Uses of FAHD1

Abstract

The present invention provides FAHD1 for use in a method for the treatment or prevention of aberrations of the energy metabolism of the nervous system, pancreas, kidney, liver, muscles or adipose tissue. Further, a method of decarboxylating an organic compound is provided, which uses FAHD1 to decarboxylate the organic compound. Additionally, a method and a kit for identifying inhibitors of FAHD1 are provided.

Claims

1. A method for the prevention or treatment of a disease comprising administering to a patient in need thereof FAHD1 or a homologue thereof, which comprises an amino acid sequence with at least 80% identity to FAHD1.

2. A method for the prevention or treatment of aberrations of the energy metabolism of the nervous system, pancreas, kidney, liver, muscles or adipose tissue comprising administering to a patient in need thereof FAHD1 or a homologue thereof, which comprises an amino acid sequence with at least 80% identity to FAHD1.

3. The method according to claim 2, wherein the aberration of the energy metabolism is type 2 diabetes mellitus, obesity, hypercholesterolemia, metabolic syndrome, epilepsy, attention deficit hyperactivity disorder (ADHD), Parkinson's disease, Alzheimer's disease, focal cerebral ischemia (stroke), lactic acidosis, psychomotor deficiencies, mental disorder or death in infancy.

4. The method according to claim 2, wherein the aberration of the energy metabolism is the reduction of the oxaloacetate concentration.

5. The method according to claim 3, wherein the aberration of the energy metabolism is metabolic syndrome or type 2 diabetes mellitus.

6. A pharmaceutical composition comprising FAHD1 or a homologue thereof, which comprises an amino acid sequence with at least 80% identity to FAHD1, and a pharmaceutical acceptable additive.

7. A pharmaceutical composition comprising an oligonucleotide derived from a gene encoding for FAHD1 a homologue thereof, which comprises an amino acid sequence with at least 80% identity to FAHD1, and a pharmaceutical acceptable additive.

8. A method of decarboxylating an organic compound, comprising the steps of: a) providing FAHD1 or a homologue thereof, which comprises an amino acid sequence with at least 80% identity to FAHD1, and a starting organic compound, b) contacting FAHD1 or the homologue thereof and the starting organic compound with each other under conditions suitable to facilitate decarboxylation of the starting organic compound and c) obtaining a decarboxylated organic compound.

9. The method according to claim 8, wherein the starting organic compound used in step a) is oxaloacetate, which is decarboxylated in step b) to pyruvate.

10. A method of identifying a compound that inhibits the catalytic activity of FAHD1 or a homologue thereof, which comprises an amino acid sequence with at least 80% identity to FAHD1, comprising: i) providing a candidate compound, ii) contacting FAHD1 or the homologue thereof with the test compound under conditions allowing for the catalytic activity of FAHD1, iii) determining whether the candidate compound inhibits the catalytic activity of FAHD1 or homologue thereof in comparison to the catalytic activity of FAHD1 or homologue thereof in absence of the candidate compound under same conditions.

11. The method according to claim 10, wherein the catalytic activity is determined by a difference in concentration of a) a catalytic substrate of FAHD1, wherein inhibition of the catalytic activity is indicated by a higher substrate concentration in comparison to the substrate concentration in absence of the candidate compound under same conditions or b) a catalytic product of FAHD1, wherein inhibition of the catalytic activity is indicated by a lower product concentration in comparison to the product concentration in absence of the candidate compound under same conditions,

12. The method according to claim 11, wherein the catalytical substrate is oxaloacetate, or the catalytical product is pyruvate.

13. A kit for identification of a FAHD1 inhibitor comprising FAHD1 or a homologue thereof, which comprises an amino acid sequence with at least 80% identity to FAHD1, a substrate of FAHD1, and an instruction to determine catalytic activity of FAHD1.

14. A method of treatment or prevention of a disease involving an aberration of the energy metabolism comprising administering FAHD1 or a homologue thereof, which comprises an amino acid sequence with at least 80% identity to FAHD1, or comprising administering an oligonucleotide derived from a gene encoding for FAHD1 or a homologue thereof, to a patient in need thereof.

Description

[0064] The figures show:

[0065] FIG. 1 shows the active site geometry of human FAHD1 (A) and FAHD-1 of C. elegans (B). By computational loop modelling, a closed structure of FAHD1 (A) was established where His30 and Glu33 complete the active site. The lid region carrying the catalytic histidine and an activating glutamate residue is closed upon binding of the inhibitor oxalate (shown as sticks). The model of the closed lid region in FAHD1 completes the active site of FAHD1 around the magnesium ion (large sphere). Conserved residues around the active site are shown as sticks and labelled. Secondary structure elements are represented as cartoons. Two water molecules (small spheres) are co-crystallized in FAHD1 at the binding site of the oxalate, suggesting a similar binding mechanism. (B) The nematode protein, referred to as FAHD-1, contains 48% identical amino acids relative to human FAHD1, shows high amino acid sequence similarity in the presumed catalytic centre, and computational modelling revealed a structure of the catalytic centre highly similar to human FAHD1.

[0066] FIG. 2 shows the characterization of in vitro ODx activity of wild-type and mutant FAHD1. (A) Effect of substrate concentration on ODx reaction rate in presence of purified FAHD1, determined by photometric analysis at RT. Nonlinear regression analysis of Michaelis-Menten kinetics was performed with Prism5 software (GraphPad Software). Data are represented as mean±SD (n=3). (B) HPLC analysis of oxaloacetate breakdown in presence (solid line) and absence (dashed line) of purified FAHD1. Retention times for oxaloacetate (1) and pyruvate (2) standards are indicated by arrows. (C) ODx activity of wild-type FAHD1 in presence/absence of 200 μM oxalate, and of mutant FAHD1. Data are represented as mean±SD (n=3).

[0067] FIG. 3 shows the comparison of oxaloacetate levels in organs of wild-type (WT) and FAHD1 knockout mice (KO). (A+B) Oxaloacetate levels determined in kidney (A) and liver (B) extracts of 3-month-old female wild-type and FAHD1 knockout mice. Data are represented as mean±SD (n=3).

[0068] FIG. 4 shows the phenotypic characterization of fahd-1 (tm 5005) C. elegans mutants. (A) Survival curves at 25° C., showing percentage of animals remaining alive over time. (B) Worms were subjected to a swimming assay in M9 buffer at 20° C. The percentage of animals still swimming is plotted against time (n=24).

[0069] FIG. 5 shows the putative role of FAHD1 in central metabolism as an antagonist of pyruvate carboxylase (PC).

[0070] FIG. 6 shows the effect of FAHD1 depletion on the plasma levels of cholesterol (A), HDL-cholesterol (B), and gycerol (C) in female and male FAHD1 knockout mice (ko) and wild-type (wt) mice after food withdrawal overnight (fasting).

[0071] FIG. 7 shows body weight development of wild-type mice (A) and FAHD1 knockout mice (B) under control diet (cd, circles) and high fat diet (hfd, squares). Relative weight gain under control diet (C) and high fat diet (D) is shown for wild-type (circles) and FAHD1 knockout mice (squares).

EXAMPLES

Example 1: Modelling of FAHD1 Active Site

[0072] Method:

[0073] The X-ray structure of human FAHD1 (PDB database code 1SAW) (Manjasetty et al., Biol Chem, 2004, 385(10)) lacks 11 highly flexible residues next to the active site (Asp29 to Leu39). This region is constructed by using the ‘Loop modeller’ tool of MOE. To allow the loop region to extend into the active site—as expected for a closed lid conformation—four water positions were deleted in the active site: HOH314, HOH316, HOH356, HOH359. The missing loop was defined between Val21 and Val43 to identify potential loop candidates from the PDB. A tolerant maximal walk step of 5 amino acids allowed varying potential anchoring points between residue 16 and residue 48. All other parameters were kept as default. 94 structured loop regions were identified as potential templates with these parameters. The best scored candidate from the putative FAH protein from Yersinia pestis (PDB code 3S52) was used as template structure for loop construction. The parameters for refined loop modelling included an adaption of the environment by side chain repacking (default parameters).

[0074] Results:

[0075] By computational loop modelling, a closed structure of FAHD1 is established where His30 and Glu33 complete the active site. The lid region carrying the catalytic histidine and an activating glutamate residue is closed upon binding of the inhibitor oxalate (shown as sticks). The model of the closed lid region in FAHD1 completes the active site of FAHD1 around the magnesium ion (large sphere). Conserved residues around the active site are shown as sticks and labelled. Secondary structure elements are represented as cartoons. Two water molecules (small spheres) are co-crystallized in FAHD1 at the binding site of the oxalate, suggesting a similar binding mechanism (see FIG. 1A).

Example 2: Bacterial Recombinant Expression and Purification of FAHD1 and FAHD1mut

[0076] N-terminally His- and S-tagged versions of human wild-type FAHD1 and a double mutant (Asp102Ala, Arg106Ala) were recombinantly expressed in E. coli and purified as reported previously (Pircher et al., J Biol Chem, 2011, 286(42)).

Example 3: Oxaloacetate Decarboxylase Assay

[0077] Method:

[0078] For measurement of oxaloacetate decarboxylase rates, 1 ml samples of 25 μM to 1 mM oxaloacetate in assay buffer (50 mM Tris-HCl, 100 mM KCl, 1 mM MgCl2, pH 7.4) containing purified FAHD1, FAHD1mut (3-60 μg, depending on substrate concentration) or no enzyme were prepared. Samples were incubated at room temperature and analysed in regular time intervals by measuring absorbance (infinite M200, Tecan) at 255 nm (ε=1070 M.sup.−1 cm.sup.−1) in disposable UV-cuvettes (Brand). Reaction mixtures containing no substrate were used as blank. For the oxalate inhibitor assay, the sample buffer was supplemented with 200 μM sodium oxalate. All rates were corrected for auto-decarboxylation under assay conditions.

[0079] Results:

[0080] The recombinantly expressed wild-type and mutant FAHD1 proteins (example 2) were tested in the above photometric assay suitable for monitoring the breakdown of oxaloacetate. The purified wild-type enzyme was able to degrade oxaloacetate with a Vmax of 0.21 μmol min.sup.−1 mg.sup.−1 and a Km value of 32 μM (FIG. 2A). To further characterize the ODx activity inherent to FAHD1, oxalate was used, an inhibitor of oxaloacetate decarboxylase Cg1485. Indeed, oxalate potently inhibited oxaloacetate decarboxylation by FAHD1, with an IC50 value of about 20 μM (FIG. 2C).

Example 4: Analysis of Oxaloacetate Decarboxylase Reaction by HPLC

[0081] Method:

[0082] 1 ml assay buffer containing oxaloacetate (1 mM) and purified recombinant FAHD1 protein (120 μg) was incubated at room temperature (RT) for 30 min. A control lacking FAHD1 was incubated analogously. The conversion mixture and control of the FAHD1 reaction were analysed by high performance liquid chromatography (HPLC) using an ÄKTA purifier system (GE Healthcare) equipped with a Bio-Rad Aminex HPX-87H column (300×7.8 mm). Detection was at 210 nm. 84 μl of sample were injected and eluted with 5 mM H2SO4 as the eluent at a flow rate of 0.5 ml min.sup.−1 at RT. Identification of peaks was based on the characteristic retention times of high purity standards (>99%) of oxaloacetate and pyruvate.

[0083] Results:

[0084] The above HPLC analysis, after incubation of the substrate in presence or absence of the purified enzyme, confirmed the conversion of oxaloacetate to pyruvate in presence of FAHD1 (FIG. 2B). Incubation without the enzyme only yielded a minor amount of pyruvate due to auto-decarboxylation, whereas the catalytically dead mutant enzyme displayed only residual ODx activity (FIG. 2C).

Example 5: Generation of a FAHD1.SUP.−/− knockout mouse

[0085] Method:

[0086] F2 generation C57BL/6 mice heterozygous for a LoxP-flanked FAHD1 gene were established by inGenious Targeting Labs. FAHD1.sup.−/− mice were generated by crossing FAHD1.sup.flox/+ with Cre.sup.0/+ transgenic mice, followed by outcrossing of Cre alleles and crossing of FAHD1.sup.−/+ mice. The knockout was verified by PCR and immunoblot. Care of experimental animals was in accordance with guidelines for mouse work at Universität Innsbruck.

[0087] Results:

[0088] A cohort of 15 male wild-type mice and 13 male knockout mice as well as 15 female wild-type and 15 female knockout mice was generated. To determine body composition, body mass and fat mass were investigated using time domain nuclear magnetic resonance (TD-NMR). The comparison of body mass and fat mass between wild-type and knockout mice displayed a slightly reduced body weight as well as decreased fat mass to some extent in male knockout mice compared to their wild-type controls. This trend is not that pronounced in female mice. Based on these findings, body composition of wild-type and knockout mice did not differ significantly. But a slight significant genotype dependent difference was observed, when taking female and male mice together displaying lower body mass of knockout mice (p=0.041). Although fat mass of wild-type and knockout mice does not differ significantly, still there is a trend towards lower fat mass in knockout mice. In addition to body composition, body surface temperature was measured to examine any discrepancies in overall body temperature. A trend to increased body temperature of male knockout mice compared to wild-type was found (less so for female mice) (p=0.091).

[0089] It was of interest to examine whether knockout mice display a different pattern in parameters involved in energy metabolism. For this purpose blood plasma parameters were controlled in mice that were fasted overnight. The screen was focusing on cholesterol, HDL-cholesterol and glycerol levels, which play an important role in lipid metabolism and as structural membrane components. Furthermore these parameters can be used as indicator for the diagnosis of the metabolic syndrome. Serum cholesterol as well as HDL-cholesterol levels of female and male knockout mice (separately) showed a trend towards decreased cholesterol and HDL-cholesterol levels (FIGS. 6A and 6B). In contrast, glycerol levels of knockout mice were slightly increased in both sexes (FIG. 6C). When comparing wild-type and knockout mice of both sexes together HDL-cholesterol and cholesterol levels were significantly decreased in knockout animals compared to control mice (p=0.018 cholesterol; p=0.016 HDL-cholesterol).

[0090] A cohort of 10 male wild-type and knockout mice each at the age of 7 months was divided into 4 groups. One group of wild-type mice obtained food containing high fat and the other was just fed with food usually used for animal keeping. The same procedure was applied for knockout mice. This special feeding was conducted over a time span of 6 weeks, including weekly measurement of body weight. As expected wild-type mice on a high fat diet showed a continuously increasing body weight differential over a time span of 3 weeks when compared to mice fed with control food. Elevated body weight was also observed in knockout mice even though the difference to control-fed mice was not significant (FIG. 7A). When concentrating on the weekly weight gain of mice fed with control diet, no genotype dependent discrepancy could be observed. Wild-type mice significantly increased their body weight 1 week upon feeding a high fat diet compared to Fand1 knockout animals, keeping this trend until the end of the experiment after 6 weeks (FIG. 7B).

Example 6: FAHD1 Western Blot

[0091] Method:

[0092] Frozen mouse organs were homogenized in PBS supplemented with protease inhibitors (1 Complete Mini EDTA-free tablet per 10 ml, Roche). Lysate supernatants (30 μg total protein) were separated by SDS-5 PAGE (12.5% acrylamide) and blotted onto a PVDF membrane. Rabbit monoclonal anti-mouse FAHD1 antibody (purpose-made, 14 μg/ml) and anti-rabbit HRP-conjugated secondary antibody (Dako P0399, 1:2,500) were applied by standard Western blot protocol. α-tubulin antibody (Sigma T5168, 1:10,000) was used for loading control with an anti-mouse HRP-conjugated secondary antibody (Dako P0447, 1:20,000). Detection was achieved by ECL Prime (GE Healthcare).

[0093] Results:

[0094] In mice, FAHD1 is predominantly expressed in kidney and liver.

Example 7: Analysis of Oxaloacetate Levels in Mouse Tissue

[0095] Method:

[0096] 3-month-old female C57BL/6 mice (wild-type and FAHD1.sup.−/− littermates) were sacrificed by cervical dislocation and the desired organs (kidney and liver) were immediately excised and shock-frozen in liquid nitrogen. Frozen organs were homogenized in ice-cold 5% perchloric acid (1 ml per 100 mg tissue) and assayed according to the method described by Parvin et al. (Anal Biochem, 104, 1980). Briefly, after hydrolysis of endogenous acetyl-CoA and subsequent neutralization, a 25 μl aliquot was included in a 200 μl reaction containing 0.6 units citrate synthase (Sigma) and 3 pmol [.sup.3H]acetyl-CoA (39 nCi, Moravek Biochemicals) to transform endogenous oxaloacetate into [.sup.3H]citrate. After adsorption of unreacted [.sup.3H]acetyl-CoA to activated charcoal (Sigma), samples were measured in a liquid scintillation counter (LS 6500, Beckman).

[0097] Results:

[0098] Metabolites were extracted from both the kidney and the liver of FAHD1.sup.−/− Mice and wild-type littermates as described above. The concentration of oxaloacetate in these extracts was determined with the above enzymatic assay utilizing the reaction with .sup.3H-labelled acetyl-CoA to form citrate. The concentration of oxaloacetate was significantly increased in both the kidney (FIG. 3A) and the liver (FIG. 3B) of FAHD1.sup.−/− mice, indicating that oxaloacetate is indeed a relevant in vivo substrate for FAHD1 in mice.

Example 8: Modelling of Nematode FAHD-1 Structure

[0099] Method:

[0100] A homology model was generated to compare the structure of FAHD-1 from C. elegans based on the sequence of ZK688.3 (NP 498715.1). As expected, sequence search revealed the FAH domain containing structures as suitable templates. Sequence identity is 46.3% for FAHD1 (PDB code 1SAW), with a similarity of 64.5%. Although this structure is the closest in sequence, the FAH protein from Yesinia pestis C092 (PDB code 3S52) was selected as template, having a better resolution and a completely resolved structure for chain A (closed and structured lid). The template and the target sequence share 39.7% sequence identity. The model was generated with MOE homology modelling tool (Chemical Computing Group, MOE release 2013.08) with chain A and D of 3S52 as templates for a dimer model using the force field option Amber12EHT. The model had unexpected cis amid configurations for Arg8 in chain A and B as well as Lys13 in chain A, which are solvent exposed or in the dimer interface respectively. They are associated with outliers in the Ramachandran plot for the neighboring Asn9 in chain B and Lys13. Additionally the distal residue Asn147 in chain B and Pro135 in both subunits have suspicious backbone configurations. However, the binding site shows no parameters indicating quality issues in the model structure. In the active site, side chains orientations of Arg100 and Glu65 were manually adapted. The initial model was complemented by water positions and co-crystallized ions from the structure of human FAHD1 (PDB code 1SAW) and not the template as the latter does not include the magnesium ion in the active site. Eight individual water molecules forming too close contacts with the model were removed. To allow water and active site adaptions to the magnesium ion, the assembly was energy minimized in several steps with decreasing positional restraints on the atoms.

[0101] Results:

[0102] The nematode protein, referred to as FAHD-1, contains 48% identical amino acids relative to human FAHD1, shows high amino acid sequence similarity in the presumed catalytic centre, and computational modelling revealed a structure of the catalytic centre highly similar to human FAHD1 (FIG. 1B).

Example 9: FAHD-1 Mutant Nematodes

[0103] The fahd-1(tm5005) mutant C. elegans strain was obtained from the Japanese ‘National Bioresource Project’ for the experimental animal ‘Nematode C. elegans’. This mutant carries a 236 bp deletion in the fahd-1 gene that removes exon 2 and leads to a frameshift in exon 3, and was confirmed by genomic PCR and Western blot, using a peptide-specific rabbit polyclonal antibody raised against FAHD-1. The strain was backcrossed six times to N2 Bristol wild-type C. elegans to eliminate any possible second-site mutations.

Example 10: C elegans Lifespan Analysis

[0104] Method:

[0105] Lifespan assays were conducted according to established methods (Artal-Sanz & Tavernarakis, 2009, Nature 461) at 25° C. Animal populations were synchronized by allowing adult hermaphrodites to lay eggs for a limited time interval (2 hours) on NGM plates seeded with E. coli OP50. These synchronized embryos developed into adulthood under controlled conditions and were then spread on fresh plates (20 worms per plate), totaling 150-200 individuals per experiment. The day of egg harvest was defined as t=0. Animals were moved to fresh plates every 1-2 days and examined daily for touch-provoked movement and pharyngeal pumping. Worms dying due to internally hatched eggs, an extruded gonad, or desiccation due to leaving the agar were censored and incorporated as such into the data set. Each survival assay was repeated at least three times. Survival curves were generated according to the product-limit method of Kaplan and Meier. Differences between survivals and p values were evaluated via the log-rank (Mantel-Cox) test. The Prism software package (GraphPad Software) was used to carry out statistical analysis and to determine lifespan values.

[0106] Results:

[0107] Deletion of FAHD-1 resulted in a significant extension of lifespan (FIG. 4A). The effect was most pronounced in worms grown at 25° C. In addition, fahd-1(tm5005) mutant animals displayed severe locomotion defects and a strongly reduced capacity for physical activity, as revealed by an endurance swimming test in liquid (FIG. 4B). Together, these results establish FAHD-1 as a novel important determinant of mitochondrial metabolism, the deletion of which impairs mitochondrial function and physical fitness in nematodes.

Example 11: Assessment of Mitochondrial Membrane Potential in C. elegans

[0108] Method:

[0109] L4 larvae of wild-type and fahd-1(tm5005) C. elegans were placed on NGM plates seeded with E. coli OP50 and containing 100 nM tetramethylrhodamine ethyl ester (TMRE, Sigma). After overnight incubation at 20° C. the worms were imaged within 3 minutes after anaesthetizing with 10 mM levamisol hydrochloride VETRANAL (Fluka), using a Nikon Eclipse TE300 microscope.

[0110] Results:

[0111] A significant reduction of mitochondrial membrane potential in fahd-1(tm5005) mutants was observed relative to wild-type worms, indicating that FAHD-1 is required for proper function of mitochondria in nematodes.