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WO2008037835A1 - Modèle animal non humain destiné à l'identification de composés pharmaceutiques régulateurs de la voie hedgehog et ses applications - Google Patents

Modèle animal non humain destiné à l'identification de composés pharmaceutiques régulateurs de la voie hedgehog et ses applications Download PDF

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WO2008037835A1
WO2008037835A1 PCT/ES2007/070164 ES2007070164W WO2008037835A1 WO 2008037835 A1 WO2008037835 A1 WO 2008037835A1 ES 2007070164 W ES2007070164 W ES 2007070164W WO 2008037835 A1 WO2008037835 A1 WO 2008037835A1
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animal model
protein
model according
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ptc
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Isabel Guerrero Vega
Ainhoa Itziar Callejo De Prado
Joaquín CULI ESPIGUL
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Consejo Superior De Investigaciones Científicas
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/60—New or modified breeds of invertebrates
    • A01K67/61—Genetically modified invertebrates, e.g. transgenic or polyploid
    • A01K67/65—Genetically modified arthropods
    • A01K67/68—Genetically modified insects
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/60—New or modified breeds of invertebrates
    • A01K67/61—Genetically modified invertebrates, e.g. transgenic or polyploid
    • A01K67/65—Genetically modified arthropods
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests

Definitions

  • Hedgehog (Hh) protein signaling pathway Activation of the Hedgehog (Hh) protein signaling pathway is key in the induction of morphogenetic patterns and in cell proliferation processes and modulates and promotes oncogenesis and other diseases in humans (Torroja et al., 2005, J Neurobiol , 64, 334-356).
  • signaling or signal transmission using the Hedgehog protein (Hh) is related to lipids in different ways.
  • the Hh protein is synthesized from a precursor that undergoes a self-protective process, being doubly modified both by the addition of a cholesterol group at its C-terminal end and by palmitoylation at its N-terminal end ( Figure 1, see Mann and Culi , 2005).
  • Hh modified by lipids takes place through the mediation of a transmembrane protein called Dispatched (Disp) (Burke et al., 1999, CeIl 99 (7), 803-815).
  • This protein has a sterol-sensitive domain (SSD) as does the Hh receptor, the Patched protein (Ptc), and other proteins involved in lipid and cholesterol metabolism and in vesicular traffic.
  • SSD sterol-sensitive domain
  • Ptc Patched protein
  • lipid modifications of Hh are essential for the interaction between Hh and heparan sulfate proteoglycans (HSPGs) to control diffusion and signaling activity of morphogens (Callejo et al., 2006, Development 133, 471-483).
  • lipoprotein particles have been proposed as transporters of lipid modified ligands from the cell surface, thus acting as transport vehicles for a wide range of elements (Eaton, 2006, Curr. Opin. Genet. Dev. 16, 17 -22). From this point of view, lipids may be necessary to load the Hh protein to these particles (Panakova et al., 2005, Nature 435, 30-33; Despande and Schell, 2005, Developmental CeIl 9, 629-638). The Hh protein modified by lipids and loaded in lipoprotein particles interacts with the Shifted protein, a new component of the extracellular matrix that collaborates with HSPGs for stabilization and diffusion through the extracellular matrix.
  • An object of the present invention is a non-human animal model useful for the identification of pharmaceutical compounds regulating the hedgehog pathway, hereinafter animal model of the invention, characterized in that it allows the identification of compounds that regulate the formation, diffusion and reception of particles. lipoproteins with Hh.
  • a particular object of the present invention is the animal model of the invention that presents an inability to synthesize sterols caused by an absence of the enzymes involved, a need that is artificially replaced in the diet of the animal, and in which the Hh and Smo road signs.
  • This inability to synthesize sterols is caused by the evolutionary absence of the biosynthetic enzymes of these compounds.
  • Another particular object of the present invention is the animal model of the invention that has a genetic modification in at least one gene of the pathway of Hedgehog signaling that participates in the formation, diffusion and reception of lipoprotein particles with Hh, belonging by way of illustration and without limiting the invention, to the following group: Ptc and Smo that block or stimulate the Hh protein signaling pathway.
  • Another object of the invention is the use of the animal model of the invention, hereinafter use of the invention, for the identification of pharmaceutical compounds regulating the formation, diffusion and reception of lipoprotein particles with Hh.
  • Another particular object of the invention is the use of the invention comprising the following steps: i) addition to the diet of the animal of the candidate compound to determine its action on the signaling of Hh, ii) an optional step of induction of the gene of interest related to the formation, diffusion and reception of lipoprotein particles with Hh, when this is not constitutive, iü) determination of a modification of the phenotype or biological parameter characteristic of a normal or altered functioning of lipoprotein particles with Hh, induced in ii), and iv) identification of a regulatory compound, either inducer or blocker, if phenotype modification occurs in iii).
  • Another object of the invention is the use of pharmaceutical compounds regulating the formation, diffusion and reception of lipoprotein particles with Hh for the preparation of a medicament for the treatment of human diseases, preferably cancer or any other where it was involved. This regulation. Detailed description
  • the present invention is based on the demonstration of the importance of the adequate interaction of the Hh protein, with lipids and with lipoproteins and the consequent formation of lipoprotein particles for the correct release and diffusion to the extracellular matrix of Hh and its subsequent internalization and / or regulation of the Ptc and Smo proteins in the recipient cells, carried out in different Hh signaling models (see Examples).
  • Hh protein in the extracellular environment is stabilized after the overproduction of lipids by overexpression of the transcription factor SREBP or the enzyme HMGCoA reductase.
  • the decrease in the levels of the products of these genes rescues the phenotype caused by the increase in Hh production ( Figure 3, 4, 6 and 9, see Example 1).
  • Lipoforins Drosophila lipoproteins
  • Hh in the wing's imaginal disk
  • a tissue that does not express Lp but obtains it from hemolymph Example 1.4
  • Lipoforin is the main lipid transporting lipoprotein in insects. Lipoforin it carries neutral lipids in its hydrophobic core such as sterols, fatty acids and sugars (Arrese et al., 2001, Insect Biochemistry and Molecular Biology 31, 7-17).
  • Lp was inhibited by interfering RNA, thus reducing the contribution of hemolymph; and by overexpression of lipoforin receptor 2 (LpR2), the main Lp receptor expressed in disk cells, in order to increase its endocytosis and thus reduce the amount of free Lp in the extracellular matrix of the discs.
  • LpR2 lipoforin receptor 2
  • the secreted Hh protein is not stable in the extracellular matrix, suggesting that the Hh protein needs to be packaged with Lp, forming lipoprotein particles, in the producing cells for proper diffusion and stabilization of the Hh protein in the extracellular matrix.
  • Patched protein (Ptc) and lipoprotein lipoprotein are capable of co-immunoprecipitating, detecting the form of 75 Kda lipoforin (Figure 11, see Example 3).
  • the Hh receptor the Patched protein (Ptc)
  • Ptc is a new lipoprotein receptor, being able to actively internalize lipoproteins (lipoforin in insects) to the endocytic compartment, which would be capable of transporting lipids into the cell where they would regulate the activity of the Smo protein ( Figure 8 and 11), in a path independent of Hh.
  • the Ptc protein acts not only as a Hh receptor but also as a lipid transporter inside the cell, which could modulate Smoothened (Smo) activity and thus become potential Pharmaceutical compounds useful for the treatment of human diseases, such as cancer (Pasca di Magliano, M. & Hebrok, M. (2003) Nat Rev Cancer 3, 903-11) or any other in which this Smo protein is involved .
  • a useful non-human animal model can be developed ( fly) for the identification of pharmaceutical compounds regulating the Hedgehog and Smo pathway, more specifically, to trace compounds that participate in the formation, diffusion and reception of lipoprotein particles with Hh and that could modulate said pathways, for example sterols, and fatty acids. It can be carried out from several approaches depending on the point of regulation of the Hh signaling analyzed, both genetically and by administration of drugs or drug candidate compounds. Another advantage of this model is that the Hedgehog pathway is essential for the morphogenesis of all fly structures, so it represents a very versatile model.
  • this non-human animal model will be based on the inhibition of the gene or genes involved in the lipid synthesis, for example, the SREBP or HMGCoA reductase protein gene, among others. That is, by mutation of the gene that prevents the expression of the proteins necessary for the synthesis of lipids in mutated animals (Drosophila).
  • the wild fly is not able to synthesize sterols, these types of compounds have to be obtained from the diet. Since cholesterol is key to its survival, the fly must have an efficient system for incorporating lipid compounds.
  • the lipid compounds to be tested dissolve in the cans containing the porridge in which Drosophila is normally grown.
  • Drosophila eggs are deposited by the mother in the porridge and once their embryonic development is over, they hatch from the egg and begin to move, eat and develop in this medium throughout the larval period.
  • the mutant Drosophila model for the gene SREBP for example, the Drosophila HLH1061ine52 model described in the present invention (Example 1), is unable to synthesize fatty acids so that not only cholesterol but fatty acids have to be supplied in the diet for the survival of these SREBP mutant flies, being able to define the composition of different lipid diets that jointly analyzing their involvement in the activity of the Hh pathway allows it to identify which specific lipid regulates, inhibiting or inducing, said pathway, which became an active principle of a pharmaceutical composition useful for the treatment of human diseases, such as cancer.
  • new fly models can be developed in which genetic manipulation (for example, using the UAS-Gal4 yeast system, crossing flies with different mutations) allows to obtain flies in which genes of the Hedgehog pathway have been altered which, as just demonstrated in the invention, are regulated by compounds in which they participate in the formation, diffusion and reception of lipoprotein particles with Hh , and that with specific tissue promoters they would only be expressed in the precursor cells of some structure of the body, without affecting the rest, making possible its viability and facilitating the use of it.
  • genetic manipulation for example, using the UAS-Gal4 yeast system, crossing flies with different mutations
  • genes of the Hedgehog pathway have been altered which, as just demonstrated in the invention, are regulated by compounds in which they participate in the formation, diffusion and reception of lipoprotein particles with Hh , and that with specific tissue promoters they would only be expressed in the precursor cells of some structure of the body, without affecting the rest, making possible its viability and facilitating the use of it.
  • the expression has been specifically manipulated on the fly wing disc, although for a person skilled in the art, that is, in the biology of the development and using the Drosophila fly as a working model, developing models in different tissues based on this invention does not represent a complication.
  • flies can be obtained that overexpress elements of the Hedgehog pathway in other tissues, for example, the eyes, and as a consequence of this anomalous expression obtain a phenotype in the eyes and not in another part of the body of the fly.
  • Patched Hedgehog pathway receptor
  • an object of the present invention constitutes a non-human animal model useful for the identification of pharmaceutical compounds regulating the hedgehog pathway, hereinafter animal model of the invention, characterized in that it allows the identification of compounds that regulate the formation, diffusion and reception of lipoprotein particles with Hh.
  • a particular object of the present invention is the animal model of the invention that has an inability to synthesize lipids caused by a genetic cancellation of the enzymes involved, a need that is artificially replaced in the diet of the animal, and in which it can be determined Hh and Smo signaling.
  • Another particular object of the present invention is the animal model of the invention that has a genetic modification in at least one gene of the Hedgehog signaling pathway that participates in the formation, diffusion and reception of lipoprotein particles with Hh, belonging by way of illustration and without limiting the invention, to the following group: Ptc and Smo that block or stimulate the Hh protein signaling pathway.
  • Another particular object of the present invention is the animal model of the invention in which the pharmaceutical compounds regulating the hedgehog pathway are useful for the preparation of a medicament for the treatment of human diseases, preferably cancer.
  • a particular embodiment of the present invention is the animal model of the invention that has an inability to synthesize lipids caused by the genetic cancellation, at least, of a protein belonging, by way of illustration and without limiting the scope of the invention, to the Next group: transcription factor SREBP and HMGCoA reductase.
  • Another particular embodiment of the present invention is the animal model of the invention that has a genetic modification, in at least one gene of the Hedgehog signaling pathway involved in the formation, diffusion and reception of lipoprotein particles with Hh, which induces the overexpression of at least one of them, belonging to illustrative title and without limiting the invention, to the following group: Ptc and Smo that blocks or stimulates the signaling pathway of the Hh protein.
  • Another particular embodiment of the present invention is the animal model of the invention where the animal belongs to the following group: Drosophila megalobaster fly, mouse, chicken and zebrafish.
  • Another particular embodiment of the present invention is the animal model of the invention where the genetic modification is tissue specific by the use of tissue or cell specific expression promoters.
  • Another particular embodiment of the present invention is the animal model of the invention where the compound object of identification belongs, by way of illustration and without limiting the scope of the invention, to the following group: lipids, sterols, fatty acids and sugars.
  • lipids for example, oxysteroles, Vitamin D3, cholesterol, ergosterol, etc. could be analyzed. and derivatives thereof
  • a more particular embodiment of the present invention is the animal model of the invention where the model is Drosophila megalobaster with a genetic modification consisting of the overexpression of Ptc, specific to the disc of the animal's wing or eye, and blocking the signaling of the Hh protein.
  • Another more particular embodiment of the present invention is the animal model of the invention. where the model is Drosophila megalobaster with a genetic modification consisting of the overexpression of Smo, specific to the disc of the wing or eye of the animal, and inducing the signaling of the Hh protein.
  • Another more particular embodiment of the present invention is the animal model of the invention where the model is Drosophila megalobaster with a genetic modification consisting of overexpression of a mutated form of Ptc, for example, PtcSSD, specific to the disc of the wing or eye of the animal, and inducer of Hh protein signaling.
  • Another more particular embodiment of the present invention is the animal model of the invention where the model is Drosophila megalobaster with a genetic modification consisting of the genetic cancellation or mutation of the SREBP transcription factor, and will be particularly the Drosophila HLH106 mutant ( HLH1061ine52).
  • Another object of the invention is the use of the animal model of the invention, hereinafter use of the invention, for the identification of pharmaceutical compounds regulating the formation, diffusion and reception of lipoprotein particles with Hh.
  • Another particular object of the invention is the use of the invention comprising the following steps: i) addition to the diet of the animal of the candidate compound to determine its action on the signaling of Hh, ii) an optional step of induction of the gene of interest related to the formation, diffusion and reception of lipoprotein particles with Hh, when this is not constitutive, iii) determination of a modification of the characteristic phenotype or biological parameter of a normal or altered operation of lipoprotein particles with Hh, induced in ii), and iv) identification of a regulatory compound, either inducer or blocker, if phenotype modification occurs in iii).
  • Another particular embodiment of the invention is the use of the invention in which the phenotype or biological parameter determined in iii) belongs to the following group: determination of the development of the imaginal disc of the wing and size of the eye of a fly.
  • Another object of the invention is the use of pharmaceutical compounds regulating the formation, diffusion and reception of lipoprotein particles with Hh for the preparation of a medicament for the treatment of human diseases, preferably cancer or any other where it was involved. This regulation.
  • FIGURES Figure 1 Scheme of production, transport and interactions of the Hh protein.
  • FIG. 1 A scheme of the proteins that share the transmembrane motif Sterol Sensing Domain is described.
  • Hh Hh, and Dispatched, a protein that secretes Hh, participate in Hh signaling.
  • Figure 4 Rescue of Hh signaling by overexpression of HMGCoA reductase (shf 2 ; UAS-HMG CoA / Hh- Gal4) in a shifted Drosophila wing mutant model in the posterior wing compartment (shf 2 ).
  • FIG. 5 The LDL receptor, ⁇ -megalin is not a co-receptor for the Hh protein in Drosophila. Study of the loss of function in Drosophila clones dor mutants, with fluorescent protein (FRT18dor8 / FRT18armLacZ; apGal4 / UAS-HhGFP); without cholesterol (FRT18dor8 / FRT18armLacZ; apGal4 / UAS-HhNGFP) and without palmitic acid
  • Figure 7 Glycerol gradient (30-15%) for the detection of ⁇ -Hh and ⁇ -lipophorin proteins. Once precipitated, the fractions were tested by Western blots to detect the presence of ⁇ -Hh and ⁇ -Lipoforin proteins using specific antibodies.
  • Figure 8.- The Hh protein is internalized in the absence of the megalin / LPR2 receptor.
  • FIG. 10 The Patched protein (Ptc) is capable of internalizing lipoprotein lipoprotein (Lp) as effectively as the lipoforin receptor performs.
  • D) Imaginary disk ApGal4 / UAS- Rab7-GFP; TubGal80ts / UAS-PtcWT showing the co-localization of Lp (ApoLI-II) (red) and Ptc (blue) proteins using specific antibodies against these proteins with Rab7-GFP (green) (early endocytic compartment marker, indicated by tips arrow in the figure).
  • E) Imaginal disc of genotype ApGal4 / UAS-PtcS2-GFP; TubGal ⁇ Ots (after 24 hours at restrictive temperature) stained by immunofluorescence with antibodies against Hh (blue) and anti-ApoLI-II (red).
  • PtcS2-GFP green also accumulates Lp (ApoLI-II) and Hh, (PtcS2 has a point mutation in the SSD).
  • F Imaginary disk of ApGal4 / UAS-Ptcl4-GFP genotype; TubGal ⁇ Ots (24 hours at restrictive temperature) that overexpresses the mutant protein Ptcl4-GFP, which is defective in internalization, it can be seen that it cannot internalize neither Lp (ApoLI-II) (red) nor Hh (blue).
  • G Immunoprecipitation (IP) and Western blot assays.
  • Mw molecular weight marker
  • lane IPl immunoprecipitation of a larval protein extract of the UAS-PtcWT-GFP genotype
  • ABlGal4 using an ⁇ -GFP antibody (mouse) and developed with an ⁇ -ApoLI-II antibody (rabbit).
  • a band of Iras of 20OkDa is observed that corresponds to the ApoLI subunit of greater molecular weight
  • lane input immunoprecipitation of a fraction of the same lysate.
  • Two bands are observed, one of 20OkDa (Apo-LI) and another of 75kDa (Apo-LII), which correspond to each of the two lipoforin monomers; lane IP2: immunoprecipitation of a larval protein extract UAS-PtcWT-GFP; ABlGal4 using ⁇ -GFP (mouse) and Western Blot using ⁇ -GFP (rabbit).
  • a band close to 20OkDa is observed that corresponds to Ptc-GFP;
  • FIG. 13 Ectopic LpR2 increases the internallization of Lp in the imaginal discs of the wings and reduces extracellular Hh levels.
  • the upper case shows the accumulation of ApoLI-II in dotted structures in the dorsal compartment.
  • Transversal sections show the location of ApoLI-II in apical, lateral and basal regions of cells and endocytic vesicles.
  • E TubGal80ts / hh-Gal4 fly wing discs, with Hh-GFP (24 hours of restrictive temperature) immunostained with an anti-Ptc antibody (red and gray). Note that a gradient of Hh-GFP (green and gray) exists in compartment A (white bar).
  • F TubGal ⁇ Ots fly wing discs; hh-Gal4, Hh-GFP / UAS-LpR2-HA stained with an anti-Ptc (red) and anti-HA antibody to detect the expression of LpR2 (blue). Note that the levels of Hh in the P compartment, the gradient of Hh (white bar) and the expression of Ptc are reduced compared to that observed in E.
  • G Staining with anti-Ptc antibody (red) of wing discs of flies TubGal ⁇ Ots / UAS-HhC85S-GFP, hh-Gal4, which overexpress a form of Hh (green and gray) without palmitic acid.
  • H Staining with an anti-HA (LpR2, blue) and anti-Ptc (red) antibody from TubGal ⁇ Ots fly wing discs; UAS-HhC85S-GFP, hh-Gal4 / UAS-LpR2-HA. Note that the overexpression of LpR2 did not alter the levels of HhC85S-GFP (green and gray) in the compartment did not change its gradient in compartment A.
  • Example 1 Regulation of the rescue of Hh signaling by lipids and their transport in the form of lipoprotein particles.
  • 1.1. Rescue of Hh signaling in mutant Drosophila flies for shifted protein that overexpress the HMGCoA reductase enzyme in the wing's imaginal disc ( Figure 3).
  • the Drosophila wing imaginal disc is a relatively simple and well-known model for the study of the relationships and activities of the Hh protein. Hh protein levels on the cell surface are reduced in shifted mutants (Gorfinkiel et al., 2006, Developmental CeIl ⁇ , 241-253). Overexpression of HMGCoA reductase in a shifted mutant model in the compartment Rear wing causes rescue of Hh signaling ( Figure 4).
  • Hh protein in the imaginal disk of the mutant that overexpresses the enzyme HMGCoA reductase (shf EY ; apGal4 / UAS-hmgcr) are similar to those observed in the control group (compare immunofluorescence staining using anti-Hh antibodies on a disk wild) ( Figure 3). In addition, it is observed that Hh signaling is recovered in these discs (see immunofluorescence staining using anti-Ptc antibodies).
  • the rescue of the Hh function in the mutants to gain it from the Hh function (Hh Mrt ) that shows an ectopic expression of Hh in the dorsoventral region of the wing imaginal disc is analyzed.
  • the "one dose" reduction of the expression of the SREBP gene (HLH1061ine52) in these Hh mutants (Hh Mrt ) entails obtaining a phenotype practically identical to that observed in the control fly (wild-type). This result indicates that the total lipid content of the fly controlled by the SREBP gene is important for Hh signaling, since by decreasing the SREBP function, the Hh function gain phenotypes are rescued.
  • the mutants in SREBP are lethal larvae in homozygosis, however, this lethality can be rescued by administering oleic acid in the porridge (Kunte AS, Matthews KA, Rawson RB. Fatty acid auxotrophy in Drosophila larvae lacking SREBP. CeIl Metab. 2006 Jun; 3 (6): 439-48).
  • These mutant flies survive without any synthesis of lipids in your body but acquiring cholesterol and oleic acid from the diet as the only lipid source. Therefore, they are a tool important to control that lipids supplied in your diet would be able to alter the path of Hh once the lethality with low doses of cholesterol and oleic acid has been rescued.
  • RNAi against ApoLI and ApoLII were expressed transiently using a heat-sensitive promoter (heat shock).
  • heat shock Two pulses of RNAi expression against Lp before disc dissection caused a decrease in Lp levels in the imaginal disc ( Figure 12E) and also reduced Hh levels in compartment P of the imaginal disc of the wings ( Figure 12G, compared to Figure 12F and Figure 12J), also affecting the range of all Hh responses ( Figure 121 compared to Figure 12H and 12K).
  • LpR2 Lp2 receptor
  • LpR2 Lp2 receptor
  • a pulse at restrictive temperature inactivates the Gal80 repressor protein, allowing Gal4 protein to activate UAS-LpR2.
  • LpR2 was transiently expressed for 24 hours in the dorsal compartment of the wing discs, the expression of activated Caspase 3, an apoptosis marker (data not shown), was not observed.
  • LpR2 was overexpressed exclusively in the recipient cells using the ptc-Gal4 vehicle. In this way it was observed that the gradient of Hh was slightly expanded compared to normal disks.
  • Example 2 The LDL receptor, ⁇ -megalin is not a co-receptor for the Hh protein in Drosophila.
  • Example 3. The Patched protein (Ptc) is capable of internalizing lipoprotein lipoprotein (Lp) as efficiently as the lipoforin receptor (LpR) performs ( Figure 11).
  • PtcS2 contains a mutation in its SSD that, by analogy with other proteins that contain this same SSD, causes the protein to be insensitive to modulation by sterols (Kuwabara & Labouesse (2002) Trends Genet. 18, 193-201). This mutant cannot repress Smo's function but is capable of internalizing Hh.
  • both forms of Ptc both the wild, which blocks the Hh pathway, and the mutated PtcSSD, which constitutively activates the Hh pathway, internalize Lp, indicating that this is a Ptc property independent of its function in transduction. of the signal Hh.
  • the second mutant used in Ptc was Ptcl4, which regulates Smo in response to Hh but is defective in the endocytosis of Hh (Torroja, C, Gorfinkiel, N. & Guerrero, I. (2004) Development 131, 2395-408).
  • UAS-LpRNAi obtained from IMP Vienna Drosophila RNAi Center (VDRC)
  • Fat-body-Gal4 Fat-body-Gal4 (Gronke, S., Beller, M., Fellert, S.,
  • the complete LpR2 cDNA (EST line GH26833, obtained from BDGP) was fused with the C-terminal HA tag and cloned into the pUAST vector.
  • Ap-Gal4 (or Ptc-Gal4) was used for the transient expression of UAS constructs; Tub Gal ⁇ Ots were obtained by keeping the crosses at 18 0 C and inactivating the Gal ⁇ Ots repressor for 16 to 24 hours at a restrictive temperature (29 0 C).
  • the clones were generated by FLP-mediated mitotic recombination. Larvae from each corresponding genotype were incubated at 37 0 C for one hour, 24- 48 hours after oviposition by the females (AEL). The genotypes used were: FLP; FRT 42D, ptcl ⁇ / FRT 42D, arm-lacZ and FLP; FRT 82, dispS037707 / FRT 82, ubi-GFP.

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Abstract

La présente invention concerne un modèle animal non humain destiné à l'identification de composés pharmaceutiques qui régulent la formation, la diffusion et la réception de particules lipoprotéiques avec Hh. Ces composés pharmaceutiques régulateurs de la formation, de la diffusion et de la réception de particules lipoprotéiques avec Hh peuvent être utilisés pour l'élaboration d'un médicament destiné au traitement de maladies humaines, de préférence le cancer.
PCT/ES2007/070164 2006-09-30 2007-09-27 Modèle animal non humain destiné à l'identification de composés pharmaceutiques régulateurs de la voie hedgehog et ses applications WO2008037835A1 (fr)

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ES200602497A ES2302625B1 (es) 2006-09-30 2006-09-30 Modelo animal no humano util para la identificacion de compuestos farmaceuticos reguladores de la via hedgehog, procedimiento de obtencion y su utilizacion en un procedimiento de identificacion de compuestos farmaceuticos reguladores de la via hedgehog.

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000076308A1 (fr) * 1999-06-14 2000-12-21 Exelixis, Inc. Modeles animaux et methodes d'analyse du metabolisme des lipides et criblage d'agents pharmaceutiques et de pesticides modulant le metabolisme des lipides
US20020026648A1 (en) * 1998-12-21 2002-02-28 Ernst Hafen Function-based small molecular weight compound screening system in drosophila melanogaster
WO2002036818A2 (fr) * 2000-11-02 2002-05-10 Consejo Superior De Investigaciones Cientificas Methode permettant de detecter des inhibiteurs de croissance tumorale

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020026648A1 (en) * 1998-12-21 2002-02-28 Ernst Hafen Function-based small molecular weight compound screening system in drosophila melanogaster
WO2000076308A1 (fr) * 1999-06-14 2000-12-21 Exelixis, Inc. Modeles animaux et methodes d'analyse du metabolisme des lipides et criblage d'agents pharmaceutiques et de pesticides modulant le metabolisme des lipides
WO2002036818A2 (fr) * 2000-11-02 2002-05-10 Consejo Superior De Investigaciones Cientificas Methode permettant de detecter des inhibiteurs de croissance tumorale

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