WO2001021645A2 - Cible de therapie antivirale - Google Patents

Cible de therapie antivirale Download PDF

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Publication number
WO2001021645A2
WO2001021645A2 PCT/GB2000/003568 GB0003568W WO0121645A2 WO 2001021645 A2 WO2001021645 A2 WO 2001021645A2 GB 0003568 W GB0003568 W GB 0003568W WO 0121645 A2 WO0121645 A2 WO 0121645A2
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fic
atom
e2nt
molecular complex
molecule
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PCT/GB2000/003568
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WO2001021645A3 (fr
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Alfred Antson
Norman Maitland
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The University Of York
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Priority to AU73008/00A priority Critical patent/AU7300800A/en
Priority to EP00960836A priority patent/EP1409524A2/fr
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Publication of WO2001021645A3 publication Critical patent/WO2001021645A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention provides a crystallised module of a nuclear phosphoprotein and an assay and method for determining interactions with human papillomavirus E2 for u: ⁇ ?e in drug design, for use particularly but not exclusively in designing antiviral agents with potential use in treating warts, proliferative skin lesions and carcinoma of the cervix.
  • HPVs Human papillomaviruses
  • HPVs have evolved a sophisticated system of control, mediated by protein:DNA and protei protein interactions, that involves both cellular and viral proteins.
  • the 45 kDalton nuclear phosphoprotein, E2 has two central roles in this control. It acts as the principal virally encoded transcription factor and, in association with the viral El protein, it creates the molecular complex at the origin of the viral DNA replication .
  • E2 has three distinct modules.
  • the N-terminal module (E2NT) of about 200 amino acids is responsible for interactions with viral and host cell transcription factors. It is followed by a flexible, proline-rich, linker module and a C-terminal module (E2CT), each of about 100 amino acids 3 (Fig. la).
  • the E2CT binds as a homodimer to DNA sites with a consensus sequence of ACCGN 4 CGGT 4 .
  • a long upstream regulatory region (UR ) precedes the viral genes and contains four spatially conserved E2 binding sites: three sites proximal to the transcription start site (p97 in HPV 16) and one approximately 500bp upstream.
  • the dimer of E2CT serves to anchor E2 protein to its recognition sites on the DNA, the function of the E2NT is to bind and localise at least three cellular transcription factors, Spl, TFIIB and AMF-1, to the transcription initiation complex.
  • E2 interacts with another viral protein, El, which has ATPase and helicase activities. El itself binds to the viral origin of replication which consists of about 100 bp and is surrounded by the three E2-binding sites, proximal to the transcription start.
  • the E2:E1 interaction greatly increases the rate of HPV genome replication 2,5,6 , Fig. la.
  • E2 is essential for the normal productive (wart) life cycle of HPV, however during malignant progression HPV DNA is integrated into the host cell genome, which usually results in disruption of the E2/E1 ORFs and loss of E2 protein, in turn leading to dysregulated expression of the viral oncogenes E6 and E7 7 .
  • E2 has been shown to direct the formation of loops in DNA containing E2 binding sites .
  • the loops were only formed with intact E2, and not with the E2CT alone.
  • the E2 binding sites did not function independently and their co-operative effect was mediated by full length E2, leading the authors to suggest that there were specific interactions mediated by E2 that bridged across the set of DNA binding sites through its N-terminal.
  • a similar DNA loop structure could also be achieved with Spl, a cellular transcription factor, which forms a complex with distally bound E2 9 ; Spl/E2 interactions are critical for transcription activation in BPV 10 .
  • dimerisation of E2NT plays an important and key role in induction of DNA loop formation, the mechanism by which distally bound transcription factors would be brought close to the site of transcription initiation. More importantly, our results raise the possibility that dimer formation serves as a molecular switch between early gene expression and viral genome replication during HPV infection.
  • the Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure.
  • the procedure used in Molecular Similarity to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalences in these structures; 3) perform a fitting operation; and 4) analyze the results.
  • Each structure is identified by a name.
  • One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures).
  • the working structure is translated and rotated to obtain an optimum fit with the target structure.
  • the fitting operation uses a least squares fitting algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
  • the screening process may begin by visual inspection of the target on the computer screen, generated from a machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within that binding pocket as defined supra. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER.
  • Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include:
  • MCSS (A. Miranker et al., "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method.” Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available from Molecular Simulations, Burlington, Mass.
  • AUTODOCK (D. S. Goodsell et al., "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins: Structure, Function, and Genetics, 8, pp. 195-202 (1990)).
  • AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.
  • DOCK (I. D. Kuntz et al., "A Geometric Approach to Macromolecule-Ligand Interactions", J. Mol. Biol., 161, pp. 269-288 (1982)). DOCK is available from University of California, San Francisco, Calif.
  • CAVEAT P. A. Bartlett et al, "CAVEAT: A Program to Facilitate the Structure- Derived Design of Biologically Active Molecules". In Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, pp. 182- 196 (1989)). CAVEAT is available from the University of California, Berkeley, Calif.
  • 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif). This area is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2145-2154 (1992).
  • inhibitory or other target-binding compounds may be designed as a whole or de novo.
  • LUDI H.-J. Bohm, "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp. 61-78 (1992)).
  • LUDI is available from Biosym Technologies, San Diego, Calif.
  • LEGEND (Y. Nishibata et al., Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from Molecular Simulations, Burlington, Mass.
  • an effective ligand will preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding).
  • the most efficient ligands should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, preferably, not greater than 7 kcal/mole.
  • Ligands may interact with the target in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free entity and the average energy of the conformations observed when the inhibitor binds to the protein.
  • An entity designed or selected as binding to a target may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme.
  • Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions.
  • the sum of all electrostatic interactions between the inhibitor or other ligand and the target, when the inhibitor is bound to the target preferably make a neutral or favourable contribution to the enthalpy of binding.
  • substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties.
  • initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided.
  • substituted chemical compounds may then be analyzed for efficiency of fit to a calcineurin-like binding pocket by the same computer methods described in detail, above. Again, all these facts are familiar to the skilled person.
  • E2 N-terminal module E2NT dimer protein or homologue thereof, for use in rationalised drug design.
  • the dimer comprises residues vital for transcriptional and replicational activities of said protein lying on opposite sides of an N-terminal domain, for use in rationalised drug design.
  • E2NT dimer protein is substantially as depicted in any of Figures 2c and/or 3a-d.
  • an in vitro method for identifying and/or selecting a candidate therapeutic agent comprising determining interaction of a E2 N-terminal module (E2NT) dimer in a sample by contacting said sample with said candidate therapeutic agent and measuring DNA loop formation.
  • E2NT E2 N-terminal module
  • the method is for use in identifying and/or selecting an antiviral candidate therapeutic agent.
  • the candidate therapeutic agent interferes or blocks interactions of E2NT so as to interfere or block viral and/or cellular transcription factors.
  • an E2NT dimerisation inhibitor in the preparation of a medicament for use in treating warts, proliferative skin lesions and/or cervical cancer.
  • a method of monitoring the efficacy of an antiviral therapy in a patient receiving a medicament for the treatment of warts, proliferative skin lesions and/or cervical cancer comprising taking a sample from said patient and measuring E2NT interactions and/or DNA loop formation.
  • a patient can be monitored at the start of therapy to test its effectiveness.
  • a patient can be monitored once a therapy has been established so as to monitor its efficacy with a view to altering a therapy if found to be unsatisfactory.
  • the human papillomavirus E2 protein controls the primary transcription and replication of the viral genome. Both activities are governed by a ⁇ 200 amino acid N-terminal module (E2NT) which is connected to a DNA binding C-terminal module by a flexible linker.
  • E2NT ⁇ 200 amino acid N-terminal module
  • the crystal structure of the E2NT module from high-risk type 16 human papillomavirus reveals an L-shaped molecule with two closely packed domains, each with a novel fold. It forms a dimer in the crystal and in solution.
  • the dimer structure is important in the interactions of E2NT with viral and cellular transcription factors and is the key to induction of DNA loops by E2. These loops may serve to target distal DNA-binding transcription factors to the region proximal to the start of transcription. The structure has implications for antiviral drug design and cervical cancer therapy.
  • the invention includes method for identifying and/or selecting a candidate therapeutic agent, comprising applying rationalised drug design to a crystal structure obtainable by crystallising E2NT, cryogenically freezing the crystals and generating the crystal structure using X-ray diffraction.
  • the method by which the E2NT crystal structure is obtainable may comprise crystallisation using hanging-drop vapour diffusion.
  • the method by which E2NT crystal structure is obtainable may comprise X-ray diffraction using uranium acetate and gold cyanide E2NT derivatives and refining with data extending to 1.9 A spacing.
  • the crystal structure may comprise the portions of amino acids Ile82, Glu90, Trp92, Lysl l2, Tyrl38, Vall45, Prol06, Lysl l l, Phel68, Trpl34, Trp33 and Leu94.
  • the rationalised drug design may comprise designing drugs which interact with the dimerisation surface of E2NT.
  • a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises or a three-dimensional representation of a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A , wherein said computer comprises:
  • a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises the structure coordinates of E2NT amino acids Ile82, Glu90, Trp92, Lysl l2, Tyrl38, Vall45, ProlO ⁇ , Lysl 11, Phel68, Trpl34, Trp33 and Leu94 according to Table 3;
  • the three-dimensional representation is of a molecule or molecular complex is defined by the set of structure coordinates according to Table 3, or wherein said three-dimensional representation is of a homologue of said molecule or molecular complex, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A.
  • An additional aspect of the invention resides in a computer for determining at least a portion of the structure coordinates corresponding to an X-ray diffraction pattern of a molecule or molecular complex, wherein said computer comprises:
  • a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises an X-ray diffraction pattern of said molecule or molecular complex
  • a central-processing unit coupled to said working memory and to said machine- readable data storage medium of (a) and (b) for performing a Fourier transform of the machine readable data of (a) and for processing said machine readable data of (b) into structure coordinates;
  • a display coupled to said central-processing unit for displaying said structure coordinates of said molecule or molecular complex.
  • a yet further aspect of the invention relates to a crystallised molecule or molecular complex
  • a crystallised molecule or molecular complex comprising a dimerisation surface defined by structure coordinates of E2NT amino acids Ile82, Glu90, Trp92, Lysl l2, Tyrl38, Vall45, ProlO ⁇ , Lysl l l, Phel68, Trpl34, Trp33 and Leu94 according to Table 3or a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A.
  • the molecule or molecular complex may be defined by the set of structure coordinates according to Table 3, or a homologue thereof, wherein said homologue has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A.
  • a machine-readable data storage medium e.g. a magnetic or optical storage medium, for example a hard disc, a floppy disc or a CD-ROM
  • a machine-readable data storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex comprising a dimerisation surface defined by structure coordinates of E2NT amino acids Ile82, Glu90, Trp92, Lysl 12, Tyrl38, Vall45, Prol06, Lysl 11, Phel68, Trpl34, Trp33 and Leu94 according to Table 3, or a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 A.
  • the molecule or molecular complex may be defined by the set of structure coordinates according to Table 3, or a homologue of said molecule or molecular complex, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5A.
  • the invention further provides a machine-readable data storage medium comprising a data storage material encoded with a first set of machine readable data which, when combined with a second set of machine readable data, using a machine programmed with instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data, wherein: said first set of data comprises a Fourier transform of at least a portion of the structural coordinates according to Table 3; and said second set of data comprises an x-ray diffraction pattern of a molecule or molecular complex.
  • the invention resides in a method for evaluating the ability of a chemical entity to associate with a molecule or molecular complex according to the invention, comprising the steps of:
  • Table 1 illustrates X-ray data and phasing statistics
  • Table 2 illustrates refinement and model correlation
  • Table 3 shows the structure coordinates of the E2NT module
  • Figure la represents functional assignments of HPV 16 E2 protein
  • Figure lb illustrates sequence alignment of E2NT modules from a subset of HPV types
  • Figure 2a illustrates a stereo view of electron density with a final model at the dimer interface of the E2NT module, viewed down the crystallographic two-fold axis;
  • Figure 2b represents a stereo ribbon diagram of the E2NT module
  • Figure 2c represents the E2NT dimer
  • Figure 3 a illustrates a schematic view of URR
  • Figure 3b illustrates a schematic view of loop formation induced by binding of E2 proteins to two cognate sites
  • Figure 3c illustrates a model of E2 dimer formation
  • Figure 3d illustrates loops within URR as shown in Figure 3b
  • Figure 4a illustrates the distribution of conserved residues on the E2NT monomer
  • Figure 4b illustrates a first cluster of conserved residues on the E2NT monomer
  • Figure 4c illustrates a second cluster of conserved residues on the E2NT monomer
  • Figure 4d illustrates conserved residues Gin 12 and Glu39.
  • a set of structure coordinates for an enzyme or an enzyme-complex or a portion thereof is a relative set of points that define a shape in three dimensions.
  • an entirely different set of coordinates could define a similar or identical shape.
  • slight variations caused by acceptable errors in the individual coordinates will have little, if any effect on overall shape. In terms of binding pockets, these acceptable variations would not be expected to alter the nature of ligands that could associate with those pockets.
  • association refers to a condition of proximity between a chemical entity or compound, or portions thereof, and a calcineurin molecule or portions thereof.
  • the association may be non-covalent— wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions— or it may be covalent.
  • Shaded blocks above the alignment indicate the experimentally determined secondary structure. Shaded blocks below the sequences indicate the minimal peptide sequences involved in proteimprotein interactions, suggested by mutation studies. Residues with more than 90% identity among 86 PV types are coloured: red for internal structural residues, green for residues within the fulcrum region, blue for surface residues.
  • FIG. 2a there is shown a stereo view of the electron density with the final model, at the dimer interface of the E2NT module, viewed down the crystallographic two-fold axis.
  • the likelihood weighted map is contoured at the 1.5 ⁇ level. Ribbons of two independent monomers are coloured blue and yellow. Side chains of ARG37 and Ile73 which are known to be critical for transactivation 4 ' 31 , are shown in dark green; side chain of other residues at the dimer interface are shown in light green. Oxygen atoms are in red, nitrogen in blue, water molecules are shown as orange spheres and hydrogen bonds as dashed sticks.
  • Figure 2b there is shown a stereo ribbon diagram of the E2NT module.
  • the Nl domain is shown in aquamarine and the N2 domain in pink, with the fulcrum in green.
  • Figure 2c there is shown the dimer of E2NT, showing the extent of the interface between the two subunits. The view is as in Figure 2a but rotated clockwise by 90°. Side chains of Glnl2 and Glu39 which are critical for interactions with El 31" ' are shown in magenta. Side chains of residues at the dimer interface are coloured as per Figure 2a.
  • FIG. 3 a there is shown loop formation in the URR of HPV 16.
  • Figure 3 a there is shown a schematic view of the URR.
  • the four E2-binding sites are represented by boxes. Numbers in italics indicate distances between individual sites upstream of the p97 promoter. Two possible E2 configurations, with separate or dimeric E2NT modules are shown.
  • Figure 3b there is shown a schematic view of loop formation induced by binding of E2 proteins to two cognate sites, based on the experiments reported by Knight et al .
  • Figure 3d there is shown the possible DNA loops within the URR as depicted in Figure 3b.
  • FIG. 4a there is shown the distribution of conserved residues on the E2NT monomer.
  • Figures 4b and 4c there is shown the two clusters of conserved residues in the fulcrum of E2NT.
  • Figure 4d there are shown conserved residues Gin 12 and Glu39. Bonds in ball-and stick models are coloured aquamarine (Nl domain), pink (N2 domain) and green (fulcrum). Hydrogen bonds are shown as dashed lines, water molecules as orange spheres, oxygen atoms are in red, nitrogen atoms in blue and sulphur atoms in yellow.
  • the structure was determined using two heavy atom derivatives and refined with data extending to 1.9 A spacing (Fig. 2a).
  • the main chain is well defined throughout with the exception of residues 125 and 126 which are in an exposed loop and are mobile. There was density for the last residue of the His-tag at the N-terminus, but none for the remainder of this entity. All amino acids lie in the allowed regions of the Ramachandran ( ⁇ , ⁇ ) plot 17 with 92.4% in most favoured regions 18 .
  • the transactivation module is composed of two domains, Nl and N2, arranged so as to give it an overall L-shaped appearance.
  • Domain Nl which forms the N-terminus of the intact E2 is composed of residues 1 to 92, which fold into three long ⁇ -helices, Figure 2 (b,c).
  • Figure 2 (b,c) There is a tight loop between ⁇ l and ⁇ 2 and a more extended one between ⁇ 2 and ⁇ 3.
  • the three helices pack antiparallel to one another in the form of a twisted plane, with angles of about 20° and 25° between the pairs of consecutive helices.
  • DALI indicated a maximum Z-score of 5.7, that could suggest a significant correlation, for colicin la, a membrane protein which contains three 80 A long ⁇ -helices arranged more or less coplanar 21 . This is the only other known protein that contains a true domain made up of such a packing of three helices. In addition there were 42 other structures which gave Z-scores above 4.0, most of which were four helix bundles, such as bacterioferritin 22 . However, in these only two of the three Nl helices superimposed simultaneously on two, not always adjacent, bundle helices as a result of a more planar arrangement of helices within Nl. The indications are that the similarities observed reflect the optimum stacking angle of antiparallel helices against one another rather than suggesting a common ancestor for the evolution of these molecules.
  • Domain N2 is made up of residues 110 to 201 and is composed almost entirely of antiparallel ⁇ structure, with only one short helical segment from residues 171 to 178, Figure 2 (b,c).
  • the secondary structure has two short three and four stranded antiparallel ⁇ pleated sheets interconnected by two stranded ⁇ ribbons.
  • DALI failed to identify any significant homologies to known structures, with a highest Z-score of only 2.1. From the analysis of Harris and Botchan 15 and the present study, the N2 fold appears to be novel.
  • the structure between the Nl and N2 domains contains two consecutive single turns of helical structure, resulting in a compact and tight turn. It packs closely against elements of both domains and is not a truly independent structural domain. Rather it forms a fulcrum in the L-shape formed by Nl and N2 where it could act as a hinge, allowing the two domains to change their relative conformation in a specific way.
  • Several of the interactions between adjacent regions of chain in the fulcrum are mediated indirectly through H-bonds involving water molecules, suggesting the possibility of flexibility.
  • the two E2NT monomers pack around the crystallographic 2-fold axis, as shown in Figure 2a.
  • the dimer interface is formed mostly by amino acids from helices ⁇ 2 and ⁇ 3 of the Nl domain and by residues 142-144 from the N2 domain.
  • the total buried surface area between the two E2NT is
  • each subunit contributes a cluster of seven equivalent residues, invariant or conserved in the 86 known sequences of E2 1 ', with many direct and water-mediated hydrogen bonds and rather few non-polar contacts, Fig. 2.
  • Analysis of the dimer forming surfaces shows that all the direct hydrogen bonds between monomers are made through these seven amino acids.
  • Arg37 all possible side-chain hydrogen bonds are made and all are well defined, Figure 2. Three of them are across the dimer interface.
  • One hydrogen bond is critical, from NH2 to the main chain carbonyl oxygen of Leu77.
  • a second hydrogen bond from NH2 is to OG1 of Thr81; in five out of 86 sequences this residue is glutamine, and modelling shows a hydrogen bond is possible to the NE of Arg37.
  • the NH1 of Arg71 H-bonds to the OE1 of residue 80, which is Glu or Gin in all but six variants.
  • At the NE of Arg37 there is an ideal H-bond to water that itself makes another strong H-bond across the dimer interface to the main-chain carbonyl oxygen of residue 142.
  • the role of the invariant Ile73 is the filling of the intersubunit non- polar volume made up of the aliphatic parts of Arg37, Gln76 and of Leu77 - in this case from both monomers.
  • the Leu77 is in a few sequences substituted by valine or isoleucine and in 9 out of 86 known sequences by methionine. Inspection of the structure shows that Leu77 is partially exposed to the solvent and therefore different hydrophobic side chains could be easily accommodated at this site.
  • Another important non-polar side chain is Ala69. Its side chain methyl packs into the surface of the other monomer at van de Waals distance from the main chain of residue 142. The only observed mutation of Ala69 is to Gly, and is easily accommodated. Gln76 is conserved or has homologous substitutions in about 2/3 of E2 sequences; in about 1/4 of the sequences there is methionine or valine at this position 11 .
  • DNA-bound E2 dimer s 25 DNA-bound E2 dimer s 25 .
  • E2CT forms dimers which bind to the multiple DNA-binding sites located within the URR of viral DNA with K d of protein:DNA interactions usually in the nanomolar range . Consequently, the local concentration of E2NT, bound to the E2CT via the non-conserved, flexible ⁇ 80 amino-acid linker, is effectively increased.
  • E2NT dimer interactions could form either between modules which are already part of a single E2 dimer, formed as a result of E2CT dimerisation interactions and bound to a single E2 binding site on the DNA (Fig. 3 a), or between two preformed E2 dimers located on different E2 binding sites (Fig. 3b).
  • the results of the electron microscopy suggest that the latter dimerisation does occur 8 .
  • no direct experimental evidence exists for the former dimerisation it does also seem possible due to the flexibility of the linker connecting the two modules.
  • E2 molecules may initially keep their N-terminal modules within their internal dimers, but swap N-terminal modules and cross link to E2 molecules bound to distant DNA binding sites to form active loop structures during transcriptional activation and / or HPV DNA replication (Figure 3d).
  • the effects of mutations on transcriptional transactivation can be explained in terms of the dimer being an essential element in this process.
  • E2 is a regulator of both transcription and viral DNA replication and thus interacts with other viral and host macromolecules in the infected cell. Indication of the possible importance of individual residues in the function comes firstly from the structure, secondly from the extensive set of sequences of the papillomaviral E2's and thirdly from mutagenesis studies on the individual proteins.
  • a further 12 of these 33 residues stand out as having a structural role in the interface of the Nl and N2 domains. They form three clusters, the first making direct interactions between the two domains (Ile82, Glu90, Trp92, Lysl 12, Tyrl38, Vall45) and two separate sets of interactions, one from N2 (Prol06, Lysl 11, Phel68, Tr l34) and the other from Nl (Trp33, Leu94) to the structure connecting them, referred to here as a fulcrum.
  • the first two clusters are shown in Figure 4 b, c and it can be seen that Lysl 11 and Lysl 12 play key roles. Their side chains point in opposite directions to one another and their terminal amino groups are involved in near ideal patterns of hydrogen bonds.
  • Glu20 lies on the top surface of Nl.
  • Asp 122 lies far away on the distal surface of N2.
  • GlulOO is completely exposed and points into the solvent at the junction of the L between the Nl and N2 domains. The functional role of these amino acids has yet to be clarified.
  • residues 74-134 and TFIIB were previously mapped onto the E2NT module ( Figure 1) using a series of deletion mutants as well as point mutations 34,35 . These sites were mutually exclusive.
  • residues 74-134 include the fulcrum, while residues 134-216 correspond to domain N2. Further biochemical and structural studies can now be planned to characterise these interactions in more detail.
  • Replication of the viral genome is initiated by binding of another viral protein, El, to the origin of DNA replication 4 which is itself flanked by two E2 binding sites, Fig. 3 a. While the function of E2CT dimers is to bind specifically to the DNA sites, E2NT interaction with El enhances the binding of El to this region. Mutational substitutions of Glu39 generally retained transcriptional activation while DNA replication was substantially reduced ' . In the structure, the conserved Glu39 makes every possible hydrogen bond by its side chain carboxyl oxygens, Fig. 4d. One hydrogen bond is to NE2 of Gin 12, which is absolutely conserved in all known sequences of E2.
  • the other three hydrogen bonds are to the water molecules which are part of an intimate net of well-defined water molecules surrounding Glu39 and mediating its interactions with adjacent residues.
  • a number of these protein interactions with water molecules are conserved as they are made to the protein backbone, including carbonyl oxygens of Glnl2, Met36 and Lys68.
  • mutation of Gin 12 in BPV1 only slightly affected both transactivation and replication, it substantially reduced cooperative origin binding 30,32 .
  • the close positioning of Glnl2 and Glu39 in the three-dimensional structure further enhances the notion that these two resides are involved in interactions with El.
  • the conserved set of interactions at Glnl2/Glu39 suggests that the main chain carbonyl oxygens of Gin 12 and Met36 and the conserved water molecules could be also involved in these interactions.
  • Glnl2/Glu39 are surrounded by Leu8, Ilel5, Met36, Tyr43, Gln57 and Lys68, which are unlikely to contribute into E2/E1 interactions, as these residues are not well conserved in E2 sequences from different papillomaviruses.
  • the Glnl2/Glu39 cluster lies on a side of the Nl domain which is opposite to the side involved in transactivation (and dimerisation), Figure 2c.
  • the spatial separation of the two functionally important surfaces suggests that E2NT module could be able to interact with El at the same time as it interacts through the dimerisation interface with another E2NT module.
  • the structure shows that the transactivation surface is involved in the formation of the E2NT dimer, which could cross-link E2 molecules bound by their E2CT modules to well-separated DNA sites. Inevitably, such dimerisation would cause DNA to form a loop structure, targeting distally bound transcription factors to regions close to the promoter. While this process has been suggested to be essential for transactivation 36 , the definition of interacting surfaces between E2 and other cellular transcription factors requires a great deal of further study. Our results suggest that the process of DNA loop formation could involve swapping of E2NT modules across E2 dimers bound at separated DNA sites (Fig. 3a-d). The polar components of the monomer-monomer interactions may favour such exchange. Domain swapping is a well-recognised phenomenon that occurs relatively frequently between two individual monomers containing domains connected by a flexible linker 39,40 . E2 is to our knowledge the first example where the swapping event is predicted to occur between dimers.
  • the dimerisation surface of E2 represents a good target for designing anti-viral drugs, since it is essential for viral transcription, there is no homologous human protein and the residues forming the interface are highly conserved among different viral strains. Dynamic interactions between transcription factors play a central role in the regulation of transcription and replication. Dimerisation, heterodimerisation and the monomer-to-dimer transition may play important roles during the control of the papillomavirus life cycle. These processes themselves can be regulated through phosphorylation, proteolysis, interaction with small ligands or changes in their intracellular concentration. It has been suggested that E2 can regulate the switch between early gene expression and viral genome replication during HPV infection 41 . It is possible that dimerisation of E2NT modules plays an essential role during this process.
  • One scenario would be to activate transcription via induction of DNA loop formation at early stages of the viral life cycle. At later stages, when the concentration of expressed E2 proteins within the cell becomes high and comparable with the K d for E2 dimer formation, free E2NT modules could compete for dimerisation with those involved in DNA loop formation and titrate them away, switching off transcription and stimulating replication. It is also possible that other protein factors could be involved in this process, including, for example, El.
  • the invention therefore includes the use of E2NT crystal structure in the design of anti-viral drugs, since it is essential for viral transcription.
  • computational analyses are therefore necessary to determine whether a molecule or the E2NT-binding portion thereof is sufficiently similar to the E2NT structure.
  • Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., Waltham, Mass.) version 3.3, and as described in the accompanying User's Guide, Volume 3 pages. 134-135.
  • the Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure.
  • the procedure used in Molecular Similarity to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalences in these structures; 3) perform a fitting operation; and 4) analyze the results.
  • Each structure is identified by a name.
  • One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures).
  • Atom equivalency within QUANTA is defined by user input and, for the purpose of this invention equivalent atoms may be defined as protein backbone atoms (N, C. alpha., C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations.
  • the working structure is translated and rotated to obtain an optimum fit with the target structure.
  • the fitting operation uses a least squares fitting algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
  • any set of structure coordinates of a molecule or molecular complex that has a root mean square deviation of conserved residue backbone atoms (N, C. alpha., C, O) of less than 1.5 .ANG. when superimposed-using backbone atoms— on the relevant structure coordinates of E2NT are considered identical. More preferably, the root mean square deviation is less than 1.0 .ANG.. Most preferably, the root mean square deviation is less than 0.5 .ANG..
  • root mean square deviation means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object.
  • the "root mean square deviation” defines the variation in the backbone of a protein from the backbone of E2NT a dimerising portion thereof, for example as defined by the structure coordinates of E2NT described herein.
  • least squares refers to a method based on the principle that the best estimate of a value is that in which the sum of the squares of the deviations of observed values is a minimum.
  • the model was completed with REFMAC (resolution 20-1.9 A) using a bulk solvent correction, to an R-factor of 23.3 % (R Free 29.7 % - for 5 % of the data).
  • REFMAC Resolution 20-1.9 A
  • R-factor 23.3 %
  • the final model contains all but two of the 201 residues of the real protein: residues 125-126 are disordered and lie in a flexible surface loop. Only one residue, HisO, of the His-tag has clear density and an ordered conformation.
  • the main statistics of the refined model are shown in Table 2.
  • E2NT was in lOmM TrisHCl pH 8.0, 5mM DTT, 0.2 mM EDTA, 300 mM NaCl. Data were obtained at rotor speeds of 12,000 and 16,000 rpm, and the time to equilibrium was 10-12 hours. All runs were carried out at 293K, and all radial scans were at a wavelength of 280 nm. Dissociation constants were obtained by nonlinear regression using the Beckman ultracentrifuge software.
  • ATOM 354 SG CYS A 40 14 .743 -10 .845 43 .945 1 .00 35 .07
  • ATOM 356 CA ALA A 41 12 .772 -14 .160 48, .820 1 .00 38 .13
  • ATOM 369 CA TYR A 43 13. 341 -18. ,254 45. ,060 1. ,00 36. ,20
  • ATOM 402 CA ALA A 46 13 .230 -22 .586 47 .356 1 .00 41 .88 ATOM 403 C ALA A 46 14 .613 -23 .167 47 .179 1 .00 41 .96
  • ATOM 418 CA GLU A 48 17, .783 -23 .090 50, .364 1 .00 48 .18
  • ATOM 426 N MET A 49 16. ,000 -24. ,730 50. ,361 1, ,00 49. .75
  • ATOM 428 C MET A 49 15. ,738 -27. ,106 49. ,543 1. .00 50. .69
  • ATOM 429 O MET A 49 15. 197 -28. ,218 49. ,620 1. ,00 51. ,52
  • ATOM 430 CB MET A 49 13. 840 -25. ,835 50. ,513 1. .00 53. .09
  • ATOM 469 CA ILE A 54 9 .598 -24 .513 41 .100 1 .00 44 .62
  • ATOM 478 C ASN A 55 7 .391 -25 .383 37 .389 1 .00 41 .70
  • ATOM 485 CA HIS A 56 9, .304 -26, .336 36. .155 1, .00 41, .80
  • fiC e ⁇ fiC fiC fit; fiC fit; fit; fit; fiC fit; fiC fiC fiC fit; fit; ⁇ cC fit; fit; cC fit; fi fiC fiC fi fiC fiC fiC ⁇
  • fiC fiC cC c fit fiC fii; fiC fit; cC fiC fiC fiC fiC fiC fiC fi£; fi fiC fiC fiC fit; fiC fit; fit; fii; fit; fi fit; fit; ⁇ fiC fiC ⁇ t; fit; fi ⁇ O
  • fiC fii ct; fiC c fiC cc fiC fit; fit; fiC fit: fiC fii; cC fiC c fiC ⁇ fit; fi fi fiC fii: fiC fi ⁇ fiC fit: fiC rt fit; fiC fi ⁇ fiC fiC o ci
  • CM i H c ⁇ n ⁇ o ⁇ cMCM j cD in ⁇ L ⁇ iD ⁇ CM ⁇ rH r ⁇ rHcn ⁇ oco , en ⁇ r ⁇ ⁇ n rH iDi ⁇ iD ⁇ rH c ⁇ c rHcn ooen iD ⁇ i ⁇ o cn cM ⁇ o ⁇ oo rH en co-o cM iD o t ⁇ ⁇ c iD iD L i iD ⁇ is r ⁇ ⁇ o co ⁇ c i c cN iD o r cn c ⁇
  • H H H H ri H C ⁇ I C CM CM H H H PI H ri H H H H H H CM N N H H H H PI r! H H H H H H H H H H rl N H ( ⁇ l H CM ( H H ⁇ n o o ⁇ ⁇ n r co o o c ⁇ o ⁇ ⁇ H H i ⁇ r H o r- ri r ⁇ o n MJi oi ⁇ n ⁇ H ⁇ cM iM M ⁇ ⁇ o o c ⁇ H O r ⁇ MO '» r ⁇ in in ⁇ cn cM w oi r n o o ⁇ r 7 i co o o co H ⁇ O H O oo rH cn o ⁇ i en rH r ⁇ cD rH in co i ⁇ e oo cM r ⁇ o ⁇ o o
  • CD CD CD O CD CD CD CD CD CD CD CD CD CD CD CD CD CD CD CD CD CD CD CD C .D.. C.D.. C.D.. .>. _> > > > > > f t; ; ⁇ a; fl; et; ⁇ ; l a: ; EH EH EH E ⁇ EH EH ⁇ EH EH EH E- ⁇ E ⁇ EH EH H EH h tH h E ⁇ H EH H EH H U e)
  • McBride, A. & Myers, G. in Human Papillomaviruses 1997 eds Myers, G., Sverdrup, F., Baker, C, McBride, A., M ⁇ nger, K. & Bernard, H.-U.
  • 111-54-111-73 Theoretical Biology and Biophysics, Los Alamos, 1997.

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Abstract

L'invention concerne un complexe moléculaire cristallisé d'une protéine dimère à module N-terminal E2 (E2NT) ou un homologue de la protéine, qui comprend des restes vitaux pour une transcription et/ou une réplication virale. L'invention concerne également l'utilisation de la protéine dimère et des interactions à sa surface de dimérisation dans la conception rationnelle de médicaments antiviraux.
PCT/GB2000/003568 1999-09-17 2000-09-18 Cible de therapie antivirale WO2001021645A2 (fr)

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WO2003006495A2 (fr) * 2001-07-12 2003-01-23 Boehringer Ingelheim International Gmbh Co-cristal de domaine/d'inhibiteur de transactivation de papillomavirus e2 humain, et coordonnees de radiographie definissant la poche de liaison avec l'inhibiteur
EP2468763A3 (fr) * 2007-05-31 2012-07-04 Academisch Ziekenhuis Leiden h.o.d.n. LUMC Épitopes HPV ciblés par des lymphocytes T infiltrant des malignités cervicales à utiliser dans des vaccins

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COOPER CHRISTOPHER S ET AL: "Identification of single amino acids in the human papillomavirus 11 E2 protein critical for the transactivation or replication functions." VIROLOGY, vol. 241, no. 2, 15 February 1998 (1998-02-15), pages 312-322, XP002162388 ISSN: 0042-6822 cited in the application *
GAUTHIER J-M ET AL: "TWO DNA-BOUND E2 DIMERS ARE REQUIRED FOR STRONG TRANSCRIPTIONAL ACTIVATION AND FOR COOPERATION WITH CELLULAR FACTORS IN MOST CELLS" NEW BIOLOGIST, vol. 3, no. 5, 1991, pages 498-509, XP000989622 ISSN: 1043-4674 cited in the application *
GHOSE A K ET AL : "DETERMINATION OF PHARMACOPHORIC GEOMETRY FOR COLLAGENASE INHIBITORS USING A NOVEL COMPUTATIONAL METHOD AND ITS VERIFICATION USING MOLECULAR DYNAMICS, NMR, AND X-RAY CRYSTAL" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY,US,AMERICAN CHEMICAL SOCIETY, WASHINGTON, DC, vol. 117, no. 16, 1995, pages 4671-4682, XP002051616 ISSN: 0002-7863 *
HARRIS SETH F ET AL: "Crystal structure of the human papillomavirus type 18 E2 activation domain." SCIENCE (WASHINGTON D C), vol. 284, no. 5420, 4 June 1999 (1999-06-04), pages 1673-1677, XP002162385 ISSN: 0036-8075 cited in the application *
HEGDE RASHMI S ET AL: "Crystal structure of the E2 DNA-binding domain from human papillomavirus type 16: Implications for its DNA binding-site selection mechanism." JOURNAL OF MOLECULAR BIOLOGY, vol. 284, no. 5, 18 December 1998 (1998-12-18), pages 1479-1489, XP002162386 ISSN: 0022-2836 cited in the application *
HEGDE RASHMI S ET AL: "Subunit rearrangement accompanies sequence-specific DNA binding by the bovine papillomavirus-1 E2 protein." JOURNAL OF MOLECULAR BIOLOGY, vol. 276, no. 4, 6 March 1998 (1998-03-06), pages 797-808, XP002162387 ISSN: 0022-2836 cited in the application *
KNIGHT J D ET AL: "THE ACTIVATION DOMAIN OF THE BOVINE PAPILLOMAVIRUS E2 PROTEIN MEDIATES ASSOCIATION OF DNA-BOUND DIMERS TO FORM DNA LOOPS" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 88, no. 8, 1991, pages 3204-3208, XP000982633 1991 ISSN: 0027-8424 cited in the application *
MCBRIDE A A ET AL: "E2 POLYPEPTIDES ENCODED BY BOVINE PAPILLOMAVIRUS TYPE 1 FORM DIMERS THROUGH THE COMMON CARBOXYL-TERMINAL DOMAIN TRANSACTIVATION IS MEDIATED BY THE CONSERVED AMINO-TERMINAL DOMAIN" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 86, no. 2, 1989, pages 510-514, XP000982632 1989 ISSN: 0027-8424 cited in the application *
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SAKAI HIROYUKI ET AL: "Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions." JOURNAL OF VIROLOGY, vol. 70, no. 3, 1996, pages 1602-1611, XP002162389 ISSN: 0022-538X cited in the application *
WIBLEY J E A ET AL: "A homology model of the three-dimensional structure of human O-6-alkylguanine-DNA alkyltransferase based on the crystal structure of the C-terminal domain of the Ada protein from Escherichia coli." ANTI-CANCER DRUG DESIGN, vol. 10, no. 1, 1995, pages 75-95, XP000989520 ISSN: 0266-9536 -& WIBLEY J E A ET AL.,: "erratum" ANTI-CANCER DRUG DESIGN, vol. 10, no. 1, 1995, page 439 XP002162457 *
YANG FAN ET AL: "Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping." JOURNAL OF MOLECULAR BIOLOGY, vol. 288, no. 3, 7 May 1999 (1999-05-07), pages 403-412, XP002162390 ISSN: 0022-2836 cited in the application *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006495A2 (fr) * 2001-07-12 2003-01-23 Boehringer Ingelheim International Gmbh Co-cristal de domaine/d'inhibiteur de transactivation de papillomavirus e2 humain, et coordonnees de radiographie definissant la poche de liaison avec l'inhibiteur
WO2003006495A3 (fr) * 2001-07-12 2003-07-31 Boehringer Ingelheim Int Co-cristal de domaine/d'inhibiteur de transactivation de papillomavirus e2 humain, et coordonnees de radiographie definissant la poche de liaison avec l'inhibiteur
EP1695980A2 (fr) * 2001-07-12 2006-08-30 Boehringer Ingelheim International GmbH Co-crystal de domaine de transactivation de papillomavirus E2 humain et d'inhibiteur, et coordonnées de radiographie définissant la poche de liaison avec l'inhibiteur
EP1695980A3 (fr) * 2001-07-12 2006-09-06 Boehringer Ingelheim International GmbH Co-crystal de domaine de transactivation de papillomavirus E2 humain et d'inhibiteur, et coordonnées de radiographie définissant la poche de liaison avec l'inhibiteur
US7167801B2 (en) 2001-07-12 2007-01-23 Boehringer Ingelheim (Canada) Ltd. Method of identifying potential inhibitors of human papillomavirus protein E2 using x-ray atomic coordinates
EP2468763A3 (fr) * 2007-05-31 2012-07-04 Academisch Ziekenhuis Leiden h.o.d.n. LUMC Épitopes HPV ciblés par des lymphocytes T infiltrant des malignités cervicales à utiliser dans des vaccins
US9764023B2 (en) 2007-05-31 2017-09-19 Academish Ziekenhuis Leiden H.O.D.N. LUMC HPV epitopes targeted by T cells infiltrating cervical malignancies for use in vaccines
US10258684B2 (en) 2007-05-31 2019-04-16 Academisch Ziekenhuis Leiden H.O.D.N. Lumc HPV epitopes targeted by T cells infiltrating cervical malignancies for use in vaccines
US10688173B2 (en) 2007-05-31 2020-06-23 Academisch Ziekenhuis Leiden H.O.D.N. Lumc HPV epitopes targeted by T cells infiltrating cervical malignancies for use in vaccines
US11458198B2 (en) 2007-05-31 2022-10-04 Academisch Ziekenhuis Leiden H.O.D.N. Lumc HPV epitopes targeted by T cells infiltrating cervical malignancies for use in vaccines

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