ES2331286A1 - Fluorescent sensor and use thereof for detection of cyclin-dependent kinase inhibitors and/or for detection of cyclins - Google Patents

Abstract

The present invention relates to a fluorescent sensor comprising a lanthanide chelating agent and a peptide sequence together with the method of obtainment and use thereof for identification of inhibitors of CDK/cyclin (kCDKCs) complexes and detection and quantification in vitro of the cyclin protein. It also relates to a kit for implementation of the identification of inhibitors of kCDKCs and the detection and quantification of cyclin in a sample.

Description

Fluorescent sensor and its use for detection of cyclin-dependent kinase inhibitors and / or for cyclin detection.

The present invention relates to a fluorescent sensor comprising a lanthanide chelator and a peptide sequence, as well as its method of obtaining and its use for the identification of inhibitors of CDK / cyclin complexes (kCDKCs) and in vitro detection and quantification of Cyclin protein In addition, it refers to a kit to implement the identification of inhibitors of kCDKCs and as well as the detection and quantification of cyclin in a sample.

Prior art

Cell division (mitosis) is one of the necessary requirements for the development of multicellular organisms. The ability of a cell to divide and differentiate allows the creation of complex multicellular structures. When there is a failure in the mechanism of cell cycle regulation, excessive cell proliferation capable of causing cancer can occur. In eukaryotic cells, the cell cycle is regulated by several kinase complexes formed by the association of a kinase unit (CDK) with a cyclin protein regulatory unit (Poon, RYC Cell. Mol. Life Sci . 2002 , 59 , 1317 -1326). The CDK-Cyclin kinase complexes (kCDKC) are in turn regulated by phosphorylation mechanisms and by interactions with different proteins. In the last 10 years, more than 80,000 articles related to cyclines have been published, which gives an idea of the biological and medical importance of these proteins.

The genetic evidence demonstrates a strong correlation between alterations in the regulation of kCDKCs and the molecular pathology of cancer (Deshpande, A. et al . Oncogene 2005 , 24 , 2909-2915), and that is why in recent years there has been a great interest in the development of inhibitors of kCDKCs. One of the strategies for obtaining these inhibitors is the design of peptide derivatives, with a cyclin binding mode, analogous to that of the p27 / Kip1 inhibitor protein, and that are capable of interfering with the process of recognition of natural substrates of the kCDKC complex. The binding site of p27 / Kip1 to cyclin is defined by a specific sequence known as CBM (Cyclin Binding Motif). Biological studies with different peptides containing the CBM have demonstrated their ability to interfere in the formation of stable protein complexes with kCDKCs, and thus induce apoptosis in tumor cells in vitro and in vivo (McInnes, C. et al . Curr. Med Chem. Anti-Cancer Agents 2003 , 3 , 57-69; Mendoza, N. et al . Cancer Res . 2003 , 63 , 1020-1024).

The biological relevance of the kCDKC system makes obtaining cyclin sensors especially interesting, substances capable of interacting specifically with cyclin and produce a signal that can be measured spectroscopically. These sensors would represent a new tool for the study of molecular mechanisms involved in cell cycle control dependent on the cyclin, and would serve as an instrument for the identification of new inhibitors of kCDKCs with possible applications Pharmacological by developing competitive trials.

Fluorescence spectroscopy offers numerous advantages for the development of sensors and systems diagnosis due to its sensitivity and the ease of essays. There are several strategies for sensor design that take advantage of different mechanisms involved in the phenomenon of fluorescence One of the most interesting is based on the use of lanthanide fluorescent complexes due to their special features: an exceptionally long lifetime long (milliseconds) and extremely narrow emission bands, which allow a better resolution of your fluorescence signal regardless of the dispersion and the background of autofluorescence in biological mixtures.

Trivalent lanthanides emit fluorescence by transitions between 4f orbitals, these transitions are prohibited by the selection rules, and therefore have an extremely low absorption coefficient (less than 10 M -1 cm -1), which means that trivalent lanthanides cannot be directly excited by normal methods. To solve this problem a method of indirect excitation known as sensitization (sensitization) or antenna effect (Gunnlaugsson, T. et al. Org. Biomol. Chem. 2007, 5, 1999-2009) is used. Sensitization generates the fluorescence of the Ln (III) cation in a three-stage process: i) light is absorbed in the immediate environment of Ln (III) by an organic chromophore (antenna); ii) the excitation energy is transferred from the organic chromophore to one or more of the excited states of the metal ion and finally; iii) the luminescent emission by the metal occurs. It is important to highlight that in the sensitization process it is not necessary the direct union of the antenna with the metallic center, but only the proximity in the space. This process can take place efficiently if both species are less than 15-20 Å.

In the design of probes based on the emission of the lanthanides, attempts are made to modulate the luminescent properties of the Ln (III) ions by processes that depend on the concentration of an analyte. This modulation can be performed using the analyte as a sensitizing species of a non-fluorescent or low fluorescent complex. There are precedents for the application of this strategy in polyaromatic molecule sensors based on complexes of
Tb (III) with a non-fluorescent cyclodextrin-DTPA (diethyltetraminopentaacetate) unit. The efficient transfer of energy from aromatic molecules to Ln (III) occurs when they bind to cyclodextrin. Nevertheless, this principle has still been little exploited in the design of sensors and examples of its application to biological systems are very scarce (Leonard, JP et al . Chem. Commun . 2007 , 129-131).

Despite the existence of some sensors, these have not been used to detect cyclin and inhibitors of kCDKCs in the manner set forth in this invention.

Currently and despite the biological importance and diagnostic relevance of cyclin-related pathologies, there are no simple tests to identify possible inhibitors. Normally, microcalorimetry or biochemical tests are used, with the consequent reproducibility problems, implementation in high-throughput tests or enzymatic activity (Gondeau, C. et al . J. Biol. Chem . 2005 , 280 , 13793-13800) .

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Explanation of the invention.

The present invention is based on the design of sensors that take advantage, as explained below, different mechanisms involved in the phenomenon of fluorescence by the use of lanthanide chelating peptides that allow a Good resolution of your fluorescent signal. These sensors are able to show the presence of cyclin.

In this sense, a first aspect of the The present invention relates to a fluorescent sensor that understands:

to.

a DOTA-lanthanide complex and

b.

a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

Being the complex DOTA-lanthanide formed by the acid compound 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and lanthanide, in the form of trivalent cation. Inside of series of these lanthanide metals (Ln (III)), Tb (III) and Eu (III) are the two cations that present The best emission properties. So, in one embodiment preferred, the lanthanide of the DOTA-lanthanide complex It is the Terbium or the Europium.

As for the peptide sequences used in the invention, these are selected from the group that comprises SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. Therefore, in A preferred embodiment the sequence is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and joins the DOTA-lanthanide complex by its N-terminal end, by its side chain of Lysine and by the side chain of the acid residue 2,3-diaminopropionic respectively, as see in figure 1 (Fig. 1).

The design of these peptides was carried out guided by the x-ray structures available at the base of Cyclin A data linked to high affinity peptides. The stabilization of the complex is mainly due to contacts that are established between the hydrophobic cyclin groove (formed by the helices α1, α3 and α4) and the side chains of Arg 1, Leu 3 and Phe 5 of the peptide, while the side chains of Arg2 and Ile4 are project out of the joint groove.

The chelating peptide is designed by trying maintain as much as possible the determining interactions for the formation of the peptide-Cyclin A complex, and minimizing the repulsions that the ligand could cause with the protein.

As described below, these sensors are capable of detecting cyclin, specifically Cyclin A. The strategy for its design is to take advantage of the presence of a Tryptophan (Trp) residue near the peptide binding site as an ion sensitizing unit. Ln (III). Said residue is capable of inducing an excitation transfer to a lanthanide complex located around 10-15 Å of Trp, inducing fluorescence of the peptide complex.
Ln (III) as a consequence of cyclin binding as shown in Figure 2 (Fig. 2).

That is, the peptide that contains an agent chelator of Ln (III) in the absence of Cyclin A should not present fluorescence emission since the metal cannot be excited in the absence of a sensitizer. However, in presence of Cyclin A, the peptide binds to the protein, leaving the Trp residue near the peptide, and the metal center, so make the sensitization process possible efficiently, and fluorescence emission takes place as seen in the Figure 3 (Fig. 3).

For all this, and taking into account the sensitivity of the sensor of the invention, another aspect is directed to the use of said fluorescent sensor to identify inhibitors of cyclin dependent kinases (kCDKCs). Preferably from cyclin A.

As described above, a strategy for the selection of these inhibitors consists of the peptide design, with a cyclin binding sequence, analogous to that of inhibitory proteins, such as, and without involving the exclusion of other inhibitors, p21, p107, E2F1, and p27, capable of interfering with the process of recognition of natural substrates of the kCDKC complex that serve to Identification of new inhibitors of kCDKCs with possible Pharmacological applications through the development of trials competitive.

On the other hand, another aspect of this invention relates to the use of the fluorescent sensor described in the preceding paragraphs for the in vitro detection and quantification of the cyclin protein. Preferably the cyclin is Cyclin A.

The detection of these proteins is performed measuring fluorescence emission levels over a length of determined wave, depending on the lanthanide used, for example, In the case of Terbium, fluorescence emission levels are measured at a wavelength between 480 and 600 nm in the that the appearance of the three typical emission peaks is observed from the Terbio complex.

In this regard, another aspect of the invention refers to a kit for the identification of inhibitors of cyclin-dependent kinases (kCDKCs) as well as for the detection and quantification of cyclin in a sample, preferably Cyclin A, which comprises:

to)

a fluorescent sensor as described in the present invention Y

b)

reagents for lysis of cells.

Understanding reagents for lysis of cells those that are capable of damaging the membrane cytoplasmic and cell wall allowing the release of intracellular content For example, chemical methods (lysis alkaline), mechanical (due to breakage of frozen material) or physical (sonication) but not limited to these examples.

In a preferred embodiment the sample is a isolated biological sample that can come from a fluid physiological such as blood, plasma, serum or urine and / or any tissue cell phone from an organism.

A fifth aspect of the present invention is a method of obtaining the fluorescent sensor comprising:

to.

Group Protection tertbutoxycarbonylmethyl compound DOTA.

b.

Synthesis of the complex peptide-DOTA-lanthanide from DOTA triproted in a), of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and halide lanthanide

As a consequence of the detection of cyclin by means of the fluorescent sensors proposed in this invention, another possible application thereof could be an in vitro method to identify pathologies that occur with the alteration in the expression of the Cyclin protein, as well as to evaluate its situation and predict its evolution in cell samples.

Also, another application could be a kit for the identification of this type of pathologies

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

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Description of the figures

Fig. 1. Schemes of the synthesized sensors.

Schemes of peptides and binding sites of the DOTA complexes [Tb (III)].

Fig. 2. Schematic representation of the fluorescence emission produced by the Tb (III) peptide complex in the presence of Cyclin A.

The union to the cyclin places the metallic center in the vicinity of the residue of Trp217 which then acts as antenna.

Fig. 3. Representation of the peptide bound in the hydrophobic cyclin groove.

It is observed how the lanthanide complex and Trp217 are nearby (approx. 14 \ ring {A}).

Fig. 4. Fluorescence spectrum of sensor 1, DOTA [Tb (III)] - GAKRRLIFE-NH2, in the absence (Pept-Tb) and in the presence of Cyclin A (Pept-Tb-CyA).

Fig. 5. Fluorescence spectrum of sensor 2, H2 N-HA-Lys (DOTA [Tb (III)]) - RRLIF-CONH2, in the absence (Pept-Tb) and in the presence of Cyclin A (Pept-Tb-CyA).

Fig. 6. Fluorescence spectrum of sensor 3, H2 N-HA-Dap (DOTA [Tb (III)]) - RRLIF-CONH2, in absence (Pept-Tb) and in the presence of Cyclin A (Pept-Tb-CyA).

Fig. 7. Displacement test by adding fractions of a solution of a peptide without a fluorescent complex, H 2 N-HAKRRLIF-CONH 2, on the mixture of sensor 1 and Cyclin A.

As aliquots of peptide 4 are added the displacement of peptide 1 from the hydrophobic groove of Cyclin A, and therefore, decreases the fluorescence signal due to said interaction between the peptide complex of Tb (III) and the protein.

Examples

The invention will be illustrated below through tests carried out by the inventors who develop the method of obtaining the fluorescent sensor, the mechanism of action of the described sensors as well as the detection of Cyclin A inhibitors.

Example one

The method by which the sensor was obtained fluorescent comprises:

to. Protection of tertbutoxycarbonylmethyl groups of DOTA compound

one

To synthesize the protected DOTA ligand (3), it was split from the hydrobromide of tris (tertbutoxycarbonylmethyl) -1,4,7,10-tetraazacyclododecane (1) which was rented with ethyl bromoacetate in the presence of K2CO3 anhydrous. The reaction was carried out in acetonitrile at reflux. Then it was necessary to remove the excess of K2CO3 by filtration and with subsequent chromatographic column purification product 2 was obtained with 99% yield.

Then the hydrolyzed selectively ethyl ester of compound 2 using a mixture of dioxane: NaOH 0.1M in a 3: 1 ratio, resulting in the desired product 3 with a yield of 61%. This product was coupled to the peptides without further purifications.

b. Synthesis of the complex peptide-DOTA-lanthanide from DOTA triproted in a), of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and lanthanide halide

For the synthesis of the sensor (1), which contains the DOTA-lanthanide at the end N-terminal peptide with sequence SEQ ID NO: 1, the triprotected DOTA was coupled in solid phase using standard Fmoc procedures as shown in the scheme next

2

Once the peptide has been synthesized with sequence SEQ ID NO: 1 in solid phase using the Fmoc / tBu strategy proceeded to check out the end amino-terminal using the conditions 20% standard piperidine in DMF. Then the DOTA derivative previously synthesized, using as agent HATU coupling in the presence of DIEA in DMF. Once done the coupling was deprotected a small amount of the resin for analysis by HPLC-MS in which the existence of a peak corresponding to the desired product, so which proceeded to its purification in reverse phase HPLC. After to lyophilize the peptide, it was redissolved in 10 mM HEPES, NaCl 100 mM, pH = 7.5 and the formation of the Terbio complex was tested adding a solution of TbCl 3 (50 mM) / HCl (1 mM). The crude The reaction was analyzed using HPLC-MS and observed the existence of a single peak corresponding to the complex Tb (III) peptide that was subsequently repurified by reverse phase HPLC.

For the synthesis of the sensors (2) and (3), the solid phase modification of the peptides with sequence SEQ ID NO: 2 and SEQ ID NO: 3 respectively, totally protected, was chosen, except in the functionality selected for the conjugation of DOTA. 2,3-Diaminopropionic acid (Dap or Dpr) or Lys residue is introduced into the peptide with its side chains protected by an alloc group that allows its orthogonal elimination and conjugation with DOTA as shown in the following scheme.

3

Once the solid phase peptide was synthesized using the FmocltBu strategy, the alloc group was removed using catalytic conditions of Pd (PPh3) 4, N-dimethylbarbituric acid and 2% H2O / CH 2 Cl 2. The previously synthesized DOTA chelating ligand was then coupled, using HATU as a coupling agent in the presence of DIEA in DMF. Once the coupling was made, a small amount of the resin was deprotected for analysis by HPLC-MS in which the existence of a peak corresponding to the desired product was observed. The amino-terminal end was then deprotected using the standard conditions of 20% piperidine in DMF and purification was carried out in reverse phase HPLC. After lyophilizing the peptide, it was redissolved in 10 mM HEPES, 100 mM NaCl, pH = 7.5 and the formation of the Terbium complex was tested by adding a solution of TbCl3 (50 mM) / HCl (1 mM). The reaction crude was analyzed by HPLC-MS and the existence of a single peak corresponding to the peptide complex of Tb (III) was observed, which was subsequently repurified by reverse phase HPLC.

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Example 2

Sensor 1 fluorescence test (DOTA [Tb (III)] - SEQ NO 1) in absence and in the presence of Cyclin A

Concentrations of 2.5 µM were used for both sensors and Cyclin A.

In Fig 4 (Fig. 4) you can see the sensor fluorescence spectrum (1), DOTA [Tb (III)] - GAKRRLIFE-NH_ {2}, in the absence and in the presence of Cyclin A and the spectrum of a target of Cyclin A. To record the fluorescence spectrum, used a 370 nm long-pass filter to eliminate the band of the harmonic of the spectrum.

In the sensor spectrum alone, between 480 and 600 nm, no significant band was observed, however at add cyclin the appearance of the three bands was observed characteristics of the complexes of Tb (III). This result indicates that the sensor peptide is interacting with Cyclin A, such that the complex of Tb (III) is maintained formed and that this is at a distance less than 20 \ ring {A} thus allowing the transfer of energy between Trp of Cyclin A and the Tb complex (III).

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Example 3

Sensor 2 fluorescence test (DOTA [Tb (III)] - SEQ NO 2) in absence and in the presence of Cyclin A

In Fig 5 (Fig. 5) you can see the fluorescence spectrum of sensor 2, H 2 N-HA-Lys (DOTA [Tb (III)]) - RRLIF-CONH 2, (in which DOTA is attached to the peptide through the chain Lysine amine type lateral, Lys), in absence and in presence of Cyclin A and the spectrum of a cyclin target. To the same as for sensor 1 the spectrum was recorded using a 370 nm long pass filter to eliminate harmonic band of the spectrum

In the peptide spectrum alone, between 480 and 600 nm no significant band was observed, however when adding Cyclin A the appearance of the three characteristic bands was observed of the complexes of Tb (III). This result indicates that the peptide is interacting with Cyclin A, such that it maintains the complex of Tb (III) formed and that it is at a distance less than 20 \ ring {A} from Trp thus allowing the transfer of energy between the antenna and the Tb complex (III).

This spectrum was compared with that of the sensor (1) and it was observed that the intensity of the characteristic bands of the Tb (III) for the sensor (2) was lower than in the case of sensor (1). This result suggests that the distance between the ion and the Trp is higher in the case of the sensor (2) than in the sensor (one).

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Example 4

Sensor 3 fluorescence test (DOTA [Tb (III)] - SEQ NO 3) in absence and in the presence of Ciclina

In Fig 6 (Fig. 6) the spectrum of the sensor 3, H 2 N-HA-Dap (DOTA [Tb (III)]) - RRLIF-CONH 2, (in which the DOTA chelating unit is bound to the acid 2,3-diaminopropionic, Dap 6 Dpr, through the amine type side chain), in the absence and in the presence of Cyclin A and the white spectrum of Ciclina A.

As in the previous cases, the peptide it only did not present any emission band between 480 and 600 nm; without however in this case, when adding Cyclin A, the appearance of the characteristic bands of the complexes of Tb (III). Possibly this is because the DOTA is coupled to a shorter side chain and this prevents interaction between peptide and Cyclin A, thus preventing energy transfer

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Example 5

Displacement test adding fractions of a solution of a peptide without fluorescent complex, H 2 N-HAKRRLIF-CONH 2, over the mixture of sensor 1 and Cyclin A

Taking into account all the results previous assessment of the sensor junction (1) and Cyclin A. To this end, aliquots of 1 µL of the stock solution of Cyclin A (37 µM) on the 1 µM solution of the sensor (1).

In Fig 7 (Fig. 7) the increase in the emission of the fluorescence signal at 545 nm. The broadcast continues the typical saturation profile and allowed to calculate the constant of dissociation (KD = 895). Under the same conditions it was carried out a displacement test adding fractions of a solution of a peptide without fluorescent complex, H 2 N-HAKRRLIF-CONH 2, (without DOTA-Tb (III) complex on the mixture of sensor 1 and Cyclin A. As aliquots of the peptide displacement of the groove sensor 1 occurred hydrophobic cyclin A, and therefore decreased the signal of fluorescence due to such interaction between the complex Tb peptide (III) and protein.

This example demonstrates that they can be detected. Cyclin A inhibitors through competitive binding of sensor 1 and the inhibitor itself to the recognition zone of the Cyclin A protein. This identification is carried out by means of of the decrease in the fluorescence level of the sensor as it increases the inhibitor concentration, as the Fluorescent sensor offset progressively.

In order to test the specificity of the sensor 1 in the most relevant biological conditions, the emission was measured of said peptide with increasing amounts of Cyclin A in lysates cellular (concentration of 290 µg / mL of total protein). He sensor 1 showed extraordinary specificity, it was not observed fluorescence emission until cyclin was added. To wing mixture. The total emission was slightly diminished under these terms.

<110> University of Santiago de Compostela

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<120> FLUORESCENT SENSOR AND ITS USE FOR THE DETECTION OF INHIBITORS OF CYCLINE DEPENDENT KINASES and / or FOR THE DETECTION OF CYCLINES

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<130> ES1596.11

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<160> 3

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<170> Patent version 3.5

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<210> 1

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<213> Artificial sequence

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<220> mat_peptide

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<223> protein analog peptide p27 / Kip1 inhibitor

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<400> 1

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\ sa {Gly Ala Lys Arg Arg Leu Ile Phe Glu}

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<221> 2,3-diaminopro acid ionic (Dpr)

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<222> third amino acid (3) of peptide

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\ sa {His Ala Dpr Arg Arg Leu Ile Phe}

Claims (13)

1. Fluorescent sensor comprising,

C.

a DOTA-lanthanide complex and

d.

a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

2. Fluorescent sensor according to claim 1, where the sequence is SEQ ID NO: 1 and joins the complex by its N-terminal end 3. Fluorescent sensor according to claim 1, where the sequence is SEQ NO: 2 and joins the complex by its Lysine side chain. 4. Fluorescent sensor according to claim 1, where the sequence is SEQ NO: 3 and joins the complex by the Side chain of the 2,3-diaminopropionic acid residue. 5. Fluorescent sensor according to any of the claims 1 to 4 wherein the lanthanide is Terbium. 6. Use of the fluorescent sensor according to any of claims 1-5 to identify cyclin dependent kinase inhibitors (kCDKCs). 7. Use of the fluorescent sensor according to any of claims 1-5 for the in vitro detection and quantification of the cyclin protein. 8. Use according to any of the claims 6 or 7 where the cyclin is Cyclin A. 9. Kit for the identification of inhibitors of cyclin-dependent kinases (kCDKCs) in a sample, which it comprises a fluorescent sensor according to any of the claims 1-5 and means for lysis of cells. 10. Kit according to claim 9 for the detection and quantification of cyclin in a sample. 11. Kit according to claims 9 and 10 wherein The cyclin is Cyclin A. 12. Kit according to any of the claims 9-11 where the sample is a biological sample isolated. 13. Method of obtaining the fluorescent sensor according to any of claims 1-5 that understands,

to.

The protection of the tertbutoxycarbonylmethyl groups of the compound DOTA

b.

The complex synthesis peptide-DOTA-lanthanide from DOTA triproted in a), of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and halide lanthanide