Crystal structure

Abstract

This invention relates to a crystallisable composition comprising a TSHR polypeptide, to crystals comparing a TSHR polypeptide and to TSHR-related applications.

1 . A crystallisable composition comprising a TSHR polypeptide. 
     
     
         2 . A crystal comprising a TSHR polypeptide. 
     
     
         3 . A crystallisable complex comprising a TSHR polypeptide and a TSHR-binding entity. 
     
     
         4 . A co-crystal comprising a TSHR polypeptide and a TSHR-binding entity. 
     
     
         5 . A crystallisable composition according to  claim 1 , or a crystal according to  claim 2 , or a crystallisable complex according to  claim 3 , or a co-crystal according to  claim 4 , in which the TSHR polypeptide includes at least a portion of the leucine-rich domain of a mammalian TSHR. 
     
     
         6 . A crystallisable composition according to  claim 1 , or a crystal according to  claim 2 , or a crystallisable complex according to  claim 3  or a co-crystal according to  claim 4 , in which the TSHR polypeptide has a human, chimpanzee, porcine, bovine, canine, feline or guinea pig sequence. 
     
     
         7 . A crystallisable composition according to  claim 1 ,  5  or  6  or a crystal according to  claim 2 ,  5  or  6  or a complex according to  claim 3 ,  5  or  6  or a co-crystal according to  claim 4 ,  5  or  6  in which the TSHR polypeptide includes amino acids 22-260 of the human wild-type sequence or corresponding amino acids from other mammalian TSHR sequences. 
     
     
         8 . A complex according to  claim 3 ,  5 ,  6  or  7  in which the TSHR polypeptide comprises the full wild-type sequence of TSHR. 
     
     
         9 . A crystallisable composition according to  claim 1 ,  5 ,  6 , or  7  or a crystal according to  claim 2 ,  5 ,  6  or  7 , a complex according to  claim 3 ,  5 ,  6 ,  7  or  8 , or a co-crystal according to  claim 4 ,  5 ,  6  or  7  in which at least one wild type TSHR amino acid has been mutated. 
     
     
         10 . A complex according to  claim 3 ,  5 ,  6 ,  7 ,  8  or  9  or a co-crystal according to  claim 4 ,  5 ,  6 ,  7  or  9  in which the TSHR-binding entity is an antibody or portion thereof. 
     
     
         11 . A complex or co-crystal according to  claim 10  in which the antibody is an autoantibody or a portion thereof or derived therefrom. 
     
     
         12 . A complex or co-crystal according to  claim 10  or  11  in which the antibody is a monoclonal antibody. 
     
     
         13 . A complex or co-crystal according to  claim 9 ,  10  or  11  in which the antibody is M22. 
     
     
         14 . A co-crystal according to any one of  claims 4  to  13  comprising a crystalline form of the TSHR polypeptide having the coordinates shown in  FIG. 9   a  or  FIG. 9   b.    
     
     
         15 . A machine-readable data storage medium encoded with data relating to at least a portion of the coordinates of TSHR polypeptide amino acids of  FIG. 9   a  or  FIG. 9   b  or a homologue thereof. 
     
     
         16 . A machine-readable data storage medium encoded with data relating to all of the TSHR polypeptide amino acid coordinates of  FIG. 9   a  or  FIG. 9   b.    
     
     
         17 . A computer system for presenting a representation of a three-dimensional structure of a TSHR polypeptide, or a homologue of such a TSHR polypeptide, in which the homologue has a root mean square deviation from the backbone atoms between 0 Å and 4 Å, the computer system including data storage means including data corresponding to TSHR polypeptide amino acid coordinates of  FIG. 9   a  or  FIG. 9   b.    
     
     
         18 . A computer system according to  claim 17 , the computer system including data storage means including data corresponding to coordinates of a chemical entity interacting with the TSHR polypeptide or homologue thereof. 
     
     
         19 . A computer system according to  claim 18 , being arranged to provide a representation of a three-dimensional structure of the TSHR polypeptide or homologue thereof interacting with the chemical entity. 
     
     
         20 . A computer system according to  claim 18  or  19 , in which the chemical entity is an antibody or portion thereof. 
     
     
         21 . A computer system according to  claim 20  in which the antibody portion is M22 Fab. 
     
     
         22 . A computer system according to any one of  claims 17  to  21 , including a display for displaying the representation of the three-dimensional structure of a TSHR polypeptide. 
     
     
         23 . A computer system according to  claim 22 , arranged to display the chemical entity interacting with the TSHR polypeptide or homologue thereof. 
     
     
         24 . An electronic representation of a three-dimensional structure of a TSHR polypeptide. 
     
     
         25 . An electronic representation according to  claim 24  which represents the leucine rich domain of a human TSHR polypeptide. 
     
     
         26 . An electronic representation according to  claim 25  which represents at least amino acids 30-257 of human TSHR. 
     
     
         27 . An electronic representation of a three-dimensional structure of a TSHR polypeptide and an antibody thereto or portion thereof. 
     
     
         28 . A method of identifying a chemical entity which will interact with the TSHR, the method comprising identifying a chemical entity which will interact with at least one amino acid of a TSHR polypeptide three-dimensional structure or homologue thereof according to a representation provided by a computer system according to any one of  claims 19  to  23  or as represented by an electronic representation according to any one of  claims 24  to  27 . 
     
     
         29 . A method according to  claim 28  in which the chemical entity is a TSHR agonist. 
     
     
         30 . A method according to  claim 28  in which the chemical entity is a TSHR antagonist. 
     
     
         31 . A method according to any one of  claims 28  to  30  in which a chemical entity is identified which will interact by forming a hydrogen bond with at least one of the TSHR amino acids: K129, E107, K58 and Y185. 
     
     
         32 . A method according to any one of  claims 28  to  31  in which a chemical entity is identified which will interact by forming van der Waals interactions with at least one of the TSHR amino acid residues R255, R80, K129, R38 and K183. 
     
     
         33 . A method according to any one of  claims 28  to  32  in which a chemical entity is identified which will interact by forming electrostatic interactions with at least one of the TSHR amino acid residues D151, K58, K129, R80, K209 and K183. 
     
     
         34 . A method according to any one of  claims 27  to  31  in which a chemical entity is identified which will interact by forming ion pair interactions with TSHR amino acid residue K209. 
     
     
         35 . A method of identifying a chemical entity which may potentially interfere with the binding of autoantibodies to the TSHR, the method comprising identifying a chemical entity which interacts with the highly positively charged ridge at the N-terminal end of the concave surface of the TSHR leucine-rich domain. 
     
     
         36 . A method according to  claim 35  in which the autoantibodies are thyroid stimulating autoantibodies. 
     
     
         37 . A method according to  claim 35  in which the autoantibodies are TSH antagonists. 
     
     
         38 . A method according to  claim 35 ,  36 , or  37  in which the chemical entity interacts with at least one of the following TSHR amino acids: R38, K58, R80, H105 and K129. 
     
     
         39 . A method of identifying a chemical entity which may potentially interfere with the binding of autoantibodies to the TSHR, the method comprising identifying a chemical entity which at least substantially fills a negatively charged cavity on the M22 surface formed by M22 H1, H2 and H3 using a computer according to any one of  claims 18  to  22  or a representation according to any one of  claims 24  to  27 . 
     
     
         40 . A method according to  claim 39  in which the autoantibodies are thyroid stimulating autoantibodies. 
     
     
         41 . A method according to  claim 39  in which the autoantibodies are TSH antagonists. 
     
     
         42 . A method according to  claim 39 , the method comprising identifying a chemical entity which will at least substantially disrupt a thyroid stimulating autoantibody binding to the TSHR residue R255. 
     
     
         43 . A method according to  claim 40  or  42 , the method comprising identifying a chemical entity which will disrupt a thyroid stimulating autoantibody binding to amino acid K209 of human TSHR. 
     
     
         44 . A method according to any one of  claims 28  to  43  in which a potential interaction of a chemical entity with other receptors is determined. 
     
     
         45 . A method according to  claim 44  in which the potential interaction is binding. 
     
     
         46 . A method according to  claim 44  or  45  in which the other receptor includes Follicle Stimulating Hormone Receptor, and/or Luteinizing Hormone Receptor. 
     
     
         47 . A method of detecting autoantibodies to TSHR including comparing binding of a putative autoantibody and a chemical entity identified as interacting with a TSHR polypeptide by a method according to any one of  claims 28  to  46  with a TSHR polypeptide. 
     
     
         48 . A chemical entity identified by a method according to any one of  claims 28  to  47 . 
     
     
         49 . A chemical compound including at least one chemical entity according to  claim 48 . 
     
     
         50 . A pharmaceutical composition comprising a chemical compound according to  claim 48  and a pharmaceutically acceptable carrier. 
     
     
         51 . A pharmaceutical composition according to  claim 50  for use in the treatment of Autoimmune Thyroid Disease. 
     
     
         52 . A pharmaceutical composition according to  claim 51  for use in the treatment of Graves' disease. 
     
     
         53 . A pharmaceutical composition according to  claim 50  for use in stimulating tissues containing the TSHR. 
     
     
         54 . A chemical entity according to  claim 48  for use in detection of autoantibodies to the TSHR. 
     
     
         55 . The use of a chemical entity according to  claim 48  or a chemical compound according to  claim 49  in the preparation of a medicament for the treatment of Autoimmune Thyroid Disease. 
     
     
         56 . The use of a chemical entity according to  claim 48  or a chemical compound according to  claim 49  in the preparation of a medicament for the treatment of Graves' disease. 
     
     
         57 . A method of removing thyroid stimulating hormone receptor autoantibodies, from a sample containing such thyroid stimulating hormone receptor autoantibodies, the method comprising providing a binding molecule that includes or mimics a thyroid stimulating hormone receptor binding site for M22 or thyroid stimulating hormone receptor autoantibodies having similar surface and binding characteristics to M22, contacting the sample with the binding molecule whereby thyroid stimulating hormone receptor autoantibodies bind to the binding molecule and are removed from the sample. 
     
     
         58 . A method according to  claim 57  in which the sample includes patient serum or plasma containing thyroid stimulating autoantibodies. 
     
     
         59 . A method according to  claim 57  or  58  in which the binding molecule is connected to a neutral protein or other tag that does not affect TSHR autoantibody binding. 
     
     
         60 . A method according to  claim 59  in which the neutral protein is maltose binding protein. 
     
     
         61 . A method according to  claim 57 ,  58 ,  59  or  60  in which the binding molecule is coupled directly to a solid support or via a tag. 
     
     
         62 . A method according to  claim 61  in which the solid support is agarose or a resin. 
     
     
         63 . A method according to any one of  claims 57  to  62  in which the sample from which antibodies have been removed is returned to the subject. 
     
     
         64 . A method according to any one of  claims 57  to  63  in which the subject is human. 
     
     
         65 . A method of treating a thyroid-related condition in a mammalian subject, the method comprising removing thyroid stimulating hormone receptor autoantibodies from a sample derived from the subject, by a method according to any one of  claims 57  to  64 . 
     
     
         66 . A method according to  claim 65  in which the thyroid-related condition is Graves' ophthalmopathy, or pre-tibial myxoedema or neonatal thyrotoxicosis.