Modeling of mPGES-1 three-dimensional structures: applications in drug design and discovery

Abstract

This invention relates to representations of prostaglandin synthase three-dimensional structures. Such representations are suitable for designing agents that modulate the activity of the enzyme by binding to the substrate binding domain.

1 . A method of identifying a set of candidate structures of a polypeptide, the method comprising:
 a) obtaining a first amino acid sequence derived from a query polypeptide;   b) obtaining a second amino acid sequence derived from a template polypeptide, wherein the second sequence comprises:
 i) a predetermined three-dimensional structure; and 
 ii) at least 50% sequence homology with the first sequence; 
   c) performing a sequence alignment between the first sequence and the second sequence, and identifying common secondary structures;   d) generating a plurality of candidate topological structures by applying predetermined geometric parameters to the secondary structures of c) and transforming each topological structure in to the amino acid residues associated with the secondary structures;   e) generating a first conformation set by screening the plurality of candidate topological structures of d) with the predetermined geometric parameters and identifying the structures that correspond to the parameters;   f) generating a second conformation set by applying energy minimization functions to the first conformation set and identifying energetically-favored conformations; and   g) generating a final conformation set by selecting those structures that exhibit an energy gradient having a root mean square deviation (RMSD) of less than 0.001 kcal mol −1  Å −1 , wherein the final conformation set represents the set of candidate structures of the query polypeptide.   
     
     
         2 . The method of  claim 1  wherein the sequence alignment is generated by ClusterW with the Blosum scoring function. 
     
     
         3 . The method of  claim 1  further including generating the sequence alignment by generating the reciprocal position of the conserved residues. 
     
     
         4 . The method of  claim 1 , wherein the query polypeptide comprises membrane-spanning regions of amino acids. 
     
     
         5 . The method of  claim 4 , wherein the query polypeptide is a member of the membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG). 
     
     
         6 . The method  claim 5 , wherein the query polypeptide is microsomal prostaglandin E synthase-1 (mPGES-1) 
     
     
         7 . The method of  claim 1 , wherein the template polypeptide is a member of the membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG). 
     
     
         8 . The method  claim 7 , wherein the template polypeptide is microsomal glutathione-S-transferase-1 (MGST-1). 
     
     
         9 . The method of  claim 1 , wherein the polypeptide comprises a substrate binding domain. 
     
     
         10 . The method of  claim 1 , wherein the secondary structures comprise alpha-helices. 
     
     
         11 . The method of  claim 1 , wherein the structural parameters comprise coordinates derived from 3D electron projection maps of MGST1. 
     
     
         12 . The method of  claim 11 , wherein the structural parameters further comprise the coordinates derived from 2D electron projection maps of mPGES-1. 
     
     
         13 . The method of  claim 1 , wherein the structural parameters correspond to the coordinates set forth in Table 1. 
     
     
         14 . The method of  claim 1 , wherein the energy minimization function comprises the Sander module of Amber7.0 program suite. 
     
     
         15 . The method of  claim 1  further comprising:
 g) modeling the interaction of each member of the final conformation set with at least one substrate, wherein the modeling comprises molecular docking using binding site searching and/or interaction energy scoring; and   h) identifying amino acid residues associated with the SBD that interact with the substrate.   
     
     
         16 . The method of  claim 15 , wherein the substrate is PGH2 or GSH. 
     
     
         17 . The method of  claim 15 , wherein the amino acid residues that interact with the substrate comprise amino acid residues Q36, R110, T114, Y130, and Q134 of mPGES-1. 
     
     
         18 . The method of  claim 15  further comprising:
 i) modifying at least one amino acid residue that interacts with the substrate; and   j) determining the effect of the modification on substrate binding to the modified polypeptide.   
     
     
         19 . The method of  claim 18 , wherein the modification is a substitution. 
     
     
         20 . The method of  claim 19 , wherein the substitution is a conservative amino acid substitution. 
     
     
         21 . The method of  claim 19 , wherein the substitution is a non-conservative amino acid substitution. 
     
     
         22 . The method of  claim 18  further comprising:
 k) producing the modified polypeptide in vivo or in vitro; and   l) assaying the activity of the modified polypeptide in vivo or in vitro.   
     
     
         23 . A representation of a three-dimensional structure of the mPGES-1 substrate binding domain (SBD) characterized in that:
 a) amino acid residues Q36, R110, T114, Y130, and Q134 of mPGES-1 are associated with the PGH2-binding site of the SBD;   b) amino acid residue Y130 of mPGES-1 is associated with the peroxy head of prostaglandin H2 (PGH2) when PGH2 occupies at least a portion of the binding site;   c) amino acid residue Y130 of mPGES-1 is associated with the —SH group of glutathione (GSH) when GSH occupies at least a portion of the binding site;   d) amino acid residues R110, T114, and Q36 of mPGES-1 are associated with the carboxyl tail of PGH2;   e) the calculated binding free energy (ΔG) for an SBD-PGH2 complex is between −5.0 kcal/mol and −9.0 kcal/mol; and   f) the calculated binding free energy (AG) for an SBD-GSH complex is between −4.0 kcal/mol and −8.0 kcal/mol.   
     
     
         24 . The representation of  claim 23 , wherein PGH2 interacts with GSH through hydrogen binding between the peroxy group of PGH2 and the —SH group of GSH. 
     
     
         25 . A representation of a three-dimensional structure of an mPGES-1 trimer characterized in that:
 a) each monomer of the trimer comprises a representation of a three-dimensional structure of the mPGES-1 substrate binding domain (SBD) as set forth in  claim 23 ;   b) the trimer comprises a C 3 -fold symmetry; and   c) the representation of the trimer comprises a homology model based on the crystallographic structure of subunit 1 of cytochrome c oxidase.   
     
     
         26 . A method of structure-based identification of candidate compounds for regulation of interactions of mPGES-1 with its cognate ligands, comprising:
 a) providing a three dimensional structure of mPGES-1, the three dimensional structure being selected from the group consisting of:
 i) the mPGES-1 substrate binding domain as set forth in  claim 23 ; and 
 ii) the mPGES-1 trimer as set forth in  claim 25 ; 
   b) identifying at least one candidate compound for interacting with the three dimensional structure of a) and performing structure based drug design.   
     
     
         27 . A machine-readable medium embedded with information that corresponds to the three-dimensional structural representation of the mPGES-1 substrate binding domain (SBD) as claimed in  claim 23 . 
     
     
         28 . A machine-readable medium embedded with information that corresponds to the three-dimensional structural representation of the mPGES-1 trimer as claimed in  claim 25 . 
     
     
         29 . A computer system comprising:
 a) a representation of the three-dimensional structure of the mPGES-1 substrate binding domain (SBD) as claimed in  claim 23 ; and   b) a user interface to view the representation.   
     
     
         30 . A computer system comprising:
 a) a representation of the three-dimensional structure of the mPGES-1 trimer as claimed in  claim 25 ; and   b) a user interface to view the representation.   
     
     
         31 . A method for conducting a biotechnology business comprising:
 a) identifying one or more candidate compounds for regulation of interactions of mPGES-1 with its cognate ligands by the method of  claim 26 ;   b) generating a machine-readable medium, or data signal embodied in a carrier wave, embedded with information that corresponds to the three-dimensional structural representation of the candidate compound; and   c) providing the medium or data signal to an end user.