# Patent

## Title
Method for assaying FTO (2-oxoglutarate dependent oxygenase) activity

## Abstract

The present invention provides a method for assaying oxygenase activity, the method comprising monitoring oxygenase activity of FTO.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national stage filing under 35 U.S.C. §371 of international application PCT/GB2008/002119, filed Jun. 20, 2008, which claims priority from Great Britain Patent Application 0711955.5, filed Jun. 20, 2007, and Great Britain Patent Application 0721944.7, filed Nov. 8, 2007.
FIELD OF THE INVENTION
The present invention relates to assays for monitoring activity of the FTO protein and, in particular, to assays for identifying inhibitors, activators and substrates of the FTO protein. The present invention also relates to the treatment or prevention of weight gain, weight loss and disorders associated with weight gain and weight loss.
BACKGROUND TO THE INVENTION
Obesity correlates with an increased risk of a range of diseases including some types of cancer, heart disease and type two diabetes. Recently it has been reported that the presence of specific alleles in the FTO gene, located on human chromosome 16, correlates with obesity and type two diabetes. Specifically, sufferers of type two diabetes were found to have an increased likelihood of a particular FTO variant (rs9939609 A allele) which correlates with an increased body weight (Frayling et al. Science (2007) 316: 889-894). Subsequent analyses involving more than 39,000 individuals revealed that the FTO allele was associated with body weight. Individuals carrying one copy of the FTO variant associated with type two diabetes had a 30% increased risk of being obese compared to an individual with no copies of that version and were on average more than 1.2 kg heavier than individuals with no copies of the disease-associated variant. Individuals with two copies of the variant (about 16% of those analyzed) had a 70% increased risk of being obese and on average weighed 3 kg more than individuals with no copies of the disease-associated variant. This study is important, as no previous work has identified a risk allele for obesity that is so prevalent. Further studies show similar results (Dina et al. Nat Genet (2007) 39: 724-726; Scott et al. Science (2007) 316: 1341-1345).
The mouse ortholog of FTO, Fatso (Fto), is a gene that is deleted in the fused toes mouse mutant (Peters et al. Mann Genome (2002) 13: 186-188; van der Hoeven et al. Development (1994) 120: 1601-2607, Grotewold & Ruther, Dev Biol (2002) 251: 129-141; Anselme et al. Dev Biol (2007) 304: 208-220). For clarity, subsequent reference to FTO includes human FTO, non-human homologues and/or any of their clinically observed variations.
Although FTO sequence variation is linked to obesity, the function of its protein product at the biochemical, cellular and physiological levels has not been reported.
Knowledge about FTO structure and its biochemical, cellular and physiological roles is needed to enable the correlation between FTO and obesity to be exploited for the treatment of diseases associated with weight gain, such as diabetes, cardiovascular disease, cancer, osteoporosis and hypertenstion.
The 2-oxoglutarate (2-OG) and ferrous iron dependent oxygenases are a superfamily of enzymes that catalyse a wide range of reactions including hydroxylations, desaturations and oxidative ring closures (Hausinger (2004), Crit. Rev. Biochem. Mol. Biol. 39, 21-68; Ryle & Hausinger (2002) Curr. Opin. Chem. Biol. 6, 193-201; and Schofield et al. (1999) Journal of Inorganic Biochemistry 74, 49-49). Substrate oxidation is coupled to conversion of 2-OG to succinate and carbon dioxide. At least in some cases, binding of oxygen is followed by the oxidative decarboxylation of 2-OG to give succinate, CO2 and a ferryl species [Fe(IV)=O] at the iron centre. This highly reactive intermediate can then oxidize an unactivated C—H bond in the prime substrate, e.g. the oxidation of prolyl or asparaginyl residues in human proteins, or effect other oxidative reactions such as oxidation of methyl groups on N-methylated versions of proteins or nucleic acids. Evidence for intermediates comes from substrate-analogue studies, model compounds and spectroscopic analyses.
The sequential binding of co-substrate and prime substrate, which is necessary to trigger oxygen binding, is probably important in limiting the generation of reactive oxidizing species in the absence of prime substrate. The generation of such species in a prime-substrate-uncoupled manner can inactivate 2-OG and the related oxygenases through self-oxidation, which sometimes leads to fragmentation. Typically, the uncoupled turnover of 2-OG occurs at approximately 5% of the rate of its coupled turnover in the presence of saturating concentrations of prime substrate, although it can also occur at a lower or higher rate.
Several 2-OG-dependent oxygenases, including procollagen prolyl hydroxylase, the hypoxia inducible factor prolyl hydroxylases, and anthocyanidin synthase, have a requirement for ascorbate for full catalytic activity. Although ascorbate might stimulate activity by reducing Fe3+, or other high valent forms of iron, to Fe2+ (either free in solution or at the active site), the stimulation of oxygenase activity by ascorbate might occur by other mechanisms, for instance, by promoting completion of uncoupled cycles. For uncoupled reaction cycles that are catalysed by procollagen prolyl hydroxylase in the absence of prime substrate, the oxidation of 2-OG to succinate has been shown to be stoichiometrically coupled to ascorbate.
Furthermore, the activity of ascorbate has been shown to stimulate the activity of 2-OG oxygenases in cells (e.g. in work on the hypoxia inducible factor (HIF) prolyl hydroxylase) and lack of ascorbate in the human diet leads to the disease scurvy due to impaired activity of the procollagen prolyl hydroxylase.
Studies with several enzymes have shown that certain substrate analogues and mutants can also stimulate uncoupled 2-OG turnover. It is also known in the literature that reducing agents other than ascorbate itself can act as reducing agents in the uncoupled turnover reaction, including derivatives of ascorbate (Zhang et al. (1995) Biochem. J. 307 (Pt 1), 77-85 and Myllyla et al. (1978) Biochem. Biophys. Res. Commun. 83, 441-8).
A number of 2-OG oxygenases are of current therapeutic interest including the transcription factor hydroxylases, e.g. the hypoxia inducible factor prolyl and asparaginyl hydroxylases, methylated nucleic acid demethylases, methylated lysyl demethylases, procollagen prolyl and lysyl hydroxylases, phytanoyl CoA hydroxylase, Mina53, NO66 and arginyl hydroxylases such as phosphatidyl serine receptor (Jmjd6). The methyl lysyl demethylases may use methylated histones as preferred substrates and may use any of all the tri, di, or mono-methylated lysine residues as preferred substrates.
Methylation of nucleic acid and nucleic acid associated acid proteins, including histones, is a known mechanism for epigenetic inheritance. Methylation of nucleic acids can also affect gene activity and expression.
SUMMARY OF THE INVENTION
The present inventors have identified FTO as a 2-oxoglutarate (2-OG) dependent oxygenase and have demonstrated for the first time that FTO is a 2-OG dependent oxygenase. The present inventors have successfully purified recombinant FTO and demonstrated that the purified recombinant FTO functions as a 2-OG dependent oxygenase and can be inhibited by known 2-OG oxygenase inhibitors.
Accordingly, the present invention provides a method for assaying FTO activity, the method comprising monitoring oxygenase activity of a FTO polypeptide.
In a method of the invention oxygen and/or a 2-oxoacid, such as 2-OG may be used as a co-substrate and/or iron may be used as a co-factor. The assay for oxygenase activity may be monitored in the presence of a reducing agent, such as ascorbate, or an analogue thereof, a thiol or a phosphine. In one embodiment, FTO oxygenase activity is measured in the presence of a substrate, such as a peptide or nucleic acid substrate.
The FTO polypeptide used in a method of the invention may be a recombinant polypeptide. In one embodiment, the FTO polypeptide comprises: the amino acid sequence of SEQ ID NO: 1; or an amino acid sequence having at least 40% identity to the amino acid sequence of SEQ ID NO: 1 over its entire length. The amino acid having at least 40% identity to the amino acid sequence of SEQ ID NO: 1 over its entire length may be an amino acid sequence as shown in any one of SEQ ID Nos: 2 to 11. Alternatively, the acid sequence having at least 40% identity to the amino acid sequence of SEQ ID NO: 1 over its entire length may be a naturally occurring variant of human FTO or a homologue of FTO from a species other than human.
In one preferred embodiment, the invention provides a method comprising: contacting a FTO polypeptide with a test agent; monitoring for oxygenase activity in the presence of the test agent; and determining whether the test agent is an inhibitor or activator of FTO activity.
The test agent may be a known inhibitor of a 2-OG oxygenase other than FTO, or an analogue or variant of such an inhibitor.
The 2-OG oxygenase other than FTO may be, for example, a prolyl, lysyl, arginyl or asparaginyl demethylase, procollagen prolyl or lysyl hydroxylases, hypoxia inducible factor prolyl hydroxylase, methylated lysyl demethylase (including histone demethylases), asparaginyl hydroxylase, phosphatidyl serine receptor (Jmjd6), AlkB or human AlkB homologues and/or gibberellin C-20 oxidase. The known 2-OG inhibitor may be an N-oxalyl amino acid such as N-oxalylglycine or a derivative thereof, a glycine or alanine derivative, a 2-oxoacid analogue, a flavonoid or flavonoid derivative such as genistein.
In a method of the invention for identifying an inhibitor or activator of FTO, the test agent may compete with 2-OG or a FTO substrate at the FTO active site and/or bind to the metal at the FTO active site. The test agent may, for example, comprise a metal ion.
In one embodiment the test agent is a reducing agent, which may be a reported activator of a 2-OG oxygenase other than FTO. For example, the test agent may be ascorbate or an analogue of ascorbate or a reducing agent of the thiol, such as a member of the dithiothreitol or phosphine chemical families.
The substrate of the FTO polypeptide used in a method of the invention may be a nucleic acid, nucleic acid derivative or analogue. For example, the substrate may be a methylated nucleic acid. The methylated nucleic acid may be associated with a gene involved in weight modulation, such as the agouti gene or the neuropeptide Y gene, the leptin gene, the proopiomelanocortin gene, the orexin gene, the galanin gene, the PYY gene, the cholecystokinin gene, the glucagon-related peptide-1 gene or the insulin gene.
In one embodiment, the invention provides a method for identifying a substrate of FTO, the method comprising: contacting a FTO polypeptide with a test substrate; monitoring for oxygenase activity; and determining whether the test substrate is a substrate of FTO. The test substrate may be a human nucleic acid sequence, such as a nucleic acid sequence containing a 3-methylthymine base, a 1-methyladenine base or a 3-methylcytosine base. Alternatively, the test substrate may be a methylated protein or peptide. An FTO inhibitor may be used to enable identification of the FTO substrate by stabilizing the interaction between FTO and the substrate prior to identification of the substrate.
In one aspect, the invention provides a method of identifying an inhibitor or activator that selectively inhibits or activates FTO or that activates or inhibits an enzyme other than FTO but does not activate or inhibit FTO, the method comprising monitoring the activity of FTO in the presence of the test agent and repeating the method using an enzyme other than FTO and determining whether the test agent selectively inhibits or activates FTO or the other enzyme.
In one embodiment of the invention, the enzyme other than FTO is typically a 2-OG oxygenase, such as a hypoxia inducible factor hydroxylase, such as a prolyl, asparaginyl or lysyl hydroxylase, a collagen or procollagen prolyl hydroxylase, a nucleic acid demethylase such as an AlkB homologue, or a protein demethylase, such as a tri-, di- or mono-methyl lysine, arginine, asparagine or proline residue demethylase. The protein demethylase may hydroxylate a methylated histone or a fragment thereof.
In further embodiments, the invention provides:

    
    
        the use of an inhibitor or activator of 2-OG oxygenase activity to modulate FTO activity;
        a modulator of FTO oxygenase activity for use in a method of treating or preventing weight gain or weight loss or treating or preventing a disease associated with weight gain or weight loss;
        a method of treating or preventing weight gain or weight loss in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of an inhibitor or activator of FTO oxygenase activity; and
        a method of treating or preventing a disease associated with weight gain or weight loss in an individual in need thereof, the method comprising administering to an individual a therapeutically effective amount of an inhibitor or activator of FTO oxygenase activity.
    
    





BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a clustalW alignment of FTO homologue sequences. Human gi|122937263, chimp gi|114662524 (*99%), Macaque gi|109128525 (*93%), dog gi|73950384 (*91%), cow gi|119910109 (*88%), opossum gi|126296336 (*65%), mouse gi|18490097 (*87%), mouse2 gi|6753916 (*87%), rat gi|89337260 (*87%), fish gi|125821796 (*48%), frog gi|62859671 (*52%) [* indicates identity with human FTO].
FIG. 2 is a homology model of FTO based on its sequence and secondary structure alignment with ABH3 (FIG. 5).
FIG. 3 shows the sequence and secondary structure alignment (secondary structure predicted for FTO) for FTO and ABH3. Roman numerals indicate the eight core DSBH strands. There is a C-terminal helical domain subsequent to the DSBH domain.
FIG. 4 shows the results of a 2-OG turnover assay for determining enzymatic activity of FTO. The assay was carried out using 11.5 μM FTO, 144 μM 20G, 16 μM 14C-20G, 80 μM (NH4)2Fe(II) (SO4)2, 4 μM ascorbate and 1 mM DTT and with one of (NH4)2 Fe(II)(SO4)2, ascorbate and DTT missing from the assay. The assay was also performed in the presence of N-oxalylglycine (NOG).
FIG. 5 shows the results of Mass Spectroscopy (MS) analyses for demethylation of oligonucleotides with recombinant FTO. The data shown is for TTX TTT TTT TTT TTT (T=thymine, X— methylated base) SEQ ID NO: 13 for the 4-charge state. +FTO=incubation with FTO with appropriate cofactors and cosubstrates. 3MeT=3-methyl thymine; 1MeA=1-methyl adenine; 3MeC=3-methylcytosine.
FIG. 6 shows the results of experiments testing the 2-OG-dependent DNA demethylase activity of FTO: (a) Demethylation of 1-meA is dependent on FTO in 2-OG decarboxylation assays; (b) Cofactor/cosubstrate dependence of FTO activity on a 3-meT substrate shown by LC-MS. Data shown represent ratios of thymine to 3-meT in ss-DNA. The oxygen control reaction was carried out in an atmosphere of <1% O2; (c) Inhibition of FTO-catalyzed 1-meA.
FIG. 7 shows: (a) the results of mass spectroscopy analyses for demethylation of single stranded DNA substrates by FTO. LC-MS data for the incubation of synthetic 15-mer of poly T, methylated at the 2-position, with FTO, cofactors and co-substrates; positions of expected peaks for demethylated substrates are indicated by dotted lines. Smaller peaks at higher masses than reactant peaks probably arise from Na+ and K+ adducts of the methylated oligonucleotides. m/z=mass-to-charge ratio, shown in units of Dalton; (b) Stoichiometry of the FTO reaction; (c) Release of formaldehyde from methylated poly(dA) and poly(dT). FTO and ABH3 were assayed for demethylase activity by incubation with CM-methylated poly(dA) or [14C]-methylated poly(dT) (total c.p.m. 1000 or 800, respectively) at 37° C. for 15 minutes. Release of ethanol-soluble [14C]-formaldehyde was monitored. FTO -∘-, -●-; ABH3 -Δ-, -▴-. [14C]-methylated poly(dA)-∘-, -Δ-; [14C]-methylated poly(dT)-●-, -▴-. In the absence of 2-OG, no significant activity was detected.
FIG. 8 shows the results of uncoupled 2-OG turnover experiments: (a) Assays used the 2-OG turnover method; no methylated DNA substrate was added. Experimental setup and procedure were as described in the Examples. Values shown are averages of two independent experiments, 100% corresponds to 130,000 counts of [14C]—CO2; (b) Reduction of uncoupled 2-OG turnover in the presence of inhibitors. DMSO=dimethylsulfoxide. Although N-oxalylglycine can chelate Fe(II), this does not fully account for its FTO inhibitory effect because N-oxalyl-D-phenylalanine (10), an inhibitor of a 2-OG dependent asparagine-hydroxylase, has similar Fe(II) chelating abilities to N-oxalylglycine but did not inhibit FTO activity. Values shown are averages of two independent experiments, 100% corresponds to 130,000 counts of [14C]—CO2; (c) Structures of inhibitors used. Compound 1 was previously identified as a potent 2-OG oxygenase inhibitor Franklin et al., 2001, Biochem J. 353:333 and synthesized using standard synthetic methodology (Banerji et al., 2005, Chem. Comm. 43:5438).



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 is the amino acid sequence of human FTO (gi|122937263).
SEQ ID NO: 2 is the amino acid sequence of chimpanzee FTO which shares 99% sequence identity with human FTO (gi|114662524).
SEQ ID NO: 3 is the amino acid sequence of macaque FTO which shares 93% sequence identity with human FTO (gi|109128525).
SEQ ID NO: 4 is the amino acid sequence of dog FTO which shares 91% sequence identity with human FTO (gi|73950384).
SEQ ID NO: 5 is the amino acid sequence of cow FTO which shares 88% sequence identity with human FTO (gi|119910109).
SEQ ID NO: 6 is the amino acid sequence of opossum FTO which shares 65% sequence identity with human FTO (gi|126296336).
SEQ ID NO: 7 is an amino acid sequence of mouse FTO which shares 87% sequence identity with human FTO (gi|18490097).
SEQ ID NO: 8 is an amino acid sequence of mouse FTO which shares 87% sequence identity with human FTO (gi|6753916).
SEQ ID NO: 9 is the amino acid sequence of rat FTO which shares 87% sequence identity with human FTO (gi|89337260).
SEQ ID NO: 10 is the amino acid sequence of fish FTO which shares 48% sequence identity with human FTO (gi|125821796).
SEQ ID NO: 11 is the amino acid sequence of frog FTO which shares 52% sequence identity with human FTO (gi|162859671).
SEQ ID NO: 12 is the amino acid sequence of human ABH3 (gi|21040275).
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have purified recombinant FTO and identified FTO as a 2-oxoglutarate (2-OG) dependent oxygenase. Accordingly, the present invention provides a method for assaying FTO activity, the method comprising monitoring oxygenase activity of a FTO polypeptide. The present invention also provides the use of a FTO polypeptide in assay methods to identify modulators of FTO oxygenase activity and substrates that are oxidised by FTO.
The present inventors have found that FTO is homologous to the Fe(II) and 2-OG dependent oxygenases with particularly strong sequence similarity to the human DNA demethylase, ABH3, which has known activity as a demethylase on 1-methyladenine and 3-methylcytosine nucleic acid bases. The FTO polypeptide for use in the invention typically binds Fe2+. Therefore, in one aspect of the invention, iron is used as a cofactor in the method for assaying FTO activity.
The FTO polypeptide for use in accordance with the invention typically has the ability to convert 2-OG and oxygen to succinate and carbon dioxide. Accordingly, in one aspect of the invention, the method for assaying FTO activity uses oxygen and/or 2-OG as co-substrates.
The FTO polypeptide may comprise the sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 40% sequence identity, for example at least 45% sequence identity, with SEQ ID NO: 1 in the double-stranded beta-helix (DSBH) domain or at least about 40%, for example at least about 50% or about 60% sequence identity with the amino acid sequence of SEQ ID NO: 1, over its entire length, typically greater than 70% or 80%, more typically greater than about 90% or 95%, such as about 99% sequence identity.
Sequence identity may be calculated using any suitable algorithm. For example, the UWGCG Package provides the BESTFIT program can be used to infer homology (for example used on its default settings) (Devereux et al. (1984) Nucleic Acids Research 12, p 387-395). The PILEUP and BLAST algorithms can be used to infer homology or line up sequences (typically on their default settings), for example as described in Latched (1993) J. Mol. Evol 36:290-300 or Latched et al. (1990) J. Mol. Biol. 215:403-10.
The FTO polypeptide may be a polypeptide encoded by any naturally occurring FTO gene. The naturally occurring FTO gene may comprise the sequence shown in SEQ ID NO: 1 or may be a variant of SEQ ID NO: 1. Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the polypeptide retains 2-OG oxygenase activity. The polypeptide also preferably has a nuclear localisation signal, in particular where the activity of the polypeptide is monitored using a cell based assay. The nuclear localisation signal may be the FTO nuclear localisation signal sequence or a nuclear localisation signal sequence from another peptide.
The FTO polypeptide may be a homologue of FTO from a non-human species such as the chimpanzee (SEQ ID NO: 2), macaque (SEQ ID NO: 3), dog (SEQ ID NO: 4), cow (SEQ ID NO: 5), opossum (SEQ ID NO: 6), mouse (SEQ ID NO: 7 or 8), rat (SEQ ID NO: 9), fish (SEQ ID NO: 10), frog homologue (SEQ ID NO: 11) of FTO or a homologue from another organism, such green algae. The homologue of FTO may be a naturally occurring protein. The FTO polypeptide may have an amino acid sequence that does not occur in nature but which contains the deletion, modification or addition of single amino acids or groups of amino acids compared to a naturally occurring FTO sequence. For example, the FTO polypeptide may have at least about 40%, such as at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence identity with any one of SEQ ID NOs: 1 to 11.
Amino acid substitutions of any one of SEQ ID NOs: 1 to 11 may be made, for example from about 1, 2 or 3 to about 10, 20 or 30 substitutions. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

















ALIPHATIC
Non-polar
G A P





I L V




Polar-uncharged
C S T M





N Q




Polar-charged
D E





K R



AROMATIC

H F W Y










Variant polypeptides within the scope of the invention may be generated by any suitable method, for example by gene shuffling techniques.
The present invention also includes use of active portions, fragments, derivatives and functional mimetic of the polypeptides of the invention. An “active portion” of a polypeptide means a peptide which is less than said full-length polypeptide, but which retains 2-OG oxygenase activity. An active fragment of FTO may typically be identified by monitoring for 2-OG oxygenase activity as described in more detail below. Such an active fragment may be included as part of a fusion protein, e.g. including a binding portion for a different i.e. heterologous ligand.
The fragment may have at least about 50 amino acids or up to about 60, 70, 80, 100, 150, 200, 400 or 500 amino acids. In particular, but not exclusively, this aspect of the invention encompasses the situation when the protein is a fragment of the complete FTO protein sequence and may represent a catalytic region, capable of converting 2-OG to succinate and carbon dioxide. The catalytic cores of human, chimpanzee, macaque, dog, cow, opossum, mouse, rat, fish and frog FTOs are shown in SEQ ID NOs: 1 to 11. In one embodiment the fragment may comprise the catalytic core and the nucleotide-recognition lid that lies adjacent to the N-terminal end of the catalytic core.
The fragment may comprise any region from about amino acid 1 to about 505 of the amino acid sequence shown in SEQ ID NO: 1, such as from amino acid 2, 3, 4, 5 or 10 to amino acid 495, 500, 501, 502, 503 or 504. Useful fragments include N-terminal truncated fragments i.e., fragments comprising an N-terminal deletion, such as fragments comprising residues 30 to 505 or 60 to 505 of the amino acid sequence shown in SEQ ID NO: 1 and fragments comprising both N-terminal and C-terminal truncations, such as fragment comprising residues 30 to 33 or 60 to 330 of the amino acid sequence shown in SEQ ID NO: 1. Other suitable fragments may readily be identified, for example by comparing the FTO amino acid sequence to the amino acid sequence of one or more known 2-OG dependent oxygenase and identifying which regions are not homologous to regions having catalytic activity. The regions having catalytic activity are typically included in the active fragments. Such fragments can be used to construct chimeric molecules.
Fragments of any FTO polypeptide having at least about 60%, such as at least about 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence column in SEQ ID NO: 1 (for example, fragments of any naturally occurring or genetically engineered sequence discussed above including fragments of any one of SEQ ID NOs: 2 to 11) which fragments have oxygenase activity may also be used in an assay of the invention and are encompassed within the term “FTO polypeptide” used herein.
The FTO polypeptides may be synthetically prepared. The polypeptides may be chemically or biochemically modified, e.g. post-translationally modified. For example, they may be glycosylated or comprise modified amino acid residues. They may also be modified by the addition of histidine residues (typically six), or other sequence tags such as a maltose binding protein tag or intein tag, to assist their purification or by the addition of a nuclear localisation sequence to promote translocation to the nucleus or mitochondria, and or by post translational modification including hydroxylation or phosphorylation. Polypeptides of the invention may be GST or other suitable fusion polypeptides. The FTO polypeptide may also be modified by addition of fluorescent tags (such as green fluorescent protein) to enable visualization within cells or organelles or to aid purification of the protein or cells expressing FTO. Such modified polypeptides fall within the scope of the term “FTO polypeptide”.
The polypeptides of the invention may be present in a partially purified or in a substantially isolated form. They may be mixed with carriers or diluents, which will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least about 90%, e.g. at least about 95%, 98% or 99%, of the proteins, polynucleotides, cells or dry mass of the preparation.
The polypeptides of the invention may be used in assays for 2-OG dependent oxygenase activity, for example to identify modulators, such as inhibitors or activators of hydroxylase activity. The inhibitors may be selective inhibitors or activators.
The FTO polypeptides may be used in assays for 2-OG oxygenase activity in the absence of a prime substrate (i.e, a non 2-OG substrate). The FTO polypeptides may also be used to determine oxygenase activity in the presence of one or more suitable substrates. In addition, the present invention provides methods for identifying substrates of FTO.
FTO used in a method of the invention may be recombinant FTO or naturally occurring FTO. Preferably, recombinant FTO is used especially where FTO is required for purposes requiring large (>1 mg) amounts of protein such as for biophysical assays or for high throughput analyses. Recombinant FTO may be produced using standard expression vectors that comprise nucleotide sequences encoding FTO. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. 1989.
Methods of the invention may utilise cells that have been modified to express a FTO polypeptide as defined herein. The FTO may also be present in a cell extract or in a partially or substantially purified form.
Methods for Obtaining Purified FTO
The present inventors have found that it is possible to express a recombinant FTO in soluble and active form using a modified expression and purification method. The inventors have also demonstrated that purified recombinant FTO is a 2-OG dependent oxygenase. Accordingly, the invention provides a method for obtaining purified FTO and a method for assaying oxygenase activity of purified FTO.
A purified FTO polypeptide may be obtained by introducing an expression vector comprising a polynucleotide encoding a FTO polypeptide into a host cell.
Expression vectors are routinely constructed in the art and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary and which are positioned in the correct orientation in order to allow full protein expression. Suitable vectors would be very readily apparent to those of skill in the art. Promoter sequences may be inducible or constitutive promoters depending on the selected assay format. The promoter may be tissue specific. Thus the coding sequence in the vector is operably linked to such elements so that they provide for expression of the coding sequence (typically in a cell). The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
The vector may be, for example, a plasmid, virus or baculovirus vector. The vector is typically adapted to be used in a bacterial cell, such as E. coli. The vector may have an origin of replication. The vector may comprise one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used to transfect or transform a host cell, for example, a bacterial host cell, fungal host cell, an insect host cell, a mammalian, e.g. human host cell or a baculovirus host cell. The bacterial host cell is preferably a strain of E. coli, for example BL21 (DE3).
A method of producing a purified FTO polypeptide is provided by the invention. The method typically comprises culturing host cells comprising an expression vector encoding a FTO polypeptide and isolating the FTO polypeptide from the cells. The host cells may typically be cultured, for example, at a temperature of from about 15° C. to about 37° C. The polypeptide may be isolated by lysing the cells and extracting the protein from the lysis buffer. The lysis buffer typically comprises from about 250 mM to about 700 mM salt, e.g. NaCl, such as from about 400 mM to about 600 mM, e.g. 500 mM. A method of producing a FTO polypeptide according to the invention may further comprise introducing a polynucleotide or vector according to the invention into the host cell. The FTO polypeptide is comprised in the soluble fraction obtained upon lysis of the cell culture. The polypeptide may be further purified from the soluble fraction, for example by affinity purification, such as via an affinity tag fused to the truncated 2-OG dependent oxygenase.
Methods for introducing polypeptides and vectors into host cells are well known in the art, and include electroporation and heat shock techniques without limitation. Expression of the truncated polypeptide may then be achieved by culturing the host cells at a suitable temperature. The cells expressing recombinant FTO are preferably kept at between about 15° C. and about 30° C., for example at about 20° C. or about 28° C. to induce expression of recombinant FTO. Where the host cells are bacteria, such as E. coli, the cells may be cultured in 2TY medium. IPTG may be added to the culture medium, either throughout the period of incubation (or growth period) or in the final stages of the incubation period.
The lysis buffer containing a high salt level is typically used to lyse the cells after centrifuging the cells to remove the cell culture medium. The buffer typically contains from about 250 mmol salt, e.g. NaCl, to about 700 mmol salt, for example from about 400 to about 600 mmol NaCl, such as about 500 mmol NaCl. The extraction buffer may comprise detergents, such as Triton X-100 and/or SDS (typically 1%), and/or lysozyme. Glycerol may be present in the lysis buffer, typically at a concentration of from about 5% to about 20%, such as about 10%. The lysis buffer typically has a pH greater than about 7.5, for example from about 7.6 to about 8.1, from about 7.8 to about 8.0, more preferably about 7.9. The lysis buffer may be suitable for sonication of the cells. Tris may also be present in the lysis buffer, for example at a concentration of from about 10 mmol to about 100 mmol, such as about 20 mmol.
After lysis, the cells may be centrifuged. After centrifugation, the supernatant represents the soluble fraction. The concentration of proteins present in the soluble fraction depends on the quantity of extraction buffer used. The FTO is present in the soluble fraction in an amount sufficient for it to be purified. This can be determined by SDS PAGE. If it is possible to detect the truncated enzyme by SDS PAGE, there is sufficient enzyme present for purification.
FTO polypeptides of the invention may be purified by standard techniques known in the art. For example, where the polypeptide comprises a His tag, it may be purified using a his-binding resin by following the manufacturer's instructions (e.g. Novagen) or by other means such as ion exchange chromatography. The purification procedure may comprise the following steps. The cells expressing a recombinant polypeptide of the invention may be pelleted and resuspended in a suitable buffer and then sonicated to break up the cells. The cell debris is separated from the soluble material by centrifugation and the soluble fraction is loaded on a his-binding column. After washing the column with binding buffer and wash buffer, the bound protein is eluted from the column using elution buffer. The binding, wash and elution buffers each typically comprise 0.5M NaCl. It is not necessary to add additional salt. The eluted protein is then concentrated and incubated with thrombin (typically at a concentration of 1 Umg−1 at 4° C. for 16 hours). The digested proteins are separated using a gel filtration column and the FTO eluted from the column is generally at least 90%, or at least 95% pure. The purified protein for use in the various assays described herein may be de-salted.
Assays
Our data show that FTO catalyses the conversion of 2-OG to succinate and carbon dioxide. This newly discovered activity of FTO means that, for the first time, assays for identifying inhibitors or stimulators/activators of FTO activity can be performed. Blocking or activating 2-OG oxygenase activity of FTO will result in weight modulation. Any suitable assay may be carried out to identify modulators of FTO oxygenase activity and in particular of 2-OG oxygenase activity. A number of different examples of suitable assays are described below. In one embodiment, the assays utilise a human FTO polypeptide as described herein. FTO polypeptides may be provided either in purified or unpurified form, for example as cellular extracts or by purification of the relevant polypeptides from such extracts. Alternatively, the relevant component can be expressed using recombinant expression techniques and purified for use in the assay. Alternatively, the components may be expressed recombinantly in a cell for use in cell-based assays.
Assay Methods
The FTO polypeptides may be used in an assay for oxygenase activity, such as 2-OG oxygenase activity. These polypeptides are also useful in assays for identifying an agent, which modulates, such as inhibits or activates, FTO oxygenase activity. The method comprises contacting a FTO polypeptide and a test substance, such as a potential inhibitor, in the presence of one or more co-substrate and optionally a prime substrate. The test substance and FTP polypeptide are typically contacted under conditions suitable for oxygenase activity. Suitable co-substrates include oxygen, for example, dioxygen, and 2-oxoacids such as 2-OG. Preferably, the co-substrate is 2-OG. In addition to oxygen or a 2-oxoacid, a reducing agent, such as ascorbate may also be used as a co-substrate. The components of the assay are contacted under conditions in which the enzyme acts on the co-substrate in the absence of the test substance and determining the extent of co-substrate modification. Alternatively, oxidation of the prime substrate may be monitored. Assays that detect binding to FTO in the absence of catalytic turnover may also be used. Such assays may employ techniques such as chromatography, NMR, MS or fluorescence spectroscopy. The co-substrate may be modified, e.g. 2-OG, or consumed, e.g. oxygen or ascorbate, by FTO. Demethylation activity may be monitored by measuring formaldehyde release Suitable methods for measuring formaldehyde release are known in the art. Such methods are, for example, described in Kleeberg and Klinger (1982) J. Pharmacol. Methods 8: 19-31, Trewick et al. (2002) Nature 419: 174-178 and Tsukada et al. (2006) Nature 439: 811-816. The assay may also be used to detect substances that increase the activity of the 2-OG dependent oxygenase by assaying for increases in activity. Suitable assays have been described in the art for other 2-OG dependent oxygenases.
Such assays of the present invention may be used to identify inhibitors of oxygenase activity and are thus preferably carried out under conditions under which FTO is active as an oxygenase including in the absence of the test substance. The FTO oxygenase activity in the presence of the test substance is compared to FTO oxygenase activity in the absence of the test substance to determine whether the test substance is an inhibitor of FTO oxygenase activity. In the alternative, the assays may be used to look for promoters of FTO oxygenase activity, for example, by looking for increased conversion of co-substrate and/or hydroxylation of substrates compared to assays carried out in the absence of a test substance. The assays may also be carried out under conditions in which oxygenase activity is reduced or absent, such as under hypoxic conditions, and the presence of or increased activity could be monitored under such conditions.
The assays of the invention may also be used to identify inhibitors or activators which are specific for FTO and which do not have activity or are less active with other 2-OG oxygenases, for example, such as the procollagen hydroxylases, human ABHs (AlkB homologues), methyl lysine demethylases, hypoxia inducible factor (HIF) asparaginyl or prolyl hydroxylases. Conversely, the assays of the invention may be used to identify inhibitors or activators specific for one or more 2-OG dependent oxygenase, for example, such as HIF asparaginyl or prolyl hydroxylases, which do not inhibit FTO.
The assays of the invention may also be used to identify inhibitors or activators which are specific for FTO activity at a particular substrate or residue within a substrate.
In medicinal applications, for example, it is often advantageous to modulate oxygenase activity of a single enzyme or group of enzymes. Assays of the invention may therefore be used to identify agents which selectively modulate activity of FTO relative to a second 2-OG dependent oxygenase, including but not limited to lysyl, prolyl, asparaginyl and arginyl demethylases, the HIF hydroxylases, including FIH, PHD1, PHD2 and PHD3, AlkB, ABH1, ABH2, ABH3, procollagen prolyl and lysyl hydroxylases, the phosphatidyl serine receptor (Jmjd6), Mina53 and 2-OG oxygenases that have been characterized as Jmj domain proteins according to the SMART database including, but not limited to lysyl demethylases.
Such selectivity screens may be used to identify selective inhibitors of FTO or selective inhibitors of other enzymes, i.e. inhibitors that are more potent inhibitors of FTO activity than of activity of the other enzyme or inhibitors that are less potent inhibitors of FTO activity than of activity of the other enzyme. Where the inhibitor is a selective inhibitor of FTO activity it may have no effect on the activity of the other enzyme or may exhibit only a low level of inhibition, such as less than about 50% inhibition on activity of the other enzyme. Where the inhibitor is a selective inhibitor of activity of the non-FTO enzyme it may have no effect on the activity of FTO or may exhibit only a low level of inhibition, such as less than about 50% inhibition of FTO activity.
The selectivity screens may be carried out with purified enzymes, partially purified enzymes (such as in crude cell lysates) or in cells.
The invention provides the use of selective inhibitors in the manufacture of a medicament for the treatment of a condition associated with altered, i.e. enhanced or reduced, 2-OG dependent oxygenase activity, such as FTO oxygenase activity.
It is also possible, using the method of the invention to identify selective inhibitors when the prime substrate of one or more of the enzymes being tested is unknown. In this embodiment, generally it will be one or more of the enzymes that it is wished not to inhibit that is an enzyme that has an unknown substrate. The effect of a test agent on activity of an oxygenase may be determined in the absence of a substrate by determining whether or not the test agent affects, for example inhibits or stimulates, the rate of turnover of 2-OG by the oxygenase.
The assays of the present invention may use a substrate that is hydroxylated or otherwise oxidised by FTO. In particular, such substrates may be used in assays to monitor for the activity of a modulator of FTO 2-OG oxygenase activity. The substrate may be a peptide or nucleic acid substrate. The nucleic acid substrate may be DNA or RNA. Oligonucleotide substrates may be used. The DNA is preferably nuclear DNA, but may be mitochondrial DNA. Preferably the nucleic acid substrate is methylated at one or more residues.
Any suitable substrate which is hydroxylated, or more generally oxidised, by FTO, with FTO typically having the amino acid sequence or a variant of SEQ ID NO: 1, may be used. Some substrates of 2-OG dependent oxygenases are well known in the art. The substrate may be a naturally occurring protein or a recombinant or synthetic protein or a nucleic acid. Fragments and variants of naturally occurring substrate proteins or nucleic acids which include the site of oxidisation by FTO may be used as substrates in the assay of the invention.
In an assay to identify a selective inhibitor of FTO, or another oxygenase, different substrates may be used for FTO and for the other oxygenase(s).
The methods of the invention may be used to detect novel substrates of FTO 2-OG dependent oxygenase activity. In such an assay a test substrate is used and the detection of hydroxylase activity indicates that hydroxylation of the test substrate has occurred and, accordingly, that the test substrate is a substrate of the FTO. Such assays may be carried out with isolated components, semi-purified extracts or in whole cells.
The test substrate may be a nucleic acid, nucleic acid derivative or analogue, such as a methylated nucleic acid, or may be a protein or peptide, such as a methylated protein or peptide. The methylated nucleic acid, protein or peptide may be associated with a gene, protein or peptide involved in weight modulation. For example, the nucleic acid may be a part of the gene or a mRNA transcribed from the gene or the peptide or protein may be encoded by the gene. Examples of genes and peptides involved with weight modulation include the agouti gene and agouti related peptide and the neuropeptide Y gene and neuropeptide Y related peptide gene and the following peptides and the genes encoding them: leptin, proopiomelanocortin, orexin, galanin, PYY, cholecystokinin, glucagon-related peptide-1 and insulin.
Inhibitors of FTO activity, including but not limited to 2-OG analogues such as N-oxoalylglycine and transition metals such as Zinc, magnesium, manganese, cobalt and nickel, may be used to enable identification of a FTO substrate by stabilising the interaction between FTO and the substrate and then identifying the substrate, or binding partner, bound to FTO. Standard methods may be used to identify the substrate, or binding partner. Examples of techniques that may be used techniques involving mass spectroscopy or antibodies or other standard reagents and methodologies used within the art of proteomics. Alternatively binding assays or cell-based assays may be used.
Methods for Monitoring Modulation
The precise format of any of the screening or assay methods of the present invention may be varied by those of skill in the art using routine skill and knowledge. The skilled person is well aware of the need to additionally employ appropriate controlled experiments. The assays of the present invention may involve monitoring for hydroxylation of a suitable substrate, monitoring for the utilisation of substrates and co-substrates, monitoring for the production of the expected products between the enzyme and its substrate. Assay methods of the present invention may also involve screening for the direct interaction between components in the system. Alternatively, assays may be carried out which monitor for downstream effects mediated by the substrate, such as substrate mediated transcription using suitable reporter constructs or by monitoring for the upregulation of genes or alterations in the expression patterns of genes known to be regulated directly or indirectly by the substrate.
Various methods for determining oxygenase activity either directly or indirectly are known in the art. Any suitable method may be used for determining 2-OG dependent oxygenase activity of FTO such as by substrate or co-substrate utilisation, product appearance such as peptide/nucleic acid hydroxylation/demethylation or down-stream effects mediated by hydroxylated/demethylated or non-hydroxylated products.
The substrate, enzyme and potential inhibitor compound may be incubated together under conditions which, in the absence of inhibitor provide for hydroxylation/demethylation of the substrate, and the effect of the inhibitor may be determined by determining hydroxylation/demethylation of the substrate. This may be accomplished by any suitable means. Small polypeptide or polynucleotide substrates may be recovered and subjected to physical analysis, such as mass spectrometry, radiography or chromatography, or to functional analysis. Such methods are known as such in the art and may be practiced using routine skill and knowledge. For example, the LC-MS assay described in the Examples may be used. Determination may be quantitative or qualitative. In both cases, but particularly in the latter, qualitative determination may be carried out in comparison to a suitable control, e.g. a substrate incubated without the potential inhibitor.
In alternative embodiments, reporter constructs may be provided in which promoters mediated by a substrate are provided operably linked to a reporter gene. Any suitable reporter gene could be used, such as for example enzymes which may then be used in colorometric, fluorometric, fluorescence resonance or spectrometric assays.
In the assay methods described herein, typically the FTO and the substrate are contacted in the presence of a co-substrate, such as oxygen and/or a 2-oxoacid, such as 2-OG and/or dioxygen. Hydroxylase/demethylase activity may be determined by determining turnover of one or more of the co-substrates, such as oxygen, 2-OG and/or ascorbate. This may be achieved by determining the presence and/or amount of reaction products, such as hydroxylated substrate or succinic acid. The amount of product may be determined relative to the amount of substrate. For example, in such embodiments the substrate may be a polypeptide and, for example, the product measured may be hydroxylated/demethylated polypeptide or nucleic acid. For example, the extent of hydroxylation/demethylation may be determined by measuring the amount of hydroxylated/demethylated polypeptide/nucleic acid, succinate, carbon dioxide, or formaldehyde generated in the reaction, or by measuring the depletion of 2-OG or dioxygen. Methods for monitoring each of these are known in the scientific literature, for example in Myllyharju et al. (1991) EMBO J. 16(6): 1173-1180 or as in Cunliffe et al. (1986) Biochem. J. 240: 617-619.
Unused 2-OG may be derivatised by chemical reagents, exemplified by but not limited to hydrazine derivatives and ortho-phenylene diamine derivatives, to give indicative chromophores or fluorophores that can be quantified and used to indicate the extent of hydroxylation of the test polypeptide. Dissolved oxygen electrodes, exemplified by but not limited to a “Clarke-type” electrode or an electrode that uses fluorescence quenching, may be used to follow the consumption of oxygen in an assay mixture, which can then be used to indicate the extent of hydroxylation of the test polypeptide in an analogous manner to the above.
The fluorescent product of the reaction of ortho-phenylenediamine (OPD) with the α-ketoacid motif of 2-OG is 3-(2-Carboxyethyl)-2(1H)-quinoxalinone. This fluorescent product can be readily detected by standard equipment such as that manufactured by for example Molecular Devices, Tecan, BMG Labtechnologies, Jasco and Perkin Elmer and there is extensive precedent demonstrating that the production of fluorescent products can be used in high-throughput screens.
The fluorescent product is generally detected with the excitation filter set as from about 300 nm to about 400 nm, preferably from about 335 to about 345 nm, most preferably at about 340 nm. The emission filter is generally at from about 400 to about 450 nm, preferably from about 415 to about 425 nm, most preferably at about 420 nm.
This assay procedure lends itself to high-throughput formats, such as multi-well plate formats e.g. 96-, 384-, or 1536-well plate formats.
Further, the nature of the fluorescent product can be tuned by modifying the nature of the derivatisation reagent used. For example, the sensitivity of the method may be increased by using either 1,2-dimethoxy-4,5-diaminobenzene, or 1,2-methylenedioxy-4,5-diaminobenzene.
The precise format of any of the screening or assay methods of the present invention may be varied by those of skill in the art using routine skill and knowledge. The skilled person is well aware of the need to additionally employ appropriate control experiments. Activity is measured by derivatisation of 2-OG with OPD or other aromatic diamines, such as 1,2-dimethoxy-4,5-diaminobenzene or 1,2-methylenedioxy-4,5-diaminobenzene, such that the derivative gives improved sensitivity compared to use of OPD (Willing et al. Journal of Chromatography B (2003) 383-392, Nakamura et al. Chem. Pharm Bull. (1987) 687-692).
The assay is carried out under conditions suitable for hydroxylation/oxidation of the substrate by the oxidase. Accordingly, 2-OG is present in the assay. The assay mixture may also contain iron, preferably ferrous iron.
Other components may be added to the assay mixtures. For example, a reducing agent such as ascorbate, a thiol such as dithiothrietol (DDT), β-mercaptoethanol, N-acetylcysteine or phenol may be added to the assay to help maintain enzyme structure and/or catalase may be added to destroy any H2O2 that might be produced. However, the assay will work in the absence of a reducing agent or catalase.
Assays are typically carried out at a temperature of from about 25° C. to about 40° C., for example at a temperature of from about 30° C. to about 39° C., or from about 35° C. to about 38° C. or about 37° C. The pH of the assay mixture is typically between about pH 7 to about pH 9, for example from about pH 7.5 to about pH 8. Suitable buffers, such as Tris or HEPES, may be used to maintain the pH of the assay mixture.
Typically, assays are carried out under normoxic conditions. The assay may also be carried out under conditions in which hydroxylation or oxidation is reduced or absent, such as under hypoxic conditions, in order to detect modulation of oxygenase activity by an agent which enhances hydroxylation/oxidation.
Alternatively, the end-point determination may be based on conversion of the substrate or substrate fragments (including synthetic and recombinant peptides or nucleic acids) derived from the polypeptide or nucleic acid substrate into detectable products. Substrates may be modified to facilitate the assays so that they can be rapidly carried out and may be suitable for high throughput screening.
For example, reverse phase HPLC (C-4 octadecylsilane column), as exemplified herein, may be used to separate starting synthetic peptide substrates from the products. Modifications of this assay or alternative assays for oxygenase activity may employ, for example, mass spectrometric, spectroscopic, and/or fluorescence techniques as are well known in the art (Masimirembwa C. et al. Combinatorial Chemistry & High Throughput Screening (2001) 4 (3) 245-263, Owicki J. (2000) J. Biomol. Screen. 5 (5) 297-305, Gershkovich A et al. (1996) J. Biochem. & Biophys. Meths. 33 (3) 135-162, Kraaft G. et al. (1994) Meths. Enzymol. 241 70-86). Fluorescent techniques may employ versions of the substrate to modified in such as way as to carry out or optimise spectroscopic or fluorescence assays.
Binding of a molecule which discriminates between the hydroxylated and non-hydroxylated or demethylated form of a polypeptide or other substrate may be assessed using any technique available to those skilled in the art, which may involve determination of the presence of a suitable label.
Assay methods of the present invention may also take the form of an in vivo assay or an assay carried out on ex vivo cells from an animal, such as a mammal (including human) or an insect. The assay may be performed in a cell line such as a yeast or bacterial strain or an insect or mammalian cell line in which the relevant polypeptides or peptides are expressed from one or more vectors introduced into the cell. Alternatively, the assay may be carried out on a mammalian cell that expresses endogenous FTO or in which FTO is over-expressed.
The FTO polypeptide used in a cell-based assay of the invention preferably comprises a nuclear localisation signal.
Test Compounds
Agents, which may be screened using the assay methods described herein, may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms, which contain several, characterised or uncharacterised components may also be used.
Combinatorial library technology (including solid phase synthesis and parallel synthesis methodologies) can provide an efficient way of testing a potentially vast number of different substances for ability to modulate an interaction. Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances. Various commercial libraries of compounds are also available. There are computational methods for screening these libraries (processes sometimes referred to as virtual screening) that can identify lead structures for inhibition.
Potential inhibitor compounds (i.e. antagonists) may be polypeptides, small molecules such as molecules from commercially available libraries, including combinatorial libraries, or the like. Small molecule compounds, which may be used, include 2-OG analogues, or substrate analogues, which inhibit the action of the enzyme. Small molecule compounds, and other types of compound, that may be used include all known 2-OG oxygenase inhibitors such as those already known to inhibit HIF hydroxylases (see for example WO02/074981 and WO03/080566) and procollagen prolyl hydroxylases.
Potential promoting agents may be screened from a wide variety of sources, particularly from libraries of small compounds, which are commercially available. Oxygen-containing compounds may be included in candidate compounds to be screened, for example 2-OG analogues. Any agents currently used for treating obesity (Cooke and Bloom, Nature Reviews Drug Discovery 2006) and type 2 diabetes may affect the activity of FTO and may be used as candidate agents.
Since naturally occurring compounds, including TCA cycle intermediates such as fumarate, are known inhibitors of 2-OG oxygenases they may inhibit FTO, possibly in a manner that is of physiological relevance, including in some cancers where fumarate is known to be upregulated as a consequence of the Warburg effect.
A test compound which increases, potentiates, stimulates, disrupts, reduces, interferes with or wholly or partially abolishes hydroxylation/oxidation of the substrate and which may thereby modulate activity, may be identified and/or obtained using the assay methods described herein.
Agents which increase or potentiate hydroxylation/oxidation (i.e. agonists), for example prolyl or asparaginyl hydroxylation, may be identified and/or obtained under conditions which, in the absence of a positively-testing agent, limit or prevent hydroxylation/oxidation. Such agents may be used to potentiate, increase, enhance or stimulate the oxygenase activity of FTO.
In various aspects, the present invention provides an agent or compound identified by a screening method of the invention to be a modulator of FTO oxygenase activity e.g. a substance which inhibits or reduces, increases or potentiates the activity of FTO.
The test agent may compete with 2-OG or a FTO substrate at the FTO active site and/or binds to the metal at the FTO active site. The test agent may comprise a metal ion such as, but not limited to, manganese, cobalt, zinc or nickel ions. Alternatively, the mode of inhibition may be via an allosteric interaction.
The test agent may be a reducing agent. Reducing agents typically act as activators of 2-OG oxygenase activity, typically in vitro. An activator of oxygenase activity may be any species that increases oxygenase activity of a FTO polypeptide either in vitro or in vivo. Reducing agents that may be used include ascorbate and analogues of ascorbate and reducing agents of the thiol chemical families, such as dithiothreitol or phosphine (e.g. triscarboxyethylphosphine).
Following identification of a modulator, the substance may be purified and/or investigated further (e.g. modified) and/or manufactured. A modulator may be used to obtain peptidyl or non-peptidyl mimetics, e.g. by methods well known to those skilled in the art and discussed herein. A modulator may be modified, for example to increase selectivity, as described herein. It may be used in a therapeutic context as discussed below.
For therapeutic treatment, the modulator may be used alone or in combination with any other therapeutically active substance or treatment.
The compounds which are acids can be present in the form of salts, such as sodium salts. The compounds may also be present in the form of derivatives such as the dimethyl ester, diethyl ester, monoethyl ester or di- or mono-amide. In certain instances these derivatives may be preferred, for example when inhibition of the enzyme within a cell of an organism is required.
Compounds which modulate 2-OG oxygenases may be useful as agents of the invention, for example, in the treatment of weight disorders as described herein, or may be used as test substances in an assay of the invention. The test compound may be known to act as an inhibitor of a 2-OG oxygenase other than FTO. For example, the test agent may be an inhibitor of procollagen prolyl hydroxylase, hypoxia inducible factor, prolyl and asparaginyl hydroxylases, collagen prolyl hydroxylase, gibberellin C-20 oxidase, a nucleic acid demethylase such as AlkB or a human AlkB homologue, a protein demethylase, such as a tri-, di-, mono-methyl lysine or arginine residue demethylase, another human or animal 2OG oxygenase involved in metabolism or regulation, or a plant 2-OG hydroxylase. Many inhibitors of 2-OG oxygenases are known in particular for human prolyl hydroxylases. N-oxaloglycine and its derivatives are suitable examples. Glycine or alanine derivatives and 2-oxoacid analogues may also be used.
Compounds which modulate 2-OG oxygenases, and families of such compounds, are known in the art, for example in Aoyagi et al. (2002) Hepatology Research 23 (1): 1-6, Aoyagi et al. (2003) Free Radical Biology and Medicine 35:410 Suppl. 1, Philipp et al. (2002) Circulation 106 (19): 1344 Suppl. S, Ivan et al. (2002) PNAS USA 99 (21): 13459-13464, Nwogu et al. (2001) Circulation 104 (18): 2216-2221, Myllyharju and Kivirikko (2001) Ann Med 33 (1): 7-21, Ohta et al. (1984) Chemical and Pharm Bulletin 32 (11): 4350-4359, Franklin et al. (2001) Biochem J. 353: 333-338, Franklin (1997) Int J. Biochem Cell Biol 29 (1): 79-89, Dowell et al. (1993) Eur J Med Chem 28 (6): 513-516, Baader et al. (1994) Biochem J. 300: 525-530, Baader et al. (1994) Eur J Clin Chem and Clin Biol 32 (7): 515-520, Bickel et al. (1998) Hepatology 28 (2): 404-411, Bickel et al. (1991) J. Hepatology 13: S26-S34 Suppl. 3, U.S. Pat. No. 6,200,974, U.S. Pat. No. 5,916,898, US Patent Applications 2003-0176317, 2003-0153503 and 2004-0053977, WO 02/074981, WO 03/080566, WO 04/035812, Cunliffe et al. (1992) J. Med. Chem. 35:2652-2658, Higashide et al. (1995) J. Antibiotics 38:285-295, Cunliffe et al. (1986) Biochem. J. 239(2):311-315, Franklin et al. (1989) Biochem. J. 261(1):127-130, Friedman et al. (2000) PNAS USA 97(9):4736-4741, Wu et al. (1999) J. Am. Chem. Soc. 121(3): 587-588, DE-A-3818850, Wang et al. (2001) Biochemistry US:15676-15683 and Lerner et al. (2001) Angew Chem. Int. Edit. 40:4040-4041.
Suitable test compounds are disclosed in WO03/080566 and WO02/074981. Other suitable compounds include inhibitors of Fibrogen HIF hydroxylase. Fibrogen HIF hydroxylase inhibitors are disclosed in United States Patent Application Publication Nos: 20070042937, 20060276477, 20060270699, 20060258702, 20060258660, 20060251638, 20060183695, 20060178317 and 20060178316.
Other suitable test compounds include compounds of formula (I):




wherein

    
    
        Y2 is selected from —OR′ and —NR′R′′ wherein R′ is hydrogen, or unsubstituted C1-4 alkyl and R′′ is hydrogen, hydroxy or unsubstituted C1-4 alkyl;
        Y1 is selected from —C—, —S— and —S(O)—;
        Z2 is selected from —C(O)— and —NR′′— wherein R′′ is selected from hydrogen, hydroxy or unsubstituted C1-4 alkyl;
        Z1 is selected from hydrogen and unsubstituted C1-4 alkyl; and
        R is a side chain of a naturally occurring amino acid.
    
    


Preferably Y1 is —C— and Y2 is —OH or —NH2. Most preferably Y1 is —C— and Y2 is —OH.
Preferably Z2 is —C(O)— or —NR′′— wherein R′′ is hydrogen, methyl or ethyl. More preferably Z2 is —C(O)— or —NH—. Preferably Z1 is hydrogen, methyl or ethyl, more preferably hydrogen. Most preferably Z2 is —C(O)— and Z1 is hydrogen, methyl or ethyl.
Preferably R is a side chain of alanine, valine, leucine or phenylalanine. Preferably R is a side chain of valine, leucine or phenylalanine. More preferably R is a side chain of phenylalanine, i.e. —CH2Ph.
L-stereoisomers or D-stereoisomers of these compounds may be used.
An exemplary synthetic scheme used to obtain test compounds of formula (I) is shown below in Scheme 1. Here an amino acid is reacted with an oxalyl chloride in order to produce a compound of formula (I). In this scheme the amino acid used is phenylalanine, although it will be apparent that the same general reaction will occur with other amino acids. The first reaction yields a protected compound of the invention (the dimethyl ester form). The diacid form is easily generated through reaction with aqueous sodium hydroxide.




Compounds in which X is —O— or —S— or Z is other than —CO—CO—OH may by synthesised as described in Mole et al. (2003) Bioorg. Med. Chem. Lett. 13, 2677-2680 and Cunliffe et al. J. Med. Chem. (1992) 35 2652-2658.
Krebs cycle intermediates such as succinate and fumarate act as inhibitors of FTO demethylase activity. Therefore analogues of succinate and fumarate may be used to inhibit FTO activity.
Therapeutic Applications
A compound, substance or agent which is found to have the ability to affect the oxygenase activity of FTO has therapeutic and other potential in a number of contexts, as discussed. In particular modulators of FTO activity may be used in the treatment or prevention of diseases associated with weight gain or weight loss. The modulators may prevent or reverse weight gain in overweight patients. The modulators may prevent weight loss or promote weight gain in underweight patients. The modulators may also be administered to individuals having a weight within the normal healthy range (typically a body mass index of about 19 to about 25) in order to prevent weight gain, for example, where those individuals have an allelic variant of the FTO gene, which allelic variant is associated with obesity. In this embodiment, the modulators are used to prevent weight gain and associated diseases or disorders.
Diseases and disorders associated with weight gain that may be treated or prevented by administering a modulator of FTO activity include obesity (a body mass index over 30), cancer (in particular colon cancer, prostate cancer, cancer of the rectum, breast cancer and endometrial cancer), cardiovascular disease (including heart attack and congestive heart failure), hypertension, high cholesterol levels, insulin resistance, type II diabetes, gallstones, sleep apnea, osteoarthritis, gout, dyslipidemia, Pickwickian syndrome and infertility
Disorders associated with weight loss that may be treated or prevented by administering a modulator of FTO activity include anorexia nervosa, bullemia, undernutrition, osteoporosis, infertility, impaired immunocompetence, AIDS or weight loss in patients on anti-cancer medication.
Modulators of FTO activity may also be used in the treatment or prevention of cancer. This is because of the ability of FTO to repair DNA.
For therapeutic treatment, an FTO modulator may be used in combination with any other active substance, e.g. for treatment in weight control, diabetes, and cardiovascular disease.
An agent identified using one or more primary screens (e.g. in a cell-free system) as having ability to modulate oxygenase activity may be assessed further using one or more secondary screens.
Generally, an agent, compound or substance which is a modulator according to the present invention is provided in an isolated and/or purified form, i.e. substantially pure. This may include being in a composition where it represents at least about 90% active ingredient, more preferably at least about 95%, more preferably at least about 98%. Any such composition may, however, include inert carrier materials or other pharmaceutically and physiologically acceptable excipients, such as those required for correct delivery, release and/or stabilisation of the active agent. As noted below, a composition according to the present invention may include in addition to a modulator compound as disclosed, one or more other molecules of therapeutic use, such as treatment in weight control, diabetes, and cardiovascular disease.
Products Obtained by Assays of the Invention
The invention further provides compounds obtained by assay methods of the present invention, and compositions comprising said compounds, such as pharmaceutical compositions wherein the compound is in a mixture with a pharmaceutically acceptable carrier or diluent. Examples of suitable carriers or diluents are given in, for example, “Harrison's Principles of Internal Medicine”. The carrier may be liquid, e.g. saline, ethanol, glycerol and mixtures thereof, or solid, e.g. in the form of a tablet, or in a semi-solid form such as a gel formulated as a depot formulation or in a transdermally administrable vehicle, such as a transdermal patch.
The invention further provides a method of treatment which includes administering to a patient an agent which interferes with FTO oxygenase activity. Such agents may include inhibitors or activators of FTO oxygenase activity.
The therapeutic/prophylactic purpose may be related to the treatment of a condition associated with reduced or suboptimal or increased FTO levels or activity, or conditions in which have normal FTO levels, but where a modulation in activity such as an increase or decrease in FTO oxygenase activity is desirable. For example, FTO activity may be modulated in the treatment of disorders associated with undesirable weight loss or gain.
A therapeutically effective amount of an agent is typically administered to a subject in need thereof. A therapeutically effective amount is an amount which ameliorates the symptoms of the condition or lessens the suffering caused to the subject by the condition. A therapeutically effective amount for treating or preventing weight gain or a disorder associated with weight gain, such as obesity, is typically an amount that reduces weight gain, for example, an amount that maintains the weight of the patient or induces weight loss. Alternatively, a therapeutically effective amount may be an amount that induces weight loss. A therapeutically effective amount for treating or preventing weight loss or a disorder associated with weight loss, such as anorexia, is typically an amount that reduces weight loss, for example, an amount that maintains the weight of the patent or alternatively induces weight gain.
Pharmaceutical Compositions
In various further aspects, the present invention thus provides a pharmaceutical composition, medicament, drug or other composition for such a purpose, the composition comprising one or more agents, compounds or substances as described herein, including inhibitors or activators of 2-OG dependent oxygenase activity, the use of such a composition in a method of medical treatment, a method comprising administration of such a composition to a patient, e.g. for treatment (which may include preventative treatment) of a medical condition as described above, use of such an agent compound or substance in the manufacture of a composition, medicament or drug for administration for any such purpose, e.g. for treatment of a condition as described herein, and a method of making a pharmaceutical composition comprising admixing such an agent, compound or substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
In one embodiment the method for providing a pharmaceutical composition may typically comprise:

    
    
        (a) identifying an agent by an assay method of the invention; and
        (b) formulating the agent thus identified with a pharmaceutically acceptable excipient.
    
    


The pharmaceutical compositions of the invention may comprise an agent, polypeptide, polynucleotide, vector or antibody according to the invention and a pharmaceutically acceptable excipient.
Whatever the agent used in a method of medical treatment of the present invention, administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.
An agent or composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated, e.g. as described above.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. In particular they may include a pharmaceutically acceptable excipient. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Liposomes, particularly cationic liposomes, may be used in carrier formulations. Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.
The substance or composition may be administered in a localised manner to a particular site or may be delivered in a manner in which it targets particular cells or tissues, for example using intra-arterial stent based delivery.
Targeting therapies may be used to deliver the active substance more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
In a further embodiment the invention provides for the use of an agent of the invention in the manufacture of a medicament for the treatment of a condition associated with increased or decreased FTO oxygenase levels or activity. The condition may, for example include diseases associated with weight loss or gain.
All the documents cited herein are incorporated herein by reference.
The following Examples illustrate the invention.
EXAMPLES
Example 1
Selection of FTO for Analysis as a 2-OG Oxygenase
A sequence alignment of Fatso with ABH3 was created using a combination of secondary structure prediction (JPRED) and sequence conservation in order to build a homology model of FTO based on the reported ABH3 crystal structure (Sundheim et al. (2006) EMBO J. 25(14) 3389-3397) with MODELLER8V2. The model of FTO was then superimposed with ABH3 and the AlkB DNA demethylase from E. coli (Yu et al. (2006) Nature, 439, 879-884) for comparison. The sequence alignments, secondary structure predictions and structural model clearly show strong similarities among these three proteins. The DSBH scaffold is clearly present in FTO and the individual active site Fe(II) and 2OG binding residues are strictly conserved (e.g. His231 FTO, His191 ABH3, His131 AlkB; Asp233 FTO, Asp193 ABH3, Asp133 AlkB; His307 FTO, His257 ABH3, His187 AlkB; Arg316 FTO, Arg269 ABH3, Arg204 AlkB). In addition, residues involved the catalytic mechanism (i.e. Arg322 FTO, Arg275 ABH3 and Arg210 AlkB; Leu213 FTO and the hydroxylated Leu177 ABH3) and aromatic residues predicted to be involved in DNA base recognition (Tyr108 FTO and Tyr143 ABH3) are present.
The FTO sequence was thus detected as a homologue of AlkB homologues (ABHs) and other known 2-oxoglutarate dependent oxygenases, some of which catalyse demethylation of methylated nucleic acid sequences, and found to contain a double-stranded beta-helix (DSBH) motif and at least one C-terminal helical domain.
The DSBH motif is characteristic of 2-OG dependent oxygenases but many proteins that are not 2-OG dependent oxygenases also contain the DSBH motif. Such proteins include, but are not limited to, the JmjC family, some but not all of which are 2-OG dependent oxygenases (Clissold & Ponting (2001) Trends Biochem. Sci. 26: 7-9). The DSBH motif is also characteristic of the functionally diverse cupin superfamily (Dunwell et al. (2004) Phytochemistry 65: 7-17). The human protein pirin (Pang et al. (2004) J. Biol. Chem. 279: 1491-1498) also contains the DSBH motif, but is not a 2-OG dependent oxygenase. One 2-OG dependent oxygenase that contains the DSBH motif is FIH. Allelic variants of FTO have previously been correlated with weight gain, but FTO has not previously been identified as a 2-OG dependent oxygenase, nor as containing a DSBH structural motif.
The C-terminal helical domain of FTO is not known to be associated with 2OG oxygenases.
The sequence analyses also demonstrated that FTO exhibits the conserved 2-His-1-carboxylate facial triad used to bind Fe(II) and the basic residue (here Arginine) characteristics of the 2-OG iron-dependent oxygenases, as well as other features that place it within the sub-family of 2-OG oxygenases typified by AlkB.
Example 2
Cloning of FTO
The cDNA sequence encoding full-length mouse fto was amplified from Image clone IMGCLO4237261 using commercially synthesized oligonucleotide primers (Sigma-Genosys) mfto 1f (sequence: 5′-GCTAGCATGAAGCGCGTCCAGACC-3′) SEQ ID NO: 14 and mftolr (sequence: 5′-GAATTCCTAGGATCTTGCTTCCAGCAG-3′) SEQ ID NO: 15 with restriction site overhangs in the following PCR reaction:
Conditions:














Template DNA
150
ng


mfto1f (10 μM)
1
μl


mfto1r (10 μM)
1
μl


MgCl2 (50 mM)
1
μl








dNTP's (10 mM each)
1 μl (New England Biolabs)









10x Polymerase buffer
5
μl








PfuTurbo DNA polymerase
1 μl (Polymerase buffer from Stratagene)


H2O
added to a total volume of 50 μl









Thermocycler Setup:



















95° C.
2 min





95° C.
1 min



50° C.
1 min
 {close oversize brace} 
25 cycles



72° C.
2.5 min  



72° C.
10 min 










The completed PCR reaction was cleaned with a QIAquick PCR purification kit (QIAGEN) and a sample run on a 1% agarose gel with TAE buffer. The PCR product was digested with NheI and EcoRI (both New England Biolabs) at 37° C. in an overnight reaction:

















DNA
1
μl



NheI (10000 units/ml)
2
μl



EcoRI (20000 units/ml)
2
μl



10x EcoRI buffer
5
μl



100x BSA
0.5
μl










H2O
added to a total volume of 50 μl










The reaction mixture was then run on a 1% agarose gel in TAE buffer, the band corresponding to the digested PCR product was excised and DNA extracted from the gel with a QIAquick gel extraction kit (QIAGEN).
The extracted DNA was ligated into similarly restricted and purified pET-28a vector (Novagen) using T4 DNA ligase (New England Biolabs) on ice over night:














Vector
50
ng


PCR product
42
ng


10xT4 buffer
2
μl


T4 DNA ligase (400000 units/ml)
0.5
μl








H2O
added to a total volume of 20 μl









4 μl of this reaction mixture was transformed into 50 μl E. coli XL10 Gold® cells (Stratagene) according to the manufacturer's instructions. Cells were grown on an LB plate containing 25 μg/ml Kanamycin at 37° C. overnight.
On the following day, 8 colonies were picked off the plate, resuspended in 5 ml 2YT medium containing 25 μg/ml Kanamycin each and incubated in an environmental shaker at 37° C. overnight.
Plasmids were isolated from these liquid cultures on the following day using a QIAprep Spin Miniprep kit (QIAGEN) and samples subjected to a control restriction digest with EcoRI and NheI at 37° C. for 3 hours:

















Miniprep DNA
3
μl



NheI (10000 units/ml)
1
μl



EcoRI (20000 units/ml)
1
μl



10x EcoRI buffer
2
μl



100x BSA
0.2
μl










H2O
added to a total volume of 20 μl










Samples were analysed by 1% agarose gel and found to contain the mfto insert. One sample was submitted for external sequencing (Geneservice) and shown to contain the desired insert. This plasmid is referred to as mfto-pET-28a.
Example 3
Expression and Purification of FTO
Mfto-pET-28a was transformed into E. coli BL21-Gold(DE3) (Stratagene) according to the manufacturer's instructions and grown on an LB plate containing Kanamycin as above. Expression trials showed moderate soluble expression at 37° C., 28° C. and 21° C. and decreasing amounts of insoluble material with decreasing temperature.
For expression, a single colony was picked from the transformation plate, resuspended in 100 ml 2YT+Kanamycin medium and grown in an environmental shaker at 37° C. over night. On the next day, 12 times 600 ml 2YT+Kanamycin were inoculated with 6 ml of the overnight culture each and grown in an environmental shaker at 37° C. until the cultures had reached OD600 1.0. At this stage, the cultures were shifted to 21° C. for an hour, after which IPTG was added to each to a final concentration of 0.25 mM. Incubation was continued at 21° C. for 9 hours, after which cells were collected (60 g total) by centrifugation and stored at −80° C.
The cell pellet was resuspended in 5 ml 10 mM HEPES pH 7.5, 0.5 M NaCl, 10 mM imidazole, 1 mM MgCl2 containing a spatula tip of DNAseI per gram of cells. 30 mg Phenylmethylsulfonylfluoride (PMSF, protease inhibitor) was added and the cells lysed by sonication on ice. After centrifugation to remove debris (Beckman AvantiTM J-25 centrifuge, JA25.50 rotor, 22000 rpm, 20 minutes, 4° C.), supernatants were decanted, filtered through a 0.45 μm Omnipore filter (Millipore) and loaded onto a 10 ml His-Bind® column (Novagen) at a flow rate of 0.5 ml/min. The column was washed with 250 ml 10 mM HEPES pH 7.5, 0.5 M NaCl, 40 mM imidazole at 2 ml/min, followed by 100 ml 25 mM HEPES pH 7.5, 0.5 M NaCl, 40 mM imidazole, 5 mM ATP dipotassium salt at 2.5 ml/min: this step should remove E. coli chaperones binding to the peptide linker between His tag and Fto protein. After another wash of 100 ml of the initial washing buffer, bound protein was eluted with an isocratic elution using 35 ml 10 mM HEPES pH 7.5, 0.5 M NaCl, 500 mM imidazole at 2.5 ml/min.
Fractions containing FTO (>90% pure by SDS_PAGE analysis) were concentrated to a final volume of 3 ml using an Amicon-15 5000 Molecular-weight-cut-off ultrafiltration device and loaded onto a 300 ml Superdex S200 gel filtration column equilibrated in 10 mM HEPES pH 7.5, 0.05 M NaCl, 1 mM DTT. The gel filtration step was run at 3 ml/min with 10 ml fractions being collected starting at an elution volume of 70 ml.
Example 4
2-oxoglutarate Decarboxylation Assays with Purified FTO
FTO was tested for enzymatic activity using a 2-OG turnover assay (Kivirikko and Myllylä (1982) Methods in Enzymology 82: 4412-4421). FTO was incubated with all necessary cofactors, in various buffers lacking a specific reagent (Fe2+, ascorbate or DTT) and in the presence of N-oxalyl-glycine (NOG), a generic Fe2+-2OG-dioxygenase inhibitor at a concentration of 0.5 mM. In addition to Fe2+ (added in the form of (NH4)2Fe(II)(SO4)2) and 2OG, dithiothreitol (DTT) and sodium ascorbate were added to the reaction mixture: DTT is a reducing agent that helps to maintain Fe2+.
Assay Components:


    11.5 μM FTO
    144 μM 2OG
    16 μM 14C-2OG
    80 μM (NH4)2Fe(II)(SO4)2 
    1 mM DTT
    4 mM Ascorbate


These were diluted to a total volume of 100 μl with 50 mM TRIS, pH 7.5. All reagents were mixed and pipetted into a 5 ml plastic screw cap tube, the FTO was added to the tube as a separate drop. A 500 μl Eppendorf tube containing 200 μl Hyamine hydroxide (Fisher Scientific, CO2 trapping agent) was added to each tube and tubes were closed with a rubber septum. After incubation in an environmental shaker at 37° C. for half an hour, 200 μl methanol was added to the contents and the tubes were put on ice for 30 minutes to quench the reaction. The Eppendorf tubes containing the hyamine hydroxide were transferred to scintillation vials, mixed with 5 ml OptiPhase Liquid Scintillation Cocktail (Fisher Scientific) and total 14C counts quantified using a Beckman L56500 Multi-Purpose Scintillation Counter.
Results of the first assay are shown in Table 1 below (whole counts—total starting 2-OG counts and in FIG. 4). The results reveal that FTO 2-OG turnover activity is stimulated by the cofactor Fe(II), and inhibited by N-oxalylglycine, which is an inhibitor of many 2-OG oxygenases. The activity is also stimulated by the addition of ascorbate which, as for other 2-OG oxygenases, may act as a surrogate for the natural substrate.
2OG uncoupled turnover assays with FTO, monitoring conversion of [1-14C]-2OG into [14C]-carbon dioxide, revealed that FTO catalysed 2OG decarboxylation, a reaction that was stimulated by ascorbate and FeSO4 (FIG. 8a). 2OG turnover was inhibited by known 2OG oxygenase inhibitors (FIG. 8b) and by the absence of Fe(II) and ascorbate.







TABLE 1







Results of 2-oxoglutarate decarboxylation assays










% whole counts
Average counts















All assay components
97.2
128559



No Fe
40.2
53152



No Asc
41.9
55413



No DTT
94.1
124389



0.5 mM NOG
57.7
76266



Whole counts
100
132199










Example 5
Use of 2-OG Oxygenase Assay to Detect FTO Inhibitors
Using the method described in Example 4 various 2-OG oxygenase inhibitors were screened for their ability to inhibit activity FTO oxygenase activity. The inhibitors tested were:

    NOG=N-oxalyl glycine
    NOFD=N-oxalyl D-phenylalanine
    P-2-4-CD—pyrdine-2,4-dicarboxylate
    P-2-4-CD—pyrdine-2,5-dicarboxylate

Compound 41 and Compound 16 have the structures set out below.







Example 6
Demethylation Assays
Using the method described in Example 2, but using only unlabelled 2OG, various potential substrates were screened for demethylation by FTO. The results are shown in FIGS. 5 and 6. The results demonstrate that, under the assay conditions used, FTO catalyses the demethylation of 3-methylthymine and 3-methylcytosine residues with a preference for 3 methylthymine residues.
Potential FTO substrates were screened including a synthetic single-stranded 1-methyl adenine (1-meA) methylated oligonucleotide, Lys-9 methylated histone H3, hypoxia inducible factor-1α (HIF-1α) subunit fragments, IκBα and coenzyme A derivatives. Only the 1-meA methylated oligonucleotide significantly stimulated turnover of 2OG (FIG. 6a). This activity was inhibited by N-oxalylglycine, fumarate and succinate, which were also inhibitors in the 2OG uncoupled turnover assays (FIG. 6c).
Using an LC-MS assay that directly monitors DNA demethylation (without the need for radiolabelled (co-)substrates or coupled assays), we demonstrated that FTO catalyses Fe(II)— and 2OG-dependent DNA demethylation. This activity was stimulated by ascorbate, as observed for other 2OG oxygenases (FIG. 6b).
Significantly reduced turnover was observed when the reaction was performed under reduced oxygen conditions. The production of succinate was verified by 1H NMR (400 MHz) analyses and that of formaldehyde was confirmed by derivatization with pentafluorophenylhydrazine.
To test the predicted role of the assigned Fe(II) binding and 2OG 5-carboxylate binding residues, His-304 and Arg-313 alanine substitution mutants were constructed. The His-304 mutant showed significantly reduced 2OG turnover whilst the Arg-313 mutant ablated activity (FIG. 6b).
FTO activity was further investigated using single-stranded oligonucleotides (ss-DNA) methylated at a single position: 1-methyladenine (1-meA), 1-methylguanine (1-meG), 3-methylcytosine (3-meC) and 3-methylthymine (3-meT) (FIG. 7b). Under the assay conditions at pH 7.5, FTO exhibited a preference for 3-meT over 1-meA or 3-meC in ss-DNA; 1-meG was not an FTO substrate (FIG. 7a). Consistent with prior reports, we found that recombinant forms of ABH2 and ABH3 exhibit a preference for 3-meC and 1-meA and 3-meT with the ss-DNA substrates, with only very low levels of 3-meT demethylation being observed under our conditions. The preference of FTO for 3-meT substrates was also observed in assays measuring the release of formaldehyde from methylated poly(dA) and poly(dT) (FIG. 7c).
Example 7
Localisation of FTO
Since FTO catalyses DNA demethylation, it was anticipated that it would be localised to the nucleus. Indeed, confocal imaging revealed that yellow fluorescent protein-tagged FTO (YFP-FTO) is concentrated in the nucleus, whereas YFP itself is present only in the cytoplasm. Interestingly, FTO localised to the nucleus even more effectively than ABH3.
2OG oxygenase catalysed post-translational hydroxylation is central to transcriptional regulation in the hypoxic response and 2OG oxygenases catalyse histone demethylation. The catalytic activity of FTO may similarly regulate the transcription of genes involved in metabolism by nucleic acid demethylation. Alternatively, it is possible that FTO, as proposed for ABH2, can act as a nucleic acid repair enzyme: there is evidence that breakdown of genomic repair processes leads to obesity and metabolic syndrome. Under the assay conditions used above at physiological pH, the preferred substrate of FTO identified was 3-methylthymine in DNA, a minor but stable lesion that is generated on exposure of DNA to methylating agents.

## Claims

1. A method for assaying FTO activity, the method comprising contacting a FTO polypeptide, with oxygen and 2-oxoglutarate as co-substrates and optionally a methylated nucleic acid substrate, and iron as a cofactor, and measuring oxygenase activity of said FTO polypeptide compared to a control, wherein said FTO polypeptide comprises:
(a) the amino acid sequence of SEQ ID NO: 1,
(b) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 over its full length, or
(c) an amino acid sequence comprising amino acids 10 to 495 of SEQ ID NO: 1.

2. The method of claim 1, wherein oxygenase activity is measured in the presence of a reducing agent.

3. The method of claim 2, wherein the reducing agent is ascorbate, or an analogue thereof, a thiol or a phosphine.

4. The method of claim 1, wherein oxygenase activity is measured in the presence of the methylated nucleic acid substrate.

5. The method of claim 1, wherein the FTO polypeptide is a recombinant polypeptide.

6. The method of claim 1, wherein the FTO polypeptide comprises
the amino acid sequence of SEQ ID NO: 1.

7. The method of claim 1, further comprising:
(i) contacting the FTO polypeptide with a test agent;
(ii) monitoring for oxygenase activity in the presence and absence of the test agent; and
(iii) determining whether the test agent is an inhibitor or activator of FTO activity.

8. The method of claim 7, wherein the test agent is a reported inhibitor of a 2-OG oxygenase other than FTO, or an analogue or variant of such an inhibitor.

9. The method of claim 8, wherein the 2-OG oxygenase is procollagen prolyl or lysylhydroxylases, hypoxia inducible factor prolyl hydroxylase, methylated lysyl demethylase, asparaginyl hydroxylase, phosphatidyl serine receptor (Jmjd6), AlkB or human AlkB homologues and/or gibberellin C-20 oxidase.

10. The method of claim 8, wherein the inhibitor is an N-oxalyl amino acid, N-oxalylglycine or a derivative thereof, a glycine or alanine derivative, a 2-oxoacid analogue, a flavonoid or flavonoid derivative.

11. The method of claim 8, wherein the test agent comprises a metal ion.

12. The method of claim 7, wherein the test agent is a reducing agent.

13. The method of claim 12, wherein the reducing agent is a reported activator of a 2-OG oxygenase other than FTO.

14. The method of claim 12, wherein the reducing agent is ascorbate or an analogue of ascorbate, or a reducing agent of the dithiothreitol or phosphine chemical families.

15. The method of claim 7, further comprising: repeating all of the method steps using an enzyme other than FTO; and determining whether the test agent selectively inhibits or activates FTO or the other enzyme.

16. The method of claim 15, wherein the enzyme other than FTO is a 2-OG oxygenase.

17. The method of claim 16, wherein the 2-OG oxygenase is a hypoxia inducible factor hydroxylase, a collagen or procollagen prolyl hydroxylase, a nucleic acid demethylase, or a protein demethylase.

18. The method of claim 17, wherein the protein demethylase hydroxylates a methylated histone or a fragment thereof.

19. The method of claim 17, wherein the hypoxia inducible factor hydroxylase is a prolyl or asparaginyl hydroxylase, the nucleic acid demethylase is an AlkB homologue, and the protein demethylase is a tri-, di- or mono-methyl lysine or arginine residue demethylase.

20. The method of claim 1, wherein the methylated nucleic acid sequence is associated with a gene involved in weight modulation.

21. The method of claim 20, wherein the gene involved in weight modulation is the agouti gene or the neuropeptide Y gene.

22. The method of claim 1, further comprising:
(i) contacting the FTO polypeptide with a test substrate;
(ii) monitoring for oxygenase activity in the presence and absence of the test substrate; and
(iii) determining whether the test substrate is a substrate of FTO.

23. The method of claim 22, wherein the test substrate is a human nucleic acid sequence.

24. The method of claim 23, wherein the nucleic acid sequence contains a 3-methylthymine base, a 1-methyladenine base or a 3-methylcytosine base.