe Ile 21 O 215 22O Lieu Lys Thr Val Lieu. Arg Ile Lys Gly Glu Gly Asp Met Ala Lys Ala 225 23 O 235 24 O Lieu. Gly Thr Cys Gly Ser His Phe Ile Lieu. Ile Leu Phe Phe Thir Thr 245 250 255 Val Lieu. Lieu Val Lieu Val Ile Thr Asn Lieu Ala Arg Lys Arg Ile Pro 26 O 265 27 O Pro Asp Val Pro Ile Lieu. Lieu. Asn. Ile Lieu. His His Lieu. Ile Pro Pro 27s 28O 285 Ala Lieu. Asn. Pro Ile Val Tyr Gly Val Arg Thr Lys Glu Ile Lys Glin 29 O 295 3 OO Gly Ile Glin Asn Lieu Lleu Lys Arg Lieu. 3. OS 310

<210s, SEQ ID NO 15 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide 22 Os. FEATURE: 223 OTHER INFORMATION: N-term Act 22 Os. FEATURE: 223 OTHER INFORMATION: C-term. NH2

<4 OOs, SEQUENCE: 15 Phe Llys Llys Ser Phe Llys Lieu. 1. 5

<210s, SEQ ID NO 16 &211s LENGTH: 13 US 8,637,259 B1 65 66 - Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 16 Arg Arg Lieu. Ile Glu Asp Ala Glu Tyr Ala Ala Arg Gly 1. 5 1O

<210s, SEQ ID NO 17 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 17 Phe Ile Lieu. Lieu. Gly 1. 5

<210s, SEQ ID NO 18 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 18 Lieu. His Thr Pro Met Tyr 1. 5

<210s, SEQ ID NO 19 &211s LENGTH: 10 212. TYPE PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 19 Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys 1. 5 1O

<210s, SEQ ID NO 2 O &211s LENGTH: 2 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2O Ser Tyr 1.

<210s, SEQ ID NO 21 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 21 Phe Ser Thr Cys Ser Ser His 1. 5

<210s, SEQ ID NO 22 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 22

Pro Met Lieu. Asn. Pro Phe 1. 5 US 8,637,259 B1 67 68 The invention claimed is: 10. The method according to claim 1, wherein said one or 1. A method for identifying an agent that modulates the more OR polypeptides is defined by the amino acid sequence function of one or more Olfactory Receptor polypeptide(s) of SEQID NOS. 2, 4, 6, 8, 10, 12 or 14. (ORs) selected from the group consisting of OR52L1, 11. The method according to claim 1, wherein said one or OR52E8, OR52B2, OR51I2, OR52E1, OR52A5 and more carboxylic acid(s) may be detectably labeled with a OR56A5 comprising the steps of: moiety selected from the group comprising: a radioisotope, a a) contacting said one or more ORS with one or more fluorophore, and a quencher of fluorescence. carboxylic acid(s) selected from the group of consisting 12. The method according to claim 1, used for detecting the of butanoic acid, isovaleric acid, pentanoic acid, hex presence of an agent that modulates the activity of one or anoic acid, 2-methylhexanoic acid, 3-methylhexanoic 10 acid, (E)-3-methly-2-hexenoic acid, 3-hydroxy-3-meth more ORS detecting carboxylic acids present in human Sweat ylhexanoic acid, heptanoic acid, 2-methylheptanoic in a sample. acid, octanoic acid, 4-ethyloctanoic acid, nonanoic acid, 13. The method according to claim 1, wherein said agent is decanoic acid, undecanoic acid and benzoic acid, in the present in a sample. presence and in the absence of said agent under condi 15 14. The method according to claim 1, in which said one or tions permitting the binding of said carboxylic acid(s) to more OR polypeptide(s) is contacted with said one or more said ORS or permitting the activation of said ORs by said carboxylic acid(s) at their EC50 concentration. carboxylic acid(s), 15. The method according to claim 1, wherein said agent is b) comparing the binding of said one or more ORS to said identified as an agent that modulates the function of ORS one or more carboxylic acid(s), or the activity of said one according to the invention, when said agent decreases the or more ORs, in the presence and in the absence of said intracellular response induced by said carboxylic acid(s), by agent, wherein a difference in binding or activity in the at least 10%. presence of said agent, relative to the binding or activity 16. The method according to claim 1, wherein the contact in the absence of the agent, identifies the agent as an ing is performed in or on a cell expressing said OR polypep agent that modulates the function of said one or more 25 tide. ORS in response to said one or more carboxylic acid(s). 17. The method according to claim 16, wherein said cell is 2. The method according to claim 1, wherein said agent is selected from: Human embryonic kidney cells (Hek293), tested for influencing the binding of said carboxylic acids to Chinese hamster cells (CHO), Monkey cells (COS), primary all 7 ORs listed therein, or for influencing the activation of all olfactory cells, Xenopus cells, insect cells, yeast or bacteria. 7 ORs listed therein by said carboxylic acids. 30 18. The method according to claim 1, wherein said con 3. The method according to claim 1, wherein said agent is tacting is performed using synthetic liposomes or virus-in tested for influencing the binding of pentanoic acid to duced budding membranes containing said OR polypeptide. OR52L1, or for influencing the activation of OR52L1 by 19. The method according to claim 1, wherein said method pentanoic acid. is performed using a membrane fraction from cells express 4. The method according to claim 1, wherein said agent is 35 ing said OR polypeptide. tested for influencing the binding of 3-hydroxy-3-methylhex 20. The method according to claim 1, wherein said method anoic acid to OR52E8, or for influencing the activation of is performed on a protein chip. OR52E8 by 3-hydroxy-3-methylhexanoic acid. 21. The method according to claim 1, wherein said mea 5. The method according to claim 1, wherein said agent is Suring is performed using a method selected from label dis tested for influencing the binding of hexanoic acid, heptanoic 40 placement, Surface plasmon resonance, fluorescence reso acid, octanoic acid, nonanoic acid, decanoic acid, or unde nance energy transfer, fluorescence quenching, and canoic acid to OR52B2, or for influencing the activation of fluorescence polarization. OR52B2 by hexanoic acid, heptanoic acid, octanoic acid, 22. The method according to claim 1, wherein said agentis nonanoic acid, decanoic acid, or undecanoic acid. selected from the group consisting of a peptide, a polypep 6. The method according to claim 1, wherein said agent is 45 tide, an antibody orantigen-binding fragment thereof, a lipid, tested for influencing the binding ofbutanoic acid, isovaleric a carbohydrate, a nucleic acid, and a small organic molecule. acid, pentanoic acid, hexanoic acid, 2-methylhexanoic acid, 23. The method according to claim 1, wherein the activity 3-methylhexanoic acid, (E)-3-methyl-2-hexanoic acid, or of said OLR polypeptide is measured by detecting a change in benzoic acid to OR51I2, or for influencing the activation of the level of a second messenger. OR51I2 by butanoic acid, isovaleric acid, pentanoic acid, 50 24. The method according to claim 1, wherein the activity hexanoic acid, 2-methylhexanoic acid, 3-methylhexanoic of said OR polypeptide is measured by measurement of gua acid, (E)-3-methyl-2-hexanoic acid, or benzoic acid. nine nucleotide binding/coupling or exchange, adenylate 7. The method according to claim 1, wherein said agent is cyclase activity, cAMP, Protein Kinase C activity, Protein tested for influencing the binding of butanoic acid to Kinase A activity phosphatidylinosotol breakdown, diacylg OR52E1, or for influencing the activation of OR52E1 by 55 lycerol, inositol triphosphate, intracellular calcium, calcium butanoic acid. flux, arachidonic acid, MAP kinase activity, tyrosine kinase 8. The method according to claim 1, wherein said agent is activity, a melanophore assay, a receptor initialization assay, tested for influencing the binding of 4-ethyloctanoic acid to or reporter gene expression. OR52A5, or for influencing the activation of OR52A5 by 25. The method according to claim 1, wherein the activity 4-ethyloctanoic acid. 60 of said ORS is measured using a fluorescence or luminescence 9. The method according to claim 1, wherein said agent is assay. tested for influencing the binding of hexanoic acid, heptanoic 26. The method according to claim 25, wherein said fluo acid, 2-methylheptanoic acid, octanoic acid, nonanoic acid, rescence and luminescence assays comprise the use of Ca"- decanoic acid, or undecanoic acid to OR56A5, or for influ sensitive fluorophores including Fluo3, Fluo4 or Fura: Ca3 encing the activation of OR56A5 by hexanoic acid, heptanoic 65 kit family or aequorin. acid, 2-methylheptanoic acid, octanoic acid, nonanoic acid, 27. The method according to claim 26, wherein said assays decanoic acid, or undecanoic acid. apply an automated fluorometric or luminescent reader Such US 8,637,259 B1 69 70 as Functional DrugScreening System (FDSS) or Fluoromet 32. A kit for performing screening methods for modulators ric Imaging Plate Reader (FLIPR). of Olfactory Receptors (ORS) detecting carboxylic acid(s) 28. The method according to claim 1, which is a high present in human Sweat, comprising: throughput Screening method. 29. The method according to claim 1, wherein the agent is a) a cell, several cells or membranes thereof expressing one part of a chemical library or animal organ extracts. or more of the OR polypeptides OR52L1, OR52E8, 30. A kit for performing screening methods for modulators OR52B2, OR51I2, OR52E1, OR52A5 and OR56A5; of Olfactory Receptors (ORS) detecting carboxylic acid(s) b) carboxylic acid(s) selected from the group of consisting present in human Sweat, comprising: of butanoic acid, isovaleric acid, pentanoic acid, hex a) isolated OR polypeptides: OR52L1, OR52E8, OR52B2, 10 anoic acid, 2-methylhexanoic acid, 3-methylhexanoic OR51I2, OR52E1, OR52A5 and OR56A5; acid, (E)-3-methly-2-hexenoic acid, 3-hydroxy-3-meth b) one or more carboxylic acid(s) selected from the group ylhexanoic acid, heptanoic acid, 2-methylheptanoic of consisting of butanoic acid, isovaleric acid, pen acid, octanoic acid, 4-ethyloctanoic acid, nonanoic acid, tanoic acid, hexanoic acid, 2-methylhexanoic acid, decanoic acid, undecanoic acid and benzoic acid; and 3-methylhexanoic acid, (E)-3-methly-2-hexenoic acid, 15 3-hydroxy-3-methylhexanoic acid, heptanoic acid, c) packaging materials therefore. 2-methylheptanoic acid, octanoic acid, 4-ethyloctanoic 33. A kit for performing screening methods for modulators acid, nonanoic acid, decanoic acid, undecanoic acid and of Olfactory Receptors (ORS) detecting carboxylic acid(s) benzoic acid; and present in human Sweat, comprising: c) packaging materials therefore. a) a cell, several cells or membranes thereof expressing one 31. A kit for performing screening methods for modulators or more of the OR polypeptides OR52L1, OR52E8, of Olfactory Receptors (ORS) detecting carboxylic acid(s) OR52B2, OR51I2, OR52E1, OR52A5 and OR56A5; present in human Sweat, comprising: b) carboxylic acid(s) selected from the group of consisting a) isolated polynucleotide’s encoding the OR polypep of butanoic acid, isovaleric acid, pentanoic acid, hex tides: OR52L1, OR52E8, OR52B2, OR51 I2, OR52E1, 25 anoic acid, 2-methylhexanoic acid, 3-methylhexanoic OR52A5 and OR56A5; acid, (E)-3-methly-2-hexenoic acid, 3-hydroxy-3-meth b) carboxylic acid(s) selected from the group of consisting ylhexanoic acid, heptanoic acid, 2-methylheptanoic of butanoic acid, isovaleric acid, pentanoic acid, hex acid, octanoic acid, 4-ethyloctanoic acid, nonanoic acid, anoic acid, 2-methylhexanoic acid, 3-methylhexanoic decanoic acid, undecanoic acid and benzoic acid; and acid, (E)-3-methly-2-hexenoic acid, 3-hydroxy-3-meth 30 ylhexanoic acid, heptanoic acid, 2-methylheptanoic c) packaging materials therefore, wherein said cell is trans acid, octanoic acid, 4-ethyloctanoic acid, nonanoic acid, formed with a polynucleotide encoding said one or more decanoic acid, undecanoic acid and benzoic acid; and OR(s). c) packaging materials therefore.