WO2009156541A1 - Fluorescent sensor and use thereof for detection of cyclin-dependent kinase inhibitors and/or for detection of cyclins - Google Patents

Fluorescent sensor and use thereof for detection of cyclin-dependent kinase inhibitors and/or for detection of cyclins Download PDF

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Publication number
WO2009156541A1
WO2009156541A1 PCT/ES2009/070246 ES2009070246W WO2009156541A1 WO 2009156541 A1 WO2009156541 A1 WO 2009156541A1 ES 2009070246 W ES2009070246 W ES 2009070246W WO 2009156541 A1 WO2009156541 A1 WO 2009156541A1
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cyclin
seq
dota
complex
fluorescent sensor
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PCT/ES2009/070246
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Spanish (es)
French (fr)
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Elena Pazos Chantrero
Anxo Vidal
José Luis MASCAREÑAS CID
M. Eugenio VÁZQUEZ SENTÍS
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Universidade De Santiago De Compostela
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Publication of WO2009156541A1 publication Critical patent/WO2009156541A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4704—Inhibitors; Supressors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • 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 the in vitro detection and quantification of The cyclin protein.
  • kCDKCs CDK / cyclin complexes
  • 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.
  • Cell division 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.
  • 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 CeII. Mol. Life Sci. 2002, 59, 1317 -1326).
  • CDK-Cyclin kinase complexes kCDKC
  • the binding site of p27 / Kip1 to the cyclin is defined by a specific sequence known as CBM (Cyclin Binding Motif).
  • 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 (Mclnnes, C. et al. Curr. Med Chem. Anti-Cancer A ⁇ ents 2003, 3, 57-69; Mendoza, N. et al. Cancer Res. 2003, 63, 1020-1024).
  • cyclin sensors substances capable of interacting specifically with cyclin and producing a signal that can be measured spectroscopically. These sensors would represent a new tool for the study of the molecular mechanisms involved in the control of the cell cycle dependent on cyclin, and would serve as an instrument for the identification of new inhibitors of kCDKCs with possible pharmacological applications through the development of competitive assays.
  • Fluorescence spectroscopy offers numerous advantages for the development of sensors and diagnostic systems due to their sensitivity and the ease of testing. There are several strategies for the design of sensors that take advantage of different mechanisms involved in the phenomenon of fluorescence. One of the most interesting is based on the use of fluorescent complexes of lanthanides due to their special characteristics: an exceptionally long life time (milliseconds) and extremely narrow emission bands, which allow a better resolution of their 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 makes the lanthanides Trivalents cannot be directly excited by normal methods.
  • an indirect excitation method known as sensitization or antenna effect is used (Gunnlaugsson, T. et al. Ora. Biomol. Chem. 2007, 5, 1999-2009).
  • Sensitization generates the fluorescence of the cation Ln (III) in a three-stage process: i) the light is absorbed in the immediate environment of the 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 is produced by the metal. It is important to highlight that in the sensitization process the direct union of the antenna with the metal center is not necessary, but only the proximity in space. This process can take place efficiently if both species are less than 15-20 A.
  • the present invention is based on the design of sensors that take advantage, as explained below, of different mechanisms involved in the phenomenon of fluorescence by means of the use of lanthanide chelating peptides that allow a good resolution of their fluorescent signal. These sensors are capable of showing the presence of cyclin.
  • a first aspect of the present invention refers to a fluorescent sensor comprising: a. a DOTA-lanthanide complex and b. a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the DOTA-lanthanide complex being formed by the compound 1, 4,7,10-tetraazacyclododecane-1, 4,7,10-tetraacetic acid (DOTA) and the lanthanide, in the form of a trivalent cation.
  • DOTA 4,7,10-tetraazacyclododecane-1, 4,7,10-tetraacetic acid
  • the lanthanide of the DOTA-lanthanide complex is Terbium or Europium.
  • the peptide sequences used in the invention are selected from the group comprising 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 Usina side chain and by the 2,3-diaminopropionic acid residue side chain respectively , as seen in Figure 1 (FIG. 1).
  • the chelating peptide is designed trying to maintain as far as possible the determining interactions for the formation of the peptide-Cyclin A complex, and minimizing the repulsions that the ligand with the protein could cause.
  • 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 A of the Trp, inducing the fluorescence of the peptide-Ln (III) complex as a consequence of the binding to the cyclin as shown in Figure 2 (FIG. 2).
  • the peptide containing an Ln (III) chelating agent in the absence of Cyclin A should not present fluorescence emission since the metal cannot be excited in the absence of a sensitizer.
  • the peptide binds to the protein, the Trp residue remaining close to the peptide, and to the metal center, so that the sensitization process is possible efficiently, and the emission of fluorescence as seen in Figure 3 (FIG. 3).
  • another aspect is directed to the use of said fluorescent sensor to identify inhibitors of cyclin-dependent kinases (kCDKCs).
  • kCDKCs cyclin-dependent kinases
  • cyclin A Preferably of cyclin A.
  • a strategy for the selection of these inhibitors consists in the design of peptides, with a cyclin binding sequence, analogous to that of inhibitor proteins, as for example, and without involving the exclusion of other inhibitors. , p21, p107,
  • E2F1, and p27 capable of interfering with the process of recognition of the natural substrates of the kCDKC complex that serve to identify new inhibitors of kCDKCs with possible pharmacological applications through the development of competitive assays.
  • another aspect of this invention refers to the use of the fluorescent sensor described in the preceding paragraphs for the in vitro detection and quantification of the cyclin protein.
  • the cyclin is Cyclin A.
  • the detection of these proteins is carried out by measuring the fluorescence emission levels in a given wavelength, depending on the lanthanide used, for example, in the case of Terbio, the fluorescence emission levels are measured in a wavelength between 480 and 600 nm in which the appearance of the three typical emission peaks of the Terbio complex is observed.
  • kits for the identification of cyclin dependent kinase inhibitors as well as for the detection and quantification of cyclin in a sample, preferably Cyclin A, comprising: a) a fluorescent sensor as described in the present invention and b) reagents for cell lysis.
  • Understanding reagents for cell lysis are those that are capable of damaging the cytoplasmic membrane and the cell wall allowing the release of intracellular content.
  • chemical (alkaline lysis) mechanical (by breaking frozen material) or physical (sonication) methods, but not limited to these examples alone.
  • the sample is an isolated biological sample that can come from a physiological fluid such as blood, plasma, serum or urine and / or any cellular tissue from an organism.
  • a fifth aspect of the present invention is a method of obtaining the fluorescent sensor comprising: to. Protection of the tertbutoxycarbonylmethyl groups of the DOTA compound.
  • FIG. 1 Synthesized sensor schemes.
  • FIG. 2 Schematic representation of the fluorescence emission produced by the peptide complex of Tb (III) in the presence of Cyclin A.
  • the binding to the cyclin places the metal center in the vicinity of the residue of Trp217 which then acts as an antenna.
  • FIG. 3 Representation of the bound peptide in the hydrophobic cyclin groove.
  • FIG. 4 Fluorescence spectrum of sensor 1, DOTA [Tb (III)] - GAKRRLIFE-NH 2 , in the absence (Pept-Tb) and in the presence of Cyclin A (Pept-Tb-CyA).
  • FIG. 5 Fluorescence spectrum of sensor 2, H 2 N-HA-LyS (DOTA [Tb (III) I) -RRLIF-CONH 2 , in the absence (Pept-Tb) and in the presence of Cyclin A (Pept-Tb-CyA ).
  • FIG. 6 Fluorescence spectrum of sensor 3, H 2 N-HA-DaP (DOTA [Tb (III)]) -RRLIF-CONH 2 , in the absence (Pept-Tb) and in the presence of Cyclin A (Pept-Tb-CyA ).
  • FIG. 7 Displacement test 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.
  • the method by which the fluorescent sensor was obtained comprises:
  • the tris (tertbutoxycarbonylmethyl) -1,4,7,10-tetraazacyclocyclodedecane hydrobromide (1) was started and rented with ethyl bromoacetate in the presence of anhydrous K 2 CO 3 .
  • the reaction was carried out in acetonitrile at reflux.
  • the ethyl ester of compound 2 was then selectively hydrolyzed using a mixture of dioxane: 0.1 M NaOH in a 3: 1 ratio, yielding the desired product 3 with a yield of 61%. This product was coupled to the peptides without further purification.
  • the tripotreated DOTA was coupled in solid phase using standard Fmoc procedures as shown in the following scheme
  • the 2,3-diaminopropionic acid (Dap or Dpr) or the Lys residue is introduced into the peptide with its side chains protected by an alloc group that allows its orthogonal elimination and conjugation with the DOTA as shown in the following scheme.
  • Fig 5 the fluorescence spectrum of sensor 2, H 2 N-HA-Lys (DOTA [Tb (lll)]) - RRLIF-CONH 2 , (in which the DOTA is attached) can be observed to the peptide through the amine-like side chain of Lysine, Lys), in the absence and in the presence of Cyclin A and the spectrum of a cyclin target.
  • the spectrum was recorded using a 370 nm long-pass filter to eliminate the harmonic band from the spectrum.
  • no significant band was observed between 480 and 600 nm, however, when Cyclin A was added, the appearance of the three characteristic bands of the Tb (III) complexes was observed.
  • FIG. 6 shows the spectrum of sensor 3, H 2 N-HA-Dap (DOTA [Tb (lll)]) - RRLIF-CONH 2 , (in which the DOTA chelating unit is attached to 2,3-diaminopropionic acid, Dap or Dpr, through the side chain of the amine type), in the absence and in the presence of Cyclin A and the target spectrum of Cyclin A.
  • the peptide only showed no emission band between 480 and 600 nm; However, in this case, when adding Cyclin A, the appearance of the characteristic bands of the Tb (III) complexes is not observed. Possibly this is due to the fact that the DOTA is coupled to a shorter side chain and this prevents the interaction between peptide and Cyclin A, thus preventing the transfer of energy.
  • Displacement test adding fractions of a solution of a peptide without fluorescent complex, H 2 N-HAKRRLIF-CONH 2 , on the mixture of sensor 1 and Cyclin A.
  • Cyclin A inhibitors can be detected by means of the competitive binding of the sensor 1 and the inhibitor itself to the recognition zone of the Cyclin A protein. This identification is carried out by means of the decrease of the fluorescence level of the sensor when increasing the concentration of inhibitor, as the displacement of the fluorescent sensor progressively occurs.

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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

SENSOR FLUORESCENTE Y SU USO PARA LA DETECCIÓN DE INHIBIDORES DE KINASAS DEPENDIENTES DE CICLINAS v/o PARA FLUORESCENT SENSOR AND ITS USE FOR THE DETECTION OF INHIBITORS OF CYCLINE DEPENDENT KINASES v / o FOR
LA DETECCIÓN DE CICLINAS.THE DETECTION OF CYCLINES.
La presente invención se refiere a un sensor fluorescente que comprende un quelatante de lantánido y una secuencia peptídica, así como su método de obtención y su uso para Ia identificación de inhibidores de complejos CDK/ciclinas (kCDKCs) y Ia detección y cuantificación in vitro de Ia proteína ciclina. Además, se refiere a un kit para implementar Ia identificación de inhibidores de kCDKCs y así como Ia detección y cuantificación de ciclina en una muestra.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 the in vitro detection and quantification of The 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.
ESTADO DE LA TÉCNICA ANTERIORSTATE OF THE PREVIOUS TECHNIQUE
La división celular (mitosis) es uno de los requisitos necesarios para el desarrollo de los organismos multicelulares. La capacidad de una célula de dividirse y diferenciarse permite Ia creación de estructuras multicelulares complejas. Cuando hay un fallo en el mecanismo de regulación del ciclo celular puede tener lugar una proliferación celular excesiva capaz de producir cáncer. En células eucariotas, el ciclo celular está regulado por varios complejos de kinasas formados por Ia asociación de una unidad de kinasa (CDK) con una unidad reguladora de Ia proteína ciclina (Poon, R. Y. C. CeII. Mol. Life Sci. 2002, 59, 1317-1326). Los complejos kinasas CDK-Ciclina (kCDKC) son a su vez regulados por mecanismos de fosforilación y por interacciones con distintas proteínas. En los últimos 10 años se han publicado más de 80.000 artículos relacionados con ciclinas, Io que da una idea de Ia importancia biológica y médica de estas proteínas.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 CeII. 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.
La evidencia genética demuestra una fuerte correlación entre alteraciones en Ia regulación de kCDKCs y Ia patología molecular del cáncer (Deshpande, A. et al. Oncoaene 2005, 24, 2909-2915), y es por ello que en los últimos años ha habido un gran interés en el desarrollo de inhibidores de kCDKCs. Una de las estrategias para Ia obtención de estos inhibidores consiste en el diseño de derivados peptídicos, con un modo de unión a Ia ciclina, análogo al de Ia proteína inhibidora p27/Kip1 , y que sean capaces de interferir con el proceso de reconocimiento de los sustratos naturales del complejo kCDKC. El sitio de unión de p27/Kip1 a Ia ciclina está definido por una secuencia específica conocida como CBM (Cyclin Binding Motif). Estudios biológicos con distintos péptidos conteniendo el CBM han demostrado su capacidad de interferir en Ia formación de complejos estables de proteínas con kCDKCs, y de esta forma inducir apoptosis en células tumorales in vitro e in vivo (Mclnnes, C. et al. Curr. Med. Chem. Anti-Cancer Aαents 2003, 3, 57-69; Mendoza, N. et al. Cáncer Res. 2003, 63, 1020-1024).The genetic evidence demonstrates a strong correlation between alterations in the regulation of kCDKCs and the molecular pathology of cancer (Deshpande, A. et al. Oncoaene 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 consists in the design of peptide derivatives, with a way of binding to cyclin, analogous to that of the inhibitor protein p27 / Kip1, and which are capable of interfering with the process of recognition of natural substrates of the kCDKC complex. The binding site of p27 / Kip1 to the 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 (Mclnnes, C. et al. Curr. Med Chem. Anti-Cancer Aαents 2003, 3, 57-69; Mendoza, N. et al. Cancer Res. 2003, 63, 1020-1024).
La relevancia biológica del sistema de kCDKC hace especialmente interesante Ia obtención de sensores de ciclina, sustancias capaces de interaccionar específicamente con Ia ciclina y producir una señal susceptible de ser medida espectroscópicamente. Estos sensores representarían una nueva herramienta para el estudio de los mecanismos moleculares implicados en el control del ciclo celular dependientes de Ia ciclina, y servirían como instrumento para Ia identificación de nuevos inhibidores de kCDKCs con posibles aplicaciones farmacológicas mediante el desarrollo de ensayos competitivos.The biological relevance of the kCDKC system makes it especially interesting to obtain cyclin sensors, substances capable of interacting specifically with cyclin and producing a signal that can be measured spectroscopically. These sensors would represent a new tool for the study of the molecular mechanisms involved in the control of the cell cycle dependent on cyclin, and would serve as an instrument for the identification of new inhibitors of kCDKCs with possible pharmacological applications through the development of competitive assays.
La espectroscopia de fluorescencia ofrece numerosas ventajas para el desarrollo de sensores y de sistemas de diagnóstico debido a su sensibilidad y a Ia facilidad de los ensayos. Existen diversas estrategias para el diseño de sensores que aprovechan diferentes mecanismos implicados en el fenómeno de Ia fluorescencia. Una de las más interesantes se basa en Ia utilización de complejos fluorescentes de lantánidos debido a sus especiales características: un tiempo de vida excepcionalmente largo (milisegundos) y bandas de emisión extremadamente estrechas, que permiten una mejor resolución de su señal de fluorescencia independientemente de Ia dispersión y del fondo de autofluorescencia en mezclas biológicas.Fluorescence spectroscopy offers numerous advantages for the development of sensors and diagnostic systems due to their sensitivity and the ease of testing. There are several strategies for the design of sensors that take advantage of different mechanisms involved in the phenomenon of fluorescence. One of the most interesting is based on the use of fluorescent complexes of lanthanides due to their special characteristics: an exceptionally long life time (milliseconds) and extremely narrow emission bands, which allow a better resolution of their fluorescence signal regardless of the dispersion and the background of autofluorescence in biological mixtures.
Los lantánidos trivalentes emiten fluorescencia por transiciones entre orbitales 4f, estas transiciones son prohibidas por las reglas de selección, y por Io tanto presentan un coeficiente de absorción extremadamente bajo (menor de 10 M"1 cm"1), Io que hace que los lantánidos trivalentes no se puedan excitar directamente por métodos normales. Para solucionar este problema se recurre a un método de excitación indirecta conocido como sensibilización (sensitization) o efecto antena (Gunnlaugsson, T. et al. Ora. Biomol. Chem. 2007, 5, 1999-2009). La sensibilización genera Ia fluorescencia del catión Ln(III) en un proceso de tres etapas: i) Ia luz es absorbida en el entorno inmediato del Ln(III) por un cromóforo orgánico (antena); ii) Ia energía de excitación se transfiere desde el cromóforo orgánico a uno o varios de los estados excitados del ion metálico y finalmente; iii) se produce Ia emisión luminiscente por parte del metal. Es importante resaltar que en el proceso de sensibilización no es necesaria Ia unión directa de Ia antena con el centro metálico, sino sólo Ia proximidad en el espacio. Este proceso puede tener lugar de manera eficiente si ambas especies se encuentran a menos de 15-20 A.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 makes the lanthanides Trivalents cannot be directly excited by normal methods. To solve this problem, an indirect excitation method known as sensitization or antenna effect is used (Gunnlaugsson, T. et al. Ora. Biomol. Chem. 2007, 5, 1999-2009). Sensitization generates the fluorescence of the cation Ln (III) in a three-stage process: i) the light is absorbed in the immediate environment of the 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 is produced by the metal. It is important to highlight that in the sensitization process the direct union of the antenna with the metal center is not necessary, but only the proximity in space. This process can take place efficiently if both species are less than 15-20 A.
En el diseño de sondas basadas en Ia emisión de los lantánidos se intentan modular las propiedades luminiscentes de los iones Ln(III) por procesos que dependan de Ia concentración de un analito. Esta modulación puede ser efectuada utilizando el analito como especie sensibilizadora de un complejo no fluorescente o poco fluorescente. Existen precedentes de Ia aplicación de esta estrategia en sensores de moléculas poliaromáticas basados en complejos de Tb(III) con una unidad de ciclodextrina-DTPA (dietiltetraminopentaacetato) no fluorescentes. La transferencia eficiente de energía desde las moléculas aromáticas al Ln(III) se produce cuando éstas se unen a Ia ciclodextrina. A pesar de todo, este principio ha sido aún poco explotado en el diseño de sensores y los ejemplos de su aplicación a sistemas biológicos son muy escasos (Leonard, J. P. et al. Chem. Commun. 2007. 129-131 ).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 Tb (III) complexes with a unit of non-fluorescent cyclodextrin-DTPA (diethyltetraminopentaacetate). The efficient transfer of energy from aromatic molecules to Ln (III) occurs when they bind to cyclodextrin. In spite of everything, 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).
A pesar de Ia existencia de algunos sensores, éstos no se han empleado para detectar ciclina e inhibidores de kCDKCs del modo expuesto en esta invención.Despite the existence of some sensors, they have not been used to detect cyclin and kCDKC inhibitors in the manner set forth in this invention.
Actualmente y a pesar de Ia importancia biológica y Ia relevancia en diagnóstico de patologías relacionadas con ciclina, no existen ensayos simples para identificar posibles inhibidores. Normalmente se recurre a ensayos de microcalorimetría o bioquímicos, con los consiguientes problemas de reproducibilidad, implementación en ensayos de high- throughput o de actividad enzimática (Gondeau, C. et al. J. Biol. Chem. 2005, 280, 13793-13800).Currently and despite the biological importance and relevance in diagnosis 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) .
EXPLICACIÓN DE LA INVENCIÓNEXPLANATION OF THE INVENTION
La presente invención se basa en el diseño de sensores que aprovechan, como se explica más adelante, diferentes mecanismos implicados en el fenómeno de Ia fluorescencia mediante Ia utilización de péptidos quelatantes de lantánido que permiten una buena resolución de su señal fluorescente. Estos sensores son capaces de mostrar Ia presencia de ciclina.The present invention is based on the design of sensors that take advantage, as explained below, of different mechanisms involved in the phenomenon of fluorescence by means of the use of lanthanide chelating peptides that allow a good resolution of their fluorescent signal. These sensors are capable of showing the presence of cyclin.
En este sentido, un primer aspecto de Ia presente invención se refiere a un sensor fluorescente que comprende: a. un complejo DOTA-lantánido y b. una secuencia seleccionada de entre SEQ ID NO: 1 , SEQ ID NO: 2 ó SEQ ID NO: 3.In this sense, a first aspect of the present invention refers to a fluorescent sensor comprising: a. a DOTA-lanthanide complex and b. a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
Siendo el complejo DOTA-lantánido el formado por el compuesto ácido 1 ,4,7,10-tetraazaciclododecano-1 ,4,7,10-tetraacético (DOTA) y el lantánido, en forma de catión trivalente. Dentro de Ia serie de estos metales de lantánidos (Ln(III)), Tb(III) y Eu(III) son los dos cationes que presentan las mejores propiedades de emisión. Así pues, en una realización preferida, el lantánido del complejo DOTA-lantánido es el Terbio o el Europio.The DOTA-lanthanide complex being formed by the compound 1, 4,7,10-tetraazacyclododecane-1, 4,7,10-tetraacetic acid (DOTA) and the lanthanide, in the form of a trivalent cation. Within the series of these lanthanide metals (Ln (III)), Tb (III) and Eu (III) are the two cations that have the best emission properties. Thus, in a preferred embodiment, the lanthanide of the DOTA-lanthanide complex is Terbium or Europium.
En cuanto a las secuencias peptídicas utilizadas en Ia invención, éstas se seleccionan de entre el grupo que comprende SEQ ID NO: 1 , SEQ ID NO: 2 ó SEQ ID NO: 3. Por ello, en una realización preferida Ia secuencia es SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3 y se une al complejo DOTA- lantánido por su extremo N-terminal, por su cadena lateral de Usina y por Ia cadena lateral del residuo ácido 2,3-diaminopropiónico respectivamente, como se observa en Ia figura 1 (FIG. 1 ).As for the peptide sequences used in the invention, these are selected from the group comprising 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 Usina side chain and by the 2,3-diaminopropionic acid residue side chain respectively , as seen in Figure 1 (FIG. 1).
El diseño de estos péptidos se llevó a cabo guiado por las estructuras de rayos X disponibles en Ia base de datos de Ia Ciclina A unida a péptidos de alta afinidad. La estabilización del complejo se debe fundamentalmente a los contactos que se establecen entre el surco hidrofóbico de ciclina (formado por las hélices α1 , α3 y α4) y las cadenas laterales de Arg1, Leu3 y Phe5 del péptido, mientras que las cadenas laterales de Arg2 e He4 se proyectan fuera del surco de unión.The design of these peptides was carried out guided by the X-ray structures available in the database of Cyclin A linked to high affinity peptides. The stabilization of the complex is mainly due to the contacts established between the hydrophobic cyclin groove (formed by the α1, α3 and α4 helices) and the side chains of Arg 1 , Leu 3 and Phe 5 of the peptide, while the chains Arg 2 and He 4 sides project out of the junction groove.
El péptido quelatante se diseña intentando mantener en Ia medida de Io posible las interacciones determinantes para Ia formación del complejo péptido-Ciclina A, y minimizando las repulsiones que pudiera ocasionar el ligando con Ia proteína. Como se describe más adelante, estos sensores son capaces de detectar ciclina, en concreto Ciclina A. La estrategia para su diseño consiste en aprovechar Ia presencia de un residuo de Triptófano (Trp) próximo al sitio de unión de los péptidos como unidad sensibilizadora del ion Ln(III). Dicho residuo es capaz de inducir una transferencia de excitación a un complejo de lantánido situado en torno a 10-15 A del Trp, induciendo Ia fluorescencia del complejo péptido- Ln(III) como consecuencia de Ia unión a Ia ciclina tal como se muestra en Ia figura 2 (FIG. 2).The chelating peptide is designed trying to maintain as far as possible the determining interactions for the formation of the peptide-Cyclin A complex, and minimizing the repulsions that the ligand with the protein could cause. 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 A of the Trp, inducing the fluorescence of the peptide-Ln (III) complex as a consequence of the binding to the cyclin as shown in Figure 2 (FIG. 2).
O sea, el péptido que contiene un agente quelatante de Ln(III) en ausencia de Ciclina A no debe presentar emisión de fluorescencia puesto que el metal no puede ser excitado en ausencia de un sensibilizador. Sin embargo, en presencia de Ciclina A, el péptido se une a Ia proteína, quedando el residuo de Trp próximo al péptido, y al centro metálico, de manera que sea posible el proceso de sensibilización de forma eficiente, y tenga lugar Ia emisión de fluorescencia como se observa en Ia figura 3 (FIG. 3).That is, the peptide containing an Ln (III) chelating agent 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 the presence of Cyclin A, the peptide binds to the protein, the Trp residue remaining close to the peptide, and to the metal center, so that the sensitization process is possible efficiently, and the emission of fluorescence as seen in Figure 3 (FIG. 3).
Por todo ello, y teniendo en cuenta Ia sensibilidad del sensor de Ia invención, otro aspecto va dirigido al uso de dicho sensor fluorescente para identificar inhibidores de kinasas dependientes de ciclinas (kCDKCs). Preferiblemente de ciclina A.Therefore, 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 of cyclin A.
Como se ha descrito anteriormente, una estrategia para Ia selección de estos inhibidores consiste en el diseño de péptidos, con una secuencia de unión a Ia ciclina, análogos al de las proteínas inhibidoras, como por ejemplo, y sin que suponga Ia exclusión de otros inhibidores, p21 , p107,As described above, a strategy for the selection of these inhibitors consists in the design of peptides, with a cyclin binding sequence, analogous to that of inhibitor proteins, as for example, and without involving the exclusion of other inhibitors. , p21, p107,
E2F1 , y p27, capaces de interferir con el proceso de reconocimiento de los sustratos naturales del complejo kCDKC que sirven para Ia identificación de nuevos inhibidores de kCDKCs con posibles aplicaciones farmacológicas mediante el desarrollo de ensayos competitivos. Por otro lado, otro aspecto de esta invención se refiere al uso del sensor fluorescente descrito en los párrafos precedentes para Ia detección y cuantificación in vitro de Ia proteína ciclina. Preferiblemente Ia ciclina es Ciclina A.E2F1, and p27, capable of interfering with the process of recognition of the natural substrates of the kCDKC complex that serve to identify new inhibitors of kCDKCs with possible pharmacological applications through the development of competitive assays. On the other hand, another aspect of this invention refers 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.
La detección de estas proteínas se realiza midiendo los niveles de emisión de fluorescencia en una longitud de onda determinada, dependiendo del lantánido utilizado, por ejemplo, en el caso del Terbio, los niveles de emisión de fluorescencia se miden en una longitud de onda comprendida entre 480 y 600 nm en Ia que se observa Ia aparición de los tres picos de emisión típicos del complejo de Terbio.The detection of these proteins is carried out by measuring the fluorescence emission levels in a given wavelength, depending on the lanthanide used, for example, in the case of Terbio, the fluorescence emission levels are measured in a wavelength between 480 and 600 nm in which the appearance of the three typical emission peaks of the Terbio complex is observed.
En relación a ello, otro aspecto de Ia invención se refiere a un kit para Ia identificación de inhibidores de kinasas dependientes de ciclinas (kCDKCs) así como para Ia detección y cuantificación de ciclina en una muestra, preferiblemente Ciclina A, que comprende: a) un sensor fluorescente como los descritos en Ia presente invención y b) reactivos para Ia lisis de células.In this regard, another aspect of the invention relates to a kit for the identification of cyclin dependent kinase inhibitors (kCDKCs) as well as for the detection and quantification of cyclin in a sample, preferably Cyclin A, comprising: a) a fluorescent sensor as described in the present invention and b) reagents for cell lysis.
Entendiéndose por reactivos para Ia lisis de células aquellos que son capaces de lesionar Ia membrana citoplasmática y Ia pared celular permitiendo Ia liberación del contenido intracelular. Como por ejemplo, métodos químicos (lisis alcalina), mecánicos (por rotura de material congelado) o físicos (sonicación).pero sin limitarse únicamente a estos ejemplos.Understanding reagents for cell lysis are those that are capable of damaging the cytoplasmic membrane and the cell wall allowing the release of intracellular content. As for example, chemical (alkaline lysis), mechanical (by breaking frozen material) or physical (sonication) methods, but not limited to these examples alone.
En una realización preferida Ia muestra es una muestra biológica aislada que puede proceder de un fluido fisiológico como sangre, plasma, suero u orina y/o cualquier tejido celular procedente de un organismo.In a preferred embodiment, the sample is an isolated biological sample that can come from a physiological fluid such as blood, plasma, serum or urine and / or any cellular tissue from an organism.
Un quinto aspecto de Ia presente invención es un método de obtención del sensor fluorescente que comprende: a. Protección de los grupos tertbutoxicarbonilmetil del compuesto DOTA.A fifth aspect of the present invention is a method of obtaining the fluorescent sensor comprising: to. Protection of the tertbutoxycarbonylmethyl groups of the DOTA compound.
b. Síntesis del complejo péptido-DOTA-lantánido a partir del DOTA triprotegido en a), de una secuencia seleccionada de entre SEQb. Synthesis of the peptide-DOTA-lanthanide complex from the DOTA triprotected in a), of a sequence selected from SEQ
ID NO: 1, SEQ ID NO: 2 ó SEQ ID NO: 3 y de haluro de lantánido.ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and lanthanide halide.
Como consecuencia de Ia detección de ciclina por mediación de los sensores fluorescentes propuestos en esta invención, otra posible aplicación de los mismos podría ser un método in vitro para identificar patologías que cursen con Ia alteración en Ia expresión de Ia proteína Ciclina, así como para evaluar su situación y pronosticar su evolución en muestras celulares.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.
Asimismo, otra aplicación podría ser un kit para Ia identificación de este tipo de patologíasAlso, another application could be a kit for the identification of this type of pathologies
A Io largo de Ia descripción y las reivindicaciones Ia palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en Ia materia, otros objetos, ventajas y características de Ia invención se desprenderán en parte de Ia descripción y en parte de Ia práctica de Ia invención. Las siguientes figuras y ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de Ia presente invención.Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from 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.
DESCRIPCIÓN DE LAS FIGURASDESCRIPTION OF THE FIGURES
FIG. 1. Esquemas de los sensores sintetizados.FIG. 1. Synthesized sensor schemes.
Esquemas de los péptidos y los sitios de unión de los complejos DOTA[Tb(III)]. FIG. 2. Representación esquemática de Ia emisión de fluorescencia producida por el complejo peptídico de Tb(III) en presencia de Ciclina A.Schemes of peptides and binding sites of DOTA complexes [Tb (III)]. FIG. 2. Schematic representation of the fluorescence emission produced by the peptide complex of Tb (III) in the presence of Cyclin A.
La unión a Ia ciclina sitúa el centro metálico en las proximidades del residuo de Trp217 que actúa entonces como antena.The binding to the cyclin places the metal center in the vicinity of the residue of Trp217 which then acts as an antenna.
FIG. 3. Representación del péptido unido en el surco hidrofóbico de ciclina.FIG. 3. Representation of the bound peptide in the hydrophobic cyclin groove.
Se observa cómo el complejo del lantánido y Trp217 se encuentran próximos (aprox. 14 A).It is observed how the lanthanide complex and Trp217 are close together (approx. 14 A).
FIG. 4. Espectro de fluorescencia del sensor 1, DOTA[Tb(III)]- GAKRRLIFE-NH2, en ausencia (Pept-Tb) y en presencia de Ciclina A (Pept-Tb-CyA).FIG. 4. Fluorescence spectrum of sensor 1, DOTA [Tb (III)] - GAKRRLIFE-NH 2 , in the absence (Pept-Tb) and in the presence of Cyclin A (Pept-Tb-CyA).
FIG. 5. Espectro de fluorescencia del sensor 2, H2N-HA-LyS(DOTA[Tb(III)I)-RRLIF-CONH2, en ausencia (Pept-Tb) y en presencia de Ciclina A (Pept-Tb-CyA).FIG. 5. Fluorescence spectrum of sensor 2, H 2 N-HA-LyS (DOTA [Tb (III) I) -RRLIF-CONH 2 , in the absence (Pept-Tb) and in the presence of Cyclin A (Pept-Tb-CyA ).
FIG. 6. Espectro de fluorescencia del sensor 3, H2N-HA-DaP(DOTA[Tb(III)]) -RRLIF- CONH2, en ausencia (Pept-Tb) y en presencia de Ciclina A (Pept-Tb-CyA).FIG. 6. Fluorescence spectrum of sensor 3, H 2 N-HA-DaP (DOTA [Tb (III)]) -RRLIF-CONH 2 , in the absence (Pept-Tb) and in the presence of Cyclin A (Pept-Tb-CyA ).
FIG. 7. Ensayo de desplazamiento añadiendo fracciones de una disolución de un péptido sin complejo fluorescente, H2N-HAKRRLIF-CONH2, sobre Ia mezcla del sensor 1 y Ciclina A.FIG. 7. Displacement test 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.
A medida que se añaden aliquotas del péptido 4 se produce el desplazamiento del péptido 1 del surco hidrofóbico de Ciclina A, y por tanto, disminuye Ia señal de fluorescencia debida a dicha interacción entre el complejo péptidico de Tb(III) y Ia proteína.As aliquots of peptide 4 are added, displacement of peptide 1 from the hydrophobic groove of Cyclin A occurs, and by therefore, the fluorescence signal due to said interaction between the peptide complex of Tb (III) and the protein decreases.
EJEMPLOSEXAMPLES
A continuación se ilustrará Ia invención mediante unos ensayos realizados por los inventores que desarrollan el método de obtención del sensor fluorescente, el mecanismo de acción de los sensores descritos así como Ia detección de inhibidores de Ciclina A.Next, the invention will be illustrated by 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 inhibitors of Cyclin A.
EJEMPLO 1EXAMPLE 1
El método por el que se obtuvo el sensor fluorescente comprende:The method by which the fluorescent sensor was obtained comprises:
a. Protección de los grupos tertbutoxicarbonilmetil del compuestoto. Protection of tertbutoxycarbonylmethyl groups of the compound
DOTA.DOTA
Figure imgf000011_0001
Figure imgf000011_0001
Esquema 1. Procedimiento sintético utilizando como sustancia de partida el hidrobromuro de tris(tertbutoxi carbonilmetil)-1 ,4,7,10-tetraazaciclododecano.Scheme 1. Synthetic procedure using tris (tertbutoxy carbonylmethyl) -1,4,7,10-tetraazacyclododecane hydrobromide as the starting substance.
Para sintetizar el ligando DOTA protegido (3), se partió del hidrobromuro de tris(tertbutoxicarbonilmetil)-1 ,4,7,10-tetraazaciclododecano (1 ) que se alquiló con bromoacetato de etilo en presencia de K2CO3 anhidro. La reacción se llevó a cabo en acetonitrilo a reflujo. A continuación fue necesario eliminar el exceso de K2CO3 mediante filtración y con una posterior purificación en columna cromatográfica se obtuvo el producto 2 con un rendimiento del 99%. A continuación se hidrolizó selectivamente el ester etílico del compuesto 2 utilizando una mezcla de dioxano:NaOH 0.1 M en una proporción 3:1 , dando lugar al producto deseado 3 con un rendimiento del 61%. Este producto fue acoplado a los péptidos sin posteriores purificaciones.To synthesize the protected DOTA ligand (3), the tris (tertbutoxycarbonylmethyl) -1,4,7,10-tetraazacyclocyclodedecane hydrobromide (1) was started and rented with ethyl bromoacetate in the presence of anhydrous K 2 CO 3 . The reaction was carried out in acetonitrile at reflux. Next, it was necessary to remove the excess of K 2 CO 3 by filtration and with a subsequent chromatographic column purification the product 2 was obtained in a 99% yield. The ethyl ester of compound 2 was then selectively hydrolyzed using a mixture of dioxane: 0.1 M NaOH in a 3: 1 ratio, yielding the desired product 3 with a yield of 61%. This product was coupled to the peptides without further purification.
b. Síntesis del complejo péptido-DOTA-lantánido a partir del DOTA triprotegido en a), de una secuencia seleccionada de entre SEQ ID NO: 1, SEQ ID NO: 2 ó SEQ ID NO: 3 y de haluro de lantánido.b. Synthesis of the peptide-DOTA-lanthanide complex from DOTA triprotected in a), of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and lanthanide halide.
Para Ia síntesis del sensor 1, que contiene el DOTA-lantánido en el extremo N-terminal del péptido con secuencia SEQ ID NO: 1 , se acopló en fase sólida el DOTA triprotegido utilizando procedimientos Fmoc estándar como se muestra en el esquema siguienteFor the synthesis of the sensor 1, which contains the DOTA-lanthanide at the N-terminal end of the peptide with sequence SEQ ID NO: 1, the tripotreated DOTA was coupled in solid phase using standard Fmoc procedures as shown in the following scheme
1. 20% Pipeπdina/DMF 2. Fmoc-aa-OH1. 20% Pipeπdina / DMF 2. Fmoc-aa-OH
^ HOBt/HBTU, DIEA/DMF . . H ^ 20% Piperidina / DMF , . H^ HOBt / HBTU, DIEA / DMF. . H ^ 20% Piperidine / DMF,. H
FmocHN— φ > ^ Fmoc— |GAKRRLIFE|-N-# »» H2N- (GAKRRLIFE|— N^FmocHN— φ> ^ Fmoc— | GAKRRLIFE | -N- # » » H 2 N- (GAKRRLIFE | - N ^
Figure imgf000012_0001
Figure imgf000012_0001
Esquema 2. Síntesis del complejo SEQ ID NO: 1-DOTA-Terbio.Scheme 2. Synthesis of the SEQ ID NO complex: 1-DOTA-Terbio.
Una vez sintetizado el péptido con secuencia SEQ ID NO: 1 en fase sólida utilizando Ia estrategia Fmoc/tBu se procedió a Ia desprotección del extremo amino-terminal utilizando para ello las condiciones estándar de 20% piperidina en DMF. A continuación se acopló el derivado del DOTA previamente sintetizado, utilizando como agente de acoplamiento HATU en presencia de DIEA en DMF. Una vez realizado el acoplamiento se desprotegió una pequeña cantidad de Ia resina para su análisis por HPLC-MS en el que se observó Ia existencia de un pico correspondiente al producto deseado, por Io que se procedió a su purificación en HPLC en fase reversa. Después de liofilizar el péptido, éste se redisolvió en HEPES 10 mM, NaCI 100 mM, pH = 7.5 y se ensayó Ia formación del complejo de Terbio añadiendo una disolución de TbC^ (50 mM) / HCI (1 mM). El crudo de reacción se analizó utilizando HPLC-MS y se observó Ia existencia de un único pico correspondiente al complejo peptídico de Tb(III) que fue posteriormente repurificado mediante HPLC en fase reversa.Once the peptide with sequence SEQ ID NO: 1 was synthesized in solid phase using the Fmoc / tBu strategy, the amino-terminal end was deprived using the standard conditions of 20% piperidine in DMF. The previously synthesized DOTA derivative was then coupled, using HATU as the coupling agent in the presence of DIEA in DMF. Once the coupling was carried out, an unprotected Small amount of the resin for analysis by HPLC-MS in which the existence of a peak corresponding to the desired product was observed, so that it was purified in reverse phase HPLC. After lyophilizing the peptide, it was redissolved in 10 mM HEPES, 100 mM NaCI, pH = 7.5 and the formation of the Terbio complex was tested by adding a solution of TbC ^ (50 mM) / HCI (1 mM). The reaction crude was analyzed using 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.
Para Ia síntesis de los sensores 2 y 3, se optó por Ia modificación en fase sólida de los péptidos con secuencia SEQ ID NO: 2 y SEQ ID NO: 3 respectivamente, totalmente protegidos, excepto en Ia funcionalidad seleccionada para Ia conjugación del DOTA. El ácido 2,3-diaminopropiónico (Dap ó Dpr) o el residuo de Lys se introducen en el péptido con sus cadenas laterales protegidas por un grupo alloc que permite su eliminación ortogonal y Ia conjugación con el DOTA como se muestra en el esquema siguiente.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, totally protected, was chosen, except in the functionality selected for the conjugation of DOTA. The 2,3-diaminopropionic acid (Dap or Dpr) or the Lys residue is introduced into the peptide with its side chains protected by an alloc group that allows its orthogonal elimination and conjugation with the DOTA as shown in the following scheme.
1 20% Piperidina/DMF1 20% Piperidine / DMF
2 Fmoc-aa-OH Pd(PPh3)4 / NDMBA HOBt/HBTU, DIEA/DMF 2% H2O/CH2CI2 2 Fmoc-aa-OH Pd (PPh 3 ) 4 / NDMBA HOBt / HBTU, DIEA / DMF 2% H 2 O / CH 2 CI 2
Fmoc— |-N— # X=K(AIlOC) o Dap(Alloc) X=K(NH2) o Dap(NH2)Fmoc— | -N— # X = K (AIlOC) or Dap (Alloc) X = K (NH 2 ) or Dap (NH 2 )
/ DMF / TIS / H2O/ DMF / TIS / H 2 O
Figure imgf000013_0001
Esquema 3. Síntesis de los complejos SEQ ID NO: 2-DOTA-Terbio y SEQ ID NO: 3-DOTA-Terbio. Nótese que el complejo DOTA-Terbio está acoplado en las cadenas laterales de Lys o Dap respectivamente. Una vez sintetizado el péptido en fase sólida utilizando Ia estrategia Fmoc/tBu se procedió a Ia eliminación del grupo alloc utilizando para ello condiciones catalíticas de Pd(PPh3)4, ácido Λ/-dimetilbarbitúrico y 2% H2O/CH2CI2. A continuación se acopló el ligando quelantante DOTA previamente sintetizado, utilizando HATU como agente de acoplamiento en presencia de DIEA en DMF. Una vez realizado el acoplamiento se desprotegió una pequeña cantidad de Ia resina para su análisis por HPLC-MS en el que se observó Ia existencia de un pico correspondiente al producto deseado. A continuación se desprotegió el extremo amino-terminal utilizando para ello las condiciones estándar de 20% piperidina en DMF y se procedió a su purificación en HPLC en fase reversa. Después de liofilizar el péptido, éste se redisolvió en HEPES 10 mM, NaCI 100 mM, pH = 7.5 y se ensayó Ia formación del complejo de Terbio añadiendo una disolución de TbCI3 (50 mM) / HCI (1 mM). El crudo de reacción se analizó mediante HPLC-MS y se observó Ia existencia de un único pico correspondiente al complejo peptídico de Tb(III) que fue posteriormente repurificado mediante HPLC en fase reversa.
Figure imgf000013_0001
Scheme 3. Synthesis of the SEQ ID NO: 2-DOTA-Terbio and SEQ ID NO: 3-DOTA-Terbio complexes. Note that the DOTA-Terbio complex is coupled to the side chains of Lys or Dap respectively. Once the solid phase peptide was synthesized using the Fmoc / tBu strategy, the alloc group was eliminated using catalytic conditions of Pd (PPh 3 ) 4, Λ / -dimethylbarbituric acid and 2% H 2 O / CH 2 CI 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 performed, 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 NaCI, pH = 7.5 and the formation of the Terbio complex was tested by adding a solution of TbCI 3 (50 mM) / HCI (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.
EJEMPLO 2EXAMPLE 2
Ensayo de fluorescencia del sensor 1 (DOTA[Tb(lll)]-SEQ NO 1) en ausencia y en presencia de Ciclina A.Sensor 1 fluorescence test (DOTA [Tb (lll)] - SEQ NO 1) in the absence and in the presence of Cyclin A.
Se utilizaron concentraciones de 2.5 μM tanto para los sensores como para Ia Ciclina A. En Ia Fig 4 (FIG. 4) se puede observar el espectro de fluorescencia del sensor 1, DOTA[Tb(lll)]-GAKRRLIFE-NH2, en ausencia y en presencia deConcentrations of 2.5 μM were used both for the sensors and for Cyclin A. In Fig 4 (FIG. 4) the fluorescence spectrum of sensor 1, DOTA [Tb (lll)] - GAKRRLIFE-NH 2 , can be observed absence and in the presence of
Ciclina A y el espectro de un blanco de Ciclina A. Para registrar el espectro de fluorescencia se utilizó un filtro de 370 nm de paso largo para eliminarCyclin A and the spectrum of a Cyclin A blank. To record the fluorescence spectrum a 370 nm long-pass filter was used to remove
Ia banda del armónico del espectro. En el especio del sensor solo, entre 480 y 600 nm, no se observó ninguna banda significativa, sin embargo al añadir ciclina se observó Ia aparición de las tres bandas características de los complejos de Tb(III). Este resultado indica que el péptido del sensor está interaccionando con Ciclina A, de tal modo que se mantiene el complejo de Tb(III) formado y que éste se encuentra a una distancia inferior a 20 A permitiendo así Ia transferencia de energía entre Trp de Ia Ciclina A y el complejo de Tb(III).The harmonic band of the spectrum. In the space of the sensor alone, between 480 and 600 nm, no significant band was observed, however when adding cyclin the appearance was observed of the three characteristic bands of the Tb (III) complexes. This result indicates that the sensor peptide is interacting with Cyclin A, so that the complex of Tb (III) formed is maintained and that it is at a distance of less than 20 A thus allowing the transfer of energy between Trp of Ia Cyclin A and the complex of Tb (III).
EJEMPLO 3EXAMPLE 3
Ensayo de fluorescencia del sensor 2 (DOTA[Tb(lll)]-SEQ NO 2) en ausencia y en presencia de Ciclina A.Sensor 2 fluorescence test (DOTA [Tb (lll)] - SEQ NO 2) in the absence and in the presence of Cyclin A.
En Ia Fig 5 (FIG. 5) se puede observar el espectro de fluorescencia del sensor 2, H2N-HA-Lys(DOTA[Tb(lll)])-RRLIF-CONH2, (en el que el DOTA está unido al péptido a través de Ia cadena lateral de tipo amina de Ia Lisina, Lys), en ausencia y en presencia de Ciclina A y el espectro de un blanco de ciclina. Al igual que para el sensor 1 el espectro se registró utilizando un filtro de 370 nm de paso largo para eliminar Ia banda del armónico del espectro. En el especio del péptido solo, entre 480 y 600 nm no se observó ninguna banda significativa, sin embargo al añadir Ciclina A se observó Ia aparición de las tres bandas características de los complejos de Tb(III). Este resultado indica que el péptido está interaccionando con Ciclina A, de tal modo que se mantiene el complejo de Tb(III) formado y que éste se encuentra a una distancia inferior a 20 A del Trp permitiendo así Ia transferencia de energía entre Ia antena y el complejo de Tb(III).In Fig 5 (FIG. 5) the fluorescence spectrum of sensor 2, H 2 N-HA-Lys (DOTA [Tb (lll)]) - RRLIF-CONH 2 , (in which the DOTA is attached) can be observed to the peptide through the amine-like side chain of Lysine, Lys), in the absence and in the presence of Cyclin A and the spectrum of a cyclin target. As with sensor 1, the spectrum was recorded using a 370 nm long-pass filter to eliminate the harmonic band from the spectrum. In the peptide space alone, no significant band was observed between 480 and 600 nm, however, when Cyclin A was added, the appearance of the three characteristic bands of the Tb (III) complexes was observed. This result indicates that the peptide is interacting with Cyclin A, so that the complex of Tb (III) formed is maintained and that it is located at a distance of less than 20 A from the Trp thus allowing the transfer of energy between the antenna and the complex of Tb (III).
Se comparó este espectro con el del sensor 1 y se observó que Ia intensidad de las bandas características del Tb(III) para el sensor 2 era menor que en el caso del sensor 1. Este resultado sugiere que Ia distancia entre el ion y el Trp es mayor en el caso del sensor 2 que en el del sensor 1. EJEMPLO 4This spectrum was compared with that of sensor 1 and it was observed that the intensity of the characteristic bands of Tb (III) for sensor 2 was lower than in the case of sensor 1. This result suggests that the distance between the ion and Trp it is greater in the case of sensor 2 than in sensor 1. EXAMPLE 4
Ensayo de fluorescencia del sensor 3 (DOTA[Tb(lll)]-SEQ NO 3) en ausencia y en presencia de Ciclina A.Sensor 3 fluorescence test (DOTA [Tb (lll)] - SEQ NO 3) in the absence and in the presence of Cyclin A.
En Ia Fig 6 (FIG. 6) se observa el espectro del sensor 3, H2N-HA- Dap(DOTA[Tb(lll)])-RRLIF-CONH2, (en el que Ia unidad quelatante DOTA se encuentra unida al ácido 2,3-diaminopropiónico, Dap ó Dpr, a través de Ia cadena lateral de tipo amina), en ausencia y en presencia de Ciclina A y el espectro del blanco de Ciclina A.Figure 6 (FIG. 6) shows the spectrum of sensor 3, H 2 N-HA-Dap (DOTA [Tb (lll)]) - RRLIF-CONH 2 , (in which the DOTA chelating unit is attached to 2,3-diaminopropionic acid, Dap or Dpr, through the side chain of the amine type), in the absence and in the presence of Cyclin A and the target spectrum of Cyclin A.
Al igual que en los casos anteriores, el péptido sólo no presentó ninguna banda de emisión entre 480 y 600 nm; sin embargo en este caso, al añadir Ciclina A no se observa Ia aparición de las bandas características de los complejos de Tb(III). Posiblemente esto se deba a que el DOTA se encuentra acoplado a una cadena lateral más corta y esto impide Ia interacción entre péptido y Ia Ciclina A, imposibilitando así Ia transferencia de energía.As in the previous cases, the peptide only showed no emission band between 480 and 600 nm; However, in this case, when adding Cyclin A, the appearance of the characteristic bands of the Tb (III) complexes is not observed. Possibly this is due to the fact that the DOTA is coupled to a shorter side chain and this prevents the interaction between peptide and Cyclin A, thus preventing the transfer of energy.
EJEMPLO 5EXAMPLE 5
Ensayo de desplazamiento añadiendo fracciones de una disolución de un péptido sin complejo fluorescente, H2N-HAKRRLIF-CONH2, sobre Ia mezcla del sensor 1 y Ciclina A.Displacement test adding fractions of a solution of a peptide without fluorescent complex, H 2 N-HAKRRLIF-CONH 2 , on the mixture of sensor 1 and Cyclin A.
Teniendo en cuenta todos los resultados anteriores se realizó una valoración de Ia unión del sensor 1 y Ia Ciclina A. Para ello se añadieron alícuotas de 1 μl_ de Ia solución madre de Ciclina A (37 μM) sobre Ia disolución 1 μM del sensor 1. En Ia Fig 7 (FIG. 7) se presenta el aumento en Ia emisión de Ia señal de fluorescencia a 545 nm. La emisión sigue el típico perfil de saturación y permitió calcular Ia constante de disociación (KD = 895). En las mismas condiciones se llevó a cabo un ensayo de desplazamiento añadiendo fracciones de una disolución de un péptido sin complejo fluorescente, H2N-HAKRRLIF-CONH2, (sin complejo DOTA-Tb(III)) sobre Ia mezcla del sensor 1 y Ciclina A. A medida que se añadieron alícuotas del péptido se produjo el desplazamiento del sensor 1 del surco hidrofóbico de Ciclina A, y por tanto disminuyó Ia señal de fluorescencia debida a dicha interacción entre el complejo peptídico de Tb(III) y Ia proteína.Taking into account all the previous results, an evaluation of the union of the sensor 1 and the Cyclin A was performed. To this end, aliquots of 1 μl of the stock solution of Cyclin A (37 μM) were added 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 is presented. The emission follows the typical saturation profile and allowed to calculate the dissociation constant (KD = 895). In the same conditions, a displacement test was carried out by adding fractions of a solution of a peptide without a 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 were added, the displacement of the sensor 1 of the hydrophobic groove of Cyclin A occurred, and therefore the fluorescence signal due to said interaction between the peptide complex of Tb (III) and the protein decreased.
Este ejemplo demuestra que se pueden detectar inhibidores de Ciclina A por medio de Ia unión competitiva del sensor 1 y el propio inhibidor a Ia zona de reconocimiento de Ia proteína Ciclina A. Esta identificación se lleva a cabo por medio de Ia disminución del nivel de fluorescencia del sensor al aumentar Ia concentración de inhibidor, al ir produciéndose el desplazamiento del sensor fluorescente progresivamente.This example demonstrates that Cyclin A inhibitors can be detected by means of the competitive binding of the sensor 1 and the inhibitor itself to the recognition zone of the Cyclin A protein. This identification is carried out by means of the decrease of the fluorescence level of the sensor when increasing the concentration of inhibitor, as the displacement of the fluorescent sensor progressively occurs.
Con el fin de testar Ia especificidad del sensor 1 en las condiciones biológicas más relevantes, se midió Ia emisión de dicho péptido con cantidades crecientes de Ciclina A en usados celulares (concentración de 290 μg/mL de proteína total). El sensor 1 mostró una especificidad extraordinaria, no se observó emisión de fluorescencia hasta que se añadió Ia Ciclina A a Ia mezcla. La emisión total se vio ligeramente disminuida bajo estas condiciones. In order to test the specificity of the sensor 1 in the most relevant biological conditions, the emission of said peptide was measured with increasing amounts of Cyclin A in cellular used (concentration of 290 μg / mL of total protein). Sensor 1 showed extraordinary specificity, no fluorescence emission was observed until Cyclin A was added to the mixture. Total emission was slightly diminished under these conditions.

Claims

REIVINDICACIONES
1. Sensor fluorescente que comprende, c. un complejo DOTA-lantánido y d. una secuencia seleccionada de entre SEQ ID NO: 1 , SEQ ID NO: 2 ó SEQ ID NO: 3.1. 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. Sensor fluorescente según Ia reivindicación 1 , donde Ia secuencia es SEQ ID NO: 1 y se une al complejo por su extremo N-terminal.2. Fluorescent sensor according to claim 1, wherein the sequence is SEQ ID NO: 1 and joins the complex at its N-terminal end.
3. Sensor fluorescente según Ia reivindicación 1 , donde Ia secuencia es SEQ NO: 2 y se une al complejo por su cadena lateral de Usina.3. Fluorescent sensor according to claim 1, wherein the sequence is SEQ NO: 2 and joins the complex through its Usina side chain.
4. Sensor fluorescente según Ia reivindicación 1 , donde Ia secuencia es SEQ NO: 3 y se une al complejo por Ia cadena lateral del residuo ácido 2,3- diaminopropiónico.4. Fluorescent sensor according to claim 1, wherein the sequence is SEQ NO: 3 and is attached to the complex by the side chain of the 2,3-diaminopropionic acid residue.
5. Sensor fluorescente según cualquiera de las reivindicaciones 1 a 4 donde el lantánido es el Terbio.5. Fluorescent sensor according to any of claims 1 to 4 wherein the lanthanide is Terbium.
6. Uso del sensor fluorescente según cualquiera de las reivindicaciones 1-5 para identificar inhibidores de kinasas dependientes de ciclinas (kCDKCs).6. Use of the fluorescent sensor according to any of claims 1-5 to identify cyclin-dependent kinase inhibitors (kCDKCs).
7. Uso del sensor fluorescente según cualquiera de las reivindicaciones 1-5 para Ia detección y cuantificación in vitro de Ia proteína ciclina.7. Use of the fluorescent sensor according to any of claims 1-5 for the detection and quantification in vitro of the cyclin protein.
8. Uso según cualquiera de las reivindicaciones 6 ó 7 donde Ia ciclina es Ciclina A. 8. Use according to any of claims 6 or 7 wherein the cyclin is Cyclin A.
9. Kit para Ia identificación de inhibidores de kinasas dependientes de ciclinas (kCDKCs) en una muestra, que comprende un sensor fluorescente según cualquiera de las reivindicaciones 1-5 y medios para Ia lisis de células.9. Kit for the identification of cyclin dependent kinase inhibitors (kCDKCs) in a sample, comprising a fluorescent sensor according to any of claims 1-5 and means for cell lysis.
10. Kit según Ia reivindicación 9 para Ia detección y cuantificación de ciclina en una muestra.10. Kit according to claim 9 for the detection and quantification of cyclin in a sample.
11. Kit según las reivindicaciones 9 y 10 donde Ia ciclina es Ciclina A.11. Kit according to claims 9 and 10 wherein the cyclin is Cyclin A.
12. Kit según cualquiera de las reivindicaciones 9-11 donde Ia muestra es una muestra biológica aislada.12. Kit according to any of claims 9-11 wherein the sample is an isolated biological sample.
13. Método de obtención del sensor fluorescente según cualquiera de las reivindicaciones 1-5 que comprende, a. La protección de los grupos tertbutoxicarbonilmetil del compuesto13. Method of obtaining the fluorescent sensor according to any of claims 1-5 comprising, a. Protection of tertbutoxycarbonylmethyl groups of the compound
DOTA. b. La síntesis del complejo péptido-DOTA-lantánido a partir delDOTA b. The synthesis of the peptide-DOTA-lanthanide complex from
DOTA triprotegido en a), de una secuencia seleccionada de entre SEQ ID NO: 1 , SEQ ID NO: 2 ó SEQ ID NO: 3 y de haluro de lantánido. DOTA triprotected in a), of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and lanthanide halide.
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Citations (5)

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US6589505B1 (en) * 1999-01-29 2003-07-08 St. Jude Children's Research Hospital Cells that lack p19ink4d and p27kip1 activity and methods of use thereof
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WO2005040802A2 (en) * 2003-10-20 2005-05-06 Cyclacel Limited Cylin binding peptides, and their use in drug screening assays
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WO2008000870A1 (en) * 2006-06-19 2008-01-03 Fundación De La Comunidad Valenciana, Centro De Investigación Principe Felipe 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

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US20050059100A1 (en) * 2003-04-28 2005-03-17 The Regents Of The University Of California Element-coded affinity tags
WO2005040802A2 (en) * 2003-10-20 2005-05-06 Cyclacel Limited Cylin binding peptides, and their use in drug screening assays
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WO2008000870A1 (en) * 2006-06-19 2008-01-03 Fundación De La Comunidad Valenciana, Centro De Investigación Principe Felipe 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

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