WO2005069013A1 - Modulation of thrombomodulin proteolysis - Google Patents

Abstract

This invention relates to the proteolytic cleavage of thrombomodulin by rhomboids such as RHBDL2. Methods and means for modulating rhomboid-dependent thrombomodulin proteolysis for a range of therapeutic purposes are provided.

Description

Modulation of Thrombomodulin Proteolysis

This invention relates to the proteolytic cleavage of thrombomodulin, and in particular to methods and means of modulating thrombomodulin cleavage.

Thrombomodulin is expressed on endothelial cells that line blood vessels and forms a high-affinity complex with the clotting factor thrombin. In doing so, it inhibits the interaction of thrombin with fibrinogen. At the same time, the thrombin-thrombomodulin complex activates protein C, which proteolyses the activated coagulation factors Va and Villa. These two activities combine to give thrombomodulin an important anticoagulation role in blood clotting ( eiler, H., and Isermann, B.H. (2003). J Thromb Haemost 1, 1515- 1524, Esmon, C.T. (2002) . J Exp Med 196, 561-564) .

In addition to its function in anticoagulation, thrombomodulin has an important regulatory function in inflammation, at least partly via a protein C-independent mechanism (Conway, E.M. et al (2002). J Exp Med 196, 565-577) . It can also mediate cellular adhesion (Huang, H.C. et al (2003) . J Biol Chem) ; and its expression in a variety of cancers provides indication that thrombomodulin also plays a role in tumorigenesis . Finally, mouse knockouts have shown that it has essential functions during embryogenesis (Weiler H. and Isermann B.H. (2003) . J Thromb Haemost 1, 1515-1524) .

The present inventors have found that the thrombomodulin is a substrate of mammalian rhomboid proteins. Modulation of the intramembrane proteolysis of thrombomodulin, for example through the inhibition or enhancement of rhomboid activity, may have a range of therapeutic applications.

One aspect of the invention provides a method for identifying and/or obtaining a compound which modulates the interaction of rhomboid and thrombomodulin, which method comprises: (a) contacting an rhomboid polypeptide and a thrombomodulin polypeptide in the presence of a test compound; and

(b) determining interaction or binding between the thrombomodulin polypeptide and the rhomboid polypeptide.

Interaction or binding may be determined in the presence and absence of test compound. A change, for example a reduction or decrease, in interaction in presence of the test compound relative to the absence of test compound is indicative of the test compound being an modulator, for example an inhibitor, of rhomboid proteolytic activity. Such a compound may modulate the interaction of rhomboid and thrombomodulin, in particular thrombomodulin polypeptide cleavage .

Binding may be determined by any of a number of techniques available in the art, both qualitative and quantitative.

The precise format of the present methods may be varied by those of skill in the art using routine skill and knowledge. For example, the interaction between the rhomboid and thrombomodulin polypeptides may be studied in vi tro by labelling one with a detectable label and bringing it into contact with the other which has been immobilised on a solid support. Suitable detectable labels include ³⁵S- methionine which may be incorporated into recombinantly produced peptides and polypeptides. Recombinantly produced peptides and polypeptides may also be expressed as fusion proteins containing an epitope which can be labelled with an antibody. The protein which is immobilized on a solid support may be immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per se . A preferred in vitro interaction may utilise a fusion protein including glutathione-S- transferase (GST) . This may be immobilized on glutathione agarose beads. In an in vitro assay format of the type described above a test compound can be assayed by determining its ability to diminish the amount of labelled peptide or polypeptide which binds to the immobilized GST-fusion polypeptide. This may be determined by fractionating the glutathione-agarose beads by SDS-polyacrylamide gel electrophoresis .

Alternatively, the beads may be rinsed to remove unbound protein and the amount of protein which has bound can be determined by counting the amount of label present in, for example, a suitable scintillation counter.

Interaction between rhomboid and thrombomodulin may be determined using a so-called two-hybrid assay method. A polypeptide or fragment of rhomboid or thrombomodulin may be fused to a DNA binding domain such as that of the yeast transcription factor GAL4. The GAL4 transcription factor includes two functional domains. These domains are the DNA binding domain (GAL4DBD) and the GAL4 transcriptional activation domain (GAL4TAD) . By fusing one polypeptide or peptide to one of those domains and another polypeptide or peptide to the respective counterpart, a functional GAL4 transcription factor is restored only when two polypeptides or peptides of interest interact. Thus, interaction of the polypeptides or peptides may be measured by the use of a reporter gene probably linked to a GAL4 DNA binding site which is capable of activating transcription of said reporter gene. This assay format is described by Fields and Song, (1989) Nature 340; 245-246 and may be used in both mammalian cells and in yeast. Numerous two-hybrid assay formats and modifications thereof are known in the art.

A compound identified and/or obtained by the present methods may modulate the activity of rhomboid and/or thrombomodulin.

In some embodiments, interaction between the thrombomodulin and rhomboid polypeptides may be determined by detecting the proteolytic cleavage of the thrombomodulin polypeptide by the rhomboid polypeptide.

Another aspect of the invention provides a method for identifying/obtaining a modulator of rhomboid-dependent thrombomodulin proteolysis, which method comprises: (a) contacting an rhomboid polypeptide and a thrombomodulin polypeptide in the presence of a test compound; and

(b) determining proteolytic cleavage of the thrombomodulin polypeptide.

The rhomboid and thrombomodulin polypeptides may be contacted out under conditions in which the rhomboid polypeptide normally catalyses the intramembrane proteolytic cleavage of the thrombomodulin polypeptide.

Cleavage of the thrombomodulin polypeptide may be determined in the presence and absence of test compound. A change, for example a reduction or decrease, in cleavage in the presence of the test compound relative to the absence of test compound may be indicative of the test compound being an modulator, for example an inhibitor, of rhomboid-mediated thrombomodulin polypeptide cleavage. Such a compound may, for example, modulate the activity of rhomboid and/or the interaction of rhomboid with thrombomodulin. Modulators of rhomboid-mediated thrombomodulin polypeptide cleavage may useful in modulating the biological activity of thrombomodulin.

The polypeptides may be contacted in a reaction medium in an isolated form or, more preferably, may be comprised in a liposome or cell membrane i.e. the rhomboid and thrombomodulin polypeptides may be membrane-bound. The rhomboid polypeptide may, for example, act on a membrane-bound thrombomodulin polypeptide to generate a soluble cleavage product comprising the extracellular domain of the thrombomodulin polypeptide. This soluble cleavage product may be detected by any convenient means.

In preferred embodiments, a thrombomodulin polypeptide is a Type 1 membrane polypeptide with a single TMD orientated with a C terminal cytoplasmic domain and an N terminal extracellular/lumenal domain.

A thrombomodulin polypeptide may comprise a cytoplasmic domain which includes or consists of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of the cytoplasmic domain (residues 540-575) of a mammalian thrombomodulin, for example mouse thrombomodulin (NP_033404, GI:6678339), rabbit (Oryctoclagus cuniculus; AAN15931, GI : 29294605) ; rat (Rattus norvegicus; NP_113959, GI:13929084) , cow (Bos Taurus; AAA30785, GI:163763) or human thrombomodulin (AAH53357, GI : 31418462) .

Preferably the cytoplasmic domain includes or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the amino acid sequence of the cytoplasmic domain (residues 540-575) of human or mouse thrombomodulin.

Amino acid identity is generally defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST or TBLASTN (which use the method of Altschul et al . (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and L±pman (1988) PNAS USA 85: 2444-2448) , or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), generally employing default parameters.

Sequence identity is generally determined over the full-length of the sequence unless context dictates otherwise.

In some preferred embodiments, the cytoplasmic domain of the thrombomodulin polypeptide may comprise or consist of the sequence of the amino acid sequence of residues 540 to 575 (i.e. the cytoplasmic domain) of human thrombomodulin.

In some embodiments, the transmembrane and extracellular domains of the thrombomodulin polypeptide may comprise heterogeneous sequences i.e. sequences not naturally associated with the sequence of the cytoplasmic domain of the thrombomodulin polypeptide. For example, the transmembrane domain of the thrombomodulin polypeptide may be any polypeptide transmembrane domain, preferably a transmembrane domain from a mammalian protein.

In other embodiments, the transmembrane domain may consist of the sequence of the transmembrane domain of human thrombomodulin (residues 515 to 539) and the extracellular domain may comprise or consist of the sequence of the extracellular domain of human thrombomodulin (residues 1 to 515) .

In some preferred embodiments, the thrombomodulin polypeptide may comprise or consist of the sequence of human thrombomodulin (residues 1 to 575 as set out in database entry AAH53357)

In some preferred embodiments, the thrombomodulin polypeptide further comprises a detectable label, such as green fluorescent protein (GFP) , luciferase or alkaline phosphatase. This is preferably located in the soluble extracellular domain, allowing convenient detection of the soluble cleaved product and is particularly useful in automated assays.

A rhomboid polypeptide suitable for use in the present methods preferably comprises residues Rill, G174, S176 and H239, more preferably residues WHO, Rill, N128, G174, S176 and H239. These residues equate to the conserved catalytic residues which were originally identified in Drosophila Rhomboid-1 (Urban et al Cell 107, 173-182 (2001) ) . The presence of these conserved residues may be used to identify rhomboid polypeptides which are variants, alleles or homologues as described above.

Amino acid residues of rhomboid and thrombomodulin are described in the present application with reference to their position in the human RHBDL2 (protein NP_060291.1, GI: 8923409; nucleic acid NM 017821 GI: 24497630) and thrombomodulin (AAH53357 (AAH53357.1) , GI: 31418462) sequences, respectively. It will be appreciated that the equivalent residues in other rhomboids and thrombomodulins, for example from other species, may have a different position and number, because of differences in the amino acid sequence of each polypeptide. Equivalent residues in other rhomboid and thrombomodulin polypeptides are easily recognisable by their overall sequence context and by their positions with respect to the TMDs.

Although an rhomboid polypeptide may be a fragment of the full length sequence, it is preferred that a rhomboid polypeptide comprises at least 4 TMDs, more preferably at least 5 TMDs, with residues N128, S176 and H239 each occurring in different TMD at about the same level in the lipid membrane bilayer.

Preferred, rhomboid polypeptides comprise a GxSx motif.

In some embodiments, the term ^rhomboid polypeptide' does not include RHBDLl (protein NP_003952 (NP_003952.1) , GI: 4506525; nucleic acid NM_O03961 (NM_003961.1) GI: 4506524) and/or RHBDL3 (Locus Tag: HGNC: 16502) .

Preferred rhomboid polypeptides for use in the present methods include RHBDL2-like polypeptides. An RHBDL2-like polypeptide is an intramenxbrane protease which comprises the catalytic residues described above and which shares a degree of sequence homology, i.e. sequence identity or similarity, with RHBDL2 polypeptide. RHBDL2- like polypeptides include RHBDL2 polypeptides, such as zebrafish RHBDL2 (Danio rerio; AAH48048, GI : 28856246) , or more preferably, mammalian RHBDL2 polypeptides, such as mouse RHBDL2 (NP_060921) , rat RHBDL2 (Rattus norvegicus XP_216520, GI : 34870972) ; or human RHBDL2 (protein: NP_060291 (NP_060291.1) , GI:8923409 , nucleic acid NM_017821 (NM D17821.2) GI : 24497630) . RHBDL2 polypeptides also include variants, alleles or homologues of any of these polypeptides . A variant, allele or homologue of an RHBDL2 polypeptide, such as human RHBDL2 (NM_017821) , may comprise or consist of a sequence which one or more substitutions, deletions or insertions relative to the native sequence of a mammalian rhomboid, such as human RHBDL2 (NM_017821) . For example, a variant, allele or homologue may have at least 40% sequence identity with the sequence, of a mammalian rhomboid, such as human RHBDL2 (NM_017821), at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. Preferably, a variant, allele or homologue comprises the catalytic residues set out above and catalyses the intramembrane cleavage of thrombomodulin.

A rhomboid, polypeptide may also comprise additional amino acid residues that are heterologous to the rhomboid sequence. For example, a rhomboid polypeptide or fragment thereof may be included as part of a fusion protein, e.g. including a binding portion for a different ligand.

The thrombomodulin polypeptide and the rhomboid polypeptide may each comprise an ER (endoplasmic reticulum) retention signal. For example, a rhomboid polypeptide may comprise a C terminal (lumenal) KDEL motif and a thrombomodulin may comprise a C terminal (cytoplasmic) KKXX motif (Jackson et al (1993) J. Cell Biol. 121(2) 317-333)

Rhomboid and thrombomodulin polypeptides may be conveniently produced for use in the methods described herein by expressing nucleic acid encoding them. A host cell containing a vector including encoding nucleic acid may be grown in a host cell under appropriate conditions which cause or allow expression of the polypeptide. Polypeptides may also be expressed in in vitro systems, such as reticulocyte lysate.

For example, a method of producing a rhomboid polypeptide may comprise: (a) expressing nucleic acid which encodes a rhomboid polypeptide in a suitable expression system to produce the polypeptide recombinantly; (b) determining the ability of the recombinantly produced polypeptide to proteolytically cleave a thrombomodulin polypeptide.

A method of producing a thrombomodulin polypeptide may comprise: (a) expressing nucleic acid which encodes a thrombomodulin polypeptide in a suitable expression system to produce the polypeptide recombinantly; (b) determining the cleavage of the recombinantly produced polypeptide by a rhomboid polypeptide .

Cleavage of the recombinantly produced polypeptide by two or more different rhomboid polypeptides may be determined. For example, the susceptibility of the recombinantly produced polypeptide to cleavage by a RHBDLl polypeptide, a RHBDL2 polypeptide and/or a RHBDL3 polypeptide may be determined.

A thrombomodulin polypeptide, for example, may be susceptible to cleavage by an RHBDL2 polypeptide but not a RHBDLl or RHBDL3 polypeptide . An RHBDLl polypeptide may include a mammalian RHBDLl polypeptide such as human RHBDLl (NM_003961) . An RHBDL3 polypeptide may include a mammalian RHBDL3 polypeptide such as human RHBDL3 (Locus Tag: HGNC:16502).

An isolated nucleic acid encoding a rhomboid and/or a thrombomodulin polypeptide may be comprised in a vector, for example operably linked to regulatory sequences. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al . , 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al . eds . , John Wiley & Sons, 1992.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others. A common, preferred bacterial host is E. coli.

Another aspect of the invention provides a host cell containing heterologous nucleic acid encoding a rhomboid polypeptide and a thrombomodulin polypeptide. Nucleic acid encoding the rhomboid polypeptide and thrombomodulin polypeptide may be present on a single nucleic acid construct or vector within the host cell or on separate constructs or vectors within the host cell. A host cell may thus comprise a heterologous rhomboid polypeptide and a heterologous thrombomodulin polypeptide.

The nucleic acid rαay be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques . The nucleic acid may be on an extra- chromosomal vector within the cell.

The introduction of nucleic acid into a host cell, which may (particularly for in vitro introduction) be generally referred to without limitation as "transformation", may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage .

Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.

The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide is produced.

Methods for identifying and/or obtaining compounds which modulate, in particular inhibit, thrombomodulin proteolysis may be in vivo cell-based assays, or in vitro non-cell-based assays.

In in vitro assays, the rhomboid polypeptide and the thrombomodulin polypeptide may be isolated or contained in a liposome. Liposome based assays may be carried out using methods well-known in the art (Brenner C. et al (2000) Meths in Enzymol. 322 243-252, Peters et al (2000) Biotechniques 28 1214-1219, Puglielli, H. and Hirschberg C. (1999) J. Biol. Chem. 274 35596-35600, Ramjeesingh, M. (1999) Meths in Enzymol. 294 227-246).

Preferably, assays according to the present invention take the form of in vivo assays. In vivo assays may be performed in a cell such as a cell (e.g. a Toxoplasma cell), a yeast strain, insect or more preferably, a mammalian cell line such as CHO, HeLa and COS cells, in which the relevant polypeptides or peptides are expressed from one or more vectors introduced into the cell.

In a preferred embodiment, the rhomboid polypeptide and the thrombomodulin polypeptide may be expressed in a host cell from heterogeneous encoding nucleic acid. Nucleic acid encoding the rhomboid polypeptide and the thrombomodulin polypeptide may be contained on a single expression vector or on separate expression vectors .

A rhomboid polypeptide may be co-expressed in a host cell with a thrombomodulin polypeptide and the rhomboid serine protease activity determined by determining cleavage of the thrombomodulin polypeptide. Cleavage may be determined by determining the presence or absence of soluble cleavage products which may be secreted into the culture medium, for example by determining the presence of a detectable label.

Methods may be carried out in the presence of an inhibitor of non- rhomboid proteases to inhibit non-specific cleavage of substrate. Suitable inhibitors include batimastat.

Persons skilled in the art may vary the precise format of methods of the invention using routine skill and knowledge.

Combinatorial library technology (Schultz, JS (1996) Biotechnol. Prog. 12:729-743) provides an efficient way of testing a potentially vast number of different compounds for ability to modulate activity of a polypeptide. Prior to or as well as being screened for modulation of activity, test compounds may be screened for ability to interact with the rhomboid polypeptide, e.g. in a yeast two- hybrid system (which requires that both the polypeptide and the test substance can be expressed in yeast from encoding nucleic acid) . This may be used as a coarse screen prior to testing a substance for actual ability to modulate protease activity of the polypeptide.

The amount of test substance or compound which may be added in a method of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about O.OlnM to lOOμM concentrations of putative inhibitor compound may be used, for example from 0.1 to lOμM. Test compounds may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants that contain several characterised or uncharacterised components may also be used.

Methods of the invention may comprise the step of identifying the test compound as a modulator, for example an inhibitor, of thrombomodulin polypeptide cleavage.

One class of putative modulator compounds can be derived from the rhomboid polypeptide and/or thrombomodulin polypeptide. Peptide fragments of from 5 to 40 amino acids, for example, from 6 to 10 amino acids of these polypeptides may be tested for their ability to disrupt such interaction or activity.

The inhibitory properties of a peptide fragment as described above may be increased by the addition of one of the following groups to the C terminal: chloromethyl ketone, aldehyde and boronic acid. These groups are transition state analogues for serine, cysteine and threonine proteases. The N terminus of a peptide fragment may be blocked with carbobenzyl to inhibit aminopeptidases and improve stability (Proteolytic Enzymes 2nd Ed, Edited by R. Beynon and J. Bond Oxford University Press 2001) .

Antibodies directed to trie site of interaction in the rhomboid polypeptide or thrombomodulin protein form a further class of putative modulator compounds. Candidate inhibitor antibodies may be characterised and their binding regions determined to provide single chain antibodies and fragments thereof which are responsible for disrupting the interaction.

Antibodies may be obtained using techniques that are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al . , 1992, Nature 357: 80-82) . Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal .

As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the proteins (or fragments) , or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.

Antibodies according to the present invention may be modified in a number of ways. Indeed, the term "antibody" should be construed as covering any binding substance having a binding domain with the required specificity. Thus the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimicks that of an antibody enabling it to bind an antigen or epitope.

Example antibody fragments, capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')₂ fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included. Antibody molecules may thus be useful in a therapeutic context to disrupt rhomboid mediated cleavage of thrombomodulin proteins.

An alternative to interfering with the interaction of rhomboid with the thrombomodulin polypeptide is regulation at the nucleic acid level to down-regulate production of rhomboid and thus reduce thrombomodulin cleavage.

For instance, expression of rhomboid may be inhibited using anti- sense or RNAi technology. The use of these approaches to down-regulate gene expression is now well-established in the art.

Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of rhomboid polypeptide so that its expression is reduced or completely or substantially completely prevented. In addition to targeting coding sequence, antisense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the antisense oligonucleotides can interfere with expression control sequences. The construction of antisense sequences and their use is described for example in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990) and Crooke, Ann. Rev. Pharmacol. Toxicol. 32:329-376, (1992).

Oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo within cells in which down-regulation is desired. Thus, double-stranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene.

The complete sequence corresponding to the coding sequence in reverse orientation need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A suitable fragment may have about 14-23 nucleotides, e.g. about 15, 16 or 17.

An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression; Angell & Baulcombe (1997) The EMBO Journal 16,12:3675-3684; and Voinnet & Baulcombe (1997) Nature 389: pg 553). Double stranded RNA (dsRNA) has been found to be even more effective in gene silencing than both sense or antisense strands alone (Fire A. et al Nature, Vol 391, (1998) ) . dsRNA mediated silencing is gene specific and is often termed RNA interference (RNAi) .

RNA interference is a two-step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs (~2nt) . The siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001)

RNAi may be also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3 ' -overhang ends (Zamore PD et al Cell, 101, 25-33, (2000)). Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologeous genes in a wide range of mammalian cell lines (Elbashir SM. et al . Nature, 411, 494-498, (2001)).

Another possibility is that nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific site - thus also useful in influencing gene expression. Background references for ribozymes include Kashani-Sabet and Scanlon, 1995, Cancer Gene Therapy, 2(3): 213-223, and Mercola and Cohen, 1995, Cancer Gene Therapy, 2(1), 47-59. Thus, a modulator of rhomboid activity and thus thrombomodulin activity may comprise a nucleic acid molecule comprising all or part of a rhomboid coding sequence or the complement thereof

Such a molecule may suppress the expression of rhomboid polypeptide and may comprise a sense or anti-sense rhomboid coding sequence or may be an rhomboid specific ribozyme, according to the type of suppression to be employed.

The type of suppression will also determine whether the molecule is double or single stranded and whether it is RNA or DNA.

A method as described herein may comprise identifying the test compound as a modulator i.e. an enhancer or inhibitor of rhomboid activity.

The ability of said test compound to alter, i.e. promote or inhibit, circulating plasma levels of soluble thrombomodulin in humans and/or animals may be determined.

In addition, the ability of said test compound to alter, i.e. promote or inhibit, a biological activity of thrombomodulin may be determined. For example, the effect of the compound on the anticoagulation activity of thrombomodulin may be determined. More generally, the ability of the compound to induce phenotypes associated with altered function or levels of thrombomodulin in a test animal and/or the effect of the compound on the condition of animals with thrombomodulin disease models may be determined.

Once identified, the test compound may be isolated and/or purifying. The compound may furthermore be produced and/or synthesised using conventional synthetic or recombinant techniques.

The compound may then be formulated into a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier. Pharmaceutical compositions are described in more detail below.

Following identification of a compound using a method of the invention described herein, a compound may be modified to optimise the pharmaceutical properties thereof.

The modification of a ^Λlead' compound identified as biologically active is a known approach to the development of pharmaceuticals and may be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Modification of a known active compound (for example, to produce a mimetic) may be used to avoid randomly screening large number of molecules for a target property.

Modification of a ^Λlead' compound to optimise its pharmaceutical properties commonly comprises several steps. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore" .

Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR.

Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the optimisation of the lead compound.

A template molecule is then selected onto which chemical groups that mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the modified compound is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The modified compounds found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Modified compounds include mimetics of the lead compound.

Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.

Rhomboid and thrombomodulin polypeptides may also be used in methods of designing mimetics which are suitable for inhibiting thrombomodulin activity.

The present invention provides a method of designing mimetics having the biological activity of inhibiting the rhomboid mediated cleavage of thrombomodulin polypeptides, said method comprising: (i) analysing a compound having the biological activity to determine the amino acid residues essential and important for the activity to define a pharmacophore; and, (ii) modelling the pharmacophore to design and/or screen candidate mimetics having the biological activity.

A suitable compound may be, for example, a rhomboid polypeptide or fragment as described herein. Suitable modelling techniques are known in the art. This includes the design of so-called "mimetics" which involves the study of the functional interactions of the molecules and the design of compounds which contain functional groups arranged in such a manner that they could reproduced those interactions.

The modelling and modification of a ^λlead' compound to optimise its properties, including the production of mimetics, is described above .

Other aspects of the invention provide a compound identified and/or obtained by a method described above and a pharmaceutical composition comprising such a compound.

The uses of such compositions are described in more detail below.

In some embodiments, a method of producing a pharmaceutical composition may comprise; identifying a compound which modulates rhomboid dependent thrombomodulin proteolysis using a method described herein; and, admixing the compound identified thereby with a pharmaceutically acceptable carrier.

In other embodiments, a method for preparing a pharmaceutical composition for treating a thrombomodulin mediated condition, for example a condition selected from the group consisting of a blood coagulation associated condition, a cancer condition and an inflammatory condition, may comprise; i) identifying a compound which modulates rhomboid dependent thrombomodulin proteolysis, for example by modulating rhomboid activity, ii) synthesising the identified compound, and; iii) incorporating the compound into a pharmaceutical composition. A compound may be identified as a rhomboid modulator using a method described above.

The identified compound may be synthesised using conventional chemical synthesis methodologies. Methods for the development and optimisation of synthetic routes are well known to a skilled person. The compound may be modified to optimise its pharmaceutical properties, as described above.

Incorporating the compound into a pharmaceutical composition may include admixing the synthesised compound with a pharmaceutically acceptable carrier or excipient. Suitable carriers and excipients are described below.

A compound or composition identified and/or obtained by a method described herein may be useful in the treatment of a thrombomodulin- associated condition or disorder, for example a cardiovascular disorder, an inflammatory disorder, a disorder associated with blood coagulation or a cancer condition.

Cardiovascular disorders include disorders such as cardiac myxoma, acute myocardial infarction, stroke, in particular hemorrhagic stroke, ischaemic (coronary) heart disease and myocardial ischaemia (angina) .

Inflammatory disorders include allergy, asthma, atopic dermatitis, Crohn's disease, Felty's syndrome, gingivitis, pelvic inflammatory disease, periodontitis, polymyositis/dermatomyositis, psoriasis, rheumatic fever, rheumatoid athritis, skin inflammatory diseases, spondylitis, systemic lupus erythematosus, ulcerative colitis, uveitis, vasculitis and inflammation caused by sepsis or ischaemia.

Disorders associated with blood coagulation may include cerebral thrombosis, cerebral embolism, coronary artery thrombolysis , arterial and pulmonary thrombosis and embolism, and various vascular disorders such as peripheral arterial obstruction, deep vein thrombosis, disseminated intravascular coagulation syndrome, thrombus formation after artificial blood vessel operation or after artificial valve replacement, re-occlusion and re-stricture after coronary artery by-pass operation, re-occlusion and re-stricture after PTCA (percutaneous transluminal coronary angioplasty) or PTCR (percutaneous transluminal coronary re-canalization) operation and thrombus formation at the time of extracorporeal circulation.

Cancer conditions include cancers, (e.g., histocytoma, glioma, glioblastoma, astrocyoma and osteoma) including lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, oral cancer, colon cancer, breast cancer, oesophageal cancer, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, skin cancer and brain cancer.

Other disorders associated with thrombomodulin include diabetes, disorders of peripheral nervous system, pneumonia, adult respiratory distress syndrome, chronic renal failure and acute hepatic failure.

A method of treatment of an individual having a thrombomodulin- associated condition, for example condition selected from the group consisting of a blood coagulation associated condition, a cancer condition and an inflammatory condition, may comprise administering a compound identified and/or obtained by a method described herein to the individual for treatment of the condition.

A compound identified by a method described herein may also be used in the manufacture of a medicament for the treatment of a thrombomodulin-associated condition, for example a condition selected from the group consisting of a blood coagulation associated condition, a cancer condition and an inflammatory condition.

Whether it is a polypeptide, antibody, peptide, anti-sense, sense or siRNA nucleic acid molecule, small molecule, mimetic or other pharmaceutically useful compound according to the present invention that is to be given to an individual, 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.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Pharmaceutical compositions as described herein, and for use in the present methods, 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. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Liposomes, particularly cationic liposomes, may be used in carrier formulations .

Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.

Another aspect of the invention relates to the use of a rhomboid polypeptide as described herein, for the proteolytic cleavage of a thrombomodulin polypeptide.

A method for proteolytically cleaving a thrombomodulin polypeptide may comprise; contacting the thrombomodulin polypeptide with an rhomboid polypeptide; and, determining the proteolytic cleavage of said thrombomodulin polypeptide by said rhomboid polypeptide.

Thrombomodulin and rhomboid polypeptides are described in more detail above. Proteolytic cleaveage of the thrombomodulin polypeptide may be determined using any convenient technique.

Another aspect of the invention provides a chimeric thrombomodulin polypeptide which is suitable for use in a method described above, wherein said chimeric polypeptide comprising a cytoplasmic domain, a transmembrane domain and an extracellular domain, wherein the cytoplasmic domain comprises an amino acid sequence having at least 70% sequence identity with the residues 540-575 of human thrombomodulin (AAH533357), and; wherein one or both of the extracellular and transmembrane domains are heterologous .

More preferably, the cytoplasmic domain includes an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the amino acid sequence of the cytoplasmic domain (residues 540-575) of human thrombomodulin.

In some preferred embodiments, the cytoplasmic domain may comprise or consist of the sequence of the amino acid sequence of residues 540 to 575 (i.e. the cytoplasmic domain) of human thrombomodulin.

One or both of the transmembrane and extracellular domains of the thromobomodulin polypeptide may comprise heterologous sequences i.e. sequences not naturally associated with the sequence of the cytoplasmic domain of the thrombomodulin polypeptide. For example, the transmembrane domain of the thromobomodulin polypeptide may be any heterologous transmembrane domain, preferably a transmembrane domain from a mammalian protein.

The chimeric thrombomodulin polypeptide may be cleaved under appropriate conditions within its transmembrane domain by a rhomboid polypeptide, such as RHBDL2. In some embodiments, the chimeric polypeptide may not be cleaved by certain rhomboid polypeptides, such as RHBDLl or RHBDL3.

The extracellular domain may comprise a detectable label, such as green fluorescent protein (GFP), luciferase or alkaline phosphatase.

Chimeric thrombomodulin polypeptides may be produced using conventional recombinant techniques as described above.

Related aspects of the invention provide an isolated nucleic acid encoding a chimeric thrombomodulin polypeptide as described above, a vector comprising such a nucleic acid and a host cell transformed with such a vector.

Nucleic acids, vectors and host cells are described in more detail above .

Aspects of the present invention will now be illustrated with reference to the following experimental exemplification, by way of example and not limitation.

Further aspects and embodiments will be apparent to those of ordinary skill in the art.

All documents mentioned in this specification are hereby incorporated herein by reference.

Figure 1 shows candidate mouse TMDs identified as having potential Spitz-like substrate motifs (Urban, S. and Freeman, M. (2003). Molecular Cell 11, 1425-1434) . These were selected from approximately 1200 signal peptide and TMD-containing genes.

Accession numbers: Drosophila Spitz M95199, mouse thrombomodulin NP_033404, mouse Pref-1 (preadipocyte factor 1) BAA04121, mouse endoglin AAH29080, mouse Jedi NPJD82736, unnamed 1 NM_028732, unnamed 2 XP_109600, mouse TEM7r (tumor endothelial marker 7 - related) AAL11998, mouse Bet BC034634, human Neurexin II BAA94075, mouse antigen 114 J04634, mouse syndecan 1 AAH10560, mouse syndecan 3 NM_011520, mouse integrin-a2 A45839

Figure 2 shows an alignment of the human thrombomodulin and Drosophila Spitz transmembrane domains. The motif that is essential for Spitz cleavage by rhomboids, and which is similar in thrombomodulin, is highlighted: small, relatively hydrophilic and x- branched residues in the top three positions (underlined) followed by a helix-destabilising pair of residues (bold) . Figure 3 shows a diagram of the position of the coordinates in mouse thrombomodulin used in mapping.

Figure 4 shows a series of TMD (top group) and juxtamembrane (bottom group) mutants of human thrombomodulin which were assayed for their ability to be cleaved by RHBDL2. Mutations are shown in bold. None were found to abrogate cleavage.

Examples

Materials and Methods DNA constructs

Mouse RHBDLl, 2 and 3 cDNAs were cloned into pcDNA3.1 (Invitrogen) for expression in mammalian cells under the CMV promoter. A triple haemaglutinin (HA) -tag was inserted after the initiating methionine codon. N-terminally deleted mouse RHBDL2 lacked cytoplasmic residues 1-59. The rhomboid chimera contained residues 1-61 from hRHBDL2 (its cytoplasmic N-terminus) and residues 103-355 from Drosophila Rhomboid-1, the junction being at a conserved proline residue. Other rhomboids have been described elsewhere (Urban, S. et al (2001) Cell 107, 173-182, Urban, S. et al (2002). EMBO J. 21, 4277-4286, Urban, S. et al (2002). Current Biology 12, 1507-1512, Urban, S. and Freeman, M. (2003) . Molecular Cell 11, 1425-1434) .

Two different N-terminally GFP-tagged human thrombomodulin constructs were generated by cloning residues 28-575 of thrombomodulin into pEGFP-C2 (Clontech) or residues 25-575 into pcDNA3.1; in both cases the Spitz signal peptide and eGFP were inserted at the 5' end of the gene. Transmembrane domain mutations (Fig. 2) were generated by Quick-Change (Stratagene) method into the pEGFP-C2-based construct. Mouse full-length thrombomodulin (including its native signal peptide) was cloned into pcDNA3.1 by introducing unique restriction endonuclease sites during PCR from the IMAGE clone 5065481, and a SacII-site (between nucleotides 634 and 635) was used to insert eGFP gene in frame with the cDNA. Truncated forms were generated by introducing stop codons at a given position in the context of either a GFP-tagged full length mouse thrombomodulin or a chimera containing the Spitz signal peptide, eGFP and residues 481-577 of mouse thrombomodulin. Human EPCR cDNA was obtained by PCR from the IMAGE clone 5471649, cloned into pcDNA3.1 vector, and GFP-tagged by inserting eGFP in frame with the cDNA between nucleotides 126 and 127 (Kpnl-site) .

Thrombomodulin chimeras comprised the signal peptide of TGF , GFP, followed by: the thrombomodulin juxtamembrane region (after the last EGF domain) to the end of its C terminal tail (residues 48Ϊ 575; chimera JC) ; its transmembrane domain to the end of its C terminal tail (residues 515-575; chimera TM+C) ; its TMD alone (residues 515- 539; chimera TM) in which the C terminal tail was from TGFα; and just the thrombomodulin C terminal tail (residues 540-575) , preceded by the TMD from Drosophila Delta (chimera C+D) or human TGFα (chimera C+T) (Urban and Freeman, 2003 supra) .

Antibodies

The following antibodies were used in this study; mouse anti-V5 (Invitrogen) ; rabbit anti-thrombomodulin (Santa Cruz) ; mouse anti- pll5 (Transduction Labs; a mammalian cell Golgi marker) ; Alexa Fluor 568 (red) and Alexa Fluor 488 (green) -conjugated secondary antibodies from Molecular Probes. Images were collected on a Radiance 2001 confocal microscope (Bio-Rad) . Western blotting was performed by standard techniques and visualised by ECL kit (Amersham Pharmacia) .

Results

Identifying mammalian rhomboid substrates

A panel of membrane-tethered EGFR ligands were tested to determine whether any were cleaved by human RHBDLl and RHBDL2. The ligands tested included TGFα, heparin-binding EGF, amphiregulin, epiregulin, betacellulin, neuregulin and epigen; each was tagged with GFP and co-transfected into COS cells with human RHBDLl or RHBDL2. The ADAM family metalloprotease inhibitor batimastat was used to prevent nonspecific background shedding of cell-surface proteins. Under these standard assay conditions, none were cleaved. The mouse genome sequence was searched for single transmembrane domain proteins with the characteristic rhomboid substrate motif, previously characterised in the Drosophila ligand Spitz. This specific conformational property is recognised by rhomboids from bacteria to humans and therefore represents a conserved substrate recognition determinant; moreover, a physiological substrate of the yeast mitochondrial rhomboid has the same requirement (McQuibban et al (2003) Nature 423, 537-541) . The substrate motif does not comprise a simple conserved primary sequence, instead appearing conformational. A manual search through about 50% of genes in the mouse genome annotated as having a TMD and signal peptide (approximately 1200 searched) revealed that only 13 appeared to be good candidates for Spitz-like substrates (Figure 1) . One of these was the endothelial cell surface protein thrombomodulin, which comprises a large N terminal domain with homology to lectins and six EGF repeats, a TMD, and a highly conserved short cytoplasmic domain with no recognisable motifs.

Thrombomodulin has been proposed to have several functions but is best characterised as forming a complex with thrombin, thereby inhibiting blood coagulation (Weiler, H., and Isermann, B.H. (2003) J Thromb Haemost 1, 1515-1524.). The thrombomodulin TMD resembles the Spitz TMD in a number of significant respects (Figure 2) so we tested whether human thrombomodulin could indeed be cleaved by human RHBDLl or RHBDL2 (Urban, S. et al (2001). Cell 107, 173-182; Pascall, J.C. and Brown K.D. (1998) FEBS Letters 429, 337-340) when the proteins were coexpressed in mammalian cells, using a previously described assay.

First, C-terminally tagged thrombomodulin was assayed by co- expressing C-terminally V5-tagged thrombomodulin in NIH3T3 cells with human RHBDLl, 5 or 100 ng of RHBDL2, or lOOng of an active site mutant of RHBDL2 (R2-SA) ; 250ng of thrombomodulin-V5 DNA was used in each assay. A cleaved band of expected size was detected in lysates from cells co-expressing RHBDL2 but not RHBDLl. The experiment was then repeated with N-terminally tagged thrombomodulin. N-terminally GFP-tagged thrombomodulin was coexpressed in NIH3T3 cells with RHBDLl, 5 or 100 ng of RHBDL2, or lOOng of an active site mutant of RHBDL2. The metalloprotease inhibitor batimastat (BB-94; 20μM) was included to inhibit background shedding of cell surface proteins. The thrombomodulin extracellular domain accumulated in the medium, again in response to RHBDL2 only. Finally, an antibody against the extracellular domain of thrombomodulin was used to detect the accumulation of untagged extracellular domains in the medium. Untagged thrombomodulin was coexpressed as before in NIH3T3 cells with rhomboids, and anti- thrombomodulin extracellular domain (Santa Cruz) was used to confirm RHBDL2-specific cleavage. A western blot was performed under non- reducing conditions in the presence of 20μM batimastat. Accumulation of untagged extracellular domains in the medium was observed to be triggered specifically by RHBDL2 but not other rhomboids.

As with other rhomboid substrates, thrombomodulin cleavage was insensitive to the broad-spectrum metalloprotease inhibitor batimastat, and was abolished when the putative catalytic serine of rhomboid was mutated to alanine. The cleavage was also insensitive to a range of protease inhibitors that target aspartyl, cysteine and classical serine/threonine proteases.

In all three of these assays, thrombomodulin was cleaved in response to RHBDL2 but its cleavage occurred at significantly lower levels than cleavage of the Drosophila substrate, Spitz. To address whether the lower level cleavage might indicate that thrombomodulin proteolysis was a non-specific artefact caused by over-expression of RHBDL2, we reduced the levels of RHBDL2 expression over a range of 10³-fold by reducing the amount of specific DNA in each transfection. 100-fold reduction of input rhomboid DNA was observed to reduce the expression of RHBDL2 to undetectable levels but thrombomodulin cleavage was barely affected; and even when input DNA was reduced 1000-fold, cleavage was still detectable. This sub-stoichiometric requirement resembled the cleavage of Spitz by Drosophila Rhomboid- 1.

These results confirm that human thrombomodulin is cleaved by RHBDL2 but not RHBDLl. The specificity of RHBDL2 for thrombomodulin was further tested by determining whether another single-TMD protein in the same functional clotting complex as thrombomodulin, the endothelial protein C receptor (EPCR) (Stearns-Kurosawa, D.J., (1996). Proc Natl Acad Sci U S A 93, 10212-10216), was cleaved by either rhomboid.

Under the same conditions, no EPCR cleavage was detected. The cleavage of Pref-1, which is a mouse protein with structural similarity to thrombomodulin (Smas CM., and Sul H.S. (1993). Cell 73, 725-734) by mouse RHBDL2 was also determined; no cleavage was detected. Moreover, the TMDs of the other 11 proteins that were identified by sequence similarity as being potential rhomboid substrates were also uncleaved by mouse RHBDL2.

Only RHBDL2-like rhomboids cleave thrombomodulin

The ability of other rhomboid proteases to cleave thrombomodulin was examined. A variety of rhomboids were cotransfected with GFP-tagged human thrombomodulin or mouse thrombomodulin and the thrombomodulin released into the medium was detected by western blot. Human thrombomodulin was cleaved by human and mouse RHBDL2 and the zebrafish orthologue of RHBDL2; however it was not cleaved by Drosophila Rhomboid-1 or the bacterial rhomboid AarA, both of which cleave Spitz (Urban, S. et al (2002). Current Biology 12, 1507- 1512) . The cleavage of mouse thrombomodulin by all of the three non-mitochondrial mouse rhomboids identifiable in the mouse genome was also tested and only RHBDL2 was found to show activity.

In all cases where cleavage occurred, it was dependent on the presence of the catalytic serine of RHBDL-2: activity was abolished when the serine was mutated to alanine. The localisation of GFPtagged mouse RHBDLl, 2 and 3 was examined using protocols previously described (Lee, J.R et al (2001) . Cell 107 161-171) . All three were localised in the secretory pathway, specifically the Golgi apparatus and the plasma membrane. Some variability was observed between cells but RHBDLl and 3 were usually located predominantly in the Golgi apparatus, whereas RHBDL2 was located more prominently on the plasma membrane. GFP-tagged thrombomodulin was also located in the secretory pathway - visible in the ER, the Golgi apparatus and the plasma membrane . These data indicate that differential compartmentalisation cannot account for the specificity of RHBDL2 for thrombomodulin.

RHBDL2 cleaves thrombomodulin near the top of its TMD The cleaved extracellular domain of thrombomodulin was present at levels in medium which were too low to allow a direct biochemical determination of the cleavage site. We therefore mapped the site of cleavage by RHBDL2 by comparing the size of the cleaved, GFP-tagged N-terminal fragment with identically-tagged artificial truncations of the protein. In the first series of experiments, full-length mouse thrombomodulin was cleaved, and the cleaved product was compared with C-terminal truncations that contained the whole N- terminal region. The cleaved fragment was larger than truncations at residue 508, but smaller than truncations at residue 528. This located the approximate site of cleavage to between approximately residues 510 and 525 (note that we predict the TMD to run from residues 517 to 539) .

A second set of experiments was performed in which most of the N- terminus had been removed, resulting in smaller protein fragments, allowing for more accurate size comparisons. This series gave results entirely consistent with the first series, and the cleaved fragment was indistinguishable from the 519 truncation. The site of thrombomodulin cleavage is therefore likely to be at residue 518, 519, or 520, corresponding to the top region of the TMD (Figure 3) . Importantly, this result strongly supports the conclusion that thrombomodulin is a direct substrate of RHBDL2, as rhomboids are the only proteases known to cleave within TMDs, near the extracellular side.

Spitz-like rhomboid substrates depend on helix destabilising residues in the top part of their TMDs, and require reasonably hydrophilic residues in the same region (Urban, S., and Freeman, M. (2003) Molecular Cell 11, 1425-1434) . Based on this requirement and our previous ability to abrogate cleavage with TMD mutations, we made an extensive set of mutations in the TMD and the juxtamembrane region of thrombomodulin (Figure 4) . None of these changes prevented or substantially reduced cleavage by RHBDL2.

This inability to block RHBDL2 cleavage is consistent with our observation that bacterial and Drosophila rhomboids cannot cleave thrombomodulin, even though they cleave Spitz efficiently. The recognition of thrombomodulin by RHBDL2 therefore seems to occur through a distinct mechanism to the cleavage of Spitz.

The cytoplasmic domain of thrombomodulin directs its cleavage by

RHBDL2

Since the TMD mutations did not prevent thrombomodulin cleavage, a series of domain swaps and deletions were used to map the determinants that allow it to be cleaved by RHBDL2. Removal of either the N-terminal portion (JC) or the whole (TM+C) of the extracellular domain, did not abrogate cleavage.

However, a chimera comprising an extracellular tag, the thrombomodulin TMD and a cytoplasmic domain from TGFα (TM) , was not cleaved. This shows that, unlike Spitz, the TMD of thrombomodulin is not sufficient to confer cleavage/recognition by RHBDL2. Furthermore, it demonstrated that, again unlike Spitz, the cytoplasmic C-terminus of the protein is necessary.

To examine the role of the cytoplasmic domain of thrombomodulin further, we tested whether it was also sufficient for RHBDL2 cleavage. Strikingly, the cytoplasmic domain of thrombomodulin was sufficient to transform the TMD of either Drosophila Delta or human TGFα - both type 1 transmembrane proteins - into RHBDL2 substrates (chimeras C+D, C+T) . Together, these experiments show that the cytoplasmic domain of thrombomodulin is both necessary and sufficient for the cleavage of the thrombomodulin TMD. It is also sufficient to direct cleavage by RHBDL2 of at least two other TMDs that are not otherwise substrates.

We investigated the participation of the cytoplasmic domains of RHBDL2 and thrombomodulin in the enzyme/substrate recognition mechanism by deleting the N-terminus of RHBDL2. This significantly reduced its activity against thrombomodulin providing indication of a function for the RHBDL2 cytoplasmic domain. However, substituting the N-terminal cytoplasmic domain of Drosophila Rhomboid-1 with the equivalent domain of RHBDL2, was not sufficient to transform Rhomboid-1 into an enzyme that could cleave thrombomodulin, although the chimeric rhomboid retained activity against Spitz.

These results show that the cytoplasmic domains of thrombomodulin and RHBDL2 are involved in the recognition of the substrate, although they also provide indication that other parts of RHBDL2 participate .

Claims

Claims :

1. A method for identifying and/or obtaining a modulator of the interaction of rhomboid and thrombomodulin, which method comprises:

(a) contacting an rhomboid polypeptide and a thrombomodulin polypeptide in the presence of a test compound; and

(b) determining the interaction between the rhomboid polypeptide and the thrombomodulin polypeptide.

2. A method for identifying and/or obtaining a modulator of rhomboid-dependent thrombomodulin proteolysis, which method comprises :

(a) contacting an rhomboid polypeptide and a thrombomodulin polypeptide in the presence of a test compound; and

(b) determining proteolytic cleavage of the thrombomodulin polypeptide.

3. A method according to claim 1 or claim 2 wherein the rhomboid polypeptide and the thrombomodulin polypeptide are membrane-bound.

4. A method according to claim 3 wherein the rhomboid polypeptide and the thrombomodulin polypeptide are at the plasma membrane of a host cell.

5. A method according to any one of claims 1 to 4 wherein the thrombomodulin polypeptide comprises a cytoplasmic domain which includes a sequence having at least 70% sequence identity with the sequence of the cytoplasmic domain of a mammalian thrombomodulin.

6. A method according to any one of claims 1 to 5 wherein the thrombomodulin polypeptide comprises a cytoplasmic domain which includes the sequence of the cytoplasmic domain of a mammalian thrombomodulin.

7. A method according to any one of the preceding claims wherein thrombomodulin polypeptide comprises the sequence of a mammalian thrombomodulin.

8. A method according to any one of claims 5 to 7 wherein the mammalian thrombomodulin is mouse, rat or human thrombomodulin.

9. A method according to any one of the preceding claims wherein thrombomodulin polypeptide comprises a detectable label.

10. A method according to claim 9 wherein the detectable label is selected from the group consisting of Green Fluorescent Protein, luciferase and alkaline phosphatase.

11. A method according to any one of the preceding claims wherein the rhomboid polypeptide is an RHBDL2 polypeptide.

12. A method according to claim 11 wherein the RHBDL2 polypeptide is mouse, rat or human RHBDL2.

13. A method according to any one of the preceding claims comprising identifying said test compound as a modulator of rhomboid activity.

14. A method according to claim 13 further comprising determining the ability of said test compound to modulate the anti-coagulation activity of a thrombomodulin polypeptide.

15. A method according to claim 13 or claim 14 comprising isolating and/or purifying said test compound.

16. A method according to any one of claims 13 to 15 comprising producing and/or synthesising said test compound.

17. A method according to any one of claims 13 to 16 comprising formulating said test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier.

18. A method according to any one of the preceding claims wherein said modulator is for use in the treatment of a thrombomodulin mediated disorder.

19. A method according to claim 18 wherein said thrombomodulin mediated disorder is selected from the group consisting of an cancer condition, an inflammatory disorder, a cardiovascular disorder and a blood clotting disorder.

20. A method of producing a pharmaceutical composition comprising; identifying a compound which modulates rhomboid-dependent thrombomodulin proteolysis using a method according to any one of claims 1 to 13; and, admixing the compound identified thereby with a pharmaceutically acceptable carrier.

21. A method according to claim 20 comprising the step of modifying the compound to optimise the pharmaceutical properties thereof.

22. A method for preparing a pharmaceutical composition for treating a thrombomodulin mediated condition in an individual, the method comprising; i) identifying a compound which modulates rhomboid-dependent thrombomodulin proteolysis, ii) synthesising the identified compound, and; iii) incorporating the compound into a pharmaceutical composition.

23. A compound identified as a modulator of a rhomboid polypeptide by a method according to any one of claims 1 to 19.

24. A pharmaceutical composition comprising a compound according to claim 23.

25. A method comprising administration of a compound according to claim 23 to a patient for treatment of a thrombomodulin mediated condition.

26. A method according to claim 25 wherein said condition is selected from the group consisting of a blood coagulation associated condition, a cancer condition and an inflammatory condition.

27. Use of a compound according to claim 23 in the manufacture of a medicament for the treatment of a thrombomodulin mediated condition

28. Use according to claim 27 wherein said condition is selected from the group consisting of blood coagulation associated condition, a cancer condition and an inflammatory condition.

29. A method of making a pharmaceutical composition comprising admixing a compound according to claim 23 with a pharmaceutically acceptable excipient

30. A method for proteolytically cleaving a thrombomodulin polypeptide comprising; contacting the thrombomodulin polypeptide with a rhomboid polypeptide; and, determining the proteolytic cleavage of said polypeptide by said rhomboid polypeptide.

31. Use of a rhomboid polypeptide for the proteolytic cleavage of a thrombomodulin polypeptide.

32. A chimeric thrombomodulin polypeptide suitable for use in a method of any one of claims 1 to 19 comprising; a cytoplasmic domain having a sequence which has at least 70% sequence identity with the cytoplasmic domain of human thrombomodulin, a transmembrane domain and; an extracellular domain, wherein one or both of the extracellular and transmembrane domains are heterogeneous .

33. A chimeric thrombomodulin polypeptide according to claim 32 wherein the extracellular domain comprises a detectable label

34. A chimeric thrombomodulin polypeptide according to claim 33 wherein the detectable label is selected from the group consisting of GFP, luciferase and alkaline phosphatase.

35. A host cell comprising a heterologous thrombomodulin polypeptide and a heterogeneous rhomboid polypeptide.

36. A host cell transformed with heterologous nucleic acid encoding a thrombomodulin polypeptide and a rhomboid polypeptide.

37. A host cell according to claim 35 or claim 36 which is a mammalian cell.

38. A host cell according to any one of claims 35 to claim 37 wherein the thrombomodulin polypeptide is a chimeric polypeptide according to any one of claims 32 to 34.