Screening methods using canine t2r receptors and pet food products and compositions identified using the same

Abstract

The presently disclosed subject matter relates to methods of screening raw materials and pet food products to manufacture a palatable pet food product. The presently disclosed subject matter also relates to methods for identifying compounds that modulate the activity and/or expression of a bitter taste receptor.

1 . A method for identifying a compound that modulates the activity of a bitter taste receptor comprising
 (a) contacting a test agent with a bitter taste receptor,   (b) determining the activity of the bitter taste receptor, and   (c) selecting as the compound, a test agent that increases or decreases the activity of the bitter taste receptor.   
     
     
         2 . The method of  claim 1 , wherein the bitter taste receptor is a canine bitter taste receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO:17-32. 
     
     
         3 . A method for identifying a compound that modulates the activity of a bitter taste receptor comprising
 (a) contacting a test agent with a bitter taste receptor,   (b) detecting an interaction between the test agent and one or more amino acids of the bitter taste receptor, and   (c) selecting as the compound, a test agent that interacts with one or more of the amino acids.   
     
     
         4 . The method of  claim 3 , wherein the method comprises detecting an interaction between the test agent and one or more amino acids in a 7 transmembrane domain (7TM) domain of the bitter taste receptor. 
     
     
         5 . The method of  claim 3 , further comprising determining the activity of the bitter taste receptor after step (a). 
     
     
         6 . The method of  claim 1 , further comprising contacting a bitter taste receptor ligand to the bitter taste receptor. 
     
     
         7 . The method of  claim 1 , wherein step (c) comprises selecting as the compound, a test agent that decreases the activity of the bitter taste receptor. 
     
     
         8 . The method of  claim 1 , wherein step (c) comprises selecting as the compound, a test agent that increases the activity of the bitter taste receptor. 
     
     
         9 . The method of  claim 3 , wherein the bitter taste receptor is a canine bitter taste receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO:17-32. 
     
     
         10 . The method of  claim 9 , wherein the bitter taste receptor is a canine T2R1 bitter taste receptor comprising an amino acid sequence described by SEQ ID NO:17, and wherein the one or more amino acids of the bitter taste receptor are selected from the group consisting of Asn89, Tyr239, Ile167, Gln174, Glu169, Phe257, Ala242, Phe177, His238, Cys260, Phe264, Leu234, Cys235, Phe85, Leu261, Leu178, Leu181, Va186, and Phe82. 
     
     
         11 . The method of  claim 9 , wherein the bitter taste receptor is a canine T2R2 bitter taste receptor comprising an amino acid sequence described by SEQ ID NO:18, and wherein the one or more amino acids of the bitter taste receptor are selected from the group consisting of Ser94, Trp90, Lys268, Tyr245, Glu180, Arg176, Met91, Asn185, Val184, Met181, Phe249, Pro155, Gln177, Lys174, Phe264, Phe93, Leu59, Met271, Phe246, and Leu188. 
     
     
         12 . The method of  claim 9 , wherein the bitter taste receptor is a canine T2R3 bitter taste receptor comprising an amino acid sequence described by SEQ ID NO:19, and wherein the one or more amino acids of the bitter taste receptor are selected from the group consisting of Asn93, Asp86, Tyr246, Phe247, Thr186, Asn189, Trp89, Arg175, Phe250, Gly185, Phe243, Thr90, Asn176, Val149, Ile154, Lys174, Met82, Ile85, Lys173, and Met69. 
     
     
         13 . The method of  claim 9 , wherein the bitter taste receptor is a canine T2R4 bitter taste receptor comprising an amino acid sequence described by SEQ ID NO:20, and wherein the one or more amino acids of the bitter taste receptor are selected from the group consisting of Ser186, Asp93, Tyr240, Ser94, Leu97, Asn95, Leu92, Ser96, Trp98, Val187, Thr247, Tyr243, Trp89, Met58, Ser269, Pro273, Ser270, Gln189, Thr144, Leu188, Val183, Leu182, Ser244, and Met90. 
     
     
         14 . The method of  claim 9 , wherein the bitter taste receptor is a canine T2R5 bitter taste receptor comprising an amino acid sequence described by SEQ ID NO:21, and wherein the one or more amino acids of the bitter taste receptor are selected from the group consisting of Ser89, Pro264, Leu58, Va188, Gln90, Ile86, Leu173, Trp165, Thr258, Ala261, Tyr234, Glu257, Met260, and Trp85. 
     
     
         15 . The method of  claim 9 , wherein the bitter taste receptor is a canine T2R10 bitter taste receptor comprising an amino acid sequence described by SEQ ID NO:23, and wherein the one or more amino acids of the bitter taste receptor are selected from the group consisting of Lys258, Leu180, Lys170, Glu172, Asn181, Phe261, Met265, Ile262, Gln169, Lys69, Met168, Ile245, Va190, Phe242, Gln94, Val184, Asn93, Trp89, and Tyr241. 
     
     
         16 . The method of  claim 9 , wherein the bitter taste receptor is a canine T2R43 bitter taste receptor comprising an amino acid sequence described by SEQ ID NO:30, and wherein the one or more amino acids of the bitter taste receptor are selected from the group consisting of Tyr241, Trp88, Thr181, Met177, Asn92, Asn184, Phe185, Gln152, His143, Phe261, Ala172, His85, Asp170, Lys265, Phe242, Leu245, Thr89, and Phe180. 
     
     
         17 . A method for identifying a compound that modulates the activity of a bitter taste receptor comprising
 (a) contacting a bitter taste receptor agonist with a bitter taste receptor,   (b) determining the activity of the bitter taste receptor,   (c) contacting a test agent with the bitter taste receptor,   (d) determining the activity of the bitter taste receptor, and   (e) selecting the test agent as the compound when the activity of (d) is greater or lesser than the activity of (b).   
     
     
         18 . A method for selecting a pet food product or component thereof, comprising
 (a) contacting a candidate pet food product or component thereof with a bitter taste receptor,   (b) determining the activity of the bitter taste receptor, and   (c) selecting as the pet food product, or component thereof, a test agent that decreases the activity of the bitter taste receptor.   
     
     
         19 . The method of  claim 18 , wherein the bitter taste receptor is a canine bitter taste receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO:17-32. 
     
     
         20 . The method of  claim 1 , wherein the bitter taste receptor is expressed by a cell, and wherein the test agent is contacted to the cell. 
     
     
         21 . The method of  claim 20 , wherein the cell expresses a calcium-binding photoprotein. 
     
     
         22 . The method of  claim 8 , further comprising contacting the compound that increases the activity of the bitter taste receptor to a surface. 
     
     
         23 . The method of  claim 17 , further comprising selecting the test agent as the compound when the activity of (d) is greater than the activity of (b), and contacting the compound that increases the activity of the bitter taste receptor to a surface. 
     
     
         24 . A composition comprising a compound that decreases the activity of a bitter taste receptor, wherein the compound is identified according to the method of  claim 7 . 
     
     
         25 . A composition comprising a compound that increases the activity of a bitter taste receptor, wherein the compound is identified according to the method of  claim 8 .