EP1622934A2 - Menschliche dbf4 verkürzte formen, komplexe mit ihren interaktionspartnern und verfahren zur identifizierung ihrer inhibitoren - Google Patents

Menschliche dbf4 verkürzte formen, komplexe mit ihren interaktionspartnern und verfahren zur identifizierung ihrer inhibitoren

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Publication number
EP1622934A2
EP1622934A2 EP04728359A EP04728359A EP1622934A2 EP 1622934 A2 EP1622934 A2 EP 1622934A2 EP 04728359 A EP04728359 A EP 04728359A EP 04728359 A EP04728359 A EP 04728359A EP 1622934 A2 EP1622934 A2 EP 1622934A2
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EP
European Patent Office
Prior art keywords
complex
dbf4
cdc7
cell
protein
Prior art date
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EP04728359A
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English (en)
French (fr)
Inventor
Barbara Valsasina
Cinzia Cristiani
Stefan Knapp
Sandrine Thieffine
Rita Perego
Maria Beatrice Saccardo
Henryk Kalisz
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VALSASINA, BARBARA
Pfizer Italia SRL
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Pharmacia Italia SpA
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Application filed by Pharmacia Italia SpA filed Critical Pharmacia Italia SpA
Publication of EP1622934A2 publication Critical patent/EP1622934A2/de
Withdrawn legal-status Critical Current

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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR

Definitions

  • the present invention generally relates to the design, expression and purification of truncated forms of the human dbf4 protein, optionally in complex with dbf4 interacting partners, such as human cdc7, and its use in the discovery, identification and characterization of inhibitors of the components of the complex, either when associated or when present as uncomplexed proteins in situ or in vitro. More specifically, the present invention relates to the utilization of specific truncated forms of human dbf4 for the production of an active, non -aggregated, monodispcrsed heterodimer of human dbf4 complcxcd to human cdc7, or other protein intcractors.
  • Replication of the cukaryotic genome is a highly coordinated process during which DNA is precisely duplicated during cell division.
  • DNA duplication is initiated at hundreds of chromosomal elements called origins of replication.
  • the process is strictly controlled at the multiple origins of replication during the cell cycle and involves the assembly of multiprotcin complexes that eventually lead to the formation of two replication forks at each origin.
  • sequences required for an origin of replication vary significantly between the different cukaryotic organisms, the identity and order of assembly of the replication factors is highly conserved from yeast to mammals (sec Kcll y & Brown, 2000, Annu. Rev. Biochcm. 69, 829 -881; Bell & Dutta, 2002, Annu. Rev. Biochem. 71, 333 -374, and references therein).
  • Eukaryotic origins of replication direct the formation of a number of protein complexes leading to the assembly of two bidircct ional DNA replication forks (Takisawa et al., 2000, Curr. Opin. Cell Biol., 12, 690 -696; Bell & Dutta, 2002, Annu. Rev. Biochcm.
  • Prc-RC prc-rcplicativc complex
  • ORC origin recognition complex
  • MCM minichromosomc maintenance
  • the first protein that binds to the origin is the hcxamcric ORC (Bell & Stillman, 1992, Nature, 357, 128 -134), to allow the sequential recruitment of CDC6, Cdtl and MCM2 -7 (reviewed in Diffley & Labib, 2002, J. Cell Sci., 1 15, 869 -872).
  • CDC6 is displaced before or during S phase.
  • MCM proteins arc displaced from chromatin during replication in S phase, and arc not chromatin-bound in G2 phase.
  • the pre-RC is activated by at least two kinases, cyclin - dependent kinase Cdk2 (Knobat et al., 1994, Cell 77, 107 -120; Krudc et al., 1997, Cell 88, 109-119; Strausfeld et al., 1996, J. Cell Sci. 109, 1555 -1563) and dumbbell former 4 (Dbf4)-dcpcndcnt kinasc Cdc7 (Patterson et al., 1986, Mol. Cell. Biol. 6, 1590 -1598; Hollingsworth & Sclafani, 1990, Proc. Natl. Acad. Sci. U.S.A.
  • Cdc7-Dbf4 functions before and independently of Cdk2 -cyclin E (Walter, 2000, Mol. Cell. Biol. 17, 553-563; Jarcs & Blow, 2000, Genes Dev. 14, 1528 -1540), whereas in Saccharomyces cerevisiae , Cdc7-Dbf4 acts downstream of Cdk2 (Nougarcdc ct al., 2000, Mol. Cell. Biol. 20, 3795 -3806). In addition to Cdc7-Dbf4 and Cdk2, MCM 10 is required for Cdc45 loading.
  • MCM 10 binding to the prc-RC is MCM2-7-dcpcndcnt but docs not require the presence of Cdc7 or Cdk2 (Wohlschlcgcl ct al., 2002, Mol. Cell 9, 233 -240).
  • the ordered assembly of additional replication factors facilitates the unwinding of the DNA at the origin, culminating in the association of multiple cukaryotic DNA polymerases with the unwound DNA (Mimura & Takisawa, 1998, F.MBO J. 17, 5699 -5707; Walter & Newport, 2000, Mol. Cell 5, 617-627; Aparicio ct al, 1999, Proc. Natl. Acad. Sci. U.S.A. 96, 9130 -9135).
  • Cdc7-Dbf4 is an essential cell cycle -regulated kinasc complex that is structurally and functionally conserved in cukaryotcs (Masai & ⁇ rai, 2000, Biochcm. Biophys. Res. Commun. 275, 228 -232).
  • Cdc7 a scrinc/thrconinc kinasc, is activated by the binding of alternative regulatory subunits, Dbf4 and Drfl (Jiang ct al., 1999, EMBO J. 18, 5703 -5713; 5 Kumagai ct al., 1999, Mol. Cell. Biol. 19, 5083 -5095; Montagnoli ct al., 2002, EMBO J.
  • Cdc7-Dbf4 also plays an important role during checkpoint responses induced by arrested replication forks (Costanzo et al., 2000, Mol. Cell 6, 649 -659; Jarcs ct al., 2000,
  • Cdc7-Dbf4 is also involved in induced mutagcncsis alter DNA damage (Hollingsworth ct al., 1992, Genetics, 132, 53 -62; Ostroff & Sclafani, 1995, Mut. Res. 329, 143-152) and in the maintenance of sister chromatid cohesion during S phase.
  • cdc7 -dbf4 kinasc is also involved in induced mutagcncsis alter DNA damage (Hollingsworth ct al., 1992, Genetics, 132, 53 -62; Ostroff & Sclafani, 1995, Mut. Res. 329, 143-152) and in the maintenance of sister chromatid cohesion during S phase.
  • cdc7 -dbf4 kinasc is also involved in induced mutagcncsis alter DNA damage (Hollingsworth ct al., 1992, Genetics, 132, 53 -62; Ostroff & Sclafani
  • Cdc7/Dbf4 Overproduction of Cdc7/Dbf4 increases induced mutagcncsis (Sclafani ct al., 1988, Mol. Cell. Biol. 8, 293 -300), with most induced mutations being produced in S phase cells (Ostroff & Sclafani, 1995, Mut. Res. 329, 143 -152).
  • Cdc7, Dbf4 and Drfl mRNA are ovcrcxprcsscd in a number of tumor cell lines and arc obscrv cd in tumor samples of different origins (Hcss ct al., 1998, Gene 211 , 133 -140; Kumagai ct al., 1999, Mol. Cell. Biol.
  • Cdc7 inhibition represents a novel mechanism of action for blocking DNA repl ication by targeting the initiation reaction.
  • Studies in yeast demonstrate that cells with impaired cell cycle checkpoints selectively undergo cell death as a consequence of inhibition of Cdc7, offering the potential of a preferential effect on tumor cells (Dohrmann ct al., 1999, Genetics 151 , 965-977).
  • cdc7 and Dbf4 have been identified in various cukaryotcs, including human, mouse, fission and budding yeasts (see Sclafani, 2000, J. Cell Sci. 113, 21 11 -2117; Masai & Arai, 2002, J. Cell. Physiol. 190, 287-296).
  • the kinasc domains of cdc7 arc highly conserved with 45% identity between human and yeast and 60% identity between fission and budding yeast cdc7's (Jiang & Hunter, 1997, Proc. Natl. Acad. Sci. U.S.A. 94, 14320-14325).
  • cdc 7 demonstrates about 46% homology to Cdk2 and casein kinasc II (Ck2) in the kinasc domain, and is believed to have evolved from Ck2 and to have diverged from Cdk's (Hunter & Plowman, 1997, Trends Biochcm. Sci. 22, 18 -22).
  • human cdc7 has a large insertion of 159 amino acids in a region corresponding to the activation loop in kinasc structures. This insertion, which is very basic and rich in cystcinc residues, is unique to Cdc7 kinasc (Jiang & Hunter, 1997, Proc. Natl. Acad. Sci.
  • Dbf4 the regulatory subunit of cdc7, shares no sequence similarity with cyclins, the regulatory subunits of Cdk's.
  • the Dbf4 protein family is also less conserved among the cukaryotcs than the cdc7 kinases, with 1 ess than 25% identity between the homologucs from budding and fission yeasts and no evident homology between the yeast and mammalian homologucs ofDbf4 ( Kumagai ct al., 1999, Mol. Cell Biol. 19, 5083 -5095; scc also Sclafani, 2000, J. Cell Sci. 113, 2111 -21 17; Masai & Arai, 2002, J. Cell. Physiol.
  • Motif N which is located in the N -terminal part of all Dbf4 -related proteins, is related to BRCA C -terminal domain motif (Masai & Arai, 2000, Bi ochem. Biophys. Res. Commun. 275, 228-232). This motif does not appear to be essential for mitotic function but plays an important role in DNA replication checkpoint functions and recovery from DNA damage (Takeda et al., 1999, Mol. Cell. Biol. 19, 5535 -5547). Motif N appears to target Cdc7-Dbf4 complexes to ORC subunits and to Rad53 protein, regulating origin activation (Dunckcr ct al., 2002, Proc. Natl. Acad. Sci.
  • Motif M is a unique prolinc -rich domain that also contains a large numb er of aromatic residues.
  • Motif C is a C 2 H 2 -type zinc finger-like domain (Masai & Arai, 2000, Biochcm. Biophys. Res. Commun. 275, 228 -232), which is often involved in protein - protein interactions (Machkay & Crosslcy, 1998, Trends Biochcm. Sci. 23, 1 -4).
  • Motifs M and C were shown to be essential for mitotic functions of fission yeast Dbf4 protein and for the full level activation of the corresponding cdc7 kinase ( Ogino ct al., 2001, J. Biol. Chem. 276, 31376-31387). Segments of fission yeast and human dbf4 containing cither motif M or motif C arc capable of interacting with and partially activating the corresponding cdc7 kinasc in vitro.
  • cdc7 kinase complcxcd to only one of these motifs is not capable of phosphorylating its exogenous substrates
  • cdc7 is capable of fully activating the cdc7 kinasc and re -establishing mitotic function of the complex.
  • the regions N-tcrminal to motif N arc highly variable in their lengths and sequences among the species and arc not expected to play important roles.
  • Distances between motifs N and M arc relatively constant among the species and the sequences in this region arc well conserved between dbf4 from fission and budding yeasts.
  • distances between motifs M and C are highly variable both in terms of length and sequence similarity. Whereas the distances between motif M and motif C of budding and fission yeasts arc 357 and 182 amino acids, respectively, in human dbf4 only 40 residues separate these two conserved motifs.
  • This region serves merely as a flexible spacer that connects motifs M and C for coordinated binding to the cdc7 (Ogino ct al., 2001 , J. Biol. Chem. 276, 31376-31387).
  • the locations of motif C in human and yeast dbf4s differ significantly. Motif C of budding and fission yeasts are located at the C -terminus of the protein, whereas motif C of human dbf4 (comprising residues 291 -331) is located close to motif M, with the region C -terminal to motif C containing more than 340 amino a cids. The function of the long C -terminal tail after motif C of human dbf4 is not known.
  • the invention provides a monodispcrscd, non -aggregated, active hctcrodimcr complex comprising a truncated form of human Dbf4 protein (the regulatory subunit of the complex) and cdc7 (the catalytic subunit of the complex).
  • Availability of such protein complex provides a powerful tool for the elucidation of the crystal structure of the complex. Elucidation of the crystal structure of the cdc7/dbf4 complex will help understanding the mechanism of interaction of dbf4 with and activation of cdc7, and to perform Structure - Based Drug Design (SBDD) to help drive Structure Activity Relationship (SAR) for the optimisation of drug efficacy and specificity.
  • SBDD Structure - Based Drug Design
  • SAR Structure Activity Relationship
  • the invention further provides methods for identifying highly sc lcctivc and efficacious inhibitors of the Cdc7 kinasc, capable of selectively blocking initiation of DNA replication and therefore tumor cell proliferation through the selective inhibition of the cdc7 activity in the cell.
  • the present invention provides a purified and isolated C -terminally truncated form of human Dbf4 protein comprising the amino acid residues between position 293 and 334 of SEQ ID NO: 1 and lacking the amino acid residues between po sition 367 and 674 of SEQ ID NO: 1.
  • the N -terminal end of the truncated human Dbf4 proteins of the invention lies in an amino acid residue located between position 1 and 293 of SEQ ID NO: 1 , whereas the C -terminal end is between residues 334 an d 366 of SEQ ID NO: 1.
  • the N -terminal end of the truncated human Dbf4 proteins of the invention lies in an amino acid residue located between position 188 and 293 of SEQ ID NO: 1, whereas the C -terminal end is between residues 334 and 366 of SEQ ID NO: 1.
  • the human dbf4 truncated forms of the invention thus lack most of the residues C - terminal to the conserved motif C. Indeed, motif C spans between position 291 and 331 of the Dbf4 full length sequence of SEQ ID NO: 1.
  • the truncated proteins of the invention arc provided as a complex with a catalytic subunit intcrating with Dbf4 (herein below, the intcrating partner) such as, e.g., human cdc7.
  • the intcrating partner such as, e.g., human cdc7.
  • Such interacting partner can be in the form of a full -length, truncated or mutated protc in, provided, however, that it is endowed with the ability of interacting with Dbf4.
  • the present invention relates to human dbf4 truncated forms defined by the following residue positions (referred to the full -length protein of SEQ ID NO: 1): 1 -366, 16-366, 188-356, 188-353, 188-334, 198-356, 198-353, 198-334, 280-334, 280-344, 288-334, 288-344, 290-334, 290-344, 293-334, 293-344, optionally in complex with full-length, truncated or mutated forms of a catalytic subunit interacting with db f4.
  • Such interacting partner is, preferentially, human cdc7.
  • other endogenous interacting partners capable of forming a complex with dbf4 arc envisaged by the present invention.
  • the truncated forms of human dbf4 may, optionally, be mutated, particularly in residue Ql 5, to increase the stabilization of the protein and of its complex with the interacting partner.
  • Other point mutations might be introduced to improve the crystallographic properties of the molecule, for example in the lysine rcsid ucs in positions 291 -294 and/or in positions 348 -350 of SEQ ID NO: 1.
  • the complex may be further stabilized by the presence of a physiological substrate of the complex itself, such as, for example, MCM2; alternatively, an analogue or a competitor of the physiological substrate can be present.
  • a physiological substrate of the complex such as, for example, MCM2
  • an analogue or a competitor of the physiological substrate can be present.
  • ATP, ATP analogues or ATP competitors can be present in addition to, or instead of, the substrate to stabilize the complex.
  • vectors or recombinant expression vectors, comprising a nucleic acid molecule encoding a complex of the invention.
  • Vectors arc used herein cither to amplify DNA or RNA encoding the complex of the invention and/or to express DNA which encodes the complex of the invention.
  • Preferred vectors include, but arc not limited to, plasmids, phagcs, cosmids, episomes, viral particles or viruses, and intcgratablc DNA fragments ( i.e., fragments intcgratablc into the host genome by homologous recombination).
  • Preferred viral particles include, b ut arc not limited to, adcnoviruscs, parvoviruscs, hcrpcsviruscs, poxviruscs, adcno -associated viruses, Scmliki Forest viruses, vaccinia viruses, and rctroviruscs.
  • Preferred expression vectors include, but are not limited to, pcDNA3 (Invitrogcn) and pSVL (Pharmacia Biotech).
  • Other expression vectors include, but arc not limited to, pSPORT vectors, pGEM vectors (Promega), pPROEX vectors (LTI, Bcthcsda, MD), Bluescript vectors (Stratagene), pQE vectors (Qiagcn), pSE420 (Invitrogcn), and pYES2 (Invitrogcn) an d the range of Gateway expression plasmids (LifcTcchnologics).
  • Preferred expression vectors arc replicable DNA constructs in which a DNA sequence encoding the complex of the invention is operably linked to suitable control sequences capable of effecting th c expression of the complex in a suitable host.
  • DNA regions arc operably linked when they arc functionally related to each other.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.
  • Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. The need for control sequences into the expression vector will vary depending upon the host selected and the transformation method chosen.
  • control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding, and sequences which control the termination of transcription and translation.
  • Preferred vectors preferably contain a promoter which is recognised by the host organism.
  • the promoter sequences of the present invention may be eith erprokaryotic, cukaryotic or viral.
  • suitable prokaryotic sequences include the P R and Pi. promoters of baeteriophagc lambda (The baeteriophagc Lambda, Hcrshey, A. D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973), which is incor poratcd herein by reference in its entirety; Lambda II, Hcndrix, R.
  • Additional promoters include, but arc not limited to, mouse mammary tumor virus, long terminal repeat of human immunodcficicnc y virus, maloncy virus, cytomcgalovirus immediate early promoter, Epstein Barr virus, rous sarcoma virus, human actin, human yosin, human hemoglobin, human muscle crcatinc, and human mctalothioncin. Additional regulatory sequences can also be included in preferred vectors.
  • suitable regulatory sequences arc represented by the Shine -Dalgarno of the rcplicasc gene of the phagc MS -2 and of the gene ell of baeteriophagc lambda.
  • the Shinc-Dalgarno sequence may be directly followed by the DNA encoding the complex of the invention and result in the expression of the complex.
  • suitable expression vectors can include an appropriate marker which allows the screening of the transformed host cells. The transformation of the selected ho st is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook ct al., supra .
  • An origin of replication can also be provided cither by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.
  • one skilled in t he art can transform mammalian cells by the method of co - transformation with a selectable marker and the DNA encoding the complex.
  • a suitable marker is dihydrofolate rcductasc (DHFR) or thymidine kinase (see, U.S. Patent No. 4,399,216).
  • Nucleotide sequences encoding the complex may be rccombincd with vector DNA in accordance with conventional techniques, including blunt -ended or staggered -ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatasc treatment to avoid undcsidcrablc joining, and ligation with appropriate ligascs.
  • Techniques for such manipulation arc disclosed by Sambrook et al., supra and are well known in the art.
  • Methods for construction of mammalian expression vectors arc disclosed in, for example, Okayama et al, Mol. Cell. Biol. , 1983, 3, 280, Cosman et al, Mol. Immunol. , 1986, 23, 935, Cosman el al, Nature, 1984, 12, 768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety.
  • Another aspect of the present invention is directed to transformed host cells having an expression vector comprising a nucleic acid molecule encoding a complex of the invention.
  • Expression of the nuclcotide scqucnc c occurs when the expression vector is introduced into an appropriate host cell.
  • Suitable host cells for expression of the polypcptidcs of the invention include, but arc not limited to, prokaryotcs, yeast, and eukaryotes. If a prokaryotic expression vcct or is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences.
  • Suitable prokaryotic cells include, but arc not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas , Streptomyces , and Staphylococcu .
  • cukaryotic cells are cells of higher eukaryotes.
  • Suitable eukaryotic cells include, but arc not limited to, non-human mammalian tissue culture cells and human tissue culture cells.
  • Preferred host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murinc 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, cds. (1973), which is incorporated herein by reference in its entirety).
  • a yeast host may be employed as a host cell.
  • Preferred yeast cells include, but arc not limited to, the genera Saccharomyces , Pichia, and Kluveromyces .
  • Preferred yeast hosts are S. cerevisiae and P. pastoris .
  • Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadcnylation, sequences for transcription termination, and a selectable marker gene.
  • ARS autonomously replication sequence
  • Shuttle vectors for replication in both yeast and E. coli are also included herein.
  • insect cells may be used as host cells.
  • the polypcptidcs of the invention arc expressed using a baculovirus expression system (see, Luckow el al, Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: A Laboratory Manual, O'Riclly et al. (Eds.), W.H. Freeman and Company, New York, 1992, and U.S. Patent No.4,879,236, each of which is incorporated herein by reference in its entirety).
  • the MAXBACTM complete baculovirus expression system can, for example, be used for production in insect cells.
  • the present invention provides a method of producing a complex of the invention, comp rising the steps of introducing a recombinant expression vector as described above into a compatible host cell, growing the host cell under conditions for expression of the complex, and recovering the complex from the host cells.
  • Eukaryotic systems arc pre ferred since they provide a variety of processing mechanisms which result in, for example, glycosylation, carboxy -terminal amidation, oxidation or dcrivatization of certain amino acid residues, conformational control, and so forth.
  • the complexes of the present invention arc preferably provided in an isolated form, are preferably substantially purified, and most preferably are purified to homogeneity.
  • Host cells arc preferably lyscd and the polypcptidc is recovered from the lysatc of the host cells.
  • the complex is recovered by purifying the cell culture medium from the host cells, preferably without lysing the host cell.
  • the complexes can be recovered and purified from recombinant cell cultures by well -known methods, including ammonium sulfa te or ethanol precipitation, anion or cation exchange chromatography, phosphocellulosc chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatitc chromatography and lcctin chromatography.
  • the pre sent invention provides a method of identifying compounds which bind to the complex of the invention, comprising contacting the complex with a compound and determining whether the compound binds the complex. Binding can be determined by binding assays whi ch arc well known to the skilled artisan, including, but not limited to, gel -shift assays, Western blots, radiolabclcd competition assay, phagc -based expression cloning, co -fractionation by chromatography, co -precipitation, cross linking, interaction trap/ two-hybrid analysis, southwestern analysis, ELISA, and the like, which arc described in, for example, Current Protocols in Molecular Biology , 1999, John Wiley & Sons, NY, which is incorporated heroin by reference in its entirety.
  • the compounds to be screened include, but arc not limited to, extracellular, intraccllular, biologic or chemical origin.
  • the complex employed in such a test may cither be free in solution, attached to a solid support, borne on a cell surface or located intraccllularly.
  • One skill cd in the art can, for example, measure the formation of aggregates with the compound being tested.
  • Another aspect of the present invention is directed to methods of identifying compounds which modulate (i.e., increase or decrease) activity of the complex of the invention comprising contacting the complex with a compound, and determining whether the compound modifies activity of the complex. The activity in the presence of the test compound is compared to the activity in the absence of the test compound.
  • the compound will have increased activity.
  • the activity of the sample containing the test compound is lowc rthan the activity in the sample lacking the test compound, the compound will have inhibited activity.
  • the present invention is particularly useful for screening compounds by using the complex of the invention in any of a variety of drug screening techniq ucs.
  • the compounds to be screened include, but arc not limited to, extracellular, intracellular, biologic or chemical origin.
  • the complex employed in such a test may be in any form, preferably, free in solution, attached to a solid support, borne on a ce 11 surface or located intracellularly.
  • One skilled in the art can, for example, measure the formation of aggregates with the compound being tested.
  • the activity of the complex of the invention can be determined by, for example, kinase activity assay, cell ular proliferation measurement and DNA replication activity, in the presence and absence of the test compound.
  • kinase activity assay for example, kinase activity assay, cell ular proliferation measurement and DNA replication activity, in the presence and absence of the test compound.
  • These measurements can, for example, be performed as described in: Jiang and Hunter, Proc. Natl. Acad. Sci. USA. (1997), 94, 14320-14325; Hunter and Scfton [Editors], Methods in Enzymology (1991), 200, Academic Press, NY; Abclson, J., Simon, Mclvin I., and Dunphy, W G. [Editors]., Methods in Enzymology (1997), 283, Academic Press, NY; Pagano, M. [Editor], Cell cycle: Materials and Methods, (1995 ), Springer -Verlag, NY.
  • methods of screening for compounds that modulate the activity of the complex comprise contacti ng the compound with the complex and assaying for the presence of aggregates between the compound and the complex. In such assays, the complex is typically labeled.
  • the free complex is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to the complex.
  • high throughput screening for compounds having suitable binding affinity to the complex is employed. Briefly, large numbers of different small pcptidc test compounds arc synthcsiscd on a solid substrate. The pcptidc test compounds arc contacted with the complex and washed. The bound complex is then detected by methods well known in the art. Purified complexes of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non -neutralizing antibodies can be used to capture the protein and immobilize it on the solid support.
  • inventions comprise using competitive screening assays in which neutralizing antibodies capable of binding a complex of the invention specifically compete with a test compound for binding to the complex.
  • the antibodies can be used to detect the presence of any pcptidc which shares one or more antigcnic determinants with the complex.
  • Radiolabclcd competitive binding studies are described in A.H. Lin el al. Antimicrobial Agents and Chemotherapy , 1997, vol. 41 , no. 10. pp. 21 27- 2131, the disclosure of which is incorporated herein by reference in its entirety.
  • the novel molecules identified by the screening methods according to the invention arc low molecular weight organic molecules, in which case a composition or pharmaceutical composition can be prepared thereof for oral intake, such as in tablets.
  • the compositions, or pharmaceutical compositions, comprising the nucleic acid molecules, vectors, polypcptidcs, antibodies and compounds identified by the screening methods described herein can be prepared for any route of administration including, but not limited to, oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intrapcritoncal.
  • the nature of the carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered. Examples of techniques and protocols that arc useful in this context arc, inter alia, found in Remington's Pharmaceutical Sciences, 16 edition, Osol, A (cd.), 1980, which is incorporated herein by reference in its entirety.
  • the dosage of these low molecular weight compounds will depend on the disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.
  • For treating human or animals between approximately 0.5 mg kg of body weight to 500 mg kg of body weight of the compound can be administered. Therapy is typically administered at lower dos ages and is continued until the desired therapeutic outcome is observed.
  • the present compounds identified by the screening methods described herein have a variety of pharmaceutical applications and may be used, for example, to treat or prevent unregulated cellular growth, such as cancer cell and tumor growth.
  • the present molecules are used in gene therapy.
  • gene therapy procedures sec e.g. Anderson, Science, 1992, 256, 808-813, which is incorporated herein by reference in its entirety.
  • the present invention also relates to the use of the specific truncated forms of human dbf4 in complex with cdc7 or other interacting partners for the structural studies of the human dbf4/cdc7 hctcrodimcr.
  • the present invention al so relates to the use of the specific truncated forms of analogues of dbf4 in complex with cdc7 or other interacting partners for the structure -based drug design of inhibitors of the human dbf4/cdc7 complex.
  • drfl is capable of binding and activating cdc7 and thus it is a regulatory subunit, possibly substituting dbf4 in the complex with cdc7.
  • the expert in the art will therefore recognize that the teachings of the present invention can be applied also to drfl and to its complex with an interacting partner such as cdc7.
  • Human Dbf4 full length (residues 1 -674)/cdc7 full length construct.
  • Human Cdc7 (full Icngth)/Dbf4 (full length) were cloned into a pFastBae dual vector (Invitrogcn) for in sect cells expression.
  • the first step was the Glutathionc S - Transfcrase (GST) -Cdc7 insertion into pFastBae dual vector under the polyhcdrin promoter. Restriction sites for Smal and Nsil were used.
  • GST-Cdc7 was amplified by PCR using the following primers: forward 5'-aaaaacccgggatggcccctatactaggttattgg-3' (SEQ ID NO: 2) [in bold is shown the Smal restriction site] reverse 5'-aaaaaatgcattcacaagctccatatctttaaaaatgg-3' (SEQ ID NO: 3) [in bold is shown the Nsil restriction site]
  • the hacmagg lutinin (HA)-Dbf4 region was inserted under the pTcn promoter, preparing the pFastBae dual vector with Cdc7 inserted, by digestion by restriction enzymes Sail and Notl.
  • PCR was performed using the following primers for Dbf4 full length: forward 5'-aaaaaagtcgacatgtacccatacgacgttccagattacgctaactccggagccatgaggatccac -3' (SEQ ID NO: 4) [in bold is shown the Sail restriction site] reverse 5'-aaaaaagcggccgcctaaaagccagtaaatgtagaagttg-3' (SEQ ID NO: 5) [in bold is shown the Notl restriction site]
  • the initial human Dbf4 truncated construct (residues 1 -366) was prepared by introducing into the pFastBae dual vector a stop (taa) codon at amino acid residue Lcu366 (aaa), by site directed mutagcncsis with the follow ing primers: forward: 5' gtttctgcaaglgtcctg taaagactgaacaaaggaa 3' (SEQ ID NO: 6) ; reverse: 5' ttcctttttgttcagtcttttacaggacacttgcagaaac 3' (SEQ ID NO: 7).
  • the truncated Dbf4 (1 -366) was generated using the QuickChangc mutagcncsis kit (Stratagcnc) with the pFastBae dual cdc7-dbf4 plasmid as template, according to the manufacturers' specification.
  • the PCR products were treated with 20 U of Dpnl restriction enzyme and the treated DNA was used to transform XL 1 - 1 Blue cells (Stratagcnc).
  • Plasmid DNA was purified using the Qiagcn miniprcp Kit and scqucnccd on an Applied Biosystcms DNA analyzer.
  • Table 1 List of Dbf4 truncated forms expressed in complex with cdc7 in insect cells.
  • the obtained dbf4 fragments contained a Sail (5 1 ) and a Notl (3') restriction site including a Kozak sequence.
  • PCR fragments were eluted from an agarosc gel using the Qiagcn gel extraction Kit and cloned into the TA TOPO vector (Invitrogcn).
  • Plasmid DNA was purified using the Qiagcn miniprcp Kit and scqucnccd on Applied Biosystcms DNA analyzer.
  • the plasmid DNAs were treated with 10 U of Sail and Notl restriction enzymes and the obtained fragments were ligated into the following vector:
  • cdc7pFastB ac-dual plasmid generated as described above were treated with 10 U of Sail and Notl restriction enzymes.
  • the digested DNA was treated with CIP phosphatase and purified from agarose gel using the Qiagen gel extraction Kit.
  • Plasmid DNA was prepared using the Qiagen miniprep Kit.
  • Human Dbf4 residues 16 -366 was obtained by PCR using the human full length Dbf4 cDNA as template using the following primers: forward:
  • Plasmid DNA was prepared using the Qiagcn miniprcp Kit and scqucnccd on Applied Biosystcms DNA analyser.
  • Example 2 Dicistronic vector preparation and expression in bacterial cells
  • the cDNA coding for human Cdc7 was amplified by PCR using the sense primer: forward: 5' CGTCGTATTAATATGGAGGCGTCTTTGGGGATT 3' (SEQ ID NO:
  • the fragment was ligated into pGEX6P2 BamHI>NdeI (Amcrsham) expression vector previously mutated to obtain a Ndel site instead of a BamHI site and digested with Ndel and Xhol.
  • the recombinant plasmid carrying Cdc7 is called pGEX -Cdc7poly.
  • the DNA region coding for human Dbf4 was amplified by PCR using the sense primer 5' CGTCGTCATATGAACTCCGG AGCCATGAG 3' (SEQ ID NO: 19) and the antisensc primer
  • Shorter regions coding for human Dbf4 determined by limited protcolysis were amplified by PCR using the following sense and antisensc p ⁇ mcrs listed in Table 2.
  • the fragments were then ligated into pGEX -Cdc7poly previously digested with the corresponding restriction enzymes following the same protocol as for the cloning of the full length.
  • This protocol applies to all cdc7/dbf4 constructs expressed in E. coli.
  • the pellet from a 6 L of E. coli culture expressing a dbf4 truncated/ GST -Cdc7 full length complex was resuspcnded in 600 ml of buffer A (50 mM Tris pH 7.4, 1 M NaCl, 20 mM dithiothreitol (DTT), "Complete” protease inhibitor cocktail (Roche), 1 tablet per 25 ml) and lysed by liquid extrusion with a Gaulin homogenizer (Niro Soavi).
  • the supernatant was loaded onto 30 ml of Glutathione Sepharosc equilibrated in the same buffer.
  • the resin was washed with 150 ml of buffer A and then with 150 ml of buffer B (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM DTT).
  • the resin was resuspcnded in 30 ml of buffer B containing 0.9 ml of PreScission protease and left overnight at 4 °C.
  • the cleaved protein complex was then eluted and the resin washed with 90 ml of buffer B.
  • the eluatc and the wash fractions containing the complc x were combined and concentrated to 1 mg/ml in an Amicon cell.
  • the concentrated protein was then loaded onto a 5 ml Hcparin Sepharosc column equilibrated in 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM DTT and eluted with a linear saline gradient up to 1 M NaCl. T he fractions containing the complex were pooled and concentrated to 2 mg/ml.
  • the pool was dcphosphorylatcd by incubation with 12,000 U lambda phosphatasc (New England Biolabs) per mg of protein in the presence of 2 M MnCU, and 5 mM DTT, at 30°C for 3 hou rs.
  • the dcphosphorylatcd protein was finally loaded onto a Supcrdcx 200 column and eluted in Buffer B. Recovery of the purified protein was 2 mg per liter of culture.
  • Example 4 Purification protocol for cdc7/ dbf4 complexes expressed in insect cells.
  • the resin was washed with 150 ml of buffer A and then with 150 ml of buffer B (50 mM Tris pH 7.4, 1 M NaCl, 5 m M DTT, 1 mM EDTA).
  • the resin was resuspendcd in 30 ml of buffer B containing 0.9 ml of PreScission protease and left overnight at 4 °C.
  • the cleaved protein complex was then eluted and resin washed with 60 ml of buffer B.
  • the cluatc and the wash containing the complex were combined and concentrated to 1 mg ml in an Amicon cell.
  • the concentrated protein was then loaded onto a 5 ml Hcparin Sepharosc column equilibrated in 50 mM Tris pH 8.0, 300 mM NaCl, 5 mM DTT and eluted with a linear saline gradient up to 1 M NaCl.
  • the fractions containing the complex were pooled and concentrated to 2 -3 mg/ml and the pH was adjusted to 7.8.
  • the pool was de -phosphorylatcd by incubation with 10,000 U lambda phosphatasc (Calbiochcm) per mg of protein in the presence of 2 mM MnC ⁇ -, and 5 mM DTT, at 30°C for 3 hours.
  • the dephosphorylatcd protein was finally loaded onto a Superdcx 200 column (16/60) and eluted in Buffer C (50 M Tris pH 7.4 150 mM NaCl, 5 mM DTT). Recovery of the purified protein ranged from 0.5 to 3.0 mg per billi on cells.
  • Dbf4 Residues 1 -365/cdc7 full-length complex.
  • the following experimental conditions were used: 2 ⁇ g of the Dbf4 (1 -365)/cdc7 complex were incubated for 20 minutes at room temperature in a buffer containing 90 mM Tris/HCl pH 8.5, 4 mM DTT, 2 mM CaCl 2 with increasing amounts of chymotrypsin (0.2 ⁇ g - 4 ⁇ g) in a final volume of 20 ⁇ l.
  • the reaction was stopped by boiling the samples in the presence of sodium dodccyl sulphate (SDS) sample buffer for 5 minutes and loaded onto a 12% BisTris gel.
  • AspN enzyme titration was carried under similar conditions, using 50 mM Tris HCl pH 7.8, 10 ⁇ M DTT as buffer and 0.01 to 3 ⁇ g of the enzyme.
  • LC ESI -MS liquid chromatography/ clectrospray ionization mass spectrometry
  • a sample volume containing 10 -25 ⁇ g total protein was loaded isocratically onto a Vydac C4 column (2.1 mm ID x 250 mm) and proteins were eluted applying a grad icnt from 5% to 75% cluent B over 70 min (elucnt A: 0.05% trifluoroacctic acid in water, cluent B: 0.05% trifluoroacetic acid in acetonitrile) at a flow-rate of 200 ⁇ l/min.
  • Mass spectra were acquired in the 600 -2000 m z range and deconvolutcd using the ChemStation deconvolution software package (Agilent).
  • Experimental molecular weight values were matched to the expected ones using the protein analysis software Paws, free version downloaded from www.protcomctrics.com .
  • Enzymatic activity of cdc7/dbf4 shorter complex was tested using a recombinant fragment of MCM2 and radioactive P 3 ATP as substrates. After in vitro phosphorylation reaction proteins were separated using SDS -PAGE and the incorporated radioactive phosphate was detected by autoradiography. The test was carried out in parallel on the full length complex as positive control.
  • Circular Dichroism (CD) spectra were recorded using an AVIV 215 CD spcctrophotomctcr and software provided by the manufacturer. A cell with a 0.1 cm
  • pathlcngth was used. Each spectrum was averaged using 2 accumulations collected in 1 nm intervals with an averaging time of 10 seconds. A blank spectrum was subtracted from the averaged data spectra to correct for buffer effects. Spectra were measured in 10 M sodium phosphate buffer, 20 mM NaCl, at 10°C. Secondary structure content of the 5 spectral data was determined using the program k2d (Andrade ct al.: Protein Engineering, vol. 6, p. 383-390; 1993).
  • Elimination of this problem alleviates the problem of constructing a hcterodimer that is suitable for crystallographie studies, which is an important prerogative for the structure -based design of selective inhibitors of the human cdc7/dbf4 complex.

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