# Patent

## Title
Mina53 assays

## Abstract

The present invention comprises a method for assaying oxygenase activity the method comprising monitoring oxygenase activity of Mina53.

## Background
SEQUENCE LISTING
The text file is ISII0105PUSA.txt, created Apr. 8, 2009, and of size 25.9 KB, filed therewith, is hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to a 2-oxoglutarate (2-OG) oxygenase, and in particular, to assays using the purified 2-OG oxygenase to identify inhibitors and substrates. Inhibitors of the 2-OG oxygenase are useful in inhibiting cell proliferation and in treating cancer.
BACKGROUND TO THE INVENTION
MYC induced nuclear antigen, isoform 2 (Mina53) has been shown to be a target gene of c-Myc, suggesting that it is involved in mamunalian cell proliferation (Tsuneoka et al. (2002) J. Biol. Chem. 277, 35450-9). Colon tumour cell lines were subsequently shown to highly express Mina53. Suppression of Mina53 expression severely suppressed proliferation of colon tumour cells in vitro and suppression of Mina53 or its activity may reduce cell proliferation and so be of use in the treatment of cancer. Elevated expression of Mina53 was therefore deemed a characteristic feature in colon cancer (Teye et al. (2004) Am. J. Pathol. 164, 205-16).
Reduction of Mina53 expression using RNAi has been shown to suppress cell proliferation in oesophageal squamous cell carcinoma (ESCC). Expression of Mina53 was also shown to be elevated in 83% of ESCC cell lines tested and patients with high expression level of Mina53 had shorter survival periods. Together, these results identify Mina53 as a potential prognostic factor for ESCC and indicate that inhibition of the unidentified role of Mina53 will be of benefit in treating cancer (Tsuneoka et al. (2004) Clin. Cancer Res. 10, 7347-56).
Mina53 was shown through immunoprecipitation to be part of a ribonucleoprotein complex where it associates with ribosomal as well as non-ribosomal proteins. It was proposed that Mina53 is involved in rRNA processing and/or ribosome assembly (Eilbract et al. (2005) Eur. J. Cell Biol. 84, 279-94). No molecular function or catalytic activity has been ascribed to Mina53.
None of the literature on Mina53 describes a method for expressing and purifying Mina53. The functional assays such as localization and immunoprecipitation carried out in the art do not require purified protein.
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. 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 to limit 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-oxoglutarate 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, also have a requirement for ascorbate for full catalytic activity. Although ascorbate might stimulate activity by reducing Fe3+, or other high valent form 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. It is believed that one role of ascorbate is to function as a surrogate reducing substrate to ‘rescue’ the enzyme in the event of the uncoupled production of a ferryl [Fe(IV)═O] intermediate.
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).
SUMMARY OF THE INVENTION
The present inventors have successfully purified recombinant Mina53 and demonstrated that the purified recombinant Mina53 functions as a 2-oxoglutarate (2-OG) dependent oxygenase. In particular, the inventors have devised a method for expressing and purifying both fall-length and truncated Mina53 in using a bacterial expression system. The inventors have also demonstrated for the first time that Mina53 is a 2-OG dependent oxygenase.
Accordingly, the present invention provides:
a method for assaying oxygenase activity, the method comprising monitoring oxygenase activity of Mina53;
a method for identifying an inhibitor of Mina53 oxygenase activity, the method comprising:
contacting a Mina53 polypeptide with a test agent under conditions suitable for oxygenase activity; and
monitoring for oxygenase activity;
a method for identifying a substrate of Mina53, the method comprising:
contacting a Mina53 polypeptide with a test substrate under conditions suitable for oxygenase activity; and
monitoring for oxygenase activity;
the use of recombinant Mina53 in an assay for oxygenase activity;
the use of an inhibitor of Mina53 oxygenase activity in the manufacture of a medicament for use in treating cancer;
a method of treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutic amount of an inhibitor of Mina53 oxygenase activity;
a method of inhibiting cell proliferation, the method comprising contacting cells in vivo or in vitro with an inhibitor of Mina53 oxygenase activity; and
a method for purifying a Mina53 polypeptide, the method comprising:
culturing host cells comprising an expression vector encoding Mina53 such that Mina53 is expressed; and
isolating Mina53 from the cells.



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A and 1B are an alignment of the amino acid sequence of Mina53, NO66 and factor inhibiting hypoxia inducible factor (1H2K).



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 is the nucleotide and amino acid sequence of human Mina53.
SEQ ID NO: 2 is the amino acid sequence of human Mina53.
SEQ ID NO: 3 is the nucleotide and amino acid sequence of human NO66.
SEQ ID NO: 4 is the amino acid sequence of human NO66.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the purification of recombinant Mina53 and to identification of Mina53 as a 2-oxoglutarate (2-OG) dependent oxygenase and provides for the use of Mina53 in assay methods to identify modulators of Mina53 oxygenase activity and substrates that are hydroxylated by Mina53.
In one aspect of the invention, Mina53 or a variant or fragment thereof for use in accordance with the invention has the ability to convert 2-OG to succinate and carbon dioxide.
Preferably, Mina53 for use in a method of the invention comprises:
(a) the amino acid sequence of SEQ ID NO: 2;
(b) a variant thereof having at least 50% identity to the central JmjC domain of Mina53 encompassed by the amino acid sequence of SEQ ID NO: 2 between amino acids 128 and 265 or 128 and 271 and having oxygenase activity; or
(c) a fragment of either thereof having oxygenase activity.
A variant or an active fragment of Mina53 may typically be identified by monitoring for 2-OG oxygenase activity as described in more detail below. The variant of Mina53 has at least 50% sequence identity, for example at least 55% sequence identity, with the sequence of Mina53 in the central JmjC domain (amino acids 128 and 265 or 128 and 271 in SEQ ID NO: 2) or double-stranded B-helix domain (amino acids 161 to 265 in SEQ ID NO: 2). The variant may have at least 35%, for example at least 50% or 60% sequence identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, typically greater than 70% or 80%, more typically greater than about 90% or 95% sequence identity.
The variant may be a homologue of Mina53 such as NO66 (Eilbracht et al. (2003), Eur. J. Biol. 84:279-294). The homologue is generally a human protein, but homologues from other species may be used.
The amino acid sequence of NO66 is shown in SEQ ID NO: 4. The Mina53 polypeptide used in an assay of the invention may be a variant or fragment of SEQ ID NO: 4, which variant or fragment retains 2-OG oxygenase activity. The variant may have at least 60%, such as at least 70%, 80%, 90% or 95% sequence identity with the amino acid sequence of SEQ ID NO: 4.
Sequence identity may be calculated using any suitable algorithm. For example, the UWGCG Package provides the BESTFIT program can be used to calculate 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 calculate homology or line up sequences (typically on their default settings), for example as described in Altschul (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol. 215:403-10.
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 peptide retains 2-OG oxygenase activity.
Amino acid substitutions 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 (molecular breeding) techniques.
The present invention also includes use of active portions, fragments, derivatives and functional mimetics 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. 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, 300 or 400 amino acids. In particular, but not exclusively, this aspect of the invention encompasses the situation when the protein is a fragment of the complete Mina53 protein sequence and may represent a catalytic region, capable of converting 2-OG to succinate and carbon dioxide. The fragment may comprise the region from about amino acid 161 to 265, for example from about amino acid 128 to about 265 or about 271 of the amino acid sequence shown in SEQ ID NO: 2. Useful fragments include C-terminal truncated fragments such as fragments comprising residues 1 to 301 shown in SEQ ID NO: 2, fragments comprising an N-terminal deletion, such as fragments comprising residues 26 to 465 of the amino acid sequence shown in SEQ ID NO: 2 and fragments comprising both N-terminal and C-terminal truncations, such as fragment comprising residues 40 to 239 of the amino acid sequence shown in SEQ ID NO: 2. Other suitable fragments may readily be identified, for example by comparing the Mina53 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. Such fragments can be used to construct chimeric molecules.
The equivalent fragments of SEQ ID NO: 4 having oxygenase activity may also be used in an assay of the invention and are encompassed within the term “Mina53” used herein.
The Mina53 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 tag 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 by post translational modification including hydroxylation or phosphorylation. Polypeptides of the invention may be GST fusion polypeptides. Such modified polypeptides fall within the scope of the term “Mina53”.
The polypeptides of the invention may be present 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, preferably inhibitors of hydroxylase activity. The inhibitors may be selective inhibitors. The polypeptides of the invention may also be used in structural analyses such as crystallography.
The Mina53 polypeptides may be used in assays for 2-OG oxygenase activity in the absence of a prime substrate. The Mina53 polypeptides may also be used to determine oxygenase activity in the presence of one or more suitable substrates.
Mina53 used in a method of the invention may be recombinant Mina53 or naturally occurring Mina53. Preferably, recombinant Mina53 is used. Recombinant Mina53 may be produced using standard expression vectors that comprise nucleotide sequences encoding Mina53. 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 Mina53 polypeptide as defined herein. The Mina53 may also be present in a cell extract or in a partially or substantially purified form.
A polypeptide in a partially or substantially purified form, is generally comprised in a preparation in which more than 50%, e.g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.
Methods for Obtaining Purified Mina53
The present inventors tried a number of standard approaches to obtain soluble, active recombinant Mina53 in purified form from E. coli. The standard approaches tested have not been successful. However, the present inventors have now found that it is possible to express a recombinant Mina53 in soluble and highly active form using a modified expression and purification method. The inventors have also demonstrated that Mina53 is a 2-OG dependent oxygenase. Accordingly, the invention provides a method for obtaining purified Mina53. Purified Mina53 obtainable by this method is also provided.
Mina53 may be obtained by introducing an expression vector comprising a polynucleotide encoding Mina53 into a host cell. The polynucleotide may comprise the coding region of the nucleotide sequence of SEQ ID NO: 1 or be a fragment or variant of the coding region of the nucleotide sequence shown in SEQ ID NO: 1. The fragment may lack one or more nucleotide from the 5′ or 3′ end of the coding region, for example which lacks at least about 150 nucleotides, for example between about 300 and about 690, between about 450 and about 600 nucleotides, between about 510 and about 570, or between about 525 and about 555. The variant typically has at least about 70%, 80%, 90%, 95%, 98% or 99% sequence identity to the coding region of the nucleotide sequence of SEQ ID NO: 1 over a region of at least about 588 contiguous nucleotides, for example 588 contiguous nucleotides at the 3′ end of the coding region shown in SEQ ID NO: 1. Sequence identity may be determined by any suitable method, for example, as described above.
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, a mammalian, e.g. human host cell or a bacillovirus host cell. The bacterial host cell is preferably a strain of E. coli, for example BL21 (DE3).
A method of producing a purified Mina53 polypeptide is provided by the invention. The method typically comprises culturing host cells comprising an expression vector encoding Mina53 and isolating the Mina53 polypeptide from the cells. The host cells may 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 Mina53 polypeptide according to the invention may further comprise introducing a polynucleotide or vector according to the invention into the host cell. The Mina53 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 Mina53 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 Mina53. 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 Mina53 is present in the soluble fraction in an amount sufficient for the truncated enzyme 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.
Mina53 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). 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-bind 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 Mina53 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 shows that Mina53 catalyses the conversion of 2-OG to succinate and carbon dioxide. This newly discovered activity of Mina53 means that, for the first time, assays for identifying inhibitors of Mina53 activity may be performed. Blocking 2-OG oxygenase activity of Mina53 will inhibit cell proliferation and hence inhibitors of 2-OG oxygenase activity will be useful in treating cancer.
We describe below in more detail a number of different assays that may be carried out to identify modulators of Mina53 oxygenase activity and in particular of 2-OG oxygenase activity. Typically, the assays utilise a human Mina53 polypeptide as described herein. Mina53 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 Mina53 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, Mina53 oxygenase activity. The method comprises contacting a Mina53 polypeptide and a test substance, such as a potential inhibitor, in the presence of one or more co-substrate and optionally a prime substrate. 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, hydroxylation of the prime substrate may be monitored. Assays that detect binding to Mina53 in the absence of catalytic turnover may also be used. Such assays may employ techniques such as NMR, MS or fluorescence spectroscopy. The co-substrate may be modified, e.g. 2-OG, or consumed, e.g. oxygen or ascorbate, by Mina53. 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 Mina53 is active as an oxygenase in the absence of the test substance. The Mina53 oxygenase activity in the presence of the test substance is compared to Mina53 oxygenase activity in the absence of the test substance to determine whether the test substance is an inhibitor of Mina53 oxygenase activity. In the alternative, the assays may be used to look for promoters of Mina53 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 activity or increase in 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 Mina53 and which do not have activity or are less active with other 2-OG oxygenases, for example, such as hypoxia inducible factor (HIF) asparagine 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 asparagine or prolyl hydroxylases, which do not inhibit Mina53.
The assays of the invention may also be used to identify inhibitors or activators which are specific for Mina53 activity at a particular substrate or residue within a substrate.
In medicinal applications, for example, it is advantageous to modulate oxygenase activity of a single enzyme or group of enzymes. Assays of the invention may therefore be use to identify agents which selectively modulate activity of Mina53 relative to a second 2-OG dependent oxygenase, including but not limited to the HIF hydroxylases, including FIH, PHD1, PHD2 and PHD3, AlkB, ABH1, ABH2, ABH3, procollagen prolyl and lysyl hydroxylases, the phosphotidylserine receptor and 2-OG oxygenases that have been characterized as JmjC proteins according to the SMART database.
The invention provides for the use of such 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 Mina53 oxygenase activity.
It is also possible, using the method of the invention to identify selective inhibitors when the 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 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 Mina53. In particular, such substrates may be used in assays to monitor for the activity of a modulator of Mina53 2-OG oxygenase activity. The substrate may be a peptide or nucleic acid substrate.
Any suitable substrate which is hydroxylated by Mina53, with Mina53 typically having the amino acid sequence of SEQ ID NO: 2, 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 Mina53 may be used as substrates in the assay of the invention.
The methods of the invention may be used to detect novel substrates of Mina53 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 Mina53.
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 control 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 know to be regulated directly or indirectly by the substrate.
Various methods for determining hydroxylation either directly or indirectly are known in the art. Any suitable method may be used for determining 2-OG dependent oxygenase activity of Mina53 such as by substrate or co-substrate utilisation, product appearance such as peptide hydroxylation or down-stream effects mediated by hydroxylated 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 of the substrate, and the effect of the inhibitor may be determined by determining hydroxylation of the substrate. This may be accomplished by any suitable means. Small polypeptide substrates may be recovered and subjected to physical analysis, such as mass spectrometry or chromatography, or to functional analysis. Such methods are known as such in the art and may be practiced using routine skill and knowledge. 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 Mina53 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 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 polypeptide. For example, the extent of hydroxylation may be determined by measuring the amount of hydroxylated polypeptide, succinate or carbon dioxide 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 Myllyhaiju J. et al. EMBO J. 16 (6): 1173-1180 (1991) or as in Cunliffe C. J. et al. Biochem. J. 240 617-619 (1986).
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 (Mühling 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 of the substrate by the hydroxylase. 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 mixture. 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.
The assay is 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, the assay is carried out under normoxic conditions. The assay may also be carried out under conditions in which hydroxylation is reduced or absent, such as under hypoxic conditions, in order to detect modulation of oxygenase activity by an agent which enhances hydroxylation.
Alternatively, the end-point determination may be based on conversion of the polypeptide substrate or peptide fragments (including synthetic and recombinant peptides) derived from the polypeptide substrate into detectable products. Peptides 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 for HIF hydroxylase from the hydroxylated products. The latter typically have a shorter retention time in the column. Modifications of this assay or alternative assays for HIF hydroxylase 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 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 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. The in vivo assay may be performed in a cell line such as a yeast or bacterial strain in which the relevant polypeptides or peptides are expressed from one or more vectors introduced into the cell.
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) provides 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.
Potential inhibitor compounds (i.e. antagonists) may be polypeptides, small molecules such as molecules from commercially available 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 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.
A test compound which increases, potentiates, stimulates, disrupts, reduces, interferes with or wholly or partially abolishes hydroxylation 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 (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. Such agents may be used to potentiate, increase, enhance or stimulate the oxygenase activity of Mina53.
In various aspects, the present invention provides an agent or compound identified by a screening method of the invention to be a modulator of Mina53 oxygenase activity e.g. a substance which inhibits or reduces, increases or potentiates the activity of Mina53.
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 selectively, 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. For example, for anti-tumour therapy another anti-tumour compound or treatment, such as radiotherapy or chemotherapy, may be used in combination with the modulator.
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, or may be used as test substances in an assay of the invention. The test compound may, for example, be an inhibitor of procollagen prolyl hydroxylase, hypoxia inducible factor prolyl and asparaginyl hydroxylases or gibberellin C-20 oxidase. N-oxaloglycine and its derivatives are suitable examples. 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. 6,200,934, 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 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.
Therapeutic Applications
A compound, substance or agent which is found to have the ability to affect the oxygenase activity of Mina53 has therapeutic and other potential in a number of contexts, as discussed. For therapeutic treatment, such a compound may be used alone or in combination with any other active substance, e.g. for anti-tumour therapy with another anti-tumour compound or therapy, such as radiotherapy or chemotherapy.
An agent identified using one or more primary screens (e.g. in a cell-free system) as having ability to modulate hydroxylase 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 an modulator compound as disclosed, one or more other molecules of therapeutic use, such as an anti-tumour agent.
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 Mina53 oxygenase activity. Such agents may include inhibitors of Mina53 oxygenase activity.
The therapeutic/prophylactic purpose may be related to the treatment of a condition associated with reduced or suboptimal or increased Mina53 levels or activity, or conditions in which have normal Mina53 levels, but where an modulation in activity such as an increase or decrease in Mina53 oxygenase activity is desirable. For example, Mina53 activity may be modulated in the treatment of proliferative disorders. One such example of a proliferative disorder is cancer. In particular, Mina53 inhibitors may be used in the treatment of colon cancer or oesophageal cancer, such as oesophageal squamous cell carcinoma. Other examples of proliferative disorders are plasma cell proliferative disorders. The plasma cell proliferative disorders may be caused by a viral infection, such as infection by hepatitis C virus or Epstein-Barr virus.
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.
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 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.
The agent may be used as sole active agent or in combination with another such agent or with any other active substance, e.g. for anti-tumour therapy another anti-tumour compound or therapy, such as radiotherapy or chemotherapy.
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 Mina53 oxygenase levels or activity. The condition may, for example, be cancer.
All the documents cited herein are incorporated herein by reference.
The following Examples illustrate the invention.
EXAMPLES
Example 1
Selection of Mina53 for Analysis as a 2-OG Oxygenase
The Mina53 sequence was detected as a homolog of factor inhibiting hypoxia inducible factor (FIH), a known 2-oxoglutarate dependent oxygenase in a sequence profile analysis employing structural and mechanistic data from 2-OG oxygenases that was carried out (FIG. 1) to look for human 2-OG iron dependent oxygenases. These analyses revealed that Mina53 contains a double-stranded β-helix (DSBH) motif that is characteristic of 2-OG dependent oxygenases. Despite the identification of Mina53 as a protein involved in human cell proliferation and extensive knowledge on 2-OG oxygenase structure, Mina53 has not been identified as a 2-OG dependent oxygenase, since many proteins other than 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, 1491-8) and FIH contain the DSBH motif, but only the latter is a 2-OG dependent oxygenase.
Mina53 exhibits the conserved 2-His-1-carboxylate facial triad used to bind Fe(II) and the basic residue (here Lysine) characteristics of the 2-OG iron-dependent oxygenases. Given its role in human cell proliferation, Mina53 was selected for analysis as a 2-OG oxygenase.
Example 2
Cloning of Full Length Mina53
In order to get a working assay system with a recombinant enzyme, it is necessary to express that gene encoding the protein and to purify it in such a way that the protein is correctly folded and functionally active.
The cDNA clone of Mina53 was supplied by the Integrated Molecular Analysis of Gene Expression (IMAGE) Consortium (cDNA clone MAGE:3916148). Mina53 was subcloned directly as a Nde I/Sal I fragment into the pet28a(+) vector from Novagen (a commonly used vector for work with E. coli that expresses the protein with a polyhistidine tag to enable purification through nickel affinity chromatography). The integrity of Mina53 was confirmed by DNA sequencing.
Example 3
Cloning of a C-Terminal Truncated Version of Mina53
A truncated version of Mina53 consisting of amino acids 1 to 301 shown in SEQ ID NO: 2 was subcloned into the pet24a+ vector from Novagen (pet24a+ has no additional residues added to the beginning of the enzyme, unlike pet28a+). The truncation was chosen on the basis of sequence homology with FIH, a known 2-oxoglutarate (2-OG) dependent enzyme. The deleted C-terminal amino acids sequence showed no obvious homology with other 2-OG enzymes, so it was postulated that they could be deleted with no loss of enzyme function.
Example 4
Cloning of a C-Terminal and N-Terminal Truncated Version of Mina53
The Mina53 amino acid sequence was assessed for homology to another protein, yxbC which is expressed in Bacillus subtilis. B. subtilis can be considered the Gram-positive equivalent of E. coli. Therefore it might be expected that a truncated Mina53 sequence bearing homology to a B. subtilis protein might express more solubly into E. coli than fall length Mina53. On this basis a doubly truncated Mina53, residues 40-329 was designed and subcloning into pet28a+ was confirmed through DNA sequencing.
Reference Example 1
Expression of Insoluble Mina53 Using Standard Methodology
Expression trials were carried out using both the full-length and C-terminal truncated forms of Mina53, at 37° C. and 28° C. (using a lower temperature gives slower cell metabolism and therefore more time for the protein to fold properly, thus increasing likelihood of getting active proteins of interest), in the presence of varying amounts of IPTG (a chemical that prevents cells from continuing growth and induces protein production). Following expression, the samples were sonicated into lysis buffer using 50 mmol TRIS buffer at pH7.5 in an effort to obtain soluble protein. This was entirely standard and normal methodology, but gave predominantly or totally insoluble Mina53. The 1-301 and 40-309 truncated versions of Mina53 also gave mainly insoluble protein.
Reference Example 2
Expression of Insoluble Mina53 at Reduced Temperatures
The experiments described in Reference Example 1 were repeated using reduced induction temperatures of 20° C. and 15° C., with the other parameters remaining the same. These conditions also gave predominantly insoluble Mina53.
Example 5
Expression of an Increased Amount of Soluble Mina53 Using Increased Salt and Glycerol Concentrations and Increased pH
In an attempt to obtain soluble Mina53, the expression trials were repeated (using induction temperatures of 28° C. and 15° C.) and the cells expressing Mina53 were lysed using lysis buffer having increased salt and glycerol concentrations to disrupt non-covalent interactions which might favour aggregation of Mina53.
Cells expressing the fall-length and 1-301 truncated forms of Mina53 were lysed in lysis buffer containing 250 mmol NaCl and glycerol at pH7.9 (normal pH of lysis buffer is pH7.5). An increased amount of soluble protein was observed.
Example 6
Further Optimisation of Lysis Buffer
The expression trials of Example 4 were repeated using (a) a lysis buffer in which the NaCl concentration was increased further, to 500 mmol and (b) a lysis buffer containing 500 mmol NaCl plus 1 mmol of the 2-OG oxygenase inhibitor N-oxalylglycine (NOG) and 1 mmol FeSO4.
The increased salt concentration gave a further improvement in solubility. NOG and FeSO4, however, made little difference.
Example 7
Purification of 1-301 Truncated Mina53
The most soluble sample of Mina53 (1-301) from Example 6(a) was purified using Q-sepharose and then phenyl resource column. The purified Mina53 obtained had 2-OG oxygenase activity.
Example 8
Overexpression and Purification of Full Length Mina53
The Mina53/pet28a(+) construct was transformed into E. coli BL21(DE3) and grown at 37° C. in 2TY media containing kanamycin (30 μg·ml−1). When the OD600 reached 0.8-1.0, the temperature was reduced to 15° C. and IPTG was added to a final concentration of 0.5 mM. The cells were harvested sixteen hours later by centrifugation at 9000 rpm for 15 min at 4° C.
E. coli cell pellets were resuspended in sonication buffer (20 mM Tris pH 7.9, 500 nM NaCl, 10% Glycerol) and broken by sonication. Cell debris was removed by centrifugation at 15000 rpm for 30 min at 4° C. The supernatant was then applied at 4° C. to a 10 ml His-Bind metal-binding chromatography resin (Novagen) following the protocol supplied by the manufacturer (the column was eluted into 5 ml fraction in collection tubes containing 5 μl of 0.5M EDTA 20 mM Tris pH7.9, to chelate any free metal leaching from the column). The sample was then desalted using a PD-10 column (Amersham Biosciences) into 20 mM Tris pH 7.9, 125 mM NaCl, 10% Glycerol. The desalted sample was then applied to a 1 ml MonoQ column (Amersham Pharmacia Biotech) with a linear 20 ml gradient of NaCl, from 0 to 0.5 M, in 20 mM Tris-HCl (pH 7.9).
Example 9
Cloning, Overexpression and Purification of Truncated Mina53 A26-V465/pNIC-Bsa4
A N-terminal truncation of Mina53 A26-V465 cloned into pNIC28-Bsa4 was supplied by the Structural Genomics Consortium Oxford. The Mina53 A26-V465/pNIC-Bsa4 construct was transformed into E. coli BL21(DE3) and grown at 37° C. in 2TY media containing kanamycin (30 μg·ml−1). When the OD600 reached 0.8-1.0, the temperature was reduced to 15° C. and IPTG was added to a final concentration of 0.5 mM. The cells were harvested sixteen hours later by centrifugation at 9000 rpm for 15 min at 4° C.
The Mina53 A26-V465/pNIC-Bsa4 was purified as per the protocol described for Mina53/pet28a(+). The N-terminal His-tag was cleaved by overnight cleavage at 4° C. with AcTEV protease (Invitrogen, 1 U per mg of Mina53). The polyhistidine tagged AcTEV protease was subsequently removed from the sample via metal chelation chromatography.
Example 10
Assay for 2-OG Oxygenase Activity
Once purified full-length His-Mina53 had been obtained it was tested for 2-OG oxygenase activity. A standard assay for this family of enzymes involves the use of [14C] labelled 2-OG co-substrate. Upon reaction, the 14C label is released as 14CO2 gas (old FIG. 2).




The 14CO2 gas is then trapped using a strong base and quantified by liquid scintillation. The level of uncoupled turnover of 2-OG oxygenases can be increased by the use of a reducing agent, e.g. ascorbate. Many other assays for 2-OG oxygenases are well known, e.g. those based on oxygen consumption, succinate production or oxidation of the “prime substrate” (McNeill et al (2005) Anal. Biochem. 336, 125-31; Sabourin & Bieber (1982) J. Biol. Chem. 257, 7460-7; and Cunliffe et al. (1986) Biochem. J. 240, 617-9).
As noted above, many of the enzymes in the family catalyse the decarboxylation of 2-OG in the absence of a suitable prime substrate, albeit at a reduced rate. This activity is sometimes termed as uncoupled turnover. The protocol used for the 2-OG assay decarboxylation is based on the method used to measure [14CO2] release by α-ketoisocaproate oxygenase (Sabourin & Bieber (1982) J. Biol. Chem. 257, 7460-7). Standard assay conditions consisted of a total volume of 100 μl, 50 mM Tris-HCl pH 7.5, 4 mM ascorbate, 160 μM 2OG (5% 1-[14C]), 80 μM FeSO4.6H2O, 0.48 mg·ml−1 catalase, 10 μM Mina53. Four stocks were made: (a) Mina53 (b) Fe(II) (c) ascorbate and, (d) all other reagents. Assays were started by mixing components (a) to (d). A tube containing 200 μl hyamine hydroxide was added and the vial sealed. The assays were incubated at 37° C. for 20 minutes then quenched with methanol (200 μl). Reaction tubes were then left on ice for 30 mins to collect [14CO2] gas, before the hyamine hydroxide was removed and treated with scintillant solution for counting (Beckman, LS6500). Assays were performed in triplicate unless otherwise stated. When potential small molecule inhibitors were added, they were mixed to stock (d) to a final concentration of 1 mM in Tris 50 mM, pH 7.9 buffer. To investigate inhibition of Mina53 by metals other than Fe(II), NiCl2 and ZnCl2 were added separately to the assay mixture to a final concentration of 80 μM.
Using this [14C] labelled 2-OG assay (16 nm of 2-OG was initially present in the assay mixture), the level of uncoupled turnover for Mina53 was measured under standard catalytic conditions (16 nm of 2-OG was initially present in the assay mixture), in the absence of Fe(II), in the absence of ascorbate and in the presence on N-oxalylglycine (NOG) a ubiquitous inhibitor for the 2-OG dependent non-haem Fe(II) oxygenases16. As shown in Table 1, the results show that His-Mina53 is able to decarboxylate 2-OG in the absence of prime substrate under fall catalytic condition. However, the decarboxylation of 2-OG is significantly reduced in the absence of either Fe(II) or ascorbate, and is inhibited by NOG.







TABLE 1





Iron, Ascorbate and 2OG dependence


of truncated His-Mina53(A26-V465).



















His-A26-V465
−
+
+
+


Fe(II)
+
+
−
+


Ascorbate
+
+
+
−


2OG turnover
0.12 ± 0.09
6.95 ± 1.72
0.51 ± 0.37
0.99 ± 0.23


(nm)









The results show that in the presence of ascorbate, Fe(II), 2OG and Mina53(A26-V465), 6.95±1.72 nm of 14CO2 gas is released. In the absence of either ascorbate, Fe(II), or Mina53(A26-V465) the amount of 14CO2 gas released is sub-nanomolar.

The inhibition of Mina53 mediated oxidation of 2OG by metals others than Fe(II) was studied. Both Ni and Zn (final assay concentration of 80 μM) were shown to inhibit 2OG turnover, with Zn appearing to be the more potent inhibitor (Table 3).







TABLE 2





Metal inhibition of truncated Mina53(A23-V465)





















A26-V465
−
+
+
+
+
+


Fe(II)
+
+
−
+
+
+


Ascorbate
+
+
+
−
+
+


NiCl2
−
−
−
−
+
−


ZnCl2
−
−
−
−
−
+


2OG turnover
0.07 ± 0.02
3.84 ± 0.03
0.76 ± 0.22
0.34 ± 0.16
2 ± 0.23
0.93 ± 0.17


(nm)









The inhibition of Mina53 mediated oxidation of 2-OG by the following small molecules (FIG. 2) was also studied: N-oxalyl D-phenylalaninie (NOFD) (known to inhibit FIH17), prohexadione (a 2-OG analog which inhibits gibberellin biosynthesis in plants18), fumarate (known inhibitor of the hypoxia inducible factor hydroxylases), and BB231.







TABLE 3







Selected 2OG analogues structures









Com-

Molecular


pound
Structure
Formula





NOG




C4H5NO5


 


Fumarate




C4H4O4


 


NOFD




C11H11NO5


 


Prohexa- dione




C10H12O5


 


BB231




C12H9ClN2O4









Prohexadione, NOG, and fumarate were shown to inhibit uncoupled 2-OG turnover, while NOFD and BB231 did not significantly reduce it (Table 4).







TABLE 4





Metal and small compound inhibition of truncated Mina53(A26-V465)




















A26-V465
+
+
+
+
+


Fe(II)
+
+
+
+
+


Ascorbate
+
+
+
+
+


NOG
+
−
−
−
−


NOFD
−
+
−
−
−


BB231
−
−
+
−
−


Prohexadione
−
−
−
+
−


Fumarate
−
−
−
−
+


2OG turnover
0.47 ± 0.12
3.86 ± 0.05
3.18 ± 0.03
0.17 ± 0.12
0.58 ± 0.09


(nm)

## Claims

1. A method for assaying oxygenase activity, the method comprising monitoring oxygenase activity of a polypeptide having greater than about 95% sequence identity with SEQ ID NO: 2.

2. The method of claim 1, wherein the polypeptide is a homologue of Mina53.

3. The method of claim 1, wherein oxygen is used as a co-substrate.

4. The method of claim 1, wherein iron is used as a cofactor.

5. The method of claim 1, wherein a 2-oxoacid is used as a co-substrate.

6. The method of claim 1, wherein 2-oxoglutarate is used as a co-substrate.

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

8. The method of claim 7, wherein the reducing agent is ascorbate, or an analogue thereof, a thiol or a phenol.

9. The method of claim 1, wherein oxygenase activity is measured in the presence of a substrate.

10. The method of claim 9, wherein the substrate is a peptide or nucleic acid substrate.

11. A method for assaying oxygenase activity, the method comprising monitoring oxygenase activity of a polypeptide having greater than about 95% sequence identity to a region of SEQ ID NO: 2 between amino acids 128 and 271, 128 and 265 or 161 and 265 and having oxygenase activity.