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WO2012080548A1 - Mutant de bak, méthode associée d'identification de substances modulatrices de bak et peptide inhibiteur de l'activité bak - Google Patents

Mutant de bak, méthode associée d'identification de substances modulatrices de bak et peptide inhibiteur de l'activité bak Download PDF

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Publication number
WO2012080548A1
WO2012080548A1 PCT/ES2011/070850 ES2011070850W WO2012080548A1 WO 2012080548 A1 WO2012080548 A1 WO 2012080548A1 ES 2011070850 W ES2011070850 W ES 2011070850W WO 2012080548 A1 WO2012080548 A1 WO 2012080548A1
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Prior art keywords
bak
polypeptide
membrane
protein
activation
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Spanish (es)
Inventor
Gorka BASAÑEZ ASUA
Olatz LANDETA DÍAZ
Ane Landajuela Larma
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Euskal Herriko Unibertsitatea
Consejo Superior de Investigaciones Cientificas CSIC
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Euskal Herriko Unibertsitatea
Consejo Superior de Investigaciones Cientificas CSIC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins

Definitions

  • Mutant of BAK associated method for the identification of BAK modulating substances and peptide inhibitor of BAK activity
  • the present invention relates to BAK derived polypeptides and methods that employ them to analyze the activation or functional inhibition of membrane-associated BAK . These methods are useful for identifying compounds of potential therapeutic application that modulate the proapoptotic activity of BAK.
  • the BCL2 (B-cell lymphoma 2) protein family, of which BAK is part ⁇ BCL2 antagonist / killer 1), is the main regulatory component of intracellular apoptotic machinery ⁇ Youle and Strasser 2008, Nature Rev Mol Cell Biol 9: 47-59). These proteins act at the point of "non-return" of the apoptotic process, modulating the permeabilization of the external mitochondrial membrane (MME) that allows the exit of various lethal factors from inside the mitochondria to the cellular cytosol. Given its crucial importance for cell viability, disorders in the function of BCL2 family proteins have been associated with a wide variety of human pathologies, including cancer and various degenerative and infectious diseases. In recent years, BCL2 family proteins have become promising therapeutic targets. Proof of this is that there are currently several compounds against BCL2 in advanced stages of clinical evaluation (G. Lessene et al. 2008, Nature Rev Drug Discovery 7: 989-1000).
  • BCL2 conserved homology domains
  • BAK / BAX proapoptotic proteins ⁇ BCL2-associated X protein
  • tBID proapoptotic "BH3-only” proteins
  • BCL2 type antiapoptotic proteins BCL2, BCL-X L ⁇ BCL2-like 1), MCL1 [myeloid cell leukemia sequence 1 (BCL2-related)), which contain the BH1 -BH4 domains and act as BAK / BAX inhibitor ligands.
  • BCL2 type antiapoptotic proteins BCL2, BCL-X L ⁇ BCL2-like 1
  • MCL1 [myeloid cell leukemia sequence 1 (BCL2-related)
  • BAK plays a fundamental and exclusive role (not shared by BAX) in the cellular resistance to apoptosis induced by multiple anticancer drugs (Wang and cois 2001, J B ⁇ ol Chem 276: 34307- 34317; Gillissen et al. 2010, J. Cell Biol. 188: 851-862), as well as in the premature or excessive induction of apoptosis associated with certain neurodegenerative processes and with infection by various pathogens (Someya 2009, Proc Nati Acad Sci USA 106: 19432-19437; Pardo et al. 2006, J Cell Biol 174: 509-519; Du et cois 2010, Mol Cell Biol 30: 3444-3452).
  • BAK BAK with multiple cell ligands both within the BCL2 family (for example tBID, MCL1, and BCL-X L ) and outside it (for example, DLP1 / Drp1 (dynamin 1 -like protein) (Montessu ⁇ t et al. 2010, Cell 142: 889-891)
  • BAK BAK with multiple cell ligands both within the BCL2 family (for example tBID, MCL1, and BCL-X L ) and outside it (for example, DLP1 / Drp1 (dynamin 1 -like protein) (Montessu ⁇ t et al. 2010, Cell 142: 889-891)
  • BAK is an integral protein of the MME
  • BAX and most of the antiapoptotic members of The BCL2 family are amphotropic proteins that are located both in the MME and in the cytosol.
  • BCL2 family proteins As noted above, a fundamental biological activity of BCL2 family proteins is to establish protein-protein interactions. Structural studies conducted with water-soluble and purified forms of amphotropic members of the BCL2 family have identified hydrophobic cavities on the surface of these molecules that act as binding sites for specific ligands. Based on these and other observations, various methods have been developed to quantify interactions between specific members of the BCL2 family and their respective ligands in an aqueous environment, using defined composition systems (WO2009042237, WO2002040530). Likewise, using these methods to analyze protein-protein interactions in aqueous media, peptides and other compounds of potential therapeutic application have been identified selectively directed against specific amphotropic members of the BCL2 family (WO2010068684, WO2010065865).
  • the present invention relates to a deletion mutant of the human BAK protein (SEQ ID NO: 1) that preserves the transmembrane segment and function and can be purified highly efficiently due to its majority presence in the soluble fraction resulting from the lysate of the cells where it is produced.
  • This mutant lacks between 4 and 6 amino acids of its carboxyl terminal end, which does not affect the function of the protein and nevertheless favors its purification.
  • the authors of the present invention have developed methods to examine in a controlled manner the functional activation of this BAK mutant, located in a suitable lipid environment, in order to identify compounds capable of specifically modulating the membrane-associated BAK proapoptotic activity. . These methods and compounds are useful in the pharmaceutical and clinical research of diseases associated with functional disorders of BAK.
  • BAK protein lacks only 4 to 6 amino acids from its terminal carboxyl end implies that it is very similar to the native protein since it contains its transmembrane domain, which greatly increases the chances that substances that demonstrate a capacity for Modulate the activity of BAK in vitro, do it also in vivo, and therefore be good candidates for use in the preparation of medications.
  • a first aspect of the invention relates to a mutant derived from the human BAK protein, which contains its transmembrane domain and preserves basic functional features of the wild protein but lacks 4 to 6 amino acids from its terminal carboxy terminus.
  • a second aspect of the invention relates to a method that allows the functional activation of this BAK mutant linked to model membranes of defined lipid composition to be analyzed.
  • a third aspect of the invention relates to the use of the method of the second aspect of the invention to identify substances that modulate the functional activation of BAK.
  • Another aspect of the invention relates to a peptide with BAK inhibitory activity, as well as a composition comprising it and the use of the peptide or the composition for the manufacture of a medicament.
  • Another aspect of the invention relates to a kit comprising the truncated BAK protein of the first aspect of the invention.
  • a first aspect of the invention relates to a polypeptide where the amino acid sequence is that of the truncated human BAK protein so that four to six amino acids are removed from its terminal carboxy terminus or a bioequivalent variant.
  • the amino acid sequence of the polypeptide is SEO ID NO: 3.
  • bioequivalent variant refers to a molecule with the same function as the described molecule, which may have slight variations with respect to the described molecule without such variations providing any technical effect. added to said molecule.
  • the polypeptide of the first aspect of the invention is conjugated (covalently linked) to a label.
  • said marker is a fluorophore. More preferably, the fluorophore is 7-nitrobenz-2-oxa-1,3-diazolylethylenediamine (NBD).
  • marker refers to a molecule that facilitates the detection of the molecule to which it binds and, therefore, the label.
  • a marker can be a molecule that emits energy (radioactivity, light, fluorescence), an enzyme capable of generating a detectable product (colored, fluorescent, luminous), a protein or an epitope easily detected by immunological methods or based on recognition and binding affinity
  • markers are radioactive isotopes, luciferase protein, fluorescent proteins or fluorophores, enzymes such as peroxidases or alkaline phosphatase, epitopes such as myc or FLAG, proteins such as GST (glutathione S transferase) or biotin, etc.
  • a fluorophore is a substance that emits light with a characteristic wavelength (emission wavelength (Aem)) when excited with a light with a characteristic wavelength (excitation wavelength (Aex)) and less than the emission wavelength.
  • a second aspect of the present invention relates to a method for identifying substances that modulate the functional activation of membrane-associated BAK, which comprises the following steps: (a) contacting the polypeptide of the first aspect of the invention with a system of membranes rich in mitochondrial lipids to form a complex,
  • step (b) contacting the product resulting from step (a) with the substance to be tested,
  • the polypeptide described in step (a) is NBD-SEQ ID NO: 3 (the polypeptide whose amino acid sequence is SEQ ID NO: 3 conjugated to NBD) or a bioequivalent variant of the same.
  • the membrane system described in step (a) is a set of liposomes.
  • the term "liposomes" indicates a set of purely lipid vesicles of defined composition.
  • the mitochondrial lipid is cardiolipin.
  • the activation or functional inhibition of the NBD-SEQ ID NO: 3 polypeptide is measured by fluorescence.
  • Another aspect of the invention relates to the use of the substances identified by the method of the second aspect of the invention for the preparation of a medicament for the treatment and / or prevention of diseases related to BAK activation resistance.
  • the disease is cancer.
  • Another aspect of the invention relates to the use of the substances identified by the method of the second aspect of the invention for the preparation of a medicament for the treatment and / or prevention of diseases related to hyperactivation of BAK.
  • diseases related to hyperactivation of BAK can be all those diseases related to an abnormal apoptotic death.
  • Some non-limiting examples of these diseases associated with excessive or premature BAK-dependent apoptosis are invasive aspergillosis caused by the bacterium Aspergillus fumigatus (Brown and cois. J Cell Biol. 2006; 1 74 (4): 509-19), Gonorrhea caused by Ne ⁇ sseria gonorrheae (Kepp et al.
  • Another aspect of the invention relates to a fragment of the polypeptide of the first aspect of the invention (hereinafter called the peptide of the invention) consisting of amino acids 164 to 185 of said polypeptide, a fragment or a derivative thereof.
  • the sequence of said fragment or peptide is SEQ ID NO: 7
  • SEQ ID NO: 7 (BAK 164 "185 ) inhibits BAK (Fig. 7) and also has a low IC50 (table 1), less than the IC50 of the sequence peptides SEQ ID NO: 8 (BAK 68 "), SEQ ID NO: 9 BAK 120" 146 , SEQ ID NO: 10 (BAK 167 " 181 ), SEQ ID NO: 1 (vBAK 164" 85 ', BAK variant or variant artificial) and SEQ ID NO: 12 (BAX 146 "66 ).
  • SEQ ID NO: 10 (BAK 167 "181 ) is a fragment of SEQ ID NO: 7 and also inhibits BAK although with a higher IC50.
  • BAK variant refers to a peptide obtained by substitution or elimination of one to three amino acid residues in the corresponding peptide the native human BAK protein
  • derivative means a product obtained from the original peptide by any synthetic or natural chemical reaction.
  • fragment means molecules obtained from the original peptide by eliminating sequences of between four and ten amino acids.
  • peptides or preferably a pharmacologically acceptable composition thereof, may find application in the treatment and / or prevention of human pathologies related to premature or excessive activation of BAK.
  • composition of the invention Another aspect of the invention relates to a composition (hereinafter called the composition of the invention) comprising the peptide of the invention.
  • the composition is a composition
  • the composition comprises a vehicle or an excipient.
  • composition refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases. In the context of the present invention it refers to a composition comprising at least the peptide of the invention.
  • the pharmaceutical composition of the invention can be used both alone and in combination with other compositions for the treatment or prevention of diseases related to BAK.
  • excipient is a component of a pharmaceutical composition that is not an active compound but a diluent, a vehicle or a filler, among others, which is considered pharmaceutically acceptable when safe, is not toxic and has no adverse effects.
  • excipient refers to a substance that helps the absorption of the compound, stabilizes it or helps the preparation of the drug in the sense of giving it consistency or providing flavors that make it more pleasant.
  • the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
  • pharmaceutically acceptable refers to the excipient being allowed and evaluated so as not to cause harm to organisms to which it is administered.
  • the excipient must be pharmaceutically suitable, that is, it must allow the activity of the compounds of the pharmaceutical composition, that is, it must be compatible with said components.
  • the "vehicle” or carrier is preferably an inert substance.
  • the function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the pharmaceutical composition. Therefore, the carrier is a substance that is used in the medicament to dilute any of the components of the pharmaceutical composition of the present invention to a certain volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and form to the medicine.
  • the pharmaceutically acceptable carrier is the diluent.
  • the pharmaceutical composition further comprises another active substance.
  • another active substance is any matter, whatever its human, animal, plant, chemical or other origin to which an appropriate activity is attributed to constitute a medicine.
  • kits of the invention characterized in that it comprises the polypeptide of the first aspect of the invention.
  • the kit of the invention comprises a set of membranes rich in mitochondrial lipids.
  • the kit comprises liposomes rich in mitochondrial lipids. More preferably, the liposomes are rich in cardiolipin.
  • the kit of the invention comprises an activator and / or a BAK inhibitor.
  • Membrane assemblies such as cardiolipin-rich liposomes, can be frozen and stored for use when needed. In addition, they may suffer at least two freeze / thaw cycles without their usefulness in carrying out the method of the invention being impaired.
  • a compound or substance is called "BAK activator" when the ratio of fluorescence intensities of the polypeptide of the invention increases to a fluorescent marker in the presence of liposomes and in the absence of liposomes.
  • a BAK trigger is tBID or DLP1.
  • the compound or substance has to increase this ratio in a dose-dependent manner and saturably.
  • a compound or substance is called "BAK inhibitor" when the ratio of fluorescence intensities of the polypeptide of the invention bound to a fluorescent marker in the presence of liposomes and in the absence of liposomes decreases.
  • a BAK inhibitor is BCLXL or MCL1.
  • the compound or substance must decrease said ratio in a dose-dependent manner and saturable, even in the presence of a physiological BAK activator such as, but not limited to, tBID.
  • the experimental results are analyzed to a non-linear function using appropriate mathematical equations well known to the person skilled in the art.
  • the described methods can be used to analyze a wide variety of substances, including, but not limited to, collections of biomolecules or libraries of small organic or natural compounds of less than 500 daltons.
  • the methods of the invention can be carried out in different formats that adapt to the number of compounds or substances to be analyzed.
  • the authors of the present invention have demonstrated the efficacy of the methods of the invention carried out in 96-well microplates, which facilitate the measurement of fluorescence robotically.
  • additional reagents can be used, such as, but not limited to, MgC and bovine serum albumin (BSA)
  • BAK activating or inhibiting compounds identified in these assays can be used in pharmaceutical and clinical research of diseases associated with functional BAK disorders, as single components or in complex mixtures.
  • Another aspect of the invention relates to the use of the kit of the invention for the identification of substances that modulate the activation of BAK.
  • FIG. 1 Scheme of the BAK protein where the ochelid regions and the conserved domains BH1, BH2, BH3 and transmembrane (TM) of the protein are indicated. Likewise, the terminal carboxyl end sequences corresponding to the wild-type human BAK protein (BAK), and the BAKAC2, BAKAC4 and ⁇ 06 deletion mutants are indicated.
  • BAK wild-type human BAK protein
  • Figure 2A shows duplicate assays of SDS-PAGE and immunoblot with anti-BAK antibody to determine the distribution of different recombinant forms of BAK in the supernatant (S) and in the pellet (P) obtained after centrifuging the bacterial lysates .
  • Figure 2B shows an SDS-PAGE gel stained with Coomassie blue containing increasing amounts of BAKAC4 purified by chromatographic procedures from the soluble fraction of the bacterial lysate.
  • Figure 3A shows duplicate assays of SDS-PAGE and immunoblot with anti-BAK antibody to determine the distribution of BAKAC4 in the supernatant (S) and in the petiet (P) obtained after centrifuging the ⁇ 04 protein incubated in the presence ( +) or in the absence (-) of isolated mitochondria.
  • Figure 3B represents the effect exerted by ⁇ 04, two natural activating ligands of BAK (tBID and DLP1 / Drp1), or combinations of both types of molecules, on the permeabilization of MME.
  • the concentrations of protein and mitochondria were 0.4 ⁇ and 1 mg protein / ml, respectively. Mean values of 2 individual measurements plus standard errors are represented.
  • Figure 4A represents duplicate assays of SDS-PAGE and immunoblot with anti-BAK antibody to determine the distribution of BAKAC4 in the supernatant (S) and in the pellet (P) obtained after centrifuging ⁇ 04 incubated in the presence (+) or in the absence (-) of liposomes enriched in cardiolipin.
  • Figure 4B depicts the effect of ⁇ 04, two natural activating ligands of BAK (tBID and DLP1 / Drp1), or combinations of both types of molecules on the release of 1, 3, 6-aminonaphthalene-tri-sulphonate (ANTS) encapsulated in liposomes enriched in cardiolipin. In all cases, the total protein and lipid concentrations were 0.1 ⁇ and 0.2 mM, respectively. Mean values of 3 to 6 individual measurements plus standard errors are represented.
  • Figure 5 Structure of ⁇ 021 resolved by X-ray crystallography, indicating the two endogenous cisterns used to mark BAKAC4 with the NBD fluorophore (7-nitrobenz-2-oxa-1, 3-diazolineletylenediamine).
  • F fluorescence.
  • Figure 7A shows the effect exerted increasing doses of the peptides BAK 68 "99 (SEQ ID NO: 8), BAK 120146 (SEQ ID NO: 9) and BAK 164- 185 (SEQ ID NO: 7) on the relative fluorescence of NBD-BAKAC4 incubated with tBID in the presence (fliposome) and in the absence (resolution) of Liposomes enriched in cardiolipin.
  • concentrations of NBD-BAKAC4, tBID and lipid were 0.1 ⁇ , 0.05 ⁇ , and 0.1 mM, respectively. Mean values of 3 to 6 individual measurements plus standard errors are represented.
  • FIG. 7B depicts the effect of the three synthetic peptides on the permeabilization of the MME induced by ⁇ 04 activated by tBID.
  • concentrations of peptide, BAKAC4, tBID, and mitochondria were, respectively, 12 ⁇ , 0.4 ⁇ , 0.2 ⁇ , and 1 mg protein / ml. Mean values of at least 2 individual measurements plus standard errors are represented.
  • Plasmids for expressing the BAKAC2 deletion mutants (SEQ ID NO: 2), ⁇ 04 (SEQ ID NO: 3), and BAKAC6 (SEO ID NO: 4) were prepared by molecular biology procedures known to those skilled in the art, including but not limited to standard polymerase chain reaction (PCR) techniques, restriction enzyme digestion, alkaline phosphatase treatment to prevent unwanted binding, ligase ligation and sequence confirmation.
  • PCR polymerase chain reaction
  • BAK6hislipoil is SEQ ID NO: 5
  • BAKAC4-6hislipoil is SEQ ID NO: 6
  • the appropriate bacterial strain preferably C41 pLys
  • IPTG isopropyl-D-thiogalactopyranoside
  • EXAMPLE 2 OBTAINING THE MUTANT PROTEIN ⁇ 04.
  • a bacterial lysate is obtained from cells expressing 6HisLipo-BAKAC4 following the protocol described in the previous section. Then, the soluble fraction of the lysate is loaded onto a Ni 2+ agarose (Qiagen) column equilibrated with buffer A (50mM Hepes, pH7.5, 500mM NaCI, 5mM imidazole, 1mM TCEP, 10% glycerol). To reduce the degree of contaminants, the column is washed with buffer A containing 20 mM imidazole. To elute the 6H ⁇ sl_ ⁇ po-BAKAC4 protein, wash the column with buffer A containing 500 mM imidazole.
  • buffer A 50mM Hepes, pH7.5, 500mM NaCI, 5mM imidazole, 1mM TCEP, 10% glycerol.
  • buffer A 50mM Hepes, pH7.5, 500mM NaCI, 5mM imidazole, 1mM TCEP
  • the eluted fraction with 6HisTEV protease is treated following the manufacturer's instructions (GE Healthcare), and the sample is passed on another N 2+ column equilibrated with buffer A without imidazole.
  • the fraction that has not bound to the column containing the BAKAC4 protein is collected, and the protein is concentrated using Amicon Y10 filters.
  • the sample is passed on an analytical filtration gel column (preferably Superdex 75) equilibrated in buffer A without imidazole, using an AKTA chromatography system (GE Healthcare).
  • the elution fractions corresponding to BAKAC4 are identified by SDS-PAGE and immunoblot with anti-BAK antibody, the protein is concentrated again, and its concentration is determined by UV light absorption spectroscopy at 280 nm.
  • EXAMPLE 3 OBTAINING PROTEIN NBD-BAKAC4.
  • NBD-BAKAC4 a procedure similar to that described in Saksena and cois was used. 2009, Cell 136: 97-109 for protein mapping with NBD. First, 1 mg of ⁇ 04 is passed through a desalting chromatography column type PD-10 (GE Healthcare) balanced at 100 mM KCI, 10 mM Hepes pH 7.5. Next, the NBD iodoacetamide derivative (IANBD, GE Healthcare) is added at a concentration 5 times higher than BAKAC4, and the mixture is incubated 2 hours at room temperature.
  • PD-10 desalting chromatography column type PD-10
  • IANBD NBD iodoacetamide derivative
  • EXAMPLE 4 Analysis of the ability of BAKAC4 to bind to mitochondria and permeabilize MME.
  • BAKAC4 preserves the basic functional traits of the wild BAK protein
  • procedures known to those skilled in the art were used with isolated yeast mitochondria which completely lack BCL2 family proteins (see LJ Siskind et al. 2002, J Biol. Chem. 283: 6622-6630).
  • the ability of BAKAC4 to associate with mitochondria was checked ( Figure 3A).
  • the ability of BAKAC4 to permeabilize MME was verified in the presence of two physiological activators of the wild BAK protein (tBID and DLP1 / Drp1) ( Figure 3B).
  • EXAMPLE 5 Manufacture of liposomes, binding assays of ⁇ 04 and release of ANTS.
  • Liposome assays to develop a large-scale search for potential BAK modulating compounds are better than mitochondrial assays due to the lower stability of mitochondria (between 3 and 5 hours) with respect to liposomes (several days and even weeks) , the greater cost and duration of mitochondrial tests with respect to liposome assays, and the greater ambiguity in the interpretation of results obtained in mitochondrial assays with respect to liposome assays, than due to their complexity Compositional, structural and dynamic increase the variability of the results.
  • Iiposomes were manufactured using methods well known in the field, preferably by extrusion through 0.2 ⁇ polycarbonate filters (see Lumps and cois. 2004 J. Biol. Chem. 279: 30081-30091).
  • the following optimal lipid composition was established to perform the tests with Iiposomes described in this invention: egg yolk phosphatidylcholine (20% mol), egg yolk phosphatidylethanolamine (20% mol), rat brain phosphatidylinositol (10% mol) and rat heart cardiolipin (50% mol).
  • egg yolk phosphatidylcholine (20% mol
  • egg yolk phosphatidylethanolamine 20% mol
  • rat brain phosphatidylinositol 10% mol
  • rat heart cardiolipin 50% mol.
  • cardiolipin is a specific lipid of the mitochondria whose levels in MME increase during apoptosis (Kagan et al. 2005, Nat Chem Biol. 1: 22
  • BAKAC4 binds to Iiposomes enriched in cardiolipin but not to liposomes without cardiolipin (Figure 4A).
  • ANTS release method (1, 3, 6-aminonaphthalene-tri-sulphonate) encapsulated in liposomes (see Lumps and cois. 2004 J. Biol. Chem. 279: 30081-30091) to evaluate modulation Functional of BAKAC4 by different compounds.
  • this method is not appropriate to examine the functional modulation of BAKAC4 by molecules of interest, such as DLP1 / Drp1 or the synthetic peptide BAK 64 "185 (table 1) which produce release of ANTS alone ( Figure 4B).
  • EXAMPLE 6 TESTS TO IDENTIFY BAK MODULATING COMPOUNDS AT MEMBRANE LEVEL.
  • FIG. 5 shows, BAK contains two tank residues exposed to the solvent, which can be used for the mapping of ⁇ 04 with the NBD fluorophore (see Example 3). This fluorophore has been used to analyze conformational changes in other membrane proteins (Johnson 2005, Traffic 6: 1078-1092).
  • Figure 6A shows the fluorescence spectra of the NBD-BAKAC4 protein after being incubated with the reaction buffer, with tBID, or with tBID together with cardiolipin-enriched liposomes. It is observed that when NBD-BAKAC4 is incubated with tBID in the absence of liposomes, no significant changes in the fluorescence spectrum of NBD occur.
  • NBD-BAKAC4 without liposomes
  • EXAMPLE 7 IDENTIFICATION OF INHIBITING PEPTIDES OF NBD- ⁇ 04 ASSOCIATED WITH LIPOSOMES. We analyze the effect on the functional activation of NBD-BAKAC4 associated with liposomes of three synthetic peptides.
  • NBD-BAKAC4 protein and cardiolipin-enriched liposomes were prepared as described above.
  • the tBID protein was prepared as described in Terrones and cois. 2004, J Biol Chem. 279: 30081-30091. Peptides were obtained from AMS Biotechnology (Oxford, United Kingdom).
  • Two types of reaction mixtures were prepared: on the one hand, NBD-BAKAC4 (0.1 ⁇ ) without liposomes; on the other hand, NBD-BAKAC4 (0.1 ⁇ ) with liposomes (0.1 mM).
  • One of the following peptide concentrations was added to each reaction mixture: 0 ⁇ , 0.25 ⁇ , 0.5 ⁇ , 1 ⁇ , 2 ⁇ , 4 ⁇ , 8 ⁇ , or 12 ⁇ .
  • reaction mixtures were incubated for 5 minutes at 37 S C.
  • a suboptimal concentration of tBID 0.5 ⁇ was added to all reaction mixtures.
  • the reaction mixtures were incubated again for 30 minutes at 37 9 C.
  • the reaction buffer was 100 mM KCI, 10 mM Hepes pH 7.5, 1 mM TCEP.
  • the temperature of the tests was 37 S C.
  • the measurements were performed in triplicate.
  • Table 1 shows the IC50 values obtained for BAK 164 " peptides.

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Abstract

La présente invention se rapporte à une forme tronquée de la protéine BAK humaine qui conserve le segment transmembrane et la fonction, à une méthode d'identification de substances modulatrices de l'activité de BAK faisant appel à ladite forme tronquée, à un peptide présentant une activité inhibitrice de l'activité BAK et à une trousse qui comprend ladite forme tronquée.
PCT/ES2011/070850 2010-12-17 2011-12-12 Mutant de bak, méthode associée d'identification de substances modulatrices de bak et peptide inhibiteur de l'activité bak Ceased WO2012080548A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996035951A1 (fr) * 1995-05-12 1996-11-14 Apoptosis Technology, Inc. Nouveaux peptides et nouvelles compositions modulant l'apoptose

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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