CA2302838C - Modulation of the sphingosine kinase signalling pathway - Google Patents

Abstract

The present invention relates to a sphingosine kinase signalling pathway via which cellular activities such as adhesion molecule expression are achieved. By regulating the expression and activity of individual components of this pathway, these cellular activities can be modulated. The present invention also contemplates a rapid, high volume assay for detecting agents exhibiting sphingosine kinase and agents that can act as agonists and antagonists of these sphingosine kinase activity.

Description

MODULATION OF THE SPHINGOSINE KINASE SIGNALLING PATHWAY
FIELD OF 'T.HE IlYVENTION

The present invention relates generally to a mechod of modulating cellular activity and agents useful for same. More partieularly, the prescnt invention contcmplates a merhod of modulating endothelial cell activity and even more particularly endothelial cell adhesion molecule expression. Most particularly, the present invention provides a method of treating coronary heart disease by preventing or rcducing cndothelial cell adhesion molecule expression.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected at the end ofthc description. Sequencc ldcntity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined immcdiatcly before the bibliography.

Atherosclerotic coronary heart disease is one of the major causes of death in the western worid (World Health Statistics Annual). An earlier s'vent in atherooenesis is the adhesion of monocytes to the endothelium via adhesion moleciales such as VCAM-1, ICAM-1 and E-selectin, all ofwhich are rapidly synthesised in responSe to cytokines. VCAM-1 is primarily involved in the adhesion of mononuclear leukocyces to the endothelium. It is rapidly induced by the inflammatory cytokines IL-1 and TNF-cc, and its induction is sustained for 48 to 72 hours.
ICAM-1 is expressed on many cells types and is involved in both monocyte and lymphocyte adhesion to activated endothelium. E-sclcotin is an cndothelial specific adhesion molecule important in capturing leukocytes from the axial stream to roll along the endothelium (Abbassi et ctl., 1993).

There is considerable evidence for the involvement of adhesion molecules in the development of early atherosclerotic lesions and in maturc atherosclerotic plaques (Van der Wal et al,, 1992).

Variable and low levels of E-selectin and VCAM-1 have been detected in the arterial endothelium over plaques (Van der Wal et al., 1992; Wood et al., 1993). VCAM-1 has also been observed in areas of neovascularization and in inflammatory infiltrates at the base of plaques, suggesting that intimal neovascularization may be an important site of inflammatory cell recruitment into advanced coronary lesions (O'Brien el al., 1993). ICAM-1 has been shown to be expressed on the endothelium overlaying atheromatous plaques (Johnson-Tidey et al., 1994).
The signals that lead tc- upregulation of cellular activities such as expression of adhesion molecules have not been defined. Elucidating these cellular signalling mechanisms is necessary for the development of therapeutic strategies to disease conditions in which said cellular activities are harmful such as coronary heart disease and inflammatory conditions.

In work leading up to the present invention, the inventors have identified a sphingosine kinase signalling pathway via which cellular activities such as adhesion molecule expression are achieved. By regulating the expression and activity of individual components of this pathway, these cellular activities can be niodulated. The inventors have also developed a rapid, high volume assay for detecting agents exhibiting sphingosine kinase activity and agents which can act as agonists and antaf;onists of sphingosine kinase activity.

SUMMARY OF THE INVEN'TION

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.

One aspect of the present invention relates to a method of modulating cellular activity in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway.

SUBSTiTUTE SHEET (RULE 26) -~-Another aspect of the present invention provides a method of modulating adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a tiime and tznder conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway.

Yet another aspect of the lpresent invention provides a method of modulating adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or both of sphingosine kinase and/or Sph-1-P.

Still another aspect of the present invention relates to a method of modulating endothelial cell adhesion molecule expression in a inammal said method comprising administering to said manunal an effective amount of an auent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway.

Still yet another aspect of the present invention provides a method of downregulating endothelial cell adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to downregulate the activity of one or both of sphingosine kinase and/or Sph-1-P.

A further aspect of the present invention relates to a method of modulating adhesion molecule expression in a mamma.l said method comprising administering to said mammal an effective amount of one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof.

Another further aspect of the present invention relates to a method of treatment or prophylaxis of a disease condition involving inflammatory mechanisms said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway wherein said modulation results in modulation of adhesion molecule expression.

SUBSTITUTE SHEET (RULE 26) Still another further aspect of the present invention provides a method of treating a mammal exhibiting coronary heart disease said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to downregulate the activity of one or more components of a sphingosine kinase signalling pathway wherein said downregulation results in downregulation of endothelial cell adhesion molecule expression.
Still yet another further aspect of the present invention provides a method of treatment or prophylaxis of a disease condition involving inflammatory mechanisms said method comprising administering an effective amount of one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof to said mammal.

In still yet another further aspect the present invention relates to the use of an agent capable of modulating the activity of one or more components of a sphingosine kinase signalling pathway in the manufacture of a medicament for the modulation of adhesion molecule expression in a mammal.

Another aspect of the present invention relates to the use of one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof in the manufacture of a medicament for the mociulation of adhesion molecule expression in a mammal.

Yet another aspect of the present invention relates to agents for use in modulating one or more components of a sphingosine kinase signalling pathway wherein modulating said components modulates adhesion mcilecule expression.

Yet another aspect of the present invention relates to one or more components of a sphingosine kinase signalling pathvray or functional equivalents thereof for use in modulating adhesion molecule expression in a mammal.

Still another aspect of the present invention relates to a pharmaceutical composition comprising an agent capable of modulating one or more components of a sphingosine kinase signalling pathway wherein said modulation results in modulation of adhesion molecule SUBSTITUTE SHEET (RULE 26) expression, together with one or more pharmaceutically acceptable carriers and/or diluents.
Still yet another further aspect of the present invention relates to a pharmaceutical composition comprising one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof together with one or more pharmaceutically acceptable carriers and/or diluents.

A further aspect of the present invention provides a method of detecting an analyte structurally or functionally reactive with a lipid, said method comprising the steps of contacting either:

(i) said analyte which analyte is radio labelled; or (ii) said anal;yte and a reporter molecule which reporter molecule is radio labelled, said analyte being contacted with said lipid simultaneously with or separately to said reporter molecule;

with said lipid in the presence of a scintillant for a time and under conditions sufficient for a lipid-radio label complex to form and to excite said scintillant, and detecting said excited scintillant.

Another further aspect of the present invention relates to a, method of detecting an analyte structurally or functionally reactive with sphingosine, said method comprising the steps of contacting either:

(i) said analyte which analyte is radio labelled; or (ii) said analyte and a reporter molecule which reporter molecule is radio labelled, said analyte being contacted with said sphingosine simultaneously with or separately to said reporter molecule;

SUBSTITUTE SHEET (RULE 26) with said lipid in the presence of a scintillant for a time and under conditions sufficient for a sphingosine-radio label complex to form and to excite said scintillant, and detecting said excited scintillant.

Still another further aspect of the present invention provides a method for detecting an analyte exhibiting sphingosine kinase activity, said method comprising the steps of contacting said analyte and 13P-ATP with sphingonsine in the presence of a scintillant, said analyte being contacted with said sphingosine simultaneously with or separately to said jjP-ATP, for a time and under conditions sufficient for jjP-sphingosine complex to form and to excite said scintillant, and detecting said excited scintillant.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation of the effect of high density lipoprotein (HDL) on tumour necrosis factor (TNF) mediated induction of adhesion proteins VCAM-1 and E-selectin. Mean fluorscence intensity is a flow cytometric measurement signifying the intensity of expression of adhesion molecules on the cell surface.

Figure 2 is a graphical representation of the effect of sphingosine kinase inhibitor on adhesion protein expresssion. Intensity of cell surface expression was measured as a percent of the mean fluorescence intensity stirnulated by 100U of TNF. Figure 2A filled symbols show the effect of the cell-permeable ceramide (C2-ceramide) on adhesion proteins. The open symbols show the effect of ceramide in the presence of IOOU/ml TNF. In Figure 2B the effect of sphingosine-1-phosphate (Sph-1-P) is shown. Figure 2C shows the effect dimethylsphingosine (DMS) on adhesion protein expression (MFI) stimulated by TNF or Sph-1-P. In Figure 2D
the effect of these agents on E-selectin mRNA is shown.

Figure 3 is a graphical representation of the time course of changes after TNF
treatment of HUVECs on key components of sphingomyelin pathway are shown. Open symbols in Figure 3A
show hydrolysis of sphingomyelin, in Figure 3B generation of ceramide, in Figure 3C sphingosine kinase activity and in Figure 3D formation of sphingosine-l-phosphate, all as percent of amount SUBSTITUTE SHEET (RULE 26) at time 0. Filled circles represent the effect of pre-treatment of HUVECs with HDL.

Figure 4 is a photographic representation of the effect of Sph-1-P, TNF and HDL on generation of ERK (Figure 4A) and NF-xB (Figure 4B). The bar graph provides a summary of three independent experiments on ERK activation ( 1 SEM). * show p value of difference from TNF
by <0.01 and t by <0.05, $ shows p <0.01 versus nil counted. Figure 4B top shows gel retardations with NF-xB. Figure 4B bottom shows supershifts with antibodies specific for p50 and p65 components of NF-KB and demonstrates the similar composition of NF-KB
stimulated by TNF and Sph-l-P.

Figure 5 is a schematic representation of the sphingosine kinase pathway by which HDL
inhibits TNFa-induced adhesiori protein expression.

Figure 6 is a schematic representation of the multi-well assay for sphingosine-kinase activity.
The basis of the assay is the phosphorylation of sphingosine bound by absorption to the walls of a multi-well plate in which the walls have been impregnated with scintillant (Flashplates, New England Nuclear). y-33P labelled ATP is incubated in the presence of the source of enzyme. If active enzyIne is present the 33P is transferred to the sphingosine. The wells are then washed to remove unbound "P. The "P bound to the sphingosine excites the scintillant and a signal is produced that is measured in a scintillation counter.

Figure 7 is a graphical representation showing that sphingosine kinase activity is linear for up to 400 minutes wheri measured in the multi-well assay. 50,ul of an endothelial cell extract (prepared as described above in example 8, containing sphingosine kinase activity) plus 50 l of an ATP solution containing 400 uM ATP/20,uCi/ml 33P-ATP was incubated with flashplates coated with phospholipid mixes either with (tilled squares) or without (open squares) sphingosine and incubated at 37 C for the indicated lengths of time before washing.
Figure 8 is a graphicail representation of a survey of tissues for sphingosine-kinase activity.
High speed "supernatant" (open bar) and "pellet" (filled bar) fractions from 50 ,u1 of extract were then assayed for sphingosine kinase activity.

SUBSTITUTE SHEET (RULE 26) DETAILED DESCItIPTION OF THE PREFERRED EMBODIMENTS

Accordingly, one aspect of the present invention relates to a method of modulating cellular activity in a rnammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway.

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

Reference to "modulatirig 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 such as, but not limited to, orie or more of chemokine production, cytokine production, nitric oxide synthesase production, adhesion molecule expression and production of other inflammatory modulators. Preferably said cellular activity is adhesion molecule expression.
Reference hereinafter to adhesion molecule expression should be read as including reference to other cellular activities.

Accordingly, there is provided a method of modulating adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway.

The term "expression" refers tc> the transcription and translation of a nucleic acid molecule resulting in the synthesis of a polypeptide.

Reference to "adhesiori molecule" should be understood as a reference to a molecule which mediates the binding of a cell to another cell or to a protein such as an extracellular matrix SUBSTITUTE SHEET (RULE 26) protein. Examples of adhesion molecules include, but are not lisnited to, integrins, selectins (e.g.
E-selectin, P-selectin), meinbers of the immunoglobulin-gene superfamily (e.g.
VCAM-1, ICAM-1) and CD44.

Reference to modulating the "activity" of sphingosine kinase signalling pathway components is a reference to modulating the functions which said components are capable of performing such as but not limited to inci-easing or decreasing the role or extent to which a given component performs its function or modifying the nature of the function which a given component performs.
Modulating said "activity" should also be understood to encompass increasing or decreasing the concentration levels of a given coinponent. Modulation of said "activity" may be achieved by:
(i) modulation of the catalytic activity of sphingosine kinase by competition with substrate (for example, sphingosine or ATP);

(ii) interference with the catalytic activity of sphingosine kinase by an allosteric mechanisms (binding to sites on the molecule other than the substrate-binding sites); or (iii) interfering with enzyme activation, such as by altering:

- post-translational covalent modification such as phosphorylation, lipid modification - non-covalent coupling to a required co-activator such as a protein, lipid or ion - subcellular localisation of the enzyme.

A "sphingosine kinase signalling pathway" is defined as a signalling pathway which utilises one or both of sphingosine k:inase and/or sphingosine-l-phosphate. Without limiting the present invention to any one theory or mode of action, it is thought a sphingosine kinase signalling pathway cascade which :results in adhesion molecule expression may take the form of:

(i) the generation of ceramide from sphingomyelin via S.Mase activity, said ceramide being converted to sphingosine;

SUBSTITUTE SHEET (RULE 26) (ii) sphingosine-l-phosphate (referred to hereinafter as "Sph-1-P") generation by stimulation of sphingosine kinase; and (iii) the activation of MEK/ERK and nuclear translocation of NF-KB
downstream froni Sph-1-P generation, said downstream events leading to adhesion molecule expression.

Accordingly, the term "components" should be understood to refer to any molecule which is or may be involved in a sphingosine kinase signalling pathway cascade and includes, but is not limited to, cellular proteins, metabolites (e.g. sphingomyelin, ceramide, sphingosine and Sph-1-P), kinases (e.g. S:Mase, sphingosine kinase, protein kinase C and ERK) and transcription factors (e.g. NF-xB). Preferably, said components are sphingosine kinase and/or Sph-l-P.
According to this preferred embodiment there is provided a method of modulating adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or both of sphine;osine kinase and/or Sph-1-P.

A key event in many disease conditions is the upregulation of cellular activities such as those which lead to inflammation. For example, upregulation of the production of inflammatory mediators such as cytokines, chemokines, eNOS and upregulation of adhesion molecule expression. Said upregulation may be induced by a number of stimuli including, for example, inflammatory cytokines such as tumour necrosis factor-a (TNF-a) and interleukin-1 (IL-1), endotoxin, oxidised or modified lipids, radiation or tissue injury. In a preferred aspect said cellular activity is endothelial cell activity.

Accordingly, the present invention relates to a method of modulating endothelial cell adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway.

SUBSTITUTE SHEET (RULE 26) Most preferably said components are sphingosine kinase and/or Sph-1-P.

While most of the components involved in a sphingosine kinase signalling pathway are stimulatory for a given cellular activity, inhibitory signalling components are also evident. For example, Sph-1-P is generated from sphingosine by sphingosine kinase.
Stimulation of endothelial cells causes a rapid and transient increase in cytosolic sphingosine kinase activity which leads to upregulation of adhesion molecule expression. The production of Sph-1-P is induced in parallel with sphingosine kinase activity. However, inhibitory components of a sphingosine kinase signalling pathway result in inhibition of adhesion molecule expression.
Accordingly, the term "niodulate" in relation to the activity of any one or more components of the sphingosine kinase sigrialling pathway refers to upregulating or downregulating or otherwise altering said activity. The preferred method is to downregulate sphingosine kinase pathway activity either by inhibiting or reducing the activity of one or more stimulatory components of a sphingosine kinase signalling pathway or by upregulating the activity of one or more inhibitory components of said pathway. However, niodulation of the activity of said components wherein adhesion molecule expression is upregulated may be desired under certain circumstances. Most preferably, the biological activity of said stimulatory components is down-regulated.
According to this most preferred embodiment, there is provided a method of downregulating endotheiial cell adhesion nlolecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to downregulate the activity of one or both of sphingosine kinase and/or Sph-1-P.

Modulation of said activity by the administration of an agent to a mammal can be achieved by one of several techniques, including but in no way limited to introducing into said mammal a proteinaceous or non-proteinaceous molecule which:

(i) modulates synthesis of said components;

(ii) functions as an antagonist to said component;
SUBSTITUTE SHEET (RULE 26) (iii) functions as an agonist to said component.

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 n:iay be chemically synthesised. The present invention contemplates chemical analogs of said components capable of acting as agonists or antagonists of said components. Chemical agonists may not necessarily be derived from said components but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic certain physiochemical properties of said components.
Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing said components from carrying out their nornaal biological functions. Antagonists include monoclonal antibodies specific for said components, or parts of said components, and antisense nucleic acids which prevent transcription or translation of genes or mRNA in mammalian cells.

The method of the present invention is exemplified herein utilising high density lipoprotein (referred to herein as "HDL"). Treatment of endothelial cells with HDL
inhibits both the amplitude and duration of TNF-a induced sphingosine kinase activation. HDL
treatment substantially blunts the amplitude and duration of Sph-1-P formation by inhibiting sphingosine kinase activity. The inhibition of Sph-1-P formation results in blunting of downstream pathway events including blunting of MEK/ERK activation and NF-KB nuclear translocation thereby reducing adhesion protein expression. Similarly, N,N-dimethyl sphingosine decreases TNF-a induced adhesion protein expression and mRNA levels by competitively inhibiting sphingosine kinase activity. Interruption of the sphingosine kinase signalling pathway at this point prevents the downstream formation of intracellular Sph-1-P thereby preventing the further downstream events of MEK/ERK aind NF-xB stimulation which lead to adhesion protein expression.

In a most preferred err.ibodiment the present invention relates to a method of downregulating endothelial cell adhesior.i molecule expression in a mammal said method comprising administering to said mammal an effective amount of HDL for a time and under conditions sufficient to downregulate sphingosine kinase activity.

SUBSTITUTE SHEET (RULE 26) An "effective amount" me;ans an amount necessary to at least partly obtain the desired response or to delay the onset or inhibit progression or halt altogether the onset or progression of adhesion molecule expression. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, 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.

Modulation of cellular adhesion molecule expression can also be achieved by the administration of one or more sphingosine kinase signalling pathway components or functional equivalents thereof.

Accordingly, another aspect of the present invention relates to a method of modulating adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of one or more components of a sphingosine kinase signalling pathway or functional equivalents th,ereof.

The term "functional equivalents ' used herein includes but is not limited to derivatives having the functional activity of said components.

Derivatives include fragments, parts, portions, chemical equivalents, mutants, homologs, mimetics from natural, synthetic or recombinant sources including fusion proteins.
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 t:he protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized 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 the sequence has been removed and a different residue inserted in its place. Additioris to amino acid sequences including fusions with other peptides, polypeptides or proteins.

SUBSTITUTE SHEET (RULE 26) The derivatives of said components include fragments having particular epitopes or parts of the entire component fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. For example, said components or derivative thereof may be fused to a molecule to facilitate its entry into a cell. Analogs of said components contemplated herein include, but are not limiteci 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 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 of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules.
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; carbannoylation of amino groups with cyanate;
trinitrobenzylation of amino groups with 2, 4, 6-tririitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride anci 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 0-acylisourea formation followed by subsequent derivitisation, 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 SUBSTITUTE SHEET (RULE 26) substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsu.lphonic 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 torm a 3-nitrotyrosine derivative.

Moditication 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-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, omithine. sarcosine, 4-aanino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list ot unnatural amino acid contemplated herein is shown in Table 1.

SUBSTITUTE SHEET (RULE 26) Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid Abu L-N-methylalanine Nmala a-amino-a-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 aminonorbornyi- 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 Dgln L-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine Dile 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-omithine 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 Nle SUBSTITUTE SHEET (RULE 26) D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr a-methyl-aminoisobutyrate Maib D-valine Dval a-methyl-y-aminobutyrate Mgabu D-a-methylalanine Dmala a-methylcyclohexylalanine Mchexa D-a-methylarginine Dmarg a-methylcylcopentylalanine Mcpen D-a-methylasparagine Dmasn a-methyl-a-napthylalanine Manap D-a-methylaspartate Dmasp a-methylpeniciliamine Mpen D-a-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-a-methylgiutamine Dmgln N-(2-aminoethyl)glycine Naeg D-a-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-a-methylisoleucine Dmile N-amino-a-methylbutyrate Nmaabu D-a-methylleucine Drnleu a-napthylalanine Anap D-a-methyllysine Dmlys N-benzylglycine Nphe D-a-methylmethionine Drnmet N-(2-carbamylethyl)giycine Ngln D-a-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn D-a-methylphenylalanine Dinphe N-(2-carboxyethyl)glycine Nglu D-a-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-a-methylserine Dmser N-cyclobutylglycine Ncbut D-a-methylthreonine Dmthr N-cycloheptylglycine Nchep D-a-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-a-methyltyrosine Dmty N-cyclodecylglycine Ncdec D-a-methylvaline Drnval 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-diphenylethyl)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-(1-hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser SUBSTITUTE SHEET (RULE 26) D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu N-methylcyclohexylalaiiine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobuty rate Nmaib D-N-methylproline Dnmpro N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen y-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-a-methylalanine Mala L-a-methylarginine Marg L-a-methylasparagine Masn L-a-methylaspartate Masp L-a-methyl-t-butylglycine Mtbug L-a-methylcysteine Mcys L-methylethylglycine Metg L-a-methylglutamine Mgln L-a-methylglutamate Mglu L-a-methvlhistidine Mhis L-a-methylhomophenylalanine Mhphe L-a-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-a-methylleucine Mleu L-a-methyllysine Mlys L-a-methylmethionine Mmet L-a-methylnorleucine Mnle L-a-methylnorvaline Mnva L-a-methylornithine Mom L-a-methylphenylalanine Mphe L-a-methylproline Mpro L-a-methylserine Mser L-a-methylthreonine Mthr L-a-methyltryptophan Mtrp L-a-methyltyrosine Mtyr L-a-methylvaline Mval L-N-methylhomophenylalanine Nmhphe SUBSTITUTE SHEET (RULE 26) N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1-carboxy-l-(2,2-diphenyl- Nmbc ethylamino)cyclopropaile 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=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain ari amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moietv such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition. peptides can be conformationally constrained by, for example, incotporation of C,j and Iy, -methylamino acids, introduction of double bonds between C,ti and C,, atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as torming an amide bond between the N and C termini, between two side chain[s or between a side chain and the N or C terminus.

The present invention is useful in r-elation to human disease conditions. For example the present invention is particularly useful, but in no way limited to, use as a prophylactic or as a therapy in relation to disease conditions which involve inflammatory mechanisms, such as coronary heart disease.

Accordingly, another aspect of' the present invention relates to a method of treatment or prophylaxis of a disease condition involving inflammatory mechanisms in a mammal said method comprising adnninistering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway wherein said modulation results in modulation of adhesion molecule expression.

Preferably modulation of said adhesion molecule expression is modulation of endothelial cell adhesion molecule expiression.

SUBSTITUTE SHEET (RULE 26) Even more preferably, said components are sphingosine kinase and/or Sph-1-P.
Most preferably said components are downregulated said downregulation resulting in downregulation of adhesion molecule expression.

In a most preferred embodiment there is provided the method of treating a mammal exhibiting coronary heart disease said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to downregulate the activity of one or more components of a sphingosine kinase signalling pathway wherein said downregulation results in downregulation of endothelial cell adhesion molecule expression.
Most preferably said components are sphingosine kinase and/or Sph-1-P.

Another aspect of the present invention provides a method of treatment or prophylaxis of a disease condition involving inflammatory mechanisms in a mammal said method comprising administering to said mammal an effective amount of one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof wherein said modulation results in modulation of adhesion molecule expression.

Administration of the agent or component or functional equivalent thereof, in the form of a pharmaceutical composition, may be performed by any convenient means. The agent or component or functional equivalent thereof 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 agent chosen. A brciad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of the agent or component or functional equivalent thereof 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 aulministered daily, weekly, monthly or at other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
The agent or component or functional equivalent thereof may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, SUBSTITUTE SHEET (RULE 26) intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules). With particular reference to use of the agent or component or functional equivalent thereof, said agent or component or functional equivalent thereof 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 foirm, the tablet may contain a binder such as tragacanth, com starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.

In a preferred embodiment of the present invention, the agent used in the method is linked to an antibody specific for coronary endothelial cells to enable specific delivery of said agent to these cells.

In yet another aspect the present invention relates to the use of an agent capable of modulating the activity of one or more components of a sphingosine kinase signalling pathway in the manufacture of a medicament for the modulation of adhesion molecule expression in a mamma].

Preferably modulation of said adhesion molecule expression is modulation of endothelial cell adhesion molecule expression.

Even more preferably, said components are sphingosine kinase and/or Sph-1-P.
Most preferably said components are downregulated said downregulation resulting in downregulation of adhesion molecule expression.

Another aspect of the present invention relates to the use of one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof in the manufacture of a medicament for the modulation of adhesion molecule expression in a mammal.

SUBSTITUTE SHEET (RULE 26) Yet another aspect of the present invention relates to agents for use in modulating one or more components of a sphingosine kinase signalling pathway wherein modulating said components modulates adhesion molecule expression.

Preferably modulation of'said adhesion molecule expression is modulation of endothelial cell adhesion molecule expression.

Even more preferably, said components are sphingosine kinase and/or Sph-1-P.
Most preferably said components are downregulated said downregulation resulting in downregulation of adhesion molecule expression. =

A further aspect of the p:resent invention relates to one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof for use in modulating adhesion molecule expression in a mammal.

Yet another further aspect of the present invention relates to a pharmaceutical composition comprising an agent capable of modulating one or more components of a sphingosine kinase signalling pathway wherein saici modulation results in modulation of adhesion molecule expression,together with one or inore pharmaceutically acceptable carriers and/or diluents.
In another further aspect, the present invention relates to a pharmaceutical composition comprising one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof together with one or more pharmaceutically acceptable carriers and/or diluents.

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. T'he carrier can be a solvent or dispersion medium containing, for SUBSTITUTE SHEET (RULE 26) 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 maintair,ied, 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 ancl 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. suigars 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 arid 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 ingreciients 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 percenitage 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 SUBSTITUTE SHEET (RULE 26) 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 g 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 com 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 tlavouring. 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 t7avouring 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 capable of modulating the expression of a component of the sphingosine kinase signalling pathway.
The vector may, for example, be a viral vector.

To facilitate the identification ot' agents suitable for use in the method of present invention, the inventors have developed a rapid, facile, high volume assay for screening for agents which are either structurally or functionally reactive with a lipid. In the context of identifying agents for use in the method of the present invention, this assay provides a means of easily and rapidly identifying agents which either mimic the activity of a component of SUBSTITUTE SHEET (RULE 26) sphingosine kinase signalling pathway or act as an agonist or antagonist to a component of sphingosine kinase sigrialling pathway.

Accordingly, another aspect of' the present invention provides a method of detecting an analyte structurally or functionally reactive with a lipid, said method comprising the steps of contacting either:

(i) said analyte, which analyte is radio labelled; or (ii) said analyte anci a reporter molecule which reporter molecule is radio labelled, said analyte being contacted simultaneously with or separately to said reporter molecule;

with said lipid in the presence of a scintillant for a time and under conditions sufficient for a lipid-radio label complex to form and to excite said scintillant, and detecting said excited scintillant.

Preferably said analyite or analyte and reporter molecule are contacted with lipid and scintillant coupled to a solid support.

Reference to "lipid" should be understood as a reference to any molecule comprising a lipid component. Preferablv said lipid is a lipid component of the sphingosine kinase signalling pathway and even more preferably sphingosine. This aspect of the present invention is exemplified herein with respect to sphingosine, however this should not be understood as imposing any limitaticin on the application of this method.

Accordingly, the present invention more particularly provides a method of detecting an analyte structurally or functionally reactive with sphingosine, said method comprising the steps of contacting either:

SUBSTITUTE SHEET (RULE 26) (i) said anal;yte which analyte is radio labelled; or (ii) said analyte and a reporter molecule which reporter molecule is radio labelled, said analyte being contacted simultaneously with or separately to said reporter molecule;

with said sphingosine in the presence of a scintillant for a time and under conditions sufficient for a sphingosine-radio label complex to form and to excite said scintillant, and detecting said excited scintillant.

Preferably, said analyte or analyte and reporter molecule are contacted with sphingosine and scintillant which sphingosine may be coupled to a solid support having a scintillant already coupled thereto using a method as described in Example 9.

Reference to an analyte: which is "structurally" reactive with a lipid should be understood as a reference to a molecule which either binds, links or otherwise associates with said lipid.
Said association may be due to the formation of a peptide bond, ionic bond, hydrogen bond or other interactive bonding mechanisms. Reference to an analyte which is "functionally"
reactive with a lipid should be understood as a molecule which, when it interacts with said lipid, directly or indirectly causes a functional process to take place. A
functionally reactive analyte may or may not also structurally interact with said lipid. An example of a functionally reactive analyte is an enzyme (for example, a sphingosine kinase equivalent) the substrate of which is a lipid (for example, sphingosine). In accordance with this example, an enzyme exhibiting sphingosine kinase activity is functionally reactive in that it will phosphorylate sphingosine in the presence of ATP. The occurrence of the structural or functional activity is detected by the method of this aspect of the present invention.

The detection of functional or structural reactivity is based on the formation, by the lipid, of a radio labelled cor.nplex. I)ue to the proximity of the complexed radio label with the scintillant, the scintillant becomes excited.

SUBSTITUTE SHEET (RULE 26) The analyte which is being assayed may be directly radio labelled. This is useful where, for example, said analyte directly associates with said lipids. Alternatively, a reporter molecule may be radio labelled. Fteference to "reporter molecule" should be understood as a reference to any molecule, other than the analyte, which detects reactivity between the analyte and lipid. For example, the reporter molecule may be an antibody which recognises one or more epitopes present on the surface of the analyte of interest. This method of detection is an indirect method of detection, commonly referred to as a "sandwich assay".
Alternatively, the reporter molecule niay be a molecule required to both facilitate reactivity between the analyte and the lipid, for example the reporter molecule may be a substrate, in addition to facilitating detection. In the method exemplified herein, the reporter molecule is 33P-ATP.
An analyte exhibiting sphingosine kinase activity will phosphorylate sphingosine in the presence of 33P-ATP resulting in the coupling of 33P to the sphingosine. In the absence of a phosphate reporter rnolecule. the analyte would be unable to phosphorylate the lipid.
Further, since the analyte does not structurally interact with the lipid sufficiently to facilitate detection, the method of detection of the functional reactivity is based on the phosphorylation of the lipid with radio labelled phosphate.

It should be understood, that even where the analyte is radio labelled and said analyte binds to the lipid, it may nevertheless be necessary that a molecule other than the analyte is present in the reaction mixture to facilitate reactivity of the analyte with the lipid.

Solid supports suitable tor use in the present invention include, but are not limited to, micro-titre plates, 96 well plates, columns and micro-beads.

Preferably, the present invention provides a method for detecting an analyte exhibiting sphingosine kinase activity, said method comprising the steps of contacting said analyte and 33P-ATP with sphingosine in the presence of a scintillant, said analyte being contacted with said sphingosine simultaneously with or separately to said 33P-ATP, for a time and under conditions sufficient fo:r 33P-sphingosine complex to form and to excite said scintillant, and detecting said excited scintillant.

SUBSTiTUTE SHEET (RULE 26) Preferably said analyte ,and 33P-ATP are contacted with sphingosine and scintillant coupled to a solid support.

Detection of the excited scintillant may be by any suitable means including, but not limited to, a scintillation counter.

The analyte which is assayed in the method of this aspect of the present invention may be in any suitable form. A tluid form is particularly useful as are tissue extracts such as lysates or homogenates. Said analyte may be a proteinaceous or a non-proteinaceous molecule derived from natural, recombinant or synthetic sources.

In another aspect, the method of the present invention should be understood to extend to the detection of agents which act as agonists or antagonists of analytes which either structurally or functi.onally react wiith a lipid. For example, the present invention is particularly useful for detecting agonists oi- antagonists of sphingosine kinase activity. This may be achieved, in accordance with this embodiment of the present invention, by contacting the sphingosine with sphingosine kinase in the presence of the potential agonistic or antagonistic agent.
Comparison of the degree of sphingosine-radio label complex formation relative to a control assay in which addition of said agent was omitted is indicative of agonistic or antagonistic agent activity. Specifically, an increase in sphingosine 33P complex formation relative to the control is indicative of agonistic activity while a decrease in sphingosine 33P complex activity is indicative of antagonistic activity. This aspect of the present invention should not in any way be taken as limited to the embodiment described above. Rather this aspect of the present invention can be applied to detect antagonists or agonists of any analyte which either structurally or functioiially react with a lipid.

Agents suitable for testing in the method of the present invention include, but are not limited to compounds isolated from chemical libraries or broths produced from fermentation of an organism.

SUBSTITUTE SHEET (RULE 26) With respect to the preferred embodiment detailed above, sources of sphingosine kinase suitable for use in the n;iethod o-P this aspect of the present invention include, but are not limited to: (i) partially purified splungosine kinase derived from mammalian tissue, (ii) crude homogenates of mammalian tissue exhibiting enzyme activity, (iii) recombinant enzyme or (iv) HUVEC crude lysalte.

Further features of the present invention are more fully described in the following examples.
It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention. It should not be understood in any way as a restriction on the broad description of the invention as set out above.

SUBSTITUTE SHEET (RULE 26) HDL isolation HDL were isolated from normal healthy adult donors by sequential ultracentrifugation in the density range of 1.07-1.21 g/rnl. Resulting preparation of HDL contained two main populations: one with particles of Stokes' diameter 10.45 nm (HDL,) and one with particles of diameter 8.6 nm (HDL3). Since the inhibition of adhesion molecules expression of HDL3 was stronger than that induced by other native or reconstituted HDL particles from purified Apo-A-1 and egg phosp;hatidylcholin or sphingomyelin, HDL3 was thus used in this study.
In the examples we have used the human HDL, subfraction (d =1.13 -1.21 g/ml) in order to minimise possible confounding effects of variations in the relative proportions of HDL2 and HDL3 in the plasrna of different subjects. As shown in Figure 1 HDIj inhibited the TNFa-induced expression of VCAM-1 and E-selectin by human umbilical vein endothelial cells (HUVECs) by > 80%.

Sphingosine kinase signalling pathway methodology Effect of HDL on TNFce-induced adhesion protein expression by HUVECs Confluent monolayers olF HUVECs were preincubated with or without HDL that was isolated as the fraction of density, 1.13-1.21 g/ml from human plasma at a concentration of 1 mg/ml of apoA-1. After 16-h preincubation, the cells were treated with TNFa (100 U/ml).
Following 4-h treatmer-t of TNFa, the cell-surface expression of VCAM-1 and E-selectin was measured by using flow cytometry.

Effect of ceramide and Sph-1-P on adhesion protein expression HUVECs were treated with an increasing concentration of CZ-ceramide with or without TNF
a(100 U/ml), or an increasing concentration of Sph-1-P for 4 h. The cell-surface expression SUBSTITUTE SHEET (RULE 26) of VCAM-1 or E-selectin was nieasured by tlow cytometry. Effect of sphingosine kinase inhibitor (DMS) on the e:xpression of VCAM-1 and E-selectin. The cells were treated with a vehicle (Nil), DMS (5 M), Sph-1-P (5 M), C,-ceramide (10 M) or TNFa (100 U/mi) for 4 h, then measure VCAM-1 or E-selectin expression. Effect of agents on E-selectin mRNA levels and the comparison between the effect of HDL and DMS. After the indicated treatment for 4 h, E-selectin rriRNA levels were measured by Northern blotting assay.
Results shown in (D) are representative of three similar experiments.

Effect of HDL on TNFa-induced sphingonrvelin hydrolysis, cerantide generation, sphingosine kinase activation, and :iph-1-P production HUVECs were labelled with [3H]sphingomyelin was measured at the desired time point of TNFa treatment. The unlabelled cells were treated with HDL and/or TNFa as indicated, cells were lysed to measure ceramide levels and sphingosine kinase activity, respectively.
The cells were permeabilized to measure the production of Sph-1-P in vivo.

Effect of HDL and sphirigosine kinase pathway on ERK and NF-KB activation After the cells were treated with an agent for 30 min, ERK activities were assayed with myeline basic protein (MBP) as substrate after immunoprecipitation with antibodies against p42/p44M^PK (Berra et al., 1995; Li et al., 1996; Lee et al., 1997). The kinase reaction products were separated on 10% SDS-polyacrylamide gels. Bar graph depicting ERK
activities were quantified by Phosphoimager. NF-xB binding activity was measured by electrophoretic mobility shift assay after 30 min treatment as indicated. The specific NF-xB
binding complexes were identified by the super-shift gel assay with anti-p50 and anti-p65 antibodies and by conipetition analyses with the addition of a 50-fold molar excess of unlabelled NF-xB oligonucleotides.

SUBSTITUTE SHEET (RULE 26) Sphingosine kinase signalling pathway To determine the role of sphingolipid metabolites in adhesion protein expression, either a cell-permeable form of ceramide (C,-ceramide) or sphingomyelinase that generates endogenous ceramide, oi- Sph-1-P were added to endothelial cells. C:-ceramide (Figure 2A) or sphingomyelinase were poor stimulators of E-selectin and VCAM-1 expression, reaching levels that were less than 10% of that stimulated by TNF-a. By contrast Sph-1-P was a potent and dose-dependent inducer of E-selectin and VCAM-1, reaching levels at 5/cM that were approximately equivalent to 100U/ml TNF-a (Figure 2B). The potency of Sph-was further shown by its induction of E-selectin mRNA (Figure 2D). To examine the role of Sph-l-P was inhibited by the competitive inhibitor of sphingosine kinase, N,N-dimethyisphingosine (DMS). DMS decreased the TNF-a-induced adhesion protein expression and mRNA levels by between 50 and 70% (Figures 2C and D). This demonstrates that sphir-gose kinase activation is an important event in the TNF-a action.
As a control, the same concentration of DMS inhibited sphingosine kinase activity and Sph-1-P formation by > 90'Yo induced either by TNF-a or PMA in endothelial cells, PMA has been shown to activate sphingosine kinase via protein kinase C activation (7).
In contrast to its effect on TNF-a DMS dici not prevent Sph-1-P induced adhesion protein expression (Figure 2C), indicating a specific effect of DMS on the generation of Sph-l-P.
These results show a new signallirig pathway for TNF-a-induced adhesion molecule expression.
Exogenous cell-permeable cerarnide was a potent inhibitor (by > 60%) of TNF-a induced adhesion protein expression (Figure 2A).

HLIL inhibition of sphingosine kinase signalling Figure 3A shows that TNFa stimulation of HUVECs rapidly reduced cellular sphingomyelin content to 40% of control within 30 min with return to near basal levels by 2 h. In parallel, the cellular ceramide levels were rapidly increased (approximately 2 fold) peaking at 30 min SUBSTITUTE SHEET (RULE 26) after TNFa treatment (Figure 3B). Treatment of endothelial cells with HDL had a potent effect: it delayed the reversion of post TNFa sphingomyelin levels to base line and sustained the increased ceramide levels after TNFa stimulation.

TNFa stimulation of HUVECs caused a rapid and transient increase in cytosolic sphingosine kinase activity, reaching a maximum of 165 13 % (p < 0.01) of basal within 5 min. HDL
pretreatment profoundly inhibited both the amplitude and duration of TNFa-induced sphingosine kinase activation (Figure 3C). The production of Sph-1-P was induced in parallel with the sphingosine kinase activity. HDL treatment again substantially blunted the amplitude and duration of Sph-1-P formation (Figure 3D).

EXA.MPLE 5 HDL inhibition of ERK cascade Figure 4A shows that both TNFa and Sph-1-P were approximately equipotent in stimulating ERK activities, whereas C2-ceramide did not. Treatment with DMS inhibited TNFa-activated ERK by 50% (p <0.02), showing a role for sphingosine kinase in the TNFa-activated ERK signal cascade. Preincubation of HUVECs with HDL also reduced TNFa-stimulated ERK activation, consistent with its effect on reducing cellular levels of Sph-1-P
(Figure 4A, bar 4).

HDL inhibition of NF-xB activation To measure the NF-iicB binding activity, electrophoretic mobility-shift assays were performed. Nuclear extracts were prepared from HUVECs treated for 30 min with vehicle or the indicated agents. The double-stranded oligonucleotides used as a probe in these experiements included 5'-GGATGCCATTGGGGATTTCCTCTTTACTGGATGT-3' (SEQ
ID NO: 1)which contains a consensus NF-KB binding site in E-selection promoter that is underlined. Gel mobility shift of a consensus NF-KB oligonucleotide was performed by SUBSTITUTE SHEET (RULE 26) incubating a;2P-labelled NF-xB probe with 4mg of nuclear proteins. The Sph-1-P
induced formation of NF-KB specific complexes were similar us that induced by TNFa.
These specific DNA-protein cornplexes were completely abolished by addition of a 50-fold molar excess of unlabeled E-selectin NF-KB oligonucleotides. The specificity of NF-KB binding complex was further identified by the super-shift analyses. Anti-p50 and Anti-p65 polyclonal antibodies (purchased from Santa Cruz Biotechnology, CA) were added prior to addition of radiolabeled NF-KB probe. The same phenotype of gel retardation was shown in the gel shift assay after the the preincubation of nuclear extracts from both Sph-1-P and TNFa-treated cells with the antibodies. Electrophoretic mobility shift assay shows that treatment of HUVECs with Sph-1-P induced a significant nuclear NF-KB
accumulation (Figure 4B). The composition of Sph-1-P induced NF-KB specific protein-DNA
complexes were identical to that induced by TNFa which was revealed to be p50/p65 heterodimer by antibody supershift assay and by competition analyses (Figure 4B, the bottom).
Treatment of cells with HDL or DMS markedly inhibited the TNFa-induced activation of NF-KB by 45-60%, but did not inhibit that induced by Sph-1-P.

RNA preparation Total RNA was prepared from HUVECs that was treated with the indicated agents for 4h.
Equal aliquots of total RNA (12,ug) were electrophoresed in a 1 % formaldehyde gel and transferred to nylon membrane. The blots were hybridized with a32P-labeled E-selection cDNA probe. The m:ltNA levels of E-selectin were quantified by Phosphoimager and normalised to radiolabeled GAPDH probe.

Spingomyelin measurement To measure sphingomyelin, the HUVECs were labeled with [3H]serine (5,uCi/ml) for 48h and preincubated with or without HDL for another 6h. The cells were then washed three SUBSTITUTE SHEET (RULE 26) times and incubated for additional 2h in the presence or absence of HDL. After the treatment with TNFa for the indicated times, cellular lipids were extracted and resolved by thin-laver chromatography (TLC) with chloroform:methanol:acetic acid:water (50:30:8:5, v/v). Sphingomyelin spots were visualized by t7uorography, quantitated by scintillation spectrometry, and norrnalized by radioactivity recovered in total cellular lipids. In the uptake of [jH]serine there are no significant differences between the cells that were preincubated with HDL and with HDL. To measure ceramide levels, cellular ceramide was extracted and quantified with the diacylglycerol kinase reaction (Kolesnick, 1991; Hannun & Bell, 1993). Sphingosine kinase activity was measured in vitro as previously described with some modification i;Mattie et al.. 1994; Ghosh et al., 1994; Choi et al., 1996). Cells were lysed by passing through 26'/2 G syringe 6 times in 0.1 M HEPES buffer (pH 7.2) containing 10mM MgCI,, 20% glycerol. 1mM mecaptoethanol. 1mM EDTA, 20,uM
ZnC121 1mM Na3 V04, 15mM NaF, 10,ug/ml leupeptin and aprotinin, 1mM PMSF and 0.5mM 4-deoxypyridoxine, Cytosolic fractions were prepared by ultracentrifugation at 105,000xg for 90min. Sphingosine kiriase activity was measured by incubating the supernatant with 20mM
sphingosine-BSA complex and j y;`P]ATP (1mM, 5,uCi/ml) for 15min at 37 C.
Labeled lipids were extracted and separated by TLC along with Sph-1-P standard.
Radioactivity of the spot corresponding to Sph-1-P was quantified using Phosphoimager system.
The measurement of Sph-1-1' formation in vivo was performed as previously described (Olivier, 1996). The cells were permeubilized for 15min at 37 C in hypotonic buffer (25mM
HEPES. 5mM MgCI2, 20pM ZnCI,, 20,uM Na3VO4, l0,ug/mi leupeptin and aprotinin, ImM
PMSF and 0.5mM 4-deoxypyridoxine, pH 7.2) with sphingosine (10,uM) and [y32P]ATP
(1,uM, IO,uCi/ml). Lipids were extracted and resolved by TLC, and [y32P]Sph-1-P was quantified as described above.

A multi-well assay for sphingosine-kinase activity.

Flashplates were coated with lipid substrate by adding 200 u1 of a mix of phosphatidylcholine (:19.5 ug/ml), phosphatidylserine (5.5 ,ug/ml), and sphingosine (25 SUBSTITUTE SHEET (RULE 26) ug/mi) in methanol and evaporating at 30 C under N, for 3 hours. A source of sphingosine kinase is added in a buffer consisting of 50mM HEPES pH 7.2/20% glycerol/lOmM
MgCI2/lmM DTT/20/cM ZnCI2/1mM Na3VO4/15mM NaF/0.5mM 4-deoxypyridoxine along with [33P]-y-ATP (Bresatech, 1,uCi/well/200 M) and incubated for up to 2 hours at 37 C.
The plates are then washed with 400 u1 of 50mM sodium pyrophosphate, 4 times and radioactivity was measured in a scintillation counter (Top count, Packard).

Preparation of honiogenates of tissues for survey of sphingosine kinase activity Tissues were collected fi-om freshly sacrificed animals and rapidly frozen in liquid nitrogen, 2-5 grams of wet tissue were homogenized with a Polytron tissue homogenizer in 2 volumes of 50mM Tris pH 7.4/20Rb glycerol/1mM DDT/lmM EDTA, containing a protease inhibitor cocktail (Boehringer-Mannheim). The material was subjected to centrifugation at 7500g for 30 minutes to remove large fragments. The resulting supernants were further centrifuged at high speed (100,000 X g for 1 hour) to generate "supernatant"
and "pellet"
fractions.

The multi-well assay is linear over time and specific.

Figure 7 illustrates the results of assaying for sphingosine kinase activity from a lysate made from human umbilical endothelial cells. It can be seen that the signal increases linearly over a period of approximately 400 minutes. This signal is specific to sphingosine kinase since omitting sphingosine from the lipid mix used to coat the plates reduces the resulting signal to background levels. This demonstrates that it is sphingosine, and not some other component of the lipid or assay mix, that is being phosphorylated in the assay. This is remarkable since this assay was done with a crude lysate which would be expected to contain a number of lipid and protein kinases.

SUBSTITUTE SHEET (RULE 26) Sphingosine-kinase agonist and antagonist activity measurement Plates are prepared as described in Example 9. Sphingosine kinase (prepared in buffer together with 33P-ATP as described in Example 9) is added in the presence of an agent to be tested for antagonistic or agonistic activity. The agent is added to a level of enzyme activity yielding a linear response with respect to time from 0 - 3 hours of incubation. Plates are incubated for two hours at 37 C, washed as in Example 9 and phosphorylation of sphingosine is determined by scintillation counting.

Inhibition (antagonstic activity) is detected as diminshed incorporation of 33P and stimulation (agonistic activity) is detected as increased incorporation of 'jP relative to a control incubation containing an equivalent amount of the vehicle solvent used to deliver the agents tested.

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 inclucles all such variations and modifications which fall within its spirit and scope. 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.

SUBSTITUTE SHEET (RULE 26) BIBLIOGRAPHY

Abbassi 0, Kishimoto T. K., McIntire I. V., Anderson D. C., Smith C. W., J.
Clin. Invest.
92:2719-2730 (1993) Berra E. et al., EMBO J. 14:6157 (1995) Choi O. H., Kim J. H. , Kinet J. P., Nature 380:634 (1996) Ghosh T. K. , Bian J.. Gill D. L. , J. Biol. Chem. 269:22628 (1994) Hannun Y. A. , Bell R.1Vi. , Adv. Lipid Res. 25:27 (1993) Johnson-Tidey R. R., MfcGregor J. L., Taylor P. R., Poston R. N., Am. J.
Pathol. 144:952-961 (1994) Kolesnick R.N., Prog. Lipid Res. 30:1 (1991) Lee F.S., Hagler J., C'hen Z.J., Maniatis T., Cell 88:213 (1997) Li Y.S. et al., Mol. Cell. Biol. 16:5947 (1996) Mattie M., Brooker G., Spiegel S., J. Biol. Chem. 269:3181 (1994) O'Brien K.D. et al., J. Clin. lnvest. 92:945 (1993) O'Brien K.D., Allen M.D., McDonald T.O., Chait A., Harlan J.M., Fishbein D., McCarty J., Furgerson M., Hudkins K.. , Benjamin C. D. , Lobb R., Alpers C. R. , J.
Clin. Invest.
92:945-951 (1993) SUBSTITUTE SHEET (RULE 26) Olivier C. et al., Nature 381:800 (1996) Van der Wal A.C., Das P.K., Tigges A.J., Becker A.R., Am. J. Pathol. 141:161-(1992) Wood K. M., Cadogan M. D. , Ranshaw A. I. , Parums D. V. , Histopathology 22:437-444 (1993) World Health Organizaition. World Health Statistics Annual. Geneva, Switzerland (1993) SUBSTITUTE SHEET (RULE 26) SEQUENCE LISTING

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Claims (12)

What is claimed is:

1. A method of screening a compound for the ability to modulate adhesion molecule expression, the method comprising assessing the ability of the compound to modulate the sphingosine kinase signalling pathway, wherein the method is selected from the group consisting of assessing the ability of the compound to disrupt the sphingosine kinase signalling pathway, assessing the ability of the compound to modulate the activity of sphingosine kinase, assessing the ability of the compound to modulate the activity of sphingosine-1-phosphate, assessing the ability of the compound to inhibit the activity of sphingosine kinase, assessing the ability of the compound to inhibit the activity of sphingosine-1-phosphate, and comparing the activity of Sph-1-P in the presence of the compound to the activity of sphingosine-1-phosphate in the absence of the compound.

2. A method as claimed in claim 1 in which the method comprises assessing the ability of the compound to inhibit adhesion molecule expression comprising assessing the ability of the compound to disrupt the sphingosine kinase signalling pathway.

3. A method as claimed in claim 1 in which the method comprises assessing the ability of the compound to modulate the activity of sphingosine kinase.

4. A method as claimed in claim 1 in which the method comprises assessing the ability of the compound to modulate the activity of sphingosine-1-phosphate.

5. A method as claimed in claim 1 in which the method comprises assessing the ability of the compound to inhibit the activity of sphingosine kinase.

6. A method as claimed in claim 1 in which the method comprises assessing the ability of the compound to inhibit the activity of sphingosine-1-phosphate.

7. A method of screening a compound for the ability to modulate adhesion molecule expression, the method comprising comparing the activity of sphingosine kinase in the presence of the compound to the activity of sphingosine kinase in the absence of the compound., wherein the method comprises the following steps:

(i) providing a solid support to which sphingosine is bound;

(ii) contacting the solid support with sphingosine kinase and 33P-ATP
in the presence or absence of the compound; and (iii) comparing the amount of bound33P produced in the presence of the compound with that produced in the absence of the compound.

8. The method as claimed in claim 1 in which the method comprises comparing the activity of Sph-1-P in the presence of the compound to the activity of sphingosine-1-phosphate in the absence of the compound.

9. A method for detecting sphingosine kinase activity in an analyte, comprising:

(a) bringing said analyte and 33P-ATP into contact with a sphingosine, in the presence of a scintillant, for a period of time and under conditions that are sufficient for and 33P-sphingosine complex to form and for said scintillant to be excited, wherein said analyte being contacted with said sphingosine simultaneously with or separately to said and 33P-ATP, (b) detecting the amount of said excited scintillant from step (a), and then (c) determining sphingosine kinase activity in said analyte, based on the amount of said excited scintillant.

10. A method according to claim 9 wherein said sphingosine and said scintillant are coupled to a solid support.

11. A method for detecting agonist or antagonist activity for sphingosine kinase in an analyte, comprising:

(a) bringing the sphingosine kinase and 33P-ATP into contact with sphingosine in the presence of a scintillant, and in the presence and absence of said analyte, respectively, for a period of time and under conditions that are sufficient for 33P-sphingosine complex to form and for said scintillant to be excited, wherein said sphingosine kinase being contacted with said sphingosine simultaneously with or separately to said and 33P-ATP

(b) detecting and comparing the amount of said excited scintillant in the presence and in the absence of said analyte, respectively, and then, (c) determining the agonist or antagonist activity of said analyte, based on increase or decreased amount of said excited scintillant.

12. A method according to claim 11 wherein said sphingosine and said scintillant are coupled to a solid support.