WO2008000870A1 - Organic compounds that inhibit the catalytic activity of the cyclin-dependent cyclin a/kinase 2 complex by means of bonding thereof to a new pharmacophoric site in the cyclin molecule - Google Patents

Abstract

Isolated and/or purified peptide-type organic molecule that inhibits the enzyme activity of the cdk2/cyclin A complex, which comprises a peptide of general formula 1 and/or derivatives thereof, stereoisomers, mixtures thereof and/or the pharmaceutically acceptable salts thereof. This invention also protects a composition containing it and use thereof in the preparation of a medicinal product for the treatment of cancerous processes.

Description

 ORGANIC COMPOUNDS THAT INHIBIT THE CATALYTIC ACTIVITY OF

CYCLINE COMPLEX A / KINASA 2 CYCLINE DEPENDENT BY MEANS

JOINING A NEW PHARMACOPHORIC SITE IN THE CYCLINE MOLECULE

FIELD OF THE INVENTION:

This invention is within the field of biochemistry. In particular, the invention relates to organic compounds (molecules) of a peptide nature, isolated and / or purified, comprising a peptide of general formula I:

A ₁ -B ₂ -C ₃ -D ₄ -E ₅ -F ₆ -NH ₂

(D as well as its derivatives, stereoisomers, mixtures thereof, and / or its pharmaceutically acceptable salts that selectively inhibit the CDK complex, or cyclin-dependent cyclin A / kinase 2 complex, a pharmaceutical composition containing them and their use in the elaboration of a medicament for the treatment of carcinogenic processes This invention also refers to the test methods used for the identification of substances that interfere in the cyclin-dependent cyclin A / kinase 2 interaction through the use of the organic molecule of peptide character comprising a peptide of general formula I, its derivatives, stereoisomers, mixtures thereof and / or pharmaceutically acceptable salts that interfere in the formation of the complex through its interaction with the new site defined by the binding of the peptide to the complex.

BACKGROUND OF THE INVENTION:

The protein-protein complexes formed between different cyclin and cyclin dependent kinases (CDK) are fundamental for the regulation of the cell cycle. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Given the specificity of individual protein-protein interactions, the modulation of these complexes is seen as a future source of new highly selective drugs [Berg, T. (2003) Angew Chemnt Ed Engl 42 (22), 2462-81; Cochran, AG (2001) Curr Opin Chem Biol 5 (6), 654-9; Cochran, AG (2000) Chem Biol 7 (4), R85-94]. Initial experience in the design of peptides and small non-peptide compounds that bind to protein contact surfaces [Berg, T. (2003) Angew Chemnt Ed Engl 42 (22), 2462-81; Cochran, AG (2001) Curr Opin Chem Biol 5 (6), 654-9; Cochran, AG (2000) Chem Biol 7 (4), R85-94] promoted the viability of protein-protein interactions as a target for drug discovery. In particular, there has been much progress in the development of small non-regulated cell growth inhibitor molecules that characterize cancer cells [Arkin, M. (2005) Curr Opin Chem Biol 9 (3), 317-24]. Cell growth in eukaryotic cells is under the control of a series of concerted molecular mechanisms defined as the cell cycle, the progression of which is rigorously governed by members of the family of cyclin-dependent kinases (CDK) [Loog, M., and Morgan , DO (2005) Nature 434 (7029), 104-8; Morgan, DO (1995) Nature 374 (6518), 131-4]. Thus, the CDK-mediated phosphorylation of the tumor suppressor protein, pRb, is required for the inactivation of the protein and for the consequent release of E2F transcription factors, thus altering the state of E2F regulated genes from completely repressed to induced giving rise to progression through the Gi to S phases [Dynlacht, BD, Flores, O., Lees, JA, and Harlow, E. (1994) Genes Dev 8 (15), 1772-86; Dyson, N. (1998) Genes Dev 12 (15), 2245-62]. The pRb-E2F pathway is frequently deregulated in cancer cells. The E2F transcription factors are preferential pRb targets, however, their DNA binding is also regulated by the activity of cdk2-cyclin A. The phosphorylation of E2F by cdk2-cyclin A inhibits its binding to DNA ₁ Io leading to the end of the transcription of genes regulated by E2F produced at the beginning of mitosis [Krek, 1/1 /., Ewen, M. £., Shirodkar, S., Arany, Z., Kaelin, WG, Jr., and Livingston, DM (1994) CeII 78 (1), 161-72; Xu, M., Sheppard, KA, Peng, CY, Yee, AS, and Piwnica-Worms, H. (1994) Mol CeII Biol 14 (12), 8420-31]. It has also been shown that overexpression of E2F increases cell proliferation but, paradoxically, also triggers apoptosis simultaneously [Qin, XQ, Livingston, DM, Kaelin, WG, Jr., and Adams, PD (1994) Proc Nati Acad Sci USA 91 (23), 10918-22; Shan, B., and Lee, WH (1994) Mol CeII Biol 14 (12), 8166-73; Kowalik, TF, DeGregori, J., Schwarz, JK, and Nevins, JR (1995) J Virol 69 (4), 2491-500]. All this information suggests that the pharmacological inhibition of the enzymatic activity of the cdk2-cyclin A complex could have a potential interest as an antineoplastic strategy, since cancer cells show an increased activity of E2F due to the inactivation of pRb. The cdk2-cyclin A complex is also required for the progression of the S phase that regulates the formation and activation of the pre-replicative complex [Fotedar, A., Cannella, D., Fitzgerald, P., Rousselle, T., Gupta, S., Doree, M., and Fotedar, R. (1996) J Biol Chem 271 (49), 31627-37; Frouin, i., Montecucco, A., Biamonti, G., Hubscher, U., Spadarí, S., and Maga, G. (2002) Embo J 21 (10), 2485-95]. In addition, cyclin A is revealed as an essential gene since its deletion results in embryonic mortality shortly after implantation [Murphy, M., Stinnakre, MG, Senamaud-Beaufort, C, Winston, NJ, Sweeney, C, Kubelka, M., Carrington, M., Brechot, C, and Sobczak-Thepot, J. (1997) Nat Genet 15 (1), 83-6; Winston, N., Bourgain-Guglielmetti, F., Ciemerych, MA, Kυbiak, JZ, Senamaud-Beaufort, C, Carrington, M., Brechot, C, and Sobczak- Thepot, J. (2000) Dev Biol 223 (1 ), 139-53]. The protein-protein complexes formed between different cyclines and cdks (hereinafter referred to as the complexes herein as CDK) are fundamental for the regulation of the cell cycle. These complexes and their natural inhibitors defined as CDK inhibitor proteins (CKI) have been the subject of extensive research. Considerable effort has been focused on the development of small competitive inhibitory molecules by CDK ATP [Huwe, A., Mazitschek, R., and Giannis, A. (2003) Angew Chem lnt Ed Engl 42 (19), 2122-38 ], however, in general, these compounds have several alternative protein targets that compromise their selectivity. Then, new strategies have been proposed for the inhibition of CDK activity by blocking the incorporation of substrates [Mclnnes, C, and Fischer, PM (2005) Curr Pharm Des 11 (14), 1845-63; Fischer, PM (2001) Curr Opin Drug Discov Devel 4 (5), 623-34] and more recently, through interference in the conformational changes protein-protein [Gondeau, C, Gerbal-Chaloin, S., Bello, P. , Aldrian-Herrada, G., Morris, M. C, and Divita, G. (2005) J Biol Chem 280 (14), 13793-800. Epub 2005 Jan 13]. The reason for incorporation of cyclin (cyclin recruitment motlf-CRM) is a binding domain for a large number of cdk 2-cyclin A substrates, such as for example the transcription factor E2F [Krek, W., Ewen, ME, Shirodkar, S., Arany, Z., Kaelin, WG, Jr., and Livingston, DM (1994) CeII 78 (1), 161-72] tumor suppressors pRb [Adams, PD, Sellers, WR, Sharma, SK, Wu, AD, Nalin, CM, and Kaelin, WG, Jr. (1996) Mol CeII Biol 16 (12), 6623-33] and p53 [Luciani, MG, Hutchins, JR, Zheleva, D., and Hupp , TR (2000) J Mol Biol 300 (3), 503-18]; CDKI p21 ^Cip1 and p27 ^Kip1 [Adams, PD, Sellers, WR, Sharma, S. K, Wu, AD, Nalin, CM, and Kaelin, WG, Jr. (1996) Mol CeII Biol 16 (12), 6623- 33; Luciani, MG, Hutchins, JR, Zheleva, D., and Hupp, TR (2000) J Mol Biol 300 (3), 503-18; Chen, J., Saha, P., Kornbluth, S., Dynlacht, BD, and Dutta, A. (1996) Mol CeII Biol 16 (9), 4673-82] and other substrates such as MDM2 [Zhang, T. , and Prives, C. (2001) J Biol Chem 276 (32), 29702-10. 2001 May 18]. All these substrates contain a highly homologous region of 12 amino acids, which includes the sequence RXLYY ', in which X, Y and Y' are any and hydrophobic amino acids, respectively responsible for binding to the CRM.

One of the problems that natural CDK inhibitors usually pose is that they either mutate or are deleted in primary tumor cells. In an attempt to regulate aberrant cell cycle pathways, new synthetic agents that can restore the functions of suppressor proteins of altered tumors, such as CKI, are fundamental targets in current cancer research.

The active sites of enzymes such as kinases have been a challenge for the development of new drugs. However, compounds similar to competitive drugs by ATP have proved difficult to define a specificity for the kinase since many such enzymes share a high degree of sequence similarity within the active ATP site. Alternative inhibitory routes derived from studies aimed at clarifying the structure and activation mechanisms of CDK were found [Brown, EJ, Beal, PA, Keith, CT, Chen, J., Shin, TB, and Schreiber, S. L (1995 ) Nature 377 (6548), 441-6. Morgan, DO (1997) Annu Rev CeII Dev Biol 13, 261-91]. In this sense, ₇ the CRM, a reason for short consensus recognition present in many cell cycle regulatory proteins that interacts with a small hydrophobic extension on the surface of the cyclines [Adams, PD, Sellers, I / I /. R., Sharma, S. K, Wu, AD, Nalin, CM, and Kaelin, WG, Jr. (1996) Mol CeII Biol 16 (12), 6623-33], inspired the design of new inhibitors. It was shown that the peptides derived from the carboxyl terminal of the CDK inhibitor p21 ^Cιp1 [Zheleva, D. L, Mclnnes, C, Gavine, A. L ₁ Zhelev, NZ, Fischer, PM, and Lañe, DP (2002) J Pept Res 60 (5), 257-70] or transcription factor E2F1 [Adams, PD, Sellers, WR, Sharma, SK, Wu, AD, Nalin, CM, and Kaelin, WG, Jr. (1996) Mol CeII Biol 16 (12), 6623-33. Mendoza, N., Fong, S., Marsters, J., Koeppen, H., Schwall, R., and Wickramasinghe, D. (2003) Cancer Res 63 (5), 1020-4] had effects antiproliferatives in multiple cell lines. More recently, Gondeau et al., [Gondeau, C, Gerbal-Chaloin, S., Bello, P., Aldrian-Herrada, G., Morris, M. C ₁ and Divita, G. (2005) J BbI Chem 280 (14), 13793-800. 2005 Jan 13] have described another strategy to inhibit CDK activity by means of the target selection of the contact surface between the cdk and the cyclin. Thus, it was shown that a peptide of 22 residues in length derived from the amino acid sequence of the α5 helix of cyclin A bound to the cdk2-cyclin A complex and in turn, was shown to block the proliferation of tumor cell lines [Gondeau, C, Gerbal-Chaloin, S., Bello, P., Aldrian-Herrada, G., Morris, M. C, and Divita, G. (2005) J Biol Chem 280 (14), 13793-800. Epub 2005 Jan 13, Berg, T. (2003) Angew Chem lnt Ed Engl 42 (22), 2462-81. Arkin, M. (2005) Curr Opin Chem Biol 9 (3), 317-24. Kontopidis, G., Andrews, MJ, Mclnnes, C, Cowan, A., Powers, H., Innes, L, Plater, A, Griffiths, G., Paterson, D., Zheleva, DI, Lañe, DP, Green , S., Walkinshaw, MD, and Fischer, PM (2003) Structure (Camb) 11 (12), 1537-46. Wu, SY, McNae, i, Kontopidis, G., McCIue, S. J, Mclnnes, C, Stewart, KJ, Wang, S., Zheleva, D. /., Marriage, H., Lañe, DP, Taylor, P., Fischer, PM, and Walkinshaw, MD (2003) Structure (Camb) 11 (4), 399-410].

DESCRIPTION OF THE INVENTION:

The object raised in the present invention consists of an organic peptide molecule comprising a peptide of general formula I, that is, a surprisingly small peptide, which has a high inhibitory selectivity of the CDK complex and is therefore highly useful as an antineoplastic. The concept of the invention also includes derivatives, stereoisomers, mixtures thereof and / or their pharmaceutically acceptable salts.

To explore alternative binding sites that can inhibit CDK activity, chemical libraries were selected under restrictive test conditions. The imposition of such selection conditions would limit the supposed targets for non-competitive molecules by ATP and that could bind to cyclin A and inhibit the productive protein-protein interaction that could become the cdk2-cyclin A complex. The NBH peptide was selected ( rwimyf-NH ₂ ) as the leading compound for the development of this new class of inhibitors (table I) (from now on if the Peptides are defined by lowercase letters will be understood to be D-stereoisomers). The peptide showed a Cl ₅₀ value of 1.1 μM for the inhibition of the activity of the cdk2-cyclin A complex while it was less active against a panel of other kinases (Table II). Kinetic analysis showed that the NBM peptide does not compete with either ATP or CRM substrates such as pRb or substrates such as histone H1 (Figures 1 and 2). In addition, it has been shown that the NBM peptide selectively binds to cyclin A (Figure 3). Therefore, it is concluded that the NBH binding site on the cyclin A may represent a new target site for the selective inhibition of the activity of the cdk2-cyclin A complex. Unlike the existing CDK peptide inhibitors, the hexapeptide NBM can prevent the formation of the cdk2-cyclin A complex. The X-ray crystalline structure of the cdk2-cyclin A complex [Jeffrey, PD, Russo, AA, Poíyak, K., Gibbs, E., Hurwitz, J., Massague, J., and Pavletich, NP (1995) Nature 376 (6538), 313-20] showed that on the contact surface of the complex several structural elements of both cdk2 and cyclin A subunits adapt to each other resulting in a buried surface area greater than 3000 A ² . Therefore, the NBH peptide must recognize and bind to a critical structural element of the cyclin A which in turn must induce a supposed change in the conformation that decreases its affinity to bind to the cdk2.

An attempt has been made to obtain a structural model of the binding of the NBM peptide to the cyclin A molecule. Figure 9 shows the structures obtained using the coordinated compounds deposited for the cdk2-cyclin A complex, and a peptide bound to the CRM (pdb code, 1OKU), and the results obtained using AutoDock

Morris, G. G., DS; Halliday, RS; Huey, R .; Hart, WE; Belew, RK; Olson, AJ. (1998) J

Comp Chem 19, 1639-1662] for the NBH peptide. In the present invention, the NBM peptide binds to a groove in the surface of the cyclin A that is important for the binding of the cyclin A to the cdk2 and could define a new site of pharmacological interest. From the performance of the coupling process it appears that the residues of GIn ²²⁸ , Asn ²²⁹ (helix α3), Asn ³¹² , GIn ³¹³ (helix α6); Met ³³⁴ (helix α7) and

Lys ⁴¹⁷ (at the Ct end) contribute to the definition of this new binding site. These results are consistent with those obtained from the SPR analysis of the minimum structural domain that binds to NBH (Figure 4).

Supporting the data obtained from in vitro model systems, the NBH in its form TAT-NBH has proven to be able to internalize in cellular systems (Figure 5 A) and have activity in living cells. In the S phase of the cell cycle, the cdk2-cyclin A complexes are important for phosphorylation and inactivation of the transcription factor E2F / DP1. The inhibition of cdk2-cyclin A results in a high concentration of E2F that leads to the arrest of the S phase and apoptosis. TAT-NB11 could inhibit cell proliferation (Figure B) in a way that depends on the concentration and time, and induced both the specific blockade of the S phase of the cell cycle (Figure 7) and apoptosis (Figure 8) in the glioblastoma (T98G), colon (HCT116) and ovarian cancer cells

TABLE II: Data of the enzymatic inhibition of NBH

(A2780) evaluated. The TAT-NBI1 peptide has also shown activity against acute myeloid leukemia (HL60) cell lines as well as in acute T-cell leukemia (Jurkat).

On the other hand, the cytotoxic activity of TAT-NB11 is much higher in tumor cells than in non-tumor cells. In fact, the Cl ₅₀ of TAT-NBH in non-tumor cells as primary cultures of human fibroblasts is much higher than the values obtained in tumor cells. It has also been shown that the cytotoxicity is not due to a toxic cell membrane effect, since the TAT-NB11 peptide can be considered as non-hemolytic, as derived from the experiments shown in Figure 6. The NBM peptide could define a new class of cdk2-cyclin A inhibitors that bind to a different site in the cyclin A molecule than other available pharmacological inhibitors. In fact, unlike most CDK inhibitors that are competitive with known substrates of CDK complexes, the mechanism of action of NBM was noncompetitive for ATP and noncompetitive for CRM substrates. The ability of NBH to specifically bind to a new binding site in cyclin A and inhibit the formation of the cdk2-cyclin A complex provides new alternatives for development of drugs, since their chemical properties to bind are different from those of compounds directed to active sites and would also allow selectivity.

Therefore, the object of the present invention is to provide an isolated or purified peptide organic molecule that inhibits the enzymatic activity of the cdk2-cyclin A complex, comprising a peptide of general formula I:

A ₁ -B ₂ -C ₃ -D ₄ -E ₅ -F ₆ -NH ₂

(D and / or its derivatives, stereoisomers, mixtures thereof, and / or its pharmaceutically acceptable salts characterized in that:

A ₁ is Arg;

B ₂ is Trp;

C ₃ is He;

D ₄ is selected from the group consisting of Met, Val and Cys;

E ₅ is selected from the group consisting of Tyr, Phe and Trp, and;

F ₆ is selected from the group consisting of, Phe, Cys, Met, He and Trp.

Where Ai ₁ B ₂ , C ₃ , D _4, E ₅ and F ₆ are characterized as D amino acids, L amino acids or mixtures thereof.

As used throughout the invention, "derivative" means any molecule that is obtained from a modification of said peptide of general formula (I), encompassing, but not exclusively, the use of natural or non-natural amino acids or derivatives thereof, cyclic peptides, peptides presenting complementary, homologous or functional equivalent sequences to that of the peptide of general formula (I), chemical analogs, dimers, multimers, etc. "Unnatural amino acids" are those molecules that, containing as the natural amino acids a free carboxyl group (-COOH) and an amino group (-NH2), do not appear naturally in proteins.

By "cyclic derivative peptide" is meant, the result of closing the normally linear structure of the peptides either by creating a covalent bond between the ends of the peptide, or by forming disulfide bridges between, for example, tank side chains giving rise to the formation of cycles that stiffen the structure and the orientation of side chains and can facilitate interaction with the target molecule.

"Homologous sequence" is understood to be that which shares the same or equal amino acids (hydrophobicity, polarity, etc.) in positions equivalent to those of the organic peptide molecule of the invention, also meaning homology up to 80% identity .

"Functional equivalent sequence" means that which causes the inhibition of the cycA / cdk2 activity, due to the binding to the site claimed in this document.

"Chemical analogue" is understood to be one whose aspect (formula Q) or function may be the same as those of the organic peptide molecule of the present invention.

By "dimer" is understood the repetition followed by two units of the original peptide, and by "multimer", the repetition followed by more than two units of the original peptide.

"Pharmaceutically acceptable carrier" means pharmaceutically acceptable molecules that facilitate the transport or intemalization of the organic peptide molecule of the invention. The pharmaceutically acceptable carrier that allows the cell membrane to pass through is that which facilitates the transport of the organic peptide molecule of the invention through the cell membrane.

By "excipient" is understood that inactive substance that can be used as a vehicle for the organic peptide molecule of the present invention.

By "pharmaceutically acceptable adjuvant" is understood that substance that, thanks to its properties, enhances the inhibitory effect of the organic peptide molecule of the present invention on the cyclin-dependent cyclin A / kinase 2 complex. "Pharmaceutically acceptable salt" is defined as that composition in which the organic peptide molecule of the invention modifies its biological properties without altering its structure.

Thus, this new molecule of a petidic nature comprising a peptide of general formula (I), and / or its derivatives, stereoisomers, mixtures thereof and / or its pharmaceutically acceptable salts, selectively inhibit the enzymatic activity of the cdk2 complex - Cyclin A. This invention also aims to provide a new binding site on the surface of the cyclin A protein that surprisingly inhibits the formation of the cdk2-cyclin A complex.

This invention also aims to provide a new type of assays, for the inhibition of the activity of the cdk2-cyclin A complex.

The NBM hexapeptide or organic peptide molecule comprising a peptide of general formula (I) object of the present invention, is characterized by inhibiting the cdk2-cyclin A complex and its use in the manufacture of pharmaceutical compositions for the treatment of cancer . In the present patent document we describe the identification of organic compounds of a peptide nature that comprise a peptide of general formula I, specifically a hexapeptide that inhibits the kinase activity of the cdk2-cyclin A complex by the selective binding to cyclin A. The characterization of the mechanism of inhibition revealed that the hexapeptide is not competitive for ATP or for histone H1. A derivative permeable to hexapeptide cells induces apoptosis and inhibits the proliferation of tumor cell lines.

Therefore in this patent document the identification and characterization of organic peptide molecules comprising a peptide of general formula I, which can be composed of (D) -amino acids, (L) -amino acids or a combination of ( D) and (L) -amino acids; specifically, the hexapeptide called NBM, composed of 6 D amino acids, is described, which inhibits the kinase activity of the kinase complex dependent on [cyclin (cdk2) -cyclin A] by means of a selective binding to cyclin A. The inhibition mechanism is not competitive for ATP and not competitive for protein substrates. Unlike the existing CDK peptide inhibitors, the organic peptide molecule comprising a peptide of general formula (I), and NBH in particular, interfere with the formation of the complex [cdk2-cyclin A]. In addition, a cell permeable derivative of NBH induces apoptosis and inhibits the proliferation of tumor cell lines. Therefore, the binding site of the organic molecule of Peptide character comprising the peptide of general formula I ₁ object of the present invention, in cyclin A can represent a new target site for the selective inhibition of the activity of the cdk2-cyclin A complex.

A second aspect of this invention is related to the identification of a new binding site on the surface of the cyclin A protein that allows interaction with small molecules, such as the NBM peptide as a concrete example of an organic peptide molecule comprising the peptide of general formula (I), cause non-formation of the cdk2-cyclin A complex.

A third aspect of this patent is related to the development of an assay for the search for inhibitors of the activity of the cdk2-cyclin A complex in which the binding site of the organic peptide molecule comprising a peptide of formula is used general (I) to the cyclin A protein, as a target site of the new inhibitors.

BRIEF DESCRIPTION OF THE FIGURES:

Figure 1. Mechanism of inhibition of cdk2 / cyclin A by the peptide NBH. Data from kinetic assays were analyzed using double reciprocal diagrams. The inhibition of the activity of cdk2 / cyclin A by NBM (A) and by the competitive compound by olomucine ATP (S) at different concentrations of ATP. The test consisted of the incubation of 3 μg of pepH1 in the absence (circles) and in the presence of 5 μM of NBH peptide (triangles) or 3 μM of olomucine (rhombuses). The inhibition of the activity of cdk2 / cyclin A by NBM (C) and by pepH1-T3V (an analogue that cannot be phosphorylated from pepH1) (D) at different concentrations of pepH1. The assay consisted of the incubation of 30 μg of ATP in the absence (squares) and in the presence of 5 μM of NBH peptide (triangles) or 3 μM of pepH1-T3V (empty circles).

Figure 2. Effect of NBH on the phosphorylation of substrates dependent and not dependent on CRM.

A, cdk2-cyclin A kinase assay using fpRb or histone H1 as substrates (2 μg / μl each) and NBM or pepp21, at 10 and 20 μM, to inhibit the reaction. B, the data from kinetic tests of inhibition of the activity of cdk2 / cyclin A by NBM and pepp21 were analyzed by means of double reciprocal diagrams. The assay consisted of the incubation of 30 μg of 30 μM ATP in the presence (circles) or in the absence (triangles) of 5 μM of NBM peptide.

Figure 3. Analysis of the binding of NBM to cyclin A and CDK2.

A, a 60 nM solution of NBH-CF in phosphate buffered saline with increasing concentrations of CycA ^{171 "432} was measured and the fluorescence polarization was measured at 3O ⁰ C. The data (mean, SD; n = 3) was recorded in millipolarization units (mP) and were adjusted to a union model in two states.

B to E, binding of the NBM peptide to cyclin A and cdk2 by SPR. Sensograms of immobilized TAT-NBI1 and TAT CM-5 TAT sensor chips, exposed to increasing concentrations (0.2, 0.5, 1 and 2 μM) of cyclin A (B) or cdk2 (C). The binding of cyclin A to TAT-NBH depends on the concentration. To analyze the effect of the free peptide NBH or cdk2 on the interaction between cyclin A and TAT-NBI1; Free NBH (D) or purified cdk2 (E) were previously incubated in different concentrations (0, 0.1, 0.2, 0.5, 1 and 2 μM according to lines 1 to 6 respectively) with 0.5 μM of cyclin A in HBS-EP buffer. Each reaction mixture was then loaded in a bleed buffer on the chip, after the dissociation period the surface chip was regenerated with 0.2% w / v SDS and the amount of cyclin A that remained bound to the TAT- was measured. NBH immobilized.

Figure 4. Analysis of the binding of NBH to fragments of cyclin A.

A, design of the six cyclin A fragments generated as GST fusion proteins.

B, Biacore sensograms representing binding experiments using a CM-5 chip containing immobilized TAT-NBH and TAT, exposed to 0.5 μM of each cyclin fragment A. The binding was demonstrated by fragments 4, 5 and 6 .

Figure 5

A, Internalization of CF-TAT NBH in Saos-2 cells. Confocal microscopy of

live cell in Saos-2 incubated in the presence of 10 μM of CF-TAT NBH at 37 ⁰ C.

B, In vivo cellular antiproliferative effect of the TAT-NB11 peptide. A, different tumor cell lines were treated with increasing concentrations of the TAT-NBH peptides (squares) or TAT (triangles) for 24 hours. Cell viability was measured by the MTT assay, and Cl ₅₀ was calculated for each line. (The values are the means SD; n = 3).

Figure 6 A ₅ In vivo cellular antiproliferative effect of the TAT-NB11 peptide. HL60 (Á) Jurkat (0) and macrophage (m) cell lines were treated with increasing concentrations of the TAT-NBH peptide. Cell viability was measured using MTT and Ia ₅₀ was calculated for each line.

B, Hemolytic activity of TAT-peptide NBIl Erythrocytes were obtained from rat blood and incubated for 1h at 37 ⁰ C with different concentrations of the compound. 100% hemolysis is evaluated in a sample treated with 0.5% Triton X-100.

Figure 7. Cell cycle arrest. The cell cycle distribution of HCT116 (A), HT29 (C), T98G (C) and A2780 (D), exposed to different doses of TAT-NB11 (black bars) or TAT (gray bars) for 24 hours, such as were evaluated by propidium iodide staining and analysis by flow cytometry. The results are represented as the average values of the percentage of cells in the different phases of the cell cycle of three independent experiments.

Figure 8

A, TAT-NBH inhibits the synthesis of DNA. HCT116 (left panel) and A2780 (right panel) were treated with 20 μM of TAT-NB11 or TAT for 24 h, and DNA synthesis was measured by incorporating [ ³ H] -thymidine. In both cases, the values are the mean ± SD from 3 independent experiments.

B, Western blot analysis of the phosphorylation of MCM2. HCT116 (upper spots) and A2780 (lower spots) were treated for 24 h with 20 μM of TAT, as a control, or three different doses of TAT-NB11 (10, 20 and 40 μM). Representative western blot using specific anti-phospho-MCM2 antibodies, total anti-MCM2 protein and antiactin loading control.

C, Separate PARP detection in cell extracts. HCT116 (upper spots) and A2780 (lower spots) were treated for 24 h with 20 μM TAT, as a control, or three different doses of TAT-NBM (5, 10 and 20 μM). Representative western blotting using anti-PARP revealed the presence of PARP cleavage products in the stain (fragment of PARP 85 kDa).

D, Detection of apoptotic morphology. The TAT-NB11 peptide induces the formation of apoptotic bodies in HCT116 and A2780 cells. The images show HCT116 and A2780 cells undergoing 48 h treatment of TAT-NBMo TAT (40 μM). The cells were stained with propidium iodide and the loss of integrity of the nuclear envelope and the visualization of apoptotic bodies (white arrows) were detected.

Figure 9. Surface diagram of the structure of cdk2 - cyclin A and location of the NBM binding site in cyclin A.

A, surface diagram of the structure of the cyclin A (soft gray) with a CRM binding peptide in red (the coordinates were extracted from the PDB code 10KU). The L-amino acid version of the NBM peptide (in red) was coupled to the cyclin structure

A. In this coupling approach, the NBH peptide skeleton was rigid while all side chains were defined as flexible using the deftors module. Cyclin A was treated as the macromolecule part of the coupling calculation since it remained rigid.

B, Surface diagram of the structure of the cdk2-cyclin A complex. The representation of the surface of NBM and CRM binding peptides is indicated in the figure.

DETAILED DESCRIPTION OF THE INVENTION:

For the identification of NBM, a combinatorial library of dual defined position peptides (PCL) synthesized according to the format initially proposed by Houghten et al. [Houghten, RA, PInIIIa, C ₁ Blondelle, S. £., Appel, JR, Dooley, CT, and Cuervo, JH (1991) Nature 354 (6348), 84-6]. The PCL consisted of 400 mixtures of separate hexapeptides having an amide at the C-terminal end and an acetylated N-terminal end. Each mixture (represented by the formula AC-OOXXXX-NH ₂ ) had the first two positions defined with one of the 20 naturally occurring amino acids (called ¹ O ') and the remaining four positions are close to equimolar mixtures of 19 of the naturally occurring amino acids (referred to as 'X'), in which the cysteine was omitted to avoid the uncontrolled formation of disulfide bridges. Peptide mixtures were selected to determine the ability to inhibit the activity of cdk2-cyclin A. As shown in Table I _{1 it} was found that several mixtures of peptides tested had significant inhibitory activity. From these tests, the mixture of AC-RWXXXX-NH ₂ peptides was selected for further deconvolution. In addition, from this initial selection it was announced that the presence of positive charge at the N-terminal end of the peptide mixtures was recognized as one of the characteristics of all the most active peptide mixtures, therefore it was decided to continue with the deconvolution process using a non-acetylated version of the initial library. In addition, due to the potential interest in conducting in vivo studies with the supposedly inhibitory peptides defined after the deconvolution process, it was decided to synthesize the peptide mixtures using the D-stereoisomers (which will be referred to herein as lowercase letters or preceded by (D)), more stable to degradation by proteases. However, before a further deconvolution stage, the mixture of rwxxxx-NH ₂ peptides was synthesized, which proved to have an inhibitory activity similar to that of the AC-RWXXXX-NH ₂ mixture.

In order to define the third amino acid of the hexapeptide, the rwoxxx-NH ₂ library (containing 20 different mixtures) was synthesized and selected. The most active mixtures were rwfxxx-NH ₂ , rwixxx-NH ₂ and rwlxxx-NH ₂ (table I). Due to the similar chemical character of the third amino acid, only rwixxx-NH ₂ was followed in the iterative process. The Leu-Phe pair is one of the most abundant residual pairs found in the CRM of cyclin A [Lowe, ED, Tews, /., Cheng, KY, Brown, NR, GuI, S., Noble, ME, Gamblin, SJ, and Johnson, L N. (2002) Biochemistry 41 (52), 15625-34] (i.e., RXLFY '). Taking into account the objective of finding ligands other than CRM together with the slightly higher activity observed for the rwixxx-NH ₂ mixture, D-IIe was selected in the third position. A new library was synthesized with the general formula rwioxx-NH ₂ and the following position was identified. The most active mixtures were rwicxx-NH ₂ , rwiixx-NH ₂ , rwimxx-NH ₂ , rwirxx-NH ₂ and rwivxx-NH ₂ (table I) which showed a similar inhibitory activity. At this point, in order to make the selection, a second test aimed at the selectivity of the kinase was incorporated. The five mixtures were examined against kinases not related to CDK CamKII and p38 SAPK. The mixtures rwicxx-NH ₂ , rwiixx-NH ₂ , rwirxx-NH ₂ and rwivxx-NH ₂ but not rwimxx-NH ₂ showed some inhibitory activity against these two kinases, then D-Met was selected in the fourth position for hexapeptide A new set of rwimox-NH ₂ mixtures was synthesized in order to define the fifth amino acid. The library and four mixtures were examined, rwiιmcx-NH ₂ , rwimfx-NH ₂ , rw¡mwx-NH ₂ and rwimcyx-NH ₂ were shown to have similar Cl ₅₀ values (table I). Therefore, the criterion used in this case to define the amino acid was based on the chemical nature of the most active amino acid that defines each sub-library. The D-tyrosine was selected as representative of the aromatic amino acids that appeared to be the most active in this particular position. Finally, a collection of 20 individual hexapeptides was synthesized. The sequences rwimyc-NH ₂ and rwimyf-NH ₂ with Cl ₅₀ values of 1.1 μg / ml proved to be the most active compounds (where r is arg, w is trp, i is ile, m is met, and is tyr c is cys and f is phe according to the single letter nomenclature system imposed in molecular genetics). The peptides were extensively purified and analyzed by HPLC and mass spectrometry. On the other hand, a sequence dimer (rwimyc) ₂ -NH ₂ was synthesized with which inhibition percentages were obtained in the same order as those obtained with the monomeric sequences. With the intention of facilitating the study carried out in the present invention, it was decided to continue said study with the purified rwimyf-NH ₂ (NBH peptide object of the present invention), since NBH does not form dimers.

A study conducted by the same researchers within this same research project showed that, an altered version of NBM (yirwfm-NH ₂ sequence, called NBIrd) and a peptide derivative of a different study that shares four of six of the amino acids of NBH (ykrwqm-NH ₂ sequence, called NBInr) did not inhibit the kinase activity of the cdk2-cyclin A complex in our experimental conditions, which suggests that the NBM inhibitory activity depends on the sequence and is specific. In addition, NBH was found to inhibit both cdk1-cyclin B1 and cdkβ-cyclin D3, although more specifically the first (Table II). On the other hand, NBH was a weak cdk2-cyclin E inhibitor and showed a surprising selectivity with respect to the representative game of other serine / threonine kinases, thus, CK2 was not inhibited while GSK3-β was only weakly inhibited and MAPKII (table II).

As a next step, the steady state kinetics of the inhibition of the enzymatic activity of the cdk2-cyclin A complex by NBH was investigated. In these experiments, phosphorylation of a histone H1-derived peptide (pepHM) was measured by the cdk2-cyclin A complex in the presence of increasing amounts of ATP and in the absence or presence of 5 µM NBH. The double reciprocal diagram revealed that NBH behaves as a non-competitive inhibitor with respect to ATP (Figure 1A). The competitive inhibitor by ATP, Olomucina, was used as a control (Figure 1B). Next, the inhibition by NBH with respect to the substrate of pepH1 was examined in the presence of saturated concentrations of ATP. The double reciprocal diagram corresponds to a non-competitive inhibition mode (Figure 1 C). A peptide derivative of pepH1 (pepH1-T3V) in which the phosphorylation-prone Thr residue was substituted as Val was used as a control for competitive inhibition (Figure 1 D). It was concluded, therefore, that the NBH peptide inhibits the catalytic activity of the cdk2-cyclin A complex, however it does not compete with either ATP or those defined as directly phosphorylated substrates such as histone Hl

As mentioned above, it is surprising that the NBH inhibitory activity seems to depend on the cyclin subunit associated with the cdk (Table II). In fact, it was found that NBM more effectively inhibited the cdk2-cyclin A complex than cdk2-cyclin E by a factor of 50 times. This result points to a supposed new type of cdk2-cyclin A inhibitors that, in addition to not being competitive for ATP, may not target CRM. To further support this observation, cdk2-cyclin A kinase assays were performed using a well-defined CRM-dependent substrate (fpRb, a pRb fragment) or a non-CRM-dependent substrate (histone H1). As expected, pepp21, a peptide derived from p21 ^Cιp1 that targets CRM, inhibited the phosphorylation of pRb but not that of histone H1. Interestingly, when the NBM peptide was included in the kinase assays, phosphorylation of both substrates was suppressed (Figure 2A). The double reciprocal diagram of the steady state kinetic analysis using fpRb as a substrate revealed that the NBH peptide behaved as a non-competitive inhibitor with respect to fpRb (Figure 2B).

To initially characterize the NBM binding site to cyclin A, a fluorescent analog of NBH (CF-NB11) was synthesized by covalent binding of the carboxyfluorescein fluorophore to the NBH hexapeptide.

Nonlimiting examples of other possible fluorophores would be made of Oregon green ^®, AlexaFluor ^®, Bodipy ^®, TAMRA ^®, Rhodamine ^®, naphthofluorescein ^®, Texas red ^®, aminomethylcoumarin ^®, Propidium Iodide ^®, dansyl ^®, NBD ^® (all of Invitrogen Molecular Probes), etc.

The CF-NBH peptide was bound to cyclin A with a dissociation constant (Kd) of 102 ± 15 nM as determined in a fluorescent polarization assay (Figure 3A). The binding profile was also analyzed, using surface plasmon resonance technology (SPR). To facilitate the analysis, the TAT-NBH peptide was synthesized, in which the NBH peptide was fused to a TAT peptide, a cellular vehicle that allows the internalization of NBH in cellular systems, where said NBH acts by inhibiting the CDK complex, through its interaction with the new binding site defined in the present invention. Said TAT peptide, well characterized, corresponds to a short basic region comprising residues 48-57 of the HIV-1 TAT protein [Dietz, GP, and Bahr, M. (2004) Mol CeII Neurosci 27 (2), 85- 131; Wadia, JS, Stan, RV, and Dowdy, S. F (2004) Nat Med 10 (3), 310-5. Epub 2004 Feb 8].

Non-exclusive examples of other possible cell vehicles, which allow crossing the cell membrane would be polymers such as polyglutamic or polyethylene glycol, liposomes, peptides such as Penetratin, TAT etc., the preferred option is that constituted by the use of the TAT peptide.

Thus, the TAT-NBH was joined to a dextran-CM5 chip with a surface density of 2000 RU. Subsequently, increasing concentrations of purified recombinant cdk2 or cyclin A were injected onto the surface of the chip and subsequently the binding was analyzed. Cyclin A (Figure 3B), but not cdk2 (Figure 3C), joined the immobilized TAT-N BH with association and dissociation rate constants that match a K _D value of 52 nM, in good agreement with the K ₀ value determined in the binding experiment based on fluorescence. The capacity of increasing concentrations of the NBH peptide to compete for the binding of cyclin A to the surface of the TAT-NBH was also measured. The results, shown in Figure 3D, indicate that when cyclin A was previously mixed with increasing concentrations of free NBH in solution, the amount of cyclin A that bound to TAT-NBI1 decreased. In contrast, the purified cdk2 could not compete in a similar experiment in which the cdk2 was previously mixed with TAT-NBH (Figure 3E). Consequently, it is demonstrated that the NBH peptide binds to cyclin A and makes it difficult to recognize it by cdk2.

In order to identify a structural domain with NBH binding capacity, the association of different cyclin A fragments with the TAT-NBH peptide was analyzed. Therefore, six different cyclin A fragments that map the entire protein sequence were subcloned, expressed in E. coli bacteria and purified (Figure 4A).

The TAT-NBH binding profile was analyzed by means of SPR (Figure 4B). The results suggest that the minimum structural domain that binds to NBH is defined by a highly conserved region between amino acids 257 and 345 that comprises the α3, α4 and α5 helices. These propellers are part of a structural domain called the cyclin box

(residues 209-310), a conserved region between members of the cyclin family and that intervenes in the interaction with the cdks [Jeffrey, PD, Russo, AA, Polyak, K., Gibbs, E., Hurwitz, J., Massague, J., and Pavletich, NP (1995) Nature 376 (6538), 313 -twenty].

NBH inhibits cell cycle progression and induces apoptosis - The effect of the hexapeptide on cell viability was then studied. To ensure efficient uptake of NBH through cell membranes, the TAT-NB11 peptide was used in cell-based assays. Preliminary studies showed that TAT-NBM had the same inhibitory activity as NBH in the in vitro assay of cdk2-cyclin A (data not shown) and was able to internalize in cellular systems (Figure 5A). Therefore, the effect of different doses of TAT-NBH or TAT on a number of tumor cell lines (HCT116, HT29, T98G, and A2780) was studied using a conventional MTT cell viability assay. The results revealed that the hexapeptide decreased the cell viability in a dose-dependent manner (Figure 5). The TAT-NBH peptide has also shown activity against acute myeloid leukemia (HL60) cell lines as well as acute T-cell leukemia (Jurkat) with Cl ₅₀ of 15 and 20 μM respectively (Figure 6 A). However, they show activity at concentrations of the order of 130 μM in macrophage lines (raw 264.7) and at concentrations of 80 μM in primary fibroblast cultures. On the other hand, in hemolysis tests, hemolysis percentages of less than 10% are observed at the concentrations in which NBH has activity in cell lines (Figure 6B)

The cytometric flow analysis of tumor cells that grew asynchronously treated with TAT-NBH resulted in a cell cycle arrest mainly in the S phase, clearly accompanied by an increase in the sub-G1 population (Figure 7). The blockade of the S phase was confirmed additionally in two cell lines, HCT116 (colon carcinoma cell line) and A2780 (ovarian cancer cell line) by measuring the speed of DNA synthesis in the presence of the TAT-NBH peptide (figure 8A). These effects were correlated with an in vivo NBH-induced decrease in the activity of cdk2-cyclin A, as measured by the low phosphorylation rate of the CDK protein substrate, MCM2 (maintenance minichromosome 2), a nuclear protein involved in the beginning of DNA replication (Figure 8 B) [Forsburg, S. L (2004) Microbiol Mol Biol Rev 68 (1), 109-31, table of contents].

The treatment of cells (HCT116 and A2780) with the hexapeptide produced apoptosis as observed by the segmentation dependent on caspase 3 of PARP (Figure 8C) and by induction of apoptotic bodies (Figure 8 D). According to a first important aspect, the present invention aims at an isolated and / or purified peptide organic molecule, inhibitor of the enzymatic activity of the cdk2-cyclin A complex, which comprises the peptide of general formula I:

A ₁ -B ₂ -C ₃ -D ₄ -E ₅ -F ₆ -NH ₂

(I)

and / or its derivatives, stereoisomers, mixtures thereof, and / or its pharmaceutically acceptable salts, characterized in that:

A ₁ is Arg; B ₂ is Trp; C ₃ is He;

D ₄ is selected from the group consisting of Met, Val, and Cys;

E ₅ is selected from the group consisting of Tyr, Phe, and Trp, and;

F ₆ is selected from the group consisting of Phe, Cys, Met, He and Trp.

Where A ₁ , B ₂ , C ₃ , D ₄ , E ₅ and F ₆ are characterized by being D or L or mixtures thereof. The term "Pharmaceutically acceptable salts" suitable for use in the invention comprises any appropriate salt known in the state of the art [P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor) ISBN: 978-3-90639-026-0 June 2002; Properíies, Selection, and Use]. As an example of acid salts, the following could be mentioned: acetic acid, adipic acid, alginic acid, L-ascorbic acid, L-aspartic acid, capric acid, caproic acid (hexanoic acid), nicotinic acid, nitric acid, oleic acid, acid oxalic, carbonic acid, cinnamic acid, citric acid, decanoic acid (see cupric acid), dodecylsulfuric acid, hydrochloric acid, isobutyric acid, lactobionic acid, lauric acid, maleic acid, (-) L-malic acid, malonic acid, acid DL- mandéllco, metasulfonic acid, oroic acid, palmitic acid, octanoic acid, etc.

According to a second important aspect, the present invention provides an organic molecule of a peptide nature, isolated or purified, inhibiting the enzymatic activity of the cdk2-cyclin A complex, which comprises a peptide of general formula I ₁ and / or its derivatives, stereoisomers, mixtures thereof and / or their pharmaceutically acceptable salts, in which all the amino acids included in the sequence of the peptide of general formula I are D-amino acids. A third important aspect of the present invention provides an organic peptide molecule comprising a peptide of general formula I ₁ as indicated in the previous paragraph in which all the amino acids included in the sequence of the peptide of general formula I They are L-amino acids.

Another important aspect of the present invention provides an organic molecule of a peptide nature, isolated or purified, inhibiting the enzymatic activity of the cdk2-cyclin A complex, which comprises a peptide of general formula I, and / or its derivatives, stereoisomers, mixtures of the same and / or their pharmaceutically acceptable salts, in which the amino acids included in the sequence of the peptide of general formula I are a mixture of D and L amino acids.

A following important aspect of the present invention provides, as described in previous paragraphs, an organic peptide molecule comprising a peptide of general formula 1, its complementary sequence, homologous sequences and / or equivalent functional sequences.

According to another important aspect, the present invention provides an organic peptide molecule comprising a peptide of general formula I ₁ object of the present invention, comprising a homologous sequence of the inhibitor peptide, which has at least 80% identity and homology with the sequence of the peptide of general formula I.

According to another important aspect, the present invention provides an organic molecule of a peptide nature, isolated or purified, inhibiting the enzymatic activity of the cdk2-cyclin A complex, which comprises a peptide of general formula I, and / or its derivatives, stereoisomers, mixtures of the same and / or their pharmaceutically acceptable salts, which binds to the cdk2-cyclin A complex by any of the amino acids of the cyclin A protein selected from the group formed by GIn ²²⁸ , Asn ²²⁹ (helix α3), Asn ³¹² , GIn ³¹³ ( helix α6); Met ³³⁴ (helix α7) and Lys ⁴¹⁷ (at the Ct end).

According to a following important aspect, the present invention aims at a pharmaceutical composition comprising a pharmaceutically effective amount of at least one organic peptide compound comprising a peptide of general formula I ₁ as described in previous paragraphs, and at least one pharmaceutically acceptable excipient or adjuvant. Non-exclusive examples of pharmaceutically acceptable excipients and adjuvants would include: Starches; sugars and celluloses such as hydroxypropyl cellulose or lactose; talc or silica cellulose dibasic calcium phosphate; glycerol; starch ethanol; lactose; saccharose; mannitol; levulose; pectin or gelatin; sodium bicarbonate and citric acid; polyols, such as sorbitol, ascorbic acid, lecithin, acetone, acetic acid, chlorobutanoi, benzyl alcohol, propylene glycol etc; acetone agar, aluminum oxide, aluminum stearate, dimethylacetamide, vinyl acetate, iron oxides, myristic acid, palmitic acid, pectin, sodium borate, sodium hyaluronate, sodium lactate, sodium sulphite, Thaumatin, Thymol, acetate zinc, erythrobic acid, hydroxypropyl starch, leucine, macrogol, neoesperidine dihydrocalcone, octyldodecanol, oleic alcohol, aluminum hydroxide, aluminum phosphate adjuvant, ammonium alginate adjuvant, boric acid, calcium alginate, disodium edetate, ethyl lactate, ethyl lactate, ethyl lactate, lactate etc, as well as other excipients known in the state of the art [David E. Bugay and W. Paul Findlay; Pharmaceutical excipient.]

Another important aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of the organic peptide compound comprising a peptide of general formula I object of the present invention, which comprises a homologous sequence of the inhibitor peptide, which has the less than 80% identity and homology with the sequence of the peptide of general formula I and at least one pharmaceutically acceptable excipient or adjuvant.

According to another important aspect, the present invention provides a use of organic peptide molecules, isolated or purified, inhibitors of the enzymatic activity of the cdk2-cyclin A complex, which comprise a peptide of general formula I ₁ and / or its derivatives, stereoisomers , mixtures thereof and / or their pharmaceutically acceptable salts, in the preparation of a medicament.

Another important aspect of the present invention provides a use of the organic peptide compound comprising a peptide of general formula I, according to the previous paragraph in the preparation of a medicament that selectively inhibits the enzymatic activity of the cdk2-cyclin A complex and that selectively inhibits the growth of cancer cells.

According to another important aspect, the present invention provides a use of the organic peptide compound comprising a peptide of general formula I ₁ as described in previous paragraphs, in the preparation of a medicament that inhibits Selectively forms the growth of cancer cells by binding to the cdk2-cyclin A complex by any of the amino acids to the cyclin A protein selected from the group consisting of GIn ²²⁸ , Asn ²²⁹ (helix α3), Asn ³¹² , GIn ³¹³ (helix α6) , Met ³³⁴ (helix α7) and Lys ⁴¹⁷ (at the Ct end).

According to another important aspect, the present invention provides a use of the organic peptide molecule, isolated or purified, inhibiting the enzymatic activity of the cdk2-cyclin A complex, which comprises a peptide of general formula I, and / or its derivatives, stereoisomers , mixtures thereof and / or their pharmaceutically acceptable salts, as a tool for assays aimed at the search for inhibitors of the enzymatic activity of the cdk2-cyclin A complex.

EXAMPLES OF REALIZATION:

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein.

Test 1: Preparation of SCL and individual peptides

The dual defined position library, Ac-0OXXXX-NH ₂ , was synthesized in an interactive format, using the method of dividing, coupling and recombining together with simultaneous peptide synthesis. All individual peptides were prepared using simultaneous multiple peptide synthesis. Mixtures of peptides and individual peptides were dissolved in 5% H ₂ O or DMSO (dimethylsulfoxide) / H ₂ O, aliquots were formed and stored at -2O ⁰ C. Individual peptides were characterized by spectroscopy analysis of masses by time of flight by laser desorption and were purified by preparative RP-HPLC.

Test 2: Expression and purification of proteins.

Cdk2 cDNA, full length cyclin A, cyclin A fragments (A1 aa 1 - were cloned)

88; A2 aa 1- 171; A3 a 1-257; A4 aa 258 - 432 and A6 aa 171 - 432) inside the plasmid

PGEX-6P; and a fragment of pRb, fpRb (aa 792-928) and p21 ^Cip1 was cloned in plasmid PGEX-KG-27. All were fusion proteins with glutathione-S-transferase (GST) and were expressed in E coli BI21 (DE ₃ ). They were grown cells at 37 ⁰ C with shaking (200 rpm) until the phase average logarithmic growth (A ₆₀₀ = 0.8). Expression was induced by the addition of IPTG (isopropyi-beta-D-galactopyranoside) at a final concentration of 0.5 mM and the culture was incubated for a further 4 h at room temperature. The cells were collected by centrifugation, and the cell pellet was resuspended in NENT buffer (20 mM Tris pH 8, 100 mM NaCI, 1 mM EDTA, 0.5% Nonidet P40 (Igepal ®, Sigma)), protease inhibitors (1 mM PMSF, 1 μg / ml aprotinin and 10 μg / ml leupeptin). Simply put, bacterial cells were used by sonication. The insoluble fraction was pelleted by centrifugation and the supernatant was incubated on a glutathione-sepharose column according to the manufacturer's instructions (Pharmacia). On several occasions, the GST was separated from the fusion proteins - GST by means of digestion with the thrombin protease (Sigma) or the Scintion protease (Amersham) according to the manufacturer.

Test 3: Kinase tests for the selection.

Kinase assays were carried out on ELISA plates that were blocked with 200 µl of PBS blocking solution (phosphate buffered saline) containing 1% BSA (bovine serum albumin), 0.02% Tween and azide 0.02% sodium) overnight at 4 ⁰ C. Then the plates were subsequently washed 3 times, 5 min each time, with 100 μl of wash solution (PBS containing 0.02% Tween and sodium azide 0.02%). The plates were then dried for 2-4 h at room temperature, and stored at 4 ⁰ C. Under these conditions, they were stable for 1 month. The tests were performed in a final volume of 60 μl of kinase buffer (25 mM HEPES pH 7.4 and 10 mM MgCl ₂ ) containing 4 μg of histone H ₁ (Boerhinger Mannheim), 30 μM ATP, 2 mM DTT, 0.2 μCi of [γ ³² P] ATP (Amersham, 3000 Ci / mmol, 10 mCi / ml), 800 mM GST-cdk2, and 800 mM GST-cyclin A. Assays were achieved in the presence or absence of different concentrations of peptide mixtures to analyze. An inhibitor control was performed by adding 800 nM of GST-p21 to the reaction medium. The mixtures were incubated for 30 min at 37 ^° C. After incubation, 50 µl of each mixture was filtered on nitrocellulose membranes placed in a spot blot. The samples were washed with 35 μl of 10% TCA, and finally with two washes of 100 μl of 10% TCA followed by 100 μl of H ₂ O. After this process, the membranes were dried at room temperature. The radioactivity associated with the membranes was detected with a radioactivity detection and quantification system (Molecular Dynamics ^® FX. BIO-RAD). Test 4: Determination of the Cl _5n of Cdk1, Cdk2 and Cdk6

Duplicate analyzes were performed in a final volume of 30 μl containing 2 μg of histone H1 or histone H1 derived peptide, pepH1 (PKTPKKAKKL) as substrate and CDK complex (25 ng of cdk2 - cyclin A ₁ 25 ng of cdk2 - cyclin E, 20 ng of cdk1 - cyclin B1 or 25 ng of cdkδ - cyclin D3), all purchased from Upstate Biotechnology, in kinase buffer (25 mM HEPES pH 7.4, 10 mM ₂ MgM, ₂ mM DTT, ATP 30 μM and 0.2 μCi of [γ ³² P] ATP). Reactions were incubated for 30 minutes at 37 ⁰ C and then stopped by the transfer of 25 .mu.l of each on a Whatman P81 phosphocellulose paper. The filters were washed three times with 1% phosphoric acid, air dried and finally counted in a liquid scintillation kit (Wallac). In competitive substrate assays the peptide inhibitor sequence (called pepH1-T3V) is PKVPKKAKKL.

In some tests, the fragment of the GST-pRb protein, fpRb (aa 792-928), was used as a substrate. In these cases, the reaction was stopped by adding 10 µl of Laemmi buffer. Samples were resolved on a 10% SDS-PAGE gel and then the gel was dried. The radioactivity associated with the gel was detected with a radioactivity detection and quantification system (Molecular Dynamics). In the trials that describe a non-competitive action due to the incorporation of the cyclin (CRM) of cyclin A, pepp21 (FYHSKRRLIFS) was used, which is a peptide derived from p21 ^Cpp1 whose target is the CRM.

Test 5: Determination of the activity of CK2:

The activity of the protein kinase CK2 was analyzed as previously described [Riera, M., Roher, N., Miro, F., Gil, C, Trujillo, R., Aguilera, J., Plana, M., and liarte, E. (1999) Mol CeII Biochem 191 (1-2), 97-104] using β-casein 4 mg-ml ^"1 , β-casein peptide only phosphorylated by CK2 as substrates. One unit of activity is defined protein kinase as the amount that catalyzes the transfer of 1 nmol phosphate from [γ- ³² P] GTP to β-casein per minute at 3O ⁰ C.

Test 6: Determination of MAPKII activity.

The activity was measured by incorporating radioactive phosphate into the myelin basic protein (MBP - myelin basic protein), catalyzed by MAP kinase 2 / Erk2 active (recombinant protein expressed in E. coli) catalog No. 14-550 Upstate Biotechnology; and the manufacturer's protocol was followed.

Test 7: Determination of the activity of the GSK3β, glycogen synthase kinase 3β.

We use the active GSk3β (recombinant protein expressed in Sf21 cells) catalog No. 14-492, Upstate Biotechnology, and peptide 2 of phosphoglycogen synthase (GSK3 substrate) catalog No. 12-241 Upstate Biotechnology, as substrate, and we proceed according to the manufacturer's protocol.

Trial 8: Surface Plasmon Resonance Experiments

All surface plasmon resonance analyzes (SPR) were performed at room temperature using a Biacore 3000 ^{® system} (Biacore International AB). TAT peptides (YGRKKRRQRRRG) or TAT-NB11 were immobilized on a carboxymethylated dextran (CM5) sensor chip using the amine coupling method as described by the manufacturer. The peptide was used to construct the reference surface. CDK2, full length cyclin A, cyclin A fragments, and the NBH peptide were diluted in various concentrations with HBS-EP buffer (Biacore International AB) and each sample was injected separately into the flow cells at a flow rate of 10 μl / min for 180 seconds. After a variable dissociation time, the final regeneration of the surfaces was performed with a brief impulse of 0.2% SDS (w / v). The interaction between the proteins and TAT-NB11 was detected and presented as a sensogram by means of the representation of the resonance units (RU) versus time. Kinetic results were calculated using BIA Evaluation ^® software, version 3.0.2. , and the binding curves were adjusted to a simple 1: 1 Langmuir reaction model.

Test 9: Fluorescence Polarization Test

Fluorescence polarization assays were performed on 96-well black plates, using a Wallac Victor2 1420 Multilabel HTS plate reader, setting as excitation wavelength of 485nm and emission 535nm. The test was performed by adding 6OnM either from CF-GG_NBI1 or from CF-TAT-GGNB11 at different concentrations of the Cyclin A protein (171-432). Test 10: Cell lines.

HCT116 and HT29 are colon carcinoma cells cultured in DMEM (Eagle medium modified by Dulbecco): F12 supplemented with 10% FCS (fetal calf serum). T98-G and A2780, a human gliobastoma and ovarian carcinoma cell line, respectively, are grown in RPMI medium supplemented with 10% FCS. All media contained 2 mM L-glutamine, 1% non-essential amino acids, 1 mM pyruvic acid, 50 units / ml penicillin and 50 μg / ml streptomycin. Acute myeloid leukemia (HL60) cell lines as well as Acute T-cell leukemia (Jurkat) was grown in RPMI medium with 10% FBS (fetal bovine serum), 2mM Glutamine and the Osteosarcoma line (Saos-2) was grown in DMEM medium 15% FBS, 2mM Glutamine . Cells were maintained at 37 ⁰ C in a humidified atmosphere containing 5% _CO2.

Test 11: Feasibility tests.

Cells were plated with 96 wells at 7x10 ³ cells / well. After 24 h, TAT or TAT-NB11 was added at different concentrations and incubated for 24 h.

After incubation, the plates were washed with PBS and then 100 µl of MTT stock solution (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyltetrazoyl bromide (0.5 mg / ml) was added . in culture medium) cultures at 37 ⁰ C were incubated for 1 h, then 200 .mu.l of DMSO was added to each well carrying out the solubilization of formazan for 1 h, the plates were read on a MRX reader (Dynatech. Laboratories), using a test wavelength of 570 nm. The results are tripled in at least three independent experiments.

Test 12: Internalization tests of CF-TAT NBH in Saos-2

The cells (1 x 10 ⁵ cells) were seeded on Xmm plates, allowed to adhere for 24h. They were incubated for 0, 45,120,150 and 180 min with 10 μM CF-TAT-NB11 at 37 ⁰ C.

Three washes were performed with fresh medium before visualization, for which a Leica confocal microscope with a blue-1-20x objective was used. The excitation was performed with an Argon laser (548,476,488,496 and 514nm) and blue diode (405nm) and the images were captured on an 8-bit gray scale and processed with the software LCS (version 2.5.1347a, Leica, Germany) that Contains macro color and 3D components. Test 13: Flow cytometry analysis.

The cells were placed in culture plates of 60 mm ² at 5 x 10 ⁵ cells / plate 24 h before the treatments. TAT or TAT-NBI1 was added for 24 or 48 h. The cells were fixed with methane! 70% for 2 h at RT, then washed with PBS and finally incubated with 50 μg / ml propidium iodide (Sigma) and 200 μg / ml RNase for 10 minutes at room temperature. DNA content analysis was performed on a FACS Calibur by Becton Dickinson. The results are tripled in at least three independent experiments.

Test 14: Determination of DNA synthesis. Thymidine incorporation.

The cells were seeded at 5 x 10 ⁵ cells / plate and allowed to grow for at least 24 h. TAT or TAT-NB11 was then added for 24 h and methyl- [ ³ H] -thymidine (4 mCi / ml; Amershman) was incorporated 1 hour before collecting the cells. DNA synthesis was determined by measuring the incorporation of methyl- [ ³ H] -thymidine into the DNA as described [Cripps-Wolfman, J., Henshaw, E. C, and Bambara, RA (1989) J Biol Chem 264 (33), 19478-86].

Test 15: Protein extraction and immunoblot analysis (Western blot).

Treated cells were washed twice with PBS, scraped, and used in lysis buffer (80 mM Tris-HCI pH 6.8, 2% SDS (Wv)). The protein concentration was determined by the Lowry method. The used ones (25 μg) were loaded on an SDS-PAGE gel and after that, the proteins were transferred to Immobilon membranes. Subsequently, the membranes were blocked in 5% skimmed milk powder in TBS buffer for 1 h at room temperature and then incubated with different primary antibodies overnight at 4 ⁰ C: antiphosphorus (S ³² ) -MCM2 1: 20000 (Abcam ab11897), anti-MCM2 1: 50 (Abcam ab6153), anti-PARP 1: 1000 (Pharmigen 556493) and clone C4 anti-actin 1: 1000 (ICN. Biomedicals). After three washes with TBST, the membranes were incubated for 45 minutes with secondary antibodies coupled to horseradish peroxidase diluted in TBS with 2.5% skimmed milk powder. This filter was then washed twice with TBST and once with TBS, and analyzed by enhanced chemiluminescence with ECL detection reagents (Amersham Biosciences). Trial 16: Apoptosis assays.

In addition to the detection by PARP immunoblotting (see above) another apoptosis analysis was performed: the detection of cell fragmentation in apoptotic bodies. The cells were seeded on slides, treated at different times, fixed with 70% methanol and subjected to DNA staining with propidium iodide (Pl).

Test 17: Hemolysis tests

Blood was obtained from Wistar rats by cardiac puncture immediately after death and was kept in a heparinized tube on ice. The erythrocytes were obtained by 3 successive washes with PBS (150Ox g, 10min at 4 ⁰ C). The final pellet was resuspended in 40 times the starting volume. 50mL of these erythrocytes were incubated in the presence of 50 mL of TAT-NBM peptide at different concentrations in PBS for 1 h at 37 ⁰ C under stirring. After centrifuging 10 min at 150Ox g at room temperature, the supernatants were transferred to a 96-well plate and the Absorbance was read at 550 nm using a Multiskan Ascent reader (Thermolab Systems). 100% hemolysis was determined in a sample with 0.5% Triton X-100 and a PBS sample was used as a negative control.

Analyzing the results of the tests carried out so far, the following was observed:

The crystallographic structure of the cdk2-cyclin A complex shows that cyclin A binds and interacts with both N and C lobes of the cdk2 to form a protein-protein contact surface [Russo, AA, Jeffrey, PD, and Pavletich, NP (1996) Nat Struct Biol 3 (8), 696-700; Jeffrey, PD, Russo, AA, Polyak, K., Gibbs, E., Hurwitz, J., Massague, J., and Pavletich, NP (1995) Nature 376 (6538), 313-20]. The structure of the cdk2-cyclin A complexed with the CKI p27 ^KIP1 protein has also been resolved by showing the interaction between the substrate and the complex in the CRM [Russo, AA, Jeffrey, PD, Paiten, AK, Massague, J., and Pavletich, NP (1996) Nature 382 (6589), 325-31]. Several groups have reported on the identification of cdk2-cyclin A inhibitors by designing molecules that bind to CRM [Mclnnes, C, and Fischer, PM (2005) Curr Pharm Des 11 (14), 1845-63; Andrews, MJ, Mclnnes, C, Kontopidis, G., Innes, L, Cowan, A., Plater, A., and Fischer, PM (2004) Org Biomol Chem 2 (19), 2735-41. Epub 2004 Sep 9 ; Fischer, PM (2004) CurrMed Chem 11 (12), 1563-83]. As stated above, the objective of this study is to identify molecules that inhibit cdk2-cyclin A complexes that do not target the binding site of ATP or CRM. With this objective combinatorial libraries were used in the appropriate experimental conditions; therefore, the ATP concentration was adjusted to be high enough to hinder competition with the ATP while minimizing the use of [γ ³² P] -ATP. Histone H1 was used as a substrate that directly phosphorylates, without requiring the binding to the CRM of the cyclin A having other substrates such as families of pRb and E2F proteins [Andrews, MJ, Mclnnes, C, Kontopidis, G., Innes, L, Cowan, A., Plater, A., and Fischer, PM (2004) Org Biomol Chem 2 (19), 2735-41. Epub 2004 Sep 9].

Claims

1. An organic molecule of a peptide nature, isolated and / or purified, inhibitor of the enzymatic activity of the cdk2-cyclin A complex, comprising a peptide of general formula I:

A ₁ -B ₂ -C ₃ -D ₄ -E ₅ -F ₆ -NH ₂

OR)

and / or its derivatives, stereoisomers, mixtures thereof, and / or its pharmaceutically acceptable salts characterized in that:

A ₁ is Arg; B ₂ is Trp;

C ₃ is He;

D ₄ is selected from the group consisting of Met, Val, and Cys; E ₅ is selected from the group consisting of Tyr, Phe and Trp, and; F ₆ is selected from the group consisting of Phe, Cys, Met, Ne, and Trp.

2. Organic peptide molecule according to claim 1, characterized in that A ₁ , B ₂ , C ₃ , D ₄ , E ₅ and F ₆ are D amino acids, L amino acids or mixtures of both.

3. Organic peptide molecule according to claim 1, characterized in that all the amino acids included in the sequence of the peptide of general formula I are D-amino acids.

4. Organic peptide molecule according to claim 1, characterized in that all the amino acids included in the sequence of the peptide of general formula I are L-amino acids

5. Organic peptide molecule according to claim 1, characterized in that the amino acids included in the sequence of the peptide of general formula I are a mixture of D and L-amino acids

6. Organic peptide molecule according to previous claims, characterized in that it comprises a complementary, homologous and / or functional equivalent sequence of the peptide of general formula I.

7. Organic peptide molecule according to claim 6 characterized in that it comprises a homologous sequence Ia which has at least 80% identity and homology with the sequence of the peptide of general formula I.

8. Organic peptide molecule according to previous claims, characterized in that the derivatives of said molecule are chemical analogues, derived cyclic peptides, dimers and / or multimers.

9. Organic peptide molecule according to previous claims characterized in that the amino acids included in the sequence of the peptide of general formula I are natural, non-natural amino acids, derivatives of natural or non-natural amino acids or mixtures thereof.

10. NBM organic molecule according to claim 1, characterized in that the peptide of general formula I is rwimyf-NH ₂

11. Chemical analog CF-NB11 of the NBH molecule according to claim 10, characterized in that it consists of the covalent binding of the carboxyfluorescein fluorophore to the NBH peptide.

12. TAT-NBH chemical derivative of the NBH molecule according to claim 10, characterized in that it consists of the TAT peptide fused to the NBH peptide that allows the internalization of NBH in cellular systems.

13. Organic peptide molecule according to previous claims, characterized in that it binds to the cdk2-cyclin A complex by at least one of the amino acids of the cyclin A protein selected from the region of the three-dimensional structure defining amino acids GIn ²²⁸ , Asn ²²⁹ (helix α3), Asn ³¹² , GIn ³¹³

(helix α6), Met ³³⁴ (helix α7) and Lys ⁴¹⁷ (at the Ct end).

14. Composition comprising the organic peptide molecule according to any of the preceding claims in combination with a pharmaceutically acceptable carrier.

15. Composition according to claim 14 characterized in that the pharmaceutically acceptable carrier allows to pass through the cell membrane.

16. Pharmaceutical composition characterized in that it comprises a pharmaceutically effective amount of at least one organic peptide molecule according to claims 1 to 13, and at least one pharmaceutically acceptable excipient or adjuvant.

17. Pharmaceutical composition according to claim 16 characterized in that the organic peptide molecule comprises a sequence Ia which has at least 80% identity and homology with the sequence of the peptide of general formula I.

18. Use of the organic peptide molecule according to claims 1 to 13 in the preparation of a medicament.

19. Use of the organic peptide molecule according to claim 18, characterized in that said medicament selectively inhibits the enzymatic activity of the cdk2-cyclin A complex.

20. Use of the organic peptide molecule according to claims 18 and 19, characterized in that said medicament selectively inhibits the growth of cancer cells.

21. Use of the organic peptide molecule according to claim 20 characterized in that the cancer cells belong to the HL60 cell types,

HCT116, HT29, T98G, and A2780, Macrophages (raw 264.7) and Jurkat, Saos-2.

22. Use of the organic peptide molecule according to claims 18 to 21, characterized in that said medicament is used in the treatment of acute myeloid leukemia, colon carcinoma, glioblastoma, ovarian carcinoma, acute T-cell leukemia and osteosarcoma.

23. Use of the organic peptide molecule according to claims 20 to 22, characterized in that said selective inhibition of the growth of cancer cells is produced by the union of the organic peptide molecule to the cdk2-cyclin A complex by any of the amino acids of Ia Cyclin A protein selected from the group consisting of GIn ²²⁸ , Asn ²²⁹ (helix α3), Asn ³¹² , GIn ³¹³ (helix α6); Met ³³⁴ (helix α7) and Lys ⁴¹⁷ (at the Ct end)

24. Use of the organic peptide molecule according to claims 1 to 13 in assays for the search for inhibitors of the activity of the cyclin-dependent cyclin A / kinase 2 complex.

25. In vitro test method for the identification of substances capable of binding to cyclin A and / or inhibiting the enzymatic activity of the cdk2-cyclin A complex comprising the following steps:

(a) Contacting the organic peptide molecule according to claims 1 to 13 and / or any candidate substance with cyclin

A, and / or with cdk2 and / or with the cdk2 complex - cyclin A and

(b) Monitor any change in the expected union between the organic peptide molecule according to claims 1 to 13 and the cyclin and / or any change in the activity of the complex.

26. In vitro test method according to claim 25 characterized in that it comprises the use of a three-dimensional model of cyclin and the candidate compound.

27. In vitro test method according to claims 25 and 26 characterized in that any of the components is labeled with a fluorophore and the signal is detected using fluorescence.

28. In vitro test method according to any of claims 25 to 27, characterized in that the detection method comprises monitoring changes in the cell cycle in the GO and / or G1 / S phases, apoptosis related to cell cycle or measurement of antiproliferative effects in vitro