Patent Application
20080181877
The present invention relates to a method for monitoring/detecting the effect of test compounds on the regulation of the forkhead transcription factor Foxc2, which compounds may be useful for the treatment of various diseases, such as obesity, type 2 diabetes, insulin resistance and diet induced insulin resistance. The screening for the compounds has been performed in recombinant mouse cells containing the Foxc2 gene and the β-galactosidase gene in a suitable targeting vector.
Obesity and related diseases are an increasing problem within the population and require a change in life-style with interventions on the individual behaviour (for example diet and exercise). For many individuals this will not be sufficient and a combination of life-style changes with anti-obesity drugs may be essential. The understanding of the molecular basis for metabolic regulation in insulin responsive tissues (e.g. adipose tissue and skeletal muscle) will help in the development of new analytic tools for defining future drug-based intervention strategies of obesity and type 2 diabetes. Numerous studies have demonstrated a relationship between obesity and insulin resistance, and recent epidemiologic studies have linked the increasing prevalence of diabetes to the obesity epidemic (see Harris et al., The Third National Health and Nutrition Examination Survey, 1988-1994. Diabetes Care 21, 518-524 (1998)).
Diabetes related diseases are caused by multiple factors and may be characterized by elevated levels of plasma glucose (hyperglycemia). In general, there are two recognized forms of diabetes. Type 1 diabetes, or insulin-dependent diabetes mellitus (IDDM), in which patients produce small amounts or no insulin, and type 2 diabetes, the so-called noninsulin-dependent diabetes mellitus(NIDDM), in which the patients produce insulin and even exhibit plasma levels of insulin, which are similar or higher than in comparison with non-diabetic subjects. Type 1 diabetes may be treated by the administration of insulin in high amounts. Patients with type 2 diabetes develop quite often the so-called “insulin resistance”, characterized by the effect that the stimulation of insulin on the glucose and lipid metabolism is diminished. It may be assumed that insulin resistance occurs primarily due to a receptor binding defect and results inter alia in an insufficient activation of the glucose uptake. Patients with type 2 diabetes exhibit at increased risk of developing different cardiovascular complications.
Diabetes may be treated by the administration of a variety of therapeutic compounds including different insulin sensitizers. The plasma level of insulin is increased by the administration of sulfonylureas or meglitinides, which stimulate the insulin secretion, and/or by injection of insulin when the above compounds get ineffective, may result in insulin concentrations high enough to stimulate insulin-resistant tissues. However, dangerously low levels of plasma glucose can result, and increasing insulin resistance due to the even higher plasma insulin levels may occur.
Other possibilities for the treatment of obesity and diabetes related diseases pertain in the identification of metabolic regulators, for example to the forkhead/winged helix transcription factors. This family of proteins has been shown to play a role in embryonic pattern formation, regulation of tissue-specific gene expression, and tumorigenesis (see Carlsson et al.; Dev Biol 250, 1-23 (2002) and Kume et al.; Genes Dev 15, 2470-2482 (2001)). One member of this protein family, Foxc2 promoted greater sensitivity of the protein kinase A (PKA)-signalling pathway, owing to altered subunit composition of PKA, as well as increased levels of β-adrenergic receptors. As a result of these changes in gene expression, Foxc2 decreased total body lipid content and levels of free fatty acids (FFA); and increased insulin sensitivity as well as thermogenesis in white adipose tissue. The expression in mice resulted in an animal protected from diet-induced obesity and insulin resistance.
The Foxc2 gene has already been used for example in the U.S. Pat. No. 6,709,860, which pertains to transgenic non-human mammalian animals capable to express the human FKHL14/Foxc2 gene in their adipose tissue. In addition, methods for identifying compounds useful for the treatment of medical conditions related to obesity or diabetes are disclosed. The compounds are capable to stimulate the expression of the human FKHL14/Foxc2 gene, or the biological activity of a polypeptide encoded by the human FKHL14/Foxc2 gene and may be used for the treatment of medical conditions related to malnutrition.
Isolated promoter regions of the mammalian transcription factor Foxc2 are known from the WO 0227008. Said document also relates to screening for agents, which are capable to modulate the expression of Foxc2 and which have a potential use for the treatment of medical conditions related to obesity.
From the WO 03064467 complexes of the Foxc2 protein with other proteins are known, which may be used likewise for the identification of agents. Said agents may be used for the treatment of medical conditions such as obesity, hypertriglyceridemia, diet-induced insulin resistance, and/or diabetes.
The prior art merely relates to possibilities for the identification of compounds which may alter the Foxc2 expression. Nevertheless, the construction of suitable assays for the identification of said compounds is not disclosed.
A problem of the present invention is, therefore, the provision of a suitable assay system capable to monitor/detect test compounds, which may alter the Foxc2 transcription and/or expression.
The present invention solves the above-mentioned problem by the provision of an assay system as set forth in claim 1, which provides a fast and reliable method for the identification of test compounds altering the Foxc2 expression, which method may be performed in a high throughput manner.
The present invention provides a method for monitoring and/or detecting the regulation of a gene construct with a test compound. Said gene construct comprises the gene encoding the forkhead transcription factor Foxc2 or parts thereof, and a reporter gene, such as a gene encoding e.g. β-galactosidase, GFP, or luciferase, or other suitable genes allowing detection, such that an in frame fusion or an operative linkage is provided between these components (the gene encoding the forkhead transcription factor Foxc2 or parts thereof, and the reporter gene) to report the transcription/expression of the construct, if the Foxc2 promoter is influenced by a chemical substance.
As the gene encoding the forkhead transcription factor Foxc2 or parts thereof any nucleic acid sequence is envisaged that comprises the promotor region of the forkhead transcription factor Foxc2 and either the enitre gene sequence or parts thereof, in particular those parts ensuing the promotor region, i.e. the 5′-region of the gene.
The method according to the present invention is characterized in that (a) said gene construct is inserted in a vector, which is preferably a targeting vector, and subsequently introduced in mouse primary cells or mouse embryonic fibroblasts, which shall be heterozygous for said gene construct and which process is also referred herein as “knock-in”. Therefore, the respective Foxc2 fusion gene with lacZ has been created in such a way that the LacZ gene is under transcriptional control of an endogenous Foxc2 promoter. After insertion of said construct in a suitable targeting vector, the vector is introduced into mouse cells.
In step (c) a cell line from said primary cells or embryonic fibroblasts is established, followed by an optional differentiation step of said cells into tissue cells. This step may be effected by any of the methods for differentition known to the skilled person. Subsequently, in step (e) the cells are exposed to a substance of interest (the test compound), which may, if desired, be carried out during an optional incubation step, which facilitates uptake and transport of the test substance in and within the cell. After a given time period, which will be selected by the skilled person depending on the assay to be run, the transcription and/or expression level of the reporter gene may be determined by using suitable means, such as e.g. Northern blot, PCR, Wetser-blots and dye and/or fluorescence detection of the reporter gene product. The compound yielding a desired result, i.e. activation or increase or decrease of the transcription or expression of the reporter gene may be selected for further investigation.
The gene construct as used herein refers to any combination of a particular gene with other nucleic acid sequences by for example ligation techniques. Said gene construct may be obtained by any recombinant technique known to the skilled person and outlined for example in Maniatis et al.; Molecular Cloning: A Laboratory Manual 2nd ed., (1989). The same applies likewise to respective techniques for introducing a gene construct in a targeting vector and the subsequent transfection of the host cells. The transfection techniques may comprise preferably electroporation.
The method according to the present invention may be surprisingly also carried out in small sample volumes, which allow the set-up of high troughput screening systems. Such a system employs for example micro-titer plates of any size having an optional coating of the wells. Micro-titer plates used for this purpose may comprise preferably 96 well micro-titer plates with gelatine coating.
The monitoring/detecting of the regulation of said gene construct is performed via a “reporter means”, which may be the product of the reporter gene, either on the transcriptional or the expression level, i.e. for example via enzymatic activity (for example by X-gal staining in case of using the lacZ gene as the reporter gene or when using luciferase applying the respective substrate) or via a protein (e.g. GFP that emits fluorescence when irradiated), or via northern blotting, realtime RT-PCR, western blotting etc. The β-galactosidase enzyme is an conventional and easy means, that catalyzes the hydrolysis of X-gal (5-bromo-3-indoyl-β-D-galactopyranoside), with the effect that cells containing a β-galactosidase gene will appear blue after incubation with X-gal. The blue cells may easily be visualized by eye or microscope. As the β-gal activity (the protein product of the LacZ gene) may be measured easily, the cells may be subjected to in vitro high through-put screens using compound libraries. Alternatively, an increase in β-galactosidase activity may be confirmed and quatified by using a luminometric Beta-glo kit (Promega) and an appropriate a luminometric plate reader. Likewise to X-gal staining, western blotting is directed to the detection of proteins, whereas northern blotting and RT-PCR are methods for locating/quantifying RNA. These methods are all well known to the skilled person.
The gene construct used for the method according to the present invention is preferably designed such that the start codon of the reporter gene, e.g. the lacZ gene, is inserted 17 amino acids downstream of the Foxc2 start codon, which enables the transcription and expression of a reporter gene product still exhibiting its native properties, while being under the control of the Foxc2 promotor. Hence modulation of the Foxc2 transcription/expression with test compounds may easily be tracked by monitoring the transcription and/or expression of the reporter gene/reporter gene product. Insulin, which is known to by an inducer of Foxc2 may be utilized as a control.
The construct containing the Foxc2 gene (or parts thereof) with the reporter gene may contain at the respective ligation site between the two components, a nuclear localization signal (nls) to ensure a distinct nuclear detection of reporter gene positive cells, for a reliable report of Foxc2 expression levels, since transcription start, cis-elements and exon/intron structure will be very similar to that of the wild type (wt) Foxc2 allele.
The targeting vector is preferably the pPGKneobpAlox2PGKDTA vector.
The test compound identified in the method according to the present invention is preferably capable to activate and/or increase and/or decrease Foxc2 expression by inducing/repressing the Foxc2 promoter and thereby, initiating the expression of the reporter gene. After further investigations such a test compound may be suitable to be used as a lead compound in clinical studies. I.e. not only a positive induction is envisaged in the present invention, since potential inhibitors of the Foxc2 expression also have suitable applications.
The capability of said test compound to activate Foxc2 expression is preferably verified in mouse models, which may comprise Foxc2:nls:β-galactosidase as already mentioned-above or other mouse models, such as knock-in mice or diabetic and obese mouse models, which are known to the skilled person.
The test compound obtained by the method according to the present invention is preferably capable to induce adaptive thermogenesis, insulin sensitivity and an increased sensitivity of the protein kinase A signaling pathway, thereby indicating inter alia the possible use of said compound to create heat in the sense of consuming stored energies in the form of for example fat.
The test compound obtained by the method according to the present invention is preferably used for the preparation of a composition for the prevention or treatment of any disease associated with Foxc2 expression levels, which may be for example selected from obesity, type 2 diabetes, insulin resistance, diet induced insulin resistance, hypertriglyceridemia, cancer and an increased plasma levels of free fatty acids.
Two test compounds, forskolin and TPA, capable of regulating a gene construct comprising an in frame fusion of the forkhead transcription factor Foxc2 with a reporter gene have been identified using the method according to the present invention. A single dose of forskolin resulted in a fast increasing of the levels of reporter gene followed by a fast decreasing to normal levels, suggesting to apply said compound in single doses of short intervals. Employing the same assay, TPA exhibited a moderate increasing of the levels of reporter gene remaining for long periods at similar levels, which suggests that said compound exhibits a repository effect rendering it suitable for long-time application. These results suggest that both compounds are suitable for prevention or treatment of any disease associated with Foxc2 expression levels, in particularly with the diseases as outlined above.
Without wishing to be bound by any theory it is presently assumed that the effect of both compounds resides in their common basic structural features, i.e. the phorbol moiety in case of TPA and the labdane-diterpene moiety in case of forskolin. It is to be expected that other molecules having a similar basic structure exhibit a similar effect as may be derived from fitting the respective molecules to the putative three dimensional structures of SVCT1 and SVCT2.
Forskolin (7 beta-acetoxy-8, 13-epoxy-1 alpha,6 beta,9 alpha-trihydroxy-labd-14-ene-11-one) is a labdane diterpene produced by e.g. the Ayurvedic herb Coleus forskohlii. Forskolin is known to raise levels of cyclic AMP. It is reported that said compound resensitizes cell receptors by activating the enzyme adenylyl cyclase and increases the intracellular levels of cyclic Adenosine Monophosphate. Forskolin may be either isolated directly from a suitable strain or produced by means of recombinant techniques well known to the skilled person. Forskolin may be included in a pharmaceutical composition in an amount of from about 0.01% to 50% by weight. Preferably, a pharmaceutical composition includes forskolin in an amount of from about 0.05% to 10% by weight, more preferably in an amount of from about 1% to 5% by weight.
12-O-tetradecanoylphorbol-13-acetate (TPA), also known as phorbol 12-myristate 13-acetate (PMA), represents a tumor promoter employed in biomedical research to activate the signal transduction enzyme Protein Kinase C (PKC). TPA belongs to the family of phorbol esters and may be isolated from the genus croton, such as Croton tiglium. It may be produced by recombinant means well known to the skilled person. TPA may be included in a pharmaceutical composition in an amount of from about 0.01% to 40% by weight. Preferably, a pharmaceutical composition includes forskolin in an amount of from about 0.05% to 10% by weight, more preferably in an amount of from about 1% to 5% by weight.
The concentration of forskolin and/or TPA in a pharmaceutical composition will nevertheless depend on verious factors, e.g. absorption, inactivation, excretion rates, the dosage schedule and amount administered as well as other factors known to those of skilled in the art. Other ingredients to produce e.g. an enteral applicable composition, such as a tablet or capsule or a liquid form, are also well known to the skilled person and may be formulated depending on the desired consistency and other properties envisaged.
The following examples illustrate the invention without limiting it thereto.
Targeting Construct
Foxc2 clones were isolated from a mouse 129/SvJ genomic library (Stratagene) using a specific Foxc2 probe. The correct identity of the clones was confirmed by sequencing. To construct the targeting vector, two fragments of the genomic DNA flanking the DNA-binding region were subcloned at convenient restriction sites into the pPGKneobpAlox2PGKDTA vector. The targeting vector contained 2.7 and 5.0 kb of homologous genomic DNA on either side of a floxed neomycin resistance cassette. The 2.7 kb short arm was fused in frame with an nls-LacZ cassette obtained from the pCH110-nls plasmid leaving the first 17 amino acids of Foxc2 open reading frame intact. This construct deleted 3.7 kb, including sequences encoding the DNA-binding winged helix domain, of endogenous Foxc2 genomic sequence.
ES Cells and Foxc2nLacZ Mutant Mice
R1 ES cells (2×107) were electroporated in Dulbecco's modified Eagle medium (DMEM) containing linearized targeting construct (30 μg). Selection was performed on neomycin resistant mouse embryonic fibroblasts with G418 (300 μg/ml) and ganciclovir (2 μM). Double-resistant colonies were screened by Southern blot analysis. DNA samples were digested with XbaI and probed with a 5′ external probe or with NcoI and probed with a 3′ external probe. Homologous recombination was detected in 0.4% (4/960) of the ES cell clones screened. The ES clones were subsequently subjected to transfection with a cre expressing plasmid, PGK-cre, to recombinate out the loxP site flanked neomycin resistance cassette. Correct recombination was screened for by Southern blot analysis. DNA samples were digested with NdeI and probed with the 3′ external probe. Four correctly targeted ES cell clones were injected into C57B16/J host blastocysts to generate chimeric animals. Male chimeras were bred with C57B16/J females and two of the cell lines were found to generate germ-line transmission, determined by Southern-blot analysis after XbaI digestion. Heterozygous mice were mated on to C57B16/J background for at least five generations. Subsequent progeny and embryos were genotyped by PCR. The PCR primers were designed to use a common 5′ primer located upstream of the ATG (5′-GTCCCTTCCTGCTCTCCTG-3′), and specific primers for the LacZ gene (5′-TCAGTTCGAGGTGCTGTT-3′) and the Foxc2 ORF (5′-GCCCGGTAGTAGTTTTGCTC-3′). These primers generate products of 221 bp specific for the mutant allele and 168 bp for the wild-type.
Generation of Foxc2nLacZ Knock-In Mice
The strategy of the generation of Foxc2nLacZ knock-in mice is shown in
MEFs and Primary Tissue Cultures
MEFs were isolated from E13.5 embryos from Foxc2nLacZ±heterozygous intercrosses and genotyped by PCR on DNA from the tail. The head, limbs and viscera were removed from embryos and the remains were minced with a razor blade and incubated in 0.25% trypsin, 0.1% EDTA for 1 h at 37° C. The cell suspension was plated in DMEM containing 10% fetal calf serum and antibiotics (100 units of penicillin and 100 μg/mL streptomycin). Once the cultures reached confluency, 1×106 cells were plated on 10-cm tissue culture plates (Falcon) and considered passage 0.
Adipocyte Differentiation of MEFs
Primary MEFs from both wild type and Foxc2nLacZ knock-in mice were maintained in DMEM supplied with 10% FBS and antibiotics. For differentiation into adipocytes the MEFs were grown until confluency and then stimulated (Day 0) with a differentiation media (DMEM, 10% FBS, antibiotics, 1 uM dexamethasone, 0.5 mM isobutyl-methyl-xanthine, 10 ug/mL insulin, 20 ng/ml FGF (all from Sigma), and 1 uM rosiglitazone (Alexis)). New differentiation media was added at day 3. At day 5 the differentiation media was removed and the cells were cultured in a media containing DMEM, 10% FBS, antibiotics, 10 ug/mL insulin, 1 uM rosiglitazone, and 20 ng/ml FGF. This media was changed every second day. From day 10 the cells were kept DMEM supplied with 10% FBS and antibiotics for 24 hours as a wash out period before initiating the experiments.
Expression of LacZ Reporter from the Foxc2 Locus
To verify the functionality of our Foxc2nLacZ reporter mice the expression of Foxc2nLacZ fusion protein was monitored by X-gal staining of whole-mount embryos and tissue pieces from adult mice, and compared with the known expression pattern for Foxc2. The Foxc2 expression pattern during embryonic development have been scrutinized fairly well, previously reported expression studies of Foxc2 using in situ hybridization demonstrated that Foxc2 is transcribed early in embryogenesis in several developing tissues such as heart, blood vessels, nasal septum, kidney, periocular mesenchyme and condensing cartilage that forms bone. Adult expression of Foxc2 has been studied much less extensive, being assessed mostly by Northern blots and realtime RT-PCR assays, in which Foxc2 expression has been demonstrated in adipose tissue and skeletal muscle. The results with X-gal staining of Foxc2nLacZ embryos and tissue pieces demonstrated a specific expression pattern. The use of whole-mounts, sectioning and X-gal staining on tissues from Foxc2nLacZ reporter mice is a very powerful tool for dissecting intricate Foxc2 expression patterns. At embryonic stage E10.5 days post-coitum (dpc) β-galactosidase activity is detected in among others presomitic mesoderm, somites, mesenchymal cells surrounding the eye (periocular mesenchyme) and the developing heart (
Monitoring Foxc2 Expression in MEFs
A cell based high-throughput screening system has been established by isolation of MEFs from heterozygous Foxc2nLacZ±E13.5 embryos as well as from wt embryos as a control. As detected by increased X-gal staining, Foxc2nLacZ protein levels increased during differentiation and moreover levels could be induced by acute treatment with insulin and TNFα (
X-Gal Staining of Whole-Mount Embryos and Tissue Pieces
Whole-mount embryos and tissue pieces were dissected in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyd (PFA) at 4° C. in dark for 1 hour or overnight. Rinsing was performed for 3×15 minutes with rinse buffer (5 mM EGTA, 0.01% Deoxycholate, 0.02% NP40, 2 mM MgCl2 in PBS), and followed by incubation in staining solution [5 mM EGTA, 0.01% Deoxycholate, 0.02% NP40, 2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 1 mg of X-gal/ml in PBS] overnight at 37° C. to detect β-galactosidase activity. β-galactosidase expressing tissues were located and photographed under a dissecting microscope.
High Through-Put Screening
Foxc2nLacZ MEFs were plated onto gelatine coated 96 well plates. Differentiation was initialized after confluence according to protocol. Day 10 adipocytes were used in the assay. After a wash out period for 24 hours the cells were subjected to different compounds for the indicated time points and subsequently induction of β-gal enzyme activity was measured using the Beta-glo kit (Promega) and a luminometric plate reader. Confluent cells differentiated into adipocytes were detected by increased X-gal staining (
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/SE2006/000358 | 3/22/2006 | WO | 00 | 11/14/2007 |
