G protein-coupled receptor structural model and a method of designing ligand binding to g protein-coupled receptor by using the structural model

Abstract

The present invention provides a method for constructing a structural model of a complex that a G protein-coupled protein receptor forms with a ligand capable of binding the G protein-coupled receptor and a three-dimensional structural model of an activated intermediate in the structural model of the complex. The present invention also provides a method for identifying, screening for, searching for, evaluating, or designing a ligand capable of binding a GPCR by using the three-dimensional model. In one specific method by the present invention, a three-dimensional structural model of a photoactivated intermediate of rhodopsin is constructed by using a molecule modeling software and by using the three-dimensional structural coordinate of the crystal structure of rhodopsin in such a manner that amino acid residues highly conserved among GPCRs are taken into consideration. The three-dimensional stractural model of the photoactivated intermediate of rhodopsin is subsequently used to construct structural models of activated intermediates of other GPCRs. The present invention further provides a method for identifying, screening for, searching for, evaluating, or designing a ligand that binds a GPCR to act as an agonist or an antagonist. This method employs the three-dimensional structural model constructed by the above-described method.

1 . A method for constructing a three-dimensional structural model of an activated intermediate of a G protein-coupled receptor for use in identifying, screening for, searching for, evaluating, or designing a ligand that binds the G protein-coupled receptor to act as an agonist or an antagonist.  
     
     
         2 . The method according to  claim 1 , wherein the activated intermediate of the G protein-coupled receptor is an intermediate of activated rhodopsin.  
     
     
         3 . The method according to  claim 1  or  2 , wherein the structural model of the intermediate of the activated rhodopsin is a structural model of metarhodopsin II.  
     
     
         4 . The method according to  claim 1  or  2 , wherein the structural model of the intermediate of the activated rhodopsin is a structural model of metarhodopsin I.  
     
     
         5 . The method according to  claim 1  or  2 , wherein the structural model of the intermediate of the activated rhodopsin is a structural model of metarhodopsin Ib.  
     
     
         6 . The method according to  claim 1  or  2 , wherein the structural model of the intermediate of the activated rhodopsin is a structural mode of metarhodopsin I 380 .  
     
     
         7 . The method according to  claim 1 , characterized in that the three-dimensional structural model of the activated intermediate of the G protein-coupled receptor is constructed based on a structural model of metarhodopsin II.  
     
     
         8 . The method according to  claim 1 , characterized in that the three-dimensional structural model of the activated intermediate of the G protein-coupled receptor is constructed based on a structural model of metarhodopsin I.  
     
     
         9 . The method according to  claim 1 , characterized in that the three-dimensional structural model of the activated intermediate of the G protein-coupled receptor is constructed based on a structural model of metarhodopsin Ib.  
     
     
         10 . The method according to  claim 1 , characteristics in that the three-dimensional structural model of the activated intermediate of the G protein-coupled receptor is constructed based on a structural model of metarhodopsin I 380 .  
     
     
         11 . A three-dimensional structural model of the activated intermediate of the G protein-coupled receptor obtained by the method according to any one of  claims 1  to  10 , or a three-dimensional coordinate for determining the structural model.  
     
     
         12 . A three-dimensional coordinate shown in Table 1 or Table 2.  
     
     
         13 . A computer storage medium that stores all or part of the three-dimensional coordinate according to  claim 11  or  12  for use in identifying, screening for, searching for, evaluating, or designing a ligand that binds the G protein-coupled receptor to act as an agonist or an antagonist.  
     
     
         14 . A method for identifying, screening for, searching for, evaluating, or designing a ligand that binds a G protein-coupled receptor to act as an agonist, the method comprises the step of using the three-dimensional strut model according to  claim 11  or the three-dimensional coordinate for determining the structural model according to  claim 11  or  12 , or the computer storage medium according to  claim 13 .  
     
     
         15 . The method according to  claim 14 , wherein the agonist is a full agonist of the G protein-coupled receptor.  
     
     
         16 . The method for identifying, screening for, searching for, evaluating, or designing the full agonist of the G protein-coupled receptor of  claim 15 , characterized in that, of the three-dimensional structural models according to  claim 11  or the three-dimensional coordinates for determining the structural models according to  claim 11  or  12 , the metarhodopsin II structural model or the three-dimensional coordinate for determining the structural model, or the structural model constructed based on the metarhodopsin II structural model or the three-dimensional coordinate for determining the structural model is used.  
     
     
         17 . The method according to  claim 14 , herein the agonist is a partial agonist of the G protein-coupled receptor.  
     
     
         18 . The method for identifying, screening for, searching for, evaluating, or designing the partial agonist of the G protein-coupled receptor of  claim 17 , characterized in that, of the three-dimensional structural models according to  claim 11  or the three-dimensional coordinates for determining the structural medals according to  claim 11  or  12 , the metarhodopsin I 380  structural model or the three-dimensional coordinate for determining the structural model, or the structural model constructed based on the metarhodopsin I 380  structural model or the three-dimensional coordinate for determining the structural model is used.  
     
     
         19 . A method for identifying, screening for, searching for, evaluating, or designing a ligand capable of binding a G protein-coupled protein to act as an antagonist, the method comprises the step of using the three-dimensional structural model according to  claim 11  or the three-dimensional coordinate for determining the structural model according to  claim 11  or  12 , or the computer storage medium according to  claim 13 .  
     
     
         20 . The method according to  claim 19 , wherein the antagonist is an inverse agonist of the G protein-coupled receptor.  
     
     
         21 . The method for identifying, screening for, searching for, evaluating, or designing the inverse agonist of the G protein-coupled protein of  claim 20 , characterized in that, of the three-dimensional structural modes according to  claim 11  or the three-dimensional coordinates for determining the structural models according to  claim 11  or  12 , the metarhodopsin I structural model or the three-dimensional coordinate for determining the structural model, or the structural model constructed based on the metarhodopsin I structural model or the three-dimensional coordinate for determining the structural model is used.  
     
     
         22 . The method for identifying, screening for, searching for, evaluating, or designing the antagonist of the G protein-coupled protein of  claim 19 , characterized in that, of the three-dimensional structural models according to  claim 11  or the three-dimensional coordinates for determining the structural models according to  claim 11  or  12 , the metarhodopsin Ib structural model or the three-dimensional coordinate for determining the structural model, or the structural model constructed based on the metarhodopsin Ib structural model or the three-dimensional coordinate for determining the structural model is used.  
     
     
         23 . A method for identifying, screening for, seeing for, evaluating, or designing a mutant of a G protein-coupled receptor, the method comprises the step of using the three-dimensional structural model according to  claim 11  or the three-dimensional coordinate for determining the structural model according to  claim 11  or  12 , or the computer storage medium according to  claim 13 .  
     
     
         24 . The method according to  claim 22 , wherein the mutant of the G protein-coupled receptor is a constitutively active mutant.  
     
     
         25 . The method according to any one of  claims 14  to  24 , wherein the G protein-coupled receptor is selected from the group consisting of rhodopsin, adrenaline receptor, muscarinergic acetylcholine receptor, histamine H2 receptor, serotonin receptor, and amine receptor.  
     
     
         26 . The method according to  claims 1  to  6 , characterized in that the structural model of the intermediate is generated by using coordinates of existing amino acid sequences and existing crystal structures of amino acids and by using an ordinary molecule modeling software in such a manner that amino acid residues highly conserved among the transmembrane helices of the G protein-coupled receptor are taken into consideration, and structural optimization is performed at 300 K according to molecular kinetics and molecular dynamics in such a manner with C α carbons of the amino acids fixed as firmly as possible.  
     
     
         27 . The method according to any one of  claims 7  to  10 , corresponding the steps of: 
 introducing amino acid substitution and insertion or deletion of amino acid residues on the loop regions by means of a three-dimensional structural model of a rhodopsin/ligand complex or a three-dimensional model of rhodopsin in the structural model of the complex based on the homology between the amino acid sequence of rhodopsin and the amino acid sequence of a G protein-coupled receptor for which to construct a model;    generating a structure using a molecule modeling software; and    performing structural optimization with C α carbons of the amino acids fixed as firmly as possible.