Obtaining an Improved Therapeutic Ligand

Abstract

Methods and associated apparatus involving designing a ligand ab initio that will bind to a binding site of a macromolecular target, or of identifying a modification to a ligand for improving the affinity of the ligand to a binding site of a macromolecular target, comprising using information about non-bonding, intra-molecular or inter-molecular atom to atom contacts extracted from a database of biological macromolecules to identify favoured regions adjacent to the binding site for particular atom types and modifying a candidate ligand to increase the intersection between atoms of the candidate ligand and the favoured regions. One or more steps of the methods may be performed by a computer.

1 . A method for designing a ligand ab initio that will bind to a binding site of a macromolecular target, or of identifying a modification to a ligand for improving the affinity of the ligand to a binding site of a macromolecular target, comprising:
 a) identifying a target list of atoms forming the surface of the target binding site;   b) identifying each atom, hereinafter referred to as a theta atom, in the target list, as a particular theta atom type;   c) extracting from a structural database of biological macromolecules, information about non-bonding, intra-molecular or inter-molecular atom to atom contacts, where the first atom in a contacting pair of atoms is of a particular theta atom type and the opposing, second atom of the pair, hereinafter referred to as an iota atom, is of a particular iota atom type, said information comprising spatial and/or contextual data about the iota atom relative to the theta atom, and said data collected for a plurality of contacts of the given theta atom type from the said database is hereinafter referred to as a theta contact set;   d) for each theta atom identified in the target list in b), superimposing in or around the target binding site data relating to a given iota atom type, or a predetermined group of related iota atom types, from the corresponding theta contact set extracted in c);   e) combining and/or parsing the superimposed data in such a way as to predict one or more favoured regions of the binding site where the given iota atom type, or the predetermined group of related iota atom types, has high theoretical propensity; and   f) with a candidate ligand notionally docked into the binding site, comparing the type and position of one or more of the atoms of the candidate ligand with the predicted favoured regions for the respective iota atom types, to identify a modification to the candidate ligand, in terms of alternate and/or additional candidate ligand atoms, that will produce a greater intersection between the alternate and/or additional candidate ligand atoms and the respective iota atom type favoured regions, leading to an improvement in the affinity of the modified candidate ligand to the binding site compared to the unmodified candidate ligand;   wherein each non-bonding intra-molecular or inter-molecular contact in the database is defined as a contact between opposing residues of a protein fold or between opposing monomer units of a macromolecular fold or between two interacting macromolecular partners and is specifically between a theta atom on one side of the fold or first interacting partner and an iota atom on the opposing side or second interacting partner; in an instance where the following condition is satisfied:   s−Rw≦t, where s is the separation between the two atoms of the contact, Rw is the sum of the van de Waals radii of the two atoms of the contact, and t is a predetermined threshold distance; and   wherein the theta atom type is identified uniquely in b) such that there is no intersection between the data of a theta contact set extracted in c) for a given theta atom type and the data of any other theta contact set extracted in c) for any other theta atom type, apart from data concerning contacts involving the given theta atom as the iota atom.   
     
     
         2 . The method according to  claim 1 , wherein for each non-bonding intra-molecular contact extracted from the structural database of protein members in c) the following condition is also satisfied:
 the theta atom and the iota atom of the contact are on different residues separated by at least four residues along the linear polypeptide or are on separate polypeptide chains.   
     
     
         3 . The method according to  claim 1 , wherein the theta atom type is identified as being one and only one of:
 the 167 non-hydrogen atoms present in the 20 natural amino acids of proteins;   the 82 non-hydrogen atoms present in the 4 nucleotides of the deoxyribonucleic acid polymer (DNA);   the 42 non-hydrogen atoms present in the methylated DNA nucleotides, cytidine phosphate and adenosine phosphate;   the 85 non-hydrogen atoms present in the 4 nucleotide phosphates of the ribonucleic acid polymer (RNA);   the 89 non-hydrogen atoms present in 2-O′-methylated ribose nucleotide phosphates of RNA;   the over 400 non-hydrogen atoms present in the commonest post-transcription base modified RNA.   
     
     
         4 . The method according to  claim 1 , wherein the information extracted in c) is collected in a secondary database comprising one and only one theta contact set for each of the theta atom types. 
     
     
         5 . The method according to  claim 4 , wherein each of said secondary database theta contact sets is sub-divided into a plurality of non-overlapping iota atom types or non-overlapping groups of related iota atom types. 
     
     
         6 . The method according to  claim 1 , wherein the iota atom type is identified as being one and only one of:
 the 167 non-hydrogen atoms present in the 20 natural amino acids of proteins;   the oxygen atom present in protein bound, structurally relevant, water molecules;   the 82 non-hydrogen atoms present in the 4 nucleotides of the deoxyribonucleic acid polymer (DNA);   the 42 non-hydrogen atoms present in the methylated DNA nucleotides, cytidine phosphate and adenosine phosphate;   the 85 non-hydrogen atoms present in the 4 nucleotide phosphates of the ribonucleic acid polymer (RNA);   the 89 non-hydrogen atoms present in 2-O′-methylated ribose nucleotide phosphates of RNA; and/or   the over 400 non-hydrogen atoms present in the commonest post-transcription modified bases of RNA.   
     
     
         7 . The method according to  claim 1 , wherein said predetermined group of related iota atom types is one of a plurality of non-overlapping groups obtained by sorting the 167 non-hydrogen atoms present in the 20 natural amino acids of proteins into groups of similar chemical type. 
     
     
         8 . The method according to  claim 7 , wherein the iota atom types are sorted into the plurality of non-overlapping groups according to one or more of the following factors: elemental nature of the atom type, hybridisation state of the atom type. 
     
     
         9 . The method according to  claim 7 , wherein the iota atom types are sorted into a plurality of non-overlapping groups comprising the following: C sp 3 , C sp 2 (aromatic), C sp 2 (non-aromatic), N sp 3 , N sp 2 , O sp 3 , O sp 2 , S. 
     
     
         10 . The method according to  claim 1 , wherein:
 said spatial data extracted in c) defines the position of each iota atom specified in the theta contact set by geometrical reference to the position of the theta atom and to the positions of third and fourth atoms;   the third atom is covalently bonded to the theta atom; and the fourth atom is covalently bonded to the third atom.   
     
     
         11 . The method according to  claim 10 , wherein:
 for each iota atom specified in the theta contact set, said spatial data extracted in c) defines the position of fifth and sixth atoms by geometrical reference to the position of the theta atom and to the positions of the third and fourth atoms;   the fifth atom is covalently bonded to the iota atom; and   the sixth atom is covalently bonded to either the fifth atom or the iota atom.   
     
     
         12 . The method according to  claim 11 , wherein the superimposition in or around the target site of (d) comprises:
 parsing the theta contact set to extract spatial data for contacts comprising the given iota atom type or one or more of the predetermined group of related iota atom types; and plotting this spatial data to determine theoretical locations representing where each   iota atom type, or each of the one or more of the predetermined group of related iota atom types, would be located if: i) the theta atom of the contact were located at the position of the corresponding theta atom in the target binding site; and ii) the third and fourth atoms of the contact were located at the positions of the third and fourth atoms of the corresponding theta atom in the target binding site.   
     
     
         13 . The method according to  claim 12 , wherein:
 the extracted spatial data is parsed against said contextual data before said plotting step.   
     
     
         14 . The method according to  claim 12 , wherein a region in which a density of theoretical locations for the given iota atom type, or for the one or more of the predetermined group of related iota atom types, is above a predetermined threshold is identified as one of the favoured regions. 
     
     
         15 . The method according to  claim 12 , wherein theoretical locations for the given iota atom type, or for one or more of the predetermined group of related iota atom types, are determined for a plurality of theta atoms on the target list and a region in which a density of the cumulative theoretical locations is above the predetermined threshold is identified as one of the favoured regions. 
     
     
         16 . The method according to  claim 12 , wherein:
 if the theoretical location of an individual iota atom intersects with the location of an atom of the target macromolecule closer than Rw−0.2 angstroms then the said iota atom is excluded from subsequent analysis.   
     
     
         17 . The method according to  claim 11 , wherein:
 the third and fourth atoms are chosen uniquely for each specified theta atom type.   
     
     
         18 . The method according to  claim 11 , wherein:
 the fifth and sixth atoms are chosen uniquely for each specified iota atom type.   
     
     
         19 . The method according to  claim 11 , wherein:
 for each favoured region, vectors are derived to describe the position of the fifth atom relative to its respective iota atom and analysis is carried out on said vectors in order to identify a favoured bond vector representing a prediction of the covalent attachment of a theoretical consensus iota atom in the said region, said identified favoured bond vector being used to refine the design of the candidate ligand or modification of the candidate ligand.   
     
     
         20 . The method according to  claim 1 , wherein:
 said contextual data extracted in (c), contains contextual information concerning the local environment of each contact pair in the theta contact set, including one or more of the following in any combination: secondary structure, amino acid types or other monomer types comprising the contact pair, adjacent monomer units and/or local geometry thereof in a polymer chain either side of the contact, adjacent amino acids in a polypeptide chain on either side of the contact, local geometry of the said adjacent monomer units or amino acids, temperature factor of the theta atom, temperature factor of the iota atom, accessible surface area of the theta atom, accessible surface area of the   iota atom, the number of different iota atom contacts for the particular theta atom and the number of other theta atoms on the same monomer unit as the theta atom.   
     
     
         21 . The method according to  claim 1 , wherein (f) comprises: identifying a modification of the candidate ligand that increases a degree of overlap between one or more atoms of the candidate ligand and a predicted favoured region or regions for an iota atom type or predetermined group of related iota atom types in the binding site. 
     
     
         22 . The method according to  claim 1 , wherein a plurality of modifications to the candidate ligand are identified in (f) and the method further comprises selecting a subset of the identified modifications based on one or both of the following: 1) the extent to which the intersection between the alternate and/or additional candidate ligand atoms and the respective iota atom type favoured regions is greater compared to the unmodified candidate ligand; and 2) the extent to which one or more factors contributing to the total energy of the complex formed by the binding of the modified candidate ligand to the binding site is/are reduced compared to the case where the unmodified candidate ligand is bound. 
     
     
         23 . The method according to  claim 1 , wherein t=2.5 angstroms 
     
     
         24 . The method according to  claim 1 , wherein t=0.8 angstroms 
     
     
         25 . The method according to  claim 1 , further comprising: out-putting data representing the modification identified in (f). 
     
     
         26 . The method according to  claim 1 , wherein the ligand is a protein. 
     
     
         27 . The method according to  claim 26 , wherein the ligand is an antibody. 
     
     
         28 . The method according to  claim 26 , wherein (f) comprises replacing each of one or more of the amino acid residues of the ligand that is/are in direct contact with the target binding site, or in close proximity to the target binding site, with each of one or more alternative residues chosen from the other 19 natural amino acids, each replacement being referred to as a residue replacement,
 wherein for each residue replacement that does not cause conflict between the replacement residue and adjacent atoms of the ligand or target, the type and position of each atom of the replacement residue is compared with the respective iota atom type favoured regions to identify whether they will produce a greater intersection than the atoms of the original residue.   
     
     
         29 . The method according to  claim 28 , further comprising:
 outputting a list of the residue replacements that are identified as producing a greater intersection than atoms of the original residue;   for each listed residue replacement, using mutation of the candidate ligand to produce a modified ligand that incorporates the residue replacement;   testing the affinity of each of the modified ligands to the target binding site in order to determine which residue replacements result in an affinity improvement that is above a predetermined threshold.   
     
     
         30 . The method according to  claim 29 , further comprising:
 modifying the candidate ligand to incorporate a plurality of the residue replacements that have been determined to result in an affinity improvement that is above the predetermined threshold.   
     
     
         31 . A computer readable medium or signal comprising computer readable instructions for causing a computer to carry out the method of  claim 1 . 
     
     
         32 . The medium or signal according to  claim 31 , wherein the computer is caused to carry out at least (c)-(e). 
     
     
         33 . The medium or signal according to  claim 31 , wherein the computer is caused to carry to carry out at least (f). 
     
     
         34 . A method of manufacturing a therapeutic ligand, comprising:
 designing a therapeutic ligand according to the method of  claim 1 ; and manufacturing the therapeutic ligand thus designed.   
     
     
         35 . A therapeutic ligand manufactured according to the method of  claim 34 . 
     
     
         36 . The method or ligand according to  claim 34 , wherein the ligand is a protein. 
     
     
         37 . The method or ligand according to  claim 36 , wherein the protein is an antibody. 
     
     
         38 . A method of generating a database for use in a method for designing a ligand ab initio that will bind to a binding site of a macromolecular target, or of identifying a modification to a ligand for improving the affinity of the ligand to a binding site of a macromolecular target, comprising:
 analysing the relative positions of atoms in each of a plurality of proteins or other biological macromolecules in order to identify instances of a non-bonding intra-molecular contact between a first atom, referred to as a theta atom, and a second atom, referred to as an iota atom, of the protein or macromolecule; and   generating a database that for each identified contact specifies: the type of the theta atom, the type of the iota atom, and the position of the iota atom relative to the theta atom;   wherein a non-bonding intra-molecular contact is defined as an instance where the following conditions are satisfied:   s−Rw≦t, where s is the separation between the theta and iota atoms, Rw is the sum of the van de Waals radii of the theta and iota atoms, and t is a predetermined threshold distance of typically 2.5 angstroms and preferably 0.8 angstroms; and   wherein in the case of proteins, the theta and iota atoms are on amino acid residues separated from each other by at least four residues on a linear polypeptide or are on separate polypeptide chains.   
     
     
         39 . The method according to  claim 38 , wherein the method comprises sub-dividing the database to form groups of identified contacts in which the theta atom is one and only one of the 167 non-hydrogen atoms present in the 20 natural amino acids of proteins and the iota atom is in one and only one of a plurality of non-overlapping groups obtained by sorting the 167 non-hydrogen atoms present in the 20 natural amino acids of proteins into groups based on chemical similarity. 
     
     
         40 . A method of generating a database for use in a method for designing a ligand ab initio that will bind to a binding site of a macromolecular target, or of identifying a modification to a ligand for improving the affinity of the ligand to a binding site of a macromolecular target, comprising:
 analysing the relative positions of atoms in each of a plurality of proteins or other biological macromolecules in order to identify instances of a non-bonding intra-molecular contact between a first atom referred to as a theta atom, and a second atom, referred to as an iota atom, of the protein or macromolecule; and   generating a database that for each identified contact specifies: the type of the theta atom, the type of the iota atom, and the position of the iota atom relative to the theta atom;   wherein a non-bonding intra-molecular contact is defined as an instance where the following condition is satisfied:   s−Rw≦t, where s is the separation between the theta and iota atoms, Rw is the sum of the van de Waals radii of the theta and iota atoms, and t is a predetermined threshold distance of typically 2.5 angstroms and preferably 0.8 angstroms; and   wherein the method comprises sub-dividing the database to form groups of identified contacts in which the theta atom is one and only one of the 167 non-hydrogen atoms present in the 20 natural amino acids of proteins and the iota atom is in one and only   one of a plurality of non-overlapping groups obtained by sorting the 167 non-hydrogen atoms present in the 20 natural amino acids of proteins into groups based on chemical similarity.   
     
     
         41 . The method according to  claim 39 , wherein:
 for each contact, the position of the iota atom is defined by geometrical reference to the position of the theta atom and to the positions of third and fourth atoms, the third atom being covalently bonded to the theta atom and the fourth atom being covalently bonded to the third atom, the method further comprising:   normalizing the coordinates of the iota atom, theta atom, third atom, and fourth atom of each contact as a group to generate a normalized coordinate group;   for each of one or more of the theta atom types, using the normalized coordinate groups for a plurality of contacts involving the theta atom type and a given iota atom type to generate a two-dimensional polar plot that represents a distribution of directions of the given iota atom, in terms of latitude and longitude, relative to the theta atom;   repeating the above for different iota atom types;   comparing the resultant two-dimensional polar plots to identify groups of iota atom types that yield similar distributions of directions and using those groups as the groups based on chemical similarity to sort the 167 non-hydrogen atoms present in the 20 natural amino acids of proteins into the plurality of non-overlapping groups.   
     
     
         42 . The method of generating a database according to  claim 38 , comprising: extracting contact information from at least 2000 proteins or other biological macromolecules, the extracted information containing information about at least two million contact atom pairs. 
     
     
         43 . The method of generating a database according to  claim 38 , comprising: extracting contact information from at least 10000 proteins or other biological macromolecules, the extracted contact information containing information about at least ten million contact atom pairs. 
     
     
         44 . The computer readable medium storing a database generated according to  claim 1 . 
     
     
         45 . The method according to  claim 1 , wherein for a given antibody-antigen complex, specific mutations to amino acid residues in or around the antibody binding site are predicted to produce higher binding affinity of the antibody to the antigen.