JP2005526809A - Methods for modulating cellular activity - Google Patents

Abstract

The present invention relates generally to methods for modulating cellular activity and agents for use therein. More particularly, the present invention provides methods for modulating cellular activity by phosphorylating sphingosine kinase and thereby modulating its activity. In a related aspect, the present invention provides a method for modulating sphingosine kinase functional activity through its phosphorylation and modulation of agents for use therein. The invention further extends to sphingosine kinase variants and functional derivatives, homologs or analogs, chemical equivalents and the like that exhibit the ability to perform reduced and / or eliminated phosphorylation. The methods and molecules of the present invention are useful, inter alia, in the treatment and / or prevention of conditions characterized by abnormal and undesirable or otherwise inappropriate cellular and / or sphingosine kinase functional activity. The invention further relates to methods for identifying and / or designing agents that can modulate sphingosine kinase phosphorylation.

Description

The present invention relates generally to methods for modulating cellular activity and agents for use therein. More particularly, the present invention provides methods for modulating cellular activity by phosphorylating sphingosine kinase and thereby modulating its activity. In a related aspect, the present invention provides a method for modulating sphingosine kinase functional activity through its phosphorylation and modulation of agents for use therein. The present invention still further extends to sphingosine kinase variants and functional derivatives, homologs and analogs that exhibit the ability to perform reduced and / or eliminated phosphorylation. The methods and molecules of the present invention are particularly useful in the treatment and / or prevention of conditions characterized by abnormal and undesirable or otherwise inappropriate functional activity of cells and / or sphingosine kinase. The invention further relates to methods for identifying and / or designing agents that can modulate sphingosine kinase phosphorylation.

BACKGROUND OF THE INVENTION Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

  Reference to any prior art herein is not, and should not be taken as, a finding or any suggestion that the prior art forms part of common general knowledge in Australia.

  Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a wide variety of mammalian cell processes (Pyne et al., 2000; Spiegel 1999). S1P is mitogenic in a variety of cell types, with a diverse range of important regulatory pathways (intracellular calcium mobilization via inositol triphosphate independent pathway (Mattie et al., 1994), phospholipase D activation (Desai et al., 1992), c-Jun N-terminal kinase (JNK) inhibition (Cuvilliver et al., 1998), caspase inhibition (Cuvilliver et al., 1998), adhesion molecule expression (Xia et al., 1998), and DNA binding activity of NF-κB ( Xia et al., 2002) and transcription factor activated protein-1 (AP-1) (including stimulation of Su et al., 1994).

  In addition to its role in cell growth and survival, S1P appears to have other functions in the cell. For example, recent studies have shown that S1P is an essential signaling intermediate in vascular endothelial cell adhesion molecule expression (Xia et al., 1998), which has a role in inflammation and atherosclerosis This suggests the possibility of

  Cell-level S1P is mediated largely by the activity of sphingosine kinase, and to a lesser extent by its degradation by S1P lyase activity (Van Veldhoven et al., 2000) and S1P phosphatase activity (Mandala, 2000). Base levels of S1P in cells are generally low (Spiegel et al., 1998), but can increase rapidly and transiently when cells are exposed to various mitogenic agents. This response is a direct result of increased sphingosine kinase activity in the cytosol and can be hampered by the addition of sphingosine kinase inhibitors. This replaced sphingosine kinase kinase, and in a central and essential role in mediating the observed effects, its activation was attributed to S1P in the cell. However, at present, little is known about the mechanisms that lead to sphingosine kinase activation.

  Sphingosine kinase can be activated very rapidly by a wide variety of cellular agonists. Responses vary between cell types, but these stimuli include: TNFα (Xia et al., 1998; Pitson et al., 2000) (FIG. 1), platelet derived growth factor (Olivera et al., 1993), epithelial proliferation Factor (Meyer zu Heringdorf et al., 1999), nerve growth factor (Rius et al., 1997), vitamin D3 (Kleuser et al., 1998), phorbol ester (Pitson et al., 2000; Buehler et al., 1996), acetylcholine (muscarinic receptor) (Meyer zu Heringdorf et al., 1998) and cross-linking of immunoglobulin receptors FcεR1 (Choi et al., 1996) and FcγR1 (Melendez et al., 1998). In all cases, this sphingosine kinase activation increases the Vmax of the reaction, while the substrate affinity (Km) remains unchanged.

  The molecular mechanisms that link these agonists to the activation of sphingosine kinase activity remain largely unknown. Thus, there is a need to elucidate these mechanisms and develop methods to regulate cellular activity via regulation of the sphingosine kinase signaling pathway.

  In studies led to the present invention, the inventors have determined that phosphorylation of sphingosine kinase is essential for its activation. In addition, the inventors have identified phosphorylation sites on the sphingosine kinase molecule. Furthermore, it was determined that phosphorylation of sphingosine kinase is carried out by proline-directed protein kinase, more specifically ERK2.

SUMMARY OF THE INVENTION Throughout the specification and claims, unless otherwise indicated in context, the term “comprise” and variations thereof (eg, “comprises” and “comprising”) It is understood that it is meant to include the integers or stages or groups of integers or stages mentioned, but not to exclude any other integers or stages or groups of integers or stages.

  This specification includes nucleotide sequence information created using the program PatentIn version 3.1, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by a numeric identifier <201> followed by a sequence identifier (eg, <210> 1, <210> 2, etc.). The sequence length, type (such as DNA) and the origin organism for each nucleotide sequence are identified by the information provided in the numeric identifier fields <211>, <212>, and <213>, respectively. The nucleotide sequences referred to herein are identified by an identifier SEQ ID NO followed by a sequence identifier (eg, SEQ ID NO: 1, SEQ ID NO: 2, etc.). The sequence identifier referred to herein correlates with the information provided in the sequence identifier numeric identifier field <400>, which is followed by the sequence identifier (e.g., <400> 1, <400> 2). That is, SEQ ID NO: 1 detailed in this specification correlates with the sequence indicated as <400> 1 in the sequence listing.

A specific mutation in an amino acid sequence is referred to herein as “Xaa ₁ nXaa ₂ ”, where Xaa ₁ is the original amino acid residue prior to the mutation, n is the residue number, and Xaa ₂ is the mutation It is an amino acid. The abbreviation “Xaa” can be a three letter or one letter amino acid code. A single letter code variation is represented, for example, by X ₁ nX ₂ , where X ₁ and X ₂ are the same as Xaa ₁ and Xaa ₂ , respectively. In terms of both the mutation and the general human sphingosine kinase protein sequence, the amino acid residues for human sphingosine kinase are numbered with the serine residue (S) in the motif KTPASPVVVQ SEQ ID NO: 1 (numbered 225) )

  One aspect of the present invention provides a method of modulating sphingosine kinase functional activity, comprising: an effective amount of an agent and the sphingosine kinase for a time and under conditions sufficient to modulate phosphorylation of the sphingosine kinase. Inducing or otherwise acting on the phosphorylation up-regulates the sphingosine kinase activity and inhibits or otherwise inhibits the phosphorylation. Antagonizing down regulates the sphingosine kinase activity.

  Another aspect of the invention provides a method of modulating human sphingosine kinase functional activity, wherein the method comprises an effective amount of the agent and the human under sufficient time and conditions to modulate phosphorylation of the human sphingosine kinase. Contacting the sphingosine kinase, wherein inducing or otherwise acting on said phosphorylation up-regulates said human sphingosine kinase activity and inhibits said phosphorylation or otherwise Antagonizing this in the manner of down regulates the human sphingosine kinase activity.

Yet another aspect of the invention provides a method of modulating sphingosine kinase functional activity, said method comprising the effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation in S ²²⁵ of the sphingosine kinase Contacting the sphingosine kinase, wherein inducing or otherwise acting on the phosphorylation up-regulates the sphingosine kinase activity and inhibits the phosphorylation or otherwise. Antagonizing this in the manner of down regulates the sphingosine kinase activity.

  Yet another aspect of the present invention is directed to a method of modulating sphingosine kinase functional activity, said method being time and under conditions sufficient to modulate phosphorylation of said sphingosine kinase catalyzed by proline-directed proline kinase. Contacting said sphingosine kinase with an effective amount of wherein said inducing or otherwise acting on said phosphorylation up-regulates said sphingosine kinase activity and said phosphorylation Inhibiting or otherwise antagonizing this down-regulates the sphingosine kinase activity.

In a related aspect, the present invention provides a method of modulating sphingosine kinase functional activity, comprising: an effective amount of an agent and the sphingosine kinase under a time and condition sufficient to modulate phosphorylation of the sphingosine kinase. The agent is bound, linked, or otherwise associated with serine ²²⁵ , wherein inducing or otherwise acting on the phosphorylation increases the sphingosine kinase activity. Regulating and inhibiting or otherwise antagonizing the phosphorylation down regulates the sphingosine kinase activity.

  Yet another aspect of the invention relates to a method of modulating cellular activity, the method comprising contacting the cell with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase. Where inducing or otherwise acting on the phosphorylation up-regulates the cellular activity and inhibiting the phosphorylation or otherwise antagonizing the above Down-regulate cell activity.

  A further aspect of the invention relates to a method of modulating cellular activity, the method comprising contacting the cell with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of human sphingosine kinase. Where said inducing or otherwise acting on said phosphorylation up-regulates said cellular activity and inhibiting said phosphorylation or otherwise antagonizing said cells Down-regulate activity.

In another aspect, the present invention relates to a method of modulating human cellular activity, the method contacts the effective amount of the drug and the cell for a time and under conditions sufficient to modulate the phosphorylation in S ²²⁵ of the sphingosine kinase Inducing or otherwise acting on the phosphorylation up-regulates the human cell activity and inhibits the phosphorylation or otherwise in this manner. Antagonizing downregulates the human cell activity.

  Yet another aspect of the invention relates to a method for treating and / or preventing a condition in a mammal, wherein the condition is characterized by abnormal, undesirable, or otherwise inappropriate cellular activity, The method includes administering to the mammal an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase, wherein the method comprises inducing the phosphorylation or other manner Acting with up-regulates the cellular activity and inhibiting the phosphorylation or otherwise antagonizing it down-regulates the cellular activity.

In yet another aspect, the present invention is directed to a method for treating and / or preventing a condition in humans, wherein the condition is characterized by abnormal, undesirable, or otherwise inappropriate cellular activity. is, the method an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation in S ²²⁵ of sphingosine kinase include the step of administering to said human, wherein, to induce the phosphorylation Alternatively, acting in another manner up-regulates the cellular activity, and inhibiting or otherwise antagonizing the phosphorylation down-regulates the cellular activity.

  Another aspect of the invention is the use of an agent as defined herein above in the manufacture of a pharmaceutical formulation for the treatment of a condition in a mammal, wherein the condition is abnormal and undesirable Characterized by inappropriate cellular activity, or wherein said agent modulates phosphorylation of sphingosine kinase and inducing or otherwise acting on said phosphorylation is said cellular activity Up-regulating and inhibiting or otherwise antagonizing the phosphorylation down-regulates the cellular activity.

  In another further aspect, the present invention contemplates a pharmaceutical composition containing a modulating agent as defined herein above in combination with one or more pharmaceutically acceptable carriers and / or diluents. These drugs are called active ingredients.

  Yet another aspect of the invention relates to an agent as defined herein above when used according to the method of the invention.

  A further aspect of the present invention provides a method for detecting an agent capable of modulating phosphorylation of sphingosine kinase or a functional equivalent thereof or a derivative thereof, wherein said method comprises said sphingosine kinase and phosphorylation catalyst or function thereof Contacting a cell containing a physical equivalent or derivative or an extract thereof with a putative agent, and detecting an altered expression phenotype associated with the phosphorylation.

Yet another aspect of the present invention relates to agents identified according to the screening methods defined herein, and the agents for use in the methods of the present invention. The agent should phosphate site of sphingosine kinase, to be especially extended to monoclonal antibodies that bind to amino acid S ²²⁵ is understood.

  A further aspect of the invention relates to a sphingosine kinase variant comprising a mutation in the region of the sphingosine kinase, the region comprising a phosphorylation site, wherein the variant is a wild type sphingosine kinase or a derivative of the sphingosine kinase variant. Exhibit a phosphorylated capacity that is eliminated or reduced compared to homologues, analogs, chemical equivalents or pneumatic.

In another aspect, the amino acid ^{S 148, S 181, Y 184} , S 225, and / or human sphingosine kinase variant comprising an amino acid sequence having single or multiple amino acid substitutions and / or deletion of T ²⁵⁰ is provided Where the variant exhibits a reduced or reduced ability to phosphorylate compared to a wild-type sphingosine kinase or a derivative, homolog, analog, chemical equivalent or mimetic of the sphingosine kinase variant. .

  In yet another aspect, the present invention extends to a genetically modified animal, which is modified to express a sphingosine kinase variant as defined herein above.

  The 1-letter and 3-letter abbreviations used throughout this specification are defined in Table 1.

(Table 1) Single-letter and three-letter amino acid abbreviations

Detailed Description of the Invention The present invention describes, in part, the determination that sphingosine kinase activation is mediated by phosphorylation events, and further the identification of the sphingosine kinase phosphorylation site itself. The inventors have further determined that sphingosine kinase phosphorylation can be mediated by members of the proline-directed protein kinase family, most preferably ERK2. These decisions now allow the rational design of therapeutic and / or prophylactic methods to treat conditions as characterized by abnormal or unwanted cellular activity and / or sphingosine kinase functional activity . Furthermore, the identification and / or design of agents that specifically modulate sphingosine kinase phosphorylation is facilitated.

  Accordingly, one aspect of the present invention provides a method of modulating sphingosine kinase functional activity, wherein the method comprises an effective amount of the agent and the sphingosine kinase for a time and under conditions sufficient to modulate phosphorylation of the sphingosine kinase. Contacting, wherein inducing or otherwise acting on the phosphorylation up-regulates the sphingosine kinase activity and inhibiting the phosphorylation or otherwise Antagonizing this down-regulates the sphingosine kinase activity.

  The term “sphingosine kinase functional activity” is understood as a reference to any one or more activities by which sphingosine kinase can exert its activity as, for example, a key regulatory enzyme in the functioning of the sphingosine kinase signaling pathway. It should be. In this regard, sphingosine kinase is thought to be central to the production of sphingosine 1-phosphate during activation of this pathway. Modulation of sphingosine kinase functional activity includes any one or more up-regulation of functional activity attributed to sphingosine kinase (eg, induction or cessation of a given activity, or a change to the level or degree of any given activity). It should be understood to include both down regulation. Without limiting the present invention to any one theory or mode of action, it is believed that sphingosine kinase exhibits two levels of catalytic activity. At the first level, sphingosine kinase exhibits baseline catalytic activity. At the second level, sphingosine kinase that exhibits baseline activity can be activated to increase the Vmax of the enzyme. In the context of a preferred embodiment of the present invention, removal or reduction of sphingosine kinase catalytic activity is achieved when baseline activity and / or activation of sphingosine kinase beyond that of baseline activity is eliminated or reduced. Preferably activation is removed or reduced, and even more preferably activation is removed.

  In a preferred embodiment, antagonizing the phosphorylation prevents activation of sphingosine kinase, and affecting the phosphorylation results in activation of the sphingosine kinase.

  The term “sphingosine kinase” should be understood to include reference to all types of sphingosine kinase proteins or derivatives, variants, homologs or analogs thereof. In this regard, “sphingosine kinase” should be understood as a molecule involved in the production of sphingosine-1-phosphate, inter alia, during activation of the sphingosine kinase signaling pathway. This includes, for example, all sphingosine kinase or derivatives, variants, homologues or analogs of protein types, such as alternative splicing of sphingosine kinase mRNA or allelic variants or polymorphisms of sphingosine kinase. Any isoform arising from a sex variant is included. Reference to a “functional” derivative, variant, homolog or analog is to be understood as a reference to a molecule that exhibits any one or more of the functional activities of sphingosine kinase. Preferably, the sphingosine kinase is human sphingosine kinase.

  Accordingly, the present invention provides a method of modulating human sphingosine kinase functional activity, wherein the method comprises an effective amount of the agent and the human sphingosine kinase under a time and condition sufficient to modulate phosphorylation of the human sphingosine kinase. Inducing phosphorylation or acting in another manner up-regulates the human sphingosine kinase activity and inhibits the phosphorylation or other manner Antagonizing this down-regulates the human sphingosine kinase activity.

  The term “phosphorylation” should be understood as a reference to the addition of a phosphate group to a hydroxyl group on a protein, in particular the side chain of the amino acids serine, threonine or tyrosine. Without limiting the invention to any one theory or mode of action, this process of phosphorylating proteins is usually referred to as protein kinases (such molecules are referred to herein as “phosphorylation catalysts”). ), Often catalyzed by specific protein kinases, using ATP acting as a phosphate donor. Protein phosphorylation is commonly found to modulate the activity of a protein of interest.

As detailed hereinabove, protein phosphorylation is an event that occurs in the context of specific amino acid residues of a protein of interest. In this regard, the inventors have generally identified S ¹⁴⁸ , S ¹⁸¹ , Y ¹⁸⁴ , S ²²⁵ , and T ²⁵⁰ as amino acid residues associated with phosphorylation of sphingosine kinase molecules. However, for human sphingosine kinase molecule, the present inventors have still further to identify S ²²⁵ as the primary physiological phosphorylation site of the molecule.

Accordingly, in a preferred embodiment, the present invention provides a method of modulating sphingosine kinase functional activity, the method 1 of the phosphorylation of sphingosine kinase ^{S 148, S 181, Y 184} , S 225 or T ^250, Contacting the sphingosine kinase with an effective amount of the agent for a time and under conditions sufficient to modulate in one or more, wherein said inducing phosphorylation or acting in another manner Up-regulating the sphingosine kinase activity and inhibiting the phosphorylation or otherwise antagonizing it down-regulates the sphingosine kinase activity.

In the most preferred embodiment, the sphingosine kinase is human sphingosine kinase and the phosphorylation regulation occurs at amino acid residue ^S225 .

  The term “modulate” either sphingosine kinase functional activity or phosphorylation event should be understood as a reference to up-regulating or down-regulating the functional activity or phosphorylation event of interest. . Reference to up-regulating and down-regulating in this regard includes in addition to reference to inducing or eliminating a functional activity or phosphorylation event in a subject, in addition to a functional activity or phosphorylation event. It should be understood to include both increasing or decreasing the level, degree, or speed at which occurs. Thus, an agent utilized in accordance with the methods of the present invention induces an activity / event, acts on an already initiated activity or event, antagonizes an already existing activity or event, or such activity or event Can be a drug that completely removes

Thus, the term “inducing or otherwise acting on” phosphorylation should be understood as a reference to the following:
(I) inducing phosphorylation of sphingosine kinase, eg, inducing the interaction of sphingosine kinase with a proline-directed protein kinase such as ERK2 that results in phosphorylation of sphingosine kinase; or (ii) existing phosphorus Up-regulate, enhance, or otherwise act on oxidative events, for example, increase the affinity of sphingosine kinase with molecules that phosphorylate, or interact with sphingosine kinase with molecules that otherwise phosphorylate Establish action.

Conversely, “inhibiting or otherwise antagonizing” phosphorylation should be understood as a reference to the following:
(I) interfere with the interaction of sphingosine kinase with the phosphorylated molecule; or (ii) phosphorylation with sphingosine kinase such that phosphorylation of sphingosine kinase is ineffective or less effective. To antagonize the interactions that exist between the molecules

  It should be understood that regulation of sphingosine kinase phosphorylation (in the sense of either up-regulation or down-regulation) can be partial or complete.

  Partial regulation occurs when only some of the sphingosine kinase phosphorylation events that normally occur in a given cell or molecule are affected by the methods of the invention, whereas full regulation occurs for all sphingosine kinase phosphors. Occurs when acid events are regulated.

Modulation of sphingosine kinase phosphorylation can be achieved by any one of a number of techniques including, but not limited to:
(I) introducing a proteinaceous or non-proteinaceous drug into the cell that antagonizes the interaction between sphingosine kinase and a phosphorylation catalyst;
(Ii) introducing into the cell a proteinaceous or non-proteinaceous agent that interacts with the sphingosine kinase and the phosphorylation catalyst;
(Iii) introducing a proteinaceous or non-proteinaceous agent that acts as a phosphorylation catalyst into the cell;
(Iv) introducing into the cell a nucleic acid molecule encoding a drug that acts as a phosphorylation catalyst.

Preferably, the phosphorylation occurs at the sphingosine kinase Ser ²²⁵ , and even more preferably at the human sphingosine kinase Ser ²²⁵ .

  The term “drug” refers to any proteinaceous or non-proteinaceous molecule that modulates (ie, upregulates or downregulates) the interaction of sphingosine kinase with a phosphorylation catalyst (eg, as described above). It should be understood as a reference to (molecules detailed in (i) to (ii)). “Drug” should also be understood as extending to molecules that themselves function as phosphorylation catalysts (eg, molecules detailed in (iii)-(iv) above). A subject agent can be linked, bound, or otherwise associated with any proteinaceous or non-proteinaceous molecule. For example, this can be associated with a molecule that allows targeting to a localized region.

  Such proteinaceous molecules can be derived from natural, recombinant, or synthetic sources, including fusion proteins or, for example, after screening of natural products. The non-proteinaceous molecules described above can be derived from natural sources such as, for example, screening for natural products, or can be chemically synthesized. For example, the invention contemplates a phosphate catalyst such as a chemical analog of a sphingosine kinase or a kinase molecule (eg, a proline-directed protein kinase such as ERK2) that can act as an agonist or antagonist of sphingosine kinase phosphorylation. . Chemical agonists may not need to be derived from sphingosine kinase or phosphorylation catalyst, but may share certain conformational similarities. Alternatively, chemical agonists can be specifically designed to mimic certain physiochemical properties of sphingosine kinase or phosphorylation catalysts. An antagonist can be any compound that can block, inhibit, or otherwise interfere with sphingosine kinase phosphorylation. Antagonists include sphingosine kinases or phosphorylation catalysts, or antibodies specific for a portion of the sphingosine kinase (eg, monoclonal and polyclonal antibodies), and antisense nucleic acids that interfere with transcription or translation of a gene or mRNA in the cell of interest. including. Regulation of expression can also be achieved utilizing an antigen, RNA, ribosome, DNAzyme, RNA aptamer, antibody, or molecule suitable for use in co-expression. Screening methods suitable for use in identifying such molecules are described in more detail herein below.

  The proteinaceous or non-proteinaceous molecule can act to modulate either the direct or indirect interaction of sphingosine kinase with a phosphorylating molecule (eg, a phosphorylating kinase). The molecule acts directly when it is associated with sphingosine kinase or phosphorylation catalyst. The molecule acts indirectly when it is associated with a molecule other than sphingosine kinase or a phosphorylation catalyst, and the other molecule either directly or indirectly interacts with the sphingosine kinase with the phosphorylation catalyst. Adjust with. Thus, the methods of the invention encompass the regulation of sphingosine kinase phosphorylation through induction of a cascade of regulatory steps.

  It should be understood that the term “phosphorylation catalyst” is intended to include both molecules that catalyze the transfer of phosphate from a molecule such as ATP to a sphingosine kinase. However, molecules that achieve phosphorylation of sphingosine kinase in other ways, such as molecules capable of transferring phosphate groups from molecules other than ATP, or phosphorylation catalysts themselves to the sphingosine kinase molecule It should also be understood to include a reference to molecules that can be moved directly.

  “Derivatives” include fragments, portions, portions, and variants from natural, synthetic, or recombinant sources, including fusion proteins. Portions or fragments include, for example, the active region of sphingosine kinase or phosphorylation catalyst. Derivatives can be derived from amino acid insertions, deletions, or substitutions. Amino acid insertional derivatives include amino and / or carboxy terminal fusions as well as intrasequence insertions of one or more amino acids. Insertion amino acid sequence variants are introduced with one or more amino acid residues at a predetermined site in the protein, but random insertions are also possible using appropriate screening of the resulting products Is. Deletion variants are characterized by the removal of one or more amino acids from the sequence. Substituted 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. An example of a substituted amino acid variant is a conservative amino acid substitution. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; , Phenylalanine and tyrosine. Additions to amino acid sequences include fusions with other peptides, polypeptides, or proteins.

  "Variant" or "variant" should be understood in this context to mean a variant or variant form of sphingosine kinase that exhibits at least some functional activity of sphingosine kinase. This variation or mutation may take any form and may be naturally occurring or non-naturally occurring.

  The term “homolog” is to be understood as a reference to nucleic acid molecules or proteins from alternative species.

  Nucleic acid molecule or protein molecule equivalents are to be understood as molecules that exhibit any one or more functional activities of these molecules, and are chemically synthesized or subjected to screening processes such as natural product screening. From any source as identified through.

  Derivatives include peptides, polypeptides, or fragments having specific epitopes of the complete protein or portions thereof fused to other proteinaceous or non-proteinaceous molecules.

  Analogs contemplated herein include modifications to side chains, incorporation of unnatural amino acids and / or their derivatives during the synthesis of peptides, polypeptides, or proteins, and the use of cross-linking reagents and proteins Other methods that impose conformational constraints on sex molecules or their analogs include, but are not limited to.

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

Examples of side chain modifications contemplated by the present invention include modification of the amino group (eg, by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ₄ ); amidation with methylacetimidate; Acylation with acetic anhydride; Carbamoylation of amino group with cyanic acid; Trinitrobenzylation of amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS); Using succinic anhydride and tetrahydrophthalic anhydride Acylation of the amino group; as well as pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH ₄ .

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

  Carboxyl groups can be modified by O-acylisourea formation followed by carbodiimide activation via subsequent derivatization (eg, up to the corresponding amide).

  Sulfhydryl groups may be carboxymethylated with iodoacetic acid or iodoacetamide; oxidation of performic acid to cysteic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride, or other substituted maleimides; Formation of mercury derivatives using 4-chloromercury benzoate, 4-chloromercury phenylsulfonic acid, phenylmercury chloride, 2-chloromercury-4-nitrophenol and other mercury agents; using cyanate at alkaline pH It can be modified by methods such as carbamoylation.

  Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide, or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halide. On the other hand, tyrosine residues can be modified by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

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

  Examples of unnatural amino acids and derivatives incorporated during protein synthesis include norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, This includes, but is not limited to, the use of phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or the D-isomer of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 2.

(Table 2)

Crosslinking reagents include, for example, homobifunctional crosslinking reagents (eg, bifunctional imide esters, glutaraldehydes, N-hydroxysuccinimide esters having (CH ₂ ) n spacer groups where n = 1 to n = 6) and , Usually used to stabilize three-dimensional conformations using heterobifunctional reagents containing an amino-reactive moiety (eg, N-hydroxysuccinimide and another group-specific reactive moiety) obtain.

Without limiting the invention to any one theory or mode of action, we have determined that serine ²²⁵ is the major physiological phosphorylation site on human sphingosine kinase 1. Alanine mutagenesis was used. Furthermore, it was determined that the proline-directed protein kinase family of kinases can phosphorylate human sphingosine kinase. Specifically, ERK1, ERK2, and CDK2 phosphorylate human sphingosine kinase to varying efficiencies. However, ERK2 was determined to show the highest efficiency in human sphingosine kinase phosphorylation.

  Thus, in a most preferred embodiment, the present invention relates to a method of modulating sphingosine kinase functional activity, said method being effective for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase catalyzed by proline-directed protein kinase. Contacting the sphingosine kinase with an amount of an agent, wherein inducing or otherwise acting on the phosphorylation up-regulates the sphingosine kinase activity and inhibits the phosphorylation Doing or otherwise antagonizing it down regulates the sphingosine kinase activity.

  Most preferably, the proline-directed protein kinase is ERK1, ERK2, or CDK2, and even more particularly ERK2.

Most preferably, the phosphorylation is regulated in S ^225.

  References to proline-directed protein kinase molecules (eg, ERK1, ERK2, and CDK2) and any other phosphorylation catalysts refer to functional derivatives, mutants, homologs, analogs, equivalents, and mimetics of these molecules Should be understood to include references to.

In a related aspect, the present invention provides a method of modulating sphingosine kinase functional activity, wherein the method modulates phosphorylation of the sphingosine kinase associated with an agent binding, linking, or otherwise associated with serine ^225. Contacting the sphingosine kinase with an effective amount of the agent for a time and under conditions sufficient to induce the phosphorylation or otherwise act to upregulate the sphingosine kinase activity. Inhibiting or otherwise antagonizing the phosphorylation down-regulates the sphingosine kinase activity.

  In a most preferred embodiment, the modulation of sphingosine kinase activity is downregulation.

  Since sphingosine kinase is a central molecule for the functioning of intracellular signaling pathways, the methods of the present invention provide a means to modulate cellular activities that are regulated or controlled by sphingosine kinase signaling. For example, sphingosine kinase signaling pathways are cellular activities that result in inflammation, cell transformation, apoptosis, cell proliferation, upregulation of production of inflammatory mediators (eg, cytokines, chemokines, eNOS), and upregulation of adhesion molecule expression It is known to regulate. The above upregulation can be induced by a number of stimuli such as tumor necrosis factor α and inflammatory cytokines such as interleukin 1, endotoxin, oxidized or modified lipids, radiation or tissue damage. In this regard, reference to “modulate cell activity” refers to the production of one or more chemokines, cytokine production, nitric oxide synthesis, adhesion molecule expression and other inflammatory mediators, for example. In reference to, but not limited to, upregulating, downregulating, or otherwise altering any one or more activities that can be performed according to sphingosine kinase signaling is there. Although the preferred method is to down-regulate sphingosine kinase activity and thereby down-regulate unwanted cellular activity, the present invention nevertheless does up-regulate cellular activity that may be desired in certain situations. It should be understood to encompass rating.

  Accordingly, yet another aspect of the invention relates to a method of modulating cellular activity, the method comprising contacting the cell with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase. Wherein inducing or otherwise acting on the phosphorylation up-regulates the cellular activity and inhibits or otherwise antagonizes the phosphorylation. Down regulates the cellular activity.

  Preferably, the present invention relates to a method for 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 phosphorylation of human sphingosine kinase. Where, inducing or otherwise acting on the phosphorylation up-regulates the cellular activity and inhibiting the phosphorylation or otherwise antagonizing the cell Down-regulate activity.

In a most preferred embodiment, the present invention is contacted to a method of modulating human cellular activity, the method can comprise administering an effective amount of a drug and the cell for a time and under conditions sufficient to modulate the phosphorylation in S ²²⁵ of sphingosine kinase Inducing or otherwise acting on the phosphorylation up-regulates the human cell activity and inhibits the phosphorylation or otherwise in this manner. Antagonizing down-regulates the human cell activity.

  Most preferably, the modulation is down regulation.

  A further aspect of the invention relates to the use of the invention for the treatment and / or prevention of disease states. Without limiting the present invention to any one theory or mode of action, a wide range of cellular functional activities that are regulated through the sphingosine kinase signaling pathway are all aspects of physiological processes in both healthy and disease states. Provides the regulation of sphingosine kinase to function the incorporated components. Thus, the methods of the present invention provide a valuable tool for modulating abnormal or otherwise undesired cellular functional activity that is regulated through the sphingosine kinase signaling pathway.

  Accordingly, yet another aspect of the invention relates to a method for treating and / or preventing a condition in a mammal, wherein the condition is characterized by abnormal, undesirable, or otherwise inappropriate cellular activity. And the method comprises the step of administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase, wherein said method comprises inducing said phosphorylation or otherwise Acting in this manner up-regulates the cellular activity, and inhibiting or otherwise antagonizing the phosphorylation down-regulates the cellular activity.

  Yet another aspect of the present invention relates to a method for treating and / or preventing a condition in a mammal, wherein the condition is characterized by an abnormal, undesirable, or otherwise inappropriate sphingosine kinase functional activity. Wherein the method comprises the step of administering to the mammal an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase, wherein the method comprises inducing the phosphorylation or Acting in other ways up-regulates the sphingosine kinase activity, and inhibiting or otherwise antagonizing the phosphorylation down-regulates the sphingosine kinase activity.

Preferably, the phosphorylation event is a phosphorylation of sphingosine kinase in S ^225.

In a most preferred embodiment, the present invention relates to a method for the treatment and / or prevention of a condition in humans, wherein the condition is characterized by abnormal, undesirable or otherwise inappropriate cellular activity, method, an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of sphingosine kinase in S ²²⁵ includes the step of administering to said human, wherein, things or other inducing the phosphorylation Acting in a manner up-regulates the cellular activity and inhibiting the phosphorylation or otherwise antagonizing down-regulates the cellular activity.

In another most preferred embodiment, the present invention relates to a method for the treatment and / or prevention of a condition in humans, wherein the condition is due to an abnormal, undesirable or otherwise inappropriate sphingosine kinase functional activity. characterized, the method an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of sphingosine kinase include the step of administering to said human in S ^225, where, induces the phosphorylation Or otherwise acting up-regulates the sphingosine kinase functional activity, and inhibiting or otherwise antagonizing the phosphorylation down-regulates the sphingosine kinase functional activity To do.

  Most preferably, the modulation is down regulation.

  The phrase “abnormal, undesirable, or otherwise inappropriate” cellular activity refers to excessive cellular activity, physiologically normal cellular activity that is inappropriate in the sense that it is undesirable, or insufficient It should be understood as a reference to cellular activity. This definition applies in a similar manner with respect to sphingosine kinase activity “abnormal, undesirable, or otherwise inappropriate”. For example, TNF production during tumor cell growth has been shown to support cell growth and provide anti-apoptotic properties to neoplastic cells. Thus, in that the cells are neoplastic, it is desirable that cell proliferation and promotion of anti-apoptotic properties be down-regulated. Similarly, diseases characterized by inflammation such as rheumatoid arthritis, atherosclerosis, asthma, autoimmune diseases, and inflammatory bowel disease are involved in cell activation by cytokines such as TNF, which It is known to result in 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 act on or otherwise induce sphingosine kinase phosphorylation to stimulate cell proliferation.

  Preferably, the condition is an inflammatory condition and the cellular activity is inflammatory mediator production, particularly down-regulation of adhesion molecule expression. Most preferably, the condition is rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease, or inflammatory bowel disease.

  In another preferred embodiment, the condition is a neoplasm and the cellular activity is a down-regulation of cell proliferation and / or anti-apoptotic properties.

  In accordance with the previous aspect of the invention, the subject agent is preferably PD98059 or U0126 or a functional derivative, variant, analog, equivalent or mimetic thereof.

  The term “mammal” as used herein is a human, primate, livestock animal (eg, sheep, pig, cow, horse, donkey), laboratory animal (eg, mouse, rabbit, rat, Guinea pigs), companion animals (eg, dogs, cats), and captured wild animals (eg, foxes, kangaroos, deer). Preferably, the mammal of the present invention is a human or laboratory animal. Even more preferably, the mammal of the present invention is a human.

  “Effective amount” means an amount necessary to at least partially obtain the desired response, or to delay onset, inhibit progression, or stop altogether, and develop a particular condition Or progression is treated. The amount depends on the health and physical condition of the individual being treated, the taxonomic group of individuals being treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation and other relevant factors Varies depending on The amount is expected to meet a relatively broad range that can be determined through routine clinical trials.

  The terms “treatment” and “prophylaxis” herein should be considered in their broadest context. The term “treatment” does not necessarily imply that a subject is treated until total recovery. Similarly, “prevention” does not necessarily mean that the subject will not eventually contract a disease state. Thus, treatment and prevention includes remission of symptoms of a particular condition, or obstructing or otherwise reducing the risk of developing a particular condition. The term “prevention” can be considered as reducing the severity or onset of a particular condition. “Treatment” can also reduce the severity of an existing condition.

  The present invention further provides for the combination of therapeutic agents, eg, subjecting the mammal to other agents, drugs, or treatments (these are subject conditions such as cytotoxic agents or radiotherapy in the treatment of cancer). Intended to be administered in conjunction with treatment).

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

  Modulators are convenient, such as by oral, intravenous (if water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal, or suppository routes, or by implantation (eg, using sustained release molecules). Can be administered in any manner. The modulator may be administered in the form of a pharmaceutically acceptable non-toxic salt such as an acid addition salt or a metal complex (eg, zinc, iron, etc., which is considered a salt for purposes of this application). . Examples of such acid addition salts are hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbine Acid salts and tartrate salts. When the active ingredient is administered in tablet form, it may contain a binder such as tragacanth, corn starch, or gelatin; a disintegrant such as alginic acid; and a lubricant such as magnesium stearate.

  The routes of administration include respiratory, intratracheal, nasopharyngeal, intravenous, intraperitoneal, subcutaneous, intracranial, intradermal, intramuscular, intraocular, intrathecal, intracerebral, intranasal, infusion, These include but are not limited to oral, rectal, intravenous drip patches, and transplantation.

  According to these methods, an agent defined according to the present invention may be co-administered with one or more other compounds or molecules. By "co-administered" means co-administration in the same formulation or in two different formulations via the same route or different routes or via sequential administration by the same route or different routes Is done. For example, a subject agent can be administered with an agonistic agent to enhance its effect. By “sequential” administration is meant a time difference of seconds, minutes, hours, or days between the administration of the two types of molecules. These molecules can be administered in any order.

  Another aspect of the present invention contemplates the use of a drug in the manufacture of a pharmaceutical formulation for the treatment of a condition in a mammal, as defined hereinabove, which condition is abnormal and undesirable. Characterized by inappropriate cellular activity, or wherein said agent modulates phosphorylation of sphingosine kinase and is capable of inducing or otherwise acting on said phosphorylation Up-regulating and inhibiting or otherwise antagonizing the phosphorylation down-regulates the cellular activity.

  Yet another aspect of the present invention contemplates the use of a medicament in the manufacture of a pharmaceutical formulation for the treatment of a condition in a mammal, as defined hereinabove, which condition is abnormal, desirable Characterized by no or otherwise inappropriate sphingosine kinase functional activity, wherein the agent modulates phosphorylation of sphingosine kinase and may induce or otherwise act on the phosphorylation Up-regulating the sphingosine kinase functional activity and inhibiting the phosphorylation or otherwise antagonizing it down-regulates the sphingosine kinase functional activity.

Preferably, the phosphorylation is phosphorylation of sphingosine kinase in S ^225.

  Even more preferably, the mammal is a human and the modulation is down regulation.

  As detailed hereinabove, and without limiting the invention to any one theory or mode of action, phosphorylation of sphingosine kinase is such that the Vmax of this enzyme is increased. It is conceivable to activate sphingosine kinase that exhibits baseline activity. Thus, screening an individual for the presence, absence, or degree of sphingosine kinase phosphorylation diagnoses, monitors, or screens the patient for a condition characterized by abnormally activated sphingosine kinase activity and / or cellular activity Provide a means for Therefore, it should be understood that the present invention extends to such diagnostic assays.

  In another further aspect, the present invention contemplates a pharmaceutical composition comprising a modulator as defined herein above with one or more pharmaceutically acceptable carriers and / or diluents. . These drugs are called active ingredients.

  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 aqueous solutions or dispersions, or This 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 surfactants. Prevention of the action of microorganisms can be brought about by various antimicrobial 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 injectable compositions can be brought about by the use in the composition of agents that delay absorption, for example, aluminum monostearate and gelatin.

  Sterile injectable solutions are prepared by incorporating the required amount of the active compound in a suitable solvent with the various other ingredients listed above, followed by filter sterilization, if necessary. Generally, dispersions are prepared by incorporating a variety of sterile active ingredients into a sterile vehicle that contains a basic dispersion medium and the required ingredients that differ 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 lyophilization techniques, which involve the active ingredient and any additional desired from its pre-sterilized filtered solution. Produce powder of ingredients.

  If the active ingredients are adequately protected, they can be administered orally, for example, with an inert diluent or an absorbable edible carrier, or it can be enclosed in a hard or soft shell gelatin capsule. Or it can be compressed into tablets or incorporated directly with dietary foods. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. . Such compositions and preparations should contain at least 1% of active compound by weight. The percentages of the compositions and preparations can of course vary and can be successfully between about 5 and about 80% by weight. The amount of active compound in such therapeutically useful compositions at such appropriate dosages is 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.

  Tablets, troches, pills, capsules and the like may also contain ingredients as listed herein below: binders such as gum arabic, corn starch, or gelatin; excipients such as dicalcium phosphate Disintegrants such as corn starch, potato starch, alginic acid, etc .; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin can be added, or peppermint, winter green oil, or cherry A fragrance such as a fragrance may be added. Where the dosage unit form is a capsule, it can contain a liquid carrier in addition to the type of material described above. A variety of other substances may be present as coatings or otherwise to 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 flavoring such as cherry or orange flavor. 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 can be incorporated into sustained-release preparations and formulations.

  A pharmaceutical composition can also include a genetic molecule, such as a vector, that can transfect a target cell when the vector has a nucleic acid molecule encoding a modulator. This vector can be, for example, a viral vector.

  Yet another aspect of the invention relates to an agent as defined herein above when used in the method of the invention.

The present invention may also be in the S ^225, it should be understood to encompass a method for screening for agents that modulate the phosphorylation of sphingosine kinase. This agent is, for example, an agent that acts or antagonizes the interaction of sphingosine kinase with a phosphorylation catalyst (eg ERK2), or an agent that itself phosphorylates sphingosine kinase, eg, by functioning as a phosphorylation catalyst It is.

  Screening for modulators as defined herein above involves contacting a cell comprising a sphingosine kinase and a phosphorylation catalyst (eg, ERK2) with the agent, as well as modulating sphingosine kinase phosphorylation or downstream sphingosine kinase cell targets (eg, NF-κB) activity or expression modulation can be achieved by any one of several suitable methods, including but not limited to any method. Detection of such modulation can be achieved using techniques such as Western blotting, electrophoretic mobility shift assays, and / or readout of sphingosine kinase activity reporters (eg, luciferase, CAT, etc.).

It is understood that sphingosine kinase or phosphorylation catalyst may be naturally present in the cell being tested, or that the gene encoding them may have been transfected into a host cell for testing purposes. Should be. Furthermore, naturally occurring or transfected genes can be constitutively expressed, thereby providing, inter alia, a model useful for screening agents that down regulate sphingosine kinase phosphorylation, or The gene may require activation, thereby providing, among other things, a useful model for screening agents that modulate sphingosine kinase phosphorylation under specific stimulation conditions. In addition, in that a sphingosine kinase nucleic acid molecule is transfected into the cell, the molecule may contain the entire sphingosine kinase gene or it may contain only a portion of the gene (eg, a portion containing Ser ²²⁵ ).

  In another example, the subject of detection may be a downstream sphingosine kinase regulatory target (eg, NF-κB) rather than sphingosine kinase itself. Yet another example includes a sphingosine kinase binding site linked to a minimal reporter. For example, modulation of sphingosine kinase phosphorylation can be detected by screening for modulation of signaling components downstream of TNF-stimulated cells. When the cell that is the subject of the screening system is a neoplastic cell, for example, modulation of sphingosine kinase phosphorylation can be detected by screening the cell for induction of apoptosis.

  Suitable agents can also be identified and / or designed utilizing well-known methods such as, for example, high throughput screening of combinatorial chemistry or recombinant libraries or natural product screening described below.

  For example, a library containing small organic molecules can be screened, where organic molecules with multiple specific parent group substitutions are used. General synthetic schemes can follow published methods (eg, Bunin BA et al. (1994) Proc. Natl. Acad. Sci. USA, 91: 4708-4712; DeWitt SH et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6909-6913). Briefly, in each successive synthesis step, one of a plurality of different selected substituents is added to each selected subset of tubes in the array. This involves, for example, the selection of tube subsets that exist to generate all possible permutations of the different substituents utilized in generating the library. One suitable permutation strategy is outlined in US Pat. No. 5,763,263.

There is currently wide interest in using combinatorial libraries of random organic molecules for the search for biologically active compounds (see, eg, US Pat. No. 5,763,263). Ligands discovered by screening this type of library can be useful in mimicking or blocking natural ligands or in interfering with naturally occurring ligands of biological targets. In the context of the present invention, for example, they can be used as a starting point for developing agonists or antagonists of sphingosine kinase phosphorylation that exhibit properties such as more potent pharmacological effects. Sphingosine kinases or functional parts thereof and / or phosphorylation catalysts may be combined libraries formed by various solid phase or liquid phase synthesis methods according to the present invention (eg, US Pat. No. 5,763,263 and cited therein). See references). Millions of new chemical and / or biological compounds can be routinely screened in less than a few weeks by using techniques such as those disclosed in US Pat. No. 5,753,187. Of the large amount of compounds identified, only those that exhibit the appropriate biological activity are further analyzed.

  For high throughput library methods, oligomeric or small molecule library compounds capable of specifically interacting with selected biological agents (eg, biomolecules, macromolecular complexes, or cells) are described above. Are screened using combinatorial library devices that are readily selected by those skilled in the art from the scope of well-known methods as described in. In such a method, each member of the library is screened for its ability to interact specifically with the selected agent. In practicing the method of the present invention, the biological agent is withdrawn into compound-containing tubes, allowing it to interact with the individual library compounds in each tube. This 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 the state is adapted depending on the desired interaction. The detection can be performed, for example, by any well-known functional or non-functional underlying method for the detection of substances.

  Accordingly, another aspect of the invention provides a method for detecting an agent capable of modulating phosphorylation of sphingosine kinase or a functional equivalent or derivative thereof, said method comprising said sphingosine kinase and phosphorylation catalyst or Contacting a cell containing the functional equivalent or derivative or an extract thereof with a putative agent and detecting an altered expression phenotype associated with the phosphorylation.

The terms “sphingosine kinase” and “phosphate catalyst” refer to either a sphingosine kinase or a phosphate-catalyzed expression product, or a part or fragment of a sphingosine kinase or phosphorylation catalyst (eg, Ser ²²⁵ sphingosine kinase phosphor It should be understood as a reference to (oxidation sites). In this regard, sphingosine kinase or phosphorylation catalytic expression products are expressed in cells. The cell can be a host cell transfected with a sphingosine kinase or phosphorylation catalytic nucleic acid molecule, or it can be a cell that naturally contains a sphingosine kinase gene. Reference to “the extract” should be understood as a reference to a cell-free transcription system.

  The term “detecting an altered expression phenotype associated with the above phosphorylation” should be understood as detecting cellular changes associated with the regulation of sphingosine kinase phosphorylation. These may be detectable, for example, as intracellular changes or changes observable extracellularly. For example, this includes, but is not limited to, detecting downstream product level or activity changes (eg, NF-κB).

Yet another aspect of the present invention relates to agents identified according to the screening methods defined herein, and the agents for use in the methods of the present invention. It should be understood that the drug extends to a monoclonal antibody that binds to the phosphorylation site of sphingosine kinase, particularly amino acid ^S225 .

  A further aspect of the invention relates to a sphingosine kinase variant comprising a mutation in the region of the sphingosine kinase, the region of which comprises a phosphorylation site, wherein the variant is a wild type sphingosine kinase or a variant of the sphingosine kinase variant. It exhibits a removed or reduced phosphorylation capacity compared to a functional derivative, homolog or analog.

  The present invention also extends to variants that exhibit enhanced or upregulated activity due to the nature of existing phosphorylation site mutations or the incorporation of additional phosphorylation sites.

  The term “mutation” is understood as a reference to any change, alteration, or other modification that modulates the ability of the sphingosine kinase to phosphorylate, whether naturally occurring or non-naturally occurring. Should be. The modulation can be up-regulation or down-regulation. The present invention preferably relates to variants that exhibit removal of the ability to activate, but the invention extends to the generation of variants that exhibit an improved level of phosphorylation capacity at additional or existing sites of phosphorylation. It should be understood. Reference to a “functional” derivative, homologue, or analog in this context should be understood as a reference to a molecule of interest that exhibits a defined regulated ability to phosphorylate.

  This change, alteration, or other modification may be a structural modification (eg, a change in the secondary, tertiary, or quaternary structure of a sphingosine kinase molecule), a molecular modification (eg, one from a sphingosine kinase protein or Additions, substitutions or deletions of multiple amino acids), or any form, including but not limited to chemical modifications. It should be understood that the subject modifications also extend to the fusion, linkage, or binding of proteinaceous or non-proteinaceous molecules to a sphingosine kinase protein or nucleic acid molecule encoding a sphingosine kinase protein. Although the subject mutation must be expressed by a sphingosine kinase expression product, the creation of the mutation can be accomplished by any suitable means (such as the expression product of the mutated nucleic acid molecule is a sphingosine kinase protein variant). It should also be understood that this can be achieved by mutating a type sphingosine kinase protein, synthesizing a sphingosine kinase variant, or modifying a nucleic acid molecule encoding a wild type sphingosine kinase protein. Preferably, the mutation is a single or multiple amino acid sequence substitution, addition and / or deletion.

In accordance with this preferred embodiment, the amino acid ^{S 148, S 181, Y 184} , S 225, and / or human sphingosine kinase variant comprising an amino acid sequence having single or multiple amino acid substitutions and / or deletion of T ²⁵⁰ is provided Wherein the variant exhibits a phosphorylated capacity that is eliminated or reduced compared to wild-type sphingosine kinase or a functional derivative, homolog or analog of the sphingosine kinase variant.

Preferably the amino acid is amino acid S ²²⁵ , and even more preferably the substitution is a S ²²⁵ Ala substitution.

  In the context of the present invention, the term “wild-type” sphingosine kinase is a reference to the form of sphingosine kinase expressed by most individuals in a given population. There can be more wild-type forms of one of the sphingosine kinases (eg due to allelic or isoform variations) and the level of phosphorylation exhibited by the wild-type sphingosine kinase molecule is within a range Can be within. However, it should be understood that “wild type” does not include a reference to the naturally occurring form of sphingosine kinase that cannot be phosphorylated. Such variant forms of sphingosine kinase may indeed constitute naturally occurring variants of sphingosine kinase that are within the context of the present invention.

  It should be understood that the term “agent” includes reference to a sphingosine kinase variant as defined herein, as defined herein above.

  In yet another aspect, the present invention extends to genetically modified animals that have been modified to express sphingosine kinase variants as defined herein above.

  The invention will be further described by reference to the following non-limiting description.

に対してウサギにおいて惹起させた。ウサギへの注射の前に、このホスホペプチドを、そのN末端システインを介して、マレイミド活性化キーホールリンペットヘモシアニン（Pierce）に結合体化した。リン酸化されていないペプチドに対して活性な抗体を、SulfoLink（商標）ビーズ（Pierce）を使用して抗血清から取り出し、そこにリン酸化されていないペプチド

を製造業者の指示書を使用して結合体化した。 Example 1
Methods Generation of phospho-hSK1-specific polyclonal antibodies Phosphopeptides designed with polyclonal antibodies from hSK1 sequences around Ser ²²⁵

Against rabbits. Prior to injection into rabbits, the phosphopeptide was conjugated to maleimide activated keyhole limpet hemocyanin (Pierce) via its N-terminal cysteine. Antibodies that are active against non-phosphorylated peptides are removed from the antiserum using SulfoLink ™ beads (Pierce) and there are non-phosphorylated peptides

Was conjugated using manufacturer's instructions.

Construction and expression of hSK variants Wild-type human SK1 (hSKWT) cDNA (Pitson et al., 2000) (Genbank accession number AF200328) was tagged with a FLAG epitope at the 3 ′ end and as previously described (Pitson Et al., 2000), and subcloned into pALTER (Promega Inc., Madison, WT) site-directed mutagenesis vector. Single stranded DNA was prepared and used as a template for oligonucleotide-directed mutagenesis as detailed in the manufacturer's protocol. The mutagenic oligonucleotides shown in Table 1 were designed to generate the required mutants, which were then sequenced to confirm incorporation of the desired modification. The mutant cDNA was then transformed into pcDNA3 (Invitrogen Corp., San Francisco) for transient transfection of pGEX-4T2 into HEK293T cells for expression as a glutathione S-transferase (GST) -fusion protein in E. coli. Diego, CA). hSK ^TB2 was generated as previously described (Xia et al., 2002).

Cell culture, transfection, and cell fractionation Human embryonic kidney cells (HEK293T, ATCC CRL-1573) were prepared from 10% fetal calf serum, 2 mM glutamine, 0.2% (w / v) sodium bicarbonate, penicillin (1.2 mg / ml). ), And Dulbecco's modified Eagle medium (CSL Biosciences, Parkville, Australia) containing gentamicin (1.6 mg / ml). Transfection was performed using the calcium phosphate precipitation method. Cells were collected 24 hours after transfection and 50 mM Tris / HCl (pH 7.4), 10% (w / v) glycerol, 0.05% (w / v) Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, Lysis was by sonication (2 watts, 30 seconds, 4 ° C.) in lysis buffer A containing 2 mM Na 3 VO 4, 10 mM NaF, 1 mM EDTA, and protease inhibitor (Complete ™; Boehriger Mannheim). Activation of hSK1 cells using either phorbol 12-myristic acid 13-acetic acid (PMA; Sigma) for 30 minutes or tumor necrosis factor-α (TNFα; R & D Systems Inc., Minneapolis, MN) for 10 minutes It was evaluated by processing. For cell fractionation, HEK293T cells were scraped into lysis buffer lacking Triton X-100, sonicated and centrifuged at 1,000 × g for 10 minutes. The supernatant was then centrifuged at 100,000 × g for 60 minutes at 4 ° C. to obtain membrane and cytoplasmic fractions. The membrane fraction is then resuspended in lysis buffer containing 0.8% Triton X-100, sonicated, left on ice for 30 minutes, and centrifuged at 10,000 xg for 10 minutes at 4 ° C. The Triton insoluble material was removed. Protein concentration in the cell homogenate was determined using Coomassie Brilliant Blue (Sigma) reagent using BSA as a standard.

Production of recombinant protein in E. coli Recombinant protein was produced and purified in E. coli as previously described (Pitson et al., 2000).

Metabolic labeling of cells with ³² P
HEK293T cells were cultured and transfected as described above. Cultures in 10 cm dishes were incubated for 30 minutes with phosphate-free, serum-free Dulbecco's Modified Eagle Medium (DMEM). The medium was then replaced with fresh phosphate-free, serum-free DMEM containing 500 μCi [ ³² P] orthophosphate, and the culture was incubated for 3 hours. The cultures were then stimulated with 1 ng / ml TNF or 100 ng / ml PMA for 30 minutes. The cells were then washed 3 times with cold PBS, 50 mM Tris / HCl (pH 7.4), 1% (w / v) Triton X-100, 1% (w / v) deoxycholic acid, 0.1% SDS, 150 mM. It was scraped into 1.2 ml lysis buffer B containing NaCl, 50 mM NaF, 10 mM sodium orthovanadate, 1 mM EDTA, 1 mM DTT, and protease inhibitor (Complete ™). Cells were subjected to 3 freeze-thaw cycles to ensure complete disruption. Cell debris was then removed by centrifugation (13,000 × g, 20 min, 4 ° C.) and the supernatant was incubated with 12 μg M2 anti-FLAG antibody (Sigma) overnight at 4 ° C. with mixing. . The immune complexes were then collected using protein A-Sepharose and washed 4 times with lysis buffer B. The immunoprecipitate was then subjected to SDS-PAGE on a 12% acrylamide gel, the gel was dried and hSK phosphorylated by Phosphorimager (Molecular Dynamics) was quantified.

In vitro phosphorylation of hSK1 Purified recombinant hSK1 (1.5 μg) was recombined in 20 mM MOPS (pH 7.2) containing 20 mM MgCl ₂ , 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM DTT. Incubated with ERK2 (0.1 μg; Upstate Biotechnology), 125 μM ATP, and 5 μCi [γ ³² P] ATP. After 20 minutes at 37 ° C, the reaction was stopped by adding SDS-PAGE sample buffer, the mixture was boiled for 5 minutes, subjected to SDS-PAGE on a 12% acrylamide gel, the gel was dried and phosphorylated by Phosphorimager Quantified hSK1.

Sphingosine kinase assay
SK activity was used as described previously (Pitson et al., 2000) using D-erythro-sphingosine (Biomol, Plymouth Meeting, PA) and [γ ³² P] ATP (Geneworks, Adelaide, South Australia) as substrates. ), Determined routinely. The unit of SK activity (U) was defined as the amount of enzyme required to produce 1 pmol S1P / min.

S1P levels To determine both intracellular and extracellular SP1 levels, HEK293 cells were metabolically labeled with [ ³² P] orthophosphate as described above. Cells were then transferred to fresh phosphate-free DMEM and treated with 1 ng ml-1 TNFα or 100 ng ml-1 PMA for 30 minutes. To determine extracellular S1P release after stimulation, the medium is then removed, centrifuged at 1,000 × g, and 2.5 ml of chloroform, 2.5 ml of methanol, and 20 μl of concentrated hydrochloric acid are added to 2.5 ml of supernatant. It was. The organic phase is then dried under suction, resuspended in chloroform, and S1P is loaded onto silica gel 60 with 1-butanol / ethanol / acetic acid / water (8: 2: 1: 2, v / v). Fractionated by TLC. Intracellular S1P levels were determined by collecting cells in 400 μl methanol containing 25 μl concentrated hydrochloric acid. Lipids were then extracted by addition of 400 μl chloroform, 400 μl KCl, and 40 μl 3M NaCl under alkaline conditions. The aqueous phase containing S1P under these conditions was then acidified through the addition of 50 μl concentrated hydrochloric acid and re-extracted with 400 μl chloroform. The organic phase was then dried under suction, resuspended in chloroform and S1P was fractionated by TLC as described above.

および

を用いてpEGFP-1（Clontech）からPCR増幅し、得られる産物をHindIIIおよびBamHIで消化し、pcDNA3-hSK1-FLAG¹⁰およびpcDNA3-hSK1^S225A-FLAGにクローニングした。HEK293T細胞を、上記のようにこれらのプラスミドを用いて形質移入し、24時間培養し、次いで8ウェルのガラスチャンバースライド（Nalge Nunc international）に1×10⁴細胞/ウェルでプレーティングした。24時間後、細胞を30分間、1μg/ml PMAまたはDMSOの対照で処理し、4％パラホルムアルデヒドで固定し、そして60×油浸対物レンズを使用して、488nmでの励起後、BioRad Radiance 2100共焦点顕微鏡に連結されたOlympusIX70倒立顕微鏡で観察した。 Confocal microscopy Wild type hSK1 and hSK1 ^S225A were tagged at the N-terminus with GFP for fluorescence microscopy. In short, EGFP and the following primers

and

Was used for PCR amplification from pEGFP-1 (Clontech) and the resulting product was digested with HindIII and BamHI and cloned into pcDNA3-hSK1-FLAG ¹⁰ and pcDNA3-hSK1 ^S225A- FLAG. HEK293T cells were transfected with these plasmids as described above, cultured for 24 hours, and then plated at 1 × 10 ⁴ cells / well on 8-well glass chamber slides (Nalge Nunc international). After 24 hours, cells were treated with 1 μg / ml PMA or DMSO controls for 30 minutes, fixed with 4% paraformaldehyde, and after excitation at 488 nm using a 60 × oil immersion objective, BioRad Radiance 2100 Observations were made with an Olympus IX70 inverted microscope connected to a confocal microscope.

Example 2
result
hSK1 is phosphorylated in HEK293 cells in response to cellular agonists. Since phosphorylation is a common mechanism that regulates the catalytic activity of many eukaryotes, HEK293 cells respond to TNFα and phorbol ester (PMA) In vivo phosphorylation of hSK1 was tested. Metabolic labeling with [ ³² P] orthophosphate in HEK293 cells overexpressing hSK1 1000-fold stably does show that hSK1 is phosphorylated and that this phosphorylation is directed against TNFα (Figure 1) and PMA It showed a rapid increase in response to cell exposure. Furthermore, this increase in hSK1 phosphorylation is highly correlated with the observed increase in sphingosine kinase assays in these cells in response to TNFα (FIG. 1) and PMA. These observations suggested that phosphorylation plays a role in hSK1 activation. Further evidence for this was also obtained using hSK1 mutants that lack its ability to bind TRAF2. This hSK1 mutant was not activated or phosphorylated in response to TNFα (Xia et al., 2002) (FIG. 2). However, PMA induces both activation (Xia, 2002) and phosphorylation of this mutant (Figure 2).

hSK1 is phosphorylated at Ser ²²⁵
Analysis of hSK1 sequences using the NetPhos phosphorylation site prediction program (Blom et al., 1999) allowed identification of several possible phosphate sites in hSK1 that were also conserved in mouse and monkey SK1 isoforms (Figure 3). Alanine mutagenesis of these possible sites suggested that hSK1 mutants containing the Ser ²²⁵ → Ala mutation were not phosphorylated (Figure 4). This suggested that Ser ²²⁵ was the only physiological phosphorylation site in hSK1. Further mutagenesis of the two potentially phosphorylable amino acids Ser ²²⁰ and Thr ²²² to alanine in the region around Ser ²²⁵ had no effect on hSK1 phosphorylation (FIG. 5). This indicates that the Ser ²²⁵ → Ala mutation does not have a secondary effect on phosphorylation at adjacent sites (ie, Ser ²²⁰ and Thr ²²² ), but directly blocks phosphorylation at Ser ²²⁵ . Indicated.

Activation of hSK1 requires phosphorylation of Ser ²²⁵
The hSK ^S225A mutant cannot be activated by cell treatment with TNFα and PMA when expressed in HEK293 cells (FIG. 6). This provides strong evidence that Ser ²²⁵ phosphorylation is essential for hSK1 activation.

ERK1, ERK2, and CDK2 specifically phosphorylate hSK1 in Ser ²²⁵ in vitro
The amino acid sequence around Ser ²²⁵ is SKTPAS ²²⁵ PVVVQ (SEQ ID NO: 3). The presence of a C-terminal proline immediately after Ser ²²⁵ suggests that a member of the proline-directed protein kinase family is responsible for hSK1 phosphorylation. In particular, the PASP sequence in this region is reminiscent of the ERK1 / 2 substrate recognition motif, PxS / TP. Where x represents a small neutral amino acid (Chen et al., 2001). Experiments examining the in vitro phosphorylation of purified recombinant hSK1 using ERK1, ERK2, and cyclin dependent kinase 2 (CDK2) showed that all three kinases can phosphorylate hSK1 (FIG. 7). ). However, among these kinases, ERK2 showed the highest efficiency of hSK1 phosphorylation. Furthermore, in vitro phosphorylation analysis using purified recombinant hSK ^S225A mutant showed that ERK2 failed to phosphorylate this protein (FIG. 8). However, ERK2 was still able to phosphorylate recombinant hSK1 mutants containing alanine mutations only at other potential proline dependent kinase phosphorylation sites (Ser ¹⁴⁸ and Thr ²²² ) in hSK1. This indicated that ERK2 is specifically phosphorylates hSK1 in Ser ^225.

Increased hSK1 phosphorylation in cells is blocked by ERK1 / 2 pathway inhibitors but not by CDK inhibitors
The fact that ERK2 efficiently phosphorylates hSK1 in vitro and does it at the correct site suggested that this enzyme could be a physiologically relevant protein kinase that phosphorylates hSK1 in vivo. To test the involvement of ERK1 / 2 in intact cells, the effects of well-characterized ERK1 / 2 pathway inhibitors PD98059 and U0126 on hSK1 phosphorylation and activation were investigated. Both of these act specifically on MEK1 / 2 and block ERK1 / 2 activation (Davies et al., 2000). Both chemical inhibitors strongly blocked TNFα and PMA-induced hSK1 phosphorylation and increased catalytic activity in cells overexpressing hSK1 (FIG. 9a). Consistent with this, the U0126 inhibitor also blocked endogenous enzyme phosphorylation (FIG. 9b) and increased catalytic activity in non-overexpressing cells. This is strong evidence for the in vivo role of ERK1 / 2 in hSK1 activation. In order to establish that there is a direct interaction between ERK1 / 2 and hSK1 in vivo, as suggested by in vitro experiments, co-immunoprecipitation of two proteins from cell extracts was performed. We examined (Figure 9c). Antibodies against either component for immunoprecipitation were used to detect the interaction between hSK1 and ERK1 / 2. Importantly, an interaction between endogenous ERK1 / 2 and endogenous hSK1 was detected, confirming that this was not an interaction forced by overexpression. If ERK1 / 2 is a kinase that activates directly for hSK1, the time course of activation of these kinases should be parallel to the activation of sphingosine kinase activity. This was found to be the case for activation of both TNFα or PMA (FIG. 9d). Apparently, activation of hSK1 by TNFα is maintained in cells overexpressing hSK1. This is only partially explained by the more sustained activation of ERK1 / 2 in the cells, which is consistent with previous studies (Lacana et al., 2002), which also indicates that overexpression of hSK1 is hSK1 It can be shown to saturate the dephosphorylation mechanism. The combined results of in vitro and in vivo experiments demonstrate the activation and association of hSK1 with ERK1 / 2 and strongly indicate that hSK1 is a true direct effector of ERK1 / 2.

  HSK1 interaction with TRAF2 is essential for TNFα-induced hSK1 activation (Xia et al., 2002). Consistent with this, TNFα-induced hSK1 phosphorylation was also found to depend on hSK1 interaction with TRAF2. The data show that TRAF2 interacts with a pre-formed hSK1-ERK1 / 2 complex. This may facilitate the recruitment of components upstream of the MAP kinase pathway to this complex following TNFα involvement, which may include localized activation of EK1 / 2 and subsequent related hSK1 phosphorylation. Allows oxidation. The role of such adapter molecules for TRAF2 allows for enhanced hSK1 activation and explains how TNFα (a relatively weak ERK1 / 2 activator) results in hSK1 activation similar to PMA .

Role of phosphorylation in the mechanism of hSK1 activation
The ability to specifically phosphorylate recombinant hSK1 in Ser ²²⁵ in vitro using ERK2 provided an opportunity to investigate the direct effect of this phosphorylation on the catalytic activity of hSK1. It has been found that phosphorylation of hSK1 results in a dramatic increase in its sphingosine kinase activity. Quantification of ³² P incorporation into recombinant hSK1 after this in vitro phosphorylation indicates that 42% of hSK1 protein is phosphorylated, and hSK1 phosphate in Ser ²²⁵ shows an approximately 14-fold increase in its catalytic activity. It means to occur (Figure 10A). Substrate kinetic analysis demonstrated that phosphorylation substantially increased the turnover number (kcat) of hSK1 (Table 4). In contrast, hSKl phosphorylated, as compared to an enzyme that is not phosphorylated, just slightly lower K _M values for ATP, and had a K _M value unchanged with respect to sphingosine (Fig. 10B, 10C, and Table 4). Thus, although hSK1 has previously been shown to have an intrinsic catalytic activity to be considered in the absence of post-translational modifications, phosphorylation of this enzyme at Ser ²²⁵ is prominent in its catalytic efficiency. A 14-fold increase directly.

Mutation of Ser ²²⁵ → Glu does not produce a constitutively activated hSK1 Due to their negative charge, the acidic amino acids glutamate and aspartate can sometimes mimic phosphorylated amino acids, a protein that resembles the activated state A conformation can be created. However, hSK1 mutant containing Ser ²²⁵ → Glu mutation had the same activity as wild type hSK1 (FIG. 11). Thus, unlike some other enzymes, this mutation does not create a constitutively activated hSK1.

Phospho-hSK1-specific polyclonal antibody
The crude polyclonal antiserum raised in rabbits against phosphopeptides centered around Ser ^{225 of} hSK1 was highly reactive in ELISA against phosphopeptides but similar reactivity towards non-phosphorylated peptides Also shown (Figure 13). However, after affinity chromatography using non-phosphorylated peptides to remove reactivity to this peptide, this antiserum showed high specificity in an ELISA for phosphopeptides (Figure 13). Furthermore, high specificity was also shown for hSK1 protein phosphorylated in Western blots (FIG. 14). In these Western blots, the antiserum was reactive against ERK2-phosphorylated hSK1, but against unphosphorylated wild-type hSK1 (Figure 14) or hSK ^S225A mutant (Figure 15). Showed little reactivity. Thus, we used this purified antiserum to show that TNFα and PMA dependence increases hSK1 phosphorylation (FIG. 15).

Phosphorylation / activation of hSK1 is responsible for its effect on proliferation and transformation. It has previously been shown that overexpression of wild-type hSK1 significantly enhances cell proliferation and results in cell transformation (Xia Et al., 2000). These effects were shown to depend on the catalytic activity of hSK1. However, since this enzyme has an intrinsic catalytic activity to consider (Pitson et al., 2000a), it was not clear from these studies whether hSK1 activation plays a role in these effects . Indeed, hSK1 activation appeared to be important in Ras-mediated transformation (Xia et al., 2000). This led to the assumption that the active form of hSK1 could also be a key factor in transformation resulting from hSK1 overexpression. Elucidation of the activation mechanism of hSK1 afforded the ability to test this further through the use of the unactivated hSK1 Ser ²²⁵ → Ala mutant. Consistent with previous findings (Xia et al., 2000), HEK293T cells overexpressing wild-type hSK1 showed enhanced and serum-independent growth (FIG. 16). In contrast, cells overexpressing the non-phosphorylated hSK1 mutant, hSK1 ^S225A , showed no difference in growth rate or serum dependence relative to control cells transfected with the empty vector (FIG. 16). . These striking differences in the observed proliferative effects of wild-type hSK1 and hSK1 ^S225A were observed even when the expression levels of both proteins were similar in these cells, as was cellular sphingosine kinase activity .

Similar results were also observed in cell transformation assays performed by colony formation in soft agar. As previously reported, NIH3T3 cells transfected with wild-type hSK1 showed significant transformation activity beyond that seen in control cells (Xia et al., 2000). However, in contrast, cells transfected with hSK1 ^S225A had significantly lower transformation activity (FIG. 17). This was despite the cells expressing similar levels of transfected protein and having similar overall sphingosine kinase activity. This data provides strong evidence that it is the phosphorylated, active form of hSK1 that is responsible for the ability of this enzyme to transform. Interestingly, hSK1 ^S225A was also found to block Ras-induced cell transformation (FIG. 17), providing further evidence for the involvement of hSK1 activation in this process. Since hSK1-transfected cells already have high levels of sphingosine kinase activity, the effect of activation on cell transformation may not only be the result of a resulting increase in the catalytic activity of this enzyme. Instead, it can also be related to activation-induced changes in the subcellular localization of activated hSK1.

Translocation of hSK1 into the membrane and increase in intracellular and extracellular sphingosine 1-phosphate levels depend on hSK1 phosphorylation Recent reports indicate that activation of hSK1 by PMA or PDGF is from the cytosolic fraction to the membrane fraction This suggests that this is accompanied by the transfer of the enzyme to To test whether this transition is dependent on agonist-dependent phosphorylation of hSK1, in the cytosolic and membrane fractions, the levels of both total and phosphorylated hSK1 were compared to wild-type hSK1. ^{Alternatively} , HEK293T cells overexpressing either phosphorylation deficient hSK1 ^S225A mutant were measured with and without stimulation by TNFα or PMA (FIG. 18a). Stimulation with PMA or TNFα consistently induced an increase in membrane-bound wild type hSK1. In contrast, the Ser ²²⁵ → Ala mutation completely blocked any increase in membrane-bound hSK1, confirming that phosphorylation at this site is required for the translocation event. Furthermore, confocal microscopy of HEK293T cells expressing hSK1-green fluorescent protein (GFP) fusion protein also confirmed the phosphate-dependent translocation of hSK1. This is because PMA induced the transfer of wild-type hSK1 from the cytoplasm to the plasma membrane, but the phosphorylation-deficient hSK1 ^S225A mutant did not induce the transfer (FIG. 18b).

Phosphorylation is not only required for increased agonist-stimulation of enzyme activity, but also, as demonstrated above, due to the activation-dependent increase of S1P, the second messenger produced by hSK1 Is also necessary (Figure 18b). Overexpression of wild-type hSK1 resulted in a great increase in both intracellular and extracellular S1P over that of cells transfected with the vector, even in the absence of stimulation. However, stimulation of these cells with either TNFα or PMA resulted in a further increase in both intracellular and extracellular S1P. This fully reflects S1P changes at lower overall levels in cells transfected with the empty vector. In contrast, mutations at the Ser ²²⁵ phosphorylation site completely eliminated any agonist-dependent changes in S1P, either intracellular or extracellular. Furthermore, reduced basal S1P levels were observed in cells overexpressing the hSK1 ^S225A mutant compared to wild type hSK1 transfected cells. This was most noticeable for extracellular S1P, even though wild-type hSK1 and hSK1 ^S225A overexpressing cells had similar base sphingosine kinase activity. Consistent with ERK1 / 2 being an activator of endogenous and overexpressed hSK1, inhibition with the ERK1 / 2 pathway inhibitor U1026 increased intracellular and extracellular S1P induced by TNFα and PMA Were blocked in both cells transfected with the vector and cells transfected with wild type hSK1. The inability of this inhibitor to reduce S1P levels to the level of hSK1 ^S225A mutant is explained by an incomplete block of basal hSK1 phosphorylation.

  Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the present invention encompasses all such variations and modifications. The present invention also includes all steps, features, compositions, and compounds mentioned or shown herein, individually or collectively, and any two or more of the above Includes any and all combinations of steps or features.

Table 3 Mutagenesis primers used for site-directed mutagenesis of hSK1

Table 4 Substrate kinetics of in vitro phosphorylated hSK1

List of references

2 is a graphical representation of hSK1 activation and corresponding phosphorylation of hSK1. TNFα stimulation of hSK1-transfected HEK293T cells resulted in in vivo phosphorylation of hSK1 corresponding to increased sphingosine kinase activity in these cells. Image of inactivation and lack of phosphorylation in response to TNFα of mutants unable to bind TRAF2. Unlike wild-type hSK1, hSK1 mutants lacking its ability to bind to hSK ^TB2 and TRAF2 (Xia et al., 2002) are not activated or phosphorylated in response to TNFα treatment of HEK293T cells. In contrast, PMA treatment of these cells results in both phosphorylation and activation of wild type hSK1 and hSK ^TB2 . FIG. 6 is a schematic diagram of prediction of amino acids phosphorylated in hSK1 using NetPhos. Circled residues represent amino acids predicted to be phosphorylated in hSK1 (and its mouse and monkey homologs) by the NetPros program. This is an image of removal of phosphorylation of hSK1 expressing Ser ²²⁵ → Ala mutation. Metabolic labeling of HEK293T cells transfected with wild-type hSK1, hSK ^S148A , hSk ^S181A , hSK ^Y184A , hSK ^S225A , hSK ^T250A , or empty vector and either treated or not treated with PMA Showed that only the Ser ²²⁵ → Ala mutation removes phosphorylation of hSK1. It is an image of phosphorylation at an adjacent site of hSK1 expressing Ser ²²⁵ → Ala mutation. The metabolic labeling of HEK293T cells transfected with wild-type hSK1, hSK ^S220A , hSK ^S225A , or hSK ^T222A and either treated or not treated with PMA is again only Ser ²²⁵ → Ala mutation. It was shown to remove phosphorylation of hSK1. This indicates that the Ser ²²⁵ → Ala mutation directly results in phosphorylation of hSK1 at Ser ²²⁵ , rather than indirectly at adjacent sites (ie, Ser ²²⁰ and Thy ²²² ). 2 is a graphical representation of inactivation of hSK ^S255A by TNFα or PMA. Unlike wild-type hSK1-transfected HEK293T cells, hSK ^S255A- transfected HEK293T cells do not show increased sphingosine kinase activity after TNFα or PMA treatment. It is an image of in vitro phosphorylation of hSK1. In vitro phosphorylation of purified recombinant hSK1 was tested using ERK1, ERK2, and CDK2. Quantification of the specific activity of phosphorylation showed that purified recombinant hSK1 phosphorylated by ERK2 had the highest efficiency. Image of in vitro phosphorylation of hSK1 by ERK2 occurring only in Ser ²²⁵ . In vitro phosphorylation of purified recombinant wild type hSK1, hSK ^S225A , hSK ^S148A and hSK ^T222A was tested using ERK2. Only HSK ^S225A was not phosphorylated by ERK2. This indicates that ERK2 is specifically phosphorylates hSK1 in Ser ^225. FIG. 2 is an image of ERK1 / 2 phosphorylation and activation of hSK1 in vivo. a and b, agonist-induced phosphorylation and activation of hSK1 in HEK292T cells is blocked by ERK1 / 2 pathway inhibitors. Following transient phosphorylation and activity (a) of hSK1 in transiently transfected HEK293T cells, Western blots and sphingosine kinase assays using a phospho-hSK1 specific polyclonal antibody (phospho-hSK1) were performed. Total hSK1 levels were determined via the FLAG epitope, whereas ERK1 / 2 activation was performed according to phospho-ERK1 / 2 and ERK1 / 2 antibodies. HSK1 phosphorylation (b) in untransfected HEK293T cells showed similar results. c, immunoprecipitation of ERK1 / 2 and hSK1. HEK293T cells overexpressing either wild type hSK1 or hSK1 ^TB2 were treated with TNFα or PMA for 30 minutes. HSK1 (via their FLAG epitope) or ERK1 / 2 from clarified cell lysates is immunoprecipitated and subjected to SDS-PAGE and directly by Western blot for ERK1 / 2, FLAG or directly for hSK1 Protein complexes were searched. d, Time course of sphingosine kinase and ERK1 / 2 activation by TNFα (●) and PMA (◯) correlates in both empty vector and hSK1-transfected cells. FIG. 10A is a graphical representation showing that hSK1 is directly phosphorylated by in vitro phosphorylation of Ser ²²⁵ using ERK2. Data shows hSK1 sphingosine kinase activity (kcat) before and after in vitro phosphorylation at Ser ²²⁵ by ERK2. Values are corrected for the percentage of hSK1 phosphorylated in the assay mixture. FIG. 10B is a graphical representation of the substrate kinetics of in vitro phosphorylated hSK1 with ATP. The data show that phosphorylation of hSK1 at Ser ²²⁵ results in a 13.6-fold increase in Kcat (93S ^-1 to 1265S ^-1 ). K _M value for ATP, for phosphorus is oxidized non hSK1 and hSK1 phosphorylated, were respectively 81 ± 12 [mu] M and 56 ± 8 [mu] M. FIG. 10C is a graphical representation of the substrate kinetics of in vitro phosphorylated hSK1 with sphingosine. The data show that phosphorylation of hSK1 at Ser ²²⁵ results in a 13.6-fold increase in Kcat (93S ^-1 to 1265S ^-1 ). K _M values for sphingosine, for phosphorus is oxidized non hSK1 and hSK1 phosphorylated, was 15 ± 4 [mu] M and 13 ± 3 [mu] M, respectively. This is a graphical representation demonstrating that the Ser ²²⁵ → Glu mutation in hSK1 does not produce constitutively activated hSK1. hSK ^WT and hSK ^S225E were expressed in HEK293T cells and the resulting sphingosine kinase activity in cell lysates was quantified with respect to protein expression levels. FIG. 2 is a schematic diagram of a target for a sphingosine kinase antagonist. 2 is an ELISA graphical representation of phospho-hSK antiserum. Crude antiserum (white symbol) and affinity-purified antiserum (black symbol) from rabbits injected with KLH-conjugated phosphopeptides designed around Ser ^{225 of} hSK1 are phosphopeptides (circles) and corresponding Analyzed by ELISA using unphosphorylated peptides (squares). FIG. 2 is an image illustrating that phospho-hSK1 antiserum reacts specifically with phosphorylated hSK1 in Western blot analysis. Western blot analysis of hSK1 using affinity purified phospho-hSK1 antiserum. Lane 1, recombinant hSK1; Lane 2, recombinant hSK1 phosphorylated in vitro by ERK2; Lane 3, recombinant hSK1 phosphorylated in vitro by ERK2, followed by dephosphorylation by alkaline phosphatase. FIG. 3 is an image illustrating that phospho-hSK1 antiserum allows in vivo hSK1 phosphorylation to continue. HEK293 cells were transfected with wild type hSK1 or hSK ^S225A , and treated with TNFα or PMA, collected, and analyzed by Western blot using phospho-hSK1 antiserum and anti-FLAG antibody. FIG. 5 is a graphical representation of proliferation of HEK293T cells in response to overexpression of wild type hSK1 and hSK1 ^S225A . Wild-type hSK1 (triangle), hSK1 ^S225A (square), or empty vector (circle), DMEM medium containing either 5% fetal calf serum (FCS), 1% FCS, or no serum (0.1% BSA) Was propagated in. Cell number was determined using the MIT assay. Figure ² is a graphical representation of transformation of NIH3T3 cells in response to overexpression of wild type hSK1 and hSK1 ^S225A . (A) Left panel shows colony formation in soft agar of NIH3T3 cells transfected with either empty vector, wild type hSK1, or hSK1 ^S225A . The right panel shows colony formation in soft agar of NIH3T3 cells co-transfected with either constitutively active V12-RA and an empty vector, wild type hSK1, or hSK1 ^S225A . (B) Quantification of formed colonies. FIG. 5 shows that hSK1 phosphorylation results in increased levels of its translocation to the membrane and levels of intracellular and extracellular sphingosine 1-phosphate. a and b, the translocation of hSK1 from the cytosol to the plasma membrane depends on its phosphorylation. Cell lysates were fractionated from the cytosol to the membrane to show a phosphorylation-dependent transition to the membrane fraction (a), and Western blots for total hSK1 (using anti-FLAG) and phospho-hSK1 Explored through. An immunoblot for the integral membrane protein E-cadherin was used to show equal loading of the membrane. b, Confocal microscopy of HEK293 cells transfected with either wild-type hSK1-GFP or hSK1 S225A-GFP with or without 1 μM PMA for 30 min, shows ^hSK1 from cytosol to plasma membrane A phosphate-dependent transition was shown. Confocal images represent> 50% of cells observed in three independent experiments. c, Intracellular and extracellular S1P levels are ^measured in HEK293T cells transiently transfected with empty vectors after TNFα and PMA treatment, or with plasmids encoding wild type hSK1 and hSK1 ^S225A , and the ERK1 / 2 pathway Determined in the presence or absence of the inhibitor U1026. Data are averages (± SD) from three experiments.

Claims (49)

  A method of modulating sphingosine kinase functional activity comprising contacting said sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of said sphingosine kinase, wherein said phosphorylation is induced Or otherwise acting up regulates the sphingosine kinase activity and inhibiting phosphorylation or otherwise antagonizing down regulates the sphingosine kinase activity.   A method of modulating cellular activity comprising contacting a cell with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase, inducing said phosphorylation or other A method wherein acting in a manner up-regulates the cellular activity and inhibiting the phosphorylation or otherwise antagonizing down-regulates the cellular activity.   The method according to claim 1 or 2, wherein the sphingosine kinase is human sphingosine kinase. Phosphorylation is regulated in S ^225, The method of any one of claims 1 to 3. Agent, binds to S ^225, accompanied by ligation or otherwise, The method of claim 4, wherein.   6. The method according to any one of claims 1 to 5, wherein the modulation of phosphorylation is modulation of phosphorylation catalyzed by proline-directed protein kinase.   The method according to claim 6, wherein the proline-directed kinase is ERK1, ERK2, or CDK2.   8. The method of claim 7, wherein the proline-directed kinase is ERK2.   9. The method according to any one of claims 1 to 8, wherein the modulation is down regulation.   10. The method of claim 9, wherein the agent is U0126.   The method of claim 9, wherein the agent is PD98059.   A method for treating and / or preventing a condition in a mammal, wherein the condition is characterized by abnormal, undesirable, or otherwise inappropriate cellular activity, the method comprising sphingosine kinase Administering to the mammal an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation, wherein inducing the phosphorylation or otherwise acting increases the cellular activity A method wherein regulating and inhibiting the phosphorylation or otherwise antagonizing down regulates the cellular activity.   A method for treating and / or preventing a condition in a mammal, wherein the condition is characterized by an abnormal, undesirable, or otherwise inappropriate functional activity of sphingosine kinase, the method comprising: Administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase, said inducing or otherwise acting on said sphingosine A method wherein up-regulating the functional activity of a kinase and inhibiting or otherwise antagonizing the phosphorylation down-regulates the functional activity of the sphingosine kinase.   14. The method according to claim 12 or 13, wherein the sphingosine kinase is human sphingosine kinase. Phosphorylation is regulated in S ^225, The method of any one of claims 12 to 14. Agent, binds to S ^225, accompanied by ligation or otherwise, 16. The method of claim 15, wherein.   17. The method according to any one of claims 12 to 16, wherein the modulation of phosphorylation is modulation of phosphorylation catalyzed by a proline-directed protein kinase.   18. The method of claim 17, wherein the proline-directed kinase is ERK1, ERK2, or CDK2.   19. The method of claim 18, wherein the proline-directed kinase is ERK2.   20. A method according to any one of claims 12 to 19, wherein the modulation is down regulation.   21. The method of claim 20, wherein the cellular activity is induced by TNF.   24. The method of claim 21, wherein the condition is a neoplastic condition and the cellular activity is TNF-induced cell proliferation and / or anti-apoptotic properties.   24. The method of claim 21, wherein the condition is an inflammatory condition and the cellular activity is production of an inflammatory mediator.   24. The method of claim 23, wherein the inflammatory mediator is adhesion molecule expression.   25. The method of claim 23 or 24, wherein the inflammatory condition is rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease, or inflammatory bowel disease.   26. The method of any one of claims 20-25, wherein the agent is U0126.   26. The method of any one of claims 20-25, wherein the agent is PD98059.   Use of an agent in the manufacture of a pharmaceutical formulation for the treatment of a condition in a mammal, wherein the condition is characterized by abnormal, undesirable, or otherwise inappropriate cellular activity, Modulating phosphorylation of sphingosine kinase and inducing or otherwise acting on the phosphorylation can upregulate the cellular activity and inhibit or antagonize the phosphorylation Use to down-regulate the cellular activity.   The use of a medicament in the manufacture of a pharmaceutical formulation for the treatment of a condition in a mammal, wherein the condition is characterized by abnormal, undesired or otherwise inappropriate sphingosine kinase activity, An agent modulates phosphorylation of sphingosine kinase and inducing or otherwise acting on the phosphorylation up-regulates the sphingosine kinase activity and inhibits or otherwise antagonizes the phosphorylation Use to down-regulate the sphingosine kinase activity.   30. Use according to claim 28 or 29, wherein the sphingosine kinase is human sphingosine kinase. Phosphorylation is regulated in S ^225, the use of any one of claims 28 to 30. Agent, binds to S ^225, connection, or associated in other ways, the use of claim 31, wherein.   33. Use according to any one of claims 28 to 32, wherein the modulation of phosphorylation is modulation of phosphorylation catalyzed by a proline-directed protein kinase.   34. Use according to claim 33, wherein the proline-directed kinase is ERK1, ERK2, or CDK2.   35. Use according to claim 34, wherein the proline-directed kinase is ERK2.   36. Use according to any one of claims 28 to 35, wherein the modulation is down regulation.   40. Use according to claim 36, wherein the cellular activity is induced by TNF.   38. Use according to claim 37, wherein the condition is a neoplastic condition and the cellular activity is TNF-induced cell proliferation and / or anti-apoptotic properties.   38. Use according to claim 37, wherein the condition is an inflammatory condition and the cellular activity is the production of inflammatory mediators.   40. Use according to claim 39, wherein the inflammatory mediator is adhesion molecule expression.   41. Use according to claim 39 or 40, wherein the inflammatory condition is rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease, or inflammatory bowel disease.   42. The method of any one of claims 36 to 41, wherein the agent is U0126.   42. The method of any one of claims 36 to 41, wherein the agent is PD98059.   A pharmaceutical composition comprising a drug when used according to the method of any one of claims 1 to 27 together with one or more pharmaceutically acceptable carriers and / or diluents, A composition wherein the agent modulates phosphorylation of sphingosine kinase.   An agent that modulates phosphorylation of sphingosine kinase when used according to the method of any one of claims 1-27.   An isolated sphingosine kinase variant containing a mutation in a region of sphingosine kinase that contains a phosphorylation site, removed or reduced compared to wild-type sphingosine kinase, or a functional derivative, homolog or analog thereof The modified body which shows the phosphorylated ability made.   An isolated sphingosine kinase variant containing a mutation in the region of sphingosine kinase containing the phosphorylation site, enhanced or up compared to wild-type sphingosine kinase, or a functional derivative, homolog or analog thereof A variant exhibiting regulated phosphorylation ability. Comprising an amino acid sequence having one or deletion of a plurality of amino acid substitutions and / or amino acids S ^225, the isolated variants of claim 46. 49. The isolated variant of claim 48, wherein the substitution is a substitution of Ser ²²⁵ to Ala.

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