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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/02—Antidotes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/795—Porphyrin- or corrin-ring-containing peptides
  • C07K14/80—Cytochromes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

• the present invention relates to the human cytochrome P450 protein 3A4, methods for its crystallization, crystals of 3A4 and their 3-dimensional structures, and uses thereof.
• Cytochrome P450s form a very large and complex gene superfamily of hemeproteins that metabolise physiologically important compounds in many species of microorganisms, plants and animals. Cytochrome P450s are important in the oxidative, peroxidative and reductive metabolism of numerous and diverse endogenous compounds such as steroids, bile, fatty acids, prostaglandins, leukotrienes, retinoids and lipids. Many of these enzymes also metabolise a wide range of xenobiotics including drugs, environmental compounds and pollutants. Their involvement in drug metabolism is extensive, it is estimated that 50% of all known drugs are affected in some way by the action of CYP450 enzymes. Significant resource is employed by the pharmaceutical industry to optimise drug candidates in order to avoid their detrimental interactions with the CYP450 enzymes. Another level of complication results from the fact that these enzymes exhibit different tissue distributions and polymorphisms between individuals and ethnic populations
• Most mammalian P450s are located in the liver, but other organs and tissues have high concentrations of certain cytochrome P450s, including the intestinal wall, lung, kidney, adrenal cortex and nasal epithelium. Mammals have about 50 unique CYP450 genes and each family member is 45-55 KDa in size and contains a heme moiety that catalyses a two-electron activation of oxygen. The source of electrons may be used to classify CYP450s.
• Those that receive electrons in a three protein chain in which electrons flow from a flavin adenine dinucleotide (FAD) containing reductase, to an iron-sulphur protein, and then to P450 belong to the group of class I P450s, and include most of the bacterial enzymes.
• Class II P450s receive electrons from a reductase containing both FAD and flavin mononucleotide (FMN), and comprise the microsomal P450s that are the main culprits of drug metabolism.
• the mammalian microsomal cytochrome P450s are integral membrane proteins anchored by an N-terminal transmembrane spanning ⁇ -helix.
• cytochrome P450 are inserted in the membrane of the endoplasmic reticulum by a short, highly hydrophobic N-terminal segment that acts as a non-cleavable signal sequence for insertion into the membrane.
• the remainder of the mammalian cytochrome P450 protein is a globular structure that protrudes into the cytoplasmic space. Hence, the bulk of the enzyme faces the cytoplasmic surface of the lipid bilayer.
• P450s require other membranous enzymatic components for activity including the flavoprotein NADPH-cytochrome P450 oxidoreductase and, in some cases, cytochrome b5.
• Cytochrome P450 oxidoreductase supports the activity of all the mammalian microsomal enzymes by interacting directly with the P450s and transferring the required two electrons from NADPH.
• Cytochrome P450s are able to incorporate one of the two oxygen atoms of an O 2 molecule into a broad variety of substrates with concomitant reduction of the other oxygen atom by two electrons to H 2 O.
• Cytochrome P450 are known to catalyse hydroxylations, epoxidation, N-, S-, and O-dealkylations, N- oxidations, sulfoxidations, dehalogenations, and other reactions.
• Homo sapiens has 17 cytochrome P450 gene families and 42 subfamilies that total more than 50 sequenced isoforms. Cytochrome P450s from families 1, 2 and 3 constitute the major pathways for drug metabolism. Many drugs rely on hepatic metabolism by cytochrome P450s for clearance from the circulation and for pharmacological inactivation. Conversely, some drugs have to be converted in the body to their pharmacologically active metabolites by P450s. Many promising lead compounds are terminated in the development phase due to their interaction with one or more P450s. One of the greatest problems in drug discovery is the prediction of the role of cytochrome P450s on the metabolism or modification of drug leads. Early detection of metabolic problems associated with a chemical lead series is of paramount importance for the pharmaceutical industry.
• CYP450 isoforms involved in drug metabolism are CYP1 A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4.
• the level of sequence identity between these family members ranges from about 20-80%, with much of the variability within the resid ⁇ es involved in substrate recognition.
• CYP450 enzymes are also present in bacteria and much of the understanding of substrate recognition is derived from crystal structures obtained of bacterial CYP450 enzymes.
• CYP3A is both the most abundant and most clinically significant subfamily of cytochrome P450 enzymes.
• the CYP3A subfamily has four human isoforms, 3A4, 3A5, 3A7 and 3A43, CYP3A4 being the most commonly associated with drag interactions.
• the CYP3A isoforms make up approximately 50% of the liver's total cytochrome P450 and are widely expressed throughout the gastrointestinal tract, kidneys and lungs and therefore are ultimately responsible for the majority of first-pass metabolism. This is important as increases or decreases in first-pass metabolism can have the effect of administering a much smaller or larger dose of drag than usual.
• CYP3A4 More than 150 drags are known substrates of CYP3A4, including many of the opiate analgesics, steroids, antiarrhythmic agents, tricyclic antidepressants, calcium-channel blockers and macrolide antibiotics. Although several substrates show age-dependent reductions in elimination, the enzyme itself does not appear to be altered. CYP3A4 is important in the metabolism of many drags including cyclosporine, codeine, tamoxifen, lovastatin, and many more, and endogenous compounds such as testosterone, estradiol and cortisol.
• Ketoconazole, itraconazole, erythromycin, clarithromycin, diltiazem, fluvoxamine, nefazodone, and dihydroxybergamottin and various substances found in grapefruit juice, green tea and other foods are potent inhibitors of CYP3 A4 and are known to be responsible for many drug interactions. These interactions can have serious clinical consequences.
• Each protein crystallizes under a unique set of conditions, which cannot be predicted in advance. Simply supersaturating the protein to bring it out of solution may not work, the result would, in most cases, be an amorphous precipitate. Many precipitating agents are used, common ones are different salts, and polyethylene glycols, but others are known. In addition, additives such as metals and detergents can be added to modulate the behaviour of the protein in solution. Many kits are available (e.g. from Hampton Research), which attempt to cover as many parameters in crystallization space as possible, but in many cases these are just a starting point to optimise crystalline precipitates and crystals which are unsuitable for diffraction analysis.
• a mathematical operation termed a Fourier transform relates the diffraction pattern observed from a crystal and the molecular structure of the protein comprising the crystal.
• a Fourier transform may be considered to be a summation of sine and cosine waves each with a defined amplitude and phase.
• it is possible to calculate the electron density associated with a protein structure by carrying out an inverse Fourier transform on the diffraction data.
• This requires amplitude and phase information to be extracted from the diffraction data.
• Amplitude information may be obtained by analysing the intensities of the spots within a diffraction pattern.
• the conventional methods for recording diffraction data do, however, mean that any phase information is lost.
• phase information must be in some way recovered and the loss of this information represents the "crystallographic phase problem”.
• the phase information necessary for carrying out the inverse Fourier transform can be obtained via a variety of methods. If a protein structure exists a set of theoretical amplitudes and phases may be calculated using the protein model and then the theoretical phases combined with the experimentally derived amplitudes. An electron density map may then be calculated and the protein structure observed.
• MIR multiple isomorphous replacement
• Non-isomorphisms Perturbations to the physical properties of the crystal are termed non-isomorphisms and prevent this type of experiment being successfully completed.
• Successful isomorphous incorporation of heavy atoms into a protein crystal results in the intensities of the spots within the diffraction pattern obtained from the crystal being modified, as compared to the data collected from an identical, unsoaked, (native) crystal.
• the diffraction data obtained from a successful isomorphous replacement experiment are termed a "derivative" dataset.
• By mathematically analysing the "native" and “derivative" datasets it is possible to extract preliminary phase information from the datasets. This phase information, when combined with the experimentally obtained amplitudes from the native dataset, enables an electron density map of the unknown protein molecule to be calculated using the Fourier transform method.
• An alternative method for obtaining phase information for a protein of unknown structure is to perform a multi- wavelength anomalous dispersion (MAD) experiment.
• MAD multi- wavelength anomalous dispersion
• Anomalous scattering by atoms within a protein will modify the diffraction pattern obtained from the protein crystal.
• a diffraction dataset (anomalous dataset) may be collected at an X-ray wavelength at which this anomalous scattering is maximal.
• a native dataset may then be collected.
• phase information necessary for the calculation of an electron density map may be determined.
• the most usual way to introduce anomalous scatterers into a protein is to replace the sulphur containing methionine amino acid residues with selenium containing seleno-methionine residues. This is done by generating recombinant protein that is isolated from cells grown on growth media that contain seleno-methionine.
• Selenium is capable of anomalously scattering X-rays and may thus be used for a MAD experiment.
• Further methods for phase determination such as single isomorphous replacement (SIR), single isomorphous replacement anomalous scattering (SIRAS) and direct methods exist, but the principles behind them are similar to MIR and MAD.
• the final method generally available for the calculation of the phases necessary for the determination of an unknown protein structure is molecular replacement.
• This method relies upon the assumption that proteins with similar amino acid sequences (primary sequences) will have a similar fold and three-dimensional stracture (tertiary structure). Proteins related by amino acid sequence are termed homologous proteins. If an X-ray diffraction dataset has been collected from a crystal whose protein stracture is not known, but a stracture has been determined for a homologous protein, then molecular replacement can be attempted. Molecular replacement is a mathematical process that attempts to correlate the dataset obtained from a new protein crystal with the theoretical diffraction pattern calculated for a protein of known structure. If the correlation is sufficiently high some phase information can be extracted from the known protein stracture and combined with the amplitudes obtained from the new protein dataset. This enables calculation of a preliminary electron density map for the protein of unknown stracture.
• an electron density map has been calculated for a protein of unknown stracture then the amino acids comprising the protein must be fitted into the electron density for the protein. This is normally done manually, although high resolution data may enable automatic model building. The process of model building and fitting the amino acids to the electron density can be both a time consuming and laborious process. Once the amino acids have been fitted to the electron density it is necessary to refine the structure. Refinement attempts to maximise the correlation between the experimentally calculated electron density and the electron density calculated from the protein model built. Refinement also attempts to optimise the geometry and disposition of the atoms and amino acids within the user-constructed model of the protein stracture. Sometimes manual re-building of the stracture will be required to release the stracture from local energetic minima.
• the scattering of the X-rays is described as "anomalous".
• the electrons are forced to undergo vibrations at the same frequency as that of the incident X-ray photon, emitting elastically scattered photons (i.e. no change in frequency) in the process.
• this frequency is far from the natural frequency of vibration of the electron there is no effect on the scattered photon from this natural vibration.
• the frequency of the incident photon is close to the natural frequency of the electron, resulting in a resonance effect, which is manifested as a dispersion (decrease in velocity, though still no change in frequency) of the photon, as well as a vibration damping effect, which is manifested as absorption (decrease in intensity) of a fraction of the incident photons.
• the anomalously scattered photon will thus have a phase angle associated with it that is retarded when compared with one being scattered normally, all other conditions being equal.
• the routine collection of X-ray data at cryo-temperatures has prolonged crystal lifetime and has made collection of multiple datasets (at different wavelengths) from a single crystal now feasible for many crystal systems. Collection and analysis of multiple datasets from a single crystal has the advantage of eliminating all effects related to non-isomorphism (variations in stracture between different crystals due to random variations in soaking and/or freezing conditions).
• the haem group that forms the site of enzymatic activity naturally contains a single iron atom. Iron has transition energies at the high energies (long wavelengths) obtainable at tunable synchrotron beamlines.
• cytochrome P450 structures had been solved by X-ray crystallography and were available in the public domain. All of the cytochrome P450s, whose structures had been solved, were expressed in E. coli.
• Six structures correspond to bacterial cytochrome P450s: P450cam (CYP101 Poulos et al., 1985, J. Biol. Chem., 260, 16122), the hemeprotein domain of P450BM3 (CYP102, Ravichandran et al, 1993, Science, 261, 731), P450terp (CYP108, Hasemann et al, 1994, J. Mol. Biol.
• the eighth stracture is that of the rabbit 2C5 isoform, the first structure of a mammalian cytochrome P450 (Williams et al. 2000, Mol. Cell. 5, 121).
• WO 03/035693 describes the crystallisation of a human 2C9 P450 protein molecule and provides an analysis of the protein crystal structure.
• mammalian cytochrome P450s have been particularly difficult to crystallize, compared to their bacterial counterparts, resides in the nature of these proteins.
• the bacterial cytochrome P450s are soluble whereas the mammalian P450s are membrane-associated proteins.
• structural studies on mammalian cytochrome P450s may use the combination of heterologous expression systems that allow expression of single cytochrome P450s at high concentration with modification of their sequences to improve the solubility and the behaviour of these proteins in solution.
• the present invention relates to the crystal structure of human 3A4, which allows the binding location of the substrates in the enzyme to be investigated and determined.
• the present inventors have obtained an electron density map for 3 A4 which is useful for the provision of atomic coordinate models of this protein, and also for other applications which are discussed in Section H below.
• the data of Table 3 herein provides stracture factor phase data, permitting others of skill in the art to solve X-ray diffraction data of 3 A4 and homologous protein crystals more readily in order to provide electron density maps.
• the invention provides a three dimensional structure of 3 A4 set out in Table 5, and uses thereof.
• the present invention is concerned with the provision of a 3 A4 stracture and its use in modelling the interaction of molecular structures, e.g. potential and existing pharmaceutical compounds, prodrugs, P450 inhibitors or substrates, or fragments of such compounds, prodrugs, inhibitors or substrates with this 3 A4 structure.
• molecular structures e.g. potential and existing pharmaceutical compounds, prodrugs, P450 inhibitors or substrates, or fragments of such compounds, prodrugs, inhibitors or substrates with this 3 A4 structure.
• Table 1 provides the data statistics
• Table 2 provides the phasing statistics.
• Table 3 ( Figure 1) provides the structure factors and phases which can be used to generate an electron density map of the 3A4 crystal stracture.
• Table 4 provides refinement statistics.
• Table 5 ( Figure 2) sets out the coordinate data of the structure of 3A4.
• Table 6 ( Figure 3) sets out one possible set of coordinate data of a loop region of 3A4.
• Table 7 details binding site residues of 3A4.
• Table 8 sets out newly identified binding site residues of 3A4.
• Figure 1 sets out Table 3.
• Figure 2 sets out Table 5.
• Figure 3 sets out Table 6.
• the present invention provides a crystal of 3A4 having an orthorhomobic space group 1222, and unit cell dimensions 78 A, 100 A, 132 A, 90°, 90°, 90°. Unit cell variability of 5% may be observed in all dimensions.
• Such a crystal may be obtained using the methods described in the accompanying examples.
• the crystal may be of a 3 A4 protein which is desirably truncated in its N-terminal region to delete the hydrophobic trans-membrane domain, and the region is replaced by a short (e.g. 8 to 20) amino acid sequence.
• a short amino acid sequence e.g. 8 to 20 amino acid sequence.
• the 3 A4 P450 may optionally comprise a tag, such as a C-terminal polyhistidine tag to allow for recovery and purification of the protein.
• a tag such as a C-terminal polyhistidine tag to allow for recovery and purification of the protein.
• N-terminal truncation a polyhistidine tag at the C-terminus.
• the N-terminal truncation and tag are both features which can be varied by those of skill in the art using routine skill.
• alternative N-terminal sequence might be utilised, for example for production in host cells other than E. coli.
• other tags may be used for purification of the protein as described below.
• These N- and C-terminal modification may be made to a 3 A4 protein which retains the core sequence of the wild type protein from the residue 17 onwards of S ⁇ Q ID NO:2 shown herein, up to the residue immediately preceding the polyhistidine tag.
• the N-terminal sequence is preferably not the full length wild-type sequence, and preferably smaller than 30, e.g. 20 residues in size. Preferably, it is shorter that the wild type sequence.
• the N-terminal region is the truncation illustrated in the accompanying examples. This type of N-terminal sequence reduces the tendency of 3 A4 to anchor to membranes and to aggregate compared to the wild type sequence. The truncation utilised here has wild-type residues 3-24 deleted.
• the C-terminal sequence is preferably no larger than 30, and preferably no larger than 10 amino acids in size.
• the 3A4 sequence may be that of the core sequence illustrated herein, or an allele thereof, or a variant which retains the ability to form crystals under the conditions illustrated herein.
• Such variants include those with a number of amino acid substitutions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids by an equivalent or fewer number of amino acids. Further examples of variants, including mutants, are discussed further herein below.
• the methodology used to provide a P450 crystal illustrated herein may be used generally to provide a human 3A4 crystal resolvable at a resolution of at least 3.0 A, and preferably at least 2.8 A.
• the invention thus further provides a 3A4 crystal having a resolution of at least 3.0 A, preferably at least 2.8 A.
• the proteins may be wild-type proteins or variants thereof, which are modified to promote crystal formation, for example by N-terminal truncations and/or deletion of loop regions, which prevent crystal formation.
• the invention provides a method for making a P450 protein crystal, particularly of a 3A4 protein comprising the core sequence of 3A4 (as defined above) or a variant thereof, which method comprises growing a crystal by vapor diffusion using a reservoir buffer that contains 0.05-0.2 M HEPES pH 7.0-7.8, 2.5-10% IP A, 0-20% PEG 4000, 0-0.3 M sodium chloride, 0-10% PEG 400, 0-10% glycerol, preferably 0.1 M HEPES pH 7.2, 5% IPA, 10% PEG 4000.
• the crystal is grown by vapor diffusion and is performed by placing an aliquot of the solution on a cover slip as a hanging drop above a well containing the reservoir buffer.
• the concentration of the protein solution used was 0.3-0.7 mM.
• Crystals of the invention also include crystals of 3A4 mutants, chimeras, homologues in the 3 A family (e.g. 3A1, 3A5, 3A7, 3A12 and 3A43) and alleles.
• 3A4 mutants e.g. 3A1, 3A5, 3A7, 3A12 and 3A43
• homologues in the 3 A family e.g. 3A1, 3A5, 3A7, 3A12 and 3A43
• alleles e.g. 3A1, 3A5, 3A7, 3A12 and 3A43
• a mutant is a 3 A4 protein characterized by the replacement or deletion of at least one amino acid from the wild type 3A4.
• Such a mutant may be prepared for example by site-specific mutagenesis, or incorporation of natural or unnatural amino acids.
• mutants wherein a “mutant” refers to a polypeptide which is obtained by replacing at least one amino acid residue in a native or synthetic 3A4 with a different amino acid residue and/or by adding and/or deleting amino acid residues within the native polypeptide or at the N- and/or C-terminus of a polypeptide corresponding to 3 A4, and which has substantially the same three-dimensional stracture as 3 A4 from which it is derived.
• having substantially the same three-dimensional stracture is meant having a set of atomic structure co-ordinates that have a root mean square deviation (r.m.s.d.) of less than or equal to about 2.0 A (preferably less than 1.55 or 1.5 A, more preferably less than 1.0 A, and most preferably less than 0.5 A) when superimposed with the atomic stracture co-ordinates of the 3A4 from which the mutant is derived when at least about 50% to 100% of the C ⁇ atoms of the 3 A4 are included in the superposition.
• a mutant may have, but need not have, enzymatic or catalytic activity.
• amino acids present in the said protein can be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophobic moment, antigenicity, propensity to form or break -helical or ⁇ -sheet structures, and so on.
• Substitutional variants of a protein are those in which at least one amino acid in the protein sequence has been removed and a different residue inserted in its place. Amino acid substitutions are typically of single residues but may be clustered depending on functional constraints e.g. at a crystal contact. Preferably amino acid substitutions will comprise conservative amino acid substitutions.
• Insertional amino acid variants are those in which one or more amino acids are introduced. This can be amino-terminal and/or carboxy-terminal fusion as well as intrasequence. Examples of amino-terminal and/or carboxy-terminal fusions are affinity tags, MBP tag, and epitope tags.
• Amino acid substitutions, deletions and additions which do not significantly interfere with the three-dimensional stracture of the 3A4 will depend, in part, on the region of the 3A4 where the substitution, addition or deletion occurs. In highly variable regions of the molecule, non- conservative substitutions as well as conservative substitutions may be tolerated without significantly disrupting the three-dimensional structure of the molecule. In highly conserved regions, or regions containing significant secondary stracture, conservative amino acid substitutions are preferred. Conservative amino acid substitutions are well-known in the art, and include substitutions made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the amino acid residues involved.
• negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
• Other conservative amino acid substitutions are well known in the art.
• mutants contemplated herein need not exhibit enzymatic activity. Indeed, amino acid substitutions, additions or deletions that interfere with the catalytic activity of the 3A4 but which do not significantly alter the three-dimensional structure of the catalytic region are specifically contemplated by the invention. Such crystalline polypeptides, or the atomic stracture co-ordinates obtained there from, can be used to identify compounds that bind to the protein.
• residues for mutation could easily be identified by those skilled in the art and these mutations can be introduced by site-directed mutagenesis e.g. using a Stratagene QuikChangeTM Site-Directed Mutagenesis Kit or cassette mutagenesis methods (see e.g. Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, and Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989)).
• the present invention contemplates "alleles" wherein allele is a term coined by Bateson and Saunders (1902) for characters which are alternative to one another in Mendelian inheritance (Gk. Allelon, one another; morphe, form). Now the term allele is used for two or more alternative forms of a gene resulting in different gene products and thus different phenotypes.
• An allele contains nucleotide changes that have been shown to affect transcription, splicing, translation, post-transcriptional or post-translational modifications or result in at least one amino acid change. These different alleles are particularly important in P450s as some confer different metabolic clearance rates of specific drugs onto the phenotype. Alleles of P450s are often only different by one or two amino acids.
• crystals of such proteins may be formed.
• the skilled person would recognize that the conditions provided herein for crystallising 3A4 may be used to form such crystals. Alternatively, the skilled person would use the conditions as a basis for identifying modified conditions for forming the crystals.
• aspects of the invention relating to crystals of 3A4 may be extended to crystals of mutant and mutants of 3A4 which result in homologue, allelic fo ⁇ n, and species variant.
• the invention provides a crystal of 3A4 having the structure factors and phases of Table 3.
• the invention also provides a crystal of P450 having the electron density map generated from the data of Table 3.
• An advantageous feature of the electron density map is that it has a resolution of about 2.8 A.
• Table 3 has eight columns. The first three columns are the indices h, k and 1 of each individual reflection. Columns four and five are the experimentally measured structure factors and the associated standard deviations of the peak wavelength, respectively. Column six is the solvent flattened stracture factor amplitude. Column seven is the solvent flattened stracture factor phase. Column eight is the solvent flattened figure of merit associated with the reflection. The data of columns six to eight were generated from the experimentally measured stracture factors and by using the phasing procedure in SHARP (see equation (2) in de la Fortelle & Bricogne, 1997) that are then used in density modification.
• the electron density map can be generated from Table 3 using columns six and seven using, for example, the FFT program which is part of the CCP4 suite of programs (Collaborative Computational Project 4.
• the CCP4 Suite Programs for Protein Crystallography, Acta Crystallographica, D50, (1994), 760-763.).
• the resulting electron density map can then be viewed, interpreted or models built into it using a crystallographic graphical viewing program such as "O" (Jones et al., Acta Crystallographica, A47, (1991), 110-119) or "QUANTA” (1994, San Diego, CA: Molecular Simulations, Jones et al., Acta Crystallography A47 (1991), 110- 119).
• Errors in electron density maps derive principally from errors in the phase angles ( ) of the structure factors used in their calculation; errors in the corresponding amplitudes (
• the expected error in the phase of a stracture factor is normally expressed as a "figure of merit" (in), which can be defined as the expected value of the cosine of the error in the "best” phase (that value of the phase which minimises the root-mean- square error ⁇ (p) in the electron density).
• phase error will vary significantly from one stracture factor to the next, partly because of the random nature of experimental error and partly because stracture factors with small amplitudes on average tend to have larger errors than those with large amplitudes (small amplitudes clearly do not contribute to the electron density summation as much as large ones; a structure factor with zero amplitude contributes nothing and so has a phase angle which is completely indeterminate).
• the phase error will tend to be greater at high resolution, because, for example, the small errors in locating the atoms used in the phase calculation have a greater effect at high resolution.
• the figures of merit and phase errors are normally binned together and averaged either according to amplitude or to resolution (we have chosen to present the averaged figures of merit by resolution).
• ⁇ (p) V "1 ( ⁇ ⁇ ⁇ jt ⁇ i ⁇ F hkl
• This electron density map will allow the placement of a large percentage of all the atoms of 3A4, and reveals for the first time the spatial arrangement of the atoms of 3A4.
• Knowledge of the spatial arrangement of these atoms has clear implications in various fields. For example, knowledge of those atoms that form the enzymatic active site of the molecule will determine the physico-chemical properties of compounds that are ligands for the enzyme. The ability to modify these properties and hence to ultimately modify the enzyme's ability to metabolise a particular compound has clear value to the pharmacological industry.
• An indicator of the quality of the phases used to generate the map is as follows: inspection of anomalous log-likelihood gradient maps within SHARP (La Fortelle, E. de and Bricogne, G.
• the data of Table 3 will in practice be used by those of skill in the art in electronic form to allow for processing of the data by computer programs such as those discussed herein. Thus in practice the programs will use all the data points of the Table. However, as indicated by the values in column 8 of the Table, the figure of merit values for some data points are relatively low. Whereas this may be taken into account in the processing of the data for the production of an electron density map, an alternative would be to ignore one or more of the data points associated with low merit values. Thus it will be understood by those of skill in the art that reference to the data of Table 3 includes the situation where a small fraction (less than 5% and preferably less than 1%, such as less than 0.5%) of the data point rows are not utilised. Once interpretation of the current map has been completed to provide an electron density map it is possible to combine the experimental phase with phases derived from the model and thus generate a new electron density map that will allow most of the crystal stracture to be defined.
• An electron density map is interpreted by placing an atomic structure in the model such that the model fits the map.
• An assessment of how the model agrees with the map can be derived by calculating a correlation coefficient between the map and the transformed model, calculation of a 2Fo-Fc map or generation of Rfactor and Free R factors by a refinement protocol. Partial interpretation of the electron density map at high resolutions (e.g. 2.5-1.0 A) can be automated in the case of high quality maps. For a lower resolution map (e.g. less than 2.5A), or maps generated from phases with less than ideal phasing statistics, interpretation is more subjective and may require manual input.
• the coordinates then obtained from this provide a measure of atomic location in Angstroms.
• the coordinates are a relative set of positions that define a shape in three dimensions, but the skilled person would understand that an entirely different set of coordinates having a different origin and/or axes could define a similar or identical shape.
• the skilled person would understand that varying the relative atomic positions of the atoms of the structure so that the root mean square deviation of the residue backbone atoms (i.e.
• the nitrogen-carbon-carbon backbone atoms of the protein amino acid residues is less than 2.0 A, preferably less than 1.55 or 1.5 A, more preferably less than 1.0 A and most preferably less than 0.5 A when superimposed on the coordinates derived from the data in Table 3 for the residue backbone atoms, will generally result in a structure which is substantially the same as the stracture derived from of Table 3 in terms of both its structural characteristics and usefulness for structure-based analysis of P450-interactivity molecular structures.
• the coordinates available from Table 3 are transposed to a different origin and/or axes; the relative atomic positions of the atoms of the structure are varied so that the root mean square deviation of residue backbone atoms is less than 2.0 A, preferably less than 1.55 or 1.5 A, more preferably less than 1.0 A, and most preferably less than 0.5 A when superimposed on the coordinates for the residue backbone atoms; and/or the number and/or positions of water molecules and/or substrate molecules is varied.
• Reference herein to the coordinate data derived from Table 3 and the like thus includes the coordinate data in which one or more individual values of the Table are varied in this way.
• root mean square deviation we mean the square root of the arithmetic mean of the squares of the deviations from the mean.
• selected coordinates of 3A4 may be used, for example at least 5, preferably at least 10, more preferably at least 50 and even more preferably at least 100 atoms such as at least 500 or at least 1,000 of the 3A4 stracture.
• the other applications of the invention described herein, including homology modelling and stracture solution, and data storage and computer assisted manipulation of the coordinates may also utilise all or a portion of the coordinates from the electron density map in Table 3 may be used.
• modifications in the 3 A4 crystal structure due to e.g. mutations, additions, substitutions, and/or deletions of amino acid residues could account for variations in the 3A4 atomic coordinates.
• atomic coordinate data of 3 A4 modified so that a ligand that bound to one or more binding sites of 3 A4 would be expected to bind to the corresponding binding sites of the modified 3A4 are, for the purposes described herein as being aspects of the present invention, also within the scope of the invention.
• Reference herein to the coordinates from the electron density map from Table 3 thus includes the coordinates modified in this way.
• the modified data define at least one 3A4 binding cavity.
• the present invention allows electron density models of other 3A4 crystals or crystals of homologous proteins to be obtained without the need to perform multiple anomalous diffraction (MAD) stracture determination or MIR.
• the invention provides a method of determining an electron density map of a target protein which is, or is homologous to, 3A4, which method comprises providing a crystal of the target protein, obtaining an X-ray diffraction of said protein, and generating an electron density map of said target protein by reference to the structure factor phase data of Table 3.
• a more general method of comparing structures is based on an analysis of electron density maps. Therefore preferably, for the purposes of this invention two maps are considered to be equivalent if the linear correlation coefficient calculated for the maps is greater than 0, and more preferably greater than 0.25 or 0.5. If the electron densities of two maps are respectively defined by the variates pi and p 2 , the linear correlation coefficient, CC, is defined as:
• a molecular (i.e. P450 3A4) mask is determined. This can be done, for example, using the CCP4 DM program to distinguish the P450 3A4 molecule from the solvent region. Each grid point within a molecular boundary is labelled ' 1 ' and each grid point outside a boundary is labelled '0'.
• One map is then transformed into maximum coincidence with the other map. This is accomplished, for example, using the CCP4 FFFEAR program to search for a best fit between two maps. During the transformation, rigid-body translations and rotations are allowed.
• One of the maps and the corresponding mask are then interpolated onto the grid points of the other map and mask. The interpolation can be performed using the Astex-ROTMAP program provided in Annex 1.
• the CC is computed for the masked maps, e.g. using the Astex-DENCOR program provided in Annex 2.
• an electron density map generated from the data of Table 3 and a set of atomic coordinates are considered to be equivalent if the CC calculated for the map generated from the data of Table 3 and a further electron density map generated from the atomic coordinate data is greater than 0, and more preferably greater than 0.25 or 0.5.
• the computation of the CC in this case can follow the procedure discussed above with the additional prior step of generating the further electron density map from the atomic coordinate data.
• the generation can be conveniently performed using the standard CCP4 programs REFMAC and FFT to respectively calculate stracture factors and then electron densities.
• the invention also provides a crystal of P450 having the three dimensional atomic coordinates of Table 5.
• the atomic coordinates of Table 5 exclude residues from a loop region (261-270), which are not as clear and amenable for unambiguous interpretation as other regions of the protein. It is not inconceivable that this loop may adopt a different conformation under different conditions e.g. data from a different crystal, upon additional of compound, and the like. Crystals of the invention will thus comprise the coordinates of Table 5, with the coordinates of the loop region optionally being as further described herein, though other atomic coordinates for this loop region are not excluded.
• An advantageous feature of the structure defined by the atomic coordinates of Table 5 is that it has a resolution of about 2.8 A. More particularly, the residues in the binding pocket are well resolved.
• a further advantage of the 3A4 structure described herein is that it is an unliganded, apo structure. This makes it particularly suitable for soaking in ligands and hence determining co- complex structures and is also ideal for homology modelling purposes as there is no conformational bias from a ligand.
• Tables 5 and 6 gives atomic coordinate data for P450 3A4.
• the third column denotes the atom, the fourth the residue type, the fifth the chain identification (in this case, chain A), the sixth the residue number (the atom numbering is with respect to the full length wild type protein), the seventh, eighth and ninth columns are the X, Y, Z coordinates respectively of the atom in question, the tenth column the occupancy of the atom, the eleventh the temperature factor of the atom, the twelfth the atom type.
• Tables 5 and 6 are set out in an internally consistent format. For example (except in the case of Tyr 25), the coordinates of the atoms of each amino acid residue are listed such that the backbone nitrogen atom is first, followed by the C-alpha backbone carbon atom, designated CA, followed by side chain residues (designated according to one standard convention) and finally the carbon and oxygen of the protein backbone.
• Alternative file formats e.g. such as a format consistent with that of the EBI Macromolecular Stracture Database (Hinxton, UK) which may include a different ordering of these atoms, or a different designation of the side-chain residues or haem molecule atoms, may be used or preferred by others of skill in the art. However it will be apparent that the use of a different file format to present or manipulate the coordinates of the Table is within the scope of the present invention.
• the coordinates of Tables 5 and 6 provide a measure of atomic location in Angstroms, to 3 decimal places.
• the coordinates are a relative set of positions that define a shape in three dimensions, but the skilled person would understand that an entirely different set of coordinates having a different origin and/or axes could define a similar or identical shape.
• the skilled person would understand that varying the relative atomic positions of the atoms of the structure so that the root mean square deviation of the residue backbone atoms (i.e.
• the nitrogen- carbon-carbon backbone atoms of the protein amino acid residues is less than 2.0 A, preferably less than 1.55 or 1.5 A, more preferably less than 1.0 A, and most preferably less than 0.5 A when superimposed on the coordinates provided in Table 5 or 6 for the residue backbone atoms, will generally result in a stracture which is substantially the same as the structure of Tables 5 or 6 in terms of both its structural characteristics and usefulness for structure-based analysis of P450-interactivity molecular structures.
• the number and/or positions of the water molecules molecules of Table 5 will not generally affect the usefulness of the stracture for structure-based analysis of P450-interacting structure.
• the Tables 5 or 6 coordinates are transposed to a different origin and/or axes; the relative atomic positions of the atoms of the structure are varied so that the root mean square deviation of residue backbone atoms is less than 2.0 A, preferably less than 1.55 or 1.5 A, more preferably less than 1.0 A, and most preferably less than 0.5 A when superimposed on the coordinates provided in Tables 5 or 6 for the residue backbone atoms; and/or the number and/or positions of water molecules is varied.
• the invention provides a crystal of P450 comprising amino acids having the atomic coordinates of Table 5, wherein the crystal additionally comprises amino acids having the atomic coordinates of Table 6.
• Protein stracture similarity is routinely expressed and measured by the root mean square deviation (r.m.s.d.), which measures the difference in positioning in space between two sets of atoms.
• the r.m.s.d. measures distance between equivalent atoms after their optimal superposition.
• the r.m.s.d. can be calculated over all atoms, over residue backbone atoms (i.e. the nitrogen-carbon-carbon backbone atoms of the protein amino acid residues), main chain atoms only (i.e. the nitrogen-carbon-oxygen-carbon backbone atoms of the protein amino acid residues), side chain atoms only or more usually over C-alpha atoms only.
• the r.m.s.d. can be calculated over any of these, using any of the methods outlined below.
• Programs for determining r.m.s.d include MNYFIT (part of a collection of programs called COMPOSER, Sutcliffe, M.J., Haneef, I., Carney, D. and Blundell, T.L. (1987) Protein Engineering, 1, 377-384), MAPS (Lu, G. An Approach for Multiple Alignment of Protein Structures (1998, in manuscript and on httpJ ⁇ ioinfol.mbfys.lu.se/TOP/maps.html)).
• the user can define the residues in the two proteins that are to be paired for the purpose of the calculation.
• the pairing of residues can be determined by generating a sequence alignment of the two proteins, programs for sequence alignment are discussed in more detail in Section G. The atomic coordinates can then be superimposed according to this alignment and an r.m.s.d. value calculated.
• the program Sequoia CM. Brans, I. Hubatsch, M. Ridderstr ⁇ m, B. Mannervik, and J.A.
• Tainer (1999) Human Glutathione Transferase A4-4 Crystal Structures and Mutagenesis Reveal the Basis of High Catalytic Efficiency with Toxic Lipid Peroxidation Products, Journal of Molecular Biology 288(3): 427-439) performs the alignment of homologous protein sequences, and the superposition of homologous protein atomic coordinates.
• the program Astex- KFIT (see Annex 4) can be used.
• the r.m.s.d. can be calculated using programs detailed above. For sequence identical, or highly identical, the structural alignment of proteins can be done manually or automatically as outlined above. Another approach would be to generate a superposition of protein atomic coordinates without considering the sequence.
• varying the atomic positions of the atoms of the stracture by up to about 0.5 A, preferably up to about 0.3 A in any direction will result in a structure which is substantially the same as the stracture of Table 5 in terms of both its structural characteristics and utility e.g. for molecular structure-based analysis.
• selected coordinates it is meant for example at least 5, preferably at least 10, more preferably at least 50 and even more preferably at least 100, for example at least 500 or at least 1000 atoms of the 3A4 stracture.
• the other applications of the invention described herein, including homology modelling and stracture solution, and data storage and computer assisted manipulation of the coordinates may also utilise all or a portion of the coordinates (i.e. selected coordinates) of Table 5.
• the selected coordinates may include or may consist of atoms found in the 3A4 P450 binding pocket, as described herein below, and particularly those of Tables 7 and more particularly those of Table 8.
• the first resolvable residue is Tyr25 and the last residue Gly498 (the protein as purified comprises residues residues 1, 2, and 25-503 of the wild type sequence (using wild type numbering from Ml 8907) and a four histidine tag as shown in SEQ ID 2).
• the overall fold of the protein is typical of all P450 structures solved to date and the secondary structure elements are named according to the convention adopted for P450s Ravichandran, K. G., Boddupalli, S. S., Hasermann, C. A., Peterson, J. A., and Deisenhofer, J. (1993) Science 261, 731-736.
• the haem sits centrally within the molecule with the single cysteine 442 coordinating and hydrogen bonds between the haem propionates and Arg 105, Tr ⁇ l26, Arg375 and Arg440.
• the FG loop comprised 34 residues (210-243) and includes helix F' and helix G', compared to the 23 residues in the FG loop of 2C9.
• the long FG loop of 3A4 is more due to the shortness of helix F than to the length of the FG loop itself.
• the B-C and F-G loops are in close proximity, forming two sides of the active site. It is widely accepted that 3A4 may bind several compound simultaneously, and can bind large compounds in excess of 1000 Da (e.g. erythromycin).
• Movement of these regions may be required to allow the compound entry and egress, and they may become more structured if in alternative conformations.
• the loops between helices G and H, and helices H and I are not clearly resolved in the electron density maps (residues 261-270, 277-290) and have been excluded from the model.
• the dominating feature of the active site of substrate-free 3A4 is the cluster of phenylalanine residues (Phe57, Phel08, Phe213, Phe215, Phe219, Phe220, Phe241, Phe304) above the haem. Of these, some have been implicated by site directed mutagenesis to play a role in cooperativity and stereoselectivity. The majority of these residues lie within substrate region sites (SRS) (Gotoh, O. (1992) JBiol Chem 267, 83-90) first identified for the CYP 2C family of proteins. Another cluster of four phenylalanine residues is found just below and to the side of the haem itself, in a position less clearly important for compound binding.
• SRS substrate region sites
• 3 A4 The kinetics exhibited by 3 A4 can be complicated, with many literature examples citing one or more compound being accommodated simultaneously within the active site of 3A4 (Domanski et al, Biochemistry 2001, 40, 10150-10160). Site directed mutagenesis suggests that different substrates may bind at different regions of the active site. There is also evidence for homotropic cooperativity (interactions between a substrate and one or more effector molecules of the same chemical stracture) and hetertropic cooperativity (where the substrate and effector molecules have different chemical structures).
• selected coordinates will comprise at least one coordinate, preferably at least one side-chain coordinate of an amino acid residue selected from either Table 7 or 8.
• the selected coordinates include the coordinates of all the atoms of Table 5 or Table 6 relating to at least one amino acid from Table 7 or 8.
• At least 2 of the selected coordinates are of side chain residues from the corresponding number of different amino acid residues. These may be selected exclusively from either of Table 7 or 8, or a combination thereof. Preferably at least one side chain residue coordinate of Table 8 is included.
• the substrate-binding pocket of P450 is generally constituted by residues that fall in the SRS regions (substrate recognition sites) defined by Gotoh (Gotoh, O, J. Biol. Chem, 267; 83-90 (1992)) and in loops of the molecule.
• aspects of the present invention therefore relate to modification of P450 proteins such that the active sites mimic those of related isoforms.
• a person skilled in the art could modify a P450 protein such that the active site mimicked that of human 3A4. This protein could then be used to obtain information on compound binding through the determination of protein/ligand complex structures using the chimaeric P450 protein.
• the present invention provides 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, and the relative positions of the plurality of atoms corresponding to the relative positions, as defined by Table 5, of the selected P450 3A4 atoms.
• 3A4 is 89% identical to 3A7, and 3A43 shares 76, 76, and 71% sequence identity on the amino acid level with CYP3A4, 3A5, and 3A7, respectively (Westlind et al, Biochemical and Biophysical Research Communications (2001), 281(5), 1349-1355; Gellner et al, Pharmacogenetics (2001), 11(2), 111-121).
• 3A4 and 3A5 are 84% identical they exhibit clear substrate specificity differences (Aoyama T; Yamano S; Waxman D J; Lapenson D P; Meyer U A; Fischer N; Tyndale R; Inaba T; Kalow W; Gelboin H N; Journal Of Biological Chemistry (1989 Jun 25), 264(18), 10388-95).
• CYP3A4 is inhibited by mifepristone and yet CYP3A5 is not.
• CYP3A4 and CYP3A5 also show different regioselectivity towards aflatoxin Bl (AFB1) biotransformation, and a site-directed mutagenesis program to understand the stractural features responsible for these differences, concluded that residues within the SRS region 2 alone were responsible for these differences (Huifen Wang, Ryan Dick, Hequn Yin, Estefania Licad-Coles, Deanna L. Kroetz, Grazyna Szklarz, Greg Harlow, James R. Halpert, and Maria Almira Correia, Biochemistry, 37 (36), 12536 -12545, 1998).
• ARB1 aflatoxin Bl
• the substrate specificity of an enzyme generally relies on only a limited number of residues located in non-contiguous parts of the polypeptide chain.
• the substrate specificities of these isoforms could be analysed by substituting these residues by site-directed mutagenesis.
• the minimal changes that would be required to convert another P450 protein into a 3A4-like chimera could be at least two amino acids selected from binding pocket, particularly the amino acid binding pocket residues of Table 7 or 8, more preferably Table 8.
• These mutations can be introduced by site-directed mutagenesis e.g. using a Stratagene QuikChangeTM Site-Directed Mutagenesis Kit or cassette mutagenesis methods (Ausubel, F.M., Brent, R., guitarist, R.E. et al. editors.
• the invention provides a chimaeric protein having one or more binding pockets defined by the residues of Table 5 and preferably including some or all of the binding pocket residues of Tables 7 or 8.
• a chimaeric 3 A4 enzyme is produced which is isoformal with another enzyme of the 3A subfamily.
• 3A4 could be turned into a 3Al-like, 3A5-like, 3A7-like, 3A12-like or 3A43-like isoform with a few amino acid changes.
• 3A5, 3A7 and 3A43 isoforms Based on the information available from the literature on the structure/activity studies performed on the human 3A4, 3A5, 3A7 and 3A43 isoforms, and the analysis of the structure of the human 3A4, we postulate that the 3A4 protein could be converted to a 3A5-like, 3 A7-like or 3 A43-like isoform with the substrate specificities attributed to 3A5, 3 A7 or 3A43, 3A5 in particular based on the references above.
• the mutations can be introduced by site-directed mutagenesis or cassette mutagenesis methods, as described herein.
• the crystallization of such chimeras and the determination of the three-dimensional structures relies on the ability of our 3A4 protein to yield crystals that diffract at high resolution.
• the aim is to modify the inside part of 3A4 to produce a new substrate binding site of 3A5, 3A7 or 3A43 without modifying the outside shell of the proteins that allow the protein to crystallize.
• the invention also provides a means for homology modelling of other proteins (referred to below as target P450 proteins).
• target P450 proteins referred to below as target P450 proteins.
• homology modelling it is meant the prediction of related P450 structures based either on X-ray crystallographic data or computer-assisted de novo prediction of structure, based upon manipulation of the coordinate data derivable from the electron density map calculated from Table 3.
• Homology modelling extends to target P450 proteins which are analogues or homologues of the 3A4 protein whose structure has been determined in the accompanying examples. It also extends to P450 protein mutants of 3A4 protein itself.
• homologous regions describes amino acid residues in two sequences that are identical or have similar (e.g. aliphatic, aromatic, polar, negatively charged, or positively charged) side-chain chemical groups. Identical and similar residues in homologous regions are sometimes described as being respectively “invariant” and “conserved” by those skilled in the art.
• the method involves comparing the amino acid sequences of the 3A4 protein of SEQ ID 2 with a target P450 protein by aligning the amino acid sequences. Amino acids in the sequences are then compared and groups of amino acids that are homologous (conveniently referred to as "corresponding regions") are grouped together. This method detects conserved regions of the polypeptides and accounts for amino acid insertions or deletions.
• Homology between amino acid sequences can be determined using commercially available algorithms.
• the programs BLAST, gapped BLAST, BLASTN, PSI-BLAST and BLAST 2 are widely used in the art for this purpose, and can align homologous regions of two amino acid sequences. These may be used with default parameters to determine the degree of homology between the amino acid sequence of the SEQ ID 2 protein and other target P450 proteins which are to be modelled.
• Analogues are defined as proteins with similar three-dimensional structures and/or functions with little evidence of a common ancestor at a sequence level.
• Homologues are defined as proteins with evidence of a common ancestor, i.e. likely to be the result of evolutionary divergence and are divided into remote, medium and close sub-divisions based on the degree (usually expressed as a percentage) of sequence identity.
• a homologue is defined here as a protein with at least 15% sequence identity or which has at least one functional domain, which is characteristic of 3A4. This includes polymorphic forms of 3A4.
• orthologues are defined as homologous genes in different organisms, i.e. the genes share a common ancestor coincident with the speciation event that generated them.
• Paralogues are defined as homologous genes in the same organism derived from a gene/chromosome/genome duplication, i.e. the common ancestor of the genes occurred since the last speciation event.
• the homlogues could also be polymorphic forms of 3A4 such as alleles or mutants as described in section (A).
• the structures of the conserved amino acids in a computer representation of the polypeptide with known structure are transferred to the corresponding amino acids of the polypeptide whose structure is unknown.
• a tyrosine in the amino acid sequence of known structure may be replaced by a phenylalanine, the corresponding homologous amino acid in the amino acid sequence of unknown stracture.
• the structures of amino acids located in non-conserved regions may be assigned manually by using standard peptide geometries or by molecular simulation techniques, such as molecular dynamics.
• the final step in the process is accomplished by refining the entire stracture using molecular dynamics and/or energy minimization.
• Homology modelling as such is a technique that is well known to those skilled in the art (see e.g. Greer, Science, Vol. 228, (1985), 1055, and Blundell et al, Eur. J. Biochem, Vol. 172, (1988), 513).
• the techniques described in these references, as well as other homology modelling techniques, generally available in the art, may be used in performing the present invention.
• Homology modelling may be performed on a three dimensional atomic coordinate model of 3 A4 obtained using the present invention.
• a preferred model is that of Table 5.
• a person of skill in the art will be able to obtain a representation of the three dimensional stracture of a crystal of cytochrome P450 3A4 by a method which comprises providing the data of at least columns 1, 2, 3, 6 and 7 of Table 3 and constructing an electron density map of said data. This method is optionally performed by reference to the data of column 8 of said Table. Having obtained an electron density map, the person of skill in the art will be able to generate an initial model of 3A4 fitted to said map, which may then be refined by reference to the data of columns 4 and 5 of said Table. Refinement may also take place of other models generated from other 3A4 crystal structures.
• the refined data may then be used in a method which comprises calculating the three- dimensional coordinates of one or more atoms of 3 A4 in said crystal to provide a first three dimensional structure of 3A4.
• the positions of one or more atoms in said first stracture may be varied to provide a second structure with three-dimensional coordinates having a r.m.s.d of less than 2.0 A from said first structure, preferably less than 1.55 or 1.5 A, more preferably less than 1.0 A, and most preferably less than 0.5 A. This may be performed for a variety of reasons, for example in the light of other P450 models, or to manually fit regions of 3A4 structures which may need to be further optimised.
• the invention provides a method of homology modelling comprising the steps of:
• steps (a) to (c) are performed by computer modelling.
• the co-ordinate data obtained from the Table 3, e.g. that of Table 5 or selected coordinates thereof, will be particularly advantageous for homology modelling of other human P450 proteins, in particular human P450s such as 2C9, 2C19, 2D6, 3A5, 3A7, 1 Al, 1 A2, 2E1 preferably 3A5, 3A7 and 3A43.
• human P450s such as 2C9, 2C19, 2D6, 3A5, 3A7, 1 Al, 1 A2, 2E1 preferably 3A5, 3A7 and 3A43.
• These proteins may be the target P450 protein in the method of the invention described above.
• the aspects of the invention described herein which utilise the P450 stracture in silico may be equally applied to homologue models of P450 obtained by the above aspect of the invention, and this application forms a further aspect of the present invention.
• a conformation of a P450 by the method described above such a conformation may be used in a computer-based method of rational drug design as described herein.
• the electron density map of the human 3A4 P450 or the atomic coordinate data of 3A4 can also be used to solve the crystal structure of other target P450 proteins including other crystal forms of 3A4, mutants, co-complexes of 3A4, where X-ray diffraction data or NMR spectroscopic data of these target P450 proteins has been generated and requires interpretation in order to provide a structure.
• this protein may crystallize in more than one crystal form.
• Tables 3 or 5, or portions thereof, as provided by this invention are particularly useful to solve the structure of those other crystal forms of 3A4. It may also be used to solve the stracture of 3A4 mutants, 3A4 co-complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of 3A4.
• the present invention allows the structures of such targets to be obtained more readily where raw X-ray diffraction data is generated.
• the electron density map of P450 may be used to interpret that data to provide a likely stracture for the other P450 by techniques which are well known in the art, e.g. phasing in the case of X-ray crystallography and assisting peak assignments in NMR spectra.
• the unknown crystal structure whether it is another crystal form of 3A4, a 3A4 mutant, a 3A4 chimera or an 3A4 co-complex, or the crystal of a target P450 protein with amino acid sequence homology to any functional domain of 3A4, may be determined using the 3A4 structure coordinates derivable from Table 3 or the coordinates of Table 5 of this invention. Furthermore, the electron density map as defined in Table 3 can be used directly for this purpose. This method will provide an accurate stractural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.
• Examples of computer programs known in the art for performing molecular replacement are CNX (Brunger AX; Adams P.D.; Rice L.M., Current Opinion in Stractural Biology, Nolume 8, Issue 5, October 1998, Pages 606-611 (also commercially available from Accelrys San Diego, CA), MOLREP (A.Nagin, A.Teplyakov, MOLREP: an automated program for molecular replacement, J. Appl. Cryst. (1997) 30, 1022-1025, part of the CCP4 suite) or AMoRe ( ⁇ avaza, J. (1994). AMoRe: an automated package for molecular replacement. Acta Cryst. A50, 157- 163).
• a method for determining the stracture of a protein comprises; providing the coordinates obtained from the electron density map of Table 3, positioning the coordinates in the crystal unit cell of said protein so as to provide a stracture for said protein.
• the coordinates are those of Table 5 or selected coordinates thereof, which may include coordinates of atoms of the amino acid residues set out in Table 7 and more preferably in Table 8.
• a method for determining the structure of a protein comprises; providing the structure factor and phases of Table 3, positioning of a search model in the crystal unit cell of said protein so as to provide a stracture for said protein.
• the invention may also be used to assign peaks of ⁇ MR spectra of such proteins, by manipulation of the data of Tables 3 or 5.
• the co-ordinates are used to solve the stracture of target 3A4 particularly homologues of 3A4 for example P450s such as 3A5, 3A7 and 3A43.
• Table 3 allows for the calculation of an electron density map using the solvent flattened phases (column 7) and the weighted stracture factors (column 6).
• the data allows for the calculation of an electron density map using the solvent flattened phases (column 7), the Figure of Merit (column 8) and the observed stracture factor amplitudes (column 4).
• the phases provided in Table 3 can also be used to calculate a map with the Figure of Merit and a different structure factor amplitude from a same or related crystal form of 3A4, or a same or related crystal form of a homologous protein. All of these maps can be used for the phased molecular replacement of other homologous proteins, as discussed above in Section G, specifically 3 A4 homologues.
• aspects of the present invention therefore are, methods of using the phases of Table 3 (reciprocal space) for: a) calculating a map together with the solvent flattened structure factor amplitude (Table 3), or b) calculating a map together with the figure-of-merit and the measured structure factor amplitude (Table 3), or c) calculating a map together with the figure-of-merit (Table 3) and structure factor amplitudes from the same or related crystal form of 3A4 or a same or related crystal form of a 3A4 homologue, and d) use of any of these resulting electron densities (real space) from step a), b) or c) for molecular replacement.
• the map calculated from these structure factors and phases could be used in cross crystal form averaging between different crystals forms of CYP 3A4. If a different crystal form of 3A4 or a crystal form of a 3A4 homologue was obtained, the data of Table 3 can be used in cross crystal averaging, in reciprocal space, to improve the phases of either crystal form.
• Complexes can be crystallized and analysed, and difference Fourier electron density maps can be calculated based on X-ray diffraction patterns of soaked or co-crystallized 3 A4 and the structure factor and phases of Table 3. The difference Fourier electron density maps can then be analysed to determine whether and where a particular compound binds to 3 A4 and/or changes the conformation of 3 A4.
• a further aspect of the invention is therefore the use of the phases of Table 3 for calculating the difference Fourier map to identify whether a ligand has bound and its mode of binding:
• the present invention provides systems, particularly a computer system, the systems containing either (a) electron density map derivable from Table 3 or co-ordinate data therefrom, said data defining the three-dimensional structure of P450 or at least selected coordinates thereof; (b) structure factor data (where a stracture factor comprises the amplitude and phase of the diffracted wave) for 3A4, said stracture factor data being the data of Table 3; (c) atomic coordinate data of a target P450 protein generated by homology modelling of the target based on the coordinate data derivable from Table 3; (d) atomic coordinate data of a target P450 protein generated by interpreting X-ray crystallographic data or NMR data by reference to the electron density map according to Table 3 or co-ordinate data therefrom; or (e) structure factor data derivable from the atomic coordinate data of (c) or (d).
• the atomic coordinate data are the data of Table 5, or selected coordinates thereof.
• the computer system may comprise: (i) a computer-readable data storage medium comprising data storage material encoded with the computer-readable data; (ii) a working memory for storing instructions for processing said computer-readable data; and (iii) a central- processing unit coupled to said working memory and to said computer-readable data storage medium for processing said computer-readable data and thereby generating structures and/or performing rational drug design.
• the computer system may further comprise a display coupled to said central-processing unit for displaying said structures.
• the invention also provides such systems containing atomic coordinate data of target P450 proteins wherein such data has been generated according to the methods of the invention described herein based on the starting data provided by Table 3.
• data are those of Table 5 or selected coordinates thereof.
• Such data is useful for a number of purposes, including the generation of structures to analyse the mechanisms of action of P450 proteins and/or to perform rational drug design of compounds, which interact with P450, such as compounds, which are metabolised by P450s.
• the present invention provides computer readable media with at least one of (a) electron density map derivable from Table 3 or co-ordinate data therefrom, recorded thereon, said data defining the three-dimensional structure of P450, or at least selected coordinates thereof; (b) structure factor data for P450 recorded thereon, the stracture factor data of Table 3; (c) atomic coordinate data of a target P450 protein generated by homology modelling of the target based on the coordinate data derivable from Table 3; (d) atomic coordinate data of a target P450 protein generated by interpreting X-ray crystallographic data or NMR data by reference to the data of Table 3; or (e) stracture factor data derivable from the atomic coordinate data of (c) or (d).
• the atomic coordinate data may be that of Table 5, or selected coordinates thereof.
• the invention provides 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 (e.g. selected coordinates as defined herein) of the structure coordinates of P450 of Table 5, or a homologue of said P450, wherein said homologue comprises backbone atoms that have a root mean square deviation from the C ⁇ or backbone atoms (nitrogen-carbon ⁇ -carbon) of Table 5 of less than 2 A, preferably less than 1.55 or 1.5 A, more preferably less than 1.0 A, and most preferably less than 0.5 A.
• 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 (e.g. selected coordinates as defined herein) of the structure coordinates of P450 of Table 5, or a homologue of said P450, wherein said homologue comprises backbone atoms that have a root mean square deviation from the C ⁇ or backbone atoms (nitrogen-carbon
• computer readable media refers to any medium or media, which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
• RASMOL Single et al., TIBS, Vol. 20, (1995), 374
• TIBS TIBS, Vol. 20, (1995), 374
• RASMOL is a publicly available computer software package, which allows access and analysis of atomic coordinate data for structure determination and/or rational drag design.
• a computer system refers to the hardware means, software means and data storage means used to analyse the atomic coordinate data derived from Table 3 (e.g. that of Table 5 or selected coordinates thereof), as well as the electron density map of Table 3 of the present invention.
• the minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means and data storage means. Desirably a monitor is provided to visualize structure data.
• the data storage means may be RAM or means for accessing computer readable media of the invention. Examples of such systems are microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems running Unix based, Windows NT or IBM OS/2 operating systems.
• the invention provides 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 (e.g. selected coordinates as defined herein) of the structure coordinates of 3A4 obtainable from the data of Table 3 (such as that of Table 5 or selected coordinates thereof), or the electron density map of Table 3, or a homologue of 3A4, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms (nitrogen- carbon ⁇ -carbon) of co-ordinate data generated from Table 3 of not more than 2.0 A, preferably less than 1.55 or 1.5 A, more preferably less than 1.0 A, and most preferably less than 0.5 A.
• 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 (e.g. selected coordinates as defined herein) of the structure coordinates of 3A4 obtainable from the data of Table 3 (such as that of Table 5 or
• the invention also provides a computer-readable data storage medium comprising a data storage material encoded with a first set of computer-readable data comprising Table 3, Table 5 or selected coordinates thereof; which, 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 electron density corresponding to the second set of machine readable data.
• a further aspect of the invention provides a method of providing data for generating structures and/or performing rational drug redesign with 3 A4, 3A4 homologues or analogues, complexes of 3A4 with a compound, or complexes of 3A4 homologues or analogues with compounds, the method comprising:
• a remote device containing computer-readable data comprising at least one of: (a) ) electron density map derivable from Table 3 or co-ordinate data therefrom, said data defining the three-dimensional stracture of 3A4, at least one sub-domain of the three-dimensional structure of 3A4, or the coordinates of a plurality of atoms of 3A4; (b) structure factor data for 3A4, said stracture factor data of Table 3; (c) atomic coordinate data of a target 3A4 homologue or analogue generated by homology modelling of the target based on the coordinate data derivable from Table 3; (d) atomic coordinate data of a protein generated by interpreting X-ray crystallographic data or NMR data by reference to the data of Table 3; and (e) structure factor data derivable from the atomic coordinate data of (c) or (d); and (ii) receiving said computer-readable data from said remote device.
• the atomic coordinate data may be that of Table 5 or selected coordinates thereof.
• the remote device may comprise e.g. a computer system or computer readable media of one of the previous aspects of the invention.
• the device may be in a different country or jurisdiction from where the computer-readable data is received.
• the communication may be via the internet, intranet, e-mail etc, transmitted through wires or by wireless means such as by terrestrial radio or by satellite.
• the communication will be electronic in nature, but some or all of the communication pathway may be optical, for example, over optical fibers. J. Uses of the Structures of the Invention.
• the crystal structures obtained according to the present invention may be used in several ways for drag design. For example, many drags or drug candidates fail to be of clinical use due to the detrimental interactions with P450 proteins, resulting in a rapid clearance of the drags from the body.
• the present invention will allow those of skill in the art to attempt to rescue such compounds from development, by following the structure-based chemical strategies detailed below.
• the crystal structure could also be useful to understand drag-drug interactions. Many examples exist where adverse reactions to drags are recorded if administered while the patient is already taking other medicines.
• the mechanism behind this detrimental and often dangerous drag-drag interaction scenario may be when one drug behaves as an inhibitor of a P450 resulting in toxic levels of the other drag building-up due to less or no metabolism occurring.
• the crystal stracture of the present invention complexed to such an inhibitor may also allow rational modifications either to modify the inhibitor such that it no longer inhibits or inhibits less, or to modify the second drag such that it could bind better to the P450 (so becoming metabolised) and so displace the inhibitor.
• P450s display significant polymorphic variations dependent on the age, gender, or ethnic origin of the patient. This can manifest itself in adverse reactions from some segments of patient populations to some drugs.
• crystal structures of the present invention to map the relevant mutation with respect to the binding mode of the drug, chemical modifications could also be made to the drug to avoid interactions with the variable region of the protein. This could ensure more consistent therapeutic value from the drag for such segments of the population and avoid dangerous side effects.
• Some pharmaceutical compounds are converted by P450s into active metabolites.
• a greater understanding of how such compounds are converted by a P450 will allow modification of the compound so that it can be converted at a different rate. For example, increasing the rate of conversion may allow a more rapid delivery of a desired therapeutic effect, whereas decreasing the rate of conversion may allow for higher doses to be administered or the development of sustained release pharmaceutical preparations, for example comprising a mixture of compounds which are metabolized at different rates to form the same active metabolite.
• the determination of the three-dimensional stracture of P450 provides a basis for the design of new compounds, which interact with P450 in novel ways. For example, knowing the three-dimensional stracture of P450, computer modelling programs may be used to design different molecules expected to interact with possible or confirmed active sites, such as binding sites or other structural or functional features of P450.
• the structure of a compound bound to a P450 may be determined by experiment. This will provide a starting point in the analysis of the compound bound to P450, thus providing those of skill in the art with a detailed insight as to how that particular compound interacts with P450 and the mechanism by which it is metabolised.
• the invention provides a method for determining the stracture of a compound bound to P450, said method comprising: providing a crystal of P450 according to the invention; soaking the crystal with said compounds; and determining the stracture of said P450 compound complex by employing the coordinate data derivable from Table 3 (e.g. that of Table 5 or selected coordinates thereof), or by employing the phases of Table 3, or by employing the electron density derivable from Table 3.
• Table 3 e.g. that of Table 5 or selected coordinates thereof
• the P450 and compound may be co-crystallized.
• the invention provides a method for determining the stracture of a compound bound to P450, said method comprising; mixing the protein with the compound(s), crystallizing the protein-compound(s) complex; and determining the stracture of said P450-compound(s) complex by reference to the coordinate data derivable from Table 3 (e.g. that of Table 5 or selected coordinates thereof), or by reference to the phases of Table 3, or by reference to the electron density derivable from Table 3.
• the analysis of such structures may employ (i) X-ray crystallographic diffraction data from the complex and (ii) a three-dimensional stracture of P450, or at least selected coordinates thereof, to generate a difference Fourier electron density map of the complex, the three-dimensional structure being defined by atomic coordinate data derivable from Table 3 (e.g. that of Table 5 or selected coordinates thereof), or by employing the phases of Table 3, or by employing the electron density derivable from Table 3.
• the difference Fourier electron density map may then be analysed.
• Such complexes can be crystallized and analysed using X-ray diffraction methods, e.g. according to the approach described by Greer et al., J. of Medicinal Chemistry, Vol. 37, (1994), 1035-1054, and difference Fourier electron density maps can be calculated based on X- ray diffraction patterns of soaked or co-crystallized P450 and the solved stracture of uncomplexed P450. These maps can then be analysed e.g. to determine whether and where a particular compound binds to P450 and/or changes the conformation of P450.
• Electron density maps can be calculated using programs such as those from the CCP4 computing package (Collaborative Computational Project 4. The CCP4 Suite: Programs for Protein Crystallography, Acta Crystallographica, D50, (1994), 760-763.). For map visualization and model building programs such as "O” (Jones et al., Acta Crystallographica, A47, (1991), 110-119) can be used.
• 3 A4 mutants may be crystallized in co-complex with known 3A4 substrates or inhibitors or novel compounds.
• the crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of the 3A4 structure from Table 3 or Table 5 or selected coordinates thereof. Potential sites for modification within the various binding sites of the enzyme may thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between 3 A4 and a chemical entity or compound.
• allelic variants For example there are alleles of 3 A4, which differ from the native 3 A4 by only 1-2 amino acid substitutions, and yet individuals who express these allelic variants may exhibit very different drag metabolism profiles.
• Polymorphisms in the human CYP3A4 genes can influence the outcome of a treatment for a range of diseases including cancer.
• the metabolism of chemotherapeutic agents used in the treatment of cancer can be investigated using the stracture provided here and the agents then altered using the methods described herein. By generating such allelic proteins and determining the co-complex with compounds a greater understanding of allelic interactions with compounds may be developed.
• All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined against 1.5 to 3.5 A resolution X-ray data to an R value of about 0.30 or less using computer software, such as CNX (Brunger et al., Current Opinion in Structural Biology, Vol. 8, Issue 5, October 1998, 606-611, and commercially available from Accelrys, San Diego, CA), and as described by Blundell et al, (1976) and Methods in Enzymology, vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985).
• computer software such as CNX (Brunger et al., Current Opinion in Structural Biology, Vol. 8, Issue 5, October 1998, 606-611, and commercially available from Accelrys, San Diego, CA), and as described by Blundell et al, (1976) and Methods in Enzymology, vol. 114 & 115, H.
• This information may thus be used to optimise known classes of 3A4 substrates or inhibitors, and more importantly, to design and synthesize novel classes of 3A4 inhibitors and design drag with modified P450 metabolism.
• a particularly preferred aspect of the invention relates to in silico methods directed to the analysis and development of compounds which interact with P450 stractures of the present invention.
• Determination of the three-dimensional structure of 3A4 provides important information about the binding sites of 3A4, particularly when comparisons are made with similar enzymes. This information may then be used for rational design and modification of 3A4 substrates and inhibitors, e.g. by computational techniques which identify possible binding ligands for the binding sites, by enabling linked-fragment approaches to drug design, and by enabling the identification and location of bound ligands using X-ray crystallographic analysis. These techniques are discussed in more detail below.
• Proteins 2002, 47, 409-443 which require accurate information on the atomic coordinates of target receptors may be used.
• the aspects of the invention described herein which utilize the P450 stracture in silico may be equally applied to both the 3A4 stracture from the data of Table 3 (e.g. that of Table 5 or selected coordinates thereof) and the models of target P450 proteins obtained by other aspects of the invention.
• Table 3 e.g. that of Table 5 or selected coordinates thereof
• the models of target P450 proteins obtained by other aspects of the invention e.g. that of Table 5 or selected coordinates thereof
• such a conformation may be used in a computer-based method of rational drag design as described herein.
• the availability of the structure of the P450 3A4 will allow the generation of highly predictive pharmacophore models for virtual library screening or compound design.
• the invention provides a computer-based method for the analysis of the interaction of a molecular structure with a P450 stracture of the invention, which comprises: providing the structure of a P450 of the invention; providing a molecular stracture to be fitted to said P450 structure; and fitting the molecular stracture to the P450 structure.
• the P450 stracture of the invention may be that of Table 5, or selected coordinates thereof.
• the method of the invention may utilize the coordinates of atoms of interest of the P450 binding region, which are in the vicinity of a putative molecular stracture, for example within 10-25 A of the catalytic regions or within 5-10 A of a compound bound, in order to model the pocket in which the structure binds. These coordinates may be used to define a space, which is then analysed "in silico".
• the invention provides a computer-based method for the analysis of molecular structures which comprises: providing the coordinates of at least two atoms of a P450 structure of the invention ("selected coordinates"); providing the stracture of a molecular stracture to be fitted to said coordinates; and fitting the stracture to the selected coordinates of the P450.
• the structure of this P450 allows the identification of a number of particular sites which are likely to be involved in many of the interactions of P450 with a drug candidate.
• the residues are set out in Tables 7 and 8.
• the selected coordinates may comprise coordinates of some or all of these residues.
• the compound structure may be modelled in three dimensions using commercially available software for this purpose or, if its crystal structure is available, the coordinates of the stracture may be used to provide a representation of the compound for fitting to a P450 stracture of the invention.
• the binding pockets of cytochrome P450 molecules are of a size which can accommodate more than one ligand. Indeed, some drag-drug interactions may occur as a result of interaction of the compounds within the binding pocket of the same P450.
• the findings of the present invention may be used to examine or predict the interaction of two or more separate molecular stractures within the P450 3A4 binding pocket of the invention.
• the invention provides a computer-based method for the analysis of the interaction of two molecular stractures within a P450 binding pocket stracture, which comprises: providing the P450 stracture of Table 5 or selected coordinates thereof; providing a first molecular stracture; fitting the first molecular structure to said P450 structure; providing a second molecular stracture; and fitting the second molecular structure to a different part said P450 structure.
• the method of analysis further comprises providing a third molecular structure and also fitting that stracture to the P450 structure. Indeed, further molecular structures may be provided and fitted in the same way.
• one or more of the molecular stractures may be fitted to one or more of the phenylalanine residues of the 3A4 binding pocket mentioned above, and one or more of the other molecular stractures may be fitted to coordinates of amino acids from another part of the P450 binding pocket, such as another part of the ligand-binding region, to the haem-binding region, or to atoms of the amino acid residues of Tables 7 or 8.
• the one or more other molecular structures may be fitted, in addition to or instead of, to the haem stracture in the P450 binding pocket.
• a person of skill in the art may seek to use molecular modelling to determine to what extent the structures interact with each other (e.g. by hydrogen bonding, other non-covalent interactions, or by reaction to provide a covalent bond between parts of the stractures) or the interaction of one stracture with 3A4 is altered by the presence of another stracture.
• the person of skill in the art may use in silico modelling methods to alter one or more of the stractures in order to design new structures which interact in different ways with 3 A4, so as to speed up or slow down their metabolism, as the case may be.
• Newly designed stractures may be synthesised and their interaction with 3 A4 may be determined or predicted as to how the newly designed structure is metabolised by said P450 stracture. This process may be iterated so as to further alter the interaction between it and the 3A4.
• fitting it is meant determining by automatic, or semi-automatic means, interactions between at least one atom of a molecular stracture and at least one atom of a P450 stracture of the invention, and calculating the extent to which such an interaction is stable. Interactions include attraction and repulsion, brought about by charge, steric considerations and the like. Narious computer-based methods for fitting are described further herein.
• Computer programs can be employed to estimate the attraction, repulsion, and steric hindrance of the two binding partners (i.e. the P450 and a compound).
• a compound may be formed by linking the respective small compounds into a larger compound, which maintains the relative positions and orientations of the respective compounds at the active sites.
• the larger compound may be formed as a real molecule or by computer modelling. Detailed stractural information can then be obtained about the binding of the compound to P450, and in the light of this information adjustments can be made to the structure or functionality of the compound, e.g. to alter its interaction with P450. The above steps may be repeated and re- repeated as necessary.
• molecular stractures which may be fitted to the P450 structure of the invention, include compounds under development as potential pharmaceutical agents.
• the agents may be fitted in order to determine how the action of P450 modifies the agent and to provide a basis for modelling candidate agents, which are metabolised at a different rate by a P450.
• Molecular structures which may be used in the present invention, will usually be compounds under development for pharmaceutical use.
• such compounds will be organic molecules, which are typically from about 100 to 2000 Da, more preferably from about 100 to 1000 Da in molecular weight.
• Such compounds include peptides and derivatives thereof, steroids, anti-inflammatory drags, anti-cancer agents, anti-bacterial or antiviral agents, neurological agents and the like.
• any compound under development in the field of pharmacy can be used in the present invention in order to facilitate its development or to allow further rational drag design to improve its properties.
• the structure of the agent and its metabolite may both be modelled and compared to each other in order to better determine residues of P450 which interact with the agent.
• the present invention provides a process for predicting potential pharmaceutical compounds with a desired activity which are metabolised by P450 at a rate different from a starting compound having the same desired activity, which method comprises: fitting a starting compound to a P450 stracture of the invention or selected coordinates thereof; determining or predicting how said compound is metabolized by said P450 stracture; and modifying the compound structure so as to alter the interaction between it and the P450.
• Modification will be those conventional in the art known to the skilled medicinal chemist, and will include, for example, substitutions or removal of groups containing residues which interact with the amino acid side chain groups of a P450 stracture of the invention.
• the replacements may include the addition or removal of groups in order to decrease or increase the charge of a group in a test compound, the replacement of a charge group with a group of the opposite charge, or the replacement of a hydrophobic group with a hydrophilic group or vice versa. It will be understood that these are only examples of the type of substitutions considered by medicinal chemists in the development of new pharmaceutical compounds and other modifications may be made, depending upon the nature of the starting compound and its activity.
• the present invention also includes developing compounds which are metabolised more rapidly than a starting compound, for example where such a compound blocks metabolism of another drug.
• the invention further includes the step of synthesizing the modified compound and testing it in a in vivo or in vitro biological system in order to determine its activity and/or the rate at which it is metabolised.
• the above-described processes of the invention may be iterated in that the modified compound may itself be the basis for further compound design.
• the above-described processes may also be used to modify a compound which interacts with a second compound within the 3 A4 binding pocket.
• compounds which dock in the 3 A4 substrate binding pocket in a manner which includes pi:pi stacking interactions with a phenylalanine side chain may be modified in order to alter their metabolism.
• such interactions may be influential in determining the rate at which the compounds undergo metabolism via movement towards, and reaction with, the haem moiety, located in the haem binding region of the 3A4 binding pocket.
• altering i.e. increasing or decreasing
• the ligand-binding region may decrease their ability to move towards the haem-binding region.
• decreasing their affinity to the ligand-binding region may be desired to decrease their affinity to this region compared to the haem binding region and hence increase their ability to move towards the haem binding region.
• compound binding to the ligand-binding pocket is a necessary prerequisite of compound binding in the haem-binding region and its subsequent metabolism by or inhibition of 3A4
• elimination of binding to the ligand-binding region may eliminate all compound metabolism by 3A4 or inhibition of 3A4.
• An alternative or additional approach is to modify such substrates to increase or decrease their affinity for residues of the haem-binding region. Changes of this type may be introduced in order to increase or decrease the turnover of the substrates.
• Some molecules are known to be effectors or activators of 3A4 metabolism. Modification of the binding between 3A4 and such a compound would mediate metabolism of the substrate.
• the present invention provides a method for modifying the stracture 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 stracture so as to increase or decrease its interaction with the ligand-binding region; wherein said ligand-binding region is defined as including at least one of the P450 residues numbered as Phe57, Phel08, Phe213, Phe215, Phe219, Phe220, Phe241 and Phe304.
• the present invention provides a method for modifying the stracture 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 stracture so as to increase or decrease its interaction with the ligand-binding region; wherein said ligand-binding region is defined as including at least one of the P450 residues of Table 7 and preferably of Table 8.
• the invention provides 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 stracture so as to increase or decrease its interaction with the haem-binding region.
• the haem binding region also optionally includes the iron ion bound to the haem molecule, and if desired, one or more of the other atoms of the haem molecule itself. In a preferred aspect of the invention, the iron ion is also included in the haem-binding region.
• coordinates from at least two, preferably at least five, and more preferably at least ten amino acid residues of the P450 (including where desired the iron ion) will be used.
• crystal stractures of the invention will also allow the development of compounds which interact with the binding pocket regions of P450s (for example to act as inhibitors of a P450) based on a fragment linking or fragment growing approach.
• the binding of one or more molecular fragments can be determined in the protein binding pocket by X-ray crystallography.
• Molecular fragments are typically compounds with a molecular weight between 100 and 200 Da (Carr et al, 2002). This can then provide a starting point for medicinal chemistry to optimise the interactions using a structure-based approach.
• the fragments can be combined onto a template or used as the starting point for 'growing out' an inhibitor into other pockets of the protein (Blundell et al, 2002).
• the fragments can be positioned in the binding pocket of the P450 and then 'grown' to fill the space available, exploring the electrostatic, van der Waals or hydrogen-bonding interactions that are involved in molecular recognition.
• the potency of the original weakly binding fragment thus can be rapidly improved using iterative structure-based chemical synthesis.
• the compound may be synthesized and tested in a biological system for its activity. This can be used to guide the further growing out of the fragment.
• a linked fragment approach may be based upon attempting to link the two fragments directly, or growing one or both fragments in the manner described above in order to obtain a larger, linked structure, which may have the desired properties.
• binding site of two or more ligands may be connected to form a potential lead compound that can be further refined using e.g. the iterative technique of Greer et al.
• Greer et al. For a virtual linked-fragment approach see Nerlinde et al., J. oj ' omputer-Aided Molecular Design, 6, (1992), 131-147, and for NMR and X-ray approaches see Shuker et al., Science, 274, (1996), 1531-1534 and Stout et al., Structure, 6, (1998), 839-848.
• the use of these approaches to design P450 inhibitors is made possible by the determination of the P450 structure.
• the invention further includes the step of synthesizing the modified compound and testing it in an in vivo or in vitro biological system in order to determine its activity and/or the rate at which it is metabolised.
• the method comprises: (a) providing 3A4 under conditions where, in the absence of modulator, the 3A4 is able to metabolise known substrates; (b) providing the compound; and (c) determining the extent to which the compound is metabolised in the presence of 3 A4 or (d) determining the extent to which the compound inhibits metabolism of a known substrate of 3A4.
• the compound is contacted with P450 under conditions to determine its function.
• the compound in the contacting step above the compound is contacted with P450 in the presence of the compound, and typically a buffer and substrate, to determine the ability of said compound to inhibit P450 or to be metabolised by P450.
• the substrate may be e.g. dibenzylfluorescein. So, for example, an assay mixture for P450 may be produced which comprises the compound, substrate and buffer.
• the invention includes a compound, which is identified by the methods of the invention described above.
• a compound may be manufactured and/or used in the preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drag. These may be administered to individuals.
• the present invention extends in various aspects not only to a compound as provided by the invention, but also a pharmaceutical composition, medicament, drag or other composition comprising such a compound.
• the compositions may be used, for treatment (which may include preventative treatment) of disease such as cancer.
• Such a treatment may comprise administration of such a composition to a patient, e.g. for treatment of disease; the use of such an inhibitor in the manufacture of a composition for administration, e.g. for treatment of disease; and a method of making a pharmaceutical composition comprising admixing such an inhibitor with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
• a further aspect of the present invention provides a method for preparing a medicament, pharmaceutical composition or drag, the method comprising:
• optimisedising the structure we mean e.g. adding molecular scaffolding, adding or varying functional groups, or connecting the molecule with other molecules (e.g. using a fragment linking approach) such that the chemical stracture of the modulator molecule is changed while its original modulating functionality is maintained or enhanced.
• optimise is regularly undertaken during drug development programmes to e.g. enhance potency, promote pharmacological acceptability, increase chemical stability etc. of lead compounds.
• Modification will be those conventional in the art known to the skilled medicinal chemist, and will include, for example, substitutions or removal of groups containing residues which interact with the amino acid side chain groups of a P450 stracture of the invention.
• the replacements may include the addition or removal of groups in order to decrease or increase the charge of a group in a test compound, the replacement of a charge group with a group of the opposite charge, or the replacement of a hydrophobic group with a hydrophilic group or vice versa. It will be understood that these are only examples of the type of substitutions considered by medicinal chemists in the development of new pharmaceutical compounds and other modifications may be made, depending upon the nature of the starting compound and its activity.
• compositions may be formulated for any suitable route and means of administration.
• Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
• conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium carbonate, and the like may be used.
• Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc, an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
• the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
• auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
• the linear correlation coefficient, CC can be used to quantify the degree of similarity between two electron density maps.
• we provide a method for comparing two molecular stractures comprising the steps of: providing respective first and second electron density maps for the molecular stractures, transforming one or both of the maps so that the two maps are in maximum coincidence with each other, and quantifying the degree of correlation between the coinciding maps.
• the degree of correlation is quantified by calculating the CC for the coinciding maps.
• a mask may be applied to the maps before the quantification step to prevent e.g. solvent molecules from contributing to the degree of correlation.
• Either or both of the electron density maps may be determined experimentally, e.g. by X-ray crystallographic analysis. Alternatively or additionally, either or both may be calculated e.g. from atomic coordinate data.
• the use of the CC has been tested for three stractural families (i.e. three different molecular types). Within each family a number of different sets of atomic coordinates were provided. Each set varied from the other sets by an r.m.s.d. of up to about 1.8 A. Electron density maps were computed for each atomic coordinate set. The aim was to confirm that the CC determined for each pair of maps correlated with the r.m.s.d. value for the corresponding pair of atomic coordinate sets (both within and across families). A number of CCP4 (Collaborative Computational Project 4. The CCP4 Suite: Programs for Protein Crystallography, Acta Crystallographica, D50, (1994), 760-763.), Unix and specially developed programs were used to perform the test.
• Annexes 5 and 6 provided respective subroutines used by the programs of Annexes 1 to 4.
• the first step was to compute, for each set of atomic coordinates, the asymmetric unit of the electron density map on a relatively fine grid (e.g. l/6 th of the minimum d-spacing). This was accomplished with weighted 2F 0 -F C coefficients using the CCP4 FFT program.
• the atomic coordinates of the molecule were extracted from the complete coordinate set using Unix GREP.
• Astex-EXTENDC see Annex 3
• the electron density map was then extended to cover the molecule thus extracted, including a minimum 3 A border to ensure that no parts of electron density for any atom of the molecule were unintentionally excluded.
• the extracted atomic coordinates were also used to generate a molecular mask with the CCP4 NCSMASK program.
• a molecular mask is a 3D array of grid points wherein each grid point covered by the molecule is labelled T and each grid point outside the molecule is labelled '0'.
• the coverage of the molecule was determined by 2 A radius spheres centred on each atomic position.
• each set of atomic coordinates was superposed onto a common reference and the rigid-body transformation was determined for r.m.s.d. minimisation between each pair of molecules.
• Each transformation was then applied, using Astex-ROTMAP (Annex 1), to the corresponding pair of electron density maps and associated masks, interpolating maps and masks onto a common unit cell and grid (e.g. at l A of the minimum d-spacing).
• the masks were interpolated linearly, whereas the electron densities were interpolated using quadratic functions.
• CCs for the pairs of transformed and interpolated electron density maps were calculated using Astex-DENCOR (Annex 2).
• the transformed and interpolated masks were used to ensure that only electron densities covered by the molecules contributed to the CCs.
• 3A4 corresponding to Ml 8907 was cloned from human liver library (Origene Technologies, Inc.).
• Primer 1 is complementary to the 5' end of the full length 3A4 cDNA.
• Primer 2 is complementary to the 3' end of the cDNA and adds a four histidine tag onto the C-terminus of the 3A4 protein.
• the expression vector pCWOri+ provided by Prof. F. W. Dahlquist, University of Oregon, Eugene, Oregon, USA, was used to express the truncated human cytochrome P450 in the E. coli strain XLl Blue (Stratagene). Full-length cDNA encoding cytochrome P450 3A4 isolated above was used as a template for PCR amplification, engineering the 5' terminus and insertion of a four Histidine tag at the C-terminus.
• N-terminal truncation of 3 A4 was carried out by PCR as outlined below, to generate the published NF10 N-terminal truncation described by Gillam (Gillam et al, Arch. Biochem. Biophys. Vol. 305, 123-131, 1993).
• l ⁇ l of product was used in a TOPO cloning reaction (vector pCR4TOPO, Invitrogen).
• the cloning reaction was used to transform E. coli XLl -blue and positive clones identified by Ndel/Sall restriction digestion of purified plasmids. Positive clones were sequenced fully and the Ndel/Sall insert subcloned into pCWori+ to yield clone p3 A4. This clone was used for protein expression.
• a single ampicillin resistant colony of XLl blue cells was grown overnight at 37 ° C in Terrific Broth (TB) with shaking to near saturation and used to inoculate fresh TB media.
• the haem precursor delta aminolevulinic acid (80 mg/1) was added 30 min prior to induction with 1 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) and the temperature lowered to 25 C.
• IPTG isopropyl- ⁇ -D-thiogalactopyranoside
• the cells were pelleted at 10000 g for 10 min and resuspended in a buffer containing 500 mM KPi, pH 7.4, 20 % glycerol (v/v), 10 mM mercaptoethanol, 0.1 % (v/v) of protease inhibitor cocktail 3 (Calbiochem), 10 mM imidazole, 40U/ml DNase 1 and 5 mM MgSO 4 .
• Detergent IGEPAL CA630 (Sigma) was added dropwise from a 10% stock solution to the lysate at a final concentration of 0.3% (v/v) and the lysate was incubated with previously washed NiNTA resin (Qiagen) overnight at 4 C, using agitation. The protein bound-NiNTA resin was pelleted by centrifugation at 2000 g for 5 min at 4 °C.
• the resin was washed with 20 resin volumes of 500 mM KPi, pH 7.4, 20% glycerol, 10 mM mercaptoethanol, 10 mM imidazole, 0.1% (v/v) of protease inhibitor cocktail, 0.3%(v/v) IGEPAL CA630 and the resin pelleted by centrifugation at 2000 g for 5 min at 4 ° C.
• the resin was then washed with 10 resin volumes of 500 mM KPi, pH 7.4, 20% glycerol, 10 mM mercaptoethanol, 20 mM imidazole, 0.1% (v/v) protease inhibitors, 0.3% IGEPAL CA630 and the resin recovered by centrifugation as described above.
• the resin was packed into a column at room temperature and the cytochrome P450 eluted with cold 500 mM KPi, pH 7.4, 20 % glycerol, 10 mM mercaptoethanol, 300 mM imidazole, 0.1% (v/v) of protease inhibitor cocktail, 0.3%(v/v) IGEPAL CA630.
• the cytochrome P450 obtained from the NiNTA column was quickly desalted into 20 mM KPi, pH 7.2, 20% glycerol, 2.0 mM DTT, 1 mM EDTA using a HiPrep 26/10 desalting column (Pharmacia), at a flow rate of 5 ml/min on a Akta FPLC system (Pharmacia).
• a watch UN command (280 nm) of greater than 750 mAu was then used to divert the desalted P450 from the HiPrep 26/10 desalting column onto a CM Sepharose column (Pharmacia), previously equilibrated with 20 mM KPi, pH 7.2, 20% glycerol, 2.0 mM DTT, 1 mM EDTA for final purification.
• the peak divert was ended when the mAu fell below 750mAu.
• CM Sepharose column wash with 10 column volumes of 20 mM KPi, pH 7.2, 20% glycerol, 2.0 mM DTT, 1 mM EDTA, followed by a wash with 6 column volumes with the above buffer with 75 mM KCl added in order to remove any trace of detergent, then eluted with the above buffer with KCl concentration increased to 500 mM.
• the protein was concentrated up to 40 mg/ml using a microconcentrator for crystallization trials.
• Crystals of the 3A4 were grown using the hanging drop vapour diffusion method. Protein at 37.4 mg/ml in 20 mM Kpi pH 7.2, 0.5 M KCl, 2mM DTT, ImM EDTA, 20% glycerol, was mixed in a 1 : 1 ratio, using 0.5ul drops, with a reservoir solution. The crystals of 3 A4 grew over a reservoir solution containing 0.15 M HEPES pH 7.5, 2.5% IP A, 10% PEG 4000.
• Crystals formed within 1-7 days at 25 °C, and were rod shaped in morphology.
• the crystals were flash frozen in liquid nitrogen, using crystallisation solution supplemented with 15% glycerol as a cryoprotectant.
• a native dataset was collected at the ESRF beamline 14.2 to a resolution of 2.7 A, from a crystal produced using the protocol above in Protein purification (1) and Crystallisation (1).
• the space group was 1222.
• the cells were pelleted at 10000 g for 10 min and resuspended in a buffer containing 500 mM KPi, pH 7.4, 20 % glycerol, 10 mM mercaptoethanol, 0.1% (v/v) of protease inhibitor cocktail 3 (Calbiochem), 10 mM imidazole, 40U/ml DNase 1 and 5 mM MgSO . Passing twice through a Constant Systems Cell Homogeniser at 10000 psi lysed the cells. The cell debris was then removed by centrifugation at 22000 g at 4 °C for 30 min.
• Detergent IGEPAL CA630 (Sigma) was added dropwise from a 10% stock solution to the lysate at a final concentration of 0.3% (v/v) and the lysate was incubated with previously washed NiNTA resin (Qiagen) overnight at 4 °C, using agitation. The protein bound-NiNTA resin was pelleted by centrifugation at 2000 g for 5 min at 4 °C.
• the resin was washed with 20 resin volumes of 500 mM KPi, pH 7.4, 20% glycerol, 10 mM mercaptoethanol, 10 mM imidazole, 0.1% (v/v) of protease inhibitor cocktail, 0.3%(v/v) IGEPAL CA630 and the resin pelleted by centrifugation at 2000 g for 5 min at 4 °C.
• the resin was then washed with 10 resin volumes of 500 mM KPi, pH 7.4, 20% glycerol, 10 mM mercaptoethanol, 20 mM imidazole, 0.1% (v/v) protease inhibitors, 0.3% IGEPAL CA630 and the resin recovered by centrifugation as described above.
• the resin was packed into a column at room temperature and the cytochrome P450 eluted with cold 500 mM KPi, pH 7.4, 20 % glycerol, 10 mM mercaptoethanol, 300 mM imidazole, 0.1% (v/v) of protease inhibitor cocktail, 0.3%(v/v) IGEPAL CA630.
• the cytochrome P450 obtained from the NiNTA column was quickly desalted into 10 mM KPi, pH 7.2, 20% glycerol, 2.0 mM DTT, 1 mM EDTA, lOmM K 2 SO 4 using a HiPrep 26/10 desalting column (Pharmacia), at a flow rate of 5 ml/min.
• CM Sepharose column (Pharmacia) previously equilibrated with 10 mM KPi, pH 7.2, 20% glycerol, 2.0 mM DTT, 1 M EDTA, lOmM K 2 SO 4 .
• the following step elution was applied: wash with 20 column volumes of 10 mM KPi, pH 7.2, 20% glycerol, 2.0 mM DTT, 1 mM EDTA, lOmM K 2 SO 4 followed by a wash with 20 column volumes of the above buffer with 75 mM KCl in order to remove any trace of detergent, then eluted with the above buffer with KCl concentration increased to 500 mM.
• the protein was concentrated up to 20 mg/ml using a microconcentrator for crystallization assays.
• Crystals of the 3A4 were grown using the hanging drop vapour diffusion method. Protein at 18.5 mg/ml in 10 mM Kpi pH 7.2, 0.5 M KCl, 2 mM DTT, 1 mM EDTA, 20% glycerol, 10 mM K2SO4 was mixed in a 1:1 ratio, using 0.5ul drops, with a reservoir solution. The crystals of 3A4 grew over a reservoir solution containing 0.1 M HEPES pH 7.2, 5% IPA, 10% PEG 4000. The crystal was frozen using the crystallization solution supplemented by glycerol to 33%.
• a native dataset was collected at the ESRF beamline 14.2 to a resolution of 2.8 A, from a crystal produced using the protocol above in Protein purification (2) and Crystallisation (2).
• the space group was 1222.
• the cells were pelleted at 10000 g for 10 min and resuspended in a buffer containing 500 mM KPi, pH 7.4, 20 % glycerol, 10 mM mercaptoethanol, 0.1% (v/v) of protease inhibitor cocktail 3 (Calbiochem), 10 mM imidazole, 40U/ml DNase 1 and 5 mM MgSO .
• Detergent IGEPAL CA630 (Sigma) was added dropwise from a 10% stock solution to the lysate at a final concentration of 0.3% (v/v) and the lysate was incubated with previously washed NiNTA resin (Qiagen) overnight at 4 °C, using agitation. The protein bound-NiNTA resin was pelleted by centrifugation, 2000 g for 5 min at 4 °C.
• the resin was washed with 20 resin volumes of 500 mM KPi, pH 7.4, 20% glycerol, 10 mM mercaptoethanol, 10 mM imidazole, 0.1% (v/v) of protease inhibitor cocktail, 0.3%(v/v) IGEPAL CA630 and the resin pelleted by centrifugation at 2000 g for 5 min at 4 °C.
• the resin was then washed with 10 resin volumes of 500 mM KPi, pH 7.4, 20% glycerol, 10 mM mercaptoethanol, 20 mM imidazole, 0.1% (v/v) protease inhibitors, 0.3% IGEPAL CA630 and the resin recovered by centrifugation as described above.
• the resin was packed into a column at room temperature and the cytochrome P450 eluted with cold 500 mM KPi, pH 7.4, 20 % glycerol, 10 mM mercaptoethanol, 300 mM imidazole, 0.1% (v/v) of protease inl ibitor cocktail, 0.3%(v/v) IGEPAL CA630.
• the cytochrome P450 obtained from the NiNTA column was quickly desalted into 10 mM KPi, pH 7.2, 20% glycerol, 2.0 mM DTT, 1 mM EDTA using a HiPrep 26/10 desalting column (Pharmacia), at a flow rate of 5 ml/min.
• CM Sepharose column Pharmacia
• the desalted cytochrome P450 was directly applied to a CM Sepharose column (Pharmacia) previously equilibrated with 10 mM KPi, pH 7.2, 20% glycerol, 2.0 mM DTT, 1 mM EDTA.
• the following step elution was applied: wash with 20 column volumes of 10 mM KPi, pH 7.2, 20%) glycerol, 2.0 mM DTT, 1 mM EDTA, followed by a wash with 20 column volumes of the above buffer with 75 mM KCl in order to remove any trace of detergent, then eluted with the above buffer with KCl concentration increased to 500 mM.
• the concentrated sample (200 ⁇ L, 7.9 mg protein) was then gel filtered using a Superdex 200 HR10/30 column (Pharmacia) in 10 mM KPi, pH7.2, 20% glycerol, 1 mM EDTA, 2 mM DTT, 500 mM KCl at a flow rate of 0.4 ml/min. Fractions of 0.5 ml were collected.
• 3A4 can alternatively be purified by gel filtration chromatography, by passage down a 26/60 Superdex 200 column equilibrated in lOmM K Pi pH 7.2, 20% glycerol, 0.5M KCl, 2mM DTT ran at 1.5mg/ml, to improve homogeneity for crystallisation.
• Crystals of the 3A4 were grown using the hanging drop vapour diffusion method. Protein at 36 mg/ml in 10 mM Kpi pH 7.2, 0.5 M KCl, 2 mM DTT, 1 mM EDTA, 20% glycerol, was mixed in a 1:1 ratio, using 0.5 ⁇ l drops, with a reservoir solution. The crystals of 3A4 grew over a reservoir solution containing 0.1 M HEPES pH 7.5, 0.025 M sodium chloride, 7.5% IP A, 10% PEG 4000.
• the crystals formed over a number of days at 25°C, and were rod shaped in morphology.
• the crystals were transferred to a cryo-solution consisting of 0.1 M HEPES pH 7.5, 0.25 M KCl, 15% PEG 4000 and 20% glycerol and then frozen in liquid nitrogen prior to data collection.
• the space group was 1222.
• Table 1 below contains the data statistics for the peak wavelength data. Table 1 : Data statistics
• Ranom Sum
• the location of the iron atom within the unit cell was determined by visual inspection of the three Harker sections of the anomalous difference Patterson map calculated using the peak anomalous data by the program FFT (part of the CCP4 suite).
• These refined parameters were obtained using the program SHARP, by refinement against the experimental data obtained from the crystal (columns 4 and 5 of Table 3). These atom parameters were then used within SHARP to generate phases for 3 A4. These phases can then be modified by density modification procedures.
• the phases from SHARP were solvent flattened using SOLOMON/DM as available through the SHARP program. The resulting solvent flattened structure factor amplitudes and phases are given in columns 6 and 7 of Table 3.
• phase set I The resulting phases (phase set I) were used in phased molecular replacement as implemented in MOLREP (A.Vagin, A.Teplyakov, J. Appl. Cryst. (1997) 30, 1022-1025, part of the CCP4 suite) and using 2C5 with the haem excluded (pdbent 1DT6) as a search model together with the sequence of SEQ ID 2. This gave an unambiguous solution where the haem moiety was consistent with the iron position obtained through inspection of the Harker sections.
• model- A The oriented and positioned model (based on 1DT6 and the sequence of SEQ ID 2), model- A, was used together with the phase set II phases in density modification as implement in SOLOMON (Abrahams J. P. and Leslie A. G. W., Acta Crystallographica D52, (1996), 30-42) through the SHARP program package.
• Table 2 contains the phasing statistics in resolution bins. The columns are:
• phase set I SHARP figure-of-merit for acentric reflections
• phase set I SHARP figure-of-merit for centric reflections
• Mass spectrometry was performed using a Bruker BioTOF II electrospray time of flight instrument. Samples were either diluted by a factor of 1000 straight from storage buffer into methanol/water/formic acid (50:48:2 v/v/v), or subjected to a reverse phase separation using a C4 Millipore 'zip-tip' or a C4 HPLC column, before being diluted into methanol/water/formic acid.
• Reaction mixtures were composed of 300 nM of 3A4 enzyme incubated with 2 units/ml purified human oxidoreductase, 2.8 ⁇ M dibenzylfluorescein and a regeneration system composed of 140 ⁇ M NADP + , 400 ⁇ M glucose-6-phosphate and 2.8 units/ml glucose-6-phosphate dehydrogenase in 100 mM potassium phosphate pH 7.8, 1 mM MgCl 2 .
• the electron density map described by Table 3 allowed a model of 3A4 to be built using the graphical program O (Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard (1991) Acta Cryst. A47, 110-119).
• This model was then refined to 2.8A resolution against the peak wavelength dataset from the iron MAD experiment (statistics of the data given in Table 1) using the programs CNX (Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse- Kunststoffleve, R. W., Jiang, J.
• CALL MAPHEAD (2, FN ( : L) // ' .map' , A, IP2, LUVW2, MUVW2, XYZ2, S, MM, CCD2, &RHOA, RHOS) IF (MM. E.2)
• CALL CCPERR(1, 'Map mode must be 2.')
• CA(I) COS(DR*AV(I) )
• SA(I) SIN(DR*AV(I) )
• ENDDO c c #### Get orthogonal rotation matrix.
• RMO(l,2) -CA(l)*CA(2)*SA(3)-SA(l)*CA(3)
• RMO (2,1) SA ( 1 ) *CA ( 2 ) *CA ( 3 ) +CA ( 1 ) *SA ( 3 )
• RMO (2 , 2 ) -SA ( 1 ) *CA ( 2 ) *SA ( 3) +CA ( 1 ) *CA ( 3 )
• RMG1 (I, J) RMG1 (I, J) +RM0 (I, K) *OMGD (K, J) ENDDO
• RMG1 ( I , J) RMG1 ( I , J) /CCG (I ) ENDDO
• TVG1 ( I ) TVO ( I ) /CCG ( I ) ENDDO c c#### Allocate memory for input maps & read in maps .
• LALC(1) (MU-LU+D* (MV-LV+1)
• LALC(2) LALC(1)
• CALL CCPALC GETDEN, 2 , TALC, LALC
• NC NC*NXYZ(I)
• VC VC*CCD(I)
• TVG2(I) 0.
• DO J l,3
• TVG2 ( I ) TVG2 ( I ) -RMG2 (I, J) *TVG1 (J) ENDDO
• LALC (3 ) LALC ( 1 ) * (MW-LW+1 )
• LALC(2) (LALC(3)+l)/2
• LALC (4 ) NXYZ (3 ) *NXYZ ( 1 )
• UVW (I) UVW (I) +RMG2 (I, J) *IXYZ (J) ENDDO
• IUVWO(I) NINT(UVW(I) )
• UVW (I) UVW (I ) -IUVWO (I )
• UVWA(I) ABS(UVW(I) )
• IUVW1(I) IUVW0(I)+INT(SIGN(1.,UVW(I) ) ) ENDDO C C#### Interpolate the mask.
• R0 IRHOR (IUO, IVO, IWO) +UVWA( 1) * ( IRHOR (IU1, IVO, IWO) - & IRHOR(IU0,IV0,IW0) )
• Rl IRHOR ( IUO, IVO, IWl ) +UVWA( 1) * (IRHOR (IU1, IVO, IWl) - & IRHOR(IU0,IV0,IWl) )
• RHOW (IZ, IX) QINT3D (LU, MU, LV, MV, LW, MW, RHOR, IUVWO, UVW) - & RHOO
• Last line of standard output is the correlation coefficient, C#### expressed as a percentage.
• LALC (1) (MU-LU+1) * (MV-LV+1)
• LALC (2) LALC ( 1)
• MUVW (3 ) LUVW ( 3) +NW-1
• the asymmetric unit of the map represents the true population of electron density values in the statistical sense and therefore the values of the mean and RMS deviation for the asymmetric unit can be considered to be those of the true population mean and population standard deviation respectively.
• Any other subset or superset of the map that is not an integral number of asymmetric units is a sample in the statistical sense, with corresponding sample mean and sample standard deviation. It may even be a biased sample because when a map is extended, density values related by the map symmetry are very likely to be generated more than once. Sample statistics, whether biased or not, are always only approximations to population statistics.
• RSF(2,NSF(NS) , S) RSF (1,NSF (NS) ,NS) ELSE
• NAP(IS) NAP(IS)+1
• ANP(NAP(IS) ,IS) A(14:27)
• INRP(NAP(IS) ,IS) NRP(IS)
• LR1 .TRUE.
• LR2 . FALSE.
• C & 'ERROR Residues have no common atoms: ' //RNP (KRF(IRF, 1) , 1) // C & ' '//RNP(KRF(IRF,2) ,2) ) ENDDO IF (NAF.LE.3) THEN
• D1 XAP (I, IA1, 1) -XC (1,1)
• D2 XAP (I, IA2, 2) -XC (1,2)
• XD(I) D1-D2
• XS(I) D1+D2 ENDDO
• DO IAF 1,NAF
• IA1 KAF(IAF, 1)
• IA2 KAF (IAF, 2 )
• D 0.
• DO 1 1,3
• NCM MAX(NCM,NCP(IA2) )
• DO IA2 1,NAP(2)
• IA1 KAP(IA2,IW) IF (lAl.GT.O) THEN
• DO 1 1,3
• LA(I) . FALSE. ENDDO ENDIF ENDDO C C IF (IT.GT.l) WRITE(6, ' () ')

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