(12) United States Patent (10) Patent No.: US 9,353,350 B2 Kobayashi Et Al

US009353350B2

(12) United States Patent (10) Patent No.: US 9,353,350 B2 Kobayashi et al. (45) Date of Patent: May 31, 2016

(54) METHOD FOR PRODUCING MULTIPOLAR 2012fO149053 A1 6, 2012 Yoshida et al. CELL 2013,0323,776 A1 12/2013 Yoshida et al. 2015. OO18286 A1* 1/2015 Kobayashi et al...... 514, 19.3 (71) Applicant: TOAGOSEICO.,LTD., Tokyo (JP) FOREIGN PATENT DOCUMENTS

(72) Inventors: Nahoko Kobayashi, Tsukuba (JP); CN 1763O82 A 4/2006 Tetsuhiko Yoshida, Tsukuba (JP); Yuki DE 102009021681 A1 11, 2010 Kobayashi, Fujisawa (JP) WO O3O24408 A2 3, 2003 WO O3O37172 A2 5, 2003 WO 2004.005472 A2 1, 2004 (73) Assignee: TOAGOSEICO. LTD., Tokyo (JP) WO 2004/020457 A2 3, 2004 WO 2007/004869 A2 1, 2007 (*) Notice: Subject to any disclaimer, the term of this WO 2007056188 A1 5/2007 patent is extended or adjusted under 35 WO 2008/081812 A1 T 2008 WO WO 2009,093692 A1 T 2009 U.S.C. 154(b) by 0 days. WO WO 2011/O13698 A1 2, 2011 (21) Appl. No.: 14/366,971 WO WO 2011/O13699 A1 2, 2011 (22) PCT Filed: Dec. 20, 2012 OTHER PUBLICATIONS Paradis-Bleau et al., “Peptide inhibitors of the essential cell division (86). PCT No.: PCT/UP2O12AO83110 protein FtsA'. Protein Engineering, Design & Selection, 2005, pp. S371 (c)(1), 85-91, vol. 18, No. 2, Oxford University Press. (2) Date: Jun. 19, 2014 Paradis-Bleau et al., “Identification of Pseudomonas aeruginosa FtsZ. peptide inhibitors as a tool for development of novel antimicro bials”, Journal of Antimicrobial Chemotherapy, Jun. 2004, pp. 278 (87) PCT Pub. No.: WO2013/094698 280. PCT Pub. Date: Jun. 27, 2013 Cho et al., “Depletion of CPAP by RNAi disrupts centrosome integ rity and induces multipolar spindles”. Biochemical and Biophysical (65) Prior Publication Data Research Communications, 2006, pp. 742-747, vol. 339. Sakaushi et al., "Live imaging of spindle pole disorganization in US 2015/OOO4697 A1 Jan. 1, 2015 docetaxel-treated multicolor cells'. Biochemical and Biophysical Research Communications, 2007, pp. 655-660, vol. 357. (30) Foreign Application Priority Data Jaiswal et al., “9-Bromonoscapine-induced mitotic arrest of cigarette Smoke condensate-transformed breast epithelial cells”. J Cell Dec. 20, 2011 (JP) ...... 2011-278975 Biochem. Apr. 15, 2009, pp. 1-20, vol. 106, No. 6. International Search Report issued in International Patent Applica (51) Int. Cl. tion No. PCT/JP2012/083110 mailed Jan. 29, 2013. CI2N5/00 (2006.01) Vollmer, “The prokaryotic cytoskeleton: a putative target for inhibi CI2N 5/02 (2006.01) tors and antibiotics?.” Applied Microbiology and Biotechnology, vol. A6 IK38/08 (2006.01) 73, pp. 37-47, 2006. A6 IK38/04 (2006.01) Huang et al., “Targeting FtsZ for anti-tuberculosis drug discovery: C07K5/00 (2006.01) non-cytotoxic taxanes as novel anti-tuberculosis agents. J. Med C07K 700 (2006.01) Chem, vol. 49, No. 2, pp. 463-466, Jan. 26, 2006. C07K 16/00 (2006.01) (Continued) C07K 17/00 (2006.01) CI2N5/09 (2010.01) A6 IK35/3 (2015.01) Primary Examiner — James H Alstrum Acevedo A6 IK35/12 (2015.01) Assistant Examiner — Thea D'Ambrosio C07K 7/06 (2006.01) (74) Attorney, Agent, or Firm — Oliff PLC (52) U.S. Cl. CPC ...... CI2N5/0693 (2013.01); A61K35/12 (57) ABSTRACT (2013.01); A61 K35/13 (2013.01); C07K 7/06 (2013.01); C12N 2501/998 (2013.01); C12N This invention provides a multipolar cell including three or 2503/02 (2013.01) more centrosomes dispersed therein and a multipolar spindle (58) Field of Classification Search formed so as to extend from the three or more respective None centrosomes. The multipolar cell production method pro See application file for complete search history. vided by the present invention supplies cultured eukaryotic cells with a synthetic peptide as a multipolarity-inducing (56) References Cited peptide, with the synthetic peptide having an amino acid sequence selected from SEQID NO: 1 to SEQID NO: 12 or U.S. PATENT DOCUMENTS an amino acid sequence formed by Substituting, deleting and/ 5,595,756 A 1/1997 Bally et al. or adding one, two or three amino acid residues in/from/to the 6,344,443 B1 2/2002 Liu et al. selected amino acid sequence. 2007/0021347 A1 1/2007 Khan et al. 2010, O297758 A1 11/2010 Yoshida et al. 2012/O122225 A1 5/2012 Kobayashi et al. 8 Claims, 5 Drawing Sheets US 9,353,350 B2 Page 2

(56) References Cited J. Rudinger, “Peptide Hormones.” University Park Press, pp. 1-7. 1976. OTHER PUBLICATIONS SIGMA, "Custom Peptide Synthesis.” pp. 1-2, 2004. Jul. 3, 2014 International Preliminary Report on Patentability issued Berendsen, "A Glimpse of the Holy Grail?” Science, vol. 282, pp. in International Patent Application No. PCT, JP2012/083109. 642-643, Oct. 23, 1998. May 29, 2015 Extended European Search Report issued in European Voet et al., “Biochemistry,” John Wiley & Sons Inc., pp. 235-241, Patent Application No. 12860097.0. 1995. Lappchen et al., “Probing FtsZ. and Tubulin with C8-Substituted GTP Ngo et al., "Computational Complexity Protein Structure Prediction, Analogs Reveals Differences in Their Nucleotide Binding Sites.” and the Levinthal Paradox.” pp. 491-494, 1994. Chemistry & Biology, vol. 15, pp. 189-199, Feb. 2008. Bradley et al., “Limits of Cooperativity in a Structurally Modular Ojima et al., “Drug discovery targeting cell division proteins, Protein: Response of the Notch Ankyrin Domain to Analogous microtubules and FtsZ.” Bioorganic & Medicinal Chemistry, vol. 22. Alanine Substitutions in Each Repeat.” Journal of Microbiology, vol. pp. 5060-5077, Mar. 5, 2014. 324, pp. 373-386, 2002. Jul. 28, 2015 Office Action issued in U.S. Appl. No. 14/267.204. Jul. 3, 2014 International Preliminary Report on Patentability issued Sporn et al., “Chemoprevention of cancer. Carcinogensis, vol. 21. in International Patent Application No. PCT, JP2012/083111. No. 3, pp. 525-530, 2000. Jun. 23, 2015 Office Action issued in U.S. Appl. No. 14/367,023. Ito et al., “Co-culture of human breast adenocarcinoma MCF-7 cells U.S. Appl. No. 14/367,023, filed Jun. 19, 2014 in the name of Nahoko and human dermal fibroblasts enhances the production of matrix Kobayashi. metalloproteinases 1, 2 and 3 in fibroblasts.” British Journal of Can U.S. Appl. No. 14/367,204, filed Jun. 19, 2014 in the name of Nahoko cer, vol. 71, pp. 1039-1045, 1995. Kobayshi. Raderschall et al., “Elevated Levels of RadS1 Recombination Protein Mar. 12, 2013 International Search Report issued in International in Tumor Cells.” Cancer Research, vol. 62, pp. 219-225, Jan. 1, 2002. Patent Application No. PCT/JP2012/083109. Neidle, "Cancer Drug Design and Discovery.” Elsevier/Academic Jan. 29, 2013 International Search Report issued in International Press, pp. 427-431, 2008. Patent Application No. PCT/JP2012/083111. Chabner et al., “Cellular and Molecular Basis of Cancer.” Merck Manual Professional, pp. 1-5, last accessed May 10, 2012. * cited by examiner U.S. Patent May 31, 2016 Sheet 1 of 5 US 9,353,350 B2

U.S. Patent May 31, 2016 Sheet 2 of 5 US 9,353,350 B2

FIG.2

U.S. Patent May 31, 2016 Sheet 3 of 5 US 9,353,350 B2

U.S. Patent May 31, 2016 Sheet 4 of 5 US 9,353,350 B2

U.S. Patent May 31, 2016 Sheet 5 of 5 US 9,353,350 B2

US 9,353,350 B2 1. 2 METHOD FOR PRODUCING MULTIPOLAR Accordingly, an objective of the present invention is to CELL provide a material and a method for efficiently providing multipolar cells as Such a cell specimen. TECHNICAL FIELD The present inventors have focused on several peptides reported in Non-Patent Document and Non-Patent Document The present invention relates to a synthetic peptide for 2. More specifically, as for peptides to inhibit the activity of inducing multipolarity in target cells. In particular, it relates the FtsZ. (Filamenting temperature-sensitive mutant Z) pro to a method for producing a cell with extra centrosomes, that tein which is present in bacteria (which are prokaryotes) and is, a multipolar cell, by Supplying the peptide to the target forms a protein assembly called the Zring involved in bacte cells. 10 rial cell division or for peptides to inhibit the activity of the The present application claims priority based on Japanese FtSA (Filamenting temperature-sensitive mutant A) protein, Patent Application No. 2011-278975 filed on Dec. 20, 2011 known to work as an to bind the FtsZ. protein to cell mem and its entire content is incorporated herein by reference. The branes when coupled to the C-terminus of the FtsZ. protein present application is a National Stage application of PCT 15 (i.e. for peptides that inhibit the GTPase activity of the FtsZ. Application No. PCT/JP2012/083110 filed on Dec. 20, 2012. protein or the ATPase activity of the Fts.A protein), they have focused on several peptides (i.e. peptides capable of acting as BACKGROUND ART FtsZ, inhibitors or Fts.A inhibitors) isolated by employing a general phage display technique. One of the characteristics of tumor cells (cancer cells) is The present inventors have found out that a supply of these that as compared to normal cells, they undergo rapid cell peptides capable of acting as FtsZ. inhibitors or Fts.A inhibi division, resulting in a significantly high cell proliferation tors to various species of tumor cells (cancer cells) leads to the rate. Thus, as anticancer agents to kill tumor cells or to inhibit presence of many centrosomes (which may be PCM) dis persed in the cells, and multipolar cells (cells with extra their proliferation, drugs that inhibit division of tumor cells centrosomes) can be produced, comprising multipolar (cancer cells) are used. For example, alkylating agents are 25 spindles which are different from dipolar spindles and are drugs that act on the DNA of tumor cells (cancer cells) to formed so as to extend from the many respective dispersed crosslink the bases between double strands, thereby inhibit centrosomes serving as spindle poles unlike the way dipolar ing DNA replication and blocking cell division. Antimetabo spindles are formed; and the present invention has been com lites such as 5-FU, etc., are drugs that inhibit DNA synthesis pleted. and block cell division. 30 In the present description, the term “tumor cells' is used to distinguish these cells from normal cells that have not yet CITATION LIST become neoplastic. Thus, cells constituting a tumor (typically a cancer) instead of normal cells are called as tumor cells Patent Literature regardless of the origin and nature of the cells. 35 To achieve the objective, the present invention provides a Patent Document 1 WO 2009/093692A method described below. In particular, disclosed herein is a Patent Document 2 WO 2011/013698A method for producing a multipolar cell having three or more Patent Document 3 WO 2011/013699A centrosomes as well as a multipolar spindle from at least one species of eukaryotic cells (typically cells of humans or ani Non-Patent Literature 40 mals belonging to mammals, or cells of birds). The method comprises culturing target eukaryotic cells, and Supplying the Non-Patent Document 1 Protein Engineering, Design & eukaryotic cells being cultured with a synthetic peptide con Selection, vol. 18(2), 2005, pp. 85-91 sisting of an amino acid sequence selected from the following Non-Patent Document 2 Journal of Antimicrobial Chemo 45 amino acid sequences that act as FtsZ, inhibitors or FtSA therapy, vol. 54(1), 2004, pp. 278-280 inhibitors: SUMMARY OF INVENTION (SEQ ID NO: 1) (1) CSSATGKSC, To elucidate at the genetic level of the mechanisms by 50 which a normal cell becomes neoplastic (cancerous) or to (SEQ ID NO: 2) analyze the state of the cell during or after the cancerous (2) CLAPSPSKC, transformation from a biochemical standpoint, it is some (SEQ ID NO : 3) times necessary to obtain a specimen of cells having abnor (3) CLGOTKMRC, malities in cell division or cell cycle unlike normal cells. 55 For example, for identifying a gene or a protein (peptide) (SEQ ID NO : 4) involved in abnormal cell division of a eukaryotic cell or for (4) CGHRPYOYC finding out a method for correcting abnormalities in cell (SEO ID NO; 5) division, it may be helpful to use, as a specimen, so-called (5) CWAFPLHHC, multipolar cells (cells with extra centrosomes or cells with 60 (SEQ ID NO : 6) multipolar spindles) in which multipolar spindles, instead of (6) CTLNSHSNC normal bipolar spindles, are formed; in other words, three or more centroSomes (which include fragments of pericentriolar (SEO ID NO: 7) materials (PCM); the same applies hereinafter) are present (7) CEISAKRTC, dispersed in a celland a spindle (multipolar spindle) is formed 65 (SEQ ID NO: 8) randomly extending from the three or more respective dis (8) CHILHAQAC, persed centrosomes serving as spindle poles. US 9,353,350 B2 4 - Continued - Continued (SEO ID NO: 9) (SEQ ID NO: 12) (9) CPRPPSLEC, (12) CLTKSYTSC; (SEQ ID NO: 10) 5 or an amino acid sequence formed by Substituting, deleting (1O) CTGHWASEC, and/or adding one, two or three amino acid residues in/from/ (SEQ ID NO: 11) to the selected amino acid sequence; and also an amino acid (11) CSYEKRPMC sequence that constitutes a nucleolar localization signal and (NoLS) and is selected from SEQID NO: 13 to SEQID NO: 10 (SEQ ID NO: 12) 20, or an amino acid sequence formed by Substituting, delet (12) CLTKSYTSC; ing and/or adding one two or three amino acid residues in/from/to the selected amino acid sequence. or an amino acid sequence formed by Substituting, deleting The present inventors have found out that amino acid and/or adding one, two or three amino acid residues in/from/ sequences known as nucleolar localization signals (NoLS) to the selected amino acid sequence. 15 (see Patent Documents 1,2 and 3) are involved intransporting According to the method of the present invention having peptides front the extracellular space into nuclei (typically Such a constitution, the Supply of the synthetic peptide (i.e. a nucleoli). By the use of a synthetic peptide constituted to multipolarity-inducing peptide) to cultured cells to a pre include the amino acid sequence (i.e. a multipolarity-induc scribed concentration can lead to formation of 3 or more ing peptide), the peptide can be introduced more efficiently (typically 5 or more, preferably 8 or more) centrosomes in the into target cells. Thus, application of the method in the cultured cells (typically in M-phase cells) and induce forma present embodiment will increase the formation efficiency of tion of a multipolar spindle having the plurality of cen multipolar cells, allowing for more efficient production and troSomes serving as spindle poles. provision of multipolar cells. Thus, the method of the present invention allows for stable 25 In the multipolar cell production method disclosed herein, production of multipolar cells (cultured cells) that contribute preferably, the eukaryotic cells to be cultured are synchro to identify a gene or a protein (peptide) involved in abnormal nized in the G2 phase or M phase and the peptide is supplied cell division of a eukaryotic cell or to find out a method for to the cultured cells that have been synchronized in the G2 correcting abnormalities in cell division. phase or M phase. Another preferable embodiment of the method disclosed 30 By synchronizing the cells to be cultured in the G4 phase or herein is for producing a multipolar cell having three or more Mphase of the cell cycle (blocking the cultured cells in the G2 centrosomes as well as a multipolar spindle from at least one phase or Niphase for a certain period of time) and Supplying species of eukaryotic cells (typically, cells of humans orani a multipolarity-inducing peptide to the target cultured cells, mals belonging to mammals). The method comprises cultur multipolarity can be induced more efficiently in the cultured ing target eukaryotic cells, and Supplying the target eukary cells. otic cells being cultured with a synthetic peptide consisting of In the multipolar cell production method disclosed herein, an amino acid sequence selected from the following amino suitably, cultured (primary cultured or subcultured) tumor acid sequences that act as FtsZ, inhibitors or Fts.A inhibitors: cells are used as the target eukaryotic cells. Particularly pref erable target cells include malignant tumor cells (cancer cells) 40 (SEQ ID NO: 1) Such as cells (cultured cells) derived from a squamous cell (1) CSSATGKSC, carcinoma. The method disclosed herein can induce multipolarity par (SEQ ID NO: 2) ticularly effectively in cultured tumor cells. Accordingly, the (2) CLAPSPSKC, present invention provides a method for inducing multipolar (SEQ ID NO : 3) 45 ity in tumor cells (i.e. a cell in which three or more cen (3) CLGOTKMRC, troSomes have formed) characterized by Supplying a syn (SEQ ID NO : 4) thetic peptide disclosed herein to cultured tumor cells (4) CGHRPYOYC The present invention can provide a multipolar cell pro duced by a method disclosed herein. Multipolar cells (cells (SEO ID NO; 5) 50 with extra centrosomes) provided by the present invention (5) CWAFPLHHC, comprises 3 or more (typically 5 or more, preferably 8 or (SEQ ID NO : 6) more) centroSomes formed therein, and preferably also a (6) CTLNSHSNC multipolar spindle formed therein having the plurality of centrosomes serving as spindle poles. The multipolar cells (SEO ID NO: 7) 55 can preferably remain viable in the multipolar state for a few (7) CEISAKRTC, or more days (typically, 5 or more days, e.g. 5 days to 14 days) (SEQ ID NO: 8) without undergoing cell division. (8) CHILHAQAC, BRIEF DESCRIPTION OF DRAWINGS (SEO ID NO: 9) 60 (9) CPRPPSLEC, FIG. 1 is a fluorescence microscope photo (image) show (SEQ ID NO: 10) ing the state of cultured cells after incubation following addi (1O) CTGHWASEC, tion of a sample peptide (Sample 9) to a culture medium of (SEQ ID NO: 11) HeLa cells (synchronized in the G2 phase (or M phase) of the (11) CSYEKRPMC 65 cell cycle by nocodazole treatment) to a peptide concentra and tion in the culture medium of 10 uM, with the photo being a merged image of a DAN nuclear stain image and a fluores US 9,353,350 B2 5 6 cence image showing the result of an immunofluorescence In the present description, the term “synthetic peptide' assay using an anti-tubulin antibody. refers to a peptide fragment that is manufactured by artificial FIG. 2 is a fluorescence microscope photo (image) show chemical synthesis or biosynthesis (i.e. genetic engineering ing the state of cultured cells after incubation following addi based production). tion of a sample peptide (Sample 12) to a culture medium of 5 In this description, the term "peptide refers to an amino HeLa cells (synchronized in the G2 phase (or M phase) of the add polymer having a plurality of peptide bonds, and is not cell cycle by nocodazole treatment) to a peptide concentra limited by the number of amino acid residues included in the tion in the culture medium of 10 uM, with the photo being a peptide chain, with the term typically referring to one having merged image of a DAPI nuclear stain image and a fluores a relatively small molecular weight with a total of no more 10 than 50 (e.g. no more than 30) amino acid residues. cence image showing the result of an immunofluorescence In this description, unless otherwise specified, the term assay using an anti-tubulin antibody. “amino acid residue' includes the N-terminal amino acid and FIG. 3 is a fluorescence microscope photo (image) show the C-terminal amino acid of a peptide chain. ing the state of a cultured cell after incubation following In this description, the term “tumor should be interpreted addition of a sample peptide (Sample 9) to a culture medium 15 in a broad sense and refers to general tumors (typically malig of HeLa cells (synchronized in the G2 phase (or M phase) of nant tumors) including carcinomas and sarcomas as well as the cell cycle by nocodazole treatment) to a peptide concen lesions of blood and hematopoietic tissue (leukemia, lym tration in the culture medium of 10 uM, with the photo being phoma, etc.). The term “tumor cells' refers to cells that form a merged image of a DAPI nuclear stain image and a fluores Such a tumor, typically referring to cells that have come to cence image showing the result of an immunofluorescence undergo abnormal proliferation with no relation to Surround assay using an anti-tubulin antibody. This image shows a cell ing normal tissue (i.e. cells that have become cancerous). with a particularly high degree of multipolarity. Thus, unless otherwise specified, cells classified as tumor FIG. 4 is a fluorescence microscope photo (image) show cells (cancer cells) instead of normal cells are called as tumor ing the state of a cultured cell after incubation following cells regardless of the origin and nature of the cells. The addition of a sample peptide (Sample 6) to a culture medium 25 concept of tumor cells referred to herein encompasses cells of A549 cells (synchronized in the G2 phase (or M phase) of that form epithelial tumors (squamous cells carcinoma, the cell cycle by nocodazole treatment) to a peptide concen adenocarcinoma, etc.), nonepithelial tumors (various types of tration in the culture medium of 10 uM, with the photo being sarcoma, osteosarcoma, etc.), various types of cytoma (neu a merged image of a DAPI nuclear stain image and a fluores roblastoma, retinoblastoma, etc.), lymphomas, melanomas, cence image showing the result of an immunofluorescence 30 and so on. assay using a fluorescence-labeled anti-tubulin antibody and In this description, the term “modified amino acid an anti-centrin antibody. sequence' regarding a certain amino acid sequence refers to FIG. 5 is a fluorescence microscope photo (image) show an amino acid sequence formed by Substituting, deleting and/ ing the state of a cultured cell after incubation following or adding (inserting) ting) one or several (e.g. two or three) addition of a sample peptide (Sample 11) to a culture medium 35 amino acid residues without impairing the function of the of A549 cells (synchronized in the G2 phase (or M phase) of certain amino acid sequence (e.g. the ability of the multipo the cell cycle by nocodazole treatment) to a peptide concen larity-inducing peptides to induce multipolarity, the NoLS’s tration in the culture medium of 10 uM, with the photo being transportability from the extracellular space into nuclei). a merged image of a DAN nuclear stain image and a fluores Typical examples of the modified amino acid sequence cence image showing the result of an immunofluorescence 40 referred to in the present description include a sequence gen assay using a fluorescence-labeled anti-tubulin antibody and erated by conservative amino acid replacement where one or an anti-centrin antibody. several (typically two or three) amino acid residues are con servatively substituted (e.g., a sequence where a basic amino DESCRIPTION OF EMBODIMENTS acid residue has been substituted with a different basic amino 45 acid residue; e.g. mutual Substitution between alysine residue Preferred embodiments of the present invention are and an arginine residue), a sequence obtained by adding (in described below. Note that technical matters other than the serting) or deleting one or several (typically two or three) matters particularly mentioned in the present description (e.g. amino acid residues in a certain amino acid sequence, and the primary structures of synthetic peptides disclosed herein) like. Thus, the multipolarity-inducing peptide disclosed which are required for carrying out the present invention (e.g. 50 herein encompasses synthetic peptides consisting of amino general matters relating to chemical peptide synthesis, cell acid sequences identical to the amino acid sequences of the cultivation, and preparation of a pharmaceutical composition respective SEQ ID NOs as well as a synthetic peptide con containing a peptide) are matters of design variation that sisting of an amino acid sequence resulting from Substitution could be apprehended by a person skilled in the art based on (e.g. the conservative Substitution), deletion and/or addition conventional art in Such fields as cell engineering, physiol 55 of one or several (typically two or three) amino acid residues ogy, medicine, pharmacology organic chemistry, biochemis in/from/to the amino acid sequence of each SEQID NO while try, genetic engineering, protein engineering, molecular biol showing comparable multipolarity-inducing activity. ogy, genetics, and the like. The present invention can be In this description, the term “polynucleotide' refers to a practiced based on the technical details disclosed in the polymer (nucleic acids) in which several nucleotides are present description and common general technical knowl 60 linked by phosphodiester bonds, but not limited by the num edge in the pertinent fields. In the following description, ber of nucleotides. The polynucleotide in the present descrip amino acids are indicated by single-letter designations (in tion encompasses DNA fragments and RNA fragments of sequence listings, by three-letter designations) in accordance various lengths. The term “artificially designed polynucle with thenomenclature foramino acids set forth in the IUPAC otide' refers to a polynucleotide whose chain the whole IUB guidelines. 65 length) does not exist by itself in nature and that is manufac The present description incorporates by reference the tured artificially by chemical synthesis or biosynthesis (i.e., entire contents of all the documents cited herein. genetic engineering-based production). US 9,353,350 B2 7 8 The present invention also provides a composition prefer The amino acid sequence of SEQIDNO: 14 corresponds to ably used for practicing the multipolar cell production an NoLS consisting of 8 amino acid residues contained in the method disclosed herein. In other words, the composition N protein (nucleocapsid protein) of IBV (avian infectious disclosed herein is a composition (which may be referred to as a “multipolarity-inducing composition hereinafter) com bronchitis virus) which belongs to the genus Coronavirus. prising at least one species of pharmaceutically acceptable The amino acid sequence of SEQ11) NO 15 corresponds to carrier, and further comprising, as an active ingredient, a an NoLS consisting of 14 total amino acid residues originat multipolarity-inducing peptide consisting of an amino acid ing from FGF2 (fibroblast growth factor 2). sequence which has been found for the first time by the The amino acid sequence of SEQIDNO: 16 corresponds to present inventors to have an activity to induce multipolarity in an NoLS consisting of 19 total amino acid residues originat certain eukaryotic cells typically, cells of mammals including 10 ing from a species of nucleolar protein (Ap LP). humans and other animals, or cells of birds, preferably cul The amino acid sequence of SEQIDNO: 17 corresponds to tured tumor cells) described earlier oran NoLS-coupled mul an NoLS consisting of 16 total amino acid residues originat tipolarity-inducing peptide constituted by coupling an NoLS ing from a protein (Y(1)34.5) of HSV-1 (herpes simplex virus to the N-terminus or the C-terminus of the amino acid type 1). sequence of the peptide. It is also referred to as a multipolar 15 ity-inducing agent. The amino acid sequence of SEQIDNO: 18 corresponds to Some of the multipolarity-inducing peptides disclosed an NoLS consisting of 19 total amino acid residues originat herein are each constituted with an amino acid sequence ing from p48 protein of HIC (human I-mfa domain-contain represented by one of SEQID NO: 1 to SEQID NO: 12 (or a ing protein). modified sequence thereof). As described earlier, the peptides The amino acid sequence of SEQIDNO: 19 corresponds to consisting of these amino acid sequences can act as FtSA an NoLS consisting a 16 total amino acid residues originating inhibitors (SEQID NOs: 1 to 10) or FtsZ, inhibitors (SEQID from MEQ protein of MDV (Marek’s disease virus). NOs: 11 to 12) against certain species of bacteria as reported The amino acid sequence of SEQIDNO:20 corresponds to in Non-Patent Document 1 or Non-Patent Document 2 listed an NoLS consisting of 17 total amino acid residues originat earlier. Heretofore, however, such various peptides capable of 25 ing from Survivin-deltaEX3 which is an apoptosis-Suppress acting as Fts.A inhibitors or FtsZ, inhibitors have never been ing protein. known to induce multipolarity in certain cells (typically By placing the NoLS species represented by one of these tumor cells), or nothing has ever Suggested this, either. SEQID NOS on the N-terminal side or the C-terminal side of Some others of the multipolarity-inducing peptides dis the amino acid sequence represented by one of SEQID NO: closed herein are each constituted with an amino acid 30 1 to SEQ ID NO 12 (or a modified sequence thereof), a sequence represented by one of SEQID NO: 1 to SEQID NO: preferable multipolarity-inducing peptide (synthetic peptide) 12 (or a modified sequence thereof) as well as an amino acid can be constituted. sequence forming an NoLS represented by one of the follow The multipolarity-inducing peptide disclosed herein pref ing SEQ ID NO: 13 to SEQ ID NO: 20 (or a modified erably has at least one amidated amino acid residue. Amida sequence thereof): 35 tion of a carboxyl group in an amino acid residue (typically the C-terminal amino acid residue of the peptide chain) may increase the structural stability (e.g., protease resistance) of (SEQ ID NO: 13) the synthetic peptide. (13) KKRTLRKNDRKKR, The multipolarity-inducing peptide disclosed herein is 40 (SEQ ID NO: 14) short in the chain length, having a relatively small number of (14) WRROARFK, amino acid residues (typically 30 or fewer). Thus, its chemi cal synthesis is facile and the multipolarity-inducing peptide (SEQ ID NO: 15) can be easily provided. With respect to the conformation (15) RSRKYTSWYVALKR, (spatial structure) of the peptide, there are no particular limi (SEQ ID NO: 16) 45 tations as long as it exhibits the multipolarity-inducing activ (16) MAKSIRSKHRROMRMMKRE, ity. From the standpoint of unlikeliness to become aan immu nogen (antigen), a preferable peptide has a linear or a helical (SEO ID NO: 17) structure. Peptides having these conformations are less likely (17) MARRRRHRGPRRPRPP, to form epitopes. From Such a standpoint, it is preferable as a (SEQ ID NO: 18) 50 peptide used for manufacturing a multipolarity-inducing (18) GRCRRLANFGPRKRRRRRR, composition disclosed herein. (SEQ ID NO: 19) It is noted that in the multipolarity-inducing peptide dis (19). RRRKRNRDARRRRRKO closed herein, all amino acid residues are preferably L-amino and acids while for as long as the multipolarity-inducing activity 55 is not lost, part or all of the amino acid residues may be (SEQ ID NO: 2O) substituted with D-amino acids. (2O) MORKPTIRRKNLRLRRK. The multipolarity-inducing peptide disclosed herein can These amino acid sequences listed are all known as NoLS, be easily manufactured according to general chemical Syn and their data can be obtained, for example, from the database thesis methodologies. For instance, any of conventional of amino acid sequences of protein provided by NCBI (Na 60 Solid-phase and liquid-phase synthetic methods can be tional Center for Biotechnology Information). employed. A preferable Solid-phase synthetic method uses Namely, the amino acid sequence of SEQID NO: 13 cor Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycar responds to an NoLS consisting of 13 amino acid residues bonyl) as the protecting group for the amino group. from, amino acid residue 491 to amino acid residue 503 of For the multipolarity-inducing peptide disclosed herein, a LIM kinase 2 present in human endothelial cells, which is a 65 peptide chain having a desired amino acid sequence and a type of protein kinase involved in intracellular signal trans portion with modification (e.g., C-terminal amidation, etc.) duction. can be synthesized by Solid-phase synthesis using a commer US 9,353,350 B2 10 cial peptide synthesizer (which is, for instance, available from kits (such as PROTEIOSTM available from Toyobo Co., Ltd. PerSeptive Biosystems, Applied Biosystems, etc.). of Japan) are commercially available. Alternatively, the multipolarity-inducing peptide may be A heretofore known method can be employed for facile biosynthesized based on genetic engineering techniques. In production (synthesis) of a single-stranded or double particular, a polynucleotide (typically a DNA strand) is syn 5 Stranded polynucleotide containing a nucleotide sequence thesized so as to have a nucleotide sequence (including the encoding the multipolarity-inducing peptide disclosed herein ATG initiation codon) encoding the amino acid sequence of and/or a nucleotide sequence complementary thereto. In the desired multipolarity-inducing peptide. Then, in accor other words, by selecting a codon corresponding to the dance with the host cells, a recombinant vector is constructed respective amino acid residues constituting the designed So as to have an expression gene construct composed of the 10 amino acid sequence, a nucleotide sequence corresponding to synthesized polynucleotide (DNA) and various regulatory the amino acid sequence of the multipolarity-inducing pep elements (including promoters, ribosome binding sites, ter tide can be easily determined and provided. Once the nucle minators, enhancers, and various cis-elements which control otide sequence is determined, by utilizing a DNA synthesizer, the expression level) to allow expression of the amino acid 15 etc., can be easily obtained a polynucleotide (single strand) sequence within the host cells. corresponding to the desired nucleotide sequence. Further By an ordinary technique, this recombinant vector is more, the target double-stranded DNA can be obtained by inserted into prescribed host cells (e.g. yeasts, insect cells, using the resulting single-stranded DNA as a template and plant cells), and the host cells, or tissue or a mass containing employing various enzymatic synthetic methods (typically these cells are cultured under specific conditions. In this way, PCR). The polynucleotide may be in the form of DNA or the target peptide can be expressed and produced intracellu RNA (mRNA, etc.). The DNA can be provided as a double larly. The target multipolarity-inducing peptide can be Strand or a single strand. When it is provided as a single obtained by isolating the peptide from the host cells (from the Strand, it may be a coding strand (sense Strand) or an anticod culture medium if Secreted) and Subjecting it to refolding, ing strand (anti-sense Strand) complementary thereto. purification, and so on as necessary. 25 The polynucleotide obtained in Such a way can be used as Methods hitherto used in the art may be directly employed a material for constructing a recombinant DNA (expression without modification as the method for constructing the cassette) for producing the multipolarity-inducing peptide in recombinant vector, introducing the constructed recombinant various host cells or in a cell-free protein synthesis system. vector into the host cell, and so on. Because Such methods The multipolarity-inducing peptide disclosed herein can themselves are not distinctive to the present invention, 30 act on at least one species of eukaryotic cells (preferably detailed description is omitted here. tumor cells of mammals or birds) causing formation of many For example, a fusion protein expression system may be centrosomes (typically, 3 or more, preferably 5 or more, par employed to allow efficient large-scale production in host ticularly preferably 8 or more, even more preferably 10 or cells. In particular, a gene (DNA) coding for the amino acid more e.g. about 3 to 20 centrosomes per cell) in the cells sequence of the multipolarity-inducing peptide of interest is 35 (typically in the M phase), thereby inducing formation of a chemically synthesized, and the synthesized gene is intro corresponding number of multipolar spindles. duced to a preferred site on a suitable fusion protein expres The multipolarity-inducing peptide may beina Saltform as sion vector (e.g. GST (glutathione S-transferase) fusion pro far as the multipolarity-inducing activity is not impaired. For tein expression vectors such as the pET series available from example, it is possible to use an acid salt of the peptide, which Novagen as well as the pGEX series available from Amer 40 can be obtained by adding a commonly used inorganic or sham Bioscience). Host cells (typically, Escherichia coil) are organic acid in accordance with an ordinary technique. Alter then transformed by the vector. The resulting transformant is natively, while the multipolarity-inducing activity is main cultured to produce the target fusion protein. This protein is tained, a different type of Salt (e.g., a metal salt) can be used. then extracted and purified. Subsequently, the purified fusion Accordingly, the scope of the "peptide' described in this protein is cleaved with a specific enzyme (protease), and the 45 description and in claims encompasses such salt forms. liberated target peptide fragments (the designed multipolar The multipolarity-inducing composition disclosed herein ity-inducing peptide) are collected by a method such as affin may contain various pharmaceutically (medically) accept ity chromatography. As necessary, it is allowed to refold by a able carriers in accordance with the preparation, as far as it Suitable method. The multipolarity-inducing peptide dis can preserve the multipolarity-inducing peptide which is closed herein can be produced by using Such a fusion protein 50 active ingredient without losing its ability to induce the mul expression system heretofore known (e.g., the GST/His sys tipolarity. For example, carriers generally used as diluents or tem available from Amersham Bioscience may be used). excipients in peptide medications can be utilized. Alternatively, the target polypeptide may be synthesized in Although the carrier may suitably vary depending or the vitro by constructing a template DNA for a cell-free protein intended purpose and form of the composition disclosed synthesis system (i.e., a synthesized gene fragment having a 55 herein (i.e. a multipolarity-inducing composition), typical nucleotide sequence that codes for the amino acid sequence examples include water, physiological buffers and various of the multipolarity-inducing peptide), and employing a cell organic solvents. The carrier may be an aqueous alcohol free protein synthesis system with use of various compounds (ethanol or the like) Solution at an appropriate concentration, (e.g., ATP RNA polymerase, amino acids, etc.) required for glycerol, or non-drying oil such as olive oil. Alternatively, it the peptide synthesis. For information concerning cell-free 60 may be liposome. Examples of secondary ingredients that protein synthesis systems, reference may be made to, for may be contained in the multipolarity-inducing composition example, Shimizu et al., Nature Biotechnology, 19, 751-755 include various fillers, hulking agents, binders, wetting (2001), and Madin et al., Proc. Natl. Acad. Sci. USA, 97(2), agents, Surfactants, dyes, fragrances and the like. 559-564 (2000). Based on the technology described in these Typical examples of the form of the multipolarity-inducing articles, many corporations have been conducting contract 65 composition (multipolarity-inducing drug) include liquid for manufacturing of polypeptides at the time when this applica mulations, Suspensions, and so on. The multipolarity-induc tion was filed. Also, wheat germ cell-free protein synthesis ing composition may be formulated as a freeze-dried product US 9,353,350 B2 11 12 or pellets to prepare a drug Solution by dissolving in Saline or Alternatively, general double thymidine block or the like a suitable buffer (e.g., PBS) just prior to use. can be employed to synchronize cultured cells it the G1 phase The process itself of preparing a composition in various of the cell cycle (i.e. to inhibit the transition from the G1 phase forms with the multipolarity-inducing peptide (primary to the S phase). ingredient) and various carriers (secondary ingredients) may Several worked examples relating to the present invention be carried out inaccordance with a heretofore known method. are described below while these examples are not intended to Because Such a preparation process itself is not distinctive to limit the scope of the invention. the present invention, detailed description is omitted here. The target cells to which the multipolarity-inducing pep Example 1 tide disclosed herein is applied are not particularly limited as 10 long as they are eukaryotic cells (cultured cells) and are Peptide Synthesis Susceptible to multipolarity induction. The composition can be applied to various types of cells of humans and nonhuman A total of 24 different synthetic peptides consisting of the mammals or cells of birds, especially to cultured tumor cells 15 respective amino acid sequences shown in Table 1 were pro (various cell lines). Examples include cells forming squa duced using a peptide synthesizer. In the following descrip mous cell carcinomas, cells forming adenocarcinomas, and tion, the 24 in total of different peptides synthesized are cells (cultured cells) forming cytomas Such as neuroblasto referred to as Samples 1 to 24. mas, retinoblastomas, pheochromocytomas, and other cyto aS. TABLE 1. No special treatment is required for the method for supply Sample Total number of ing the multipolarity-inducing peptide (multipolarity-induc No. Amino acid sequence amino acid residues ing composition) disclosed hereinto target cultured cells. For example, a suitable amount of the multipolarity-inducing 1. CSSATGKSC (SEO ID NO : 1) 9 peptide (or a composition comprising the peptide as the pri 25 2 CLAPSPSKC (SEO ID NO: 2) 9 mary component) can be added to a culture (typically a cul ture medium) in which the target cells (including a cell aggre 3 CLGOTKMRC (SEO ID NO : 3) 9 gation, tissue oran organ removed from a living organism) are 4. CGHRPYOYC (SEO ID NO : 4) 9 being cultured. The amount of the peptide per dose and the number of 30 5 CWAFPLHHC (SEO ID NO : 5) 9 doses can be decided by considering efficiency of multipo 6 CTLNSHSNC (SEQ ID NO : 6) 9 larity induction of the cells in the culture, and are not particu larly limited as they may vary in accordance with conditions 7 CEISAKRTC (SEO ID NO : 7) 9 such as the typo of tumor cells to be cultured, cell density (cell 8 CHILHAQAC (SEQ ID NO: 8) 9 density at the incubation start), passage number, incubation 35 conditions, type of culture medium, and so on. It may be 9 CPRPPSLEC (SEO ID NO: 9) 9 adjusted so that the concentration of multipolarity-inducing O CTGHWASEC (SEO ID NO : 10) 9 peptide in the culture medium is in a range of about 1 uM or higher, but lower than 20LM or preferably 5uM or higher, but 40 1. CSYEKRPMC (SEO ID NO : 11) 9 100 uM or lower. Addition of an excessively small amount of 2 CLTKSYTSC (SEO ID NO : 12) 9 the peptide is not preferable because the multipolarity induc tion rate of the cells in the culture will be too low. On the other 3 CSSATGKSCKKRTLRKNDRKKR 22 hand, addition of an excessively large amount of the peptide (SEQ ID NO: 21) is not preferable because an excessive level of multipolarity 45 4. CLAPSPSKCKKRTLRKNDRKKR 22 induction will cause cell death prematurely. (SEQ ID NO: 22) The multipolar cell production method (multipolarity induction method) disclosed herein is preferable since the 5 CLGOTKMRCKKRTLRKNDRKKR 22 target cultured cells are synchronized in the cell cycle in (SEQ ID NO: 23) advance and by Supplying a desirable multipolarity-inducing 50 6 CGHRPYOYCKKRTLRKNDRKKR 22 peptide to the synchronized cells, the multipolar level of the (SEQ ID NO: 24) cells in the culture can be increased. In particular, it is pref 7 CWAFPLHHCKKRTLRKNDRKKR 22 erable that cells in a culture are synchronized in the G2 phase (SEQ ID NO: 25) or M phase. Herein, “being synchronized in the G2 phase or M phase' 55 8 CTLNSHSNCKKRTLRKNDRKKR 22 means that by Subjecting cultured cells to a Suitable treatment (SEQ ID NO: 26) (typically, addition of a drug Such as nocodazole, etc., gener 9 CEISAKRTCKKRTLRKNDRKKR 22 ally used for cell cycle synchronization), the transition of the (SEO ID NO: 27) cultured cells from the G2 phase to the Mphase or from the M phase to the G1 phase is inhibited and the cells are arrested in 60 2O CHILHAOACKKRTLRKNDRKKR 22 the G2 phase or M phase. For example, nocodazole, colchi (SEQ ID NO: 28) cine, colcemid, Vincristine or the like can be added to a cell 21 CPRPPSLECKKRTLRKNDRKKR 22 culture (typically a culture medium) to a Suitable concentra (SEQ ID NO: 29) tion to adjust the cell cycle, thereby synchronizing the target 22 CTGHWASECKKRTLRKNDRKKR 22 cells in the G2 phase or M phase. It is particularly preferable 65 (SEQ ID NO: 30) to Supply a multipolarity-inducing peptide in Synchrony with the G2 phase. US 9,353,350 B2 14 TABLE 1 - continued After 6 hours of incubation, the presence of multipolarity was observed by immunofluorescence under fluorescence Sample Total number of microscope. In particular, after 6 hours of incubation follow No. Amino acid sequence amino acid residues ing the addition of a sample peptide, the cultured cells were 23 CSYEKRPMCKKRTLRKNDRKKR 22 subjected to nuclear staining with DAPI (4,6-diamidino-2- (SEQ ID NO: 31) phenylindole), and using C-tubulin as a marker for spindles, 24 CLTKSYTSCKKRTLRKNDRKKR 22 the cells were tested for the presence of tubulin (i.e., the (SEQ ID NO: 32) presence of spindles) by immunofluorescence with an anti tubulin antibody (primary antibody) to identify the tubulin 10 and a fluorescent dye (ALEXA FLUOR (registered trade Each sample is constituted to have an amino acid sequence mark) 555)-labeled secondary antibody to identify the pri shown in Table 1 (also in the sequence listing). More specifi mary antibody. cally, Samples 1 to 12 are peptides capable of acting as FtSA Table 2 shows the multipolar levels (% of the number of inhibitors (SEQID NOs: 1 to 10) or FtsZ, inhibitors (SEQID multipolar cells in which a spindle (multipolar spindle) was NOs: 11 to 12) disclosed in Non-Patent Document 1 or in 15 found randomly extending from each of 3 or more cen Non-Patent Document 2; and each one is a synthetic peptide trosomes relative to the number of M-phase cells observed consisting of 9 total amino acid residues, characterized by the under fluorescence microscope) computed based on the fluo N-terminal and C-terminal amino acid residues being cys rescence microscope observations. teine residues. Samples 13 to 24 are synthetic peptides each consisting of TABLE 2 22 total amino acid residues, characterized by the amino acid Sample No. Number of Number of Multipolar level sequence of each peptide of Samples 1 to 12 being coupled at (Concentration) M-phase cells multipolar cells (%) the C-terminal side with the amino acid sequence constituting 1 (10M) 84 7 8.3 the NoLS of SEQID NO: 13. 25 4 (10M) 60 6 1O.O All peptides were synthesized by solid-phase synthesis 8 (10 M) 103 11 10.7 (Fmoc chemistry) using a commercial peptide synthesizer (an 9 (10 M) 102 19 18.6 12 (10 M) 1OO 13 13.0 Intavis AG system) in accordance with its operation manual. 14 (10 M) 74 10 13.5 Because the mode of using the peptide synthesizer itself is not Control 115 4 3.5 distinctive to the present invention, detailed description is 30 (no added peptide) omitted here. Each sample synthesized was dissolved in PBS (phosphate As shown in Table 2, when compared to the multipolar buffered saline) to prepare a stock Solution having a peptide level of 3.5% in the control group with no added sample concentration of 1 mM. peptide, the multipolar levels increased (8.3 to 18.6%) in the 35 treated groups with an added sample peptide. In particular, in Example 2 the group supplemented with Sample 4, 8, 9, 12 or 14, mul tipolarity was observed in 10% or more (preferably 15% or Multipolarity-Inducing Activity Assay 1 for Each more) of the M-phase cells. Synthetic Peptide 40 Example 3 Some of the sample peptides obtained in Example 1 were tested for multipolarity-inducing activity against target cul Multipolarity-Inducing Activity Assay 2 for Each tured tumor cells. Details of the assay are as described below. Synthetic Peptide As the tumor cells tested, a HeLa cell line (HeLa S3) was used. The cell line was cultured beforehand in a DMEM 45 The same sample peptides as those used in Example 2 medium (i.e. Dulbecco MEM medium (DMEM medium, a above were used and tested for multipolarity-inducing activ Gibco product) containing 10% fetal bovine serum (FBS, a ity against cultured tumor cells synchronized in the G2 phase Gibco product), 2 mM of L-glutamine, 50 unit/mL of peni (or M phase). Details of the assay areas described below. cillin, and 50 ug/mL of streptomycin), and seeded at about As the tumor cells tested, a HeLa cell line (HeLa S3) was 3x10" cells per well in an 8-well slide glass. Thymidine was 50 used. The cell line was cultured under the same conditions as added to the culture medium to a final concentration of 2.5 Example 2 and seeded at about 3x10" cells per well in an mM. Incubation was carried out at 37°C. under 5% CO for 8-well slide glass. Thymidine was added to the culture 16 hours. The culture medium was exchanged to remove medium to a final concentration of 2.5 mM. Incubation was thymidine and incubation was carried out for 8 more hours. carried out at 37°C. under 5% CO, for 18 hours. The culture Subsequently, thymidine was added again to a final concen 55 medium was exchanged to remove thymidine and incubation tration of 2.5 mM and incubation was carried out for 16 hours. was carried out for 6 more hours. Subsequently, nocodazole By such double thymidine block, the cells being cultured was added to a final concentration of 0.1 ug/mL and incuba were synchronized in the G1 phase of the cell cycle. After tion was carried out for 16 hours. By such addition of nocoda Such synchronization treatment, the culture medium was Zole, the cells being cultured were synchronized in the G2 exchanged to remove thymidine and incubation was carried 60 phase (M phase) of the cell cycle. out for 2 more hours. Subsequently, one of the sample pep After the 16 hours of incubation, without exchanging the tides was added to each well. In this example, one of Samples culture medium (i.e. with nocodazole being contained), one 1, 4, 8, 9, 12 and 14 was added to a peptide concentration in of the sample peptides was added to each well to a peptide the well of 10 uM. concentration in the well of 10 uMandincubation was carried After a sample peptide was added as described above, the 65 out for 4 hours. Subsequently, the culture medium was 8-well slid glass was incubated under the aforementioned exchanged and incubation was continued for 2 more hours conditions for 6 hours. without nocodazole or the sample peptide. US 9,353,350 B2 15 16 After the 2 hours of incubation, in the same manner as body) to identify the centrin-2 and a fluorescent dye-labeled Example 2, the presence of multipolarity was observed by secondary antibody to identify the primary antibody. immunofluorescence under fluorescence microscope. Table 4 shows the multipolar levels of the number of mul Table 3 shows the multipolar levels (% of the number of tipolar cells in which a spindle multipolar spindle) was found multipolar cells in which a spindle (multipolar spindle) was 5 randomly extending from each of 3 or more centroSomes found randomly extending from each of 3 or more cen relative to the number of M-phase cells observed under fluo trosomes relative to the number of M-phase cells observed rescence microscope) computed based on the fluorescence under fluorescence microscope) computed based on the fluo microscope observations. rescence microscope observations. 10 TABLE 4 TABLE 3 Sample No. (Concentration) Multipolar level (%) Sample No. Number of Number of Multipolar level (Concentration) M-phase cells multipolar cells (%) 1 (10M) 42 2 (10M) 38 1 (10M) 75 47 62.7 15 3 (10 M) 44 4 (10M) 75 47 62.7 4 (10M) 36 8 (10 M) 75 51 68.0 5 (10 M) 41 9 (10 M) 75 55 73.3 6 (10M) 30 12 (10M) 75 48 64.O 7 (10 M) 36 14 (10M) 75 46 61.3 8 (10 M) 36 Control 75 2 2.7 9 (10 M) 43 (no added peptide) 10 (10 M) 31 11 (10M) 61 12 (10M) 45 As shown in Table 3, when compared to the multipolar 14 (10M) 38 level of 2.7% in the control group with no added sample 19 (10 M) 44 peptide, significantly higher multipolar levels (61.3 to 73.3%) 25 Control s3 were observed in all the treated groups with an added sample (no added peptide) peptide. This indicates that synchronization of target culture cells in the G2 phase (or Mphase) leads to significantly higher As shown in Table 4, when compared to the multipolar multipolarity-inducing activity by a multipolarity-inducing level of 3% or lower in the control group with no added 30 sample peptide, higher multipolar levels (30 to 61%) were peptide. As some examples, the results offluorescence micro observed in all the treated groups with an added sample Scope observations relating to Samples 9 and 12 are shown in peptide. In particular, in the cells supplemented with the FIG. 1 to FIG. 3. As shown in the fluorescence microscope peptide of Sample 1, 3, 5, 9, 11 or 12, or with the NoLS images of the treated groups with added Sample 9 (FIG. 1, coupled peptide of Sample 19, the multipolar level was sig FIG. 3) and Sample 12 (FIG. 2), 3 or more centrosomes nificantly high at 40% or above. formed in many cells and many cells were accordingly 35 AS Some examples, the results of fluorescence microscope observed to have multipolar spindles formed therein. observations relating to Samples 6 and 11 are shown in FIG. 4 and FIG. 5. As shown in the fluorescence microscope Example 4 images of the treated groups with added Sample 6 (FIG. 4) and Sample 11 (FIG. 5), 3 or more (typically 4 or more, e.g. Multipolarity-Inducing Activity Assay 3 for Each 40 8 or more) centrosomes formed in many cells and many cells Synthetic Peptide were accordingly observed to have multipolar spindles formed therein. Hence, the peptide disclosed herein can suit Some of the sample peptides obtained in Example 1 were ably induce multipolarity in cultured tumor cells (e.g. cells tested for multipolarity-inducing activity against target cul derived from a squamous cell carcinoma) and allows for tured tumor cells. Details of the assay are as described below. 45 stable production of multipolar cells (cultured cells). As the tumor cells tested, cells forming a human squamous cell carcinoma (human alveolar basal epithelial adenocarci INDUSTRIAL APPLICABILITY noma cells, A549) were used. The cell line was cultured in the same manner as Example 2 described above. The resulting As described above, according to the multipolarity-induc cultured tumor cells were synchronized in the G2 phase (or M 50 ing peptide disclosed herein, it is possible to frequently phase) in the same manner as Example 3 described above. induce multipolarity in target cultured cells (typically tumor Similarly to Examples 2 and 3 described above (although the cells), and to provide multipolar cells that can contribute as a number of M-phase cells was 100), the presence of multipo research material to various research purposes (e.g. search for larity was observed by immunofluorescence under fluores a factor involved in cell division or mechanism elucidation). cence microscope. In addition, using centrin-2 as a marker for 55 Sequence Listing Free Text centrosomes (centrioles), the cells were tested for the pres SEQ ID NOs: 1 to 12, 21 to 32 synthetic peptides ence of centrin-2 (i.e. the presence of centrosomes) by immu Sequence Listing nofluorescence with an anti-centrin-2 antibody (primary anti TG 12-007PCT ST25.txt

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 32

<21 Os SEQ ID NO 1 &211s LENGTH: 9 US 9,353,350 B2 17 18 - Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide <4 OOs, SEQUENCE: 1 Cys Ser Ser Ala Thr Gly Lys Ser Cys 1. 5

<210s, SEQ ID NO 2 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide <4 OOs, SEQUENCE: 2 Cys Lieu Ala Pro Ser Pro Ser Lys Cys 1. 5

<210s, SEQ ID NO 3 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 3 Cys Lieu. Gly Glin Thr Lys Met Arg Cys 1. 5

<210s, SEQ ID NO 4 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide <4 OOs, SEQUENCE: 4 Cys Gly His Arg Pro Tyr Glin Tyr Cys 1. 5

<210s, SEQ ID NO 5 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 5 Cys Trp Ala Phe Pro Leu. His His Cys 1. 5

<210s, SEQ ID NO 6 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 6 Cys Thr Lieu. Asn Ser His Ser Asn Cys 1. 5

<210s, SEQ ID NO 7 &211s LENGTH: 9 212. TYPE: PRT US 9,353,350 B2 19 20 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OO > SEQUENCE: 7 Cys Glu Ile Ser Ala Lys Arg Thr Cys 1. 5

<210s, SEQ ID NO 8 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide <4 OOs, SEQUENCE: 8 Cys His Ile Lieu. His Ala Glin Ala Cys 1. 5

<210s, SEQ ID NO 9 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 9 Cys Pro Arg Pro Pro Ser Lieu. Glu. Cys 1. 5

<210 SEQ ID NO 10 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 10 Cys Thr Gly His Trp Ala Ser Glu. Cys 1. 5

<210s, SEQ ID NO 11 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide <4 OOs, SEQUENCE: 11 Cys Ser Tyr Glu Lys Arg Pro Met Cys 1. 5

<210s, SEQ ID NO 12 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 12 Cys Lieu. Thir Lys Ser Tyr Thr Ser Cys 1. 5

<210s, SEQ ID NO 13 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 9,353,350 B2 21 - Continued

22 Os. FEATURE:

<4 OOs, SEQUENCE: 13 Llys Lys Arg Thir Lieu. Arg Lys Asn Asp Arg Llys Lys Arg 1. 5 1O

<210s, SEQ ID NO 14 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Avian infectious bronchitis virus 22 Os. FEATURE:

<4 OOs, SEQUENCE: 14 Trp Arg Arg Glin Ala Arg Phe Lys 1. 5

<210s, SEQ ID NO 15 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE:

<4 OOs, SEQUENCE: 15 Arg Ser Arg Llys Tyr Thr Ser Trp Tyr Val Ala Lieu Lys Arg 1. 5 1O

<210s, SEQ ID NO 16 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens & 22 O FEATURE;

<4 OOs, SEQUENCE: 16 Met Ala Lys Ser Ile Arg Ser Lys His Arg Arg Glin Met Arg Met Met 1. 5 1O 15 Lys Arg Glu

<210s, SEQ ID NO 17 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: herpes simplex virus 1 22 Os. FEATURE:

<4 OOs, SEQUENCE: 17 Met Ala Arg Arg Arg Arg His Arg Gly Pro Arg Arg Pro Arg Pro Pro 1. 5 1O 15

<210s, SEQ ID NO 18 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE:

<4 OOs, SEQUENCE: 18 Gly Arg Cys Arg Arg Lieu Ala Asn. Phe Gly Pro Arg Lys Arg Arg Arg 1. 5 1O 15

Arg Arg Arg

<210s, SEQ ID NO 19 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Marek's disease gammaherpesvirus MKT-1 22 Os. FEATURE:

<4 OOs, SEQUENCE: 19 US 9,353,350 B2 23 24 - Continued

Arg Arg Arg Lys Arg Asn Arg Asp Ala Arg Arg Arg Arg Arg Lys Glin 1. 5 1O 15

<210s, SEQ ID NO 2 O &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE:

<4 OOs, SEQUENCE: 2O Met Glin Arg Llys Pro Thir Ile Arg Arg Lys Asn Lieu. Arg Lieu. Arg Arg 1. 5 1O 15 Lys

<210s, SEQ ID NO 21 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 21 Cys Ser Ser Ala Thr Gly Llys Ser Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 Asn Asp Arg Llys Lys Arg 2O

<210s, SEQ ID NO 22 <211 LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 22 Cys Lieu Ala Pro Ser Pro Ser Lys Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 Asn Asp Arg Llys Lys Arg 2O

<210s, SEQ ID NO 23 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 23 Cys Lieu. Gly Glin Thr Lys Met Arg Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 Asn Asp Arg Llys Lys Arg 2O

<210s, SEQ ID NO 24 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 24 Cys Gly His Arg Pro Tyr Glin Tyr Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 US 9,353,350 B2 25 26 - Continued Asn Asp Arg Llys Lys Arg 2O

<210s, SEQ ID NO 25 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 25 Cys Trp Ala Phe Pro Lieu. His His Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 Asn Asp Arg Llys Lys Arg 2O

<210s, SEQ ID NO 26 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 26 Cys Thr Lieu. Asn. Ser His Ser Asn. Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 Asn Asp Arg Llys Lys Arg 2O

<210 SEQ ID NO 27 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 27 Cys Glu Ile Ser Ala Lys Arg Thr Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 Asn Asp Arg Llys Lys Arg 2O

<210s, SEQ ID NO 28 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide <4 OOs, SEQUENCE: 28 Cys His Ile Lieu. His Ala Glin Ala Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 Asn Asp Arg Llys Lys Arg 2O

<210s, SEQ ID NO 29 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 29 Cys Pro Arg Pro Pro Ser Lieu. Glu. Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 US 9,353,350 B2 27 28 - Continued

Asn Asp Arg Llys Lys Arg 2O

<210s, SEQ ID NO 3 O &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide

<4 OOs, SEQUENCE: 30 Cys Thr Gly. His Trp Ala Ser Glu. Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 Asn Asp Arg Llys Lys Arg 2O

<210s, SEQ ID NO 31 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide <4 OOs, SEQUENCE: 31 Cys Ser Tyr Glu Lys Arg Pro Met Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 Asn Asp Arg Llys Lys Arg 2O

<210s, SEQ ID NO 32 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide <4 OOs, SEQUENCE: 32 Cys Lieu. Thir Lys Ser Tyr Thr Ser Cys Llys Lys Arg Thr Lieu. Arg Llys 1. 5 1O 15 Asn Asp Arg Llys Lys Arg 2O

45 The invention claimed is: - Continued 1. A method for producing from at least one species of (SEQ ID NO: eukaryotic cells a multipolar cell having three or more cen CWAFPLHHC, troSomes and a multipolar spindle, the method comprising (SEQ ID NO: culturing target eukaryotic cells, and 50 CTLNSHSNC Supplying the eukaryotic cells being cultured with a syn thetic peptide consisting of an amino acid sequence (SEQ ID NO: Selected from the following amino acid sequences that CEISAKRTC, act as FtsZ inhibitors or Fts.A inhibitors: (SEQ ID NO: 55 CHILHAQAC,

(SEQ ID NO: (SEQ ID NO: 1) CPRPPSLEC, (1) CSSATGKSC, (SEQ ID NO: 10) (SEQ ID NO: 2) 60 (2) CLAPSPSKC, (10) CTGHWASEC, (SEQ ID NO : 3) (SEQ ID NO: 11) (3) CLGOTKMRC, (11) CSYEKRPMC, and (SEQ ID NO : 4) (4) CGHRPYOYC 65 (SEQ ID NO: 12) (12) CLTKSYTSC. US 9,353,350 B2 29 2. A method for producing from at least one species of - Continued eukaryotic cells a multipolar cell having three or more cen (SEQ ID NO: 11) troSomes and a multipolar spindle, the method comprising (11) CSYEKRPMC, culturing target eukaryotic cells, and and Supplying the target eukaryotic cells being cultured with a synthetic peptide consisting of an amino acid sequence (SEQ ID NO: 12) (12) CLTKSYTSC; Selected from the following amino acid sequences that and act as FtsZ inhibitors or Fts.A inhibitors: and 10 an amino acid sequence that constitutes a nucleolar local (SEQ ID NO: 1) ization signal (NoLS) and is selected from SEQID NO: (1) CSSATGKSC, 13 to SEQID NO: 20. 3. The multipolar cell production method according to (SEQ ID NO: 2) (2) CLAPSPSKC, claim 1, wherein the eukaryotic cells to be cultured are syn 15 chronized in the G2 phase or M phase and the peptide is (SEQ ID NO : 3) supplied to the cultured cells that have been synchronized in (3) CLGOTKMRC, the G2 phase or M phase. 4. The multipolar cell production method according to (SEQ ID NO : 4) claim 1, wherein cultured tumor cells are used as the eukary (4) CGHRPYOYC otic cells. (SEO ID NO; 5) 5. The multipolar cell production method according to (5) CWAFPLHHC, claim 4, wherein the tumor cells are cells derived from a squamous cell carcinoma. (SEQ ID NO : 6) 6. The multipolar cell production method according to (6) CTLNSHSNC claim 2, wherein the eukaryotic cells to be cultured are syn (SEO ID NO: 7) 25 chronized in the G2 phase or M phase and the peptide is (7) CEISAKRTC, supplied to the cultured cells that have been synchronized in the G2 phase or M phase. (SEQ ID NO: 8) 7. The multipolar cell production method according to (8) CHILHAQAC, claim 2, wherein cultured tumor cells are used as the eukary (SEO ID NO: 9) 30 otic cells. (9) CPRPPSLEC, 8. The multipolar cell production method according to claim 7, wherein the tumor cells are cells derived from a (SEQ ID NO: 10) (1O) CTGHWASEC, squamous cell carcinoma. k k k k k