US2015057166A1PendingUtilityA1

Method for analyzing the three dimensional structure of a gpcr

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Assignee: CONFOMETRX INCPriority: Aug 30, 2010Filed: Oct 17, 2014Published: Feb 26, 2015
Est. expiryAug 30, 2030(~4.1 yrs left)· nominal 20-yr term from priority
Inventors:Brian Kobilka
G06F 19/16C40B 30/02G16B 15/00G16C 20/64G16B 15/20C12Y 302/01017C07K 2299/00C07K 2319/21C07K 2319/00C07K 2319/24G16B 35/00C12N 9/2462C07K 2319/43C07K 14/705C07K 2319/23C07K 2319/35C12N 2799/026C07K 14/723G16C 20/60
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Claims

Abstract

Certain embodiments provide a method for crystallizing a GPCR. The method may employ a fusion protein comprising, from N-terminus to C-terminus: a) a first portion of a family C G-protein coupled receptor (GPCR), wherein the first portion comprises the TM1, TM2 and TM3, regions of the GPCR; b) a stable, folded protein insertion; and c) a second portion of the GPCR, wherein the second portion comprises the TM4, TM5 TM6 and TM7 regions of the GPCR.

Claims

exact text as granted — not AI-modified
1 - 17 . (canceled) 
     
     
         18 . A method for analyzing the three dimensional structure of a GPCR on a computer system, comprising:
 a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein said atomic coordinates are produced by subjecting crystals of a GPCR fusion protein to X-ray diffraction analysis, wherein said GPCR fusion protein comprises, from N-terminus to C-terminus:
 i. a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises the TM1, TM2 and TM3 regions of said GPCR; 
 ii. a stable protein insertion that aids in the formation of lattice contacts; and 
 iii. a second portion of said GPCR, wherein said second portion comprises the TM4, TM5 TM6 and TM7 regions of said GPCR; 
   b) modeling said atomic coordinates on said computer system using said modeling program to produce a model of the three dimensional structure of at least a portion of the GPCR; and   c) displaying the model of said three dimensional structure on the computer system.   
     
     
         19 . The method of  claim 18 , wherein the GPCR fusion protein is co-crystallized with an allosteric modulator of GPCR. 
     
     
         20 . The method of  claim 18 , wherein said stable, folded protein insertion element is a polypeptide that folds autonomously and is stable in its tertiary folded form. 
     
     
         21 . The method of  claim 18 , wherein said stable, folded protein insertion comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of a wild type protein. 
     
     
         22 . The method of  claim 18 , wherein said stable, folded protein insertion comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of T4 lysozyme. 
     
     
         23 . The method of  claim 18 , further comprising identifying a potential modulator of said GPCR. 
     
     
         24 . The method of  claim 18 , wherein said method comprises identifying a binding site for a modulator of said GPCR. 
     
     
         25 . The method of  claim 18 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a naturally occurring GPCR. 
     
     
         26 . The method of  claim 18 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a non-naturally occurring GPCR. 
     
     
         27 . The method of  claim 18 , wherein the amino acid sequences of said first and second portions of said GPCR are least 80% identical to amino acid sequences of a naturally occurring GPCR. 
     
     
         28 . The method of  claim 18 , wherein the GPCR of said fusion protein is a family C GPCR. 
     
     
         29 . The method of  claim 18 , wherein said GPCR is a GABAB receptor. 
     
     
         30 . The method of  claim 18 , wherein said GPCR is a metabotropic glutamate receptor. 
     
     
         31 . The method of  claim 18 , wherein said GPCR comprises an amino acid sequence that is at least 95% identical to a calcium-sensing receptor (CASR), GPRC6A (GPRC6A), GABAB receptor 1 (GABBR1); GABAB receptor 2 (GABBR2), GPR156 (GPR156), mGluR1 (GRM1), mGluR2 (GRM2), mGluR3 (GRM3), mGluR4 (GRM4), mGluR5 (GRM5), mGluR6 (GRM6), mGluR7 (GRM7) mGluR8 (GRM8), RAIG1 (GPRC5A), RAIG2 (GPRC5B), RAIG3 (GPRC5C), RAIG4 (GPRC5D), taste receptor, type 1, member 1 (TAS1R1), taste receptor, type 1, member 2 (TAS1R2), taste receptor, type 1, member 3 (TAS1R3), GPR158 (GPR158), GPR179 (GPR179); bride of sevenless protein and vomeronasal receptor, type 2. 
     
     
         32 . A method for selecting a modulator of a GPCR, comprising:
 a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein said atomic coordinates are produced by subjecting crystals of a GPCR fusion protein to X-ray diffraction analysis, wherein said GPCR fusion protein comprises, from N-terminus to C-terminus:
 i. a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises the TM1, TM2 and TM3 regions of said GPCR; 
 ii. a stable protein insertion that aids in the formation of lattice contacts; and 
 iii. a second portion of said GPCR, wherein said second portion comprises the TM4, TM5 TM6 and TM7 regions of said GPCR; 
   b) modeling said atomic coordinates on said computer system using said modeling program to produce a model of the three dimensional structure of at least the ligand binding site of the GPCR;   c) determining, using said computer system, whether a test compound docks with said binding site, wherein a test compound that docks with said binding site is a GPCR modulator; and   d) displaying on the computer system a model of said ligand binding site and said GPCR modulator, docked with said ligand binding site.   
     
     
         33 . The method of  claim 32 , wherein the GPCR is co-crystallized with an allosteric modulator of GPCR. 
     
     
         34 . The method of  claim 32 , wherein said stable, folded protein insertion comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of a wild type protein. 
     
     
         35 . The method of  claim 32 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a naturally occurring GPCR. 
     
     
         36 . The method of  claim 32 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a non-naturally occurring GPCR. 
     
     
         37 . The method of  claim 32 , wherein the amino acid sequences of said first and second portions of said GPCR are least 80% identical to amino acid sequences of a naturally occurring GPCR. 
     
     
         38 . The method of  claim 32 , wherein the GPCR of said fusion protein is a family C GPCR. 
     
     
         39 . The method of  claim 32 , wherein said GPCR comprises an amino acid sequence that is at least 95% identical to a calcium-sensing receptor (CASR), GPRC6A (GPRC6A), GABAB receptor 1 (GABBR1); GABAB receptor 2 (GABBR2), GPR156 (GPR156), mGluR1 (GRM1), mGluR2 (GRM2), mGluR3 (GRM3), mGluR4 (GRM4), mGluR5 (GRM5), mGluR6 (GRM6), mGluR7 (GRM7) mGluR8 (GRM8), RAIG1 (GPRC5A), RAIG2 (GPRC5B), RAIG3 (GPRC5C), RAIG4 (GPRC5D), taste receptor, type 1, member 1 (TAS1R1), taste receptor, type 1, member 2 (TAS1R2), taste receptor, type 1, member 3 (TAS1R3), GPR158 (GPR158), GPR179 (GPR179); bride of sevenless protein and vomeronasal receptor, type 2. 
     
     
         40 . A method for analyzing the three dimensional structure of a GPCR on a computer system, comprising:
 a) accessing a file containing atomic coordinates of a GPCR using a computer system that comprises a modeling program, wherein said atomic coordinates are produced by subjecting crystals of a GPCR to X-ray diffraction analysis, wherein said GPCR comprises an IC2 loop containing a substitution that comprises the amino acid sequence of a stable protein insertion that aids in the formation of lattice contacts; and   b) modeling said atomic coordinates on said computer system using said modeling program to produce a model of the three dimensional structure of at least a portion of the GPCR; and   c) displaying the model of said three dimensional structure on the computer.   
     
     
         41 . The method of  claim 40 , wherein said crystals comprise a ligand for said GPCR, and the method further comprises identifying the binding site for said ligand in said GPCR using said model. 
     
     
         42 . The method of  claim 41 , wherein the analyzing comprises identifying amino acids that form polar contacts with said ligand in said binding site, using said model. 
     
     
         43 . The method of  claim 42 , further comprising determining whether a test compound docks with said binding site using said model. 
     
     
         44 . The method of  claim 43 , further comprising analyzing the packing of the test compound and amino acids in said binding site, using said model. 
     
     
         45 . The method of  claim 40 , wherein said method further comprises making said test compound.

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