WO2008154432A2 - Complexes topoisomérase/adn cristallins - Google Patents

Complexes topoisomérase/adn cristallins Download PDF

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WO2008154432A2
WO2008154432A2 PCT/US2008/066194 US2008066194W WO2008154432A2 WO 2008154432 A2 WO2008154432 A2 WO 2008154432A2 US 2008066194 W US2008066194 W US 2008066194W WO 2008154432 A2 WO2008154432 A2 WO 2008154432A2
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seq
polypeptide
topoisomerase
nucleic acid
crystal
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PCT/US2008/066194
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WO2008154432A3 (fr
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James M. Berger
Ken Dong
Allyn J. Scheoffler
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The Regents Of The University Of California
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment

Definitions

  • the present invention pertains to the crystallization of DNA-modifying enzymes, particularly as topoisomerase enzymes.
  • a topoisomerase-DNA complex in crystalline form, methods of forming crystals comprising topoisomerase, methods of using crystals comprising topoisomerase, a crystal structure of topoisomerase, and methods of using the crystal structure.
  • DNA transformations performed by DNA topoisomerases are accomplished by the cleavage of either a single strand or both strands.
  • the unit change in the Linking number (Lk) resulting from such transformations is the best operational distinction between the two classes of topoisomerases (P. O. Brown & N. R. Cozzarelli, Science 206:1081-1083 (1979)).
  • the linking number (Lk) is the algebraic number of times one strand crosses the surface stretched over the other strand.
  • Type I and type II enzymes catalyze topological changes in DNA by transiently breaking one strand or two strands of the DNA helix, respectively.
  • the relaxation of superhelical DNA is a characteristic reaction catalyzed by a topoisomerase I, while a topoisomerase II catalyzes the passing of two DNA segments in a manner leading to such topoisomerization reactions of DNA as supercoiling/relaxation, knotting/unknotting and catenation/decantenation.
  • DNA topoisomerases whose reactions proceed via a transient single-stranded break and changing the Lk in steps of one are classified as type 1, while enzymes whose reactions proceed via double-stranded breaks and changing the Lk in steps of two are classified as type 2.
  • the family of type 1 topoisomerases comprises bacterial topoisomerase I, E. coli topoisomerase III, S. cerevisiae topoisomerase III (R. A. Kim & J. C. Wang, J. Biol. Chem. 267: 17178-17185 (1992), human topoisomerase III (Hanai et al, Proc. Natl. Acad. Sci. 93:3653-3657 (1996)), the type 1 topoisomerase from chloroplasts that closely resembles bacterial enzymes (J. Siedlecki et al., Nucleic Acids Res. 11 : 1523-1536 (1983), thermophilic reverse gyrases (A.
  • topoisomerases can be divided into two groups.
  • Group A consists of enzymes that require a divalent cation for activity, and form a transient covalent complex with the 5'- phosphoryl termini (prokaryotic type 1 topoisomerases, S. cerevisiae topoisomerase III, and human topoisomerase III).
  • Group B includes type 1 topoisomerases that do not require a divalent cation for activity, and bind covalently to the 3'-phosphoryl termini (nuclear topoisomerases I, enzymes from mitochondria and poxviruses commonly called eukaryotic topoisomerases I).
  • Type I topoisomerases can carry out the following topological reactions: they relax supercoiled DNA (except of reverse gyrases), catenate (or decatenate) single- stranded circular DNAs or duplexes providing that at least one of the molecules contains a nick or gap, or interact with single-stranded circles to introduce topological knots (type 1- group A topoisomerases).
  • Reverse gyrase belonging to type 1 -group A topoisomerases, is the only topoisomerase shown to be able to introduce positive supercoils into cDNA.
  • topoisomerase family include DNA gyrase, bacterial DNA topoisomerase IV, T-even phage DNA topoisomerases, eukaryotic DNA topoisomerase II, and thermophilic topoisomerase II from Sulfolobus acidocaldarius (see: A. Kikuchi et al., Syst. Appl. Microbiol. 7: 72-78 (1986); J. Kato et al., J. Biol. Chem. 267: 25676-25684 (1992); W. M. Huang in DNA Topology and Its Biological Effects (N. R. Cozzarelli and J. C.
  • Type HA DNA topoisomerase enzymes control the topology of DNA over the course of conformational and topological changes which occur during many cellular processes. For example, DNA topoisomerases are involved in DNA replication, RNA transcription and recombination.
  • Topological reactions catalyzed by type 2 topoisomerases include introduction of negative supercoils into DNA (DNA gyrase), relaxation of supercoiled DNA, catenation (or decatenation) of duplex circles, knotting and unknotting of DNA.
  • Type HA DNA toposiomerases are established targets for inhibitors with valuable clinical properties, including chemotherapy (human topoisomerase II, or topo II), and antibiotics (bacterial DNA gyrase and toposiomerase IV (topo IV)). Many of these agents target the DNA bound state of the enzyme, causing the topoisomerase to form cytotoxic double strand DNA breaks.
  • chemotherapy human topoisomerase II, or topo II
  • antibiotics bacterial DNA gyrase and toposiomerase IV (topo IV)
  • Many of these agents target the DNA bound state of the enzyme, causing the topoisomerase to form cytotoxic double strand DNA breaks.
  • Mechanistic understanding of drug interactions with the topoisomerase-DNA complex, and attempts to improve drug efficacy or overcome drug resistance have been hindered by an absence of atomic resolution information on topoisomerase/DNA complexes and topoisomerase/DNA/drug complexes. Research on DNA topoisomerases has progressed from DNA enzymology to developmental
  • Bacterial DNA topoisomerase II is an important therapeutic target of quinolone antibiotics; mammalian DNA topoisomerase II is the cellular target of many potent antitumor drugs (K. Drlica, Microbiol. Rev. 48: 273-289 (1984) and Biochemistry 27: 2253-2259 (1988); B. S. Glisson & W. E. Ross, Pharmacol. Ther. 32: 89-106 (1987); A. L. Bodley & L. F. Liu, Biotechnology 6: 1315-1319 (1988); L. F. Liu, Annu. Rev. Biochem. 58: 351-375 (1989)). Mammalian topoisomerase I is the cellular target of the antitumor drug topotecan (U.S. Pat. No. 5,004,758), which also traps the covalent reaction intermediate.
  • DNA topoisomerase II has been implicated as the chemotherapeutic target for a diverse group of antitumor agents, including epipodophyllotoxins, anthracyclines, acridines, anthracenediones and ellipticines. See Macdonald et al, in DNA TOPOISOMERASES IN CANCER 199-214 (Oxford University Press 1991) (hereafter "Macdonald (1991)”), the contents of which are hereby incorporated by reference. Under the influence of such drugs, topoisomerase II is believed to cleave DNA and form a concomitant covalent association with the broken strand(s) of duplex DNA.
  • a general approach to designing inhibitors that are selective for a given protein is to determine how a putative inhibitor interacts with a three dimensional structure of that protein. For this reason it is useful to obtain the protein in crystalline form and perform X- ray diffraction techniques to determine the protein's three-dimensional structure coordinates.
  • Various methods for preparing crystalline proteins are known in the art.
  • crystallographic data can be generated using the crystals to provide useful structural information that assists in the design of molecules, e.g., small molecules, that bind to the active site of the protein and inhibit the protein's activity in vivo.
  • the protein is crystallized as a complex with a ligand, one can determine both the shape of the protein's binding pocket when bound to the ligand, as well as the amino acid residues that are capable of close contact with the ligand. By knowing the shape and amino acid residues comprised in the binding pocket, one may design new ligands that will interact favorably with the protein. With such structural information, available computational methods may be used to predict how strong the ligand binding interaction will be. Such methods aid in the design of inhibitors that bind strongly, as well as selectively to the protein
  • the present invention provides a series of tools reagents, and approaches for stabilizing type HA topoisomerase/DNA complexes, and determining their structures by X-ray crystallography, spectroscopic or optical methods, e.g., NMR or EM.
  • a set of atomic coordinates obtained by this practice for one such complex serves as a template for pursuing additional structures, substrate bound, and drug-inhibitor-bound states for all enzyme members of the type HA topoisomerase family SUMMARY OF THE INVENTION
  • the present invention provides, for the first time, complexes of a topoisomerase and a nucleic acid in crystalline form.
  • the crystals are appropriate for structure determination by X-ray crystallography or another method (e.g., NMR or EM).
  • the crystals are of use in elucidating the structure of a topoisomerase-DNA complex and this complex when it has a ligand bound to a component of the complex.
  • the crystals are of use in the discovery of drugs modulating topoisomerase activity, and, ideally, expediting this process.
  • the invention provides a composition comprising, in crystalline form, a complex between a nucleic acid and a polypeptide.
  • the polypeptide has topoisomerase activity, or includes at least one region that has at least 90% sequence identity with a topoisomerase.
  • the polypeptide comprises a nucleic acid-binding and cleavage domain having at least 90% sequence identity with a wild type topoisomerase nucleic acid-binding and cleavage domain of a topoisomerase polypeptide.
  • the nucleic acid-binding and cleavage domain is a G-segment binding domain of a topoisomerase polypeptide.
  • the topoisomerase polypeptide comprises a polypeptide sequence according to SEQ. ID. NO.: 2 or SEQ. ID. NO.: 2A.
  • the polypeptide comprises a sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a sequence according to SEQ. ID. NO.: 2 or SEQ. ID. NO.: 2A.
  • the polypeptide can be from essentially any species and can be expressed in any convenient expression system without limitation.
  • the polypeptide is a Saccharomyces polypeptide.
  • the polypeptide is a wild type topoisomerase polypeptide and is a Saccharomyces polypeptide.
  • the nucleic acid can be of essentially any structure with the proviso that is form a complex with the polypeptide such that the resulting complex can be crystallized.
  • An exemplary nucleic acid component of the crystalline complexes of the invention is a nucleic acid having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 1, SEQ. ID. NO.: IA, SEQ. ID. NO.: IB, SEQ. ID. NO. 1C, a complement of SEQ. ID. NO.: 1, a complement of SEQ. ID. NO.: IA, a complement of SEQ. ID. NO.
  • the invention provides a complex as described hereinabove, in which the complex further comprises a ligand interacting with a component of the complex, which is selected from the polypeptide, the nucleic acid and combinations thereof.
  • ligands are small molecules, having a molecular weight of less than about 2500 Daltons, less than about 1000 Daltons, less than about 700 Daltons, less than 600 Daltons, less than 500 Daltons, or less than 400 Daltons.
  • Exemplary small molecules include but are not limited to podophyllotoxins and camptothecan analogs, which are also known as topoisomerase inhibitors and are used in certain types of chemotherapy.
  • Additional examples include but are not limited to the anthracycline antibiotics, e.g. doxorubicin (DXR) and analogs thereof. (O'Shaughnessy, et al., Combination paclitaxel (taxol) and doxorubicin therapy for metastatic breast cancer, Semin. Oncol. 21 : 19-23 (1994)).
  • the antineoplastic anthracedediones e.g.
  • mitoxantrone an anthracenedione with significant cytostatic activity against a number of experimental tumors and human malignancies (Alberts, D.S., et al., Phase I clinical and pharmacokinetic study of mitoxantrone given to patients by intraperitoneal administration. Cancer Res. 48: 5874-5877(1988)).
  • a method comprising: forming a crystallization volume comprising a precipitant solution and a complex between a nucleic acid and a polypeptide.
  • the nucleic acid has at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 1, SEQ. ID. NO.: IA, SEQ. ID. NO.: IB, SEQ. ID. NO. 1C, a complement of SEQ. ID. NO.: 1, a complement of SEQ. ID. NO.: IA, a complement of SEQ. ID. NO. IB, and a complement of SEQ. ID.
  • the polypeptide has at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 2, SEQ. ID. NO.: 2A and SEQ. ID. NO.: 3.
  • the polypeptide-nucleic acid complex crystal formed has a crystal lattice in an 1222 space group.
  • the method further comprises storing the crystallization volume under conditions suitable for crystal formation of the polypeptide nucleic acid complex.
  • the invention provides a method of identifying a compound which is a potential inhibitor of a polypeptide with topoisomerase activity.
  • the method includes (a) crystallizing a crystal of a polypeptide comprising a nucleic acid-binding and cleavage domain of a topoisomerase polypeptide complexed to a nucleic acid; (b) obtaining the atomic coordinates of the crystal of the topoisomerase polypeptide -nucleic acid complex of (a); (c) using the atomic coordinates to define potential ligand binding sites of the topoisomerase polypeptide- nucleic acid complex; and (d) identifying a compound which binds to one or more of the potential ligand binding site; wherein the compound which binds to the one or more potential ligand binding site is a potential inhibitor of a polypeptide with topoisomerase activity.
  • the data regarding the ligand's ability to complex with the polypeptide-nucleic acid complex is confirmed by (e) assessing the ability of the compound identified in step (d) to inhibit topoisomerase.
  • the invention provides a method for identifying a ligand of a topoisomerase polypeptide complexed with a nucleic acid.
  • the method includes a) soaking one or more crystals of a nucleic acid-binding and cleavage domain of a topoisomerase polypeptide complexed with a nucleic acid in a solution containing a collection of compounds generated in situ or separate from the crystal; b) obtaining an X-ray crystal diffraction pattern of the soaked crystal; and c) using the X-ray crystal diffraction pattern to identify a compound bound to the soaked crystal.
  • the compound being a ligand of the said topoisomerase polypeptide complexed with said nucleic acid.
  • the ligand so identified is an inhibitor of topoisomerase.
  • a complex between a topoisomerase polypeptide, a nucleic acid and a ligand for the complex or at least one component of the complex are co-crystallized to form a three component crystal.
  • a crystal formed in this manner can be used for elucidation of the interaction of the ligand with the topoisomerase polypeptide-nucleic acid complex as described herein or as generally recognized in the art.
  • FIG. IA is a schematic illustration of the domain arrangement of S. cerevisiae Topo II.
  • FIG. 2 is a representation of Topo II showing the Topo II active site.
  • FIG. 3 shows data collection and refinement statistics for the TopoII-DNA complex.
  • FIG. 4 is a representation of Topo II showing the two-gate model for DNA transport in type HA topoisomerases.
  • FIG. 5 is a representation of DNA cleavage by the Topo II (408-1177) fragment.
  • FIG. 6 is a representation of the DNA substrate used to crystallize Topo II.
  • FIG. 7 is a representation of SEQ. ID. NO.: 1.
  • FIG. 8 is a representation of SEQ. ID. NO. : IA.
  • FIG. 9 is a representation of SEQ. ID. NO.: IB
  • FIG. 10 is a representation of SEQ. ID. NO.: 1C.
  • FIG. 11 is a representation of SEQ. ID. NO.: 2.
  • FIG. 12 is a representation of SEQ. ID. NO. : 2A
  • FIG. 13 is a representation of SEQ. ID. NO.: 3.
  • nucleic acid means DNA, RNA, single-stranded, double- stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
  • Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates), sugar modifications, 5- position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases, isocytidine and isoguanidine and the like.
  • Nucleic acids can also include non- natural bases.
  • Base as used herein includes those moieties which contain not only the known purine and pyrimidine heterocycles and the invention pyrimidines, but also heterocycle analogs and tautomers thereof.
  • Purines include adenine, guanine and xanthine and exemplary purine analogs include 8-oxo-N 6 -methyladenine and 7-deazaxanthine.
  • Pyrimidines include uracil and cytosine and their analogs such as 5-methylcytosine, 5-methyluracil and 4,4- ethanocytosine. This term also encompasses non-natural bases.
  • Non-natural bases include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, S'-methoxycarboxymethyluracil, 5-methoxyurac
  • the inventive compounds include pyrimidines derivatized at the 5-position.
  • the derivatives are 1-alkenyl-, 1-alkynyl-, heteroaromatic- and 1-alkynyl-heteroaromatic modifications.
  • 1-Alkenyl means an olefmically-unsaturated (double bond containing) acyclic group.
  • 1-Alkynyl means an acetylenically-unsaturated (triple bond containing) acylic group.
  • Heteroaromatic means a compound having at least one heterocyclic ring, 5 or 6 ring atoms, having physical and chemical properties resembling compounds such as an aromatic group.
  • Heteroaromatic also means systems having one or more rings, including bicyclic moieties such as benzimidazole, benzotriazole, benzoxazole, and indole.
  • a base also includes heterocycles such as 2-aminopyridine and triazines.
  • 1- Alkynyl-heteroaromatic means 1-ethynyl-heteroaryl wherein heteroaryl is as defined above.
  • nucleoside means a subset of nucleic acid in which a base is covalently attached to a sugar or sugar analog and which may contain a phosphite or phosphine.
  • nucleoside includes ribonucleosides, deoxyribonucleosides, or any other nucleoside which is an N-glycoside or C-glycoside of a base.
  • the stereochemistry of the sugar carbons can be other than that of D-ribose.
  • Nucleosides include those species which contain modifications of the sugar moiety, for example, wherein one or more of the hydroxyl groups are replaced with a halogen, a heteroatom, an aliphatic groups, or are functionalized as ethers, amines, thiols, and the like.
  • the pentose moiety can be replaced by a hexose or an alternate structure such as a cyclopentane ring, a 6-member morpholino ring and the like.
  • Nucleosides as defined herein are also intended to include a base linked to an amino acid and/or an amino acid analog having a free carboxyl group and/or a free amino group and/or protected forms thereof.
  • Sugar modification as used herein means any pentose or hexose moiety other than 2'-deoxyribose.
  • Modified sugars include D-ribose, 2'-O-alkyl, 2'-amino, 2'-halo functionalized pentoses, hexoses and the like.
  • Exemplary sugar modifications include those sugars in which one or more of the hydroxyl groups are replaced with a halogen, a heteroatom, an aliphatic groups, or are functionalized as ethers, amines, and the like.
  • the pentose moiety can be replaced by a hexose or an alternate structure such as a cyclopentane ring, a 6-member morpholino ring and the like.
  • Nucleosides as defined herein are also intended to include a base linked to an amino acid and/or an amino acid analog having a free carboxyl group and/or a free amino group and/or protected forms thereof. Sugars having a stereochemistry other than that of a D-ribose are also included.
  • Phosphodiester group modification means any analog of the native phosphodiester group that covalently couples adjacent nucleomonomers.
  • Substitute linkages include phosphodiester analogs, e.g. such as phosphorothioate and methylphosphonate, and nonphosphorus containing linkages, e.g. such as acetals and amides.
  • Nucleic acid modification also include 3' and 5' modifications such as capping with a quencher, a fluorophore, intercalator, minor groove binder, a conformationally assisted stabilizing group or another moiety.
  • the capping group is covalently conjugated to the oligomer through a linker group.
  • Polypeptide refers to a polymer in which the monomers are amino acids and are joined together through amide bonds, alternatively referred to as a polypeptide.
  • the amino acids are ⁇ -amino acids
  • either the L-optical isomer or the D-optical isomer can be used.
  • unnatural amino acids for example, ⁇ -alanine, phenylglycine and homoarginine are also included.
  • Commonly encountered amino acids that are not gene- encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D - or L -isomer.
  • the L -isomers are generally preferred.
  • other peptidomimetics are also useful in the present invention.
  • Polypeptides as used herein, further includes containing amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques which are well known to the art. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
  • polypeptides of the present are, to name an illustrative few, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well.
  • the amino terminal residue of polypeptides made in E. coli or other cells, prior to proteolytic processing almost invariably will be N-formylmethionine.
  • a methionine residue at the NH 2 -terminus may be deleted. Accordingly, this invention contemplates the use of both the methionine-containing and the methionineless amino terminal variants of the protein of the invention.
  • polypeptides made by expressing a cloned gene in a host for instance, the nature and extent of the modifications in large part will be determined by the host cell posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence.
  • glycosylation often does not occur in bacterial hosts such as, for example, E. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell.
  • Insect cells often carry out the same posttranslational glycosylations as do mammalian cells and, for this reason, insect cell expression systems have been developed to express efficiently mammalian proteins having native patterns of glycosylation. Similar considerations apply to other modifications. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized by expressing a polynucleotide in a host cell.
  • nucleic acids in the complexes described herein can be variants of the natural nucleic acid substrate of the topoisomerase polypeptide.
  • polypeptide in the complex of the invention can be a variant of a wild type topoisomerase polypeptide.
  • Variant(s) of polynucleotides or polypeptides are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. With reference to polynucleotides, generally, differences are limited such that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.
  • changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type, a variant will encode a polypeptide with the same amino acid sequence as the reference. Also as noted below, changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • variants are closely similar overall and, in many region, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Ligand refers to molecules or ions which bind or interact specifically with a topoisomerase polypeptide or a polynucleotide forming a complex with a topoisomerase polypeptide, including, for example enzyme substrates, such as supercoiled DNA, cell membrane components and small molecules.
  • Ligands also include antibodies and antibody-derived reagents that bind specifically to a member selected from the polypeptide, the nucleic acid complexed to the polypeptide or combinations thereof.
  • Identity is a relationships between two polypeptide sequences or two polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between two polypeptide or two polynucleotide sequences as determined by the match between two strings of such sequences. Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al, Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. MoI. Biol. 215: 403 (1990)).
  • Topicalisomerase includes but is not limited to enzymes that are members of the topoisomerase type I, II, III, or IV groups, e.g. bacterial gyrase.
  • Members of type II topoisomerase family include DNA gyrase, bacterial DNA topoisomerase IV, T-even phage DNA topoisomerases, eukaryotic DNA topoisomerase II, and thermophilic topoisomerase II from Sulfolobus acidocaldarius (see: A. Kikuchi et al., Syst. Appl. Microbiol. 7: 72-78 (1986); J. Kato et al., J. Biol. Chem.
  • the present invention advances type II topoisomerase function by determining the structure of DNA-binding and cleavage core of a type HA topoisomerase bound to prospective G-segment DNA.
  • the present invention is concerned with identifying or obtaining agent compounds (especially inhibitors of the DNA binding domain of type IIA topoisomerase) for inhibiting topoisomerase HA activity, and in preferred embodiments identifying or obtaining actual agent compounds/inhibitors.
  • Crystal structure information presented herein is useful in designing potential inhibitors and modeling them or their potential interaction with the DNA binding domain of type IIA topoisomerase.
  • Potential inhibitors may be brought into contact with type HA topoisomerase to test for their ability to interact with the type IIA DNA binding domain.
  • Actual inhibitors may be identified from among potential inhibitors synthesized following design and model work performed in silico.
  • An inhibitor identified using the present invention may be formulated into a composition, for instance a composition comprising a pharmaceutically acceptable excipient, and may be used in the manufacture of a medicament for use in a method of treatment.
  • the present invention is at least partly based on overcoming several technical hurdles involved with the production of crystals of a topoisomerase /nucleic acid complex, e.g. a type IIA topoisomerase/DNA complex.
  • the complex is of suitable quality for performing X-ray diffraction analyses.
  • X-ray diffraction data is collected from the complex.
  • the three-dimensional structures of the complexes is determined.
  • the information is used to identify new ligands that are better inhibitors of a topoisomerase, e.g. the editing domain of a type IIA topoisomerases.
  • a first aspect of the invention provides a co-crystal type IIA topoisomerase along with.
  • the crystal has the three dimensional atomic coordinates of FIG. 5.
  • An advantageous feature of the structural data according to FIG. 5 is that they have a high resolution. In an exemplary embodiment, the resolution is about 3 Angstroms.
  • the present invention provides, for the first time, complexes of a topoisomerase and a nucleic acid in crystalline form.
  • the crystals are appropriate for structure determination by X-ray crystallography or another method (e.g., NMR or EM).
  • the crystals are of use in elucidating the structure of a topoisomerase-DNA complex and this complex when it has a ligand bound to a component of the complex.
  • the crystals are of use in the discovery of drugs modulating topoisomerase activity, and, ideally, expediting this process.
  • the invention provides a composition comprising, in crystalline form, a complex between a nucleic acid and a polypeptide.
  • the polypeptide has topoisomerase activity, or includes at least one region that has at least 90% sequence identity with a topoisomerase.
  • the polypeptide comprises a nucleic acid-binding and cleavage domain having at least 90% sequence identity with a wild type topoisomerase nucleic acid-binding and cleavage domain of a topoisomerase polypeptide.
  • the nucleic acid-binding and cleavage domain is a G-segment binding domain of a topoisomerase polypeptide.
  • the polypeptide can be from essentially any species and can be expressed in any convenient expression system without limitation.
  • the polypeptide is a Saccharomyces polypeptide.
  • the polypeptide is a wild type topoisomerase polypeptide, or a portion thereof, and is a Saccharomyces polypeptide.
  • the nucleic acid can be of essentially any structure with the proviso that it forms a complex with the polypeptide such that the resulting complex can be crystallized.
  • an exemplary nucleic acid component of the crystalline complexes of the invention is a nucleic acid having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 1, SEQ. ID.
  • the invention provides a complex as described herein above, in which the complex further comprises a ligand interacting with a component of the complex, which is selected from the polypeptide, the nucleic acid and combinations thereof.
  • ligands are small molecules, having a molecular weight of less than about 700 Daltons, less than 600 Daltons, less than 500 Daltons, or less than 400 Daltons.
  • Exemplary small molecules include but are not limited to podophyllotoxins and camptothecan analogs, which are also known as topoisomerase inhibitors and are used in certain types of chemotherapy. Additional examples include but are not limited to the anthracycline antibiotics, e.g. doxorubicin (DXR) and analogs thereof.
  • DXR doxorubicin
  • An exemplary polypeptide-nucleic acid, e.g. DNA, complex of the invention has a crystal lattice in an 1222 space group.
  • the invention provides a composition comprising, in crystalline form, a complex between a nucleic acid and a polypeptide.
  • the polypeptide comprises a nucleic acid-binding and cleavage domain.
  • An exemplary component of the crystalline complexes of the invention is a nucleic acid-binding and cleavage domain having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 2, SEQ. ID. NO.: 2 A and SEQ. ID. NO.: 3.
  • An exemplary component of the crystalline complexes of the invention diffracts X-rays for a determination of structure coordinates to a resolution greater than or equal to 5.0 Angstroms, 4.0 Angstroms or 3.0 Angstroms.
  • Topoisomerases in general consist of at least three functional domains: a GHKL type ATPase region ("ATPase"), a Mg 2+ ion binding TOPRIM fold (also known as the "B'" domain), and a DNA binding region that contains a winged-helix domain and the active site tyrosine used for DNA cleavage (the "A" region).
  • ATPase GHKL type ATPase region
  • Mg 2+ ion binding TOPRIM fold also known as the "B'” domain
  • A DNA binding region that contains a winged-helix domain and the active site tyrosine used for DNA cleavage
  • the ATPase and the B' domains reside in the GyrB or ParE subunits, while the DNA binding elements are all required for full topoisomerase activity.
  • all domains are linked into a single polypeptide chain in the order: N-terminus-ATPase-B'-DNA binding -C -terminus.
  • the ATPase, B' and DNA binding elements are all required for full topoisomerase activity.
  • the B' and DNA binding elements (sometimes referred to as the BVA' region or DNA binding and cleavage core) are necessary and sufficient for DNA binding and cleavage.
  • Some type IIA toposisomerases are appended with auxiliary DNA binding or regulatory domains at the C-terminus of their A regions; removing these domains or elements creates and "A;" fragment still capable of binding and cleaving DNA.
  • the A' domain still supports type II topo strand passage activity in conjunction with functional ATPase and B' domains (Caron et al, 1994, Corbett et al, 2005; Kampranis and Maxwell, 1996).
  • the reaction cycle for type IIA topoisomerases is believed to employ a "two-gate" mechanism for strand passage (FIG. 4) (Roca, J., Berger, J. M., Harrison, S. C. & Wang, J. C. DNA transport by a type II topoisomerase: direct evidence for a two-gate mechanism. Proc. Natl Acad. Sci. USA 93, 4057-4062 (1996)).
  • the T-segment enters through a protein gate controlled by the reversible, dimerization of two ATPase domains on one side of the enzyme (the N-gate), and exits through a C-terminal dimerization interface on the other (the C-gate).
  • Passage of the T-segment through the cleaved G-segment is further coordinated by a third dimer interface (the DNA-gate) in the interior of the topoisomerase, formed by two winged-helix domains (WHDs) that contain the catalytic tyrosines responsible for DNA cleavage and separation (Williams, N. L. & Maxwell, A. Probing the two-gate mechanism of DNA gyrase using cysteine cross-linking. Biochemistry 38, 13502-13511 (1999)).
  • WTDs winged-helix domains
  • a metal-binding TOPRIM domain composed of a triad of conserved acidic residues is believed to assist formation of the covalent protein»DNA intermediate by coordinating magnesium ions essential for DNA cleavage (Berger, J. M., et al., Structure and mechanism of DNA topoisomerase II. Nature 379, 225-232 (1996); Aravind, L., et al., TOPRIM— a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins. Nucleic Acids Res. 26, 4205-4213 (1998); Noble, C. G. & Maxwell, A., The role of GyrB in the DNA cleavage-religation reaction of DNA gyrase: a proposed two metal-ion mechanism. J. MoI. Biol. 318, 361-371 (2002)).
  • Crystal Structure The reaction cycle for type HA topoisomerases is believed to employ a "two-gate" mechanism for strand passage 4-7 (FIG. 4).
  • the T- segment enters through a protein gate controlled by the reversible, dimerization of two ATPase domains on one side of the enzyme (the N-gate), and exits through a C-terminal dimerization interface on the other (the C-gate).
  • Passage of the T-segment through the cleaved G-segment is further coordinated by a third dimer interface (the DNA-gate) in the interior of the topoisomerase, formed by two winged-helix domains (WHDs) that contain the catalytic tyrosines responsible for DNA cleavage and separation.
  • WTDs winged-helix domains
  • a metal-binding TOPRIM domain composed of a triad of conserved acidic residues, is believed to assist formation of the covalent protein»DNA intermediate by coordinating magnesium ions essential for DNA cleavage.
  • TOPRIM folds also form part of the catalytic centers of type IA topoisomerases and bacterial primases.
  • type II topoisomerase function remains unresolved.
  • type HA topoisomerases are known to preferentially associate with DNA crossovers and the apices of plectonemic supercoils, yet the molecular basis for this discrimination is not understood (Keck, J. L., Roche, D. D., Lynch, A. S. & Berger, J. M. Structure of the RNA polymerase domain of E. coli primase. Science 287, 2482-2486 (2000); Howard, M. T., Lee, M. P., Hsieh, T. S. & Griffith, J. D. Drosophila topoisomerase II-DNA interactions are affected by DNA structure. J. MoI.
  • the present invention provides, in an exemplary embodiment, the structure of the DNA-binding and cleavage core of a type HA topoisomerase (Topo II) bound to prospective G-segment DNA.
  • the crystals are appropriate for structure determination by X-ray crystallography or another method (e.g., NMR or EM).
  • the crystals are of use in elucidating the structure of a topoisomerase- DNA complex crystals.
  • the structures are of use in the discovery of drugs modulating topoisomerase activity.
  • An exemplary S. cerevisiae Topo II fragment of use in the present invention contains the metal-binding TOPRIM fold and primary DNA-binding domain (FIG. IA), and can associate with and cleave DNA in vitro (FIG. 5). It is noted that the topoisomerase sequences are also intended to encompass isoforms, mutants and fusion proteins of these sequences.
  • An exemplary fusion protein includes a 6 residue [N] -terminal tag (6 residues are histidine) that may be used to facilitate purification of the protein.
  • the structure is determined by X-ray crystallography to 3 Angstrom resolution in complex with a double-stranded DNA oligonucleotide containing complementary, four-base overhangs (FIG. 6).
  • the Topo II DNA binding and cleavage core forms a 180 kDa dimer with overall dimensions of 100 ⁇ 105 ⁇ 380 Angstroms.
  • the largest inter-subunit contacts occur through a predominantly polar and electrostatic set of interactions between the TOPRIM domain of one subunit and the DNA-binding domain of the other, burying -2150 Angstroms of solvent- accessible surface area per protomer.
  • a previously disordered linker region (amino acids 656-674) connecting the TOPRIM and WHD elements is now clearly visible, forming a short three-helix bundle that makes extensive contacts with both DNA and the rest of the protein.
  • DNA binding characteristics Each 15 bp DNA oligonucleotide duplex binds a single Topo II protomer. Contacts to the DNA are established within the confines of a large, positively charged groove that spans the width of the dimer and buries -2,900 Angstroms 2 of DNA surface area. The binding orientation of the DNA allows base pairing to occur between the four-base overhangs in the central portion of the DNA-binding site, generating a continuous, but doubly nicked, 34 bp duplex that crosses the enzyme dyad symmetry axis (FIG. 2; FIG. 6). The blunt duplex DNA ends emanating from each Topo II dimer stack against a second complex to form a compact 68 bp DNA circle in the crystal lattice.
  • the invention provides a method of forming a crystal.
  • the method is used to form a crystallization volume.
  • the crystallization volume comprises a precipitant solution.
  • the crystallization volume comprises a complex.
  • the complex is between a nucleic acid and a polypeptide.
  • the nucleic acid can be of essentially any structure with the proviso that it forms a complex with the polypeptide such that the resulting complex can be crystallized.
  • An exemplary nucleic acid component of the crystalline complexes of the invention is a nucleic acid having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 1, SEQ. ID. NO.: IA, SEQ. ID. NO.: IB, SEQ. ID. NO. 1C, a complement of SEQ. ID. NO.: 1, a complement of SEQ. ID.
  • the nucleic acid may include additional nucleic acid sequences added to the ends of a nucleic acid with a member selected from SEQ. ID. NO.: 1, SEQ. ID. NO.: IA, SEQ. ID. NO.: IB, SEQ. ID. NO. 1C, a complement of SEQ. ID. NO.: 1, a complement of SEQ. ID. NO.: IA, a complement of SEQ. ID. NO. IB, and a complement of SEQ. ID. NO.: 1C.
  • the additional nucleic acid sequences may be between 1 and 5 nucleotides in length.
  • the polypeptide has at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 2, SEQ. ID. NO.: 2A and SEQ. ID. NO.: 3.
  • the polypeptide is a topoisomerase polypeptide.
  • the polypeptide has a nucleic acid-binding and cleavage domain.
  • the polypeptide has a G-segment binding domain.
  • the G-segment binding domain is that of a wild type topoisomerase polypeptide.
  • the G-segment binding domain is a variant of a wild-type topoisomerase peptide.
  • the polypeptide is selected from the wild type DNA- binding and cleavage domain of a Saccharomyces topoisomerase.
  • the polypeptide shares at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98% or about 100% sequence identity to a topoisomerase from S. cerevisiae.
  • the polypeptide is selected from the polypeptides of insects, birds, fish, and mammals.
  • the polypeptide is selected from human polypeptides.
  • An exemplary component of the crystalline complexes of the invention diffracts X-rays for a determination of structure coordinates. In an exemplary embodiment the resolution is greater than or equal to 5.0 Angstroms, 4.0 Angstroms or 3.0 Angstroms. In another exemplary embodiment the crystallization volume is stored under conditions suitable for crystal formation of the polypeptide nucleic acid complex.
  • An aspect of the present invention is a method of identifying ligands of a topoisomerase polypeptide.
  • the polypeptide is complexed with a nucleic acid.
  • the crystals of a polypeptide complexed with a nucleic acid are soaked in a solution.
  • the solution contains a collection of compounds.
  • the compounds are generated in situ.
  • the compounds are generated separately from the crystal.
  • the solution is prepared without purification of the collection of compounds.
  • X-ray crystal diffraction pattern is used to identify a compound bound to the soaked crystal.
  • the polypeptide has at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 2, SEQ. ID. NO.: 2A and SEQ. ID. NO.: 3.
  • the nucleic acid has at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 1, SEQ. ID. NO.: IA, SEQ. ID. NO.: IB, SEQ. ID. NO. 1C, a complement of SEQ. ID. NO.: 1, a complement of SEQ. ID.
  • the polypeptide has a nucleic acid-binding and cleavage domain.
  • the method forms a crystal comprising a ligand bound to the polypeptide.
  • the crystal comprises a ligand bound to the nucleic acid. In another exemplary embodiment, the crystal comprises a ligand bound to both the nucleic acid and the polypeptide.
  • An aspect of the present invention is a method of identifying a compound which is a potential inhibitor of a polypeptide with topoisomerase activity.
  • the invention includes crystallizing a crystal of a polypeptide comprising a nucleic acid-binding and cleavage domain of a topoisomerase polypeptide complexed to a nucleic acid.
  • the invention includes obtaining the atomic coordinates of the crystal of the topoisomerase polypeptide-nucleic acid complex.
  • the atomic coordinates are used to define potential ligand binding sites of topoisomerase.
  • the invention provides a method of using the atomic coordinates to define potential ligand binding sites of the polypeptide-nucleic acid complex.
  • the topoisomerase polypeptide has at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 2, SEQ. ID. NO.: 2 A and SEQ. ID. NO.: 3.
  • the nucleic acid has at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a member selected from SEQ. ID. NO.: 1, SEQ. ID. NO.: IA, SEQ. ID.
  • compounds are identified which bind to one or more potential ligand binding site.
  • the compound is a potential inhibitor of a polypeptide with topoisomerase activity.
  • the topoisomerase polypeptide comprises a polypeptide sequence according to SEQ. ID. NO.: 2 or SEQ. ID. NO.: 2 A.
  • the polypeptide comprises a sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a sequence according to SEQ. ID. NO.: 2 or SEQ. ID. NO.: 2A.
  • the topoisomerase crystalline complex can be used to identify ligands that are potential inhibitors.
  • ligands are small molecules, having a molecular weight of less than about 2500, less than 1000, less than 700 Daltons, less than 600 Daltons, less than 500 Daltons, or less than 400 Daltons.
  • Exemplary small molecules include but are not limited to podophyllotoxins and camptothecan analogs, which are also known as topoisomerase inhibitors and are used in certain types of chemotherapy. Additional examples include but are not limited to the anthracycline antibiotics, e.g. doxorubicin (DXR) and analogs thereof.
  • DXR doxorubicin
  • antineoplastic anthracedediones e.g. mitoxantrone (MXN), an anthracenedione with significant cytostatic activity against a number of experimental tumors and human malignancies (Alberts, D. S., et al., Phase I clinical and pharmacokinetic study of mitoxantrone given to patients by intraperitoneal administration. Cancer Res. 48: 5874- 5877(1988)).
  • Another exemplary embodiment of the invention involves assessing the ability of a compound to inhibit a topoisomerase peptide.
  • Suitable methods can be used to identify chemical moieties, fragments or functional groups which are capable of interacting favorably with a topoisomerase peptide. These methods include, but are not limited to: interactive molecular graphics; molecular mechanics; conformational analysis; energy evaluation; docking; database searching; pharmacophore modeling; de novo design and property estimation. These methods can also be employed to assemble chemical moieties, fragments or functional groups into a single inhibitor molecule.
  • Interactive molecular modeling techniques can be applied by one skilled in the art to visually inspect the quality of the fit of a candidate inhibitor modeled into the binding site. Suitable visualization programs include INSIGHTII (Molecular Simulations Inc., San Diego, Calif), QUANTA (Molecular Simulations Inc., San Diego, Calif), SYBYL (Tripos Inc., St Louis, Mo.), RASMOL (Roger Sayle et al., Trends Biochem. Sci.
  • a polypeptide of known structure is used to identify ligands that are potential inhibitors of a polypeptide of unknown structure.
  • the atomic coordinates of the crystal of the polypeptide-nucleic acid complex are obtained.
  • the atomic coordinates are used to define potential ligand binding sites of topoisomerase.
  • a compound which binds to one or more of the potential ligand binding sites is identified.
  • the ligand is a potential inhibitor of a topoisomerase polypeptide, is identified.
  • the topoisomerase polypeptide comprises a polypeptide sequence according to SEQ. ID. NO.: 2.
  • the polypeptide comprises a sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a sequence according to SEQ. ID. NO.: 2.
  • the method of the current invention is used to identify a ligand of a topoisomerase polypeptide complexed with a nucleic acid.
  • An exemplary embodiment comprises soaking one or more crystals of a nucleic acid-binding and cleavage domain of a topoisomerase polypeptide complexed with a nucleic acid in a solution containing a collection of compounds.
  • the compounds are generated in situ or separate from the crystal.
  • Another exemplary embodiment comprises obtaining an X-ray crystal diffraction pattern of the soaked crystal.
  • Another exemplary embodiment comprises using the X-ray crystal diffraction pattern to identify a compound bound to the soaked crystal.
  • Another exemplary embodiment comprises the compound being a ligand of the topoisomerase polypeptide complexed with the nucleic acid.
  • the solution containing a collection of compounds generated in situ or separate from the crystal is prepared without purification of the collection of compounds.
  • the method of the current invention is used to identify a ligand of a topoisomerase polypeptide complexed with a nucleic acid.
  • the method of the current invention comprises soaking one or more crystals of a nucleic acid-binding and cleavage domain of a topoisomerase polypeptide having at least a 90% sequence identity with a member selected from SEQ. ID. NO.: 2, SEQ. ID. NO.: 2A and SEQ. ID. NO.: 3 complexed to a nucleic acid having at least a 90% sequence identity with a member selected from SEQ. ID. NO.: 1, SEQ. ID. NO.: IA, SEQ. ID. NO.: IB, SEQ.
  • the compounds are generated in situ or separate from the crystal.
  • Another embodiment comprises obtaining an X-ray crystal diffraction pattern of the soaked crystal.
  • Another embodiment comprises using the X-ray crystal diffraction pattern to identify any compound bound to the soaked crystal, the compound being a ligand of the topoisomerase polypeptide complexed with the nucleic acid.
  • the topoisomerase polypeptide comprises a polypeptide sequence according to SEQ. ID.
  • the polypeptide comprises a sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or about 100% sequence identity with a sequence according to SEQ. ID. NO.: 2 or SEQ. ID. NO.: 2A.
  • 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.
  • polypeptide may be produced in whole or in part by chemical synthesis.
  • selection of amino acids available for substitution or addition is not limited to the genetically encoded amino acids. Indeed, mutants may optionally contain non-genetically encoded amino acids.
  • Conservative amino acid substitutions for many of the commonly known non-genetically encoded amino acids are well known in the art. Conservative substitutions for other amino acids can be determined based on their physical properties as compared to the properties of the genetically encoded amino acids.
  • the gene encoding topoisomerase can be isolated from RNA, cDNA or cDNA libraries. In this case, the portion of the gene encoding amino acid residues 408-1117 was isolated and is shown as SEQ. ID NO.: 2. It is noted that the gene was modified to include a [N]-terminal hexa-histidine tag.
  • Construction of expression vectors and recombinant proteins from the DNA sequence encoding topoisomerase may be performed by various methods well known in the art. For example, these techniques may be performed according to Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor, N. Y. (1989), and Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, Stockton Press, New York (1990).
  • topoisomerase A variety of expression systems and hosts may be used for the expression of topoisomerase. Once expressed, purification steps are employed to produce topoisomerase in a relatively homogeneous state. Typical purification methods include the use of centrifugation, partial fractionation, using salt or organic compounds, dialysis, conventional column chromatography, (such as ion exchange, molecular sizing chromatography, etc.), high performance liquid chromatography (HPLC), and gel electrophoresis methods (see, e.g., Deutcher, "Guide to Protein Purification” in Methods in Enzymology (1990), Academic Press, Berkeley, Calif).
  • Topoisomerase may optionally be affinity labeled during cloning, preferably with a N-terminal six-histidine tag and rTev cleavage site, in order to facilitate purification.
  • affinity label With the use of an affinity label, it is possible to perform a one-step purification process on a purification column that has a unique affinity for the label.
  • the affinity label may be optionally removed after purification.
  • Crystallization In an exemplary embodiment, crystallization is performed in volumes commonly used in the art, for example typically 15, 10, 5, 2 microliters or less. It is noted that the crystallization volume optionally has a volume of less than 1 microliter, optionally 500, 250, 150, 100, 50 nanoliters or less.
  • crystallization may be performed by any crystallization method including, but not limited to batch, dialysis and vapor diffusion (e.g., sitting drop and hanging drop) methods. Micro, macro and/or streak seeding of crystals may also be performed to facilitate crystallization. Crystallizations may be performed by batch, dialysis, and vapor diffusion (sitting drop and hanging drop) methods.
  • a detailed description of basic protein crystallization setups may be found in McRee, D., Practical Protein Crystallography, 2.sup.nd Ed. (1999), Academic Press Inc. Further descriptions regarding performing crystallization experiments are provided in Stevens, et al. (2000) Curr. Opin. Struct. Biol.: 10(5):558-63, and U.S. Pat. Nos. 6,296,673, 5,419,278, and 5,096, 676.
  • crystals comprising topoisomerase and crystals comprising topoisomerase polypeptide according to the invention are not intended to be limited to the wild type, full length topoisomerase shown in SEQ. ID No. 3, fragments comprising residues of SEQ. ID NO.: 3. Rather, it should be recognized that the invention may be extended to various other fragments and variants of wild-type topoisomerase as described above. For example, several variants of the topoisomerase II polypeptide have shown resistance or hypersensitivity to inhibitors and are thus useful in understanding drug interactions. It should be understood the present invention encompasses crystal structures of these variants as well, nucleic acid complexes of these variants and crystal and nucleic acid- ligand complexes of these variants.
  • topoisomerase is optionally complexed with one or more ligands and one or more copies of the same ligand.
  • the ligand used to form the complex may be any ligand capable of binding to topoisomerase.
  • the ligand is a natural substrate.
  • the ligand is an inhibitor.
  • crystals comprising topoisomerase are formed by mixing substantially pure topoisomerase with an aqueous buffer containing a precipitant at a concentration just below a concentration necessary to precipitate the protein.
  • a precipitant for crystallizing topoisomerase is polyethylene glycol (PEG), which combines some of the characteristics of the salts and other organic precipitants (see, for example, Ward et al., J. MoI. Biol. 98:161, 1975, and McPherson, J. Biol. Chem. 251 :6300, 1976).
  • crystallization water is removed by diffusion or evaporation to increase the concentration of the precipitant, thus creating precipitating conditions for the protein.
  • crystals are grown by vapor diffusion in hanging drops or sitting drops. According to these methods, a protein/precipitant solution is formed and then allowed to equilibrate in a closed container with a larger aqueous reservoir having a precipitant concentration for producing crystals. The protein/precipitant solution continues to equilibrate until crystals grow.
  • Crystals comprising topoisomerase have a wide range of uses. For example, now that crystals comprising topoisomerase have been produced, it is noted that crystallizations may be performed using such crystals as a nucleation site within a concentrated protein solution. According to this variation, a concentrated protein solution is prepared and crystalline material (microcrystals) is used to "seed" the protein solution to assist nucleation for crystal growth. If the concentrations of the protein and any precipitants are optimal for crystal growth, the seed crystal will provide a nucleation site around which a larger crystal forms.
  • the crystals so formed can be used by this crystallization technique to initiate crystal growth of other topoisomerase comprising crystals, including topoisomerase complexed to other ligands.
  • Crystals may also be used to perform X-ray or neutron diffraction analysis in order to determine the three-dimensional structure of topoisomerase and, in particular, to assist in the identification of its active site.
  • Knowledge of the binding site region allows rational design and construction of ligands including inhibitors. Crystallization and structural determination of topoisomerase variants having altered bioactivity allows the evaluation of whether such changes are caused by general structure deformation or by side chain alterations at the substitution site.
  • Computational modeling Various computational methods may be used to determine whether a particular protein or a portion thereof (referred to here as the "target protein"), typically the binding pocket, has a high degree of three-dimensional spatial similarity to another protein (referred to here as the "reference protein") against which the target protein is being compared.
  • target protein typically the binding pocket
  • reference protein another protein
  • Equivalent residues or atoms can be determined based upon an alignment of primary sequences of the proteins, an alignment of their structural domains or as a combination of both. Sequence alignments generally implement the dynamic programming algorithm of Needleman and Wunsch (J. MoI. Biol. 48: 442-453, 1970).
  • a rigid body fitting operation is performed where the structure for the target protein is translated and rotated to obtain an optimum fit relative to the structure of the reference protein.
  • the fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square deviation of the fit over the specified pairs of equivalent atoms is an absolute minimum.
  • MOE Computer Computing Group Inc.
  • RMSD root mean square deviation
  • an RMSD value refers to a calculated value based on variations in the atomic coordinates of a reference protein from the atomic coordinates of a reference protein or portions of thereof.
  • the structure coordinates for topoisomerase, provided in FIG. 3, are used as the reference protein in these calculations.
  • RMSD root mean square deviation
  • the three-dimensional crystal structure of the present invention may be used to identify topoisomerase binding sites, be used as a molecular replacement model to solve the structure of unknown crystallized proteins, to design mutants having desirable binding properties, and ultimately, to design, characterize, identify chemical compounds or structures capable of interacting with topoisomerase and other structurally similar proteins as well as other uses that would be recognized by one of ordinary skill in the art.
  • Such compounds may be chemical or protein in nature.
  • chemical compounds refers to chemical compounds, complexes of at least two chemical compounds, and fragments of such compounds.
  • topoisomerase structure coordinates are useful for screening and identifying drugs that inhibit topoisomerase and other structurally similar proteins.
  • the structure encoded by the data may be computationally evaluated for its ability to associate with putative substrates or ligands. Such compounds that associate with topoisomerase may inhibit topoisomerase, and are potential drug candidates.
  • the structure encoded by the data may be displayed in a graphical three- dimensional representation on a computer screen. This allows visual inspection of the structure, as well as visual inspection of the structure's association with the compounds.
  • the computer model may not necessarily directly use the structure coordinates. Rather, a computer model can be formed that defines a surface contour that is the same or similar to the surface contour defined by the structure coordinates.
  • the structure coordinates provided herein can also be utilized in a method for identifying a ligand of a protein comprising an topoisomerase-like binding pocket.
  • One embodiment of the method comprises: using all or a portion of the structure coordinates provided herein to generate a three-dimensional structure of an topoisomerase -like binding pocket; employing the three-dimensional structure to design or select a potential ligand; synthesizing the potential ligand; and contacting the synthesized potential ligand with a protein comprising an topoisomerase -like binding pocket to determine the ability of the potential ligand to interact with protein.
  • DNA binding and cleavage domain was expressed in yeast and subsequently purified using hexa-histidine affinity tag, ion exchange, and size exclusion chromatography.
  • topo II construct An open reading frame encoding the DNA- binding and cleavage core of S. cerevisiae topoisomerase II (topo II ⁇ NC, aa 408-1177) was amplified by PCR using primers to add an N-terminal hexa-histidine tag and an accompanying TEV protease site. This DNA was cloned into a pGAL vector used for expressing full-length topo II (pGlT2)39, and the resulting plasmid transformed in BCY123 yeast cells.
  • topo II ⁇ NC S. cerevisiae topoisomerase II
  • topo II ⁇ NC Purification of topo II ⁇ NC: For lysis, cells were crushed under liquid nitrogen using a mortar and pestle, the powder resuspended in buffer A (20 mM Tris 8.5 and 10% glycerol) plus 20 mM imidazole and 300 rnM KCl, and the lysate clarified by centrifugation. Clarified extract was loaded onto a pair of tandemly-coupled nickel affinity and cation- exchange columns (nickel Chelating SepharoseTM resin and Hi-Trap S, GE-Amersham Biosciences), which were then washed with buffer A plus 20 mM imidazole and 300 mM KCl to remove unbound proteins.
  • buffer A (20 mM Tris 8.5 and 10% glycerol) plus 20 mM imidazole and 300 rnM KCl
  • Clarified extract was loaded onto a pair of tandemly-coupled nickel affinity and cation- exchange columns (nickel Chelating SepharoseTM resin and Hi-
  • Topo II ⁇ NC was eluted from the nickel column directly onto the ion-exchange column using buffer A plus 100 mM KCl and 200 mM imidazole, after which the columns were uncoupled, and the protein eluted from the ion-exchange column in Buffer A using a 100-500 mM KCl gradient.
  • Purified enzyme fractions were pooled, mixed with His6-tagged, TEV-protease 40 at a 10:1 molar ratio of protein:protease, and incubated at 4 0 C overnight while concentrating in an Amicon Centriprep-30 concentrator to remove the His-tag.
  • TEV protease and uncleaved topo II were removed by passing the concentrated solution over a second nickel-affinity column Buffer A with 500 mM KCl and 20 mM imidazole.
  • the flow-through from this step (which contained TEV-cleaved topo II ⁇ NC) was further purified over an S300 sizing column (GE Amersham) in 500 mM KCl, 50 mM Tris 7.5, 5 mM MgC12, and 10% glycerol, prior to concentrating and storing at 4 0 C.
  • the final concentration of topo II ⁇ NC varied, but was always >10 mg/ml. Similar to ATP-free topo II, the purified topo II ⁇ NC fragment protein exhibits a low but demonstrable DNA cleavage activity in vitro (FIG. 5).
  • Oligonucleotides were ordered from Operon Biosciences, resuspended in 100% formamide, and run on a 8.75 M urea, 10% polyacrylamide gel for 1 hr at 350 V. DNA bands were identified by UV-shadowing, cut out, and the separated oligonucleotides eluted by crushing and soaking in 10 mM magnesium acetate, 500 mM ammonium acetate, and 1 mM EDTA overnight at room temperature.
  • DNA/protein complexes were formed by dialysis and crystals grown by hanging drop vapor diffusion.
  • 7.5 mg/mL of purified topo II ⁇ NC was first mixed with 1 mM freshly prepared sodium orthovanadate (Sigma) and annealed DNA duplexes at a 1 :1.3 monomer:DNA ratio in a starting buffer of 50 mM Tris 7.5, 500 mM KCl, and 2 mM MgC12.
  • Sodium orthovanadate was added in an attempt to capture a transition state complex 41, but examination of electron density maps revealed no evidence for vanadate at the 3 '-5' junctions between oligonucleotide ends.
  • Vandate-treated topo II ⁇ NC'DNA solutions were dialyzed step-wise against 50 mM Tris 7.5, 2 mM MgC12, and decreasing amounts of KCl (400-200 mM) using IK micro DispoDI AL YZERs (Harvard) to a final solution of 200 mM KCl, 50 mM Tris (pH 7.5), 2 mM MgC12 over the course of 18 h at 4 0 C.
  • a high-molecular weight osmolyte 2.5 % (w/v) PEG 5000 monomethyl ether was also included in the dialysis buffer to minimize sample dilution.
  • the dialyzed sample was mixed with well solution (12-20% PEG-1000, 100-250 mM MgC12, and 100 mM sodium cacodylate pH 7.0) at a 1 :1 ratio, and subjected to hanging drop vapor diffusion at 4 0 C.
  • well solution (12-20% PEG-1000, 100-250 mM MgC12, and 100 mM sodium cacodylate pH 7.0) at a 1 :1 ratio
  • rod-shaped crystals belonging to the space group 1222 grew in heavy precipitate; despite extensive attempts to optimize growth, only 5-10 screenable crystals of variable quality could be obtained from 10-15 mg of starting material out of each 6 L yeast prep. Crystals were harvested by transferring the crystals into 25% glycerol plus well solution prior to looping and flash- freezing in liquid nitrogen.
  • the structure was solved by molecular replacement (MR) with PHASER, as implemented under the CCP4i program suite, using the individual structures of the core DNA binding and dimerization domain of S. cerevisiae topo II (PDB ID: IBGW, amino acids 683-1009 and 1151-1178) 8 and the conserved secondary structural elements of the TOPRIM fold of E. coli topoisomerase III (PDB ID: 1I7D, amino acids 2-39 and 82-141) as search models.
  • MR molecular replacement
  • PHASER molecular replacement

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Abstract

La présente invention concerne des cristaux d'un polypeptide de topoisomérase et d'un acide nucléique. Les cristaux sont utilisés pour élucider la structure de complexes topoisomérase-acide nucléique. De plus, les cristaux sont utilisés dans les découvertes de ligands qui interagissent avec les complexes topoisomérase-acide nucléique et modulent l'activité de tels complexes par cette interaction.
PCT/US2008/066194 2007-06-06 2008-06-06 Complexes topoisomérase/adn cristallins WO2008154432A2 (fr)

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TWI461433B (zh) * 2011-06-20 2014-11-21 Univ Nat Taiwan 人類第二型拓樸異構酶二元複合體之晶體、其製備方法及其應用

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130018180A1 (en) * 2011-06-20 2013-01-17 National Taiwan University Crystals of human topoisomerase ii-dna binary complex, methods for preparing the same and uses thereof
US8765443B2 (en) * 2011-06-20 2014-07-01 National Taiwan University Crystals of human topoisomerase II-DNA binary complex, methods for preparing the same and uses thereof
TWI461433B (zh) * 2011-06-20 2014-11-21 Univ Nat Taiwan 人類第二型拓樸異構酶二元複合體之晶體、其製備方法及其應用

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