WO2009156541A1 - Capteur fluorescent et utilisation de celui-ci pour détecter des inhibiteurs de kinase dépendantes des cyclines et/ou pour détecter des cyclines - Google Patents

Capteur fluorescent et utilisation de celui-ci pour détecter des inhibiteurs de kinase dépendantes des cyclines et/ou pour détecter des cyclines 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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WIPO (PCT)
Prior art keywords
cyclin
seq
dota
complex
fluorescent sensor
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PCT/ES2009/070246
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English (en)
Spanish (es)
Inventor
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/fr

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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

La présente invention concerne un capteur fluorescent comprenant un agent chélateur de lanthanides et une séquence peptidique, ainsi qu'un procédé de production de celui-ci et son utilisation pour identifier des inhibiteurs de complexes CDK/cyclines (kCDKCs) et pour détecter et quantifier in vitro la protéine cycline. L'invention concerne également une trousse permettant de mettre en oeuvre l'identification d'inhibiteurs de kCDKCs, ainsi que la détection et la quantification de cycline dans un échantillon.
PCT/ES2009/070246 2008-06-26 2009-06-24 Capteur fluorescent et utilisation de celui-ci pour détecter des inhibiteurs de kinase dépendantes des cyclines et/ou pour détecter des cyclines WO2009156541A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP200801912 2008-06-26
ES200801912A ES2331286B2 (es) 2008-06-26 2008-06-26 Sensor fluorescente y su uso para la deteccion de inhibidores de kinasas dependientes de ciclinas y/o para la deteccion de ciclinas.

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WO2009156541A1 true WO2009156541A1 (fr) 2009-12-30

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20050059100A1 (en) * 2003-04-28 2005-03-17 The Regents Of The University Of California Element-coded affinity tags
WO2005040802A2 (fr) * 2003-10-20 2005-05-06 Cyclacel Limited Peptides
US20050260730A1 (en) * 2003-10-20 2005-11-24 Fischer Peter M CDK2/cyclin A crystals and uses thereof
WO2008000870A1 (fr) * 2006-06-19 2008-01-03 Fundación De La Comunidad Valenciana, Centro De Investigación Principe Felipe Composés organiques inhibant l'activité catalytique du complexe cycline a/kinase 2 dépendant de la cycline par sa liaison avec un nouveau site pharmacophorique dans la molécule cycline

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20050059100A1 (en) * 2003-04-28 2005-03-17 The Regents Of The University Of California Element-coded affinity tags
WO2005040802A2 (fr) * 2003-10-20 2005-05-06 Cyclacel Limited Peptides
US20050260730A1 (en) * 2003-10-20 2005-11-24 Fischer Peter M CDK2/cyclin A crystals and uses thereof
WO2008000870A1 (fr) * 2006-06-19 2008-01-03 Fundación De La Comunidad Valenciana, Centro De Investigación Principe Felipe Composés organiques inhibant l'activité catalytique du complexe cycline a/kinase 2 dépendant de la cycline par sa liaison avec un nouveau site pharmacophorique dans la molécule cycline

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PAZOS ELENA ET AL.: "Cyclin A Probes by Means of Intermolecular Sensitization of Terbium- Chelating Peptides", J. AM. CHEM. SOC., vol. 130, 1 July 2008 (2008-07-01), pages 9652 - 9653 *

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ES2331286B2 (es) 2010-10-25

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