Crystal structure of cytochrome P450
AssigneeAstex Technology Ltd.
InventorsIan Tickle, Clemens Vonrhein, Dijana Vinkovic, Stewart Kirton, Pamela Williams, Harren Jhoti
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Abstract
The invention provides the crystal structure of the cytochrome P450 3A4 protein molecule. The structure is set out in Tables 1-4. The structure may be used in to model the interaction of compounds such as pharmaceuticals with this protein, and to determine the structure of related cytochrome P450 molecules.
Claims66
A computer-based method for the analysis of the interaction of a molecular structure with a P450 structure, which comprises:
- providing a structure comprising a three-dimensional representation of P450 3A4 or a portion of P450 3A4, which representation comprises all or a portion of the coordinates of any one of Tables 1-4±a root mean square deviation from the Cα atoms of not more than 1.5 Å;
- providing a molecular structure to be fitted to said P450 3A4 structure or selected coordinates thereof;
- and
- fitting the molecular structure to said P450 3A4 structure.
A method of obtaining a structure of a target P450 protein of unknown structure, the method comprises the steps of:
- providing a crystal of said target P450;
- obtaining an X-ray diffraction pattern of said crystal,
- calculating a three-dimensional atomic coordinate structure of said target, by modelling the structure of said target P450 of unknown structure on the 3A4 P450 structure of any one of Tables 1-4±a root mean square deviation from the Cα atoms of not more than 1.5 Å or selected coordinates thereof.
A computer-based method for the analysis of molecular structures which comprises:
- (a) providing the coordinates of at least two atoms of a P450 3A4 structure as defined in any one of Tables 1-4±a root mean square deviation from the Cα atoms of less than 1.5 Å (“selected coordinates”);
- (b) providing the structure of a molecular structure to be fitted to the selected coordinates;
- and
- (c) fitting the structure to the selected coordinates of the P450 3A4 structure.
A computer-based method of rational drug design comprising:
- (a) providing the coordinates of at least two atoms of a P450 3A4 structure as defined in any one of Tables 1-4±a root mean square deviation from the Cα atoms of less than 1.5 Å (“selected coordinates”);
- (b) providing the structures of a plurality of molecular fragments;
- (c) fitting the structure of each of the molecular fragments to the selected coordinates;
- and
- (d) assembling the molecular fragments into a single molecule to form a candidate modulator molecule.
A method for identifying a candidate modulator of P450 3A4 comprising the steps of:
- (a) employing a three-dimensional structure of P450 3A4, at least one sub-domain thereof, or a plurality of atoms thereof, to characterise at least one P450 3A4 binding cavity, the three-dimensional structure being defined by atomic coordinate data according to any one of Tables 1-4±a root mean square deviation from the Cα atoms of less than 1.5 Å;
- and (b) identifying the candidate modulator by designing or selecting a compound for interaction with the binding cavity.
A method for determining the structure of a protein, which method comprises;
- providing the co-ordinates of any one of Tables 1-4±a root mean square deviation from the Cα atoms of not more than 1.5 Å or selected coordinates thereof, and
- either (a) positioning said co-ordinates in the crystal unit cell of said protein so as to provide a structure for said protein, or (b) assigning NMR spectra peaks of said protein by manipulating said co-ordinates.
A method for determining the structure of a compound bound to P450 protein, said method comprising:
- providing a crystal of P450 protein;
- soaking the crystal with the compound to form a complex;
- and
- determining the structure of the complex by employing the data of any one of Tables 1-4± a root mean square deviation from the Cα atoms of not more than 1.5 Å or a portion thereof.
A method for determining the structure of a compound bound to P450 protein, said method comprising:
- mixing P450 protein with the compound;
- crystallizing a P450 protein-compound complex;
- and
- determining the structure of the complex by employing the data of any one of Tables 1-4±a root mean square deviation from the Cα atoms of not more than 1.5 Å or a portion thereof.
A method for modifying the structure of a compound in order to alter its metabolism by a P450, which method comprises:
- fitting a starting compound to one or more coordinates of at least one amino acid residue of the ligand-binding region of the P450;
- modifying the starting compound structure so as to increase or decrease its interaction with the ligand-binding region.
A method for modifying the structure of a compound in order to alter its metabolism by a P450 3A4, which method comprises:
- fitting a starting compound to one or more coordinates of at least one amino acid residue of the haem-binding region of the P450;
- modifying the starting compound structure so as to increase or decrease its interaction with the haem-binding region.
A method for modifying the structure of a compound in order to alter its, or another compounds, metabolism by a P450, which method comprises:
- fitting a starting compound to one or more coordinates of at least one amino acid residue of the peripheral binding region of the P450;
- modifying the starting compound structure so as to increase or decrease its interaction with the peripheral binding region;
- wherein said peripheral binding region is defined as the P450 residues numbered as:
- 213, 214, 219.
A method for designing the structure of a compound which binds to the peripheral binding region, in order to alter another compounds metabolism by a P450, which method comprises:
- fitting a starting compound to one or more coordinates of at least one amino acid residue of the peripheral binding region of the P450;
- modifying the starting compound structure so as to increase or decrease its interaction with the peripheral binding region;
- wherein said peripheral binding region is defined as the P450 residues numbered as:
- 213, 214, 219.
A method of obtaining a representation of the three dimensional structure of a crystal of cytochrome P450 3A4, which method comprises providing the data of any one of Tables 1-4 or selected coordinates thereof, and constructing a three-dimensional structure representing said coordinates.
A computer system, intended to generate structures and/or perform optimisation of compounds which interact with P450, P450 homologues or analogues, complexes of P450 with compounds, or complexes of P450 homologues or analogues with compounds, the system containing computer-readable data comprising one or more of:
- (a) 3A4 co-ordinate data of any one of Tables 1-4, said data defining the three-dimensional structure of P450 or at least selected coordinates thereof;
- (b) atomic coordinate data of a target P450 protein generated by homology modelling of the target based on the coordinate data of any one of Tables 1-4;
- (c) atomic coordinate data of a target P450 protein generated by interpreting X-ray crystallographic data or NMR data by reference to the co-ordinate data of any one of Tables 1-4;
- (d) structure factor data derivable from the atomic coordinate data of (b) or (c). and
- (e) atomic coordinate data of any one of Tables 1-4±a root mean square deviation from the Cα atoms of not more than 1.5 Å or selected coordinates thereof.
A method of providing data for generating structures and/or performing optimisation of compounds which interact with P450, P450 homologues or analogues, complexes of P450 with compounds, or complexes of P450 homologues or analogues with compounds, the method comprising:
- (i) establishing communication with a remote device containing
- (a) computer-readable data comprising atomic coordinate data of any one of Tables 1-4±a root mean square deviation from the Cα atoms of not more than 1.5 Å or selected coordinates thereof;
- (b) atomic coordinate data of a target P450 homologue or analogue generated by homology modelling of the target based on the data (a);
- (c) atomic coordinate data of a protein generated by interpreting X-ray crystallographic data or NMR data by reference to the data of any one of Tables 1-4 and
- (d) structure factor data derivable from the atomic coordinate data of (d) or (e);
- and
- (ii) receiving said computer-readable data from said remote device.
A computer-readable storage medium, comprising a data storage material encoded with computer readable data, wherein the data are defined by all or a portion of the structure coordinates of the P450 protein of any one of Tables 1-4 or a homologue of P450, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of said any one of Tables 1-4 respectively of not more than 1.5 Å.
A computer-readable storage medium comprising a data storage material encoded with a first set of computer-readable data comprising a Fourier transform of at least a portion of the structural coordinates for the P450 protein defined by the structure of any one of Tables 1-4±a root mean square deviation from the Cα atoms of not more than 1.5 Å or selected coordinates thereof;
- which data, when combined with a second set of machine readable data comprising an X-ray diffraction pattern of a molecule or molecular complex of unknown structure, using a machine programmed with the instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data.
A crystal of P450 3A4.
A co-crystal of P450 3A4 and a ligand.
A crystal of P450 3A4 having an orthorhomobic space group I222.
A crystal of P450 3A4 having a space group space group P21212.
A crystal of P450 3A4 protein having a resolution better than 3.1 Å.
A crystal of P450 protein having the structure defined by the co-ordinates of any one of Tables 1-4±a root mean square deviation from the Cα atoms of not more than 1.5 Å.
A method of predicting three dimensional structures of P450 homologues or analogues of unknown structure, the method comprises the steps of:
- aligning a representation of an amino acid sequence of a target P450 protein of unknown three-dimensional structure with the amino acid sequence of the P450 of any one of Tables 1-4±a root mean square deviation from the Cα atoms of not more than 1.5 Å to match homologous regions of the amino acid sequences;
- modelling the structure of the matched homologous regions of said target P450 of unknown structure on the corresponding regions of the P450 structure as defined by said any one of Tables 1-4 respectively±a root mean square deviation from the Cα atoms of not more than 1.5 Å;
- and
- determining a conformation for said target P450 of unknown structure which substantially preserves the structure of said matched homologous regions.
A chimaeric protein having a binding cavity which provides a substrate specificity substantially identical to that of P450 3A4 protein,
- wherein the chimaeric protein binding cavity is lined by a plurality of atoms which correspond to selected P450 3A4 atoms lining the P450 3A4 binding cavity, the relative positions of said plurality of atoms corresponding to the relative positions, as defined by any one of Tables 1-4±a root mean square deviation from the Cα atoms of not more than 1.5 Å, of said selected P450 3A4 atoms.
A method of assessing the ability of a compound to interact with P450 3A4 protein which comprises:
- obtaining or synthesising said compound;
- forming a crystallised complex of a P450 3A4 protein and said compound, said complex diffracting X-rays for the determination of atomic coordinates of said complex to a resolution of better than 2.8 Å;
- and
- analysing said complex by X-ray crystallography to determine the ability of said compound to interact with the P450 3A4 protein.
A computer-based method for identifying a candidate modulator of P450 3A4 comprising the steps of:
- employing a three-dimensional structure of P450 3A4, or selected co-ordinates thereof, the three-dimensional structure being defined by atomic coordinate data according to any one of Tables 1-4±a root mean square deviation from the Cα atoms of less than 1.5 Å;
- identifying the candidate modulator by designing or selecting a compound for interaction with the binding cavity.
Dependencies are inferred from the claim text.