Methods of identifying, isolating and using odorant and aroma receptors

Abstract

Provided here are new methods to identify specific families of mammalian odorant receptors for odorants or aroma, particularly indole and skatole malodors and their use in assays that may be used to discover compounds that modulate (blocking, enhancing, masking or mimicking compounds) their activity. Orphan mouse odorant receptors are identified from olfactory sensory neurons that respond to target compounds. The resulting receptors as well as their human counterparts can be screened in assays against test compounds to confirm their identity as odorant or aroma receptors, particularly malodor receptors and subsequently discover for example modulators that inhibit the perception of the malodor in humans.

What is claimed is: 
     
         1 . A method of identifying olfactory receptors that are activated by an odor and/or aroma compound comprising:
 a) dissociating an isolated olfactory epithelium containing native olfactory neurons into a single cell from a non-human mammal species wherein each neuron expresses the olfactory receptor;   b) loading the olfactory cells with an indicator dye that allows for the measurement of malodor receptor binding activity of the olfactory receptors;   c) contacting the olfactory receptors with malodor compounds;   d) measuring changes of odorant-induced neuronal activity;   e) isolating one or more olfactory neurons that were activated by the malodor compound;   f) isolating or isolating and amplifying the mRNA of the isolated olfactory receptors;   g) sequencing at least a portion of the entire transcriptome of the mRNA by Next-Generation Sequencing; and   h) identifying a group of malodor olfactory receptors by comparing the sequence of the transcriptome to a reference genome sequence of the same species.   
     
     
         2 . The method of  claim 1 , further comprising identifying a human receptor that is most closely related to a malodor olfactory receptor identified in  claim 1 . 
     
     
         3 . The method of  claim 1 , further comprising modifying a cell line to express at least one malodor olfactory receptor identified in  claim 1 . 
     
     
         4 . The method of  claim 2 , further comprising modifying a cell line to express at least one human receptor identified in  claim 2 . 
     
     
         5 . The method of  claim 3 , further comprising contacting the malodor olfactory receptor with a test compound to determine if the test compound binds to the malodor olfactory receptor. 
     
     
         6 . The method of  claim 4 , further comprising contacting the human receptor with a test compound to determine if the test compound binds to the receptor. 
     
     
         7 . An expression vector comprising
 a) a nucleic acid comprising a nucleotide sequence having at least 90%, 95%, 98%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, or SEQ ID NO: 85; or   b) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, or SEQ ID NO: 86.   
     
     
         8 . A cell that is recombinantly modified to express an olfactory receptor polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, or SEQ ID NO: 86; or that comprises the expression vector of  claim 7 . 
     
     
         9 . The cell of  claim 7 , wherein the cell is selected from the group consisting of HEK293, CHO,  Xenopus oocytes , COS, yeast, bacteria and cells derived from the olfactory placode. 
     
     
         10 . A method for identifying a compound that blocks, inhibits, modulates, and/or enhances the activity of an olfactory receptor selected from the group consisting of odorant or aroma receptors that is activated by a compound comprising indole and/or skatole comprising
 a) culturing at least one cell of  claim 8  under conditions conducive to expressing at least one olfactory receptor;   b) contacting a compound with the at least one receptor;   c) measuring the extent to which the compound blocks, inhibits, modulates, or enhances the activity of the receptor by measuring the response of the olfactory receptor in the presence and absence of the compound.   
     
     
         11 . The method of  claim 10 , further comprising identifying a compound that blocks, inhibits, modulates, or enhances the response of the olfactory receptor on the basis of the response that was measured in the presence and absence of the compound; and selecting the identified compound as a compound that blocks, inhibits, modulates, or enhances the response of the olfactory receptor. 
     
     
         12 . The method of  claim 10 , wherein the olfactory receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, or SEQ ID NO: 86.