Oligopeptide compounds and their applications
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to the field of biotechnology, namely to obtaining oligopeptide compounds, containing a motive, interacting with a proliferating cell nuclear antigen (PCNA) and can be used in medicine. The oligopeptide compound consists of 14-70 amino acids and contains. a PCNA-interacting motive, representing [K/R]-[F/Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R], at least one signal sequence of nuclear localisation and at least one signal sequence of penetration into a cell, with the PCNA-interacting motive being located towards an N-end relative to the signal sequence.
EFFECT: invention makes it possible to carry out the efficient treatment of hyperproliferative disorders by the application of the oligopeptide compound in cyctostatic therapy or in radiotherapy as a sensitising substance.
34 cl, 6 dwg, 4 tbl, 8 ex
Field of the INVENTION
The present invention relates to new agents, pharmaceutical compositions and their use in therapy, in particular in any therapy where it is desirable or advantageous to reduce or prevent the proliferation or growth of cells, for example in the treatment of hyperproliferative diseases or in fact any condition which needs to cytostatic therapy or susceptible to it. The invention is based on the establishment of new interactions between nuclear antigen of proliferating cells (PCNA) and various proteins involved in DNA repair, maintenance and regulation of the cell cycle and the subsequent identification of a new Pentapeptide motif responsible for these interactions, which the inventors have named APIM. Thus, the present invention particularly relates to peptides or their mimetics containing this motif and is able to interact with PCNA, to pharmaceutical compositions containing such agents, and to the use of such agents in therapy, particularly therapies, including reduction or prevention of cell proliferation, as described above. Also proposed therapeutic methods that include the use of an agent containing the motif, binding of PCNA, preferably in combination with a cytostatic agent.
Human and animal cells are exposed to several factors that cause DNA damage, such as reactive oxygen species, ultraviolet radiation, x-rays and endogenous or exogenous cytotoxic agents.
Cytostatic agents are agents that suppress or inhibit cell growth and/or reproduction (proliferation/replication), for example by damaging DNA or by acting on the mechanism of cell replication. Alkylating agents are a class of cytotoxic agents, some of which use clinically or for research purposes.
Alkylating agents cause DNA damage by modifying the base atoms are N or O. the Type of damage depends on the type of agent, most agents cause-specific modification of DNA. Damage include DNA alkylation adducts and minitalia of crosslinking that can lead to incorrect coding during replication and/or locks replication with subsequent dvuhlistovymi breaks or translational synthesis.
Human and animal cells have different systems of DNA repair, including excisional repair of grounds, excision repair nucleotide and repair incorrectly paired nucleotides. An example is the oxidative DNA demethylase human ABH2, which turns 3-methylcytosine (3meC) back in cytosine and 1-methyladenine (1meA) to adenine by oxidative demethylation.
Close coordination between DNA repair and DNA replication regulated the cell cycle, is of great importance for the integrity of the genome. It is important that in the presence of damaged DNA replication stopped to repair the damage, otherwise it emerge and are reproduced mutations. Known one protein included in DNA replication and in DNA repair represents a nuclear antigen of proliferating cells (PCNA).
PCNA is a member of a family of proteins sliding engagement, which are functionally preserved from bacteria to higher eukaryotes, and whose main function is to offer replication polymerases with high processionally needed to copy the genome. In living cells S-phase PCNA-labeled green fluorescent protein (GFP), which forms distinct foci representing sites of replication. Thus, it can be used as a marker of S-phase.
Numerous proteins involved in cellular processes such as DNA repair, chromatin Assembly, epigenetic and chromatin remodeling, the cohesion of sister chromatids, cell cycle control and survival, localized in the so-called replicating zones �will win more than a dozen replication forks. Many of these proteins interact with PCNA through conservative peptide sequence that interacts with PCNA, called PIP-box (QxxL/I/MxxF/DF/Y), where x can be any amino acid. Alternative motif, binding of PCNA, called Kah-Boxing, identified using the peptide library display, but the importance of this motif for interaction with PCNA in vivo has not been confirmed.
Various proteins interact with PCNA, and it was proved that some of these proteins, including hABH2 localized with PCNA in replication foci. However, simultaneous localization does not in itself imply that between co-localized proteins occurs either direct or indirect interaction. Indeed, in the absence of hABH2 PCNA-binding motif, such as PIP-box or Ported box, suggests that hABH2 does not interact with PCNA.
Description of the INVENTION
In the work leading to the present invention, the inventors unexpectedly found that different proteins interact with PCNA through a new motif that interacts with PCNA. In one of these proteins, hABH2, this motif is located at N-end. The inventors have found that this motif is both necessary and sufficient for interaction with PCNA (see Example 1).
In the Examples below is explained in detail that the study� function of this motif, interacts with PCNA in DNA repair in case of damage by alkylation cell line expressing a recombinant peptide containing this motif were exposed to different doses of MMS (methylmethanesulfonate), which is an alkylating agent SN2, causing the formation of 3-methylcytosine and 1-methyladenine. It was found that the expression of a recombinant peptide containing the motif, increased the sensitivity of cells to DNA damage caused by MMS, indicating that the recombinant peptide containing the motif, competitively inhibited the interaction between PCNA and hABH2.
Also tested other agents, including BCNU (bis-metilhlorizotiazolinon), temozolomide (TZM) and mitomycin C (MMC), which cause other types of damage to DNA, and to my big surprise, the inventors found that the recombinant peptide containing the motif, also increased the sensitivity of cells to damage caused by such agents. It was totally unexpected, as BCNU represents About6-chloranilide agent that mainly causes magnetewan lateral relations and to some of monobasic cyclic adducts (1,N(6)ethenoadenine), it is reported that TZM is O6G-meteorous agent and MMS causes the formation miniawy cross-linking via N-�of alkilirovanny guanine in CpGs, and not hABH2 fixes these types of DNA damage. Alternatively, there are other enzymes that fix this kind of damage, for example damage by TZM fix directly O-methylguanine-DNA transferase (MGMT). The data obtained show that the recombinant peptide containing this motif, not only inhibits the interaction between hABH2 and PCNA, and that may be involved other proteins, i.e. other proteins can interact with PCNA through a new motive.
The inventors also found that the expression of a recombinant peptide containing the motif, enhanced the cytotoxic effect of the cytotoxic agents (especially MMS) above the observed in cell lines from knockout mice AVN, that is, mice that do not have OWN, it was also indicated that the recombinant peptide containing the motif, has a wider effect, and may inhibit other proteins in addition to hABH2.
Such unexpected data allowed the authors of the invention offer therapeutic use of the peptide containing the motif, binding of PCNA.
New PCNA-binding motif according to the invention, called APIM, has been described and can be defined as follows:
X1X2X3X3'X1'(SEQ ID NO:1),
where X1and X1'independently selected from the group of basic amino acid�t, X2is a lipophilic amino acid, and X3and X3'is independently selected from the group of uncharged, preferably non-polar amino acids.
Peptide (Oligopeptide or compound) capable of interacting with PCNA, may include or contain such a motif peptide (or sequence). Thus, the Oligopeptide compound capable of interacting with PCNA and containing this motif, disclosed in this description of the invention, and may represent some aspects of the present invention.
For example, in one embodiment of the present invention can be offered Oligopeptide compound that are capable of interacting with PCNA, and which comprises a motif X1X2X3X3'X1'(SEQ ID NO:1), where X1and X1'independently selected from the group of basic amino acids, X3is a lipophilic amino acid, and X3and X3'is independently selected from the group of uncharged, preferably non-polar amino acid, where the Oligopeptide compound additionally differs by at least one of the following:
(1) the Oligopeptide compound contains at least 11 amino acids or equivalent subunits;
(2) X2is phenylalanine;
(3) Oligopeptide compound contains at least one D-amino acid�;
(4) Oligopeptide compound contains at least one signal sequence, namely the sequence that directs Oligopeptide compound in a specific place, for example in cell (for example the sequence for entering the cell, which directs Oligopeptide compound in a cell) and/or in particular cell compartment (for example, a nuclear localization signal that directs Oligopeptide compound in the core); and
(5) the compound contains Oligopeptide motif [K/R]-F-[L/I/V]-[L/I/V]-[K/R] (SEQ ID NO:27).
In particular, in this embodiment the Oligopeptide compound contains a signal sequence for nuclear localization. In another embodiment the compound contains Oligopeptide sequence for entering the cell (peptide penetration into the cell). In yet another embodiment the compound contains Oligopeptide sequence entering the cell and a nuclear localization sequence.
Thus, it can be seen that in such embodiments the compound according to the invention can take the form of structures containing (comprising) Oligopeptide compound, which contains a motif that interacts with PCNA, as defined above, together with at least one signal sequence. In this aspect the invention is, therefore, rassmotrev�as you offer the design, containing Oligopeptide compound, which is capable of interacting with PCNA, and which comprises a motif X1X2X3X3'X1'(SEQ ID NO:1), where X1and X1'independently selected from the group of basic amino acids, X2is a lipophilic amino acid, and X3and X3'independently selected from the group of uncharged, preferably non-polar amino acids, together with at least one signal sequence.
As stated above, it was determined that a new motive of the invention mediates the interaction Oligopeptide compounds (e.g. peptides) or protein containing this motif, with PCNA.
The interaction may be direct or indirect, and may involve direct binding of the motif with PCNA, or the motive to communicate indirectly, for example, binding may be mediated by another molecule. This reference to the "PCNA-interaction" or "PCNA-binding" may, therefore, include any form of interaction and both direct and indirect binding.
Any reference in this description of the invention on "motive" should be understood to mean X1X2X3X3'X1'as defined in this specification.
Preferably, X1and X1'independently selected from lysine (K), arginine (R), gisti�ina (I), ornithine (Om), methyllysine (MEK) and acetylglycine (ASC), and more preferably K, R and H, or R;
X2preferably is an aromatic amino acid, more preferably it is selected from phenylalanine (F), tryptophan (W), tyrosine (Y), tert-butylglycol, cyclohexylamine, tert-butylbenzylamine, biphenylamine and tri-tert-butylthiophene (in some embodiments, this list may be deleted (F), in particular F, W and Y, or W and Y, F and Y, or F, and W, or in specific embodiments of X2can represent F, or W, or Y;
X3and X3'preferably represent an aliphatic amino acid, and can, for example, be independently selected from leucine (L), isoleucine (I), valine (V), alanine (A), methionine (M) and norleucine (Nor);
Preferably, X3and X3'are not both A, more preferably X3and X3'is selected from L, I, V and M, even more preferably from L, I and V.
The binding motif of PCNA can be improved in some embodiments, when X2is a W or Y. Thus, in one embodiment, X2not an F. However, as noted above, in other embodiments it may be an F.
Thus, the invention may be asked Oligopeptide compound comprising the motif [K/R]-[F/Y/W]-[L/I/V/AM]-[L/I/V/A/M]-[K/R] (SEQ ID NO:28), where the specified Oligopeptide�e compound capable of interacting with PCNA.
In another embodiment the motif may be defined as: [K/R]-[Y/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R] (SEQ ID NO:29).
In another embodiment the motif may be defined as: [K/R]-[F/Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R] (SEQ ID NO:30).
In another embodiment the motif may be defined as: [K/R]-[Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R] (SEQ ID NO:31).
In another embodiment the motif may be defined as: [K/R]-[F/W]-[L/I/V/A/M]-[L/I/V/A/M]-[K/R] (SEQ ID NO:32).
In another embodiment the motif may be defined as: [K/R]-[F/W]-[L/I/V/A]-[L/I/V/A]-[K/R] (SEQ ID NO:33).
In another embodiment the motif may be defined as: [K/R]-[F/W]-[L/I/V]-[L/I/V]-[KR] (SEQ ID NO:34).
In another embodiment the motif may be defined as: [K/R]-[F/Y/W]-[L/I/V]-[L/I/V]-[K/R] (SEQ ID NO:35).
In yet another embodiment the motif may be defined as: [K/R]-[Y/W]-[L/I/V]-[L/I/V]-[K/R] (SEQ ID NO:36).
In yet another embodiment the motif may be defined as: [K/R]-F-[L/I/V]- [L/I/V]-[K/R] (SEQ ID NO:37).
Oligopeptide compound preferably is a dedicated connection.
In a preferred embodiment the Oligopeptide compound is or contains a sequence RFLVK (SEQ ID NO:2). In other preferred embodiments the Oligopeptide compound is or contains a sequence selected from KFLLR (SEQ ID NO:3), KYLLR (SEQ ID NO: 4), KWLLR(SEQ ID NO: 5), KYILR (SEQ ID NO: 6), KYVLR (SEQ ID NO: 7), RFLLR (SEQ ID NO: 8), RYLLR (SEQ ID NO: 9), RWLLR (SEQ ID NO: 10), RYILR (SEQ ID NO: 11), RYVLR (SEQ ID NO: 12), RFLIR (SEQ ID NO: 13), RYLVR (SEQ ID NO: 14) RWLMR (SEQ ID NO: 15), RYVLR (SEQ ID NO: 16), RYVIR (SEQ ID NO: 17), RWLVK (SEQ ID NO: 18), RYLVK (SEQ ID NO: 19), RWLIK (SEQ I NO: 20), RWIVK (SEQ ID NO: 21), RWVVK (SEQ ID NO: 22), RWAVK (SEQ ID NO: 23), RYVVK (SEQ ID NO: 24), RYLIK (SEQ ID NO: 25) or RYLMK (SEQ ID NO: 26). These specific sequences are listed as examples and are not intended to limit the scope of the present invention.
In one preferred embodiment of the Oligopeptide compound according to the invention also contains a signal sequence that directs the motif on the specific type of cells, facilitates the penetration of compounds into the cell, and/or localizes the connection to a specific intracellular compartment, preferably the kernel.
Thus, the signal sequence can be considered as any sequence that affects the localization, or in other words, the direction of translocation or Oligopeptide transport connection at any desired position, for example at any desired cell or subcellular position. In preferred embodiments the desired position is a cell (i.e. the inner space of the cell) and/or the nucleus of the cell.
Thus, the signal sequence may be a sequence, which affects the Oligopeptide transport connections in the cell or across the cell membrane (inside the cell). It can be, thus, the so-called sequence "of penetration in a cage" or more specifically "peptide penetration into the cell"), also known in the art as a domain of a protein transduction (PTD) or the sequence of a protein transduction.
Thus, as indicated above, the preferred embodiment of the invention is a structure containing (1) Oligopeptide compound comprising the APIM motif (i.e. PCNA-interacting motif), as defined in this description of the invention, and (2) the sequence of penetration into the cell (more specifically, the peptide penetration into the cell).
The technology of peptide penetration into the cell (CPP) in recent years developed to a considerable extent, and is known for a large variety of peptides entering the cell described in the art, and of course, a number of such peptides are commercially available. The peptides entering the cell can vary widely in size, sequence, and charge, and of course on the mechanism of their action (which is currently unknown for certain peptides and not fully elucidated for others), but share a common ability to move through the plasma membrane and deliver the attached or associated group (the so-called "goods") to the cytoplasm, or even in some cases in the nucleus of the cell. The RAF are, thus, peptide delivery vectors.
The CPP can be made from naturally available protein, cat�who are able to move through the cell membrane, such as homeobox protein antennapedia Drosophila (transcription factor), viral proteins such as HIV-1 (human immunodeficiency virus - 1), a transcription factor TAT and capsid protein VP22 of HSV-I (herpes simplex virus - 1), and/or they can be produced synthetically, such as chimeric proteins or synthetic polypeptides, such as polyalanine. As stated above, there is no single mechanism responsible for the effect of transduction, and therefore the design of the CPP can be based on different structures and sequences. The peptides entering the cell considered in Jarver et al. 2006 Biochimica et Biophysica Acta 1758, pages 260-263, and Table 2 below lists the various typical peptides. In US 6645501 additionally describes various peptides entering the cell, which can be used.
|The Antp class||SEQUENCE||LINK|
|Penetratin||RQIKIWFQNRRMKWKK (SEQ ID NO:38)||Bolton (2000) Eur. J. Neuro. 12:287|
|Derivatives penetratin||RRMKWKK (SEQ ID NO:39)||US 6472507|
|NRRMKWKK (SEQ ID NO:40)||EP4855781|
|QNRRMKWKK (SEQ ID NO:41)||WO 97/12912|
|FQNRRMKWKK (SEQ ID NO:42)|
|RREKWKK (SEQ ID NO:43)|
|RRQKWKK (SEQ ID NO:44)|
|KRMKWKK (SEQ ID NO:45)|
|RKMKWKK (SEQ ID NO:46)|
|RROKWKK (SEQ ID NO:47)|
|RRMKQKK (SEQ ID NO:48)|
|RRMKWFK (SEQ ID NO:49)|
|RORKWKK (SEQ ID NO:50)|
|RRMWKKK (SEQ ID NO:51)|
|RRMKKWK (SEO ID NO:52)|
|(using standard single letter designation for amino acids, ornithine (O), diaminobutane acid (In), norleucine (N)|
|D-penetratin||rqikiwfqnrrmkwkk (SEQ ID NO:53)||Rouselle, C. et al.(2000) Mol. Pharm 57: 679|
|Puhelin (Syn)||RGGRLSYSRRRFSTSTGR (SEQ ID NO:54)||Rouselle, C. et al. (2000) Mol. Pharm 57: 679|
|Class of HIV-TAT|
|HIV-TAT||GRKKRRQRRRPPQ (SEQ ID NO:55)||Vives E. J Biol, Chem 1997, 272: 16010 Snyder (2004) PLOS 2: 186|
|47-57 OF HIV-TAT||YGRKKRRORRR (SEO ID NO:56)||Potocky et al. (2003)JBC|
|VP22||DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO:57)||Elliott g. Cell 1997, 88: 223 to 233|
|MAP||KLALKLALKALKAALKLA (SEQ ID NO:58)||Morris MC., Nat Biotechnol. 2001, 19: 1173-1176|
|Transportan||GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:59)||Pooga M, FASEB J 1998, 12: 67-77|
|Transportan-10||AGYLLGKNLKALAALAKKIL (SEQ ID NO:60)||Soomets U, Biochim Biophys Acta 2000, 1467: 165-176|
|KALA||WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO:61)||Oehike J., Biochim Biophys Acta 1998, 1414: 127-139|
|PEP-1||KETWWETWWTEWSQPKKKRKV (SEQ ID NO:62)||Wyman Biochemistry 1997, 36: 3008-3017|
|PEP-2||KETWFETWFTEWSQPKKKRKV (SEQ ID NO:63)|
|MPG||GALFLGFLGAAGSTMGAWSQPKSKRKV (SEQ ID NO:64)||Wagstaff KM Curr Med Chem 2006, 13: 1371-1387|
|Peptides Vectocell||VKRGLKLRHVRPRVTRMDV (SEQ ID NO:65)||Coupade (2005) Bochem. J. 407|
|SRRARRSPRHLGSG* (SEQ ID NO:66)|
|LRRERQSRLRRERQSR* (SEQ RD NO:67)|
|GAYDLRRRERQSRLRRRERQSR CSEO TH NO:68)|
|*indicates the addition of cys for conjugation with cargo|
|Wr-T conveyor||KETWWETWWTEWWTEWSQ-GPG-rrrrrrrr (SEQ ID NO:69) r =D-enantiomer of arginine||Kondo (2004) Mol. Can. Thera 1623|
|R7||RRRRRRR (SEQ ID NO:70)||Rothbard et al., Nat. Med 6 (2000) 1253-1257|
The SRF produced from Antennapedia (Antp class) are a particularly interesting class, based on about 16 amino acids of the sequence penetratin, as shown in Table 2, which correspond to the third loop of the protein antennapedia, and have been shown to be responsible for translocation of the protein. Penetratin actively developed as a carrier, including in particular pharmaceutical use, and was proposed and described a wide range of derivative penetratin and modified sequences. Reference may be made in particular to WO 91/1891, WO 00/1417, WO 00/29427, WO 2004/069279 and US 6080724. Thus, a 16-amino acid sequence Penetratin can be modified and/or shortened, or the peptide may be chemically modified or may be obtained retro-inverted - or retro-inverse analogues that still maintains the activity of entering the cell.
Another group of peptides entering the cell, which can be used successfully, based on the sequence of HIV-TAT, HIV-TAT and fragments constitute a preferred class of the RAF for use according to the present invention. Different the RAF on the basis of TAT is described in US 5656122. Typical peptide HIV-TAT, which is used in the Examples below, presented�is RKKRRQRRR (SEQ ID NO:71), but it is also true that you can use longer or shorter fragments of TAT.
As mentioned above, there are no specific signs of structural or sequence motifs that are common to all of the RAF. However, different classes of the RAF can be identified by specific characteristics such as, for example, peptides which are amphipathicity and have a positive net charge. Other groups of the RAF may have a structure with a high content of α-helices. Another group may represent peptides with high content of basic amino acids. The RAF may, thus, constitute or may contain oligomers of basic amino acids such as arginine, for example from 5 to 20, from 6 to 15 or 6 to 12 R-residue, e.g., R7(SEQ ID NO:70), R8(SEQ ID NO:72), or R11(SEQ ID NO:73), or QSR8(SEQ ID NO:74).
Proline-rich amphipathicity peptides are another class of CPP, and such peptides, characterized by the presence of pyrrolidinone rings from prolinol described in Pujals et al. 2008 Advanced Drug Delivery Reviews 60, pages 473-484.
Others have successfully developed the CPP include pVEC (Elmquist et al. 2003 Biol. Chem 384, pages 387-393; Holm et al. 2005 Febs Lett. 579, pages 5217-5222) and peptides produced from calcitonin (Krauss et al. 2Q04 Bioorg. Med. Chem. Lett., 14, pages 51-54).
Commercially available CPP include Chariot on the basis of peptide PEP-1 (Active Motif, Franc, vectors Syn-B-based peptide protegrin PG-1 (Syntem, France) and Express-si Delivery on the basis of the MPG peptide from Genospectra, USA.
In addition to the public and described the CPP can be designed and synthesized new peptides or derivatives of the RAF on the basis of known or described criteria (for example known sequences or signs of CPP, such as the content of essential amino acids, the content of α-helix and so forth, as discussed above). In addition, a randomly-constructed or other peptides can be subjected to screening for the activity of the RAF, for example by attaching or attachment of this peptide containing a reporter molecule, for example defined by a label or marker, such as a fluorescent label to the desired load (Oligopeptide compound of the present invention) and test to make sure you move this design through the cell membrane, for example by the addition of these peptides to the living cells with the subsequent study of the cellular import; for example, using confocal microscopy.
Indeed, while the RAF will usually penetrate or to enter into virtually any cell type, and in some cases it is possible to observe that successful or effective delivery may depend, or may vary depending on the exact nature of the goods (for example, Pepto�Noah sequence of cargo and/or used by the RAF. In the ordinary qualifications of the specialist in the art to determine the optimal peptide sequences and combinations and so on, and test it and/or modify the goods and/or sequence of the RAF or structure and so on.
As mentioned above, the signal sequence, which may be contained in the Oligopeptide compounds (or structures) of the invention, may represent a signal peptide, which directs the connection (or design) in a particular subcellular compartment, and in particular the core. The nuclear localization signals (NLS) are also well known in the art and widely described in the literature, and can be used any known or functional NLS.
Thus, another preferred embodiment of the invention is a construct containing (1) Oligopeptide compound comprising the APIM motif (motif interacting with PCNA), as defined in this description of the invention, and (2) a nuclear localization signal.
NLS can vary in length and/or sequence and was described a wide range of specific sequences of NLS. In General, however, it was found that peptides containing positively charged amino acids (particularly lysine (K), arginine (R) and/or histidine (H)) can function as an NLS. Type�CNY NLS maybe thus, to provide a peptide, for example from 4 to 20, more specifically from 4-15, 4-12, 4-10, or 4-8 amino acids, where at least 4 amino acids (and more specifically at least 60, 70, 75, 80, 85 or 90% amino acid residues in the peptide NLS) are a positively charged amino acid, preferably selected from K, R or N. A typical NLS may, for example, to have or contain the sequence RKRH (SEQ ID NO:75).
The nuclear localization signals, including how real the experimental and predicted or proposed NLS sequences, and strategies of identification NLS described in Lange et a.l, J. Biol. Chem. 2007, 282 (8), 5101-5105 ; Makkerh et al., Current Biology 1996, 6 (8), 1025-1027; Leslie et al., Methods 2006, 39, 291-308; and Lusk et al. Nature Reviews MCB 2007, 8, 414-420.
Classical NLS consists of either one (single) or two (double) plots of basic amino acids. Example of a single NLS can be SV40 large T-antigen NLS (126PKKKRKV132[SEQ ID NO:76]) and double - nucleoplasmic NLS (155KRPAATKKAGOAKKKK170[SEQ ID NO:77]). Proposed consensus sequence of a single NLS K-[K/R]-X-[K/R] (SEQ ID NO:78), and, thus, the NLS of the present invention may in one embodiment contain or consist of such a consensus sequence (where X represents any amino acid).
A typical double NLS according to the invention may have posledovatel�of KR-[X] 5-20-KKKK (SEQ ID NO:79), for example KR-X10-KKKK (SEQ ID NO:80) (where X represents any amino acid).
Alternative typical double NLS may take the form RKRH-[X]2-10-KK (SEQ ID NO:81), for example RKRH-X2-KK (SEQ ID NO:82), for example RKRH-II-KK (SEQ ID NO:83).
NLS oncoprotein c-myc is different from the classical NLS that only 3 of the 9 amino acid residues are basic (PAAKRVKLD [SEQ ID NO:84]), indicating that NLS does not necessarily correspond to a consensus or classic sequences given above. Makkerh et al. (above) describes the NLS sequence in which the cluster of basic amino acids (for example KKKK [SEQ ID NO:85]) flanked by neutral and acidic residues, for example PAAKKKKLD (SEQ ID NO:86).
Other potential NLS sequences that may be given by way of example, include: PKKKRKVL (SEQ ID NO:87), KKKRK (SEQ ID NO:88), KKKRVK (SEQ ID NO:89), KKKRKVL (SEQ ID NO:90) and RKKRKVL (SEQ ID NO:91). You can use any NLS, which is a derivative of the known NLS, such as SV40 NLS, nucleophosmin, UNG2 or c-myc NLS.
The implied, suggested or predicted NLS sequence can be tested on the activity of the NLS using the principles and analyses, known and described in the art. For example, a candidate NLS sequence can be attached to the desired "load" (in this case Oligopeptide according to the invention, as defined in Yes�dimensional description of the invention), and design can be provided to the designated reporter molecule (such as a label or marker that can be visualized, for example a fluorescent label) and put in contact with the test cell. You can then determine the distribution of structures in the cell.
Thus, as a generalization, the specialist is aware of suitable signal sequences, but as an example in this description of the invention is referred to the following. Examples of peptide sequences entering the cell include Penetratin™, a 16-amino acid peptide corresponding to the third helix of homeodomain squirrel Antennapedia, R-enriched markers, such as RB-Penetratin (where arginine residues added to the N-end Penetratin) and the derived protein HIV Tat, such as GRKKRRQRRRPPQQ (SEQ ID NO:92). Examples of nuclear localization sequences include derived protein SV40 KKKRK (SEQ ID NO:93).
The preferred design of the present invention contains (1) Oligopeptide compound comprising the APIM motif, as defined in this description of the invention, (2) a nuclear localization signal, and (3) the signal sequence of penetration into the cell.
Individual elements or components of the structure according to the present invention may contain or be present in any order, but preferably in the manner outlined above (�reamer Oligopeptide compound APIM-CPP; Oligopeptide compound APIM-NLS; Oligopeptide compound APIM-NLS-CPP).
In addition, the Oligopeptide or the connection structure according to the invention can contain more than one motif that interacts with PCNA. Thus, in alternative, the design of the present invention may contain more than one Oligopeptide compounds containing motif that interacts with PCNA. Design or Oligopeptide compound may, for example, contain 1-10, for example 1-6, or 1-4, or 1-3, or one or two motives. The design also contains a signal sequence, such motives can be positioned or placed as desired, for example they can be grouped together, or they may be separated by elements of the signal sequence, for example the motif-NLS-motif-CPP; or motive-NLS-motive-motive-CPP; or the motive-motive-NLS-CPP and so on.
Components or subassemblies according to the invention can be attached or connected to each other by any desired or convenient by methods well known in the art. Thus, the components or parts may be connected or anywherevery chemically, for example using known methods of chemical binding, or these structures may be formed in the form of a whole, using the methods of genetic engineer�and, for example means for the formation of fusion proteins, or they can simply be synthesized in whole, for example using methods of synthesis of peptides.
Individual parts or components can be associated directly with each other, or they can be connected indirectly by means of one or more of the linker (or the GS spacer) sequences. Thus, the linker sequence can fill in the gaps or divide two or more separate parts of the design or split the elements of the Oligopeptide motif in the design. The exact nature of the linker sequence is not critical, and it may have a different length and/or sequence, for example it may have 0-40, more particularly 0-20, 0-15, 0-12, 0-10, 0-8 or 0-6, 0-4 or 0-3 residues, for example 1, 2 or three and more balance. As typical examples of the linker sequence, if present, can have 1-15, 1-12, 1-10, 1-8, 1-6 or 1-4 residue and so on. Nature of residues is not critical and may, for example, represent any amino acid, such as neutral amino acid, or aliphatic amino acid, or alternatively, they may be hydrophobic, or polar, or charged, or structural, such as Proline. Was shown to be applicable a number of different linker sequences, including short�e (e.g. 1-6) sequence neutral and/or aliphatic amino acids.
Typical linker sequence, thus, include any single amino acid residue, for example A, I, L, V, G, R, Q, T or W, or di-, tri-, Tetra-, Penta -, or Hexapeptide consisting of such balances.
As typical linkers may mention I, II, IL, R, W, WW, WWW, RIL, RIW, GAQ, GAW, VAT, IILVI (SEQ ID NO:94), IILVIII (SEQ ID NO:95) and so on.
The linkers between the different elements can be the same or different.
In one embodiment it is proposed Oligopeptide compound having or containing a sequence MDRWLVKRILVATK (SEQ ID N0: 96) or MDRWLVKRILKKKRKVATKG (SEQ ID NO:97).
Other typical compounds (or more specifically structures) of the invention include
MDRWLVKGAQPKKKRKVLRQIKIWFQNRRMKWKK (SEQ ID NO:98),
MDRWLVKGAWKKKRVKIIRKKRRQRRRK (SEQ ID NO:99),
MDRWLVKGAWKKKRKIIRKKRRQRRRG (SEQ ID NO:100),
MDRWLVKGAWKKKRKIIRKKRRQRRRK (SEQ ID NO:101),
MDRWLVKJUWKKKRKIIRKKRRQRRRK (SEQ ID NO:102),
MDRWLVKWWWKKKRKIIRKKRRQRRRK (SEQ ID NO:103),
MDRWLVKWWRKRHIIKKRKKRRQRRRK (SEQ ID NO:104),
MDRWLVKillWKKKRKIIRRRRRRRRRRRK (SEQ ID NO:105),
MDRWLVKJirWKKKRKIIRQIKIWFQNRRMKWkk(SEQ ID NO:106),
MDRFLVKGAWRKRHIIKKRKKRRQRRRK (SEQ ID NO:107),
MDRWLVKWKKKRKIRRRRRRRRRRRK (SEQ ID NO:108),
MDRWLVKWKKKRKIRKKRRQRRRK (SEQ ID NO:109),
MDRWLVKWRKRHIRKKRRQRRRK (SEQ ID NO:110),
Ac-MDRWLVKGAWRKRHIRKKRRQRRRK (SEQ ID NO:111),
Ac-MDRWLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO:112),
Ac-MDRALVKWKKKRKIRRRRRRRRRRR (SEQ ID NO; 113),
Ac-MDRWLVKKKKRKRRRRRRRRRRRK (SEQ ID NO:114),
Ac-MDRWLVKKKKRKRRRRRRRRRRR (SEQ ID NO:115),
MDRWLVKRIWKKKRKIIRWLVKWWWRKKRRQRRRK (SEQ ID NO:116),
KRRRQRRKKRIIKRKKKWWWKVLWRDM (SEQ ID NO:117).
Oligopeptide compounds having the sequence as shown� in SEQ ID NO:98-117, shown in Table 3 in Example 6 below, which shows the individual components that make up the design (motif-containing sequence, linker, NLS, CPP and so on) Thus, it can be seen that SEQ ID NO:98-117 are structures containing at least one motif-containing sequence, NLS and the RAF, in some cases associated linker sequences, which may vary in sequence as indicated. SEQ ID NO:117 (RI-MDR26-3) is a retro-inverted peptide composed of D-amino acids. Use NLS sequences based on SV40 or UNG2 NLS sequences, and the RAF-sequence-based Penetratin, HIV-TAT or R-enriched peptides.
In another aspect of the invention provides a nucleic acid molecule encoding a peptide having or containing (for example) SEQ ID NO:1, as defined above. It also offers a complement of such nucleic acid molecules. Preferably, a nucleic acid molecule that contains a promoter sequence functionally linked to a sequence encoding a peptide having or containing (for example) SEQ ID NO:1. In a preferred embodiment, a nucleic acid molecule also encodes a signal sequence, as defined above.
The nucleic acid molecule according to the invention with�contains at least 15 nucleotides and preferably by more than 800 nucleotides, more preferably not more than 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 nucleotides. The nucleic acid molecule preferably is a molecule selected.
Another aspect relates to a vector containing a nucleic acid molecule as defined in this description of the invention. The vector may also contain additional elements, usually located in the vector such as a replication origin, a selectable marker, such as antibiotic resistance, and/or a multiple cloning site. Additionally, the vector may be an expression vector, and can contain additional elements, such as elements for transcriptional and/or translational control or regulatory elements for expression of the nucleic acid molecules. These control elements such as promoters, binding sites of ribosomes, enhancers, terminators, and so forth, are well known and widely described in the art.
The vector may be a plasmid or a virus, preferably it is selected from a retrovirus, adenovirus and virus associated with adenovirus.
In another aspect proposes a recombinant cell host containing a nucleic acid molecule and/or vector as described above. A host is an animal cell, preferably� cell of a mammal, most preferably the cell of the rat, mouse or human.
By "recombinant" is meant a nucleic acid molecule and/or vector introduced into a host cell. A host may naturally contain or not contain endogenous copy of the nucleic acid molecule, but it is a recombinant, was introduced as exogenous or extra endogenous copy of the nucleic acid molecule and/or vector.
In yet another aspect of the proposed pharmaceutical composition containing Oligopeptide compound, as defined in this description of the invention, the nucleic acid molecule as defined in this description of the invention and/or the vector as defined in this description of the invention, together with pharmacologically (or pharmaceutically) acceptable excipient.
Excipient may include any excipient known in the art, for example, a suitable carrier or diluent or any other ingredient or agent, such as a buffer, antioxidant, chelating agent, binding agent, covering agent, disintegrant, filler, aromatic substance, a colorant, moving a substance, a lubricant, a preservative, a sorbent and/or a sweetener and so on
Excipient can be selected, for example, from dairy sour�s, dextrose, metabisulphite sodium, benzyl alcohol, polyethylene glycol, polypropylene glycol, microcrystalline cellulose, lactose, starch, chitosan, pre-jellied starch, calcium carbonate, calcium sulphate, dextrates, dextrin, dextrose, dibasic dihydrate calcium phosphate, tribasic calcium phosphate, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, powdered cellulose, sodium chloride, sorbitol and/or talc.
The pharmaceutical composition may be offered in any form known in the art, for example in the form of tablets, capsules, coated tablets, liquids, suspensions, records, Sasha, implant, means for inhalation, powder, pills, emulsions, lyophilized powder, effervescent beverage, spray, salve, emulsion, balm, patch, or any mixtures thereof. It may be asked, for example, in the form of the drug, resistant to gastric fluids, and/or in the form of prolonged action. It can represent a form suitable for oral, parenteral, local, rectal, genital, subcutaneous, transurethral, transdermal, intranasal, intraperitoneal, intramuscular and/or intravenous and/or for administration by inhalation.
In a typical embodiment, the pharmaceutical comp�a new exhibition is in the form suitable for liposomal injection, so that preferably offers liposomes containing the pharmaceutical composition. If using liposomes, may not be required to include an additional excipient thus also provides liposomes containing Oligopeptide compound, as defined in this description of the invention, the nucleic acid molecule as defined in this description of the invention and/or the vector as defined in this specification.
Using the database search, the inventors discovered the presence of a new PCNA-binding motif in more than 200 other proteins, many of which are involved in DNA repair, maintenance and cell cycle regulation, such as transcription, replication, phosphorylation, ubiquitination, translationy synthesis, the cohesion of sister chromatids and cell cycle regulation (see Table 1 and Example 4). Such proteins include the following:
- protein with unknown function that contains conservative domain I, N-Terminus, was detected in the elongation factor TFIIS (transcription factor IIS), an important protein for the development stopped transcription, so the inventors called it a TFIIS-like protein. This protein contains a motif within its 7 N-terminal amino acids.
- multifunctional transcription factor TII-I, which is critical to controlling cell cycle and proliferation. Cells, sverkhekspressiya TFII-I, have increased resistance of the foci of Y-H2AX (a marker duhsitelya DNA breaks) that assumes the role of TFII-I in DNA repair. This protein contains 4 of the motif.
- DNA-topoisomerase II alpha (Toro II α), which operates in postreplication separation of DNA and DNA segregation. This protein contains a single motif.
a key protein of nucleotide excision repair system STORAGE, which recognizes spiral loop. This protein contains a single motif.
- RAD51 In homologous recombinant protein which, as shown, is important for proper functioning of the centrosome and chromosome segregation. This protein contains a single motif.
- nuclear protein complex of the Fanconi anemia (FA), FANCC. Nuclear FA complex, as shown, is involved in the signaling pathway that activates DNA damage by regulating the repair of DNA cross-linking agents. This protein contains a single motif.
Additional proteins that have been found to contain at least one motif are listed in Table 1.
Not wanting to be bound by theory, the inventors suggest that by preventing the interaction of PCNA with at least one of his usual partners, you can increase the sensitivity of cells to the action of qi�astiticeski agents. Thus, the effect of the cytotoxic agent can be modulated. For example, the repair protein interaction with PCNA can be inhibited (for example hABH2), thereby inhibiting DNA repair, and consequently increasing the effect of the cytotoxic agent in DNA damage.
Thus, in another aspect provides a method of treatment of a disorder or condition in a particular disorder or condition where it is desirable to inhibit the growth of cells, for example, hyperproliferative disorders, or any condition which needs to cytostatic therapy or responds to it, including the introduction (in particular the introduction of an effective amount) Oligopeptide compounds, as defined in this description of the invention, nucleic acid molecules, as defined in this description of the invention and/or a vector as defined in this description of the invention, to a subject in need of it.
In another aspect, it is proposed Oligopeptide compound, as defined in this description of the invention, the nucleic acid molecule as defined in this description of the invention and/or the vector as defined in this description of the invention, for use in therapy, in particular for use in the treatment of a disorder or condition in which it is desirable to inhibit the growth of cells, for example, hyperproliferative �of ustroystva, or any treatment which involves cytostatic therapy (i.e. the use of the cytotoxic agent). Thus, the connection and so on may be used in the treatment of any condition which needs to cytostatic therapy or responds to it.
In another aspect is provided the use of Oligopeptide compounds, as defined in this description of the invention, nucleic acid molecules, as defined in this description of the invention and/or a vector as defined in this description of the invention in the manufacture of a medicament for use in the treatment of a disorder or condition in which it is desirable to inhibit the growth of cells, for example, hyperproliferative disorders, or treatment which involves cytostatic therapy.
As indicated above, one unexpected observation, leading to the present invention, is that the action of a number of different cytotoxic drugs can amplify or potentiate by application of a peptide having a motif that interacts with PCNA, thereby to inhibit the interaction of PCNA presumably with a wide variety of proteins, such as proteins involved in the repair and DNA replication and passage of the cell cycle and so on. This leads to the General assumption that any molecule interacting�Yu with PCNA, can be used in combination with a cytotoxic agent to enhance the effect of such a cytotoxic agent, or to increase the sensitivity of cells to the action.
Thus, in another aspect provides a method of treatment of a disorder or condition in which it is desirable to inhibit the growth of cells, for example a hyperproliferative disorder, or a method of treatment which involves cytostatic therapy, including the introduction of Oligopeptide compounds capable of interacting with PCNA or a nucleic acid molecule that contains a nucleotide sequence encoding the Oligopeptide compound capable of interacting with PCNA, and separate, simultaneous or sequential administration of cytotoxic agent to a subject in need of it.
Alternatively, it is proposed Oligopeptide compound capable of interacting with PCNA or a nucleic acid molecule containing the nucleotide sequence encoding the specified Oligopeptide compound, for use in combination with a cytostatic agent in the treatment of a disorder or condition in which it is desirable to inhibit the growth of cells, for example, hyperproliferative disorders, or treatment which involves cytostatic therapy.
Thus, it is proposed the use of oligota�tidnyj connection capable of interacting with PCNA or a nucleic acid molecule containing the nucleotide sequence that encode the specified Oligopeptide compound, in the manufacture of a medicament for use in combination with a cytostatic agent in the treatment of a disorder or condition in which it is desirable to inhibit the growth of cells, for example, hyperproliferative disorders, or treatment which involves cytostatic therapy.
Thus, in one embodiment the medicament may further comprise a cytotoxic agent.
The drug can be in the form of a single composition containing both Oligopeptide compound or nucleic acid molecule and the cytotoxic agent, or it may be in the form of a package or product containing them to separate (for example joint or sequential) administration.
Thus, also provided the use of Oligopeptide compounds capable of interacting with PCNA or a nucleic acid molecule containing the nucleotide sequence that encodes the specified Oligopeptide compound in the manufacture of a medicine for the treatment of disorders of cells, for example a hyperproliferative disorder, or in the treatment, which includes cytotoxic t�therapy, where a drug is administered separately, together or alternately with a cytotoxic agent.
In another aspect of the invention proposes a product containing Oligopeptide compound capable of interacting with PCNA or a nucleic acid molecule containing the nucleotide sequence that encode the specified Oligopeptide compound, together with a cytotoxic agent, as a combined preparation for separate or sequential use in the treatment of a disorder or condition in which it is desirable to inhibit the growth of cells, for example, hyperproliferative disorders, or treatment which involves cytostatic therapy.
Oligopeptide compound capable of interacting with PCNA, preferably Oligopeptide compound comprising or having SEQ ID NO:1, can be used to modulate or potentiate the action of the cytostatic agent.
Oligopeptide compounds (including designs) according to the invention, therefore, therapeutically useful in any condition or clinical situation where it is desirable (or where it may be useful to inhibit the growth of cells.
The term "inhibit" is used in a broad sense and includes any reduction or decrease of cell growth and the prevention or elimination of cell�about growth. "Inhibition", thus, includes the reduction or prevention of cell growth. It can be defined by any suitable or convenient means, such as determining or estimating the number of cells of size (e.g. the size of the tissue, which contains cells), cell viability and/or cell death and so on, and you can also define methods, well known in the art.
"Growth" of cells, as it is referred to in this description of the invention, is also used in a broad sense and it includes every aspect of cell growth, including in particular cell proliferation.
Oligopeptide compounds, therefore, can be used in the treatment of any condition (used in this description of the invention in a broad sense and includes any disorder or any clinical situation), which responds to the reduction of cell growth (in particular cell proliferation). Oligopeptide compounds, thus, find application in any therapy (or treatment), which focuses on cellular growth (or proliferation). In other words, the compounds can be used in any therapeutic application where it is desirable or advantageous to inhibit cell proliferation.
The term "treating" when used in this description of the invention refers in a broad sense� to any act or stage (or intervention), favorable for the monitoring of clinical condition, and, thus, includes both therapeutic and prophylactic treatment. Treatment may include reducing, relieving, improving, slowing the development or elimination of the condition or one or more of the symptoms being treated, compared to the condition or symptom to treatment, or improve in any way the clinical status of the subject. Treatment may include any or clinical stage of the intervention that contribute to, or is part of a therapeutic program or scheme. Prophylactic treatment may include slowing, limitation, reduction, or prevention of a condition or initial condition, or one or more symptoms, for example concerning the condition or symptom to preventive treatment. Prevention, therefore, clearly includes both the absolute prevention of the emergence or development of a state, or its symptoms, and any slowdown at the beginning or development of a condition or symptom, or a reduction or limitation of development or progression of a condition or symptom. Treatment according to the invention, thus, includes the elimination, inhibition or slowing down of cell growth, or increase in size of an organism or population of cells (e.g. tissue, tumors or culture), reducing the number of cells Il� the prevention of the proliferation of cells (e.g. in another anatomical area), the decrease in the cell growth and so on. The term "treatment" does not involve healing or fully eliminated or cell growth, or cell growth.
Since therapeutic application and use of the present invention can in General include inhibition of cell proliferation, any proliferating cell may be targeted in therapies and uses disclosed and included in this description of the invention. These proliferating cells can include healthy or diseased cells and the cells of any tissue, in which there has been a proliferation. For example, such cells may include, in particular, neoplastic cells, including both malignant and non-malignant neoplastic cells and cells of the immune system (immune cells), cells of the hematopoietic system in General, or skin cells.
Disorder or condition comprising abnormal or unwanted cell growth, can be treated with cytotoxic agents, and cytostatic agents can be used in any situation where it is desirable to reduce or prevent cell growth and proliferation, including situations in which it is desirable to kill or remove the cells. Thus, as an alternative to the above, Oligopeptide compounds (including structures) of the present invention �can be used in any method of treatment, which means (or includes) the use of a cytotoxic agent. It may include treatment of any condition responsive to a cytotoxic agent, or any condition which can be treated with the cytostatic agent or need in this application.
Treatment of hyperproliferative disorders is a particularly interesting aspect. The term "hyperproliferative disorder" is widely used in this description of the invention and includes any disorder or condition that includes increased, adverse or unwanted cell proliferation. Thus included not only the state in which cell proliferation is increased, for example relative to a normal or healthy cells or cells in the absence of the state in question (for example compared to or relative to healthy or control of the subject, or compared to, or relative to cells derived from healthy or undamaged tissue from the same subject), but also the condition in which cell proliferation is not increased (or slightly or greatly increased) compared to normal, but in which the proliferation is adverse or undesirable, either in General or in a specific context. It may include, for example, I�p, adverse or unwanted cell proliferation, which may occur during "normal" response, such as immune response or inflammatory response, and so on (in other words a "normal" response, which may take place in particular (e.g. normal) context, but which may, nevertheless, be undesirable). Such unwanted proliferative response may, for example, be a proliferation of cells, leading to undesirable inflammatory response, or undesirable immune response, such as an autoimmune response or an allergic reaction, and so on.
Hyperproliferative disorders that can be treated according to the present invention, thus clearly include inflammation (more specifically, an inflammatory disorder or condition, or condition that includes inflammation, or associated with inflammation, or characterized by inflammation and an autoimmune disorder or condition, or disorder or condition, which have an autoimmune component.
Hyperproliferative disorder may include (without limitation) the proliferation of cells that have the capacity for Autonomous growth, i.e. the cells that exist and reproduced regardless of normal regulatory mechanisms. Hyperproliferative disorder may �thus, to represent a neoplastic disorder, and as indicated above, it may be malignant or non-malignant disorder.
Hyperproliferative cells can be classified as pathological (i.e., with deviations from normal cells and associated with a painful condition) or non-pathological (i.e., with a deviation from normal but not associated with a painful condition).
Pathological Hyperproliferative cells can be associated with, or characterized by, the following painful conditions or disorders, restenosis, diabetic nephropathy, hyperplasia of the thyroid gland, graves ' disease, psoriasis, benign prostatic hyperplasia, syndrome Li-Fromenty, and cancers (including any tumor or malignancy).
Examples of non-pathological hyperproliferative cells include the epithelial cells of the ducts of the mammary gland during development, lactation and also cells associated with wound healing.
Compounds of the invention may be useful in the treatment of such disorders and diseases and others, including diabetic retinopathy and peripheral vascular disease.
Hyperproliferative disorders can be, as mentioned above, malignant or n�malignant neoplastic disorders. Also included precancerous and neoplasticism disorders. Examples of precancerous or neoplastically or non-malignant hyperproliferative disorders include myelodysplastic disorders, cervical carcinoma in situ, familial intestinal polyposis (such as Gardner syndrome), oral leukoplakia, histiocytosis, keloids, hemangiomas, hyperproliferative arterial stenosis, inflammatory arthritis, hyperkeratosis and papulosquamous precipitation, including arthritis. Also included is caused by a virus hyperproliferative diseases, such as warts caused by a virus and Epstein-Barr disease (e.g. infectious mononucleosis), scarring and the like.
Hyperproliferative disorder may, therefore, represent any Hyperproliferative disorder, for example selected from neoplastic disorders such as cancer, psoriatic arthritis, rheumatoid arthritis, gastric hyperproliferative disorders, such as inflammatory bowel disease, skin disorder, including psoriasis, Reiter's syndrome, red scalp pityriasis, and hyperproliferative variants of disorders of keratinization.
Cancer is a particularly interesting Hyperproliferative disorder, and included all types of cancer, e.g. solid tumors and GE�etologicheskie cancer. Typical types of cancer include cervical cancer, uterine cancer, ovarian cancer, pancreatic cancer, kidney cancer, gallbladder cancer, liver cancer, head and neck, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate cancer, testicular cancer, lung cancer, non-small cell lung cancer, nahodkinskuju lymphoma, multiple myeloma, leukemia (such as acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, and chronic myelogenous leukemia), brain cancer (e.g. astrocytoma, glioblastoma, medulloblastoma), neuroblastoma, sarcoma, colon cancer, rectal cancer, stomach cancer, anal cancer, bladder cancer, pancreatic cancer, endometrial cancer, plasmacytoma, lymphoma, retinoblastoma, Wilms tumor, Ewing sarcoma, melanoma and other skin cancers.
You can also mention sinus tumors, cancer of the urethra and urinary system, cancer of the esophagus, melanoma, myeloma, endocrine cancer, osteosarcoma, angiosarcoma, and fibrosarcoma, and any tumor of the peripheral or Central nervous system, malignant or benign, including gliomas and neuroblastomas.
Autoimmune disorders or diseases are also of particular interest to States, and include, for example, I�p, rheumatoid arthritis, multiple sclerosis, immune disorders such as systemic lupus erythematosus (SLE; lupus) or false bulbar palsy.
Also of interest, in General, hematologic disorders, or diseases of the blood or bone marrow that are not necessarily malignant or cancerous (e.g. different dyscrasia, or dysplasia, non-cancerous hyperplasia, granuloma or MGUS (monoclonal gammopathy of unknown significance). Thus, any condition that includes unwanted, or adverse, or abnormal proliferation of blood cells or bone marrow, or their precursors, can be treated according to the present invention.
Other conditions that may especially be mentioned include neoplastic meningitis and myeloproliferative diseases, such as true polycythemia Vera (which occurs when excess production of red blood cells).
Different States may also arise as a result of inflammation or autoimmune diseases, or can be otherwise associated with them. Such conditions may also be treated according to the present invention. Especially may be mentioned scleromyxedema and papular Mucins, amyloidosis, and Wegener's granulomatosis.
As indicated above, the compounds according to the invent�NIJ may enhance or potentiate the effects of cytotoxic agent. Thus, it is easily applied in either a therapeutic use, where to use cytotoxic agent. It can include any situation where it is desirable to kill or remove the cells, which may include not only the diseased cells. In particular, this situation occurs when it is desirable to remove the bone marrow prior to transplantation. Compounds according to the invention can thus be used in myeloablative, and in particular myeloablation prior to transplantation, which may, for example, be a bone marrow transplant or, more generally, transplantation of hematopoietic stem cells (HSCT) (as well as from bone marrow, hematopoietic stem cells can be derived or produced from blood, e.g., peripheral blood).
Transplantation of stem cells may be used in the treatment of diseases or conditions of the blood or bone marrow (hematologic conditions or disorders) that can be malignant or non-malignant, and some other types of cancer, including solid tumor cancers, such as neuroblastoma, templatecache finely-kruglosutochny cancer, Ewing's sarcoma and choriocarcinoma. Hematologic cancers include leukemias, lymphomas (Hodgkin's and non-Hodgkin) and myeloma. Desloca�significant hematological disorders include phagocytic disorders (such as myelodysplasia), anemia (such as severe aplasia or aplastic anemia), and myeloproliferative disorders (for example true polycythemia Vera and essential thrombocytosis). Other acquired conditions that can be treated using stem cell transplantation include metabolic disorders, such as amyloidosis and diseases caused by the environment, such as radiation poisoning. Stem cell transplant can also be used in the treatment of congenital disorders, including lysosomal storage diseases, immunodeficiency States and non-malignant hematologic disorders such as anemia, cytopenia, hemophagocytic syndromes, hemoglobinopathies, sickle cell disease and a large β-thalassemia.
Radiotherapy (also known as radiation therapy and radiation Oncology) can be used in the treatment of various conditions, including hyperproliferative disorders described above. "Radiotherapy" refers to the use of ionizing radiation which can damage DNA cells through direct or indirect ionization of atoms, which form the DNA chain. Indirect ionization happens as a result of ionization of water, free radicals, particularly hydroxyl radicals, which then damage the DNA. In most RA�widespread forms of radiotherapy the greatest effect of the radiation is achieved by means of free radicals.
Radiotherapy is useful in the treatment of cancer and to remove abnormal tissue from the cytotoxic effects that are caused by persistent dvuhlistovymi breaks DNA or activation of programmed cell death. Ionizing radiation causes cell death hyperproliferating cells, such as tumor and cancer cells by apoptosis both in vivo and in vitro.
Unfortunately, radiotherapy is often unsuccessful for the complete elimination of cancer cells of the patient, as often it is not capable of delivering a high local dose of radiation to kill tumor cells without unacceptably high risk of damage to surrounding normal tissue. It is also known that cells exhibit a widely varying sensitivity to induced radiation of cell death, and ionizing radiation can also activate the response mechanism of survival through pathways of signal transduction phosphatidylinositol-3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase (MARK). Thus, there is a need to enhance the effectiveness of radiotherapy by increasing the sensitivity of cells to the action of ionizing radiation.
Thus, the compounds according to the invention can be used to obtain this effect sensitization, in other words, for pileni� (or, alternative speaking, to enlarge, increasing or potentiating) effects of radiotherapy, or to make the subject (or, more specifically, cells that may be present in the subject) are more sensitive to the effects of radiotherapy. Thus, they can be used in any therapeutic treatment using radiotherapy. It can include any situation where it is desirable to kill or to eliminate cells, which may include not only the diseased cells.
Thus, the compounds according to the invention can be used as a sensitizer of cells to DNA-damaging effects of ionizing radiation. "Sensitizer" means the use of the compounds according to the invention for amplification of DNA-damaging effect of ionizing radiation on cells. It is possible to achieve inhibition of endogenous mechanisms of repair of cellular DNA.
Thus, the present invention includes an Oligopeptide compound comprising PCNA-interacting motif (more specifically, Oligopeptide compound containing PCNA-interacting motif, as defined in this description of the invention), or a nucleic acid molecule containing a sequence encoding specified PCNA-interacting motif, for use in combination with radiotherapy, where soedineniya separately together or sequentially with radiotherapy. Radiotherapy, together with the connection, you can enter in the treatment of any condition which responds to radiotherapy or who needs it. Compounds or constructs according to the invention can thus be used in the treatment of a disorder or condition in which it is desirable to inhibit the growth of cells, for example, hyperproliferative disorders, or any treatment which includes radiotherapy.
Alternative determining, the invention proposes Oligopeptide compound containing PCNA-interacting motif (more specifically, Oligopeptide compound containing PCNA-interacting motif, as defined in this description of the invention), or a nucleic acid molecule containing a sequence encoding specified PCNA-interacting motif, as a sensitizer for radiotherapy, where the compound is administered separately, simultaneously or sequentially with radiotherapy.
In these aspects of the invention also provides a method of sensitizing a subject (or, more specifically, cells or tissue of the specified entity to radiotherapy, including the introduction of a specified subject Oligopeptide compounds according to the invention, as defined in this description of the invention, in particular such amount of the compound, the cat�Roy is effective to increase the sensitivity of a specified entity (or such cells or tissue) to radiotherapy.
As you can see, this aspect of the invention also provides a method of treatment of a subject, comprising administering radiotherapy to the specified subject, together with Oligopeptide compound according to the invention, as defined in this description of the invention. More specifically, this method may be a method of treatment of a disorder or condition that responds to radiotherapy, or who needs it, or disorder or condition in which it is desirable to inhibit the growth of cells, or a method of treatment which includes radiotherapy.
The invention involves all types of radiotherapy, including but without limiting them, conventional external beam radiotherapy, stereotactic radiotherapy, virtual simulation, 3-dimensional conformal radiotherapy, adjustable intensity radiotherapy and radioisotope therapy (RIT).
Thus, in one preferred embodiment of any of the aspects listed in this description of the invention, the Oligopeptide compound, nucleic acid molecule and/or a vector as defined in this description of the invention are used together (jointly, separately or follower) with radiotherapy.
In yet another preferred embodiment of any of the aspects listed in this description of the invention, the Oligopeptide compound, molecule n�kleinova acid and/or vector as defined in this description of the invention are used together (jointly, separately or sequentially) with a cytostatic agent.
By "cytotoxic agent" refers to an agent that can inhibit or slow the growth and/or reproduction (replication/proliferation) of animal cells.
Cytotoxic agents include cytotoxic agents, antineoplastic agents, and any agent that may be indicated for oncological or hematological use. Thus, the agents used in chemotherapeutic treatment protocols ("chemotherapeutic agents").
Cytostatic agents are typically grouped into different classes according to their mechanism of action, and all of these classes are discussed in this description of the invention. Thus, the cytotoxic agent can be an alkylating agent, cross-linking agent, intercalating agent, a nucleotide analogue, inhibitor of the formation of the mitotic spindle and/or an inhibitor of topoisomerase I and/or II. Other types or classes of agent include antimetabolites, plant alkaloids and terpenoids or antitumor antibiotic. Preferably it is an alkylating agent.
Alkylating agents modify DNA using alkylating nucleosides, which lead to the prevention �correct DNA replication. Nucleotide analogs are incorporated into DNA during replication and inhibit DNA synthesis. Inhibitors of the formation of the mitotic spindle disrupt the formation of the mitotic spindle, leading to a delay in mitosis during metaphase. Intercalating agents are inserted between the bases of DNA, thereby inhibiting DNA synthesis. Inhibitors of topoisomerase 1 or II affect the twist of the DNA, thereby preventing DNA replication.
Suitable cytotoxic agents are known in the art, but as an example in this description of the invention the specified actinomycin D, BCNU (carmustine), carboplatin, CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosoanatabine), computacin (CPT), cantharidin, cisplatin, cisplatine, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, DTIC ((dimethylthiazolidine carboxamide), epirubicin, etoposide, gefinitib, gemcitabine, ifosfamide, irinotecan, ionomycin, melphalan, methotrexate, mitomycin C (MMC), datasetreportparam, oxaliplatin, paclitaxel (Taxol), an inhibitor of PARP-I (poly(ADP-ribose) polymerase), Taxotere, temozolomide (TZM), teniposide, topotecan, treosulfan, vinorelbine, vincristine, vinblastine, 5-azacytidine, 5,6-dihydro-5-azacytidine and 5-fluorouracil. The specialist is aware of the suitable intervals of dosage for any such cytotoxic agent, and in one embodiment the cytotoxic and�UNT is present in the pharmaceutical composition, or administered to a subject, in the typical dose range.
In the preferred embodiment may be present/used a lower dose of the cytotoxic agent, as Oligopeptide compound, nucleic acid molecule or vector according to the invention increase the sensitivity of cells to cytotoxic agents, and therefore, when used in combination with Oligopeptide compound, nucleic acid molecule or vector according to the invention a lower dose of the cytotoxic agent will have the same or a comparable therapeutic effect as a higher dose of the cytotoxic agent itself. Oligopeptide compound, nucleic acid molecule or vector according to the invention thus make possible the treatment of subjects who have low or below average, tolerability of cytotoxic agents, such as elderly people, infants or small children, or people who are immunocompromised, for example, due to disease, malnutrition and the like.
One problem encountered in the use of cytotoxic agents for treatment of hyperproliferative disorders, is that usually not all kill the affected cells. It is assumed that, when the cytotoxic agent and Oligopeptide compound, nucleic acid molecule or vector according to the invention is used�form together to kill a higher percentage of affected cells, and in one embodiment is used, the dose of the cytotoxic agent above normal along with Oligopeptide compound, nucleic acid molecule or vector according to the invention, to achieve the killing of a very large part diseased cells, for example at least 50, 60 70 or 80%, preferably at least 85, 90 or 95%, most preferably to essentially kill all the cancer cells.
As stated above, when an Oligopeptide compound, a nucleic acid molecule and/or a vector as defined in this description of the invention, used in conjunction with a cytostatic agent, then the agent may be present in the same pharmaceutical composition, or they can be entered separately. Separate the introduction may include the introduction of on the merits at the same time but by different routes of administration or by introducing different areas. Separate the introduction may also include an introduction to different times, for example at intervals of up to 1, 2, 3, 4, 5, 6 or 12 hours.
The subject is an animal (that is, any human or animal that is non-human, preferably a mammal, most preferably human.
Specialist well known suitable methods of introducing Oligopeptide compounds, molecules nuclein�Oh acid and/or of the vector into the cells. As an example, several suitable methods are briefly discussed below. As discussed in detail above, can be used-mediated peptide delivery methods, in particular the peptides entering the cell (CPP), which, as discussed above, are short, in some cases, poly, sequences that can facilitate cell capture of peptides, proteins or nucleic acid molecules that contain the CPP or to which is attached the CPP, for example by enhancing capture in endosomes of mammalian cells. Microencapsulation provides a simple and cost effective method of incorporating bioactive substances in semipermeable polymer membrane to protect bioactive substances and release of incorporated substances or their products in a controlled way. When photochemical internalization (PC1) and representing the interest of the molecule and a photosensitizing compound are captured by the cell to the lysosome or endosome. Then the cell is exposed to light of appropriate wavelength to activate the photosensitizing compound, forcing photosensitizing compound to destroy the membrane of the lysosomes or endosomes, thereby releasing interest of a molecule into the cytosol of the cell.
Other methods include microinjection, merger, those mediated�s cells, liposome fusion, osmotic lysis of the pines, introduction by scraping, electroporation, transfection mediated by calcium phosphate and the virus, and the use of a copolymer of media.
Chitosan and water-soluble derivatives of chitosan, in particular glycolbased are the preferred carriers of drugs because of their biocompatibility and ability to biodegrade in vivo. A preferred example is glycolbased, hydrophobic modified 5 β-holonovel acid.
In this description of the invention using standard one-letter amino acid code, thus, means To lysine (Lys), I is isoleucine (IIe) and the like.
Oligopeptide compound according to the invention may include one or more, e.g. at least 1, 2, 3, 4 or 5 amino acids have a side chain that is not encoded by the standard genetic code, referred to in this description of the invention "unencrypted amino acids". They can be selected from amino acids, which are formed in metabolic processes, such as ornithine or taurine, and/or artificially modified amino acids, such as 9H-fluoren-9-ylmethoxycarbonyl (Fmoc),-(t) - (b)scrap(o)XI(K)arbonyl (Boe), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc)-protected amino acids, or amino acids having benzyloxy�onilne (Z) group. Preferably, when there are such unencrypted amino acids, they are not in motive, but in one embodiment, one or more unencrypted amino acids are present in the motif.
The stability of Oligopeptide compounds according to the invention in vitro and/or in vivo can be improved or strengthened, using a stabilizing or protective agents known in the art, such as adding protective or stabilizing groups, the introduction of amino acid derivatives or analogs or chemically modified amino acids. Such protective or stabilizing group can be, for example, be added to the N - and/or C-Terminus. An example of such a group is the acetyl group, but other protecting groups or groups that can stabilize the peptide known in the art.
Oligopeptide compounds according to the invention usually contain only amino acids having the L-configuration, but can contain one or more amino acids having the D-configuration. Preferably the Oligopeptide compound contains at least 1, 2, 3, 4 or 5 D-amino acids and they are preferably in the motif, but in another embodiment, D-amino acids are present only outside the motif. Oligopeptide compound may be linear or cyclic.
Thus, it includes, in particular, inverse Oligopeptide compounds �whether inverse Oligopeptide analogues of the compounds according to the invention (and more specifically the inverse peptides).
Also included retro-Oligopeptide compounds (or retro-peptides) in which residues (e.g. amino acid residues) are arranged in the opposite direction to the parent or the comparative compound (e.g. peptide).
Retro-inverted Oligopeptide compounds include D-amino acids in reverse (opposite) order to the sequence of the parent or the parent compound. Retro-inverse analogue, thus, has reversed the ends and reversionary order, for example, peptide bonds, while approximately maintaining the topology of the side chains as in the parent or the comparative sequence.
Compounds of the invention may include a partial inverse, retro or retro-inverse sequence.
Under "Oligopeptide compound" means a compound that consists of amino acids or equivalent subunits that are linked together with peptide or equivalent ties. Thus, the term "Oligopeptide compound" includes peptides and peptidomimetics.
"Equivalent subunit" refers to a subunit, which is structurally and functionally similar amino acid. Skeletal grouping subunit may differ from the standard amino acids, for example, it may include one or more atoms and�OTA instead of one or more carbon atoms.
"Peptidomimetics" imply a compound that is functionally equivalent or similar to the peptide, and which can take three-dimensional structure that is similar to its peptide analogs, but which does not consist of amino acids, connected by peptide bonds. A preferred class of peptidomimetic are peptide, i.e. N-substituted glycine. Peptide are closely related to their natural peptide counterparts, but differ chemically in that their side chains attached to the nitrogen atoms along the skeleton of the molecule and not to the α-carbons, as in amino acids.
In a preferred embodiment at least part of the motive Oligopeptide compound contains only peptide bonds and preferably it is formed only coded amino acids. Most preferably, the Oligopeptide compound is a peptide.
Oligopeptide compound may include di-amino acids and/or β-amino acids, but at least part of the motive is preferably formed only of α-amino acids. Most preferably the Oligopeptide compound consists of α-amino acids.
The prefix "oligo" is used to denote a relatively small number of subunits such as amino acids, that is less than 200, preferably less than 100, 90, 80, 70 60 or 50 subunits. Oligopeptide with�connection according to the invention may, thus, to contain at least 5 and not more than 200 subunits. Preferably it contains at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 subunits. According to an alternative definition, it contains not more than 40, 35, 30, 29, 28, 27, 26 or 25 subunits. Typical ranges subunits, thus, include 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-12, 5-10 and so forth, are preferred 5-20 and 5-30.
When the Oligopeptide compound contains more than 5 subunits, then the nature of the subunits outside of the motif is not critical, i.e. outside of the subunit motif, for example, can be a subunit found in the native protein, such as hABH2, or they may represent the remains of alanine or any other suitable residues.
Peptidomimetics typically have a longer half-life of the human body, so they are preferable in embodiments where it is desirable to more lasting effect. This can help to reduce the frequency with which the composition should be administered repeatedly.
However, in other embodiments from considerations of biological safety may be preferable to a shorter half-life; in such preferred embodiments are peptides.
Oligopeptide compound according to the invention may form part of larger units, for example it can be merged � a polypeptide with obtaining recombinant fused protein or attached to the matrix with obtaining a peptide aptamer. Thus, fusion proteins or aptamers, comprising the Oligopeptide compound according to the invention form further aspects of the present invention. Some aspects include pharmaceutical compositions comprising such fusion proteins, or aptamers, and the use of such fusion proteins or aptamers in therapy or in a method of treatment as described above.
Not wishing to be bound by theory, it appears that for optimal DNA repair, maintain and/or cell cycle regulation, some proteins need to interact with PCNA, and that Oligopeptide compounds according to the invention is able to compete with proteins that have a consensus motif for interaction with PCNA. Examples of proteins that may need to interact with PCNA through a new motif for optimal DNA repair, maintain and/or regulation of the cell cycle, are shown in Table 1, so that the protein may be a DNA-floor and marasu, DNA ligase, topoisomerase, DNA repair protein, proteins associated/interacts with a DNA repair protein involved in the cohesion of sister chromatids, chromatin remodeling DNA binding, processing of ubiquitin or SUMO processing, ubiquitin ligase E3, a transcription factor, a regulator of cell cycle, protein kinase, transferase, acetyl-transferase, antig�n, associated with cancer, structural protein or kinesin localization of centrosome.
If in the cell there is a sufficient level Oligopeptide compounds according to the invention, then the activity of one or more of these proteins decreases or even disappears as a result of such competitive inhibition.
I believe that Oligopeptide compound, nucleic acid molecule or a vector as defined in this description of the invention, in themselves do not have enzymatic activity and are non-toxic to cells (see Example 2). Thus, it was shown that the expression of the peptide according to the invention, contains a PCNA-interacting motif, as defined in this description of the invention, has no effect or has only minor effects on cell growth, in which it is expressed. It may depend on the expression level. However, in some situations, as I believe, Oligopeptide compounds according to the invention can be cytotoxic and thus they can be used as a cytotoxic (or cytotoxic) agents themselves. Thus, the compounds according to the invention can be used as cytotoxic agents in the treatment of conditions, as discussed in this description of the invention, and not necessarily always in combination with a separate cytotoxic agent or radiotherapy�th.
The experiments showed that the Oligopeptide compounds introduced into the cells may have cytotoxic effects on the cell.
The cytotoxic effect may vary depending on the exact nature of the connection, such as a sequence or composition. In particular, the cytotoxic effect may be obtained by using the constructs containing the NLS and/or the RAF, and was observed with constructs containing both NLS and the EPS. Was observed convincing evidence of cytotoxicity in the case of constructions with nuclear localization.
Oligopeptide compounds that exhibit cytotoxic effect may be inverted, retro or retro-inverse and so on
More specifically, it was additionally noted that the enhanced cytotoxic effect can be obtained for compounds or structures that contain more than one PCNA-interacting motif of the present invention.
In another aspect of this description of the invention a set, or a pharmaceutical product containing
(1) Oligopeptide compound, as defined in this description of the invention, the nucleic acid molecule as defined in this description of the invention and/or the vector as defined in this description of the invention; and
(2) a cytostatic agent.
In another aspect, the offered prod�CT, contains (1) Oligopeptide compound, as defined in this description of the invention, the nucleic acid molecule as defined in this description of the invention and/or the vector as defined in this description of the invention, and (2) cytotoxic agent, as a combined preparation for joint, sequential or separate use in the treatment of a disorder or condition in which it is desirable to inhibit the growth of cells, for example, hyperproliferative disorders, or treatment which involves cytostatic therapy.
It is also assumed in vitro introduction Oligopeptide compound, nucleic acid molecule and/or vector as defined in this description of the invention in a cell or cell culture. Such in vitro methods may be used to study DNA repair, maintain and/or cell cycle regulation. In a preferred aspect, the in vitro method used for the identification of new cytotoxic agents. It can provide more rapid identification of cytotoxic agents or the identification of agents that are only weak cytotoxic drugs, when used by themselves, but which are useful cytotoxic activity when used in combination with Oligopeptide compound, nucleic acid molecule or vector according to the invention.<> New PCNA-interacting motif, as defined in this description of the invention, can be used in diagnosing or monitoring disorder or condition in which it is desirable to inhibit the growth of cells, for example, hyperproliferative disorders, or treatment which involves cytostatic therapy or radiotherapy.
The inventors have discovered that certain associated with cancer antigens have motive, PCNA binding (see Table 1). Thus, it seems possible that or other hyperproliferative disorder, as discussed above, may be diagnosed or development can be observed by determining the level of expression and/or localization of the protein containing the motif, where an aberrant level and/or localization of the protein indicated the disorder, for example a hyperproliferative disorder.
By "aberrant levels" is meant an increased level of protein, such as above 10, 20, 30 or 40% compared with the level in healthy cells of the same cell type, or a decreased level of protein, for example below 10, 20, 30 or 40% compared with the level in the healthy cells of the same cell type.
In a preferred embodiment of an elevated level of a protein containing the motif indicates the disorder, for example a hyperproliferative disorder.
the Level of motif-containing protein can be analyzed, using any known method of protein detection. Preferably using an antibody specific to this motif. The antibody must be specific enough to motive (as compared to the control protein, such as bovine serum albumin) for use in the diagnostic method.
The antibody may be a monoclonal or polyclonal antibody and may be a whole antibody, such as IgG, IgA, IgE, IgM or IgD, or antibody fragment, such as Fab, Fab', F(ab')2, scFv, Fv, dsFv, ds-scFv, Fd, dAbs.
The measurement can be performed in vivo or in vitro, for example in a tissue, or a cell or sample of biological fluid, such as cell lysate, serum or blood.
The definition can facilitate the accession (i.e., physically linking) the antibody to the designated agent (i.e. the introduction of the label in the antibody). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, contrast agents for NMR and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, luciferase, beta-galactosidase, acetylcholinesterase, glucose oxidase, lysozyme, melindagates and the like; examples of suitable prosthetic complexes of groups include� streptavidin/Biotin and avidin/Biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, a fluorescein isothiocyanate, rhodamine, dichlorotriazinyl fluorescein, ancillary or phycoerythrine; an example of a luminescent substance includes luminol; examples of bioluminescent substances include luciferase, luciferin, and acorin, and examples of suitable radioactive substances include1251,1311,35S or3H. In the case of direct visual marker, you can use colloidal metallic or non-metallic particle.
Preferably the proposed method of diagnosing or monitoring the development of the subject's disorder or condition in which it is desirable to inhibit the growth of cells, for example, hyperproliferative disorders, which includes stages:
(1) the harmonization of test samples taken from the specified subject (e.g. mammal), into contact with an antibody specific to the motive, under conditions that allow formation of a complex of antibody-antigen;
(2) measuring the amount of a complex of antibody-antigen in the sample; and
(3) comparing the amount of complex antibody-antigen in the sample with the control.
Control can be a healthy cell, taken from the same subject, such as healthy fibroblast cell.
In another aspect, the invention relates to antibodies, �pacificrim to the motive.
In addition the invention includes a kit for diagnosing or monitoring disorder or condition in which it is desirable to inhibit the growth of cells, for example, hyperproliferative disorders,
where the specified set contains the antibody specific to the motive, and instructions for their use for diagnosing the disorder or condition. Preferably, the antibody attached to a detectable substance as described above, or the kit includes such a detectable substance.
An alternative method of diagnosing includes determining aberrant level of nucleic acid molecules, which encode the motif. In this method, the level of nucleic acids see, using a suitable determination method, such as polymerase chain reaction (PCR) or hybridization methods using appropriately labeled probe. Hereinafter the invention is further described with reference to the following non-limiting Examples and the drawings, where:
Fig. 1 shows images obtained using confocal microscopy, which show that hABH2 and PCNA localized together, and that the 10 N-terminal amino acids hABH2 are necessary and sufficient for such joint localization. Different designs hABH2 (full-size hAHB2 with residues 1-261, truncated hABH2 with the remains 11-261 and N-end�Oh fragment NAVN, consisting of residues 1-10), labeled with EYFP were tested in the co-localization with ECFP-PCNA labeled (see Example 1). On the bar on the left shows cells transfetsirovannyh one hABH2, while on the other three bands shown cells, together transfetsirovannyh design hABH2 and PCNA.
Fig. 2 is a diagram showing the results of the analysis of FRET (fluorescent resonance energy transfer). Normalized FRET measurements (NFRE) shown between EYFP (yellow fluorescent protein)/ESR (cyan fluorescent protein) (lane 1, the background due to the dimerization labels), EYFP-PCNA/ECFP-PCNA (lane 2, positive control, since it binds to PCNA PCNA), hAHB2-EYFP/ECFP-PCNA (lane 3) and 1-10N hABH2-EYFP/ECFP-PCNA (lane 4).
Fig. 3 presents diagrams showing the effect of various cytotoxic agents to cells expressing hABH21-10-EYFP, or expressing EYFP as a control. Treated cells are shown on the left, untreated on the right. Processing was performed using 10 µm MMS, 40 μm BCNU, 1 µm MMC or 600 μm TMZ for 4 days (Fig.3 a-d, respectively). Presents data of one typical experiment of at least 3 experiments, cell growth at different doses were tested in the 8 parallel holes.
Fig.4 shows the alignment of sequences of the 10 N-terminal amino acids AVN-homologs from Homo apiens (NP 001001655.1), Bos Taurus (NP 001019687.1), Rattus non/egicus (XP 222273.3), Mus musculus (NP 778181.2), Gallus gallus (XP 415188.2) and Strongylocentrotus purpuratus (XP 797704.1) using Clustal W. the Sequences were obtained from public databases.
Fig.5 shows the alignment of protein sequences identified in Example 4, from a variety of different types.
Fig.6 presents diagrams showing the results of analysis of cytotoxicity with different peptides as described in Example 7 (Fig.6(A)-(H)). HeLa cells were seeded in 96-well plates (6000 cells/well) and incubated for 3 hours. Different doses of peptides were added into the wells in the presence (filled diamonds) or in the absence of serum (shaded square) in the environment. After 1 h the wells were added an equal volume of medium with 10% or 20% serum to the medium containing no serum). Cells were incubated for 48 hours, then measured cell survival by MTT analysis (analysis using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Graphic materials show cell growth (OD (optical density)750 nm) versus peptide concentration (μm).
Experimental methods used in Examples
Construction of expression
Cloning of fluorescently labeled structures in the expression of ECFP-PCNA and hABH21-261-EYFP was described previously (Aas et al., 2003). Using as matrices� hABH2 1-261-EYFP, using PCR generated hABH21-10-EYFP and hABH211-261-EYFP. The amplicon was cloned into pEYFP-NI (Clonetech) using /VcteI/AgeI and AgeI/EcoRI, respectively. The PCR product from EST (Image clone 5176979 (S) RZPD) was cloned into pEYFP-CI (HindIII/Acc65I) with an EYFP-TFIIS-L. Design EYFP-XPA created by switching EYFP to EGFP (fragment NheI/BsrGI) in His9-HA-EGFP-XPA (Rademakers et al., 2003), generously offered by Dr. Wim Vermeulen (Department of Cell Biology and Genetics, Rotterdam). TFII-I-EYFP was generated by PCR-aplicatia TFII-I from pI3CX-TFII-I (Roy et al., 1993), generously offered by Dr. Robert G. Roeder (Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York), and cloning in EYFP-N1 (SacI/ApaI). EYFP-Topo-IIα created label switching EGFP (EcoRI blunt/NheI) on the label EYFP (XhoI blunt/NheI) in EGFP-Topo-IIα (pT104-1) (Mo and Beck, 1999), generously offered William T. Beck (Division of Molecular Pharmacology, Department of Molecular Genetics, University of Illinois, Chicago). Design hABH21-7-EYFP, including the F4 mutants, were obtained by annealing oligo with "sticky" end of the XhoI/EcoRI, followed by cloning into EYFP-N1, mutated in codon ATG. All point mutations were created using site-directed mutagenesis in accordance with the guide QuickChange®II. Restriction enzymes and calf intestinal alkaline phosphatase (CIP) was obtained from England Biolabs®Inc. and oligonucleotides were obtained from MedProbe, Eurogentech (Oslo, Norway). All constructs were confirmed by sequencing.
Confocal imaging and FRET measurements Living HeLa cells studied che�from 16-24 hours after transient transfection (using Fugene 6 (Roche Inc.) in accordance with the manufacturer's recommendations) merged designs ECFP and EYFP. Fluorescent images were obtained using a laser scanning microscope Zeiss LSM 510 Meta equipped with an oil-immersion objective Plan-Apochromate h/1,4. Enhanced cyan fluorescent protein (ECFP) was excited at λ=458 nm and was determined at λ=470-500 nm, and enhanced yellow fluorescent protein (EYFP) was excited at λ=514 nm and was determined at λ=530-600 nm, using continuous scanning. The slice thickness was 1 µm.
Fluorescence resonance energy transfer (FRET) occurs if label (EYFP and ECFP) are at a distance of less than 100 Å (10 nm). The authors of the invention was determined by FRET using the sensitized emission, measuring the emission of the acceptor (EYFP) after excitation of the donor (ECFP). The inventors received FRET, when the intensity of light emitted from EYFP after excitation of ECFP-fluorochrome was stronger than the light emitted by ECFP or EYFP-labeled proteins after excitation of EYFP and ECFP-lasers, respectively (seepage) specified by the equation: FRET=I2-I1(ID2/ID1)-I3(IA2/IA3was >0. FRET normalized to the expression levels using the equation: NFRET=fret/(I1×b)1/2. NFREwas calculated from the average of the intensities (I) in a region of interest (ROI) containing more than 25 pixels where all pixels had intensities below 250, and the average intensity Rav�ALIS 100-200 designs for both the donor and acceptor. Channel 1 (ECFP) and 3 (EYFP) was measured as described above to obtain the image, and channel 2 (FRET) was excited at λ=458 nm and was determined at λ=530-600 nm. ID1, D2, D3and IA1, A2, A3was determined for cells transfected only ECFP and EYFP, with the same settings and the same fluorescence intensities as for the co-transfected cells (I1and I3). ECFP-PCNA and EYFP-PCNA were included as positive controls, and due to the coupling together of a murine labels, proteins ECFP and EYFP expressed with empty vector were included as negative controls in all experiments.
Cultivation of cell lines and receiving cellular extracts
HeLa cells (cervical cancer) and Nasal (spontaneously transformed keratinocyte) stably expressing interest structures, were obtained by transfection (using Fugene 6) with subsequent sorting of cells or cloned by serial dilution and prolonged cultivation in selective (using geneticin, G418, 400 µg/ml, Invitrogen) modified Dulbecco medium Needle with high glucose 4.5 g/l (DMEM) (BioWhittaker®with the addition of 10% fetal calf serum (FCS), amphotericin b (250 µg/ml, Sigma-Aldrich), gentamicin (100 µg/ml, Gibco) and glutamine (1 mm, BioWhittaker®). Cell to�have Litvinovo at 37°C in 5% carbon dioxide, humid atmosphere. Fractionated cell extracts from HeLa were obtained by resuspending cellular residue in the 1x hematocrit (PCV) in buffer I (10 mm Tris-HCl, pH 8.0 and 50 mm KCl) and 1x PCV in buffer II (10 mm Tris-HCl, 100 mm KCI, 20% glycerol, 0.5% Nonidet P-40, 10 mm EGTA, 10 mm MgCl2, 1 mm DTT, 1x complete protease inhibitor (Roche), a mixture of phosphatase inhibitors (PIC I and PIC II, Sigma). Cells were incubated with constant shaking for 30 min at 4°C. the Collected supernatant (soluble fraction). The precipitate (containing nuclei) was resuspended in 1x PCV with buffer III (10 mm Tris-HCl, pH 8.0 and 100 mm KCl) and 1x PCV buffer II, and in a short time was treated with ultrasound until the destruction of all cores. 750 mcg fractions containing nucleoli, centrifugally, and the precipitate (chromatin-binding fraction) were resuspended in buffer II and III. Chromatin-binding fraction were incubated with a mixture of Tnkase/RNase (2 µl restriction endonuclease Omnicleave®(200 U/ál, Epicentre®Biotecnologies, WI), 2 µl DNase (10 U/µl, Roche Inc.), 2 ál benzonase (250 U/µl, Novagene, Ge), 2 µl of nuclease of micrococci (100-300 U/ál, Sigma - Aldrich) and 2 ál of RNase (10 mg/ml, Sigma-Aldrich) for 30 min at room temperature and 1 h at 37°C. 750 mg of the soluble fraction was incubated with 2 µl Omnicleave®overnight at 4°With during the IP.
Joint immunoprecipitate (co-IP) and Western analysis (WB). Purified rabbit polyclonal�nye antibodies with internal affinity, obtained against GFP, which also detects proteins EYFP and ECFP, covalently linked to paramagnetic granules protein A (Dynal®) according to the procedure from New England Biolabs®Inc (hereinafter called granules α-GFP). Each fraction was incubated with granules a-GFP (10 µl) under constant rotation at 4°C overnight (IP). After the IP pellets were washed 4 times using 200 μl of 10 mm Tris-HCl, 50 mm KCl (pH 7.5), with 5 min incubation on ice in between. Then the pellets were resuspended in loading buffer NuPAGE®(Invitrogen) and 1 mm DTT, heated, and separated on gels of 10% or 4-12% bis-Tris-HCl NuPAGE®) and transferred to PVDF membrane (Immobilon®, Millipore). Membranes were blocked for 1 h in 5% dry milk with low fat content in PBST (PBS with 0.1% Tween®20). Primary antibodies, a-PCNA (PCIO, Santa Cruz biotechnology Inc.) and α-GFP were diluted in 1% dry milk in PBST and incubated for 1 h, followed by 1 h incubation with secondary antibodies, polyclonal rabbit antibodies against mouse IgG/HRP and polyclonal antibodies pigs against rabbit IgG/HRP (DakoCytomation, Denmark). The membrane was treated with chemiluminescence reagent (SuperSignal®West Femto Maximum, PIERCE), and proteins were visualized on a Kodak Image Station 2000R.
For analysis of the initial sequence used database Swiss-Prot and Findings to detect proteins with �UB-sequences, similar sequences for regions of interest. In databases requested motifs in PROSITE format. For alignment for sequences used Clustal W. the Conservative motifs are listed in additional file 1, were identified by comparison of orthologs of genes. The data files for Inparanoid version 5.1 was downloaded from the web server Inparanoid <http://inparanoid.sbc.su.se/> for a typical subgroups of organisms. The sequence of man used as a comparison, processed and searched Inparanoid fasta file with regular expression APIM motif, using the local instrument. Used a slightly broader definition of motive, where Ala was allowed in position 3 or position 4 of the motif, in addition to lie, Val and Leu, but not in both positions simultaneously. Of the total number 22218 protein sequences, there were 636 sequence with at least one match to the APIM motif. These input data were compared with the experimental and predicted subcellular localization in the database eSLDB downloaded from the web server <http://gpcr.biocomp.well as from UNIBO.it/esldb/>, and removed 349 input data without specifying targeting the core. For the remaining 287 input data identified relevant Inpranoid orthologs corresponding sequences were extracted from the fasta file, and the library of sequences exp�univali using Clustal W.
Input 24 sequences without orthologs in Inparanoid were excluded from the analysis. Two different methods were used in parallel to identify conservative areas. In the first method (consensus) consensus sequence was determined from a multiple alignment for each matching position in the sequence of a person. When determining consensus equivalent characters in conservative APIM motif (without Ala) was treated as equivalent symbols to determine consensus, so that, for example, Ile, Val and Leu were treated as remnants of the same type. Normal expression was tested a second time at a consensus before the recognition of the matching position. In an alternative method (individual) normal expression were tested for each ontologically supposedwholesale corresponding to the matching position in the sequence of a person, and were recognized only those provisions where at least 50% of the orthologs of the same expression. For this assessment were excluded supposedwholesale, consisting only of spaces, believing that they can be, for example, alternative splice-variants. These two methods gave almost identical results, and the total yield are shown in Additional file 1. In the end, this method was excluded 37 input data, Stausee�I 226 input data were organized and analyzed. The description of proteins used at the output were taken from Inparanoid unprocessed human (untreated, for humans) fasta file and EnsembI release 45. The output file is presented in html format and can be opened using a standard web browser.
Dot-blot analysis predicted PCNA-binding peptides
Sheet amino-PEG500-UC540 (kislotozawisimah with enhanced stability containing spots 28 nmol peptide (stained with Ponceau to visualize spots), obtained in the laboratory of peptide synthesis in the Centre of biotechnology, University of Oslo, Norway. The membrane was tested using 1 μg/ml of PCNA within 2 h, and Then tested with the primary antibody (α-PCNA, PCIO) and expressed as described above for WB (Western blot).
Analysis of cell survival
HeLa cells and Nasal sown in 96-well plates (4000 cells/well) and incubated for 4 hours. The wells were added various doses of MMS (methylmethanesulfonate, Acros), BCNU (1,3-bis(2-chloroethyl)-1-nitrosoanatabine, Sigma), temosolomida (4-methyl-5-oxo-2,3,4,6,8-pentatonica-[4.3.0] Nona-2,7,9-trien-9-carboxamide, TZM, Sigma) and mitomycin C (6-amino-1,1 a,2,8,8 a,8b-hexahydro-8-(gidroximetil)-8A-methoxy-5-methyl-azirine[2',3',3,4]-imidazo[1,2-a]indole-4,7-dione-carbamate, MMC, Sigma). U20S cells were exposed only MMS and TMZ. Cells were exposed continuously until harvest. Cells were collected every day for 4 days, ispolzuyet-analysis (Mosmann, 1983). OD (optical density) was measured at 570 nm, and the average value of at least 6 wells were used to calculate cell survival. The data provided are the growth in one typical experiment and were replicated at least 2 times.
The study described in this Example, examines the localization PAWN in focus replication and identifies the direct interaction between PAWN and PCNA, and the plot PAWN responsible for this interaction.
In living cells S-phase PCNA-labeled green fluorescent protein (EGFP) forms a clear focus, representing the areas of replication and, thus, it can be used as a marker of S-phase.
PCNA labeled with a blue fluorescent protein (ECFP) together expressively with various deletion constructs hABH2, fused with yellow fluorescent protein (EYFP). It was found that the deletion of the 10 N-terminal amino acids in PAWN (hABH211-261-EYFP) mA joint localization with PCNA in replication focus. Suddenly, what if these 10 amino acids were fused with EYFP (with obtaining construction, named hABH21-10-EYFP), they were sufficient for co-localization with PCNA. (Fig.1). In particular, the co-expression of ECFP-PCNA increased localization of the full-sized PAN (hABH21-261-EYFP) and hABH21-10-EYFP in the core focus on with�avanyu with cells, expressing only design PAWN. This indicates a direct interaction between PCNA and hABH2, mediated 10 N-terminal amino acids hABFO.
To study the degree of closeness of hABH2 and PCNA was measured with fluorescence resonance energy transfer (FRET). Both full-sized hABH2-EYFP and hABH21-10-EYFP created a positive FRET from ECFP-PCNA, showing that the distance between the fluorescent labels is less than 100 Å. This suggests that the options hABH2 directly interact with the same complex as ECFP-PCNA or are in it (Fig.2).
To confirm the direct interaction of the studied joint immunoprecipitation using protein extracts from cells stably expressing hABH2-EYFP, hABH211-261-EYFP, hABH21-10-EYFP or EYFP. Anti-GFP antibody was used for immunoprecipitation of the respective fusion proteins. Subsequent Western blotting analyses showed that endogenous PCNA weakened hABH21-216-EYFP and hABH21-10-EYFP, but not hABH211-261-EYFP or EYFP. Taken together, these results indicate that hABH2 directly interacts with PCNA and that the binding sequence is contained within the 10 N-terminal amino acids hABH2.
Tested the ability hABH2i.io to inhibit hABH2.
Cell lines expressing only hABH21-10-EYFP or EYFP, were subjected to Vosges�vey alkylating agents: MMS (methylmethanesulfonate), BCNU (carmustine), temosolomida (TZM) or mitomycin C (MMC). MMS is an alkylating agent SN2, resulting in 3-methylcytosine (3meC) and 1-methyladenine (1meA) repair using hABH2, while BCNU represents About6-chloranilide agent that mainly causes magnetewan lateral relations and to some of monoammonium cyclic adducts (1,N(6)etendering). Report that TZM is a O6G-metymirumi agent, while MMS initiates minitalia communication by N-alkylation of guanine in CpG.
Overexpression hABH21-10-EYFP or EYFP does not interfere with the rate of growth of untreated cells; however, it was found that the expression of hABH21-10-EYFP increased the sensitivity of HeLa cells to the treatment with the help of MMS. Also unexpectedly found that the expression of hABH21-10-EYFP increased the sensitivity of HeLa cells to all other tested cytotoxic agents (Fig.3). This indicates that the increased sensitivity was due not only to inhibition of hABH2, which is thought mainly repairwe 3meC and 1meA.
From previous studies it is known that murine cells Abh2-/-then there is a knock-out cells AVN demonstrate increased sensitivity to MMS, but not to BCNU. Thus, the increased sensitivity of cells expressing hABH21-10-EYFP, to cytotoxic agents, such as BCNU, cannot be explained only by inhibition of hABH2.
Cells Nasat (spontaneously transformed keratinocyte) stably expressing hABH21-10-EYFP, were also hypersensitive to MMS and TMZ.
Running and comet analysis of cells after treatment with different alkylating agents have shown that drugs affect the cell cycle in different ways and induced different levels and structures of intermediates in DNA repair (sites to remote bases, SSB (single strand breaks), DSB (duhsitelya breaks)), as determined by the comet assay. MMS leads to a delay in S-phase (1 day) and gives the highest levels of intermediates in DNA repair after 4 hours. However, cells treated with MMS, showed a normal distribution of the cell cycle and the absence of elevated levels of intermediates of DNA repair in 2 days. Like MMS, TMZ also leads to the highest levels of repair intermediates after 4 hours, but the cells are delayed in G2/M up to 3 days, indicating more damage and the nature of reparations. TMZ also induced more strand breaks than all three other tested agent. This is unexpected, as it was assumed that TMZ induces mainly O6METI�- which repair mechanism direct reparation with the help of MGMT, not involving the removal of any grounds or breaks threads. The BCNU treatment leads to a temporary inhibition of G2/M in 1 day and to the very low levels of the intermediates of repair, indicating that most cells containing cross-links, were killed. Processing of MMC, on the other hand, leads to inhibition of S-phase on the 1st and 2nd day, and this was more pronounced for cells expressing hABH21-10-EYFP than for control cells. The cells were still slow at G2/M for 3 days. In the comet assay after treatment with MMS was not observed significant differences between cell lines, but the number of intermediate compounds reparations reached the upper limit of 2 a day.
In General, using comet analysis, it was impossible to determine any significant differences in the number of repair intermediates between cells expressing EYFP and hABH21-10-EYFP. Comet and flow analysis show that used different agents induce different responses from the cell cycle and different characters reparations. However, all agents had a higher cytotoxicity in cells expressing APIM, compared with cells expressing EYFP, thus confirming the role of some proteins containing APIM, regulate� between repair or cell death.
Alignment of sequences AWN from different species showed that 7 N-terminal amino acids are highly (Fig. 4). To identify binding sequences studied the importance of these amino acids for the interaction of peptide-PCNA using dot-blot analysis. Incidentally, it was found that Arg3 and Lys7 could replace each other, and that Leu5 and Val6 could be replaced by one another or other aliphatic amino acids, such as Not and Ala, with no effect on the apparent affinity to PCNA. Moreover, a fully conservative Phe4 aromatic amino acid can be replaced with Tyr, while the Ala in this position significantly reduced the binding of PCNA.
Replacement of amino acids 1-2 and 8-10 on Ala does not affect the binding of PCNA, allowing to assume that the Pentapeptide RFLVK (SEQ ID NO:2) is a sequence that interacts with the kernel.
Further analyses showed that this Pentapeptide in itself was sufficient to bind PCNA, but the additional flanking amino acids increased the interaction.
Then amino acids 1-7 in hABH2 and variants of the sequence in which Phe4 replaced by Tyr, Trp or Ala, expressively in fusion with EYFP and tested in co-localization with PCNA in vivo. Like discovered in the dot-blot analysis, fusion proteins containing aromatic amino acid in the p�Appendix 4, were co-localized with ECFP-PCNA, while proteins with Ala in this position were not together localized.
Database Swiss-Prot and Findings used to search for proteins with supposedwholesale similar to the sequence that was identified as responsible for the binding of hABH2 with PCNA. Using consensus [KR]-[FYW]-[LIVA]-[LIVA]-[KR] (SEQ ID NO:30) as a query, got 226 matches (a summary of the results see Table 1), of which several human proteins were selected for further analysis.
The first was a protein that is similar hABH2 above contains a consensus sequence within its 7 N-terminal amino acids. This protein also contains a conservative N-end domain I found in elongation factor TFIIS, an important protein for the development stopped transcription. The inventors have named this protein TFIIS-like protein (TFIIS-L). The function of this protein is unknown.
The second protein was a multifunctional transcription factor TFII-I, which contains 4 consensus sequences. TFII-I is critical for regulating cell cycle and proliferation, and cell, sverkhekspressiya TFII-I, are more resistant focus Y-H2AX (a marker duhsitelya DNA breaks), which confirms the role of TFII-I in DNA repair.
Also the study�and DNA topoisomerase II alpha (Toro II α), contains a single consensus sequence. Toro II α functions in postreplication disengagement of DNA and DNA segregation.
The consensus sequence is also found within a key protein nucleotide excision repair (NER) XPA (Xeroderma pigmentosum group A), which recognizes spiral curves.
A consensus sequence found in RAD51B, a homologous recombinant protein which has been shown is important for correct English centrosome and segregation of chromosomes.
Another protein was a protein nuclear complex Fanconi anemia, FANCC, which, as observed, contains a single consensus sequence. Fanconi anemia (FA) is a rare genetic disorder characterized by aplastic anemia, increased susceptibility to leukemia and hypersensitivity to crosslinking agents. It has been shown that nuclear FA complex is included in the signaling pathway that activates the DNA damage by regulating the repair of DNA crosslinking agents.
In all of these proteins was discovered that the alleged PCNA-binding motif is conservative for different species (Fig. 5). It was reported that among these five proteins only Toro II α and FANCC (after damage) are localized together with BRCAI in the magic nuclei of S-phase, and Toro II α represents the only long p�respectively protein containing potential PIP-box (QttLaFkp and.to. 1277-84). Now, the inventors have proved that fused with EYFP proteins, without exception, all of these proteins are localized together with ECFP-PCNA tricks in S-phase.
Other interesting APIM-containing proteins are members of a family of poly(ADP(adenosine diphosphate)-ribose) (PARP-1, 2 and 4) involved in some processes preserve the DNA, including DNA repair, partner PARP-1 and Toro II α isoforms of DNA topoisomerase II beta, involved in the resolution of topological problems caused by replication forks. In addition, APIM detected in the subunit REV3L translational polymerase ζ, involved in both point mutation and large-scale genomic stability, DNA-ligase I and IV, involved in replication and DNA repair, in four ubiquitin protein-ligases E3 (UHRF1 and UHRF2/NIRF, UBR1 and 2), all of which are involved in the regulation of the cell cycle, the preservation of genome integrity and also in some other ubiquitin-E3 ligases (table 1). Interestingly, the ubiquitin-ligase ITHOUT often shown to be genetically and expression modified during the formation of breast cancer tumors. Also containing APIM is N-acetyltransferase ESC01/EF01, protein, whose yeast ortholog binds PCNA through its truncated PIP-box, and which is involved in the right cohesion of nursing chromate�, and preservation of structural chromosomal protein 5 human hSMC5, which, as shown, is involved in the repair of duhsitelya DNA breaks by HR and to the preservation of telomeres in cells of the ALT (alternative lengthening of telomeres). Finally, it was found that some major subunit of transcription factor II and III, subunit of RNA polymerase II and serine/threonine-protein kinases contain the APIM motif (table 1).
|Type/group of proteins||Proteins containing APIM|
|DNA polymerase||The catalytic subunit of Pol zeta (hREV3L)|
|DNA ligase||DNA ligase I1, DNA ligase IV|
|Topoisomerase||Topo II alpha and Topo II beta2|
|Protein DNA repair||hABH2*, XPA*, PARP-132 and 4, RAD51B*,FANCC*4|
|Proteins associated/interacting with DNA repair||STORAGE binding protein 2, BRCA1/VRSA-containing complex subunit 45 (prot-BRE), protein 1 associated with resistance to x-rays|
|Cohesion of sister chromatids||N-acetyltransferase ESCOI/EFO11, hSMCS5|
|Proteins, DNA-bound proteins and chromatin remodeling||Bromodomain-helicase-DNA-binding protein 3, 4 and 5, subunit R RSF (remodeling factor and spacing) chromatin remodeling complex protein 2, binds to the telomeric repeat (TRF2)6|
|Ubiquitin-ligase E3||UHFR1, UHRF2, UBR1, UBR2, proteins with a ring finger 3, 17 and 151, probable E3 ubiquitin-protein ligase MYCBP2|
|Processing of ubiquitin||ubiquitin-specific processorsa protease (FAF-X)|
|Processing SUMO (small ubiquitin-like protein)||Sentry/SUMO-specific protease SENP2|
|Transcription factors||TFIIS-L*, TFII-I* TFIIE-alpha, transcription factor 2 (SREBF2), linking sterelny regulatory element, alpha subunit TFIIIC, a subunit of TFIID 100 kDa (TAF5), subunit of TFIIIC 102 kDa (TF3C gamma), protein MRG15, similar transcription factor, and X (protein 1 and 2, similar to mortality factor 4), transcription factor E2F/tr>|
|Regulators of the cell cycle||Associated with the cell division cycle 2, Bcl2-interacting mediator of cell death protein 2 gene, regulated testicular apoptosis of spermatocytes|
|Protein kinase||Serine/threonine (S/T) protein kinase SRPK1 and 2, 33 and MST4, S/T-protein kinase 1 leucine-rich repeat, STK23 (S/T-protein kinase 23), S/T-protein kinase PLK3, S/T-protein kinase 1 associated with microtubules, P110 subunit gamma P13-kinase, an activator of the interferon-inducible dvuhruchevoj RNA-dependent protein kinase, FYVE finger-containing phosphoinositide-kinase, phosphoinositide-3-kinase-C2-beta, phosphatidylinositol-4-phosphate-5-kinase type II alpha and beta isoforms of the catalytic subunit alpha phosphatidylinositol-4,5-biphosphate 3-kinase, MARKAR-kinase 2 and 5, mitogen-activated protein kinase 15 (MAP 15)|
|Methyltransferase||H3 lysine-specific MLL3, H3-K9 methyltransferase 5, the estimated rRNA-methyltransferase 3|
|Antigens associated with cancer|
|Structural proteins||Lamin-B1 and B2, actin-like protein 2|
|The centrosome, kinesins||Centrosomal protein 110 kDa (Ser), centrosomal protein|
|192 kDa, kinesiology motor, aimed at the plus-end of microtubules (KIF3A), a heavy chain of kinesin (UKHC), protein 1 associated with kinetochores|
Bold: proteins, localized to replication foci under normal conditions or after DNA damage. This study* or elsewhere:
1G. L. Moldovan, B. Pfander, and S. Jentsch, Cell 129 (4), 665 (2007).
2Z. Lou, K. Minter-Dykhouse, and J. Chen, Nat Struct Mol In//12 (7), 589 (2005); A. Niimi, N. Suka, M. Harata et al., Chromosome 110 (2), 102 (2001).
3C. M. Simbulan-Rosenthal, D. S. Rosenthal, S. Iyer et al., Molecular and cellular biochemistry 193 (1-2), 137 (1999).
4S. Jacquemont and T. Taniguchi, BMC biochemistry Q SuppI I, SIO (2007).
5P. R. Potts, M. H. Porteus, and H. Yu, Embo J25(14), 3377 (2006).
6P. L. Opresko, M. Otterlei, J. Graakjaer et a., Mol Cell 14 (6), 763 (2004).
Experimentally studied the function of the consensus sequences in proteins, studied in Example 4. Since the replacement of the aromatic amino acids Phe in the consensus sequence to Ala abrogated interaction with PCNA in vitro and joint localization with PCNA in vivo, studied, does the corresponding mutation is similar to the effect on the full-size proteins. Mutation Phe4 at Ala in full-length hABH2 cancelled joint localization with PCNA. In TFII-I, containing 4 of the motif, residues Phe (F431A, F536A, F641 A and F803A) were replaced separately and together at Ala. One mutation did not reduce joint localization with PCNA, but mutations in all consensus sequences in TFII-I is significantly reduced joint localization with PCNA in replication foci, it was indicated that some of the motifs of the consensus sequences present in the same protein, can contribute to optimal PCNA-interaction.
Aas, PA, Otterlei, M., Falnes, P. O., Vagbo, C. B., Skorpen, F., Akbari, M., Sundheim, O., Bjoras, M., Slupphaug, G., Seeberg, E., and Krokan, H. E. (2003). Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421, 859-863.
Mp, Y. Y., and Beck, W. T. (1999). Association of human DNA topoisomerase ll-alfa with mitotic chromosomes in mammalian cells is independent of its catalytic activity. Experimental cell research 252, 50-62.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods 65, 55-63.
Rademakers, S., Volker, M., Hoogstraten, D.,Nigg, A. L, Mone, M. J., Van Zeeland, A. A., Hoeijmakers, J. FL, Houtsmuller, A. B., and Vermeulen, W. (2003). Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions. Mol Cell Biol 23, 5755-5767.
Roy, A. L., Malik, S., Meisterernst, M., and Roeder, R. G. (1993). An alternative pathway for transcription initiation involving TFII-I. Nature 365, 355-359.
Obtaining and testing of structures containing motif ("APIM") - containing peptide with signal sequences
Peptides SEQ ID NO:98-117 synthesized using standard methods and introduced fluorescent labels, where indicated, again using standard methods. The peptides shown in Table 3, which presents the amino acid sequence for each peptide, and also separately lists the individual components that make up each peptide (motive ("ARM")-containing peptide), NLS, CPP and linkers, as appropriate, and used the label, where it is contained). Peptides SEQ ID NO:98-116 composed of L-amino acids. Peptide RI-MDR26-3 of SEQ ID NO:117 is a retro-inverted peptide composed of D-amino acids. All peptides shown in Table 3, have at least one APIM-peptide, NLS and the RAF.
Have been made various studies to demonstrate the influence of the peptides on cells. Thus, cells were incubated with peptides for determination of localization of the peptides in the cells, using methods based on the methods described in the Examples above. In brief, peptides, yasanacak labeled with fluorescent labels, incubated with cells (HeLa cells), and studied the cellular import using confocal microscopy.
The action of peptides sensitized cells to the cytotoxic effects of drugs were studied using MTT analysis and clonogenic assay (CFU (analysis of colony-forming units), as described below.
The cytotoxicity of the peptides was studied using MTT analysis, as described below, using only the peptide in the absence of cytostatic drugs. The data on cytotoxicity were also received from the controls used in MTT assays cytotoxic drugs (control peptide, but without cytostatic medicines), where the control peptides was monitored over a longer period (4 days).
Membrane toxicity was studied again as described below.
The stability of the peptides was studied using M8(mass spectrometric) analysis of peptides after incubation of the peptides in the medium containing serum.
HeLa cells were seeded in 96-well plates (6000 cells/well) and incubated for 3 hours. The wells were added various doses of MMS and cisplatin. After 24 hours, the peptides were added to cells in medium containing no serum, and incubated for 1 h. Added fresh medium with cytostatic drugs and cell collec�and through another 24, 48 and 72 hours. To the cells was added MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and OD was measured at 570 nm , and the average value of at least 6 wells were used to calculate cell survival. Data are presented as the increase in one typical experiment and were replicated at least 2 times.
Clonogenic (CFU) analysis
750 HeLa cells were seeded in 10 cm culture dish of cells in 11 ml of growth medium containing cytostatic drugs (MMS). On the 2nd day, the cells were treated with peptides for one hour in conditions that do not contain serum. To the cells was added with fresh medium containing fresh cytostatic medicines for an additional incubation for 10 days. Then cells were fixed in 6% glutaraldehyde in PBS for 15 min at room temperature, washed once in PBS, stained with crystal violet and counted colony-forming units. Included only colonies consisting of at least 50 cells.
Analysis of cytotoxicity
The cytotoxicity of the peptides was measured after 48 hours using MTT analysis. HeLa cells were seeded in 96-well plates (6000 cells/well) and incubated for 3 hours. Different doses of peptides were added into the wells in the presence or in the absence of serum in the medium. After 1 h was added an equal volume of medium, 1X �Lee 2 (to the environment not containing serum) and cells were incubated for 48 hours before addition of MTT (see above)
Toxicity for membranes, determined by using flow cytometry
Cells were treated with different concentrations (2, 4 and 8 μm) of peptide for 1 hour. Was then added reflects the iodide (PI) (50 μg/ml in PBS), which stains DNA when the cell membrane permeable. The analysis was performed within 10 minutes on a flow cytometer FACS Canto (BD-Life Science).
The results are shown in Table 4, which shows that all of the labeled marker tested peptides, which are essentially design APIM-containing peptide with the CPP and NLS localized in the nucleus. This effect is found in peptides with different linker sequences connecting the individual components of the design, the linkers of different lengths, and peptides without linker sequences. This shows that the presence of linker sequences is not significant, and the linker sequence may vary.
The results of MTT and CFU-experiments with cytotoxic agents show the effect of peptides in the increase of inhibitory growth effects of cytostatic agents. Thus, the peptides are capable of increasing the sensitivity of cells to the action of cytotoxic agents. This effect is similar to that which COO�the look settled in Example 2 above for the transfected cell lines, expressing APIM-containing peptide.
Table 4 also shows the cytotoxic effect of several peptides. An additional pilot study (data not shown) showed higher cytotoxic effect was observed for peptides, which are localized in the nucleus, compared with those that are not localized in the nucleus. Experiment on membrane toxicity, the peptide (MDR2; SEQ ID NO:98), indicates that membrane toxicity is low, it may indicate that the cytotoxic effect observed for peptides that are not associated with membrane effect. It was found that in some cases, the cytotoxic effect may be observed only in the case of higher concentrations of the peptide (e.g., 1 μm of peptide cytotoxicity could not be observed (although there is the effect of sensitization of cells to cytotoxic agent) and 2 μm of the peptide is observed cytotoxic effect of the peptide itself, as well as sensitizing effect).
The results in Table 4 also show that the effects of the peptides to enter the cell and localize in the nucleus, on the sensitization of cells to cytotoxic agents and the cytotoxicity can be obtained with different NLS sequences and/or the CPP and, although the magnitude or distribution of the effect may vary, the effects do not depend� from specific sequences of NLS and/or the RAF.
In some peptides, as shown in Tables 3 and 4, on the N-end of the included AC-group. It was included to stabilize peptides in serum and cytosol. Peptide MDR26-72-0 (SEQ ID NO:112) showed good stability and good activity in CFU - and MTT-assays and cytotoxicity tests. MDR26-72-0 contains an R-rich sequence as the RAF. It is believed that equally good results may be obtained with equivalent peptides, in which the CPP is replaced by the CPP derived from penetratin or HIV-TAT or based on them, where NLS may be a SV40 or be derived from UNG2).
Additional data on cytoxicity
This Example presents a more detailed test data on cytotoxicity. The cytotoxicity analysis was performed as described in Example 6 above. The results are presented in Fig. 6, which shows the results of MTT analysis of cytotoxicity of peptides MDR26-0 (SEQ ID NO:100), MDR26-3 (SEQ ID NO:103), MDR26-4 (SEQ ID NO:104), MDR26-8 (SEQ ID NO:106), MDR26-7 (SEQ ID NO:105), MDR26-72-0 (SEQ ID NO:112), MDR26 - 72-01 (SEQ ID NO:115) and MDR34 (SEQ ID NO:116).
As you can see, can be observed cytotoxicity of the peptides, with more significant effect manifested in the absence of serum. Cytotoxicity is observed for all variant peptides MDR26, cat�who have one APIM motif. The increased cytotoxicity observed for variants MDR34 that have two APIM-motive. Similar results demonstrating the cytotoxicity, get to peptides corresponding to MDR34 (SEQ ID NO:116), which do not have labels (MDR34-0), or which are the inverse (I-MDR-34) or retainership (RI-MDR-34) equivalents MDR34. MDR34-2 that does not have a NLS sequence, and which instead had an elongated linker sequence IILVIII (SEQ ID NO:95) as the linker 2, showed a reduced cytotoxicity.
Were performed additional experiments, which confirm the interaction between PCNA and APIM-containing peptides. In experiments on joint immunoprecipitation (co-IP) showed that both endogenous PCNA and EYFP-PCNA from cells stably expressing EYFP-PCNA, were able to reduce hABH2. The significant interaction observed between hABH2 and PCNA in fractions enriched in the chromatin, indicating posttranslational modifications of PCNA or hABH2. In these experiments was demonstrated by co-IP for hABH2 from cells stably expressing EYFP-PCNA, using the magnetic bead connected with antibodies against α-EYFP. The membrane was probed with α-hABH2 and re-probed using antibodies to α-PCNA. It was also demonstrated by co-IP hABH2 from cells expressing only endogenous proteins, and�use of magnetic granules, associated with antibodies against α-PCNA. The membrane was probed using a-hABH2 and re-probed with α-PCNA antibodies.
Additional experiments showing the stitching in vivo, confirm direct binding between ARM and PCNA. Stitched and sewn in the reverse direction FLAG fusion proteins from cells stably expressing hABH21-7-EYFP and 3xFLAG hABH21-7-F4A-EYFP 3xFLAG was immunoprecipitated using affine gel α-FLAG. IP (immunoprecipitation) - eluted fractions were analyzed via Western blotting using α-PCNA or α-FLAG antibodies.
1. Oligopeptide compound comprising PCNA (nuclear antigen of proliferating cells) - interacting motif for use in the treatment which involves cytostatic therapy, or as a sensitizer for radiotherapy,
where PCNA-interacting motif is a
(SEQ ID NO: 30), and
where the compound contains Oligopeptide 14-70 amino acids and contains at least one signal nuclear localization sequence and at least one signal sequence entering the cell, where in the compound of PCNA-interacting motif is situated towards the N-Terminus relative to the signal sequence.
2. Oligopeptide compound according to claim 1, wherein the PCNA-interacting�th the motive is:
(SEQ ID NO: 31);
(SEQ ID NO: 33);
(SEQ ID NO: 34);
(SEQ ID NO: 35);
(SEQ ID NO: 36); or
(SEQ ID NO: 37).
3. Oligopeptide compound according to claim 1, wherein the PCNA-interacting motif has the sequence represented in SEQ ID NO.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24 25.
4. Oligopeptide compound according to claim 1, where the signal sequence entering the cell is an HIV-TAT or penetratin or a fragment or derivative.
5. Oligopeptide compound according to claim 1, wherein the cytotoxic therapy involves the use of cytotoxic agent where specified Oligopeptide compound are used together, simultaneously, separately or sequentially with the specified cytotoxic agent.
6. Oligopeptide compound according to claim 5, where the specified cytotoxic agent selected from MMS (methylmethanesulfonate), bis-chloroethylnitrosourea, carboplatin, CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosoanatabine), cisplatin, cyclophosphamide, cytarabine, dacarbazine, doxorubicin, gemcitabine, irinotecan, melphalan, mitomycin C (MMC), oxaliplatin, temosolomida (TZM), treosulfan, 5-azacytidine, 5,6-dihydro-5-azacytidine and 5-fluorouracil.
7. Oligopeptide compound according to claim 5, �de specified cytotoxic agent is an alkylating agent.
8. Oligopeptide compound according to claim 1 for use in the treatment of hyperproliferative disorders or myeloablative.
9. Oligopeptide compound according to claim 8, where the specified hyperproliferative disorder selected from malignant, predislocation or non-malignant neoplastic disorder, inflammation, autoimmune disorders, hematologic disorders, skin disorders caused by a virus hyperproliferative disorders, myelodyplastic disorder or myeloproliferative disorders.
10. Oligopeptide compound according to claim 8, where the specified hyperproliferative disorder selected from cancer, benign tumors, psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease, psoriasis, Reiter's syndrome, red hairy pitiriasi, hyperproliferative variants of disorders of keratinization, restenosis, diabetic nephropathy, hyperplasia of the thyroid gland, graves ' disease, benign prostatic hyperplasia, syndrome Li-Fromenty, diabetic retinopathy, peripheral vascular disease, cervical carcinoma-in-situ, familial intestinal polyposis, oral leukoplakia, histiocytosis, keloids, hemangiomas, hyperproliferative arterial stenosis, inflammatory�about arthritis hyperkeratosis, papulosquamous rashes, including arthritis, warts, and EBV (Epstein-Barr) - induced disease, scarring, multiple sclerosis, systemic lupus erythematosus (SLE; lupus), severe myasthenia gravis, non-cancerous hyperplasia, granuloma, MGUS (monoclonal gammopathy of uncertain significance), neoplastic meningitis, polycythemia Vera, scleromyxedema, papular mucinosis, amyloidosis and granulomatosis Wegener.
11. Oligopeptide compound according to claim 1, containing a PCNA-interacting motif, presented in SEQ ID NO: 18, signal nuclear localization sequence presented in SEQ ID NO: 125, and a signal sequence for entering the cell, is presented in SEQ ID NO: 73.
12. Oligopeptide compound according to claim 11, containing the sequence represented in SEQ ID NO: 112.
13. Oligopeptide compound comprising PCNA (nuclear antigen of proliferating cells) - interacting motif, signal sequence, nuclear localization signal sequence entering the cell, for use in the treatment of a disorder or condition in which it is desirable to inhibit the growth of cells, where the compound is characterized by the sequence represented in any of SEQ ID NO: 100, 103-106, 112 or 114-116.
14. The use Oligopeptide�of compounds according to claim 13 in the manufacture of a medicament for use in the treatment of a disorder or condition in which it is desirable to inhibit cell growth.
15. The use of Oligopeptide compounds according to any one of claims. 1-12 in the manufacture of a medicament for use in treatment which involves cytostatic therapy.
16. Method of treatment of a disorder or condition in which it is desirable to inhibit cell growth, comprising administering Oligopeptide compound according to claim 13 to a subject in need of it.
17. A method for the treatment, including cytotoxic therapy or radiotherapy, comprising administering Oligopeptide compounds according to any one of claims. 1-12 to a subject in need of it.
18. Pharmaceutical composition for use in the treatment of a disorder or condition in which it is desirable to inhibit the growth of cells containing an effective amount Oligopeptide compound according to claim 13 together with at least one pharmacologically acceptable carrier or excipient.
19. Pharmaceutical composition for use in treatment, including cytotoxic therapy, or as a sensitizer for radiotherapy, containing an effective amount of the Oligopeptide compounds according to any one of claims. 1-12 together with at least one pharmacologically acceptable nositelj�m or excipients.
20. Pharmaceutical composition according to claim 18 or 19, as proposed in a form suitable for oral, parenteral, local, rectal, genital, subcutaneous, transurethral, transdermal, intranasal, intraperitoneal, intramuscular and/or intravenous and/or for administration by inhalation.
21. A kit for use in the treatment of a disorder or condition in which it is desirable to inhibit the growth of cells, or in a treatment which involves cytostatic therapy, or as a sensitizer for radiotherapy, comprising:
(1) Oligopeptide compound according to claim 1 and
(2) a cytostatic agent.
22. The kit according to claim 21, where the specified cytotoxic agent selected from MMS (methylmethanesulfonate), bis-chloroethylnitrosourea, carboplatin, CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosoanatabine), cisplatin, cyclophosphamide, cytarabine, dacarbazine, doxorubicin, gemcitabine, irinotecan, melphalan, mitomycin C (MMC), oxaliplatin, temosolomida (TZM), treosulfan, 5-azacytidine, 5,6-dihydro-5-azacytidine and 5-fluorouracil.
23. The kit according to claim 21, where the specified cytotoxic agent is an alkylating agent.
24. Pharmaceutical combined preparation for simultaneous, sequential or separate use in the treatment of a disorder or condition�,
in which it is desirable to inhibit the growth of cells, or in a treatment which involves cytostatic therapy, containing:
(1) Oligopeptide compound according to claim 1; and
(2) cytotoxic agent
25. Pharmaceutical combined preparation according to claim 24, where the specified cytotoxic agent selected from MMS (methylmethanesulfonate), bis-chloroethylnitrosourea, carboplatin, CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosoanatabine), cisplatin, cyclophosphamide, cytarabine, dacarbazine, doxorubicin, gemcitabine, irinotecan, melphalan, mitomycin C (MMC), oxaliplatin, temosolomida (TZM), treosulfan, 5-azacytidine, 5,6-dihydro-5-azacytidine and 5-fluorouracil.
26. Pharmaceutical combined preparation according to claim 24, where the specified cytotoxic agent is an alkylating agent.
27. A nucleic acid molecule that encodes the Oligopeptide compound according to claim 1.
28. The expression vector containing the nucleic acid molecule according to claim 27.
29. The use of Oligopeptide compounds according to any one of claims. 11-13 or combined pharmaceutical preparation according to any one of claims. 24-26 in the manufacture of a medicament for use in the treatment of inflammation.
30. The use of Oligopeptide compounds according to any one of claims. 11-13 or combined pharmaceutical preparation according to any one of claims. 24-26 in the manufacture lekarstvennoj� funds for cancer treatment.
31. The use according to claim 30, where the specified cancer is selected from bladder cancer, prostate cancer, or hematological cancer, such as multiple myeloma or leukemia.
32. A method of treating inflammation, comprising administering Oligopeptide compounds according to any one of claims. 11-13 or combined pharmaceutical preparation according to any one of claims. 24-26 to a subject in need of it.
33. A method for treating cancer, comprising administering Oligopeptide compounds according to any one of claims. 11-13 or combined pharmaceutical preparation according to any one of claims. 24-26 to a subject in need of it.
34. A method according to claim 33, where the specified cancer is selected from bladder cancer, prostate cancer, or hematological cancer, such as multiple myeloma or leukemia.
SUBSTANCE: on paramagnetic particles bearing the immobilised bacterial protein G of the family Streptococcus, with the blocked solution Denhardt-DNA the protein BoNT/A is adsorbed using specific highly affinity polyclonal antibodies. The formation of the protein complex BoNT/A is detected with a biotin-conjugated antibody by a noncovalent conjugate of DNA fragments with neutravidin. PCR amplification of the DNA matrix is carried out with the fluorescence detection of the signal in real time. Registration of the presence of BoNT/A in the samples under study is carried out by the change in the level of fluorescence compared to the control ones.
EFFECT: effective method of determining the presence of the compound.
5 dwg, 1 tbl, 5 ex
SUBSTANCE: invention relates to the field of medicine, namely to a method of predicting the development of severe sepsis. The essence of the method consists in the following: on the first day after acute poisoning with substances of narcotic action the content of procalcitonin, interleukin-6 and interleukin-8 is determined in blood serum. If the value of procalcitonin is from 0.5 ng/ml to 5 ng/ml, interleukin-6 from 20 pg/ml to 150 pg/ml, interleukin-8 from 50 pg/ml to 300 pg/ml, the development of severe sepsis is predicted.
EFFECT: application of the claimed method ensures the high reliability of prediction of severe sepsis development.
1 tbl, 3 ex
SUBSTANCE: invention relates to the field of molecular genetics, genosystematics and pharmacognosy and is intended for the identification of the species affinity of common heather (Calluna vulgaris (L.) Hull.). Claimed is a set of synthetic oligonucleotides for PCR with the fragment ITS2 of nuclear DNA, including forward and reverse primers and a degradable probe.
EFFECT: set possesses high sensitivity and specificity and makes it possible to carry out the identification of a medicinal plant in a fast and reliable way.
1 dwg, 1 ex
SUBSTANCE: invention aims at detecting benign and malignant new growths in human thyroid. Involved thyroid and reference adjacent intact tissues are sampled; micro-RNA is recovered from the samples; that is followed by conducting a reverse transcription reaction, measuring an expression level of microRNA-21, -221, -222, -155, -205 by real-time RNA followed by a comparative analysis of the microRNA expression according to the norm and thyroid tumour involvement, and stating the presence and type of the new growth. If the above microRNA expression varies by no more than 4 times to the higher and lower figures of expression in relation to the reference, the benign new growth is stated. The malignant new growth is shown by the measured microRNA expression by more than 4 times.
EFFECT: effective detection of the benign and malignant thyroid new growths that promotes improving the further therapeutic approach.
5 dwg, 1 tbl, 4 ex
SUBSTANCE: present group of inventions refers to medicine, namely to cardiology, and concerns diagnosing various forms of the left ventricular hypertrophy. That is ensured by determining an amount of a necrosis marker - cardiac troponin T, cardiac function marker - NT-proBNP and one of an inflammation marker - GDF-15. Comparing their amounts to the reference values enables diagnosing a physiological or pathological left ventricular hypertrophy. Besides, the presence of the pathological hypertrophy requires determining the relation between the cardiac function marker and inflammatory marker, as well as between the necrosis marker and inflammatory marker, and comparing these relations to the reference enables stating if the individual suffers from hypertrophic nonobstructive cardiomyopathy, or hypertrophic obstructive cardiomyopathy, or hypertrophy accompanying pressure overload.
EFFECT: improving the diagnostic procedure.
7 cl, 3 tbl, 3 ex
SUBSTANCE: method of detection of intracellular proteins using fluorescein-5-isothiocyanate (FITC) in various types of mammalian cells is provided, which are characterised by different levels of the synthetic process, using the method of confocal laser microscopy. The mammalian cells are fixed by paraformaldehyde (PFA) preventing the extraction of proteins in subsequent stages of staining, permeabilised with detergent, then stained for 2 h with FITC in a concentration of 1 mcg/ml. The cells are enclosed in Mowiol 4-88 adding DABCO (1,4-diazabicyclo[2.2.2] octane). The resulting preparations are used to analyse the localisation and the content of the protein component of the cells by confocal microscopy.
EFFECT: invention enables to obtain information and to investigate the location of proteins in the cells and to carry out a comparative analysis of protein content in cells with different levels of metabolism.
7 dwg, 7 ex
SUBSTANCE: if observing clinical risk factors of thromboembolia of the pulmonary artery, venous blood 3 ml is taken from the patient to analyse polymorphisms -174 G>C of IL-6 gene and -308 G>A of TNFalpha gene by polymerase chain reaction. The genetic typing results are determined by assigning certain points: if observing GG genotype of IL-6 gene, 3 points are assigned, GC - 1 point, CC - 0 points; the GG genotype of TNFalpha gene, 3 points are assigned, GA - 1 point, AA - 0 points, the total score of certain patient is derived. If the total score is 4 and more, the presence of individual predisposition to thromboembolia of the pulmonary artery is stated.
EFFECT: invention provides the effective assessment of genetic predisposition to thromboembolia of the pulmonary artery.
2 tbl, 2 ex
SUBSTANCE: observing no development of ventriculomegalia on the 5th day is combined with the postnatal seromucoid concentration of 0.136-0.166 absorbance units measured in supernatant of biological nasopharyngeal aspirate, whereas ventriculomegalia developing on the 5th day is shown by the postnatal seromucoid concentration of seromucoid concentration of 0.167-0.196 absorbance units measured in supernatant of biological nasopharyngeal aspirate.
EFFECT: early diagnosis of ventriculomegalia accompanying moderate cerebral ischemia caused by the intrauterine cytomegaloviral-herpetic infection in newborns.
2 tbl, 3 ex
SUBSTANCE: claimed group of inventions relates to the field of medicine, in particular to oncology and molecular biology. Claimed are a method and a set of primers and a probe with sequences SEQ ID NO: 1, 2 and 3 for the realisation of a polymerase chain reaction in a real time mode to diagnose clear cell renal cell carcinoma (CCRCC). The quantitative content of mRNA of the NETO2 gene is evaluated. In case of an increased content of mRNA in a supposedly cancer-affected human tissue in comparison with the quantity of mRNA in a healthy tissue, CCRCC is diagnosed.
EFFECT: claimed group of inventions makes it possible to diagnose CCRCC with high reliability, including an early stage of the tumour disease.
4 cl, 1 dwg, 5 tbl, 6 ex
SUBSTANCE: claimed group of inventions relates to the field of medicine, in particular to oncology and molecular biology. Claimed are a method and a set of primers and a probe with sequences SEQ ID NO: 1, 2 and 3 for the realisation of a polymerase chain reaction in a real time mode to diagnose clear cell renal cell carcinoma (CCRCC). The quantitative content of mRNA of the ACY1 gene is evaluated. In case of a reduced content of mRNA in a supposedly cancer-affected human tissue in comparison with the quantity of mRNA in a healthy tissue, CCRCC is diagnosed.
EFFECT: claimed group of inventions makes it possible to diagnose CCRCC with high reliability, including an early stage of the tumour disease.
4 cl, 1 dwg, 5 tbl, 6 ex
SUBSTANCE: invention relates to biochemistry. Disclosed is a polypeptide essentially consisting of amino acid sequence SEQ ID NO: 23 for inducing immune response in a mammal against meningococcal bacteria. Provided is a nucleic acid which codes said polypeptide. The invention also provides a plasmid which contains a nucleotide sequence and a host cell which is transformed by said plasmid, which are meant for expressing said polypeptide. Provided is a membrane vesicle containing the polypeptide for use as a medicament for preventing meningococcosis in a mammal. Disclosed is an immunogenic composition which contains an effective amount of the polypeptide or vesicle.
EFFECT: invention enables to induce immune response in a mammal against meningococcal bacteria.
14 cl, 5 tbl
FIELD: genetic and cellular engineering, veterinary science and animal husbandry.
SUBSTANCE: invention proposes a capsule for implantation in animal representing a semi-penetrating membrane made of alginate-poly-L-lysine-alginate wherein rat myoblast cells are included (L6). These cells secrete porcine somatostatin owing to the expression cassette incorporated into cells consisting of a nucleotide sequence encoding the insulin secretory signal and a nucleotide sequence encoding active form of porcine somatostatin operatively associated with its. Invention provides the development of the safety and highly technological method for provides animal with necessary amount of the growth hormone involving intramuscular administration of one or more capsules in the pig neck region or ear base.
EFFECT: improved administration method, valuable properties of capsule.
4 cl, 13 dwg, 3 tbl, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to antibodies including human antibodies and their antigen-binding portions, which specifically bind to CCR2, in particular to human CCR2, and can act as CCR2 inhibitors. Anti-CCR2 antibodies are those binding to first and/or second extra-cellular CCR2 loops. The present invention also refers to human anti-CCR2 antibodies and to their antigen-binding portions. The present invention refers to the recovered heavy and light chains of immunoglobulin initiated from human anti-CCR2 antibodies, and to nucleic acid molecules coding such immunoglobulins. The present invention also refers to methods for preparing human anti-CCR2 antibodies and their antigen-binding portions, to compositions containing such antibodies or their antigen-binding portions, and to methods for using antibodies and their antigen-binding portions, and compositions for diagnosing and treating.
EFFECT: invention refers to methods for gene therapy with the use of nucleic acid molecules coding molecules of heavy and light chains of immunoglobulin, wherein the above molecules contain anti-CCR2 antibodies and their antigen-binding portions.
25 cl, 24 dwg, 8 tbl, 17 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, specifically to a fused protein containing a variant of rodostomin, and can be used in medicine. An ανβ3 integrin selective polypeptide consisting of an amino acid sequence SEQ ID NO:1 conjugated on the N terminal by a linker amino acid sequence containing a combination of the amino acids glycine and serine with a variant of a human serum albumin (HSA) with SEQ ID NO:4.
EFFECT: invention enables the higher therapeutic effectiveness in the diseases related to ανβ3 integrin.
12 cl, 14 dwg, 2 tbl, 7 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and immunology. Presented are antibodies targeting integrin α2β1 containing humanised anti-integrin alpha-2 (α2) antibodies, as well as a method of treating by the integrin α2 antibodies. The humanised integrin α2 antibodies comprise a variable region of a light chain domain, a constant human light chain domain and a variable constant heavy chain domain of human IgG1, which exhibit the altered effector function. The variable constant heavy chain domain of human IgG1 comprises an S324N substitution. The invention can be used in medicine.
EFFECT: antibodies exhibit complement-dependent cytotoxicity, improved antibody-dependent cell-mediated cytotoxicity and improved CDC and ADCC.
33 cl, 3 dwg, 1 tbl, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and immunology. What is described is a pharmaceutical composition used for treating and/or preventing pathological bone metabolism and containing this antibody. The invention can be used in medicine.
EFFECT: antibody and its functional fragment specifically recognising human Siglec-15 and possessing the osteoclast inhibitory activity are described.
73 cl, 57 dwg, 4 tbl, 33 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and immunology. What is described is a recovered human antibody or its antigen-binding fragment. The antibody binds to human interleukin-4 alpha-receptor (hlL-4R). There are also described a nucleic acid molecule coding this antibody, an expression vector, a host cell, a method for producing such antibody and a therapeutic composition containing this antibody.
EFFECT: presented group of inventions can be used in medicine for treating asthma and atopic dermatitis.
15 cl, 3 tbl, 3 ex
SUBSTANCE: invention refers to biotechnology, in particular to tumour-specific promoters, and can be used in the anti-cancer therapy. There are constructed the broad-spectrum tumour-specific promoters providing the therapeutic gene expression inside a cancer cell. The invention also involves expression cassettes, expression vectors, pharmaceutical compositions, methods of treating cancer and using the expression cassettes and vectors.
EFFECT: promoters of the present invention provide a high expression level of the operatively linked therapeutic gene in the cancer cells of different origin, wherein the normal cell expression is absent or low.
29 cl, 19 dwg, 4 tbl, 20 ex
SUBSTANCE: claimed invention relates to nucleic acids, coding functional AID mutants, to vectors and cells, including said nucleic acids. Claimed invention also relates to methods of applying mutants of AID protein.
EFFECT: obtaining functional mutants of protein of activation induced cytidine deaminase (AID), which possess higher activity in comparison with wild type AID protein.
39 cl, 11 dwg, 14 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to virology and implies using the similar mini-antibodies for the prevention and therapy of influenza. What is declared is a similar mini-antibody specifically binding to a certain epitope of influenza A (H5N2) hemagglutinin and suppressing the influenza A (H5N2) infection. What is created is an adenovirus viral vector expressing the similar mini-antibody, which can effectively bind to the certain epitope of influenza hemagglutinin and thereby block the influenza progression. What is presented is a composition containing an effective amount of the similar mini-antibody and the viral vector expressing this similar mini-antibody.
EFFECT: what is presented is a method for the prevention and therapy of the influenza A (H5N2) infection providing administering the preventive or therapeutic effective amount of the pharmaceutical composition intranasally in the form of drops or spray into the patient in need thereof.
9 cl, 10 dwg, 2 tbl, 10 ex