WO2014016605A1 - Dosage - Google Patents

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WO2014016605A1
WO2014016605A1 PCT/GB2013/051992 GB2013051992W WO2014016605A1 WO 2014016605 A1 WO2014016605 A1 WO 2014016605A1 GB 2013051992 W GB2013051992 W GB 2013051992W WO 2014016605 A1 WO2014016605 A1 WO 2014016605A1
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jmjd4
activity
erfl
protein
inhibitor
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PCT/GB2013/051992
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English (en)
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Mathew Louis COLEMAN
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Isis Innovation Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

Definitions

  • the present invention relates to assays for monitoring activity of lysyl-hydroxylases, as well as to the control of translational termination, the treatment of cancer and the treatment of disorders caused by nonsense mutations.
  • 2-Oxoglutarate (20G) and Fe(II)-dependent dioxygenases (20G-oxygenases) are known to catalyse the hydroxylation of protein, lipid and nucleic acid substrates. They are involved in a variety of medicinally important biological processes including collagen biosynthesis, chromatin modification and hypoxia signalling. These hydroxylation reactions can result in either stable incorporation of a hydroxyl group, or in the case of N-methyl group hydroxylation, the formation of an unstable intermediate that decomposes into formaldehyde. The latter reaction is employed by 20G- oxygenases that catalyse N-demethylation reactions of nucleic acids and proteins.
  • the human genome contains approximately 60 genes predicted to encode 20G- oxygenases, many of which are not characterised in terms of their substrates. Proteins of this gene family can be subdivided into groups based on similarity within the catalytic motif.
  • the Hypoxia Inducible Factor (HIF) prolyl hydroxylases consists of three 20G-hydroxylases (PHD 1-3 or EGLN1-3 enzymes) that target the Hypoxia Inducible transcription Factor (HIF) for proteolytic degradation.
  • the JmjC histone demethylase subfamily members catalyse the demethylation of specific lysine residues within the N-terminal tails of histone proteins and are involved in the regulation of chromatin structure and gene expression.
  • JMJD4 is a 20G-dioxygenase of the JmJC-only family of which comparatively little is known.
  • JMJD4 is a 20G oxygenase which plays a role in cancer cell proliferation and survival.
  • JMJD4 modulation therefore represents a new target for the treatment of cancer.
  • JMJD4 hydroxylates the translation termination protein eRFl, specifically hydroxylating the protein at K63. Inhibition of the lysyl- hydroxylation activity of JMJD4, and hence K63 hydroxylation of eRFl offers a way to reduce translational termination efficiency and so stimulate stop codon read through. A new way to modulate translational termination has therefore been provided.
  • the invention also provides assays for JMJD4 activity. Particularly such as assay employing eRFl, or a polypeptide therefrom, as a substrate.
  • the present invention provides a method for assaying JMJD4 activity, the method comprising contacting a peptide comprising a lysyl residue, with a JMJD4 polypeptide and determining whether the lysyl residue in said peptide is hydroxylated.
  • the invention also provides a method for identifying an inhibitor of JMJD4 activity, the method comprising contacting a JMJD4 polypeptide and lysine containing peptides with a test agent under conditions suitable for oxygenase activity, and monitoring for hydroxylation of the lysine of said peptide to give a hydroxylysyl residue. Additionally, the invention provides a method for identifying a modulator of protein translation and/or stop codon readthrough, the method comprising contacting a cell which expresses JMJD4 with a test agent and determining whether the test agent modulates the JMJD4 mediated regulation of protein translation and/or stop codon readthrough.
  • the invention further provides an inibitor or activator of 20G oxygenase activity for use in modulating lysyl hydroxylation by JMJD4 of a translation termination protein comprising a lysyl residue.
  • the invention also provides an inhibitor of JMJD4 activity for use in a method of treating cancer.
  • the invention provides an inhibitor of JMJD4 activity for use in a method for treating conditions caused by premature stop codons (nonsense mutations).
  • the invention additionally provides a method for introducing hydroxylysyl residue into a peptide or protein comprising contacting a peptide or protein containing a lysyl residue with a JMJD4 polypeptide.
  • JMJD4 Primary sequence alignment of JMJD4 with the lysyl hydroxlase JMJD6, the primary sequences of human JMJD4 and JMJD6 were aligned using ClustalW and shaded using Boxshade software. The catalytic domain of JMJD4 shares 34% sequence identity with that of JMJD6.
  • Figure 2 Showing relative JMJD4 mRNA expression levels. Assembled using biogps.org.
  • FIG. 3 Characterisation of JMJD4 expression in proliferating cells.
  • Cell lines were serum-starved for 24h before stimulation with 10% serum for 6 and 24h prior to immunoblot for JMJD4, eRFl and ⁇ -actin loading control.
  • a coomassie stain of the membrane is also provided to demonstrate equal loading, as ⁇ - actin was serum responsive in HEK293T cells.
  • FIG. 4 JMJD4 siRNA inhibits HeLa cell proliferation - Results shown for proliferation assay with HeLa cervical carcinoma cells treated with control, FIH (negative control) or one of two independent JMJD4 siRNAs prior to proliferation MTS assay (Promega).
  • JMJD4 siRNA induces a Gl -phase cell cycle arrest - results shown for FACS analysis for A549 cells treated with control, JMJD4 siRNA #31 or FIH (negative control) in triplicate and stained with propidium iodide. Data from triplicate samples are averaged and are presented in the bar graph, illustrating the increase in Gl -phase cells, and corresponding decrease in S-phase cells , in JMJD4 siRNA treated cells.
  • FIG. 6 JMJD4 interacts with the eRFl/eRF3Atranslational termination complex in an enzyme-dependent manner.
  • A Immunoblot of extracts from HEK293T cells stably expressing empty vector, FLAG-FIH (control), FLAG-JMJD4, or an inactive Fe(II) binding mutant (H189A) which were immunoprecipitated with anti-FLAG resin prior to immunoblot for endogenous eRFl and eRF3A.
  • Input cell extract prior to immunoprecipitation (IP).
  • HEK293T cells were transiently transfected with eRFl-V5 and eRF3A-HA in the presence or absence of FLAG-JMJD4 prior to anti-V5 immunopurification of the complex and in-solution protease digestion and MS. 5% of the purified material was visualised by SDS-PAGE and coomassie blue staining.
  • FIG. 7 endogenous eRFl is hydroxylated at K63.
  • A549 cells were transfected with either control or JMJD4 siRNA prior to endogenous eRF 1 purification and LCMS quantification of K63 hydroxylation in newly
  • Figure 8 shows JMJD4 directly catalyses hydroxylation of eRFl K63.
  • Recombinant human eRFl and JMJD4 were purified from E.coli and analysed by SDS-PAGE and coomassie blue staining.
  • D) JMJD4 is a 20G and Fe(II)-dependent dioxygenase. In vitro enzyme assays were performed in the presence and absence of the indicated cofactors and inhibitors (NOG; N-Oxalylglycine).
  • Figure 9 Graphical representation of the readthrough reporter vectors used.
  • the pEF l V5-HisA series were based on tandem Beta-galactosidase and firefly luciferase cDNA's separated by a UGA, UAG or UAA stop codon embedded within a minimal leaky termination signal derived from the replicase cistron of the plant tobacco mosaic virus (TMV) ('leaky UGA/UAG/UAA').
  • TMV tobacco mosaic virus
  • the positive control for luciferase activity consisted of the same TMV sequence but with the stop codon mutated to a Glutamine codon to allow constitutive readthrough from Beta-galactosidase to luciferase ('readthrough').
  • a non-leaky termination sequence with UAA stop codon was also included for comparison ('non-leaky UAA').
  • Figure 10 shows eRFl and JMJD4 are required for efficient translational termination.
  • A) Translational termination requires eRFl .
  • A549 cells were transfected with control or eR l siRNA and stop codon readthrough reporter vectors, prior to enzymatic assay of Beta-galactosidase and firefly luciferase. Reduced translational termination and increased stop codon readthrough are indicated by an increase in the ratio of luciferase:Beta-galactosidase activity.
  • B) Translational termination requires JMJD4. A549 cells were transfected with control or JMJD4 siRNA and stop codon readthrough reporter vectors prior to assay as above.
  • FIG 11 Structural analyses of human eRFl .
  • Previously solved crystal structures of human eRFl (PDB's 1DT9 and 3E1Y) were analysed using UCSF Chimera molecular graphics software.
  • eRFl has a Y-shaped topology that is thought to mimic tRNA, and includes the N- (NIKS and YxCxxxF motifs for stop-codon recognition), M- (GGQ motif for peptidyl-tRNA hydrolysis) and C-domains (eRF3 binding) (right).
  • Exploded view of the NIKS motif and hydroxylation target of JMJD4 (K63: highlighted in white), located on the apex of a large alpha-helical hairpin in the N-domain (left).
  • SEQ ID NO: 1 provides the nucleic acid sequence of splice variant 1 from the human JMJD4 gene mRNA encoding the full length JMJD4 protein (Accession Number NM_023007.2).
  • SEQ ID NO: 2 provides the human JMJD4 splice variant 1 protein encoded by SEQ ID No: 1 (Accession No: NP_075383.2).
  • SEQ ID NO: 3 provides the nucleic acid sequence of splice variant 2 from the human JMJD4 gene mRNA as well as the accompanying encoded protein sequence
  • SEQ ID NO: 4 provides the human JMJD4 splice variant 2 protein encoded by SEQ ID No: 3 ( Accession No: NP_001154937.1).
  • SEQ ID NO: 5 provides the amino acid sequence of human eRFl (Accession No: P62495).
  • SEQ ID NO: 6 provides the amino acid sequence of human eRF3A (Accession No: P15170).
  • SEQ ID NO: 7 provides the amino acid sequence of human JMJD6.
  • SEQ ID No: 8 provides the nucleotide sequence of the control siRNA against FIH used in the Examples of the present application.
  • SEQ ID No: 9 provides the amino acid sequence of a possible peptide substrate for JMJD4 derived from the eRFl sequence.
  • SEQ ID No: 10 provides the amino acid sequence of a possible further peptide substrate for JMJD4 derived from the eRFl sequence.
  • SEQ ID No: 11 provides the amino acid sequence of a possible further peptide substrate for JMJD4 derived from the eRFl sequence.
  • SEQ ID No: 12 provides the amino acid sequence of a possible further peptide substrate for JMJD4 derived from the eRFl sequence
  • SEQ ID NO: 13 provides the amino acid sequence of the portion of the FIH protein indicated in Figure 1.
  • SEQ ID NO: 14 provides the amino acid sequence of the portion of the N066 protein indicated in Figure 1.
  • SEQ ID NO: 15 provides the amino acid sequence of the portion of the Mina53 protein indicated in Figure 1.
  • SEQ ID NO: 16 provides the amino acid sequence of the portion of the JMJD5 protein indicated in Figure 1.
  • SEQ ID NO: 17 provides the amino acid sequence of the portion of the HSPBAP1 protein indicated in Figure 1. Detailed Description of the Invention
  • JMJD4 plays a role in cancer and that the inhibition of JMJD4 can inhibit cancer cell proliferation. Further, the inventors have also identified that JMJD4 catalyses lysyl-hydroxylation of the protein eRFl , which, when playing its role in vivo, is in a complex of eRFl/eRF3A, eRFl being involved in the termination of translation. Inhibition of JMJD4 therefore offers a new way to modulate translational termination and so, for instance, bring about read-through of stop codons.
  • JMJD4 is a 20G oxygenase and hence that the enzyme may be inhibited by known 20G oxygenase inhibitors.
  • the present invention therefore provides a method for assaying JMJD4 activity, the method comprising contacting a J JD4 polypeptide with a peptide containing a lysine residue (or analogue thereof), and determining either directly or indirectly whether the peptide is hydroxylated at the lysine-residue.
  • JMJD4 polypeptides in accordance with the present invention are typically human JMJD4 or a homologue thereof, variants thereof which retain lysyl-hydroxylase activity, or a fragment of any thereof which retains lysyl-hydroxylase activity.
  • the sequence of human JMJD4 is described in SEQ ID NO: 1.
  • Homologues thereof may be derived from other species, including in particular mammalian species. Exemplary species include orangutan, cow, rat and mouse.
  • the JMJD4 polypeptide may comprise the sequence shown in SEQ ID NO: 1, or may be a fragment or variant of SEQ ID NO: 1 having lysyl hydroxylase activity.
  • the JMJD4 polypeptide may have an amino acid sequence having at least about 60% sequence identity, for example at least about 70% sequence identity, with SEQ ID NO: 1 over its entire length or over an active fragment thereof, typically greater than about 80% or 90%, such as about 95% or about 99% sequence identity.
  • the JMJD4 polypeptide employed in the invention may have any such levels of sequence identity.
  • the transcript from the human JMJD4 gene is the subject of alternative splicing.
  • SEQ ID No: l provides the nucleic acid sequence encoding the full length JMJD4 protein, whose amino acid sequence is provide by SEQ ID No:2.
  • any embodiment described herein for SEQ ID Nos: 1 and 2 may also be performed with SEQ ID Nos: 3 and 4.
  • any of the assays herein may employ SEQ ID No:4.
  • variants, fragments, levels of sequence identity, levels of sequence homology and so on in relation to SEQ ID Nos: 1 and 2 the equivalent for SEQ ID Nos: 3 and 4 may also be employed.
  • any instance disclosed herein for SEQ ID No: 1 and/or 2 is also disclosed where SEQ ID No: 3 and/or 4 are employed as alternatives to SEQ ID Nos: land 2, or in some instance in addition to SEQ ID Nos: 1 and 2.
  • Sequence identity may be calculated using any suitable algorithm.
  • 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, p387-395).
  • 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; Latched et al. (1990) J. Mol. Biol. 215:403- 10.
  • the JMJD4 polypeptide may be polypeptides encoded by any naturally occurring JMJD4 gene in humans or other organisms.
  • the naturally occurring JMJD4 gene may encode the sequence shown in SEQ ID NO: 1 or may encode a variant or homologue.
  • 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 lysyl hydroxylase activity.
  • the JMJD4 polypeptide employed may be that of SEQ ID No:4.
  • Amino acid substitutions of SEQ ID NO: 1, or fragments thereof 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. The same modifications may be made for SEQ ID No:4. ALIPHATIC Non-polar GA P
  • 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 lysyl-hydroxylase activity.
  • An active fragment of JMJD4 may typically be identified by monitoring for 20G oxygenase or hydroxylase activity as described in more detail below. Such an active fragment may be included as part of a fusion protein.
  • the JMJD4 fragment may have up to about 200, 250, 300, 350, 400, 450, or 463 amino acids.
  • the JMJD4 fragment may comprise any region from the amino acid sequence shown in SEQ ID NO: 1 , such as from amino acid 2, 3, 4, 5 or about 10 to about amino acid 400, 410, 420, 430, 440, 450, or 463.
  • Useful fragments include N-terminal (or C- terminal) truncated fragments i.e., fragments comprising an N-terminal deletion, such as fragments comprising residues 10 to 463, 20 to 463 or 25 to 463 of the amino acid sequence shown in SEQ ID NO: 1.
  • Useful fragments also include fragments comprising C-terminal truncations such as fragments comprising residues 1 to 462, 1 to 450, 1 to 440 or 1 to 430 of the amino acid sequence shown in SEQ ID NO: 1. Useful fragments also include fragments comprising both N-terminal and C-terminal truncations, such as fragment comprising residues 10 to 463, 20 to 450 or 25 to 430 of the amino acid sequence shown in SEQ ID NO: 1. Where possible equivalent modifications may also be made to SEQ ID No. -4.
  • JMJD4 fragment will comprise the catalytic domain indicated in Figure 1 for JMJD4 or such a domain with one of the levels of sequence identity indicated herein.
  • fragment or polypeptide will comprise one or preferably all of the Fe(II) and 20G binding residues indicated in Figure 1.
  • JMJD4 may readily be identified, for example by comparing the amino acid sequence to the amino acid sequence of one or more Icnown 20G oxygenases and identifying which regions are 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 chimerical molecules. Fragments of any JMJD4 polypeptide having at least approximately 60% sequence identity (such as at least approximately 70%, 80%, 90%, 95% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2) that have lysyl hydroxylase activity may also be used in an assay of the invention and are
  • JMJD4 polypeptides encompassed within the term "JMJD4 polypeptides" used herein.
  • the JMJD4 polypeptides may comprise one or more particular site directed mutations.
  • the JMJD4 polypeptide may be synthetically prepared.
  • the polypeptides may be chemically or biochemically modified, e.g. post-translationally modified. For example, they may be glycosylated, phosphorylated or contain modified amino acid residues. They may also be modified by the addition of additional 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. In a preferred instance the JMJD4 polypeptide is found in the cytoplasm.
  • Polypeptides of the invention may be GST or other suitable fusion polypeptides.
  • the JMJD4 polypeptide may also be modified by addition of fluorescent tags (such as green or yellow fluorescent protein) to enable visualisation within cells or organelles or to aid purification of the protein or cells expressing JMJD4. Such modified polypeptides fall within the scope of the term "JMJD4 polypeptide”.
  • the JMJD4 polypeptides of the invention may be present in a partially purified or in a substantially isolated form.
  • the polypeptide may be mixed with carriers or diluents, which will not interfere with its intended use and still be regarded as substantially isolated.
  • the polypeptide may also be in a substantially purified form, in which case it 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 JMJD4 polypeptides used in a method of the invention may be recombinant JMJD4 or naturally occurring JMJD4.
  • Naturally occurring JMJD4 may be obtained from any organism that produces a JMJD4 polypeptide.
  • recombinant JMJD4 is used especially where JMJD4 is required for purposes requiring large (>20 mg) amounts of protein such as for biophysical assays or for high throughput analyses.
  • Recombinant JMJD4 may be produced using standard expression vectors that comprise nucleotide sequences encoding JMJD4.
  • JMJD4 polypeptide may be present in a cell, including, but not limited to, human- derived cells.
  • methods of the invention may utilise cells that have been modified to express JMJD4 polypeptides as defined herein.
  • the JMJD4 may also be present in a cell extract or in a partially or substantially purified form.
  • a JMJD4 polypeptide of the invention may localize to the cytoplasm of a cell.
  • Purified JMJD4 polypeptides may be obtained by introducing an expression vector comprising a polynucleotide encoding JMJD4 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.
  • 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).
  • 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.
  • JMJD4 polypeptides may be purified by lysing the host cells and extracting JMJD4 from the soluble fraction, for example by affinity purification, such as via an affinity tag fused to truncated JMJD4 polypeptides.
  • JMJD4 polypeptides 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 methods of the present invention typically use a peptide containing a lysyl residue as a substrate (or binding agent) for the JMJD4 polypeptides.
  • the general peptide's length used in the screen is typically at least 15 amino acids in length, preferably of at least 20 amino acids in length and even more preferably of at least 25, 30, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 or 260 amino acids in length. In a preferred instance the peptide is at least 50 amon acids in length.
  • the protein may, in some instances, be 280, 300, 320, 340, 360, 380, 400, 420 or 437 amino acids in length.
  • a full-length protein which is a substrate for the JMJD4 polypeptide can be used, for example an eRFl protein particularly a human eRFl protein.
  • the polypeptide may comprise at least 90%, 95%, 98% or 99% of the full length sequence.
  • the eRFl protein is provided as a complex with eRF3A, particularly where both proteins are the human.
  • eRFl is not provided as a complex with eRF3A as JMJD4 is capable of hydroxylating eRFl without eRF3A also being present.
  • Any suitable peptide can be used, so long as the peptide contains a lysyl residue (or analogue thereof) capable of hydroxylation by JMJD4 (or of binding to the active site of JMJD4).
  • the peptide may be modified, e.g. by the presence of a group to facilitate assays such as a fluorescent group; many such modifications are routinely used and described in the scientific literature.
  • the peptide used in the assays is a substrate for JMJD4 in vivo, or a homologue, variant or fragment thereof.
  • the present inventors have identified the protein eRFl to be a substrate for JMJD4.
  • one preferred target derived peptide containing a lysyl residue for use in accordance with the present invention is SEQ ID NO: 5.
  • Amino acid substitutions of SEQ ID NO: 5, or of fragments thereof 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.
  • JMJD4 has been shown to hydroxylate the lysine residue at position K63 of eRFl of SEQ ID NO: 5.
  • a variant or homologue of SEQ ID NO: 5 includes a residue equivalent to the lysyl residue at position 63 of SEQ ID NO: 5.
  • the substrate may, in some instances, be a variant of eRFl with any of the levels of sequence identity specified herein.
  • the assays of the present invention also include the use of fragments of SEQ ID NO: 5 or fragments of the variants thereof as defined above. Such fragments are typically of at least 20 amino acids in length and incorporate an lysyl equivalent to lysine at position K63 of SEQ ID NO: 5.
  • the peptide typically comprises the sequence
  • the peptide may comprise the sequence
  • the peptide employed has the lysine residue to be hydroxylated at the center of the peptide.
  • the peptide may comprise, consist of, or consist essentially of the sequence
  • a peptide substrate for use in accordance with the present invention consists of or comprises either of those sequences, or variants thereof having 1, 2 or 3 substitutions therein, and retaining a lysine residue (or analogue thereof) equivalent to K63 of SEQ ID NO: 5.
  • the substrate may in particular comprise the NIKS motif of eRFl and hence have that four residue sequence.
  • the substrate may have a NIKS motif.
  • the substrate may have the NIKS motif corresponding to that in the sequence of eRFl.
  • the present invention also provides assays for JMJD4 activity.
  • the method of the invention may be used to identify modulators of JMJD4 activity.
  • the assay may be carried out in the presence of a test agent to determine whether the test agent is a modulator of JMJD4 activity. Such assays may use purified materials or be carried out in cells. Any suitable assay may be carried out to identify modulators of JMJD4 lysyl-hydroxylase activity. A number of different examples of suitable assays are described below. Assays of the invention may be used to identify an agent which modulates, such as inhibits or activates, J JD4 lysyl hydroxylase activity.
  • the assay preferably measures hydroxylation of eRFl .
  • the assay employs eRFl without eRF3 release factor and particularly without being part of a complex with the eRF3 release factor complex.
  • eRFl is provided with eRF3, particularly as a complex with eRF3.
  • the assay measures hydroxylation of eRFl at K63 of eRFl.
  • JMJD4 lysyl hydroxylase activity may be assayed by monitoring oxygenase activity of JMJD4 polypeptides in the presence of substrate.
  • the substrate is eRFl, and in particular the human eRFl (in other instances, other JMJD4 substrates containing a lysyl-residue, may be employed).
  • the JMJD4 polypeptide hydro xylates K63 of the eRFl protein, or fragment or analogue thereof.
  • the substrate and the JMJD4 polypeptide, and optionally the test agent, are typically contacted under conditions suitable for oxygenase
  • Suitable co-substrates include oxygen, for example, dioxygen, and 2-oxoacids such as 2-oxoglutarate (20G) or 20G analogues (such as 2-oxoadipate).
  • the co- substrate is 20G.
  • a reducing agent such as ascorbate may also be used as a cofactor.
  • the translation termination protein or analogue or fragment thereof and JMJD4 polypeptide are contacted in the presence of Fe(II), oxygen and 2-oxoglutarate and optionally in the presence of a reducing agent.
  • eRFl is contacted with JMJD4.
  • the eRFl is not part of a complex with eRF3, in some instances it may though be part of such a complex.
  • Hydroxylation of the substrate may be assayed directly or indirectly.
  • Such assays may employ techniques such as chromatography, NMR, MS or fluorescence spectroscopy.
  • the co-substrate may be modified, e.g. 20G, consumed, e.g. oxygen, or produced, e.g. succinate or carbon dioxide, by JMJD4 polypeptide.
  • the components of the assay are preferentially contacted under conditions in which JMJD4 has lysyl
  • the assay may also be used to detect agents that increase or decrease the activity of JMJD4 activity by assaying for increases or decreases in activity including in whole organisms. Suitable assays have been described in the art for other 20G enzymes. Other assay configurations may rely on methods for assessing binding, e.g. by displacement of an appropriately labelled JMJD4 binding peptide from the JMJD4 active site. Cell-based assays in which the hydroxylation status of, for instance, eRFl is assessed either by mass spectrometry or by use of appropriate antibodies are also suitable.
  • Such assays have been developed for the HIF prolyl hydroxylases and OGFOD1 and are amenable to the study of JMJD4 activity in animals including humans, and in different tissue types (healthy and diseased). Any of the techniques disclosed herein for mass spectroscopy may be, for instance, employed. Measurement of JMJD4 activity is of particular interest with respect to investigating the
  • Assays of the present invention may be used to identify inhibitors of oxygenase activity and are thus preferably, but not necessarily, carried out under conditions under which JMJD4 is active as an oxygenase (a lysyl-hydroxylase) in the absence of the test agent.
  • the JMJD4 oxygenase activity in the presence of the test agent is compared to JMJD4 oxygenase activity in the absence of the test substance to determine whether the test substance is an inhibitor of JMJD4 oxygenase activity.
  • the assays may be used to look for promoters of JMJD4 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, either with purified materials in cells or in animals, under conditions in which JMJD4 oxygenase activity is reduced or absent, such as under hypoxic conditions, and the presence of or increased activity can be monitored under such conditions.
  • the assays of the invention may also be used to identify inhibitors or activators that are specific for lysyl hydroxylases, such as JMJD4 (or homologues of JMJD4) and which do not have activity or are less active with other 20G oxygenases, including other human 20G oxygenases, for which assays have been reported or which are amenable to reported assays for 20G oxygenases.
  • the assays of the invention may be used to identify inhibitors or activators specific for one or more 20G oxygenases which do not inhibit JMJD4 activity. Human 20G oxygenases that may be tested in such a method of the invention are listed in Table 1.
  • Such 20G oxygenases include, but are not limited to: argininyl, prolyl, and asparaginyl demethylases, hypoxia inducible factor (HIF) asparaginyl or prolyl hydroxylases, including FIH, PHD1, PHD2 and PHD3, and nucleic acids modifying enzymes including ABH1, ABH2, ABH3 and ABH8, procollagen prolyl and lysyl hydroxylases, methyl arginine demethylases, the fat mass and obesity protein, the epidermal growth factor hydroxylases, AlkB, TauD, and other 20G oxygenases that have been characterized as JmjC domain proteins according to the SMART database including, but not limited to argininyl demethylases.
  • HIF hypoxia inducible factor
  • the present invention also provides a method for identifying a selective inhibitor of JMJD4 (or JMJD4 homologue), or an inhibitor that is selective for another 20G oxygenase over JMJD4.
  • This method comprises: (i) contacting an JMJD4 substrate, such as eRFl respectively, or fragment thereof comprising a lysyl residue, with an JMJD4 polypeptide in the presence of a test agent and determining whether the protein or fragment thereof is hydroxylated; (ii) determining whether the test agent modulates activity of a 20G oxygenase other than JMJD4, thereby determining whether the test agent selectively modulates JMJD4 activity or selectively modulates activity of the 20G oxygenase other than JMJD4.
  • Oxygenase activity of the 20G oxygenase other than JMJD4 may be determined by contacting a substrate of the 20G oxygenase with the 20G oxygenase in the presence of a test agent and determining whether the substrate is hydroxylated or demethylated or otherwise oxidized. In an assay to identify a selective inhibitor of JMJD4, or another oxygenase, different substrates may be used for JMJD4 and for the other oxygenase(s).
  • oxygenase activity of the 20G oxygenase other than JMJD4 may be determined in the absence of a prime substrate (i.e., a non-20G substrate).
  • a prime substrate i.e., a non-20G 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 20G by the oxygenase.
  • the invention also provides methods for screening for compounds that do not inhibit JMJD4.
  • Such compounds are of use with respect to developing inhibitors that are selective for 20G oxygenases other than JMJD4.
  • JMJD4 inhibitors that do not inhibit the HIF prolyl or asparaginyl hydroxylases are of interest.
  • Standard methods can be used to develop selective inhibitors including examples of developing selective 20G oxygenase inhibitors, for example, for the HIF prolyl hydroxylases over the HIF asparaginyl hydroxylase.
  • the development of selective inhibitors may employ structural methods that identify differences in the active sites between the enzymes of interest (e.g. between JMJD4 and the HIF prolyl hydroxylases and or the human collagen prolyl hydroxylases).
  • the assays of the invention may also be used to identify inhibitors or activators, which are specific for JMJD4 activity at a particular substrate or residue within a substrate.
  • Such selectivity screens may be used to identify selective inhibitors of JMJD4 or selective inhibitors of other enzymes, i.e. inhibitors that are more potent inhibitors of JMJD4 activity than of activity of the other enzyme or inhibitors that are less potent inhibitors of JMJD4 activity than of activity of the other enzyme.
  • the inhibitor is a selective inhibitor of JMJD4 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.
  • the inhibitor is a selective inhibitor of the activity of the enzyme other than JMJD4, it may have no effect on the activity of JMJD4 or may exhibit only a low level of inhibition, such as less than about 50% inhibition of JMJD4 activity.
  • the selectivity screens may be carried out with purified enzymes, partially purified enzymes (such as in crude cell lysates) or in cells, or in animals including humans, and employ the assays methods listed above or other methods.
  • the invention provides for 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 JMJD4 oxygenase activity.
  • the precise format of any of the assay or screening 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 the 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. For instance, the effect on translation and in particular stop codon readthrough, may be measured. Suitable reporter constructs (e.g. readthrough reporters) may be employed in the assays.
  • Any suitable method may be used for determining 20G oxygenase activity of JMJD4 such as by substrate or co-substrate utilisation, product appearance such as peptide hydroxylation (or demethylation for some 20G oxygenases) or down- stream effects mediated by hydroxylated products (or demethylated or non- hydroxylated products for some 20G oxygenases).
  • the substrate, enzyme and potential inhibitor compound may be incubated together under conditions which, in the absence of inhibitor provide for hydroxylation (or demethylation for some 20G oxygenases) of the substrate, and the effect of the inhibitor may be determined by determining hydroxylation (or demethylation for some 20G oxygenases) 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. Any such methods described herein, for instance, may be employed. 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.
  • reporter constructs may be provided.
  • the reporter constructs may be used to measure translation and in particular stop codon readthrough.
  • Any suitable reporter gene may be used, such as for example enzymes which may then be used in colorimetric, fluorimetric, fluorescence resonance or spectrometric assays.
  • the JMJD4 polypeptide and the substrate are contacted in the presence of a co-substrate, such as oxygen and/or a 2- oxoacid, such as 20G (or analogue thereof).
  • Hydroxylase activity may be determined by determining turnover of one or more of the co-substrates, such as oxygen or 20G. This may be achieved by determining the presence and/or amount of reaction products, such as hydroxylated substrate, carbon dioxide or succinic acid.
  • the amount of product may be determined relative to the amount of substrate.
  • the product measured may be hydroxylated peptide or protein.
  • the extent of hydroxylation may also be determined in cells, e.g.
  • the extent of hydroxylation may be determined by measuring the amount of hydroxylated peptide/protein, succinate, carbon dioxide, or formaldehyde generated in the reaction, or by measuring the depletion of 20G 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. An assay that measures oxygen consumption such as that described by Ehrismann et al. Biochem J. (2007) may be used.
  • an enzyme activity assay that measures 14 C0 2 generated from the decarboxylation of [ ! C]-20G coupled to hydroxylation (Kivirikko KI, Myllyla R. Methods Enzymol (1982) may also be used.
  • 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.
  • Use of ion-exchange chromatography to separate [ 14 C]- succinic acid and [5- 14 C]-20G or separation using 2,4-dinitrophenylhydrazine to precipitate [5- 14 C]-20G may also be used.
  • the formation of a hydroxylated peptide fragment can be determined directly, e.g. by using either LC/MS analysis, Li D, Hirsila M, Koivunen P, et al. J Biol Chem (2004), or matrix-assisted laser desorption ionization, time-of-flight mass spectrometer or by other assay monitoring hydroxylation.
  • Monitoring the consumption of a reducing agent such as potassium ferrocyanide (replacing ascorbate) FibroGen, Inc.
  • Antibody based methods may also be used by employing an antibody selective for a hydroxylated product or non-hydroxylated substrate. Antibody based methods may be enhanced such that they are more efficient for modulator screening, e.g. by use of homogenous time resolved fluorescence (HTRF) methods which measure the energy transfer between a labelled dye (e.g., via biotin-streptavidin complex) to hydroxyl-proline peptide fragment substrate, and europium, which is tagged to a hydroxyl-proline specific antibody similar to methods described in Dao JH, Kurzeja RJM, Morachis JM, et al. Anal Biochem (2009). Assays that measure displacement of a substrate from JMJD4 may also be employed - these may employ the use of suitably tagged reagents and antibodies.
  • HTRF homogenous time resolved fluorescence
  • the amount of unused 20G may be determined, e.g., by spectroscopy or derivatisation by chemical reagents, exemplified by but not limited to hydrazine derivatives and ort/zo-phenylene diamine derivatives, to give indicative chromophores or fluorophores that can be quantified and used to indicate the extent of hydroxylation of the substrate. Suitable methods are described in McNeill et al. (2005) (Anal. Biochem. 366: 125- 131).
  • the fluorescent product of the reaction of or ⁇ o-phenylenediamine (OPD) with the -ketoacid motif of 20G is 3-(2-carboxyethyl)-2(lH)-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.
  • a reducing agent such as ascorbate, a thiol such as dithiothreitol (DDT), ⁇ -mercaptoethanol, tris(2- carboxyethyl)phosphine hydrochloride (TCEP), N-acetylcysteine or phenol
  • DDT dithiothreitol
  • TCEP tris(2- carboxyethyl)phosphine hydrochloride
  • phenol tris(2- carboxyethyl)phosphine hydrochloride
  • catalase may be added to destroy any H 2 0 2 that might be produced.
  • 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.
  • as assay for enzymatic activity employed in the invention may, for instance, comprise employing recombinant HIS-eRFl as a substrate, for instance in an amount of 2 g, and a JMJD4 modulator of the invention, for instance in an amount of 200ng.
  • the two may, for instance, be incubated at 37°C, for example in a reaction volume of about 200 ⁇ 1 of assay buffer (on suitable reaction buffer comprises 50mM Tris-HCl pH 7.4, 0.8mM L-ascorbate, 16 ⁇ FeS0 4 , 160 ⁇ 20G and ImM DTT). After the incubation time the reaction may be quenched, for instance with 0.1% formic acid.
  • Proteins may then, for example, be extracted using methanol-chloroform, digested with trypsin and eRFl K63 hydroxylation quantified by mass spectroscopy.
  • assays are carried out under normoxic conditions, but may be carried out at oxygen concentrations above or below atmospheric levels.
  • 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.
  • 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.
  • reverse phase HPLC C-4 octadecylsilane column
  • 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.
  • 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, such as an antibody, which discriminates between the hydroxylated and non-hydroxylated forms of a peptide or protein 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 endogenously or from one or more vectors introduced into the cell.
  • Such assays may employ the use of antibodies specific for hydroxylated or non-hydroxylated forms of JMJD4 substrates, or may employ proteomic mass spectrometry-based methods based on protease-catalysed digestions or analyses on intact proteins.
  • the invention further provides a method for identifying a modulator of protein production and/or fidelity, the method comprising contacting a cell (an organism) which expresses JMJD4 with a test agent and determining whether the test agent modulates JMJD4 regulation of protein translation and stop codon readthrough.
  • the invention further provides a method for distinguishing between cells that are hypoxic and normoxic. This is because the JMJD4 activities are dependent on oxygen.
  • the degree of hydroxylation of JMJD4 substrates e.g. eRFl
  • the degree of hydroxylation of JMJD4 substrates e.g. eRFl
  • the invention thus further discloses a way of selectively targeting hypoxic cells (such as cancer cells) by use of compounds that preferably inhibit translation termination mediated by JMJD4.
  • hypoxic cells such as cancer cells
  • JMJD4 Many inhibitors are available and some are used as antibiotics and some possess anti-tumor activity. Modification of these, or other inhibitors, will allow selective recognization of a hydroxylated or non-hydroxylated eRFT.
  • JMJD4 may be over-expressed in cells.
  • JMJD4 may be over- expressed in a cell in vitro or in vivo by any suitable method, typically by introducing an expression vector encoding a JMJD4 polypeptide into the cell.
  • Protein translation (or stop codon readthrough) may be monitored in the cell over-expressing JMJD4 and compared to protein translation in a control cell that does not over-express JMJD4.
  • the cell over-expressing JMJD4 may be contacted with a test agent and protein translation and termination may be monitored in the presence of the test agent.
  • JMJD4 By comparing stop codon readthrough observed in the presence and absence of the test agent and in the presence and absence of JMJD4 over-expression, it may determine whether the test agent modulates JMJD4-mediated regulation of protein translation and termination. Levels of JMJD4 catalysed hydroxylation in cells may be determined by use of antibodies or by mass spectrometric methods as routinely used in proteomic analyses. In another embodiment, JMJD4 may be under-expressed in cells. JMJD4 may be under-expressed in cells in vitro or in vivo by any suitable method, for example by using RNAi/siRNA technology to knock down the JMJD4 protein.
  • siRNA may be used, in particular one capable of inhibiting expression of JMJD4 from the polynucleotide sequence of SEQ ID NO: 1.
  • Protein translation and stop codon readthrough may be monitored in the cell under-expressing JMJD4 and compared to protein translation and stop codon readthrough in a control cell that does not under- express JMJD4.
  • the cell under-expressing JMJD4 may be contacted with a test agent and protein translation and stop codon readthrough may be monitored in the presence of the test agent. By comparing the protein translation and stop codon readthrough observed in the presence and absence of the test agent and in the presence and absence of JMJD4 under-expression, it may be determined whether the test agent modulates JMJD4-mediated regulation of protein translation and stop codon readthrough.
  • the methods of the invention may entail seeing what effect modulating JMJD4 has on translation when used in combination with a known modulator of translational termination, such as a known stimulator of readthrough of translational termination.
  • the effect on translational termination may be examined for the two individually and in combination.
  • Methods for monitoring protein translation rate, accuracy and termination are well known in the art.
  • protein translation and stop codon readthrough may be monitored using a reporter construct.
  • the cell in a method for identifying a modulator of protein translation and stop codon readthrough according to the invention, the cell may comprise a protein translation or stop codon readthrough reporter construct and the method may comprise determining whether JMJD4-mediated regulation of protein translation and stop codon readthrough of the reporter constructs is modulated by the test agent.
  • An assay of the invention may measure translation termination and may measure readthrough.
  • an assay may comprise a construct with a stop codon, where readthrough of the stop codon can be detected.
  • the construct may have two detectable markers, where the first is present before the stop-codon so is expressed irrespective of readthrough and the second is only expressed with readthrough of the stop codon.
  • Any suitable markers may be used and in particular any suitable pair of markers may be employed.
  • the two markers may be selected from beta-galactosidase, renilla luciferase and firefly luciferase.
  • the two markers may, for example, be one of beta-galactosidase and renilla luciferase paired with firefly luciferase, for instance beta galactosidase and firefly luciferase may be used as the pair of markers or renilla luciferase and firefly luciferase.
  • the assays may, for instance measure the relative expression of each and hence the amount of readthrough may be determined.
  • Red Fluorescent Protein (RFP) and/or Green Fluorescent Protein (GFP) are used as markers and in a particularly preferred instance, RFP and GFP may be used together as the two markers in an assay of the invention.
  • the stop codon used in assays of the invention may for instance be one characterized as a leaky stop codon or a strong stop codon. The ability of a test agent to modulate JMJD4 may be measured by such an assay.
  • Assays may include in vitro assays of stop codon readthrough and/or translational termination efficiency. Any suitable assay for translation, for instance for translational termination and in particular readthrough of stop codons, may be used.
  • An example of an in vitro assay which may be used is one performed is an in vitro assay of stop codon readthrough performed with rabbit reticulocyte lysate. Addition of eRFl to such a lysate usually reduces the amount of readthrough, for instance, as measured by tandem reporter constructs separated by a stop codon.
  • a further possible assay which may be employed is an in vitro assay for translational termination efficiency performed using a reconstituted eukaryotic translation system (for instance using purified ribosomal subunits, initiation, elongation, and termination factors, and aminoacyl tR As).
  • Such assays may use eRFl exposed to a JMJD4 polypeptide of the invention, followed by a measure of the ability of the eRFl to either inhibit stop codon readthrough or promote translational termination.
  • Such assays may be, for instance, used as a way to gauge the effect of a test agent on JMJD4 activity, with measurement being performed in the presence and absence of the test agent and the results compared.
  • Assays of the invention may look at the ability of a modulator of JMJD4 to bring about readthrough in cells from a subject with a disorder involving nonsense mutations.
  • illustrative assays may be based on readthrough in cancer cells, such as in: colon carcinoma cells LoVo cells (APC Rl 114 stop mutation), where, for instance, APC activity may be measured using the pTOPGlow reporter to measure beta-catenin transcriptional activity;
  • breast carcinoma HDQ-P1 cells (p53 R213 stop mutation), where, for instance, p53 activity can be measured using a concatamerized p53 responsive element upstream of luciferase;
  • lung adenocarcinoma A549 cells where, for instance, LKB1 activity is measured by probing for phosphroylation of an LKB1 substrate, AMPK; or
  • VHL Q132 stop mutation - renal carcinoma SKRC-7 cells
  • Assays of the invention may also be based on non-cancer cells. For instance, readthrough may be measured in cells from any condition which involves nonsense mutations, including cells from any of such conditions named herein.
  • 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 synthesis 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.
  • peptide antagonists may be polypeptides, peptides, small molecules such as molecules from commercially available libraries, including combinatorial libraries, or the like.
  • the peptide may be a cyclic peptide and may contain non proteinogenic residues.
  • Small molecule compounds, which may be used include 20G 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 20G oxygenase inhibitors such as those already known to inhibit HIF hydroxylases (see for example WO03/080566, WO02/074981, WO2007/146483, WO2007136990, WO2007/103905, WO2007/150011, US2007/0299086,
  • Potential promoting agents may be screened from a wide variety of sources, particularly from libraries of small compounds, which may be commercially available.
  • Candidate compounds to be screened may include 20G analogues, compounds that chelate iron or known families of 20G oxygenases inhibitors.
  • TCA cycle intermediates such as fumarate and succinate
  • JMJD4 Since naturally occurring compounds, including TCA cycle intermediates such as fumarate and succinate, are known inhibitors of 20G oxygenases they may inhibit JMJD4 possibly in a manner that is of physiological relevance, including in some cancers where fumarate is known to be upregulated.
  • 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 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 JMJD4.
  • the present invention provides an agent or compound identified by a screening method of the invention to be a modulator of JMJD4 oxygenase activity e.g. a substance which inhibits or reduces, increases or potentiates the activity of JMJD4.
  • a modulator of JMJD4 oxygenase activity e.g. a substance which inhibits or reduces, increases or potentiates the activity of JMJD4.
  • the test agent may compete with 20G or an JMJD4 substrate at the JMJD4 active site and/or binds to the active site of JMJD4 or to metal at the JMJD4 active site.
  • the test agent may comprise a metal ion such as, but not limited to, manganese, cobalt, zinc or nickel ions as inhibitors or iron (II), iron (III) as activators.
  • the mode of inhibition may be via competition with the substrate or by an allosteric interaction.
  • the test agent may be a reducing agent.
  • Reducing agents typically act as activators of 20G oxygenase activity, typically in vitro.
  • An activator of oxygenase activity may be any species that increases oxygenase activity of a JMJD4 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).
  • 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.
  • the modulator may be alone or used in combination with any other therapeutically active substance or treatment including, but not limited to, metal ions or succinate or fumarate (Chen et al. J Biol Chem 2010).
  • 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 a dimethyl ester, diethyl ester, monoethyl ester or di- or mono-amide, or other prodrug form rendering suitable pharmacokinetic properties. 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 20G oxygenases may be useful as agents of the invention, for example, in the treatment of disorders as described herein, or may be used as test substances in an assay of the invention.
  • the test compound may be Icnown to act as an inhibitor of a 20G oxygenase other than JMJD4.
  • the test agent may be a described 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 20G oxygenase involved in metabolism or regulation, or a plant 20G hydroxylase.
  • Many inhibitors of 20G oxygenases are known in particular for human prolyl hydroxylases and histone demethylases.
  • N-Oxalylglycine and its derivatives are such examples, but there are many others, which one of skilled in the art of oxygenases may test as JMJD4 inhibitors, glycine or alanine derivatives and 2-oxoacid analogues may also be used.
  • Compounds which modulate 20G 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.
  • HIF hydroxylase inhibitors are disclosed in United States Patent Application Publication Nos: 20070042937, 20060276477, 20060270699, 20060258702, 20060258660, 20060251638, 20060183695,
  • the present invention further provides for the use of an inhibitor or activator of 20G oxygenase activity to modulate lysyl-hydroxylation of translation termination proteins by JMJD4, particularly eRFl .
  • an inhibitor or activator of 20G oxygenase activity to modulate lysyl-hydroxylation of translation termination proteins by JMJD4, particularly eRFl .
  • Such use may be, for instance, in vitro, in vivo and/or ex vivo.
  • a compound, substance or agent which is found to have the ability to affect the oxygenase (lysyl-hydroxylase) activity of JMJD4 has therapeutic and other potential uses in a number of contexts, as discussed.
  • the modulator of JMJD4 lysyl-hydroxylase activity may be a known inhibitor of a 20G oxygenase, such as an N-oxalyl amino acid such as N-oxalylglycine (NOG) or a derivative thereof, a glycine or alanine derivative, a 2-oxoacid analogue, a bipyridyl derivative, a diacylhydrazine, a catechol or catechol derivative such as gallic acid, or pyridine-2,4-dicarboxylic acid or FG2216.
  • the inhibitor may be a selective inhibitor of JMJD4 activity compared to other 20G oxygenases.
  • 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.
  • 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.
  • 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.
  • 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 term "therapy” includes use of the agents of the invention described herein for the benefit of a human or animal patient. Specifically this term includes therapeutic treatment, prophylactic treatment, and diagnosis. This list is provided by way of illustration only, and is not intended to be limiting. In a particularly preferred instance, the subject to be treated is human, though the subject may be any of the species referred to herein.
  • the agents of the present invention may be administered to a patient using any one or more of a number of modes of administration.
  • modes of administration are well known in the art and may include parenteral injection (e.g. intravenously, subcutaneously, intraperitoneally, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intradermal, intrathecal, intranasal, ocular, aural, pulmonary or other mucosal administration.
  • parenteral injection e.g. intravenously, subcutaneously, intraperitoneally, intramuscularly, or to the interstitial space of a tissue
  • rectal oral, vaginal, topical, transdermal, intradermal, intrathecal, intranasal, ocular, aural, pulmonary or other mucosal administration.
  • the precise mode of administration will depend on the disease or condition to be treated.
  • the invention is particularly relevant to the treatment of cancer, given the identification herein that JMJD4 plays a role in cancer cell proliferation and survival. JMJD4 therefore represents a target for cancer therapy.
  • the invention hence provides a method of treating an animal suffering from cancer comprising administering to said animal a modulator of JMJD4 in a therapeutically effective amount and in particular administering an inhibitor of JMJD4 activity.
  • the cancer treated via the invention may be, in one instance, a cancer involving a nonsense mutation.
  • the invention may be used to promote readthrough of the stop codon and help treat a cancer involving a nonsense mutation.
  • the invention may be, for example, used to treat any of the type of cancer mentioned herein where the subject has a nonsense mutation causing, giving rise to,
  • the invention may also be used to treat cancers in general, including those cancers where a nonsense mutation is not involved.
  • cancer examples include a carcinoma, a sarcoma, or a blastoma.
  • the invention contemplates the treatment of cancers of any organ including but not limited to cancers of the breast, lung, ovaries, pancreas, testes, skin, colon, brain, liver or cervix, as well as melanoma.
  • Other cancers which may be treated include bladder cancer, renal cancer, prostate cancer, pancreatic cancer, stomach cancer, bone cancer, skin cancer, including melanoma, malignant soft tissue sarcoma, leukemia, including myeloma, and the like.
  • the agent or composition of the inventon may be used as a
  • chemotherapeutic agent for the treatment of cancer.
  • the agent or composition of the invention may be used as a chemotherapeutic agent in combination with another anticancer agent or treatment. Such combinations may, for instance, be administered simultaneously, sequentially or separately.
  • an anti-mitotic agent such as docetaxel, paclitaxel
  • a platinum- based chemotherapeutic compound such as cisplatin, carboplatin, iproplatin, oxaliplatin, or other conventional cytotoxic agent, such as 5-fluorouracil (5-FU), capecitabine, irinotecan, leucovorin, gemcitabine
  • inhibitors of receptor tyrosine kinases and/or angiogenesis such as ErbB inhibitors, RTK class III inhibitors, VEGFR.
  • the JMJD4 inhibitor is used in combination with one or more other agents that modulate stop codon readthrough.
  • the JMJD4 inhibitor may be administered simultaneously, sequentially or separately with the other inhibitor, for instance they may be administered in the same composition or in two separate compositions.
  • Assays of the invention may assess the efficacy of JMJD4 modulators to act in tandem with other modulators of readthrough.
  • an assay of the invention may involve a step of screening compounds identified as inhibitors of JMJD4 for their ability to modulate readthrough together with a second compound able to bring about readthrough.
  • Other agents which may be used in combination with the JMJD4 modulators include agents known to modulate protein translation, such as mTOR inhibitors, and agents known to regulate the degradation of mis-folded proteins by the proteasome
  • any suitable agent known to induce stop codon readthrough may be used in conjunction with a JMJD4 modulator of the invention, such agents include aminoglycoside antibiotics (gentamicin, tobramycin, amikacin, paromomycin, G418, lividomycin, streptomycin, hygromycin B, neomycin, kanamycin A, and Isepamicin), dipeptide antibiotics (negamycin) and non-antibiotics (PTC 124).
  • aminoglycoside antibiotics gentamicin, tobramycin, amikacin, paromomycin, G418, lividomycin, streptomycin, hygromycin B, neomycin, kanamycin A, and Isepamicin
  • dipeptide antibiotics negamycin
  • PTC 124 non-antibiotics
  • the invention may result in decreasing, reducing, or halting tumor growth in a subject in need thereof.
  • the invention may also be used to bring about cell cycle arrest, for instance by inhibiting JMJD4.
  • the treatment of the invention when used in combination with other cancer treatments may result in reduced toxicity of the treatment, compared to the other treatment alone. For instance, because the amount of the other treatment may be lowered.
  • the invention further provides a method of treatment which includes administering to a patient an agent which modulates JMJD4 oxygenase activity.
  • agents may include inhibitors of JMJD4 oxygenase activity.
  • the agent may modulate the lysyl hydroxylase activity of JMJD4 and preferably inhibit such activity. Any of the modulators specified herein may be used.
  • the invention also provides for the use of a substance or composition of the invention to suppress translational termination.
  • modulators of JMJD4 may be used for the treatment of diseases caused by premature stop-codons.
  • the invention may therefore be used to bring about stop codon readthrough.
  • JMJD4 inhibitors include, but are not limited to, cancer, cystic fibrosis (CF), haemophilia, retinitis pigmentosa, Duchene muscular dystrophy (DMD), Thalassaemia, cystinosis, spinal muscular atrophy, type 1 Usher syndrome, Factor VII deficiency, Familial atrial fibrillation, Hailey-Hailey disease, Hemophilia, ataxia- telangiectasia, Infantile neuronal ceroid lipofuscinosis. McArdle disease, X-linked nephrogenic diabetes insipidus, and Hurler syndrome. Such agents may also be used to combat retroviruses, such as HIV.
  • Cancer may also be treated by ensuring efficient translational termination using a modulator of JMJD4 activity, particularly of nonsense mutation(s) in tumour suppressor genes comprising such mutations.
  • Xenograft tumour models may be used to assess the efficacy of particular modulators of JMJD4 in treating cancer.
  • a therapeutically effective amount of an agent is typically administered to a subject in need thereof in the methods discussed herein.
  • the modulator of JMJD4 employed may, for instance, be any of those discussed herein, including in particular a small molecule modulator or an siRNA.
  • the modulator will be an inhibitor.
  • the present invention thus enable the development of pharmaceutical compositions, medicaments, drugs or other compositions for modulating JMJD4 for any of the purposes discussed herein.
  • the composition may, for instance, comprise one or more agents, compounds or substances as described herein, including a modulator of, particularly an inhibitor of, JMJD4 activity.
  • the invention includes 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
  • a pharmaceutical composition comprising admixing such an agent, compound or substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
  • agents may be useful as anti-microbial agents, for example for use as antibiotics to treat bacterial infection in an individual.
  • the invention also provides for the use of an inhibitor of JMJD4 to reduce or inhibit cell proliferation, particularly where the cell is a cancer cell.
  • Anti-sense and siRNA inhibitors may, for instance be used, as may antibodies against JMJD4. Any of the inhibitors referred to herein may be employed.
  • the method for providing a pharmaceutical composition may typically comprise:
  • compositions of the invention may comprise an agent, polypeptide, polynucleotide, vector or antibody according to the invention and a pharmaceutically acceptable excipient.
  • an anti-sense nucleic acid or an siRNA capable of modulating JMJD4 is provided in a pharmaceutical composition of the 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.
  • compositions according to 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.
  • 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.
  • 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.
  • 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.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • 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.
  • 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.
  • the substance or composition may be delivered locally to a tumour.
  • 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.
  • the therapy is specifically targeted to a tumour or cancer cells.
  • HEK293T, A549, HeLa, and U20S cells were cultured in DMEM supplemented with 10% fetal calf serum, penicillin and streptomycin under standard conditions.
  • Stable HEK293T and HeLa cell lines overexpressing FLAG-JMJD4 or -FIH were cultured as above, in the presence of lOg/ml puromycin.
  • RNA interference (Mission SiR A, Sigma) was performed using three consecutive transfections using Oligofectamine (for HEK293T, A549 and HeLa; Invitrogen) or N-TER (for U20S; Sigma) reagent at 25nM per transfection.
  • Transient transfections were performed using Fugene6 (HEK293T; Roche) or Turbofect (A549; Fermentas) according to the manufacturer's instructions. Endogenous eRFl hydroxylation was measured in SILAC-labelled A549 cells (see below) treated as follows. Hypoxia was performed at 1 or 0.2% oxygen for 16 hours using a Ruskinn hypoxia station, followed by cell lysis (see below) in the presence of ImM N-oxalylglycine (NOG; Santa Cruz Biotechnology). Cellular inhibition of 20G dioxygenases was achieved by treating cells with ImM dimethyl -N-oxalylglycine (DMO G; Sigma) for 16 hours.
  • Expression constructs Expression constructs
  • eRFl or eRF3A cDNA's were cloned into pEF6 by PCR to incorporate a V5- or HA-epitope tag at the N-terminus, respectively.
  • SiRNA resistant wildtype and inactive H189A JMJD4 cDNA's were created by introducing four single base pair silent mutations into the sequence targeted by siRNA #31 (Sigma): these cDNA's were subsequently cloned into the RFP site of the doxycycline-inducible pTRIPZ vector (Open Biosystems). Site-directed mutagenesis was performed by PCR using standard procedures.
  • Human JMJD4 and FIH cDNA's were cloned into the Not-I/Cla-I restriction sites of the pGIPZ vector (Open Biosystems) using PCR primers incorporating N-terminal 3XFLAG epitope tags, thereby replacing the GFP cDNA with the required enzyme cDNA.
  • Human JMJD4 cDNA was cloned into the pTriEx2 recombinant expression vector platform (Merck) by PCR. Stop codon readthrough reporters were designed to encode the Beta-Galactosidase cDNA upstream of a recoding window and in-frame with the firefly luciferase cDNA.
  • Plasmids were constructed using a two step PCR cloning strategy: the Beta- galactosidase coding sequence was inserted into pEFl/V5-HisA (Invitrogen) via Kpnl and BamHI sites, and the firefly luciferase gene subsequently inserted via BamHI and NotI restriction sites with a 5' extension on the forward primer containing the recoding sequences.
  • pEFl/V5-HisA Invitrogen
  • the firefly luciferase gene subsequently inserted via BamHI and NotI restriction sites with a 5' extension on the forward primer containing the recoding sequences.
  • the nuclear pellet was then resuspended in 50 ⁇ 1 of buffer NB (20mM HEPES pH7.9, 420mM NaCl, 1.5mM MgC12, 0.2mM EDTA, 0.5mM DTT, 0.5mM PMSF, IX Complete protease inhibitors) and homogenised for 30 minutes on ice with a magnetic stirrer. Samples were spun at 15000xg- for 15 minutes at 4°C and the nuclear fraction supernatant collected. Samples were equalised for protein content (BioRad DC reagent) prior to SDS-PAGE.
  • buffer NB 20mM HEPES pH7.9, 420mM NaCl, 1.5mM MgC12, 0.2mM EDTA, 0.5mM DTT, 0.5mM PMSF, IX Complete protease inhibitors
  • Membranes were blocked in 5% (w/v) milk powder in PBS/0.1% Tween-20 prior to probing with the following primary antibodies; anti-HA-HRP (Roche), anti-FLAG-HRP (Sigma), anti- JMJD4 (Sigma), anti-eRFl (Sigma), anti-eRF3A (Sigma), and anti-actin-HRP (Abeam).
  • HRP-conjugated secondary antibodies were purchased from Dako. Signals were developed using SuperSignal West Pico, Dura, or Femto chemiluminescent substrates (Thermo Fisher Scientific).
  • JMJD4 proteomic pulldowns to identify candidate substrates was performed by immunoprecipitating extracts derived from control, FLAG-JMJD4 or FLAG-JMJD4 H189A overexpressing cells with anti-FLAG affinity resin (Sigma) overnight at 4°C.
  • Identification of hydroxy lation sites in the eRFl/eRF3A complex was performed by first transiently transfecting HEK293T cells with V5-eRFl and HA-eRF3a in the presence or absence of HA-JMJD4 for 48 hours, prior to immunoprecipitation of extracts with anti-V5 affinity resin (Sigma) overnight at 4°C.
  • MS/MS spectra were extracted from raw files by ProteoeWizard MSConvert (http://proteowizard.sourceforge.net/) using the 200 most intense peaks in each and converted into MGF files.
  • the peaklists were searched against the IPI human database (v3.87, 91464 entries) using Mascot (http://www.matrixscience.com/) v2.3.01, allowing one missed cleavage and 20ppm/0.5 Da mass deviations in MS/MSMS.
  • Carbamidomethylation of cysteine was a fixed modification. Oxidation of methionine and lysine as well as deamidation of asparagine and glutamine were used as variable modifications. Annotation of oxidized methionines and lysines was performed manually and assisted with ModLS
  • Human HIS-eRFl was purified as described previously (3).
  • Human HIS- JMJD4 was purified from bacterial lysates using Ni-NTA resin (Qiagen) following standard procedures .
  • Recombinant HIS-eRFl (2ug) and JMJD4 (200ng) were incubated at 37°C in 200 ⁇ 1 of assay buffer (50mM Tris-HCl pH 7.4, 0.8mM L-ascorbate, 16 ⁇ FeS0 4 , 160 ⁇ 20G and ImM DTT. After the indicated incubation time, the reaction was quenched with 0.1% formic acid. Proteins were extracted using methanol-chloroform, digested with trypsin and eRFl K63 hydroxylation quantified by LC-MS.
  • A549 cells were transfected with reporter vectors for 48 hours before washing in ice cold PBS and lysis in passive lysis buffer (Promega). Samples were freeze-thawed once before assaying for firefly luciferase activity using luciferin (Promega) and beta-galactosidase activity using the FluorAce kit from BioRad. Both assays were performed on a Safire2 microplate reader (Tecan). RESULTS
  • JMJD4 The expression pattern of JMJD4 was determined in normal tissues and also in cancer cells, including a panel of tumours. The results obtained are presented in Figure 2. As can be seen from Figure 2, JMJD4 is ubiquitously expressed in normal tissues. Figure 2 shows that the protein is expressed in every line of the NCI60 panel of tumour cell lines. Figure 2 shows that the protein is over-expressed in tumour versus normal tissue. Hence over-expression of JMJD4 is associated with cancer.
  • eRFl The expression of eRFl in a variety of normal and corresponding tumours is depicted in Figure 3B and Figure 4B. Similar to JMJD4, eRFl was also found to be overexpressed in tumours. Further, eRFl was also found to be mitogen induced. Further work showed that JMJD4 is localised to the cytoplasm. A549, Hela and 293T cells were fractionated into nuclear and cytoplasmic fractions prior to immunoblot for endogenous JMJD4 and JMJD6. The immunoblot obtained showed that JMJD4 localised to the cytoplasmic fraction, whereas JMJD6, the enzyme most closely related to JMJD4, is not localised to the cytoplasm (data not shown). The different localization reflects the different roles the proteins play. In particular, the role of JMJD4 elucidated here by the inventors and the role of JMJD6 in splicing in the nucleus.
  • JMJD4 localizes to the cytoplasm. Localization of FLAG-JMJD4 in stable HEK293T cells was determined by confocal immunofluorescence microscopy and also of nuclear FLAG-JMJD5 for comparison. Consistent with an interaction between JMJD4 and a cytoplasmic complex (eRFl/eRF3A), JMJD4 was found to be cytoplasmically expressed. Requirement of JMJD4 activity for cancer cell proliferation
  • JMJD4 activity is required for cancer cell proliferation and survival. This was achieved via siRNA knockdown experiments. The results obtained are depicted in Figures 3 and 4.
  • siRNAs for JMJD4 Two independent siRNAs for JMJD4, a negative control siRNA for FIH and non- targetting siRNA were transfected into HeLa cells.
  • the two siRNAs against JMJD4 were obtained from Sigma Aldrich and sold under product codes SASI_Hs01_00053631 and SASI_Hs01_0005363.
  • Figure 4C shows the rate of cell proliferation, as well as a Western blot confirming the level of JMJD4 expression. As can be seen, the two JMJD4 siRNAs resulted in much lower cell proliferation, indicating that JMJD4 normally promotes cell proliferation.
  • JMJD4 enzymatic activity may promote proliferation/survival in tumour cells and be responsible for cell cycle progression. It therefore represents a new target for the treatment of cancer.
  • JMJD4 is related to the lysyl hydroxylase JMJD6 (see Figure 1A) and contains critical residues required for activity (see Figure 1C).
  • substrate(s) we screened for substrate(s) by identifying proteins that interact with the enzyme in an activity-dependent manner. Affinity purification of FLAG-tagged JMJD4 followed by mass spectrometry (MS) did not co- purify known JMJD6 substrates such as U2AF65 or other RS-domain proteins (data not shown), consistent with an independent function of JMJD4 from JMJD6. Rather, the most abundant activity-dependent JMJD4 interactors were the translational termination factors eRFl and eRF3A. Table 1 below shows the results obtained.
  • the Table below provides a summary of proteins interacting with JMJD4 in an activity-dependent manner.
  • Proteomic pulldown MS data from empty vector, FLAG-JMJD4 and FLAG-JMJD4 H189A (inactive) were cross-referenced and then filtered to highlight those proteins only identified in active JMJD4 complexes.
  • Thirteen candidate substrates were identified and are tabulated. These candidates were ranked according to MS parameters that are proportional to abundance in the sample; peptide spectral matches (PSMs), peptides and percentage coverage of the protein.
  • PSMs peptide spectral matches
  • the only two proteins demonstrated significant activity-dependent abundance were eRFl and eRF3A: binding partners that form the translational termination complex. The cellular localisation of each candidate is indicated (where known) for comparison to JMJD4.
  • Figure 6 A shows extracts from HEK293T cells stably expressing empty vector, FLAG-FIH (control), FLAG-JMJD4, or an inactive Fe(II) binding mutant (HI 89 A) immunoprecipitated with anti-FLAG resin prior to immunoblot for endogenous eRFl and eRF3A.
  • the left panel shows "Input" which corresponds to the cell extract prior to immunoprecipitation (IP).
  • IP immunoprecipitation
  • JMJD4 is sufficient for eRFl and/or eRF3A hydroxylation we overexpressed them in HEK293T cells in the presence or absence of exogenous FLAG- JMJD4.
  • Anti-V5 purification of the V5-eRFl/HA-eRF3A complex was performed from HEK293T cells.
  • HEK293T cells were transiently transfected with eRFl-V5 and eRF3A-HA in the presence or absence of FLAG-JMJD4 prior to anti-V5 immunopurification of the complex.
  • 5% of the purified material was visualised by SDS-PAGE and coomassie blue staining and the results obtained are shown Figure 6B.
  • eRFl was immunopurified from the tumour cell lines prior to trypsinolysis and LC-MS analyses.
  • Rabbit reticulocyte lysate (RRL) was diluted with lysis buffer before immunopurification of eRFl and in-gel trypsinolysis and MS analyses.
  • Mouse tissues were snap-frozen in liquid N 2 prior to homogenisation in lysis buffer and immunopurification of eRFl and MS analyses as described in the supporting methods. The results obtained indicate that eRFl K63 hydroxylation is abundant (>93%), ubiquitous and conserved across species (data not shown).
  • eRFl was immunopurified from the indicated human tumour cell lines prior to trypsinolysis and LC-MS analyses.
  • JMJD4 is a 2QG-dioxygenase
  • JMJD4 The ability of JMJD4 to act as a 20G and iron-dependent-dioxygenase, and direct lysyl hydroxylase of eRFl, was confirmed by in vitro assay. Incubation of E.coli expressed and purified recombinant human JMJD4 and eRFl in the presence of the 20G dioxygenase cofactors iron, 20G, and ascorbate resulted in complete hydroxylation of eRFl (see Figure 8B). This hydroxylation was time-dependent (see Figure 8C).
  • the 20G analogue inhibitor N-Oxalylglycine (NOG) also significantly impaired eRFl K63 hydroxylation indicating that JMJD4 is amenable to small molecule inhibition.
  • Termination of eukaryotic protein translation is mediated by either of three stop codons (UGA, UAG and UAA) and by eRF3 in complex with eRFl .
  • eRFl is a substrate for JMJD4.
  • the eRFl terminates translation by triggering peptidyl- tRNA hydrolysis at the peptidyl-transferase center following recognition of a stop codon in the ribosomal A-site.
  • Stop codon recognition is conferred by highly conserved motifs within the 'N-domain' of eRFl, including the 'NIKS' sequence (see Figure 9). The lysine within the NIKS motif is thought to be directly involved in binding and recognition of the invariant uridine of stop codons.
  • K63 This lysine is the residue that we have shown is hydroxylated by JMJD4. Therefore, we tested whether K63 hydroxylation is required for normal eRFl function and that if its inhibition impairs stop codon recognition and leads to 'readthrough' translation.
  • reporter vectors consisting of tandem beta-galactosidase and firefly luciferase cDNA's separated by a UGA, UAG or UAA stop codon embedded within a 'leaky' termination sequence from tobacco mosaic virus (see Figure 9).
  • JMJD4 siRNA was also sufficient to increase readthrough of a 'strong' stop codon in the absence of a leaky termination sequence (see Figures 10B and IOC). These effects were found to be specific, since JMJD4 knockdown did not affect luciferase activity from a control vector lacking a stop codon between the beta- galactosidase and luciferase cDNA's (see Figure IOC). Furthermore, similar assays in siRNA-resistant JMJD4 rescue cells confirmed that effects of JMJD4 siRNA on readthrough were 'on-target' and dependent on JMJD4 lysyl hydroxylase activity (see Figure IOC).
  • JMJD4 promotes cell proliferation/survival and is a lysyl hydroxylase of the translational -termination factor eRFl .
  • the over-expression of JMJD4 in tumours and the fact JMJD4 is required for tumour cell proliferation indicates that the enzyme is important in cancer and represents a therapeutic target for the treatment of cancer.
  • the work done shows that the JMJD4-eRFl pathway is, in particular, a novel chemotherapeutic target, particularly for two reasons, both of which are predicted on the fact that JMJD4 positively regulates eRFl function.
  • tumourigenesis coordinated activation of protein synthesis pathways is a critical feature of tumourigenesis. Therefore, a corresponding increase in translational-termination activity may be required in order to maintain high-fidelity protein synthesis.
  • numerous TSGs are inactivated by premature stop-codon mutations. Therefore, tumour cells driven by such mutations may depend on JMJD4 and efficient translational-termination to maintain TSG inactivation.
  • the protein eRFl found here to interact with JMJD4, terminates translation by triggering peptidyl-tRNA hydrolysis at the ribosomal peptidyl transferase center.
  • Structures of eRFl indicate a 'Y-shaped' topology consisting of N-, M- and C-domains, which are thought to mimic tRNA (Fig-6A). Whereas the C-domain mediates eRF3A binding, the N-domain mimics the tRNA anti-codon arm.
  • the N-domain contains highly conserved residues that mediate stop-codon recognition, including the 'NIKS' motif (Fig-6A). Cross-linking experiments show the NIKS motif Lysine contacts the invariant uridine of stop-codons.
  • this Lysine is the residue that we have now found here to be hydroxylated by JMJD4. Since endogenous eRFl is abundantly hydroxylated in rapidly proliferating cells the modification may promotes stop-codon recognition, efficient translational-termination, and high fidelity protein synthesis. Hence, we have identified a potential way to modulate those processes.
  • JMJD4 inhibition represents an additional opportunity for stimulating readthrough of premature stop codons and treatment of diseases caused by nonsense mutations.
  • 'Readthrough agents' such as aminoglycoside antibiotics and PTC 124 induce stop-codon readthrough and restore expression of nonsense-mutated genes responsible for inherited diseases. These agents have also been used to restore the function of the nonsense-mutated TSGs APC and p53. Therefore, as JMJD4 positively regulates translational-termination, its inhibition may represent an alternative approach to restore expression of nonsense-mutated disease genes.
  • the results presented here show a direct role for eRFl K63 hydroxylation in translational termination.
  • K63 hydroxylations promote intra- and inter- molecular interactions via hydrogen-bonding. Since K63 hydroxylation regulates the efficiency of eRFl to terminate at all three stop codons it may well be that the mechanism involves an interaction fundamental to the termination process. It is possible therefore that eRFl K63 hydroxylation regulates the interaction with itself, or with a neighbouring molecule such as a ribosomal protein, rRNA, or mRNA. Evidence for the latter comes from structural and biochemical cross-linking studies indicating that K63 binds nucleotide and interacts with the invariant uridine of stop codons, respectively. 2
  • JMJD4 inhibition therefore represents a new opportunity for stimulating readthrough of premature stop codons and treatment of diseases caused by nonsense mutations.

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Abstract

La présente invention concerne une méthode de traitement du cancer par modulation de l'activité du JMJD4. L'invention concerne également des dosages permettant de surveiller l'activité des activités du JMJD4, en particulier des dosages servant à identifier des modulateurs des activités du JMJD4. L'invention concerne en outre des dosages de surveillance de l'activité lysyl hydroxylase du JMJD4 sur son substrat, la protéine de terminaison de la traduction eRFl. L'invention permet également l'introduction de résidus hydroxylysyle dans des peptides et des protéines. L'invention consiste en outre en une façon de moduler la terminaison de la traduction et ainsi de stimuler la translecture de mutations non-sens associées à la maladie.
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WO2004031413A2 (fr) * 2002-09-30 2004-04-15 Oncotherapy Science, Inc. Technique de diagnostic de cancers bronchopulmonaires « non a petites cellules »
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WO2004031413A2 (fr) * 2002-09-30 2004-04-15 Oncotherapy Science, Inc. Technique de diagnostic de cancers bronchopulmonaires « non a petites cellules »
WO2005005601A2 (fr) * 2003-06-09 2005-01-20 The Regents Of The University Of Michigan Compositions et methodes de traitement et de diagnostic du cancer

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