ES2302625B1 - NON-HUMAN ANIMAL MODEL USEFUL FOR THE IDENTIFICATION OF PHARMACEUTICAL COMPOUNDS REGULATING THE VIA HEDGEHOG, PROCEDURE OF OBTAINING AND ITS USE IN A PROCEDURE OF IDENTIFICATION OF PHARMACEUTICAL COMPOUNDS REGULATING THE VIA HEDGEHOG. - Google Patents

Abstract

Non-human animal model useful for identification of pharmaceutical compounds regulating the pathway hedgehog, procurement procedure and its use in a pharmaceutical compound identification procedure hedgehog track regulators.

The present invention describes the key role of the Hedhehog protein receptor, Patched (Ptd), in the internalization of lipid compounds that modulate Intracellularly the activity of Smoothened. Some of these compounds would have antitumor activity so They would represent an important therapeutic advance. The present invention describes an animal model in Drosophila to identify said compounds.

Description

Non-human animal model useful for identification of pharmaceutical compounds regulating the pathway hedgehog, procurement procedure and its use in a pharmaceutical compound identification procedure hedgehog track regulators.

Technical sector

Identification of pharmaceutical compounds by nonhuman animal models, in particular in Drosophila. Biotechnology and pharmaceutical sector.

State of the art

Activation of the signaling pathway of the Hedgehog (Hg) protein is key to inducing morphogenetic and in the processes of cell proliferation and modulates and promotes oncogenesis and other diseases in humans (Torroja et al., 2005, J Neurobiol , 64, 334-356; Gorfinkiel et al ., 2006, Developmental Cell 8, 241-253). On the other hand, signaling or signal transmission using the Hedgehog (Hh) protein is related to lipids in different ways. The Hh protein is synthesized from a precursor that undergoes an auto-proteolytic process, being doubly modified both by the addition of a functional cholesterol group at its C-terminal end and by palmitoylation at its N-terminal end (Figure 1). Hh secretion in the posterior lipid-modified cell compartment takes place through the mediation of a transmembrane protein called Dispatched (Disp) (Burke et al ., 1999, Cell 99 (7), 803-815). This protein has a sterol-sensitive domain (SSD) such as the Hh receptor, the Patched protein (Ptc), and other proteins involved in the metabolism of lipids and cholesterol and in vesicular traffic. On the other hand, Hh lipid modifications are essential for the interaction between Hh and heparan sulfate proteoglycans (HSPGs) to control the diffusion and signaling activity of morphogens (Callejo et al ., 2006, Development 133, 471-483).

Recently, 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 charge the Hh protein to these particles (Panakova et al. , 2005, Nature 435, 30-33; Despande and Schell, 2005, Developmental Cell 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. (Gorfinkiel et al ., 2006, Developmental Cell 8, 241-253; Glise et al ., 2005, Developmental Cell 8, 255-266). Lipid modifications in the Hh protein are also required for optimal interaction and recognition of Ptc during its intracellular reception (Callejo et al ., 2006, Development 133, 471-483). On the other hand, it has been suggested that the LDLR receptor might be associated with the receptor
Ptc

In the absence of cholesterol, the Ptc protein and the Smoothened (Smo) protein are held together and there are no signs of activation of this pathway. The covalent binding of cholesterol to Hh facilitates the binding of this to the membrane and allows recognition to Ptc which allows the Smo protein to be released and can give the signal for activation. The mechanism of Smo inhibition remains unresolved, although it has recently been observed that sterols or steroids could mediate Smo inhibition at the same time that the enzymes for cholesterol biosynthesis have been suggested could be involved in the regulation of inhibition of Smo by Ptc (Biljsma et al., 2006, PLoS Biology 4 (8), 1397-1410). In the case of the antagonistic role of steroid compounds on the Smo protein, the mechanism of action remains unsolved.

In this sense, the increase in knowledge of lipid modulation in protein interaction Hh with its membrane or intracellular receptors or with the Smo protein would allow progress in the development of tools useful for the diagnosis and treatment of human tumors.

Description of the invention

Thus, to demonstrate the importance of lipids in Hh protein signaling (can be considered as a morphogen), the activity of the enzymes that control the production of lipids in the disc Drosophila's imaginal. In this sense, it is known that SREBP (Sterol-regulatory element binding protein) is a transcription factor that controls multiple genes involved in lipid metabolism, specifically cholesterol, like the gene of the HMGCoA reductase or of the LDL receptor, making it an enzyme key in sterol biosynthesis. Recently, it has been suggested which regulates the gradient Hh (Despande and Schell, 2005, Developmental Cell 9, 629-638).

In the present invention the effect on the secretion, diffusion and reception of Hh in the loss or Function gain in mutated cells for these two genes. From in particular, the inhibition of the SREBP gene by mutation of the gene prevents the expression of the proteins necessary for the synthesis of sterols in mutated animals (Drosophila) of the present invention, so they must be fed conveniently by adding the necessary lipids to that act as substitutes for the lack caused.

It has been found that the Hh protein in the extracellular environment stabilizes after overproduction of lipids by overexpression of the transcription factor SREBP or the enzyme HMGCoA reductase. In addition, the decrease in the levels of the products of these genes rescue the phenotype caused by the increase in Hh production (Figure 3, 4, 6 and 9). These data show the importance of lipids in the stabilization and diffusion of the Hh protein in the matrix extracellular, suggesting that the Hh protein travels bound to lipoproteins

In addition, the involvement of the LDL receptors (megalin) in Hh protein signaling and it has found that none of them are involved in the signaling and internalization of the Hh protein (Figure 5) and that it is able to internalize in the cells without the need for megalin (Figure 8).

In addition, it was observed that the Patched protein and lipoprotein lipoprotein are capable of co-immunoprecipitating, detecting the form of 75 Kda lipoforin (Figure 10). Lipoforin is the main lipid transporting lipoprotein in insects, being considered a high density lipoprotein (HDLp). Lipoforin carries neutral lipids such as sterols, fatty acids and sugars in its hydrophobic core (Arrese et al ., 2001, Insect Biochemistry and Molecular Biology 31, 7-17).

Finally, and as a result of this invention it has been shown that the Patched receiver is capable of actively internalize lipoproteins (particularly, the lipoforin), which would be able to transport lipids inside of the cell where they would regulate the activity of the Smo protein (Figure 8 and 11).

In conclusion, in the present invention proposes that the Ptc protein acts not only as an Hh receptor but also as a lipid transporter inside the cell, which could modulate the activity of Smoothened (Smo) and thus become potential useful pharmaceutical compounds for the treatment of human diseases, as per the Cancer.

Based on this discovery and the previous results indicating sterols and other lipids may regulate Smo activity and given certain characteristics of the fly Drosophila can develop an animal model useful for identification of pharmaceutical compounds regulating the pathway Hedgehog and Smo. The mutant Drosophila model for the gene SREBP, particularly the Drosophila HLH106line52 model described in the present invention, it is unable to produce sterols so that these should be administered in the diet of this clone, being able to define the composition of different lipid diets that jointly analyzing their involvement in the activity of the Hh pathway would identify what specific lipid regulates, inhibiting or inducing said route.

Thus, an object of the present invention is it constitutes a non-human animal model useful for identification of pharmaceutical compounds regulating the hedgehog pathway, in further animal model of the invention, characterized in that said model has an inability to synthesize lipids caused by a genetic cancellation of the enzymes involved, need that is artificially replaced in the diet, and in the can determine the signaling of the Hh and Smo path after transport by Patched of lipids inside the cell.

A particular object of the invention is the animal model of the invention that is constituted by the Drosophila fly mutated in the SREBP transcription factor gene, and more particularly the Drosophila mutant HLH106 (HLH106line52) and where the characteristic to be determined from the via Hh as an identifying parameter of a regulatory compound is the determination of the development of the imaginal disk of the Drosophila wing. The parameters that can be used as identification of a regulation of the Hh and Smo pathway are widely known by researchers in the area of developmental biology, and those described by the inventors themselves can be used as a reference (Torroja et al ., 2005, J Neurobiol, 64, 334-356).

Another object of the present invention is constitutes the use of the animal model of the invention, in further use of the animal model of the invention, in a procedure for identifying regulatory compounds, preferably lipids, of the Hh and Smo pathway useful as compounds Pharmacists for the treatment of human diseases, particularly cancer.

Description of the figures

Figure 1.- Scheme of production, transport and interactions of the Hh protein .

Figure 2 a schematic of proteins that share the transmembrane domain motif Sterol Sensing (SSD) is described. The NPC1, HMG CoA reductase and SCAP proteins are involved in the transport of sterols, traffic and metabolism, while Patched, Hh protein receptor, and Dispatched, Hh secreting protein, participate in Hh signaling.

Figure 3.- Rescue of Hh signaling in mutant Drosophila flies for shifted protein that overexpress the enzyme HMGCoA reductase in the wing's imaginal disc .

Figure 4.- Rescue of Hh signaling by overexpression of HMGCoA reductase in a shifted mutant model of the Drosophila wing in the posterior compartment of the wing .

Figure 5.- The LDL receptor , α- megalin is not a co-receptor for the Hh protein in Drosophila .

Figure 6.- Hh function gain phenotypes are rescued by decreasing lipid synthesis .

Figure 7.- Glycerol gradient (30-15%) for the detection of Hh and lipoforin 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 .

Figure 9.- Effect of decreased lipid synthesis on extracellular levels of the Hh protein .

Figure 10.- Immunoprecipitation assay of the Ptc-GFP protein and lipoprotein lipoprotein . Lane C: consists of a protein extract obtained from a Drosophila AB1-Gal4 / UAS-PtcGFP mutant as a positive lipoforin control. IP lane: Salivary gland protein extract from a Drosophila mutant of AB1-Gal4 / UAS-PtcGFP immunoprecipitated with a polyclonal antiGFP antibody. IPC lane: Drosophila AB1-Gal4 / UAS-PtcGFP mutant salivary gland protein extract immunoprecipitated with polyclonal anti bGal antibody as a negative control. An anti lipoforin antibody was used for Western blot analysis of the samples. The form of lipoforin 75 Kda was detected in the immunoprecipitation assay (arrowhead).

Figure 11.- The Patched protein (Ptc) is capable of internalizing lipoprotein lipoprotein as efficiently as it is carried out by the lipoforin receptor (LpR) .

Figure 12.- Scheme of the diffusion and gradient model of morphogen Hedhehog .

Examples of the invention

Example one

The Hedhehog (Hh) receptor, Patched, is the protein involved in the internalization of lipoproteins

- Rescue of Hh signaling in Drosophila flies mutants for shifted protein that overexpress the enzyme HMGCoA reductase in the wing's imaginal disk (Figure 3).

Drosophila's imaginal disk 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 shfEY mutants (Gorfinkiel et al ., 2006, Developmental Cell 8, 241-253). The levels of the Hh protein in the imaginal disc that overexpresses the enzyme HMGCoA reductase (HMGCR) are similar to those observed in the control group (wild-type, anti-Hh staining). In addition, Hh signaling is recovered on these disks (anti-Ptc staining).

In addition, overexpression of HMGCoA reductase in a shifted mutant model of the Drosophila wing in the posterior compartment of the wing causes rescue of Hh signaling (Figure 4).

- Hh function gain phenotypes are rescue by decreasing lipid synthesis (Figure 6).

In this case the rescue of the function is analyzed Hh in Drosophila wings mutants due to the ectopic expression of Hh in the dorsoventral region of the imaginal disc. "One reduction dose "of the expression of the SREBP gene that takes place in the line HLH106line52 mutant fly involves obtaining a phenotype practically identical to that seen in the control fly (wild-type).

- Effect of decreased synthesis of lipids in the extracellular levels of the Hh protein (Figure 9).

Overexpression of Hh is observed in the posterior compartment in Drosophila control (wild-type) with the vector hhGal4 and Drosophila HLH106 (HLH106line52) mutants in the imaginal disk, while the reduction of the Hh protein in the region is observed dorsoventral.

- The LDL receptor, α-megalin is not a co-receptor for Hh protein in Drosophila (Figure 5).

Next, we proceeded to analyze the role of the LDL receptor as a possible Sonic-Hh co-receptor, proposed in the literature by studying the loss of function in Drosophila dor mutant clones. The intense orange protein (Deep orange, Dor) is a protein involved in lysosomal cell degradation machinery. In the mutant clones dor, Hh protein, and its mutated forms (HHN cholesterol, and C85S without palmitic acid) accumulates in the rear endocytic compartment. It is also observed that the Megalin protein is increased inside these mutant clones, and is placed with Hh.

- The Hh protein is internalized in the absence of megalin / LPR2 receptor (Drosophila lipoforin receptor) (Figure 8).

The internalization of the Hh protein in the double dor and megalin mutant clones (α-megalin) has been studied. The accumulation of Hh and HhN proteins (form of Hh without cholesterol) is observed within the double mutant Drosophila clone, due to the internalization of the protein and despite the absence of? -Megaline or any other member of the family of LDL receivers (data not shown).

- Patched protein (Ptc) is capable of internalize lipoprotein lipoprotein as effectively as what performs the lipoforin receptor (LpR) (Figure 11).

Overexpression of wild Patched (Pct-wt) and lipoforin receptor (LpR) was performed in the dorsal compartment of the wing's imaginal disc, using Gal4 / Gal80 ts for 14 h at a restrictive temperature. Thus, it was observed that the wild ectopic protein Ptc-GFP (wild-type) is capable of internalizing Hh and lipoforin. On the contrary, the expression of the Ptc14 protein (Torroja et al ., 2004, Development 131, 2395-2408), a mutated form of internally defective Patched, was not able to accumulate the Hh protein nor lipoforin ( anti-Hh and anti-Lp staining).

Claims (5)

1. Non-human animal model useful for the identification of pharmaceutical compounds regulating the hedgehog pathway characterized in that said model has an inability to synthesize lipids caused by a genetic cancellation of the enzymes involved in its synthesis, either by a mutation of the transcription factor SREBP or the enzyme HMGCoA reductase, a need that is artificially replaced in the diet, and in it the signaling of the Hh and Smo pathway can be determined after the transport by Patched of lipids into the cell. 2. Animal model according to claim 1 characterized in that it is constituted by the Drosophila fly. 3. Animal model according to claim 2 characterized in that the Drosophila fly is a mutated clone in the SREBP transcription factor gene, and more particularly the Drosophila HLH106 mutant (HLH106line52). 4. Animal model according to claims 1 to 3, characterized in that the characteristic to be determined of the Hh pathway as an identifying parameter of a regulatory compound is the determination of the development of the imaginal disk of the Drosophila wing. 5. Use of the animal model according to claims 1 to 4 in a method of identifying regulatory compounds, preferably lipids, of the Hh pathway and Saint.