WO2006135968A1

WO2006135968A1 - Modulation of sphingosine kinase signalling

Info

Publication number: WO2006135968A1
Application number: PCT/AU2006/000866
Authority: WO
Inventors: Stuart M. Pitson; Tamara Leclercq; Mathew Alexander Vadas
Original assignee: Medvet Science Pty. Ltd.
Priority date: 2005-06-21
Filing date: 2006-06-21
Publication date: 2006-12-28
Also published as: EP1910524A1; EP1910524A4; JP2008546721A; CA2612644A1

Abstract

The present invention relates generally to a method of modulating cellular activity and to agents for use therein. More particularly, the present invention provides a method of modulating the level of sphingosine kinase functional activity. In a related aspect, the present invention provides a method of modulating sphingosine kinase mediated signalling via modulation of its intracellular level of activity. The present invention still further extends to novel molecules which exhibit the capacity to induce sphingosine kinase activity. The methods and molecules of the present invention are useful, inter alia, in the treatment and/or prophylaxis of conditions characterised by aberrant, unwanted or otherwise inappropriate cellular functional activity and/or aberrant, unwanted or otherwise inappropriate sphingosine kinase mediated signalling. The present invention is further directed to methods for identifying and/or designing agents capable of modulating the level of sphingosine kinase activity.

Description

MODULATION OF SPHINGOSINE KINASE SIGNALLING

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Sphingosine kinases (SK) are enzymes that catalyse the phosphorylation of sphingosine to generate the bio-active phospholipid, sphingosine 1 -phosphate (SlP) (Pyne & Pyne, 2002, Biochim. Biophys. Acta 1582:121-131; Spiegel & Milstien, 2003, Nat. Rev MoI Cell Biol. 4:397-407). SlP can affect many biological processes, including calcium mobilization, mitogenesis, apoptosis, atherosclerosis, inflammatory responses and cytoskeleton rearrangement (Spiegel & Milstien, 2000, FEBS Lett. 476:55-57). As SK is the main regulator of SlP levels in mammalian cells (Spiegel & Milstien, 2003, supra), the activity and activation of this enzyme plays a central and crucial role in controlling the observed effects attributed to S IP in the cell.

There is now considerable evidence implicating sphingosine kinase and SlP in tumourigenesis through enhancing cell proliferation and cell survival. Xia et al. (2000, Curr. Biol. 10:1527-1530) have demonstrated overexpression of SKl in NIH 3T3 fibroblasts induced neoplastic cell transformation as measured by foci formation, cell growth in soft agar, and the formation of tumours in NOD/SCID mice. Additionally, inhibition of SKl by treatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SK or through the use of a dominant-negative SK mutant which blocked transformation mediated by oncogenic H-Ras (Xia et al, 2000, supra). Other studies have shown that, hSKl mRNA levels are higher in certain human tumours, including cancers of the breast, colon, lung, ovary, stomach, uterus, kidney and rectum (French et ah, 2003, Cancer Res. 63:5962-5969). Recently developed SKl inhibitors have also been shown to block breast tumour growth in mice (French et ah, 2003, supra). This is further supported by other studies that have also shown that SKl activation is important in promoting estrogen- dependent tumour formation in breast cancer cells (Nava et ah, 2002, Expt. Cell Res. 281 :115-127; Sukocheva et al, 2003, MoI. Endocrinol. 17:2002-2012).

However, in addition to its role in cell growth and survival SKl and SlP appears to be involved in other aspects of cellular regulation. It has been shown that SlP may be involved in inflammation and atherosclerosis through the induction of adhesion molecule expression on vascular endothelial cells (Xia et al, 1999, J Biol. Chem. 274:34499- 34505). Other studies suggest its involvement in hypertension since high levels of SKl can enhance blood vessel constriction (BoIz et al, 2003, Circulation 108:342-347; Coussin et al, 2002, Circ. Res. 91:151-157). Also, SKl has been shown to involved in TNFα-induced activation of the pro-inflammatory transcription factor, NF-κB (Xia et al, 2002, J. Biol Chem. 277:7996-8003). Similarly, SlP appears to be associated with asthma as it was found to enhance constriction of airway smooth muscle cells and histamine release (Jolly et al, 2001, MoI Immunol. 38:1239-1245).

It has previously been shown that hSKl has intrinsic catalytic activity that is independent of post-translational modifications of the protein (Pitson et al 2000a, Biochem. J. 350:429-441). Using a combination of techniques including chemical inhibition, co- immunoprecipitation and in vitro phosphorylation, we have also shown that ERK1/2 phosphorylates hSKl in vivo (Pitson et al, 2003, EMBO J. 22:1-10) This phosphorylation of hSKl results directly in a 14-fold increase in the &_cat of the enzyme, while having no significant effect on Ku values for either sphingosine or ATP. In general, activation of hSKl activity correlates with its translocation from the cytosol to the plasma membrane (Rosenfeldt et al, 2001, FASEB J. 15:2649-2659; Young et al, 2003, Cell calcium 33:119-128). The phosphorylation at Ser225 not only directly increases the catalytic activity of hSKl, but it was also shown to be necessary for agonist-induced translocation of the protein to the plasma membrane (Pitson et al, 2003, supra). Translocation of SKl may be important in localizing it to its potential substrate and hence generate localized signalling of SlP or its secretion to engage cell-surface SlP receptors (Pitson et al, 2003, supra). While this study has shown that activation of SKl occurs by ERKl/2-mediated phosphorylation, many aspects of the regulation of this phosphorylation are not yet known, including the possibility that other phosphorylation-independent SKl activation mechanisms may also exist.

Two human sphingosine kinase isoforms exist (1 and 2), which differ in their tissue distribution, developmental expression, catalytic properties, and somewhat in their substrate specificity (Pitson et al, 2000, supra; Liu et al, 2000, J. Biol. Chem. 275:19513- 19520). A number of studies have shown the effects of sphingosine kinase 1 in enhancing cell proliferation and suppressing apoptosis (Olivera et al, 1999, J. Cell Biol. 147:545- 558; Xia et al, 2000, Curr. Biol 10:1527-1530; Edsall et al, 2001, J. Neurochem. 76:1573-1584). Furthermore, overexpression of human sphingosine kinase 1 (hSKl) in NIH3T3 fibroblasts results in acquisition of the transformed phenotype and the ability to form tumors in nude mice, demonstrating the oncogenic potential of this enzyme (Xia et al, 2000, supra). More recent work has shown the involvement of hSKl in estrogen- dependent regulation of breast tumor cell growth and survival (Nava et al, 2002, Expt. Cell Res. 281:115-127; Sukocheva etal, 2003, MoI Endocrinol. 17:2002-2012), while other studies have shown elevated hSKl mRNA in a variety of human solid tumors and inhibition of tumor growth in vivo by sphingosine kinase inhibitors (French et al, 2003, Cancer Res. 63:5962-5969). Thus, the involvement of hSKl in cell growth, survival and tumorigenesis is now well established.

In addition to SphKl, another isoform SphK2 has been cloned and characterized (Liu et al, 2000, J Biol Chem, 275,:9513-19520). Although both SphKl and SphK2 contribute to total cellular SphK activity, these two isoforms demonstrate distinct enzyme kinetics and expression patterns (Kohama et al., 1998, J Biol Chem, 273:23722-23728; Liu et al, 2000, supra). While SphKl is described as a cytoplasmic enzyme, SρhK2 was found to localize in the nuclei through its nuclear localization signal sequence (Igarashi et al., 2003, J Biol Chem, 278:46832-46839). Additional functional distinctions between Sphkl and Sρhk2 are also evident. For example, Sphkl enhances cell survival and proliferation, whereas Sphk2 inhibits DNA synthesis (Liu et ah, 2000, supra) and induces apoptosis potentially through a BH3 domain and Bcl-xL interactions (Liu et al, 2003, J Biol Chem, 278:40330-40336). A recent study using Drosophila SphK, suggests that SphK2 is the more primitive of the two mammalian enzymes involved in sphingolipid catabolism (Herr et al, 2004, J Biol Chem, 279:12685-12694). As there is no evidence that SphK2 expression or activity are regulated, the ability of SphKl to be regulated at both transcriptional and post- transcriptional levels in response to a variety of physiological stimuli may be a relatively recent evolutionary step, facilitating a signaling role in mammalian cells.

Accordingly, as detailed above, although the central role of sphingosine kinase in the context of its regulation of a wide variety of cellular activities is well established, the precise mechanisms by which this occurs have been only partially determined. Accordingly, there is an ongoing need to both elucidate those mechanisms and identify molecules which can regulate those mechanisms in order to provide better means for developing methods of regulating cellular activities via regulation of the sphingosine kinase signalling pathway.

In work leading up to the present invention it has been determined that the interaction of sphingosine kinase with eEFl A results in an increase in the intrinsic catalytic activity of sphingosine kinase. Still further, it has been determined that to effect binding to sphingosine kinase, the subject eEFl A must not be GTP bound. In fact, a truncated form of eEFl A which cannot be bound to GTP, corresponding to prostate tumour inducer (PTI), continues to exhibit sphingosine kinase binding activity and the capacity to increase its catalytic activity. The identification of this cellular signalling mechanism has now enabled the development of simple and streamlined methods of modulating, in particular upregulating, sphingosine kinase mediated cellular functioning based on modulating the interaction of sphingosine kinase with eEFl A molecules. Accordingly, this has provided for the development of highly effective methods for therapeutically or prophylactically treating conditions characterised by unwanted or inappropriate cellular functioning.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The subject specification contains nucleotide sequence information prepared using the programme Patentln Version 3.1, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <201> followed by the sequence identifier (eg. <210>l, <210>2, etc). The length, type of sequence (DNA, etc) and source organism for each nucleotide sequence is indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO: 1 , SEQ ID NO:2, etc.). The sequence identifier referred to in the specification correlates to the information provided in numeric indicator field <400> in the sequence listing, which is followed by the sequence identifier (eg. <400>l, <400>2, etc). That is SEQ ID NO:1 as detailed in the specification correlates to the sequence indicated as <400>l in the sequence listing.

One aspect of the present invention provides a method of modulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with eEFIA or functional derivative, variant, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.

Another aspect of the present invention provides a method of modulating sphingosine kinase 1 mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase 1 with eEFIA or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase 1 catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.

Yet another aspect of the present invention provides a method of modulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with truncated eEFl Al or derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.

Still another aspect of the present invention provides a method of upregulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of eEFIA or functional derivative, homologue or mimetic thereof for a time and under conditions sufficient to induce the interaction of sphingosine kinase with eEFIA and thereby upregulate sphingosine kinase catalytic activity.

Yet still another aspect of the present invention is directed to a method of modulating cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with eEFIA or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.

Still yet another aspect of the present invention is directed to a method of modulating cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase 1 with eEFIA or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.

A further aspect of the present invention is directed to a method of modulating human cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with tr.eEFl Al wherein inducing or otherwise agonising said association up-regulates said human cellular activity and inhibiting or otherwise antagonising said association down-regulates said human cellular activity.

Still another further aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with eEFIA or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.

Yet another further aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate interaction of sphingosine kinase with eEFIA or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said association down-regulates said sphingosine kinase activity. Still yet another further aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a human, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, said method comprising administering to said human an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with tr.eEF IAl, wherein inducing or otherwise agonising said interaction up-regulates said cellular activity and inhibiting or otherwise antagonising said interaction down-regulates said cellular activity.

Yet still another further aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a human, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, said method comprising administering to said human an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with tr.eEF IAl wherein inducing or otherwise agonising said interaction up-regulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said interaction down-regulates said sphingosine kinase functional activity.

Another aspect of the present invention contemplates the use of an agent, as hereinbefore defined, in the manufacture of medicament for the treatment of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, wherein said agent modulates the interaction of sphingosine kinase and wherein inducing or otherwise agonising said interaction up-regulates said cellular activity and inhibiting or otherwise antagonising said interaction down-regulates said cellular activity.

Still another aspect of the present invention contemplates the use of an agent, as hereinbefore defined, in the manufacture of medicament for the treatment of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, wherein said agent modulates the interaction of sphingosine kinase with eEFl A or functional derivative, homologue or mimetic thereof and wherein inducing or otherwise agonising said interaction upregulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said interaction downregulates said sphingosine kinase functional activity.

In yet another further aspect, the present invention contemplates a pharmaceutical composition comprising the modulatory agent as hereinbefore defined together with one or more pharmaceutically acceptable carriers and/or diluents.

Yet another aspect of the present invention relates to the agent as hereinbefore defined, when used in the method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on both the determination that eEFl A molecules upregulate the intrinsic catalytic activity of sphingosine kinase and the determination that only the GDP form of eEFl A, including the PTI truncated form, will achieve this outcome. These determinations now permit the rational design of therapeutic and/or prophylactic methods for treating conditions characterised by aberrant or unwanted cellular activity and/or sphingosine kinase functional activity. Further, there is facilitated the identification and/or design of agents which specifically modulate the interaction of eEF 1 A with sphingosine kinase.

Accordingly, one aspect of the present invention provides a method of modulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with eEF 1 A or functional derivative, variant, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.

Reference to "sphingosine kinase mediated signalling" should be understood as a reference to a signalling pathway in which the sphingosine kinase molecule forms a functional component. In this regard, it is thought that sphingosine kinase is central to the generation of sphingosine- 1 -phosphate during activation of this pathway. It should be understood that modulation of sphingosine kinase mediated signalling encompasses both up and downregulation of the signalling events, for example the induction or cessation of a given signalling event or a change to the level or degree of any given signalling event.

In accordance with the present invention antagonising the interaction of sphingosine kinase with eEFl A (for example, where this interaction has either occurred naturally by virtue of endogenously expressed eEFl A or has resulted from a non-natural event such as the administration of a treatment protocol) prevents the completion of a sphingosine kinase mediated signalling event while agonising or otherwise inducing the interaction of sphingosine kinase with eEFl A promotes sphingosine kinase mediated signalling. It should also be understood that the degree or level of a sphingosine kinase mediated signalling event can be modulated by increasing or decreasing the concentration of interacting molecules. Accordingly, the modulation of signalling need not necessarily equate to the onset or inhibition of signalling but may be designed to regulate the level of sphingosine kinase mediated signalling which occurs.

Reference to "sphingosine kinase" should be understood to include reference to all forms of sphingosine kinase protein and derivatives, variants, homologues or mimetics thereof. In this regard, "sphingosine kinase" should be understood as being a molecule which is, inter alia, involved in the generation of sphingosine- 1 -phosphate during activation of the sphingosine kinase signalling pathway. This includes, for example, all protein forms of sphingosine kinase and its functional derivatives, variants, homologues or mimetics thereof, including, for example, any isoforms which arise from alternative splicing of sphingosine kinase niRNA or allelic or polymorphic variants of sphingosine kinase.

Without limiting the present invention to any one theory or mode of action, two human sphingosine kinase isoforms exist (1 and 2), which differ in their tissue distribution, developmental expression, catalytic properties, and somewhat in their substrate specificity (Pitson et at, 2000, supra; Liu et ah, 2000, supra). A number of studies have shown the effects of sphingosine kinase 1 in enhancing cell proliferation and suppressing apoptosis (Olivera et al, 1999, supra; Xia et at, 2000, supra; Edsall et al, 2001, supra). Furthermore, overexpression of human sphingosine kinase 1 (hSKl) inNIH3T3 fibroblasts has been shown to result in acquisition of the transformed phenotype and the ability to form tumors in nude mice, demonstrating the oncogenic potential of this enzyme (Xia et al, 2000, supra).

Reference to a "functional" derivative, variant, homologue or mimetic thereof should be understood as a reference to a molecule which exhibits any one or more of the functional activities of sphingosine kinase or eEFl A. Preferably, the present invention provides a method of modulating sphingosine kinase 1 mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase 1 with eEFIA or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase 1 catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.

Reference to "eEFIA" (elongation factor IA) should be understood as a reference to all forms of this protein. This includes, for example, any isoforms which arise from alternative splicing of eEFIA mRNA or functional mutants or polymorphic variants of this molecule. Without limiting the present invention to any one theory or mode of action, the canonical role of eEFIA resides in the process of mammalian peptide elongation during protein synthesis, specifically, the non-ribosomal translation elongation machinery that facilitates peptide chain elongation during mRNA translation (Anderson et al. 2003). As well as this role, however, eEFIA has also been implicated in seemingly unrelated processes including cytoskeletal rearrangement, cellular signalling and tumourigenesis. Two isoforms of eEFIA have been identified in the human, these being eEFlAl and eEFl A2 (Ejiri, 2002; Thornton et al. 2003). Both forms can upregulate the catalytic activity of sphingosine kinase. Still further, eEFIA functional derivatives, such as the truncated version of eEFl Al, which is missing 67 (with the addition of 3) N-terminal amino acids) similarly upregulate sphingosine kinase catalytic activity and therefore fall within the scope of the definition of "eEFIA". This truncated version of eEFl Al is also known as prostate tumour inducer (PTI) but is herein referred to as truncated eEFl Al (tr.eEFlAl).

Still without limiting the present invention in any way, it has been shown that each of the two eEFIA isoforms interact with hSKl and directly enhance its activity in cells and in vitro approximately three-fold. Notably, the artificial truncated eEFl Al form (PTI) also interacts with hSKl and enhances its activity, demonstrating that the SKl -interacting and functional region of eEF 1 A resides in the non-GTP binding region of eEF IAl. Although the molecular mechanism whereby eEF IA mediates this enhanced sphingosine kinase activity is not currently known, substrate kinetic analysis indicates that this effect results from a direct increase in the catalytic efficiency of sphingosine kinase, rather than an alteration in its affinity for either ATP or sphingosine. The involvement of the guanidine nucleotide bound state of eEF IA is another possibility for regulating the interaction and effect of eEFIA on sphingosine kinase. In cells, eEFl A (which contains a Ras-like G protein domain) exists in either GTP or GDP bound forms (Ejiri, 2002). Transition between these two forms results in a large conformational change in eEFIA (Ejiri, 2002). Like the small G proteins, the GTP/GDP bound state of eEFIA is actively regulated by its GTPase activity, as well as specific guanine nucleotide exchange factors (GEFs) and guanidine nucleotide dissociation inhibitors (GDIs) (Cans et ah, 2003). It has been found that the guanidine nucleotide bound state of eEFIA, while not affecting its interaction with sphingosine kinase, does modulate the effect of eEFl A on the catalytic activity of sphingosine kinase. Thus, this represents a novel mechanism to dynamically regulate the effect of eEFIA on cellular sphingosine kinase activity. In contrast, GTP bound eEFIA had no effect on SKl or SK2 activity. Consistent with this finding, the PTI truncated form of eEFl Al, lacking much of the G protein domain and therefore unable to bind GTP, retains the ability to bind sphingosine kinase and enhance its catalytic activity.

The present invention therefore more particularly provides a method of modulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with truncated eEFl Al or derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.

Preferably, said sphingosine kinase is sphingosine kinase 1 or 2 and most preferably sphingosine kinase 1. Most preferably, said sphingosine kinase mediated signalling is upregulated by inducing or agonising the interaction of sphingosine kinase with eEFIA or truncated eEFlAl or derivative, homologue or mimetic thereof.

Elucidation of both the role of eEFIA as an upregulator of the catalytic activity of sphingosine kinase and the region of interaction between these two molecules now provides a means for modulating sphingosine kinase mediated cellular activity. By "modulated" is meant upregulated or downregulated. For example, inducing or otherwise agonising the interaction of sphingosine kinase with eEFIA provides a means of increasing the level, degree or rate at which the signalling event occurs, in addition to including reference to inducing the subject signalling event thereby effectively inducing, upregulating or sustaining the subject cellular activity. Conversely, to the extent that a eEFIA mediated sphingosine kinase signalling event is unwanted (for example, where it is sought to reduce or terminate a treatment protocol by virtue of which a patient was administered eEFIA or where it is sought to downregulate a naturally occurring interaction) the present invention extends to decreasing the level, degree or rate at which the signalling event occurs, in addition to including reference to ablating the subject signalling event, thereby effectively ablating or downregulating the subject cellular activity. Accordingly, the agent which is utilised in accordance with the method of the present invention may be an agent which induces the subject event, agonises an event which has already undergone onset, antagonises a pre-existing event or entirely prevents the onset of such an event.

Reference to "inducing or otherwise agonising" should be understood as a reference to:

(i) inducing the interaction of sphingosine kinase with eEF 1 A, in particular the region corresponding to truncated eEFlAl; or

(ii) up-regulating, enhancing or otherwise agonising a sphingosine kinase/eEFIA interaction subsequently to its initial induction. Conversely, "inhibiting or otherwise antagonising" the interaction of sphingosine kinase with eEFl A, in particular the region corresponding to tr.eEFl Al is a reference to:

(i) preventing the interaction of sphingosine kinase with eEFl A (for example, in the context of a prophylactic capacity where endogeneous eEFl A is known to upregulate sphingosine kinase activity); or

(ii) antagonising an existing interaction of sphingosine kinase with eEFl A such that it is ineffective or less effective.

It should be understood that modulation of the interaction between sphingosine kinase and eEFl A (either in the sense of up-regulation or down-regulation) may be partial or complete. Partial modulation occurs where only some of the sphingosine kinase/eEFIA interactions which may normally occur in a given cell are affected by the method of the present invention (for example, the agent which is contacted with the subject cell is provided in a concentration insufficient to saturate the intracellular sphingosine kinase/eEFIA interactions) while complete modulation occurs where all sphingosine kinase/eEFIA interactions are modulated.

Modulation of the interaction between sphingosine kinase and eEFl A may be achieved by any one of a number of techniques including, but not limited to:

(i) introducing into a cell a nucleic acid molecule encoding eEFl A or derivative, homologue or mimetic thereof or introducing the proteinaceous form of eEFl A or derivative, homologue or mimetic thereof in order to modulate the intracellular concentrations of eEFl A which is available for signalling purposes.

(ii) introducing into a cell a proteinaceous or non-proteinaceous molecule which modulates the transcriptional and/or translational regulation of the eEFl A gene. (iii) introducing into a cell a proteinaceous or non-proteinaceous molecule which antagonises the interaction between a eEFl A and sphingosine kinase, such as a competitive inhibiter or antibody.

(iv) introducing into a cell a proteinaceous or non-proteinaceous molecule which agonises the interaction between eEFl A and sphingosine kinase.

Reference to "agent" should be understood as a reference to any proteinaceous or non- proteinaceous molecule which modulates the interaction of sphingosine kinase with eEFl A and includes, for example, the molecules detailed in points (i) - (iv), above. The subject agent may be linked, bound or otherwise associated with any proteinaceous or non- proteinaceous molecule. For example, it may be associated with a molecule which permits its targeting to a localised region. In a preferred embodiment, the subject agent is eEFl A itself, or functional derivative, homologue or mimetic thereof, which is introduced to upregulate sphingosine kinase activation.

According to this preferred embodiment, there is therefore provided a method of upregulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of eEFl A or functional derivative, homologue or mimetic thereof for a time and under conditions sufficient to induce the interaction of sphingosine kinase with eEFl A and thereby upregulate sphingosine kinase catalytic activity.

Said proteinaceous molecule may be derived from natural, recombinant or synthetic sources including fusion proteins or following, for example, natural product screening. Said non-proteinaceous molecule may be derived from natural sources, such as for example natural product screening or may be chemically synthesised. For example, the present invention contemplates chemical analogues of eEFl A capable of acting as agonists or antagonists of the sphingosine kinase interaction. Chemical agonists may not necessarily be derived from sphingosine kinase or eEFl A but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic or upregulate certain physiochemical properties of sphingosine kinase or eEFl A. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing sphingosine kinase and eEFl A from interacting. Antagonists include antibodies (such as monoclonal and polyclonal antibodies) specific for sphingosine kinase or eEFl A, or parts of said sphingosine kinase or eEFl A. Reference to antagonists also includes antigens which competitively inhibit sphingosine kinase/eEFIA interaction, siRNA, antisense molecules, ribozymes, DNAzymes, RNA aptamers, or molecules suitable for use in co- suppression. The proteinaceous and non-proteinaceous molecules referred to in points (i)- (iv), above, are herein collectively referred to as "modulatory agents".

Screening for the modulatory agents hereinbefore defined can be achieved by any one of several suitable methods including, but in no way limited to, contacting a cell comprising sphingosine kinase and eEFl A with an agent and screening for the modulation of sphingosine kinase/eEFIA functional activity (such as a specific cellular activity) or modulation of the activity or expression of a downstream sphingosine kinase or eEFl A cellular target. Detecting such modulation can be achieved utilising techniques such as Western blotting, electrophoretic mobility shift assays and/or the readout of reporters of sphingosine kinase or eEFl A activity such as luciferases, CAT and the like.

It should be understood that the sphingosine kinase or eEFl A protein may be naturally occurring in the cell which is the subject of testing or the genes encoding them may have been transfected into a host cell for the purpose of testing. Further, the naturally occurring or transfected gene may be constitutively expressed - thereby providing a model useful for, inter alia, screening for agents which down-regulate sphingosine kinase/eEFIA interactivity or the gene may require activation - thereby providing a model useful for, inter alia, screening for agents which modulate sphingosine kinase/eEFIA interactivity under certain stimulatory conditions, such as phage-display and yeast two- or multi-hybrid screening. Further, to the extent that a sphingosine kinase or eEFl A nucleic acid molecule is transfected into a cell, that molecule may comprise the entire sphingosine kinase or eEFl A gene or it may merely comprise a portion of the gene such as the eEFl A region which binds to sphingosine kinase. In another example, the subject of detection could be a downstream sphingosine kinase regulatory target, rather than sphingosine kinase itself. Yet another example includes sphingosine kinase or eEFl A binding sites ligated to a minimal reporter. For example, modulation of sphingosine kinase/eEFl A interactivity can be detected by screening for the modulation of a downstream signalling component. This is an example of a system where modulation of the molecules which sphingosine kinase and eEFl A regulate the activity of, are monitored. These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as the proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical or natural libraries.

The agents which are utilised in accordance with the method of the present invention may take any suitable form. For example, proteinaceous agents may be glycosylated or unglycosylated, phosphorylated or dephosphorylated to various degrees and/or may contain a range of other molecules fused, linked, bound or otherwise associated with the proteins such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins. Similarly, the subject non-proteinaceous molecules may also take any suitable form. Both the proteinaceous and non-proteinaceous agents herein described may be linked, bound otherwise associated with any other proteinaceous or non-proteinaceous molecules. For example, in one embodiment of the present invention said agent is associated with a molecule which permits its targeting to a localised region, such as a specific tissue.

The term "expression" refers to the transcription and translation of a nucleic acid molecule. Reference to "expression product" is a reference to the product produced from the transcription and translation of a nucleic acid molecule. Reference to "modulation" should be understood as a reference to upregulation or downregulation.

"Derivatives" of the molecules herein described (for example sphingosine kinase, eEFl A or other proteinaceous or non-proteinaceous agents) include fragments, parts, portions or variants from either natural or non-natural sources. Non-natural sources include, for example, recombinant or synthetic sources. By "recombinant sources" is meant that the cellular source from which the subject molecule is harvested has been genetically altered. This may occur, for example, in order to increase or otherwise enhance the rate and volume of production by that particular cellular source. Parts or fragments include, for example, active regions of the molecule. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.

Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in a sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins, as detailed above.

Derivatives also include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. For example, sphingosine kinase, eEFl A or derivative thereof may be fused to a molecule in order to facilitate cell membrane localisation. Analogs of the molecules contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogs.

Derivatives of nucleic acid sequences which may be utilised in accordance with the method of the present invention may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules. The derivatives of the nucleic acid molecules utilised in the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules. Derivatives of nucleic acid sequences also include degenerate variants.

A "variant" of sphingosine kinase or eEFl A should be understood to mean a molecule which exhibits at least some of the functional activity of the form of sphingosine kinase or eEFl A of which it is a variant. A variation may take any form and may be naturally or non-naturally occurring. A mutant molecule is one which exhibits modified functional activity.

By "homologue" is meant that the molecule is derived from a species other than that which is being treated in accordance with the method of the present invention.

"Mimetics" should be understood as molecules exhibiting any one or more of the functional activities of the subject molecule, which functional equivalents may be derived from any source such as being chemically synthesised or identified via screening processes such as natural product screening. For example chemical or functional equivalents can be designed and/or identified utilising well known methods such as combinatorial chemistry or high throughput screening of recombinant libraries or following natural product screening. These methods may also be utilised to screen for any of the modulatory agents which are useful in the method of the present invention.

For example, libraries containing small organic molecules may be screened, wherein organic molecules having a large number of specific parent group substitutions are used. A general synthetic scheme may follow published methods (eg., Bunin et al. (1994) Proc. Natl. Acad. ScI USA, 91 :4708-4712; De Witt et al. (1993) Proc. Natl. Acad. ScI USA, 90:6909-6913). Briefly, at each successive synthetic step, one of a plurality of different selected substituents is added to each of a selected subset of tubes in an array, with the selection of tube subsets being such as to generate all possible permutation of the different substituents employed in producing the library. One suitable permutation strategy is outlined in US. Patent No. 5,763,263. There is currently widespread interest in using combinational libraries of random organic molecules to search for biologically active compounds (see for example U.S. Patent No. 5,763,263). Ligands discovered by screening libraries of this type may be useful in mimicking or blocking natural ligands or interfering with the naturally occurring ligands of a biological target. In the present context, for example, they may be used as a starting point for developing sphingosine kinase/eEFIA agonists or antagonists. Sphingosine kinase and/or eEFl A or a relevant part thereof may, according to the present invention, be used in combination libraries formed by various solid-phase or solution-phase synthetic methods (see for example U.S. Patent No. 5,763,263 and references cited therein). By use of techniques, such as that disclosed in U.S. Patent No. 5,753,187, millions of new chemical and/or biological compounds may be routinely screened in less than a few weeks. Of the large number of compounds identified, only those exhibiting appropriate biological activity are further analysed.

With respect to high throughput library screening methods, oligomeric or small-molecule library compounds capable of interacting specifically with a selected biological agent, such as a biomolecule, a macromolecule complex, or cell, are screened utilising a combinational library device which is easily chosen by the person of skill in the art from the range of well-known methods, such as those described above. In such a method, each member of the library is screened for its ability to interact specifically with the selected agent. In practising the method, a biological agent is drawn into compound-containing tubes and allowed to interact with the individual library compound in each tube. The interaction is designed to produce a detectable signal that can be used to monitor the presence of the desired interaction. Preferably, the biological agent is present in an aqueous solution and further conditions are adapted depending on the desired interaction. Detection may be performed for example by any well-known functional or non-functional based method for the detection of substances.

"Analogues" of sphingosine kinase, eEFl A or agonistic or antagonistic agents contemplated herein include, but are not limited to, modifications to side chains, incorporating unnatural amino acids and/or derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the analogues. The specific form which such modifications can take will depend on whether the subject molecule is proteinaceous or non- proteinaceous. The nature and/or suitability of a particular modification can be routinely determined by the person of skill in the art.

For example, examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using

4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with

N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate .

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aniinohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 1.

TABLE 1

Non-conventional Code Non-conventional Code amino acid amino acid . α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine DgIn L-N-methylnorvaline Nmnva

D-glutamic acid DgIu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine DiIe L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine NIe

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl- -aminobutyrate Mgabu D-α-methylalamne Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine NgIu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3 -aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine NgIn

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diρhenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3 -guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(I -hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine NaIa D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(I -methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamine MgIn L-α-methylglutamate MgIu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine MnIe L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- 1 -(2,2-diphenyl-Nmbc ethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.

It should be understood that the method of the present invention may be performed in the context of a cellular source which is located either in vitro or in vivo.

Since sphingosine kinase is a molecule which is central to the functioning of an intracellular signalling pathway, the method of the present invention provides a means of modulating cellular activity which is regulated or controlled by sphingosine kinase signalling. For example, the sphingosine kinase signalling pathway is known to regulate cellular activities, such as those which lead to inflammation, cellular transformation, apoptosis, cell proliferation, up-regulation of the production of inflammatory mediators such as cytokines, chemokines, eNOS and up-regulation of adhesion molecule expression. Said up-regulation may be induced by a number of stimuli including, for example, inflammatory cytokines such as tumour necrosis factor α and interleukin 1, endotoxin, oxidised or modified lipids, radiation or tissue injury. In this regard, reference to "modulating cellular activity" is a reference to up-regulating, down-regulating or otherwise altering any one or more of the activities which a cell is capable of performing pursuant to sphingosine kinase signalling such as, but not limited, one or more of chemokine production, cytokine production, nitric oxide synthesis, adhesion molecule expression and production of other inflammatory modulators. Although the preferred method is to down- regulate sphingosine kinase activity, thereby down-regulating unwanted cellular activity, the present invention should nevertheless be understood to encompass up-regulating of cellular activity, which may be desirable in certain circumstances.

Accordingly, yet another aspect of the present invention is directed to a method of modulating cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with eEFl A or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.

Preferably, the present invention is directed to a method of modulating cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase 1 with eEFl A or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.

In a most preferred embodiment the present invention is directed to a method of modulating human cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with tr.eEFlAl wherein inducing or otherwise agonising said association up-regulates said human cellular activity and inhibiting or otherwise antagonising said association down-regulates said human cellular activity.

Most preferably, said modulation is upregulation of cellular activity which is achieved by inducing or agonising the interaction of sphingosine kinase with eEFl A or tr.eEFlAl.

Most preferably, said agent is eEFl A or tr.eEFlAl itself.

A further aspect of the present invention relates to the use of the invention in relation to the treatment and/or prophylaxis of disease conditions. Without limiting the present invention to any one theory or mode of action, the broad range of cellular functional activities which are regulated via the sphingosine kinase signalling pathway renders the regulation of sphingosine kinase functioning an integral component of every aspect of both healthy and disease state physiological processes. Accordingly, the method of the present invention provides a valuable tool for modulating aberrant or otherwise unwanted cellular functional activity which is regulated via the sphingosine kinase signalling pathway. Accordingly, yet another aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with eEFl A or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.

Still another aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate interaction of sphingosine kinase with eEFl A or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said association down-regulates said sphingosine kinase functional activity.

In a most preferred embodiment, the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a human, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, said method comprising administering to said human an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with tr.eEFlAl, wherein inducing or otherwise agonising said interaction up-regulates said cellular activity and inhibiting or otherwise antagonising said interaction down-regulates said cellular activity.

In another most preferred embodiment, the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a human, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, said method comprising administering to said human an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with tr.eEFl Al wherein inducing or otherwise agonising said interaction up-regulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said interaction down-regulates said sphingosine kinase functional activity.

Most preferably, said modulation is upregulation and said agent is eEFl A or tr.eEFl Al or functional derivative, homologue or mimetic thereof.

Reference to "aberrant, unwanted or otherwise inappropriate" cellular activity should be understood as a reference to overactive cellular activity, to physiologically normal cellular activity which is inappropriate in that it is unwanted or to insufficient cellular activity. This definition applies in an analogous manner in relation to "aberrant, unwanted or otherwise, inappropriate" sphingosine kinase activity. For example, to the extent that a cell is neoplastic, it is desirable that the promotion of cellular proliferation and anti- apoptotic characteristics be down-regulated. Similarly, diseases which are characterised by inflammation, such as rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease arid inflammatory bowel disease, are known to involve cellular activation leading to the synthesis and secretion of inflammatory mediators, such as adhesion molecules. In such situations, it is also desirable to down-regulate such activity. In other situations, it may be desirable to agonise or otherwise induce sphingosine kinase activation in order to stimulate cellular proliferation, for example in order to promote angiogenesis.

The term "mammal" as used herein includes humans, primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys), laboratory test animals (eg. mice, rabbits, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. foxes, kangaroos, deer). Preferably, the mammal is human or a laboratory test animal Even more preferably, the mammal is a human.

An "effective amount" means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity or onset of a particular condition. "Treatment" may also reduce the severity of an existing condition.

The present invention further contemplates a combination of therapies, such as the administration of the agent together with subjection of the mammal to other agents, drugs or treatments which may be useful in relation to the treatment of the subject condition such as cytotoxic agents or radiotherapy in the treatment of cancer.

Administration of the modulatory agent, in the form of a pharmaceutical composition, may be performed by any convenient means. The modulatory agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the modulatory agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of modulatory agent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.

The modulatory agent may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules). The modulatory agent may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.

Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeal^, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, infusion, orally, rectally, via IV drip patch and implant.

In accordance with these methods, the agent defined in accordance with the present invention may be coadministered with one or more other compounds or molecules. By "coadministered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. For example, the subject agent may be administered together with an agonistic agent in order to enhance its effects. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.

Still another aspect of the present invention contemplates the use of an agent, as hereinbefore defined, in the manufacture of medicament for the treatment of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, wherein said agent modulates the interaction of sphingosine kinase with eEF 1 A or functional derivative, homologue or mimetic thereof and wherein inducing or otherwise agonising said interaction upregulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said interaction downregulates said sphingosine kinase functional activity.

Preferably, said interaction is interaction with tr.eEFl Al .

Even more preferably, said mammal is a human, and said modulation is upregulation.

In yet another further aspect, the present invention contemplates a pharmaceutical composition comprising the modulatory agent as hereinbefore defined together with one or more pharmaceutically acceptable carriers and/or diluents. These agents are referred to as the active ingredients.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule encoding a modulatory agent. The vector may, for example, be a viral vector.

The present invention is now described with reference to the following non-limiting examples. EXAMPLE 1

SPHINGOSINE KINASE ACTIVATION THROUGH INTERACTION WITH eEFIA

Materials and Methods

Cell culture and transfection

Human embryonic kidney cells (HEK-293T, ATCC CRL- 1573) cells were cultured in Dulbecco's modified Eagle's medium (CSL Biosciences, Parkville, Australia) containing 10% fetal bovine serum (CSL Biosciences), 2 niM glutamine, 0.2% (w/v) sodium bicarbonate, penicillin (1.2 mg/ml), and streptomycin (1.6 mg/ml). Cells were transiently transfected using the calcium phosphate precipitation method, harvested 24 h later by scraping into cold PBS, and lysed by sonication (3 watts for 30s at 4⁰C) in extraction buffer containing 5OmM Tris/HCl pH 7.4, 15OmM NaCl, 2mM Na₃VO₄, 1 OmM NaF,

ImM EDTA, 10% glycerol, 0.05% Triton X-100, 10 mM β-glycerophosphate, 1 mM DTT and protease inhibitors (Complete; Roche Molecular Biochemicals). Protein concentrations of cell lysates were determined with Coomassie Brilliant Blue reagent (Sigma) using BSA as standard.

Yeast two-hybrid screen

Yeast two-hybrid screening was performed using the Matchmaker Gal4 Two-Hybrid System 3 (Clontech) according to the manufacturer's instructions. Full-length hSKl cDNA (Genbank accession number AF200328) was cloned into pGBKT7 (Clontech) in-frame with the Gal4 DNA-binding domain. This bait construct was then transformed into the yeast strain AHl 07 together with a human leukocyte cDNA library in pACT2 (Clontech). A total of 1 x 10⁶ independent clones were screened. Cloning ofeEFlAl and eEFl A2 and generation oftrunc-eEFlAl

Primers for PCR amplification of the full-length human eEFl Al coding region were designed using the published eEFl Al cDNA sequence (306; Genbank accession number NM001402). The eEFlAl cDNA was amplified from a human foreskin fibroblast cDNA and HA-epitope tagged at the C-terminus with primers 5'-

CCGGATCCGCCACCATGGGAAAGGAAAAGACTCATA-3' (SEQ ID NO:1) and 5'- GGCTCGAGTCAAGCGTAATCTGGAACATCGTATGGGTATTTAGCCTTCTGAGC TTTCTG-3' (SEQ ID NO:2). The PCR product was then cloned into pcDNA3 (Invitrogen) for mammalian expression and pGEX4T-l (GE Health) for bacteria expression following digestion with BamHI and Xhol. Sequencing verified the integrity of the human HA- eEFl Al cDNA sequence. An JV-terminal truncated eEFl Al (tr.eEFl Al) modelled on the sequence of the naturally occurring PTI-I protein (292; Genbank accession number L41490) was generated by PCR from the pcDNA3-eEFlAl-HA plasmid using the primer 5'-TAGAATTCGCCACCATGCAGTCGGAACGTGGTATCACCATTGAT-S' (SEQ ID NO:3). The PCR product was then cloned into pcDNA3 and ρGEX4T-l following digestion with EcoRI and Xhol. Primers for PCR amplification of the eEFlA2 coding region were designed using the published murine eEFl A2 cDNA sequence (307; Genbank accession number NM007906). The murine eEFl A2 cDNA was amplified from mouse brain cDNA and HA-epitope tagged at the C-terminus with primers 5'-

TAGAATTCCGGCCACCATGGGCAAGGAGAAGACACA-3' (SEQ ID NO:4) and 5'- TAGAATTCAAGCGTAATCTGGAACATCGTATGGGTACTTGCCCGCTTTCTGAGC -3' (SEQ ID NO:5). The PCR product was then cloned into both ρcDNA3 and ρGEX4T-2 following digestion with EcoRI. Sequencing verified the orientation and integrity of the mouse eEFlA2 cDNA sequence.

Generation of GST-fusion proteins

eEFlAl, tr.eEFl Al and eEFlA2 cDNAs were expressed in E. coli BL21 in bacteria as glutathione 5-transferase (GST)-fusion proteins. Overnight cultures were grown with shaking (200 rpm) at 37°C in Luria broth containing 100 mg/L ampicillin. The culture was then diluted 1 in 30 into fresh Luria broth and grown with shaking at 37°C to a OD_60O of 0.6-1.0. Expression of the GST-fusion proteins was then induced with 0.1 mM IPTG and the cultures incubation with shaking at 37°C for a further 90 min. The bacterial cells were then harvested by centrifugation at 6000 g for 20min at 4⁰C, resuspended in 20 ml of cold PBS, and lysed by sonication (three pulses of 5 watts for 30 s on ice with 30 s cooling between each pulse). Triton X-100 was then added to the bacterial lysates to a final concentration of 1%, lysates mixed well, and then centrifugation at 50000 g for 25 min at 4°C. The resultant clarified bacterial lysate was then incubated with GSH-Sepharose 4B for 2 h at 4°C with constant mixing. After this time the GSH-Sepharose beads (with bound protein) were pelleted by centrifugation at 3000 g for 5 min at 4⁰C and washed twice in cold PBS. Protein attached to the GSH-Sepharose was quantitated with Coomassie brilliant blue staining following SDS-PAGE using BSA as standard. These beads were then either used directly in pull-down analysis, or the GST-fusion proteins eluted by incubation with cold PBS containing 10 mM GSH for 30 min with constant mixing. GST-hSKl fusion protein was produced as previously described (Pitson et ah, 2000).

Sphingosine kinase assays

Sphingosine kinase activity was routinely determined using D-eryt/zro-sphingosine (Biomol, Plymouth Meeting, PA) and [γ³²P] ATP (PerkinElmer, Melbourne, Australia) as substrates, as described previously (Roberts et ah, 2004, Anal. Biochem. 331 : 122-129). A unit (U) of sphingosine kinase activity is defined as the amount of enzyme required to produce 1 pmol SlP / min. Substrate kinetics were analysed using Michaelis-Menten kinetics with the non-linear regression program, Hyper 1.1s.

In vitro phosphorylation ofhSKl andeEFIA

In vitro phosphorylation of His- tagged hSKl in solution was performed as described previously (Pitson et al, 2003). In vitro phosphorylation of GST-hSKl was also performed while the protein remained bound to the GSH-Sepharose beads by incubating these beads (2μg GST-hSKl) with 60 units of ERK2 (Calbiochem) and 1 mM ATP in ERK assay buffer (9 mM MOPS, 11 mM β-glycerophosphate, 2.2 niM EGTA and 0.4 mM sodium orthovanadate) for 60 min at 3O⁰C. The beads were then washed with 50 mM Tris/HCl buffer containing 150 mM NaCl and 10% glycerol. In vitro phosphorylation of GST- eEFl Al bound to GSH-Sepharose was performed in a similar manner by incubating these beads (containing 1 μg GST-eEFlAl) with 0.1 units of S6 kinase (Upstate), 3.5 mM

[γ³²P]ATP (70 nCi/μl) in buffer (7 mM MOPS, 0.1 mM EDTA₅ 4 μM β-glycerophosphate, 0.2 μM DTT, 0.15mM orthovanadate, 0.9 mM EGTA, pH 7.4) for 30 min at 37 °C. The beads were then washed three times with cold PBS.

Immunoprecipitation and western blotting

Lysates from cells expressing the hSKl(FLAG) or hSK2(FLAG) alone and/or in combination with HA-eEFlA isoforms were centrifuged at 13,000 g for 10 min at 4 ⁰C to remove insoluble material. Anti-HA monoclonal antibodies (Sigma), M2 anti-FLAG monoclonal antibodies (Sigma), or rabbit anti-hSKl antibodies (Pitson et ah, 2003) were added to the lysates and incubated at 4 ⁰C for 3 hr with constant agitation. The immune complexes were then captured by incubation with protein A-sepharose (Amersham Pharmacia Biotech) for 3 hr at 4 ⁰C, washed with cold extraction buffer, subjected to SDS- PAGE and the proteins transferred to nitrocellulose membranes. hSKl was quantitated with either the monoclonal M2 anti-FLAG antibody (Sigma), or polyclonal chicken or rabbit anti-hSKl antibodies (Pitson et ah, 2003). eEFl Al was determined with either anti- HA antibodies (12CA5; Sigma) or anti-eEFIA antibodies (Upstate). The immunocomplexes were detected with HRP-conjugated anti-mouse (Pierce), anti-rabbit (Pierce) or anti-chicken IgG (IMVS, Adelaide, Australia) using an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech).

GST fusion protein binding analyses

Lysates from transiently transfected HEK-293T cells were incubated with GSH-sepharose beads containing 1 μg of either GST, GST-eEFIA, GST-tr.eEFlAl, or GST-hSKl for 2 h at 4⁰C with constant mixing. The beads were then washed three times in 15 mM Tris/HCl, pH 7.4, containing 40 mM NaCl and 10% glycerol, subjected to SDS-PAGE, and associated proteins detected by western blotting with either anti-FLAG or anti-HA antibodies. Interaction of GST-eEFl Al with purified recombinant hSKl or hSK2 was performed in a similar manner with GST-eEFl Al bound to GSH-sepharose incubated with 1 μg of His-tagged recombinant hSKl (Pitson et ah, 2002, J. Biol. Chem. 277:49545- 49553) or hSK2 (Roberts et ah, 2004, supra) for 3 h at 4⁰C with constant mixing. The beads were then washed as described above, subjected to SDS-PAGE, and associated proteins detected by western blotting with anti-His antibodies (Santa Cruz Biotechnology). The effect of guanidine nucleotides on the interaction of eEFl Al with hSKl and hSK2 was analysed by pre-incubation of 1 μg GST-eEFlAl or GST-trunc-eEFlAl bound to GSH-sepharose beads with 0.1 mM GTPγS, 10 mM GTP or 10 mM GDP in 10 mM Tris/HCl, pH 7.4, containing 20 mM MgCl₂ for 30 min at 4°C with constant mixing. The guanidine nucleotide loaded proteins were then isolated by centrifugation (3000 g for 5 min at 4⁰C) and assessed for their ability to bind recombinant hSKl and hSK2 as described above.

Results:

eEFl Al is a hSKl interacting protein:

In an attempt to understand the mechanisms regulating the activity and function of hSKl a yeast two-hybrid screen was performed to identify proteins that interact with hSKl. One partial cDNA that was isolated in this screen encoded the C-terminal 312 amino acids of elongation factor IAl (eEFlAl).

To confirm the interaction between eEFl Al and hSKl, bacterial and mammalian expression constructs encoding the full-length eEFl Al cDNA were generated by PCR from human foreskin fibroblast cDNA. The interaction of eEFlAl with hSKl was first assessed by pull down experiments using GST-eEFl Al or GST alone bound to glutathione sepharose and lysates from HEK-293T cells overexpressing FLAG-epitope tagged hSKl . The results (Fig IA) demonstrate a specific interaction of hSKl with GST-eEFlAl and not with GST alone. Further, reverse pull down experiments were performed using GST- hSKl or GST alone bound to glutathione sepharose and HEK-293T cell lysates overexpressing HA-epitope tagged eEFl Al . Again, the results (Fig IB) demonstrated the specific interaction of GST-hSKl with eEFl Al. To further confirm the interaction between eEFl Al and hSKl, co-immunoprecipitation studies were performed on lysates from HEK-293T cells co-expressing HA-eEFlAl and hSKl-FLAG. The presence of hSKl in the anti-HA (eEFl Al) immuno-complexes was observed (Fig 1C), further indicating an interaction between hSKl and eEFl Al. Finally, we were able to demonstrate the physiological interaction between hSKl and eEFl Al through the co- immunoprecipitation of the endogenous proteins in untransfected HEK-293T cell lysates (Fig ID) using anti-hSKl (Pitson et al, 2003) and anti-eEFl Al antibodies.

eEFl Al directly enhances hSKl activity in vitro:

Although the canonical role for eEFlAl is in polypeptide elongation during protein synthesis (Browne and Proud, 2002; Ejiri, 2002; Abbott and Proud, 2004) various other apparently unrelated cellular functions have been attributed to eEFl Al, including roles in signal transduction, cytoskeletal organization, apoptosis and most importantly oncogenic transformation (Ejiri, 2002; Thornton et ah, 2003; Tatsuka et al, 1992).

SK activity assays using recombinant GST-eEFl Al or GST alone with recombinant hSKl (rec-hSKl) were performed. While under these conditions GST alone had no effect on hSKl activity, GST-eEFl Al enhanced the catalytic activity of hSKl by 2-3 fold (Fig 2A). This effect was not as a result of eEFl Al increasing the stability of rec-hSKl in this enzyme assay, since under these assay conditions rec-hSKl was shown to be stable with linear reaction kinetics (data not shown). Taken together, these results indicate that eEFl Al has a direct stimulatory effect on the activity of hSKl. To examine if this in vitro effect of eEFlAl on hSKl activity was dose dependent, the effect of increasing concentrations of GST-eEFlAl on rec-hSKl activity was determined. Results showed that hSKl activity continued to increase with increasing amounts of eEFl Al, with a significant effect seen with a half fold molar excess of eEFl Al and maximum stimulation with a ten fold molar excess of eEFlAl (Fig 2A).

Despite these in vitro effects, when eEFl Al was overexpressed in HEK-293T cells no increase in cellular SK activity was observed (refer Fig 8C). This result was not surprising since eEFl Al is one of the most highly expressed proteins in cultured cells (Dapas et ah, 2003). Thus, overexpression of eEFl Al is likely to only result in a modest increase in cellular eEFl Al levels.

Effect of eEFl Al on hSKl substrate kinetics:

As eEFl Al directly increases the activity of rec-hSKl, the effect of eEFlAl on hSKl substrate kinetics was examined. The results show that the Ku values of hSKl for both sphingosine and ATP were unaltered by the presence of eEFl Al (Fig 2B). In contrast, the £_cat for hSKl was increased approximately two to three fold by the presence of eEFl Al (Fig 2B). Taken together these results indicate that eEFl Al does not increase the binding affinity of hSKl for its substrates but does enhance the rate of catalysis.

eEFl Al interacts with and enhances hSK2 activity:

Since eEFl Al interacts with hSKl and has a direct effect on its activity, its effect on the other human SK isoform, hSK2 was examined. The interaction between eEFl Al and hSK2 was investigated using GST-eEFlAl or GST alone bound to glutathione sepharose and rec-hSK2. Like hSKl, hSK2 was able to specifically interact with GST-eEFlAl (Fig 3A).

Having demonstrated that eEFl Al and hSK2 interact, the effect of the interaction on the activity of hSK2 was examined. Purified GST-eEFlAl and rec-hSK2 were incubated in vitro and the resultant SK activity was measured. As with hSKl, GST-eEFlAl was shown to increase hSK2 activity by 2-3 fold (Fig 3B). eEFlA2 is an hSKl interacting protein:

A second isomer of eEFlAl has been identified in humans and is referred to as eEFlA2. While the sequence homology between these two proteins is extremely high (greater than 95% nucleotide sequence identity) (Thornton et ah, 2003), some differences have been observed in their distribution within human tissues (Thornton et ah, 2003; Abbott and Proud, 2004), with eEFl Al ubiquitously expressed, and eEFl A2 only present in heart, brain and skeletal muscle cells (Thornton et al., 2003). Given the sequence homology of the two proteins, an examination was made as to whether SKl also interacted with eEFlA2. Co-immunoprecipitations were performed using lysates from HEK-293T cells coexpressing HA-eEFlAl and SKl-FLAG , demonstrating that as with eEFlAl, eEFlA2 also associates SKl (Fig 4A).

In agreement with the eEFl Al results, in vitro studies have shown that GST-eEFl A2 is able to increase the activity of rec-hSKl two to three fold (Fig 4B). As with HA-eEFl Al, however, ectopic expression of HA-eEFlA2 in HEK-293T cells had no effect on endogenous SK activity (refer Fig 8C).

hSKl - eEFl Al interaction is not regulated by phosphorylation

The interaction between eEFl Al and various proteins is known to be regulated by phosphorylation, either of eEFl Al (Yang and Boss, 1994) or its target proteins (Ejiri, 2002; Chang et ah, 2002). Therefore, an analysis was performed as to whether the phosphorylation state of hSKl or eEFl Al affected their ability to interact. The effect of hSKl phosphorylation by phosphorylating GST-hSKl bound to glutathione sepharose using recombinant ERK2 was first investigated. ERK2 is known to specifically phosphorylate hSKl at Ser225 in vitro, which appears to be the only physiological phosphorylation site in this protein (Pitson et ah, 2003). This phosphorylated GST-hSKl was then used in pull down assays with lysates from HEK-293T cells overexpressing HA- eEFl Al . Both non-phosphorylated GST-hSKl and phosphorylated GST-hSKl interacted with HA-eEFl Al in a comparable manner (Fig 5A), The phosphorylation of GST-hSKl also did not affect the ability of GST-eEFlAl to increase its activity. To confirm this result pull down experiments using GST-eEFl Al bound to glutathione sepharose and lysates from HEK-293T cells overexpressing either wild type hSKl, known to be phosphorylated (Pitson et ah, 2003), or its non phosphorylatable counterpart hSKl (S225A) (Pitson et ah, 2003) were performed. The results of the pull down experiments showed that both forms of hSKl interacted with eEFl Al to a comparable extent (Fig 5B). Together, these results definitively show that, unlike some other eEFl Al associated proteins, the phosphorylation state of hSKl does not affect its ability to interact with eEFl Al.

Next, the effect of eEFl Al phosphorylation on its ability to interact with hSKl was examined. eEFl Al is known to be phosphorylated in vitro by S6 kinase (S6K) (Thornton et ah, 2003), PKC (Kielbassa et ah, 1995) and Rho-associated kinase (RhoK) (Ejiri, 2002). Since S6K phosphorylates.eEFl Al at multiple sites including those sites phosphorylated by PKC and RhoK (Thornton et ah, 2003; Ejiri, 2002), GST-eEFlAl bound to glutathione sepharose was phosphorylated with S6K (Fig 6A) and then used in pull down experiments with rec-hSKl and rec-hSK2. The results indicate that both non-phosphorylated and phosphorylated GST-eEFlAl are able to interact with hSKl and hSK2 to a similar extent (Fig 6B). The phosphorylated GST-eEFlAl was also able to activate rec-hSKl to the same extent as non-phosphorylated GST-eEFl Al . Therefore, the results from these phosphorylation experiments show that unlike the situation with some other eEFl Al interacting proteins, the phosphorylation state of eEFl Al or SK has no effect on the interaction of these two proteins.

hSKl-eEFlAl interaction is not regulated by GTP although hSKl activity is;

In cells, eEFl Al exists in two states; a GTP bound form, and a GDP bound form. Interconversion between these two forms is mediated by both the low GTPase activity of eEFl Al and other eEFl subunits that act as guanidine nucleotide exchange factors in a comparable manner to that observed with the small G proteins (Ejiri, 2002; Lamberti et ah, 2004). During this conversion between the GTP bound and GDP bound forms eEFl Al undergoes a large conformational change which alters its ability to bind to aa-tRNA (Ejiri, 2002). The possibility that the guanidine nucleotide bound state of eEFlAl may alter its interaction with SK and/or its effect on SK activity was therefore investigated. To investigate this we initially examined the ability of rec-hSKl and rec-hSK2 to interact with GST-eEFl Al bound to glutathione sepharose that had been pre-treated with GTPγS. Results from these pull down experiments indicate that hSKl or hSK2 does not have a higher binding affinity for one form of eEFlAl over the other (Fig 6A). However, when the effect of the guanidine nucleotide state of eEFlAl on SK activity was examined it was found that the eEFlAl.GTP was unable to increase rec-hSKl and rec-hSK2 activity as previously seen. In stark contrast, nil treated eEFlAl retained the ability to enhance rec- hSKl and hSK2 activity two to three fold (Fig 6B). Taken together these results indicate that while the guanidine nucleotide bound state of eEFl Al does not affect its ability to interact with hSKl or hSK2, it does affect the ability of this protein to enhance SK activity.

To further examine the guanidine nucleotide state of eEFlAl on SK activity, an artificially truncated version of eEFl Al was generated, missing 67 (with the addition of 3) iv-terminal amino acids, based on the naturally occurring truncated eEFl Al protein, prostate tumour inducer (PTI). This truncated version of eEFl Al (PTI) lacks most of the Ras-like G protein domain including residues essential for GTP binding (Mansilla et ah, 2005). As with full length eEFlAl, PTI was shown to interact with both SKl and SK2 (Fig 8A). This result was not surprising as the partial eEFl Al cDNA obtained from the yeast two- hybrid screen generated a version of eEFl Al lacking even more of the w-terminus, but clearly retained binding to hSKl . Further examination of the effect of PTI on the in vitro activity of rec-hSKl and rec-hSK2 activity revealed that this GTP -binding deficient version of eEFl Al retained the ability to enhance SK activity 2-3 fold (Fig 8B).

Furthermore, unlike wildtype eEFlAl, when PTI was overexpressed in HEK-293T cells a 2-3 fold increase in endogenous SK activity was observed compared to vector control (Fig 8C).

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Claims

CLAIMS:

1. A method of modulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with eEFl A or functional derivative, variant, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.

2. A method of modulating sphingosine kinase-mediated cellular activity said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with eEFl A or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.

3. The method according to claim 1 or 2 wherein said eEFl A is eEFl Al, eEFlA2 or tr.eEFlAl.

4. The method according to any one of claims 1-3 wherein said sphingosine kinase is human sphingosine kinase.

5. The method according to claim 4 wherein said human sphingosine kinase is sphingosine kinase 1.

6. The method according to claim 4 wherein said human sphingosine kinase is sphingosine kinase 2.

7. The method according to claim 1 or 2 wherein said modulation is upregulation of the level of sphingosine kinase catalytic activity and said upregulation is achieved by introducing into said cell a nucleic acid molecule encoding eEFlAl, eEFl A2 or tr.eEFlAl or functional equivalent, derivative or homologue thereof or the eEFl Al, eEFl A2 or tr.eEFlAl expression product or functional derivative, homologue, analogue, equivalent or mimetic thereof.

8. The method according to claim 7 wherein said eEFl Al or eEFlA2 is GDP bound or nucleotide free.

9. The method according to claim 1 or 2 wherein said modulation is upregulation of the level of sphingosine kinase activity and said upregulation is achieved by contacting said cell with a proteinaceous or non-proteinaceous molecule which functions as an agonist of the sphingosine kinase/eEFl A interaction.

10. The method according to claim 1 or 2 wherein said modulation is downregulation of the level of sphingosine kinase activity and said downregulation is achieved by contacting said cell with a proteinaceous or non-proteinaceous molecule which functions as an antagonist of the sphingosine kinase/eEFl A interaction.

11. The method according to claim 10 wherein said antagonist is an eEFl A competitor.

12. The method according to claim 11 wherein said competitor is GTP bound eEFl A.

13. The method according to claim 10 wherein said antagonist is an antibody directed to eEFlA.

14. The method according to claim 13 wherein said antibody is directed to the eEFl A region defined by tr.eEFl A.

15. The method according to claim 10 wherein said antagonist is an antisense nucleic acid molecule, siRNA or nucleic acid molecule suitable to induce co-suppression, which molecules are directed to eEFl A.

16. A method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase-mediated cellular activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with eEFl A or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.

17. A method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with eEFl A or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said association down-regulates said sphingosine kinase functional activity.

18. The method according to claim 16 or 17 wherein said condition is a neoplastic condition or other unwanted cellular proliferation and said sphingosine kinase- eEFlA interaction is downregulated.

19. The method according to claim 16 or 17 wherein said condition is an inflammatory condition and said sphingosine kinase-eEFlA interaction is downregulated.

20. The method according to claim 16 wherein said unwanted cellular activity is inflammatory mediator secretion or adhesion molecule expression and said sphingosine kinase-eEFIA interaction is downregulated.

21. The method according to claim 19 wherein said inflammatory condition is associated with rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease or inflammatory bowel disease.

22. The method according to claim 16 or 17 wherein said modulation is upregulation of the level of sphingosine kinase catalytic activity and said upregulation is achieved by introducing into said cell a nucleic acid molecule encoding eEFlAl, eEFl A2 or tr.eEFlAl or functional equivalent, derivative or homologue thereof or the eEFl Al, eEFl A2 or tr.eEFlAl expression product or functional derivative, homologue, analogue, equivalent or mimetic thereof.

23. The method according to claim 22 wherein said eEFlAl or eEFlA2 is GDP bound or nucleotide free.

24. The method according to claim 16 or 17 wherein said modulation is upregulation of the level of sphingosine kinase activity and said upregulation is achieved by contacting said cell with a proteinaceous or non-proteinaceous molecule which functions as an agonist of the sphingosine kinase/eEFl A interaction.

25. The method according to any one of claims 16-21 wherein said modulation is downregulation of the level of sphingosine kinase activity and said downregulation is achieved by contacting said cell with a proteinaceous or non-proteinaceous molecule which functions as an antagonist of the sphingosine kinase/eEFl A interaction.

26. The method according to claim 25 wherein said antagonist is an eEFl A competitor.

27. The method according to claim 26 wherein said competitor is GTP bound eEFl A.

28. The method according to claim 25 wherein said antagonist is an antibody directed to eEFlA.

29. The method according to claim 28 wherein said antibody is directed to the eEFIA region defined by tr.eEFl A.

30. The method according to claim 25 wherein said antagonist is an antisense nucleic acid molecule, siRNA or nucleic acid molecule suitable to induce co-suppression, which molecules are directed to eEFIA.

31. The method according to any one of claims 1-30 wherein said mammal is a human.

32. Use of an agent in the manufacture of a medicament for the treatment of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, wherein said agent modulates the interaction of sphingosine kinase with eEFIA and wherein inducing or otherwise agonising said interaction up-regulates said cellular activity and inhibiting or otherwise antagonising said interaction down-regulates said cellular activity.

33. Use of an agent in the manufacture of a medicament for the treatment of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, wherein said agent modulates the interaction of sphingosine kinase with eEFIA and wherein inducing or otherwise agonising said interaction up-regulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said interaction downregulates said sphingosine kinase functional activity.

34. Use according to claim 32 or 33 wherein said condition is a neoplastic condition or other unwanted cellular proliferation and said sphingosine kinase-eEFl A interaction is downregulated.

35. Use according to claim 32 or 33 wherein said condition is an inflammatory condition and said sphingosine kinase-eEFIA interaction is downregulated.

36. Use according to claim 32 wherein said unwanted cellular activity is inflammatory mediator secretion or adhesion molecule expression and said sphingosine kinase- eEFIA interaction is downregulated.

37. Use according to claim 35 wherein said inflammatory condition is associated with rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease or inflammatory bowel disease.

38. Use according to claim 32 or 33 wherein said modulation is upregulation of the level of sphingosine kinase catalytic activity and said upregulation is achieved by introducing into said cell a nucleic acid molecule encoding eEFlAl, eEFlA2 or tr.eEFlAl or functional equivalent, derivative or homologue thereof or the eEFlAl, eEFlA2 or tr.eEFlAl expression product or functional derivative, homologue, analogue, equivalent or mimetic thereof.

39. Use according to claim 38 wherein said eEFl Al or eEFl A2 is GDP bound or nucleotide free.

40. Use according to claim 32 or 33 wherein said modulation is upregulation of the level of sphingosine kinase activity and said upregulation is achieved by contacting said cell with a proteinaceous or non-proteinaceous molecule which functions as an agonist of the sphingosine kinase/eEFl A interaction.

41. Use according to claim 32-37 wherein said modulation is downregulation of the level of sphingosine kinase activity and said downregulation is achieved by contacting said cell with a proteinaceous or non-proteinaceous molecule which functions as an antagonist of the sphingosine kinase/eEFl A interaction.

42. Use according to claim 41 wherein said antagonist is a eEFl A competitor.

43. Use according to claim 42 wherein said competitor is GTP bound eEFlA.

44. Use according to claim 41 wherein said antagonist is an antibody directed to eEFl A.

45. Use according to claim 44 wherein said antibody is directed to the eEFl A region defined by tr.eEFl A.

46. Use according to claim 41 wherein said antagonist is an antisense nucleic acid molecule, siRNA or nucleic acid molecule suitable to induce co-suppression, which molecules are directed to eEFl A.

47. Use according to any one of claims 32-46 wherein said mammal is a human.

48. A pharmaceutical composition comprising an agent which agent modulates the interaction of sphingosine kinase with eEFl A, together with one or more pharmaceutically acceptable carriers and/or diluents when used in accordance with the method of any one of claims 1-31.

49. An agent, which agent modulates the interaction of sphingosine kinase with eEFl A, when used in accordance with the method of any one of claims 1-31.