JP2008546393A - Regulation of sphingosine kinase signaling - Google Patents

Abstract

The present invention relates generally to methods for modulating cellular activity and agents for use in the methods. More specifically, the present invention provides a method for regulating the level of functional activity of sphingosine kinase. In a related aspect, the present invention provides a method of modulating sphingosine kinase-mediated signaling through modulation of its intracellular activity level. The present invention still further extends to novel molecules that exhibit the ability to induce sphingosine kinase activity. The methods and molecules of the present invention are particularly useful for abnormal, undesirable, or otherwise inappropriate cellular functional activity, and / or abnormal, undesirable, or otherwise inappropriate sphingosine kinase-mediated Useful for the treatment and / or prevention of conditions characterized by signal transduction. The present invention is further directed to methods for identifying and / or designing agents capable of modulating sphingosine kinase activity levels.

Description

The present invention relates generally to methods for modulating cellular activity and agents for use in the methods. More specifically, the present invention provides a method for regulating the level of functional activity of sphingosine kinase. In a related aspect, the present invention provides a method of modulating sphingosine kinase-mediated signaling through modulation of its intracellular activity level. The present invention still further extends to novel molecules that exhibit the ability to induce sphingosine kinase activity. The methods and molecules of the present invention are particularly useful for abnormal, undesirable, or otherwise inappropriate cellular functional activity, and / or abnormal, undesirable, or otherwise inappropriate sphingosine kinase-mediated Useful for the treatment and / or prevention of conditions characterized by signal transduction. The present invention further relates to methods for identifying and / or designing agents capable of modulating the level of sphingosine kinase activity.

BACKGROUND OF THE INVENTION Bibliographic information of the publications referred to by this inventor are collected alphabetically at the end of the detailed description.

  Reference to any prior art herein is not an admission that the prior art forms part of the general knowledge in Australia or suggests it in any way, and so on. Should not be interpreted.

  Sphingosine kinase (SK) is an enzyme that catalyzes phosphorylation of sphingosine and produces sphingosine 1-phosphate (S1P), a biologically active phospholipid (Pyne & Pyne, 2002, Biochim. Biophys. Acta 1582: 121-131; Spiegel & Milstien, 2003, Nat. Rev Mol. Cell Biol. 4: 397-407). S1P can affect many biological processes including calcium migration, mitogenesis, apoptosis, atherosclerosis, inflammatory response, and cytoskeletal rearrangement (Spiegel & Milstien, 2000, FEBS Lett. 476: 55 -57). Since SK is a major regulator of S1P levels in mammalian cells (Spiegel & Milstien, 2003, supra), the activity and activation of this enzyme is the action observed in cells due to S1P Play a central and important role in controlling

  There is remarkable evidence today that sphingosine kinase and S1P are involved in tumorigenesis through enhanced cell proliferation and cell survival. Xia et al. (2000) show that overexpression of SK1 in NIH3T3 fibroblasts induces neoplastic cell transformation as measured by foci formation, cell growth in soft agar, and tumor formation in NOD / SCID mice. Demonstrated. Furthermore, inhibition of SK1 by treating cells with the SK inhibitor N, N-dimethylsphingosine (DMS) or through the use of dominant negative SK mutants is mediated by oncogenic H-Ras Transformation was blocked (Xia et al, 2000, supra). Other studies have shown that hSK1 mRNA levels are elevated in certain human tumors including breast cancer, colon cancer, lung cancer, ovarian cancer, stomach cancer, uterine cancer, kidney cancer, and rectal cancer (French et al., 2003, Cancer Res. 63: 5962-5969). Recently developed SK1 inhibitors have also been shown to block breast tumor growth in mice (French et al., 2003, supra). This is further supported by other studies that also show that SK1 activation is important in promoting estrogen-dependent tumorigenesis in breast cancer cells (Nava et al., 2002, Expt. Cell Res. 281: 115-127; Sukocheva et al., 2003, Mol. Endocrinol. 17: 2002-2012).

  However, in addition to its role in cell growth and survival, SK1 and S1P appear to be involved in other aspects of cell regulation. It has been shown that S1P can be involved in inflammation and atherosclerosis through induction of adhesion molecule expression in vascular endothelial cells (Xia et al., 1999, J. Biol. Chem. 274: 34499-34505). Other studies suggest involvement in hypertension because high levels of SK1 can enhance vascular stenosis (Bolz et al., 2003, Circulation 108, 342-347; Coussin et al., 2002, Circ. Res 91: 151-157). SK1 has also been shown to be involved in TNFα-induced activation of the pro-inflammatory transcription factor NF-κB (Xia et al., 2002, J. Biol. Chem. 277: 7996-8003). Similarly, S1P appears to be associated with asthma since it was found to enhance airway smooth muscle cell stenosis and histamine release (Jolly et al., 2001, Mol. Immunol. 38: 1239- 1245).

hSK1 has been previously shown to have an intrinsic catalytic activity that is not dependent on post-translational modification of the protein (Pitson et al. 2000a, Biochem. J. 350: 429-441). The inventors have also shown that ERK1 / 2 phosphorylates hSK1 in vivo using a combination of techniques including chemical inhibition, co-immunoprecipitation and in vitro phosphorylation (Pitson et al., 2003, EMBO J. 22: 1-10). This phosphorylation hSK1 increases directly 14 times the k _cat of this enzymatic, no significant effect on K _M values for either the sphingosine or ATP. In general, activation of hSK1 activity correlates with its translocation from the cytosol to the plasma membrane (Rosenfeldt et al., 2001, FASEB J. 15: 2649-2659; Young et al., 2003, Cell calcium 33: 119-128). Phosphorylation at Ser225 not only directly increased hSK1 catalytic activity, but was also shown to be essential for agonist-induced translocation of this protein to the plasma membrane (Pitson et al., 2003, previous Out). The translocation of SK1 may be important in bringing SK1 close to its potential substrate, thereby causing localized signaling that causes S1P or its secretions to bite into cell surface S1P receptors (Pitson et al. ., 2003, supra). Although this study showed that SK1 activation occurs by ERK1 / 2-mediated phosphorylation, many aspects of this phosphorylation control also exist for other phosphorylation-independent SK1 activation mechanisms. It is not yet known including the possibility to get.

  There are two human sphingosine kinase isoforms (1 and 2) that differ in their tissue distribution, developmental expression, catalytic properties, and somewhat but their substrate specificity (Pitson et al., 2000, previous Liu et al., 2000, J. Biol. Chem. 275: 19513-19520). Many studies have shown the effect of sphingosine kinase 1 in promoting cell proliferation and inhibiting apoptosis (Olivera et al., 1999, J. Cell Biol. 147: 545-558; Xia et al., 2000 Curr. Biol. 10: 1527-1530; Edsall et al., 2001, J. Neurochem. 76: 1573-1584). Furthermore, overexpression of human sphingosine kinase 1 (hSK1) in NIH3T3 fibroblasts has been shown to acquire the ability to form tumors in the transformed phenotype and nude mice, which Formability was demonstrated (Xia et al., 2000, supra). More recent studies have shown that hSK1 is involved in estrogen-dependent control of breast tumor cell growth and survival (Nava et al., 2002, Expt. Cell Res. 281: 115-127; Sukocheva et al. , 2003, Mol. Endocrinol. 17: 2002-2012), while other studies have shown that hSK1 mRNA is increased in various human solid tumors and that sphingosine kinase inhibitors inhibit tumor growth in vivo. (French et al., 2003, Cancer Res. 63: 5962-5969). Thus, the involvement of hSK1 in cell growth, survival and tumor formation has already been well confirmed.

  In addition to SphK1, another isoform SphK2 has been cloned and characterized (Liu et al., 2000, J Biol Chem, 275: 19513-19520). Both SphK1 and SphK2 contribute to total SphK activity, but these two isoforms display distinct enzyme kinetics and expression patterns (Kohama et al., 1998, J Biol Chem, 273: 23722-23728; Liu et al. al., 2000, supra). Although SphK1 has been shown to be a cytoplasmic enzyme, SphK2 was found to be localized in the nucleus by its nuclear localization signal sequence (Igarashi et al., 2003, J Biol Chem, 278: 46832 -46839). Additional functional differences between Sphk1 and Sphk2 have also been revealed. For example, Sphk1 enhances cell survival and proliferation, whereas Sphk2 inhibits DNA synthesis (Liu et al., 2000, supra) and induces apoptosis, possibly through the interaction of BH3 domain and Bcl-xL (Liu et al., 2003, J Biol Chem, 278: 40330-40336). Recent studies with Drosophila SphK suggest that SphK2 is the more primitive of the two mammalian enzymes involved in sphingolipid metabolism (Herr et al., 2004, J Biol Chem, 279: 12685-12694). Since there is no evidence that SphK2 expression or activity is regulated, SphK1's ability to be regulated at both transcriptional and post-transcriptional levels in response to various physiological stimuli is a relatively recent evolutionary stage, It may assist in the role of signal transduction in animal cells.

  Thus, as noted above, the central role of sphingosine kinase in relation to the regulation of a wide range of cellular activities has been well identified, but the exact mechanism by which it occurs has only been partially determined. Therefore, in order to provide a better means for constructing a method for controlling cellular activity through control of the sphingosine kinase signal transduction pathway, there is now a need for both elucidation of the mechanism and identification of molecules that can control the mechanism. Is also present.

  In the work leading up to the present invention, it was determined that the interaction of SKAM1 molecules with sphingosine kinase increases the intrinsic catalytic activity of sphingosine kinase. Still further, a sphingosine kinase binding region in SKAM1 was identified. The identification of cellular signaling mechanisms and binding motifs enables the construction of simple and rational methods for the regulation of sphingosine kinase-mediated cellular functions based on the regulation of the interaction between SKAM1 molecules and sphingosine kinase, particularly up-regulation became. This has therefore led to the construction of a highly effective method for the therapeutic or prophylactic treatment of conditions characterized by undesirable or inappropriate cellular function.

SUMMARY OF THE INVENTION Unless the context requires otherwise throughout this specification and the appended claims, the word “comprise” and variations such as “comprises” and “comprising” It is to be understood that includes the meanings encompassing the described integers or stages or integer groups or stages, but does not imply any other integer or stages or meanings that exclude integer groups or stages.

  This specification contains nucleotide and amino acid sequence information generated using the program PatentIn version 3.1, which is placed after bibliographic information herein. Each nucleotide or amino acid sequence is identified in the sequence listing by a numerical code <201> followed by a sequence identifier (eg, <210> 1, <210> 2, etc.). The sequence length, type (DNA, protein, etc.) and biological source for each nucleotide or amino acid sequence are indicated by the information provided in the numerical code columns <211>, <212> and <213>, respectively. The nucleotide and amino acid sequences referred to herein are identified by the code 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 numeric code field <400> in the sequence listing and the subsequent sequence identifier (eg, <400> 1, <400> 2, etc.). That is, SEQ ID NO: 1 detailed in the present specification correlates with the sequence represented by <400> 1 in the sequence listing.

  One aspect of the present invention is to provide an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, variant, homolog or mimetic thereof, and sphingosine kinase. Inducing or otherwise agonizing up-regulates the catalytic activity of sphingosine kinase, and inhibiting or otherwise antagonizing down-regulating the catalytic activity. Provide a method of modulating sphingosine kinase-mediated signaling.

  Another aspect of the present invention is to contact an effective amount of a sphingosine kinase for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, homolog or mimetic thereof, and sphingosine kinase 1. Inducing or otherwise agonizing up-regulates the catalytic activity of sphingosine kinase 1, and inhibiting or otherwise antagonizing down-regulates the catalytic activity of sphingosine kinase 1 Methods of modulating mediated signaling are provided.

  Yet another aspect of the present invention is to provide an effective amount of sphingosine kinase for a time and under conditions sufficient to modulate the interaction of the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1 with sphingosine kinase. Inducing or otherwise agonizing upregulates the catalytic activity of sphingosine kinase, and inhibiting or otherwise antagonizing downregulating the catalytic activity. Methods of modulating mediated signaling are provided.

  Yet another aspect of the invention is to contact sphingosine kinase with an effective amount of SKAM1, or a functional derivative, homolog or mimetic thereof, for a time and under conditions sufficient to induce the interaction of SKAM1 with sphingosine kinase A method is provided for upregulating sphingosine kinase-mediated signaling, comprising steps, thereby upregulating the catalytic activity of sphingosine kinase.

  A further aspect of the invention involves contacting a cell with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, homolog or mimetic thereof, and sphingosine kinase. Inducing, otherwise agonizing, or otherwise agonizing up-regulates cell activity, and inhibiting or otherwise antagonizing down-regulates cell activity.

  A further aspect of the invention is the step of contacting a cell with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of SKAM1 or a functional derivative, homolog or mimetic thereof with sphingosine kinase 1. Induction of the association or otherwise agonizing up-regulates cellular activity, and inhibition of the association or otherwise antagonizing down-regulates cellular activity.

  Another further aspect of the present invention is to provide cells in an effective amount of a drug for a time and under conditions sufficient to modulate the interaction of the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1 with sphingosine kinase. Inducing or otherwise agonizing up-regulation of human cell activity, or inhibiting or otherwise down-regulating human cell activity of sphingosine kinase-mediated The present invention relates to a method for regulating human cell activity.

  Yet another further aspect of the invention relates to the use of the invention in connection with the treatment and / or prevention of disease states. Although the present invention is not limited to any one theory or mode of action, for a wide range of cellular functional activities that are regulated through the sphingosine kinase signaling pathway, control of sphingosine kinase function is a healthy and disease state. Is an essential element in all aspects of both physiological processes. Thus, the methods of the present invention provide a valuable tool for modulating abnormal or otherwise undesirable cellular functional activity controlled through the sphingosine kinase signaling pathway.

  Yet another further aspect of the invention provides an effective amount of an agent in a mammal for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, homolog or mimetic thereof, and sphingosine kinase. Inducing or otherwise agonizing up-regulation of cellular activity, and inhibiting or otherwise down-regulating cellular activity of abnormal, undesirable, Or relates to methods for the treatment and / or prevention of conditions in mammals that are otherwise characterized by inappropriate cellular activity.

  Yet another aspect of the invention is to provide an effective amount of an agent to a mammal for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, homolog or mimetic thereof, and sphingosine kinase. Administration, wherein the induction of the association or otherwise agonizing upregulates sphingosine kinase activity, inhibition of the association or otherwise antagonizing downregulates sphingosine kinase activity, abnormal, undesirable Or a method for the treatment and / or prevention of a condition in a mammal characterized by an otherwise inappropriate sphingosine kinase functional activity.

  Another aspect of the invention is to administer an effective amount of an agent to a human for a time and under conditions sufficient to modulate the interaction of the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1 with sphingosine kinase Inducing or otherwise agonizing the interaction up-regulates cellular activity, and inhibiting or otherwise antagonizing down-regulating cellular activity, Or relates to methods for the treatment and / or prevention of conditions in humans that are otherwise characterized by inappropriate cellular activity.

  Yet another aspect of the present invention is to provide an effective amount of an agent in humans for a time and under conditions sufficient to modulate the interaction of the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1 with sphingosine kinase. Inducing or otherwise upregulating the functional activity of sphingosine kinase, inhibiting the interaction or otherwise down-regulating the functional activity of sphingosine kinase Relates to a method for the treatment and / or prophylaxis of conditions in humans characterized by abnormal, undesirable or otherwise inappropriate functional activity of sphingosine kinase.

  Yet another aspect of the present invention is as defined above herein in the manufacture of a medicament for the treatment of a condition in a mammal characterized by abnormal, undesirable or otherwise inappropriate cellular activity. The agent modulates the interaction of sphingosine kinase, induction of the interaction or otherwise agonizing up-regulates cellular activity, inhibition of the interaction or otherwise Antagonization is intended for use, down-regulating cellular activity.

  Yet another aspect of the present invention relates to the manufacture of a medicament for the treatment of a condition in a mammal characterized by abnormal, undesirable or otherwise inappropriate sphingosine kinase functional activity. Wherein the agent modulates the interaction of SKAM1, or a functional derivative, homolog or mimetic thereof, with sphingosine kinase, induction of the interaction or otherwise Agonize is intended for use in up-regulating the functional activity of sphingosine kinase, and inhibiting or otherwise antagonizing down-regulates the functional activity of sphingosine kinase.

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

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

  A still further aspect of the invention includes a region corresponding to residues 51-132 of SEQ ID NO: 1 or a region exhibiting at least 40% similarity thereto and interacts with sphingosine kinase, It relates to SKAM1 mimics that can up-regulate intrinsic catalytic activity.

Detailed Description of the Invention The present invention, in part, both determined that the SKAM1 molecule upregulated the intrinsic catalytic activity of sphingosine kinase and identified a SKAM1 binding motif responsible for the interaction with sphingosine kinase. based on. These determinations allow the rational design of therapeutic and / or prophylactic methods for treating conditions characterized by abnormal or undesirable cellular activity and / or functional activity of sphingosine kinase. Furthermore, it will be easier to identify and / or design agents that specifically modulate the interaction between sphingosine kinase and SKAM1.

  Accordingly, one aspect of the present invention is to provide an effective amount of a drug for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, variant, homolog or mimetic thereof, and sphingosine kinase. Contacting sphingosine kinase, wherein induction of the association or otherwise agonizing upregulates the catalytic activity of sphingosine kinase, inhibition of the association or otherwise antagonizing downregulates the catalytic activity, Methods of modulating sphingosine kinase mediated signaling are provided.

  Reference to “sphingosine kinase-mediated signaling” should be understood as a reference to the signaling pathway in which the sphingosine kinase molecule forms a functional component. In this regard, sphingosine kinase is considered to be the center of sphingosine-1-phosphate production during activation of this pathway. Modulation of sphingosine kinase-mediated signaling includes both up- and down-regulation of signaling events, such as inducing or stopping a given signaling event or changing to any given signaling event level or degree Should be understood.

  In accordance with the present invention, antagonism of the interaction between SKAM1 and sphingosine kinase (eg, this interaction occurs naturally or occurs as a result of non-natural phenomenon, eg, enforcement of a treatment protocol). Preventing the completion of mediated signaling events, agonizing or otherwise inducing the interaction of SKAM1 and sphingosine kinase promotes sphingosine kinase-mediated signaling. It should also be understood that the extent or level of sphingosine kinase-mediated signaling events can be modulated by increasing or decreasing the concentration of interacting molecules. Thus, the modulation of signaling need not necessarily coincide with the initiation or inhibition of signaling, and can be designed to control the level at which sphingosine kinase-mediated signaling occurs.

  It should be understood that reference to “sphingosine kinase” includes reference to all forms of sphingosine kinase protein and derivatives, variants, homologues or mimetics thereof. In this regard, “sphingosine kinase” should be understood to be a molecule involved in the production of sphingosine-1-phosphate, particularly upon activation of the sphingosine kinase signaling pathway. This includes, for example, alternative splicing of sphingosine kinase mRNA or any isoform arising from allelic or polymorphic variants of sphingosine kinase, including all protein forms of sphingosine kinase and functional derivatives, variants thereof Includes body, homologue, or mimetic.

  The present invention is not limited to any one theory or mode of action, but two human sphingosines differing in their tissue distribution, developmental expression, catalytic properties, and somewhat but their substrate specificity Kinase isoforms (1 and 2) exist (Pitson et al., 2000, supra; Liu et al., 2000, supra). A number of studies have shown the effect of sphingosine kinase 1 in promoting cell proliferation and suppressing apoptosis (Olivera et al., 1999, supra; Xia et al., 2000, supra; Edsall et al. , 2001, supra). Furthermore, overexpression of human sphingosine kinase 1 (hSK1) in NIH3T3 fibroblasts has been shown to acquire the ability to form tumors in the transformed phenotype and nude mice, which Formability was demonstrated (Xia et al., 2000, supra).

  It should be understood that a reference to its “functional” derivative, variant, homolog or mimetic is a reference to a molecule that exhibits any one or more of the functional activities of sphingosine kinase or SKAM1.

  Preferably, the invention contacts sphingosine kinase with an effective amount of the agent for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, homolog or mimetic thereof, and sphingosine kinase 1 Sphingosine kinase 1 mediated, wherein the induction of the association or otherwise agonizing upregulates the catalytic activity of sphingosine kinase 1, and the inhibition of the association or otherwise antagonizing downregulates the catalytic activity Methods of modulating sexual signaling are provided.

It should be understood that reference to SKAM1 is a reference to all forms of this protein. This includes, for example, alternative splicing of SKAM1 mRNA or any isoform resulting from a functional mutant or polymorphic variant of this molecule. Although the present invention is not limited to any one theory or mode of action, SKAM1 is a protein present in many organisms ranging from humans to worms. At present, the structure and function of this protein have not yet been elucidated. Furthermore, no domain can be found in SKAM1 that can be identified by comparison with other known proteins. The results of an initial domain survey using the SMART database (Schultz et al., 1998) revealed that SKAM1A may have an AAA domain. AAA ( A TPases a ssociated with diverse cellular a ctivities) proteins are involved in ATP-dependent protein folding, protein complex formation, protein transport through membrane fusion, and protein degradation (Ogura & Wilkinson, 2001) . However, more detailed sequence analysis shows that although SKAM1A shows a certain sequence similarity to the AAA domain, the Walker A (GXXXXGK (T / S)) motif and Walker are essential for ATP binding and classification into AAA domain families. Clarified that it does not have the B ((D / E) XX) motif. In addition, SKAM1A also has no other highly conserved amino acid sequences characteristic of the AAA family, such as the second region of homology (SRH) or sensor-1 motif and proline-rich region (Karata et al., 1999). Nevertheless, four known SKAM1 isoforms, SKAM1A (SEQ ID NO: 1), SKAM1B (SEQ ID NO: 2), SKAM1C (SEQ ID NO: 3) and SKAM1D (SEQ ID NO: 4) Each can upregulate the catalytic activity of sphingosine kinase. Still further, functional derivatives of SKAM1, such as truncated SKAM1Y (SEQ ID NO: 6), also upregulate the catalytic activity of sphingosine kinase, and thus are also within the scope of the definition of “SKAM1”.

Furthermore, although the present invention is not limited in any manner, each of the four SKAM1 isoforms has been shown to interact with hSK1 and directly enhance its activity in cells and in vitro by 2-4 fold. It was done. The artificially shortened SKAM1Y isoform also interacts with hSK1 and enhances its activity, so that the SK1 interaction region and functional region of SKAM1 are between amino acids 51-132 of SEQ ID NOs: 1-4 Note also that this has been demonstrated. Thus, an 82 amino acid SKAM1 polypeptide is sufficient to enhance SK activity. To further test the interaction of SKAM1 / hSK1, the effect of SKAM1 on hSK1 substrate affinity was determined. The finding that SKAM1 had no effect on hSK1 K _m values for ATP or sphingosine but increased k _cat values, SKAM1 increased enzyme kinetics but altered hSK1 binding affinity to substrates Indicates not to. SKAM1 is also the first protein reported to interact with hSK2 and show regulatory effects on this enzyme. SKAM1 is also the first protein shown to bind to both hSK1 and hSK2. The activation level of hSK2 by different SKAM1 isoforms is similar to hSK1, suggesting that SKAM1 acts on SK2 in the same manner as for hSK1. SK2 is much larger than SK1 (Spiegel & Milstien, 2003), with different tissue distribution, developmental expression (Kohama et al., 1998; Liu et al., 2000) and subcellular localization (Liu et al., 2000; Pitson et al., 2003; Igarashi et al., 2003; Inagaki et al., 2003). SKAM1 enhances both SK isoforms. In addition, SK2 also has a role for SK1 in growth promotion and survival (Payne et al., 2002; Olivera et al., 1999; Edsall et al., 2001; Osawa et al., 2001, J. Immunol. 167 : 173-180), SK2 appears to exhibit a different cellular function than SK1, as reported to have a pro-apoptotic role (Liu et al., 2003).

  Thus, the present invention more specifically provides an effective amount of a drug for a time and under conditions sufficient to modulate the interaction of the sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1. Contacting sphingosine kinase, wherein induction of the association or otherwise agonizing upregulates the catalytic activity of sphingosine kinase, inhibition of the association or otherwise antagonizing downregulates the catalytic activity, Methods of modulating sphingosine kinase mediated signaling are provided.

  Preferably, the sphingosine kinase is sphingosine kinase 1 or sphingosine kinase 2, most preferably sphingosine kinase 1.

  Most preferably, the sphingosine kinase-mediated signaling is upregulated by inducing or agonizing the interaction of the sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1.

  The elucidation of both the role of SKAM1 as an up-regulator of sphingosine kinase catalytic activity and the site of interaction between these two molecules provides a means to regulate sphingosine kinase-mediated cellular activity. “Adjustment” means upward control or downward control. For example, inducing or otherwise agonizing the interaction of SKAM1 and sphingosine kinase includes reference to the induction of a subject's signaling events that effectively induce, up-regulate or sustain subject's cellular activity, It provides the meaning of increasing the level, extent or speed at which signal transduction occurs. Conversely, unless a SKAM1-mediated sphingosine kinase signaling phenomenon is desired (eg, if it is sought to reduce or terminate the treatment protocol for administering SKAM1 to a patient or down-regulate spontaneous interactions) In addition to including reference to removal of a subject's signaling phenomenon that effectively eliminates or down-regulates the subject's cellular activity, the present invention includes the level, extent or Ranges in speed reduction. Thus, an agent used according to the method of the present invention is an agent that induces a phenomenon of interest, agonizes an already initiated phenomenon, antagonizes an already existing phenomenon, or completely prevents the initiation of such a phenomenon It can be.

References to “guidance or otherwise agonize”
(I) induction of the interaction of SKAM1, in particular the region defined by residues 51 to 132 of SEQ ID NO: 1, with sphingosine kinase to effect cellular activity; or (ii) sphingosine kinase following its first induction It should be understood that this is a reference to up-regulation, enhancement or otherwise agonizing of the / SKAM1 interaction.

Conversely, “inhibition or otherwise antagonize” of the interaction between SKAM1, particularly the region defined by residues 51-132 of SEQ ID NO: 1, and sphingosine kinase is:
(I) disruption of the interaction between SKAM1 and sphingosine kinase (eg, in the context of the prophylactic nature where endogenous SKAM1 is known to upregulate sphingosine kinase activity); or (ii) its effect A reference to antagonizing the existing interaction between SKAM1 and sphingosine kinase, which eliminates or reduces its effects.

  It should be understood that regulation of the interaction between sphingosine kinase and SKAM1 (in the sense of either up-regulation or down-regulation) can be partial or complete. Partial modulation occurs when only a portion of the sphingosine kinase / SKAM1 interaction that naturally occurs in a given cell is affected by the methods of the present invention (eg, the agent that is contacted with the cell of interest is intracellular sphingosine kinase / SKAM1). Full regulation occurs when all sphingosine kinase / SKAM1 interactions are regulated, provided at a concentration insufficient to saturate the interaction).

Regulation of the interaction between sphingosine kinase and SKAM1
(I) introduction of a nucleic acid molecule encoding SKAM1, or a derivative, homologue, or mimetic thereof into a cell, or a proteinaceous form of SKAM1, to regulate the intracellular concentration of SKAM1 utilized for signaling purposes Or the introduction of a derivative, homologue or mimetic thereof,
(Ii) introduction of a proteinaceous or non-proteinaceous molecule that regulates transcriptional and / or translational control of the SKAM1 gene into cells,
(Iii) introduction of proteinaceous or non-proteinaceous molecules, such as competitive inhibitors or antibodies, into cells that antagonize the interaction between SKAM1 and sphingosine kinase,
(Iv) can be achieved by any one of a number of techniques including, but not limited to, introducing into the cell a proteinaceous or non-proteinaceous molecule that agonizes the interaction between SKAM1 and sphingosine kinase.

  It should be understood that a reference to “drug” is a reference to any proteinaceous or non-proteinaceous molecule that modulates the interaction of SKAM1 and sphingosine kinase, eg, (i) to (iv) above The molecules detailed above are included. A subject agent can be linked, bound or otherwise associated with any proteinaceous or non-proteinaceous molecule. For example, the agent of interest can be associated with a molecule that allows targeting to a localized area. In a preferred embodiment, the subject agent is SKAM1 itself introduced to upregulate sphingosine kinase activation, or a derivative, homologue or mimetic thereof.

  Thus, according to this preferred embodiment, contacting sphingosine kinase with an effective amount of SKAM1, or a functional derivative, homolog or mimetic thereof, for a time and under conditions sufficient to induce the interaction of SKAM1 and sphingosine kinase And thereby upregulates the sphingosine kinase catalytic activity, a method of upregulating sphingosine kinase mediated signaling is provided.

  The proteinaceous molecule can be derived from natural sources, recombinant sources, or synthetic sources including fusion proteins, or can be, for example, by screening natural products. The non-proteinaceous molecule may be obtained from a natural source, such as a natural product screen, or may be chemically synthesized. For example, the present invention contemplates chemical analogs of SKAM1 that can act as agonists or antagonists of sphingosine kinase interactions. Chemical analogs are not necessarily derived from sphingosine kinase or SKAM1, but may share certain conformational similarities. Alternatively, chemical agonists that mimic or up-regulate specific physiochemical properties of sphingosine kinase or SKAM1 can be specifically designed. An antagonist can be any compound that can block, inhibit or otherwise prevent the interaction of sphingosine kinase and SKAM1. Antagonists include sphingosine kinase or SKAM1, or antibodies (eg, monoclonal and polyclonal antibodies) specific for sphingosine kinase or a portion of SKAM1. Reference to an antagonist also includes an antigen, siRNA, antisense molecule, ribozyme, DNAzyme, RNA aptamer, or molecule suitable for use in co-suppression that competitively inhibits the sphingosine kinase / SKAM1 interaction. The proteinaceous and non-proteinaceous molecules mentioned in (i) to (iv) above are collectively referred to herein as “modulating agents”.

  Screening for modulatory agents as defined herein above includes contact of cells with sphingosine kinase and SKAM1, and modulation of sphingosine kinase / SKAM1 functional activity (eg, specific cell activity) or sphingosine kinase or SKAM1. It can be achieved by any one of several suitable methods including, but not limited to, screening for modulation of downstream cell target activity or expression. Detection of such modulation can be accomplished using techniques such as Western blots, electrophoretic mobility shift assays and / or readings of sphingosine kinase activity or SKAM1 activity reporters such as luciferase, CAT, and the like.

  It should be understood that sphingosine kinase or SKAM1 protein may be naturally occurring in the cell under test, or it may be a host cell transfected with the gene encoding them for the purpose of the test. . Furthermore, naturally occurring or transfected genes may be constitutively expressed, thereby providing a useful model for screening agents that specifically downregulate sphingosine kinase / SKAM1 interactions, and Genes require activation, thereby providing a useful model for screening for agents that specifically modulate sphingosine kinase / SKAM1 interactions under specific stimulation conditions, such as phage display and yeast two-hybrid or multi-hybrid screening Can be a thing. Furthermore, as long as the sphingosine kinase or SKAM1 nucleic acid molecule is transfected into the cell, the molecule may contain the entire sphingosine kinase or SKAM1 gene, or a gene such as the SKAM1 region that binds to the sphingosine kinase. It may include only a part.

  In another embodiment, the detection target can be a control target downstream of sphingosine kinase, rather than sphingosine kinase itself. Yet another embodiment includes a sphingosine kinase or SKAM1 binding site linked to a minimal reporter. For example, modulation of sphingosine kinase / SKAM1 interaction can be detected by screening for modulation of downstream signaling components. This is an example of a system that observes the regulation of molecules whose activity is controlled by sphingosine kinase and SKAM1. These methods provide a mechanism for performing high-throughput screening of putative regulatory agents, such as proteinaceous or non-proteinaceous agents, including synthetic libraries, combinatorial libraries, chemical libraries or natural libraries.

  Agents used in accordance with the methods of the present invention can take any suitable form. For example, a proteinaceous agent may be glycosylated or non-glycosylated, phosphorylated or dephosphorylated to varying degrees, and / or a range that is fused, linked, bound or otherwise associated with the protein. Other molecules such as amino acids, lipids, saccharides or other peptides, polypeptides or proteins. Similarly, the subject non-proteinaceous molecule may also take any suitable form. Both proteinaceous and non-proteinaceous agents described herein can be linked, bound or otherwise associated with any other proteinaceous or non-proteinaceous molecule. For example, in one embodiment of the invention, the agent is associated with a molecule that allows targeting to a localized area, such as a specific tissue.

  The term “expression” refers to the transcription and translation of a nucleic acid molecule. Reference to “expression product” is a reference to the product resulting from transcription and translation of a nucleic acid molecule. It should be understood that reference to “regulation” is a reference to up-regulation or down-regulation.

  “Derivatives” of the molecules described herein (eg, sphingosine kinase, SKAM1 or other proteinaceous or non-proteinaceous agents) include fragments, portions of either natural or non-natural sources. (Part), section, or variant. Non-natural sources include, for example, recombinant or synthetic sources. “Recombinant source” means that the cell source from which the molecule of interest was obtained has been genetically altered. This can be done, for example, to increase or otherwise enhance the production rate and output by that particular cell source. Portions or fragments include, for example, the active region of the molecule. Derivatives can be obtained by amino acid insertions, deletions or substitutions. Amino acid insertional derivatives include single and multiple amino acid amino and / or carboxy terminal fusions as well as intrasequence insertions. Inserted amino acid sequence variants are those in which one or more amino acid residues are introduced into a given site in a protein, but random insertion is also possible by appropriate screening of the resulting product. . 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. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins as described above.

  Derivatives also include fragments having specific epitopes or portions of full-length proteins fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. For example, sphingosine kinase, SKAM1, or a derivative thereof can be fused with a molecule to promote localization to the cell membrane. Analogs of molecules contemplated in the present invention include side chain modifications, the incorporation of unnatural amino acids and / or their derivatives during peptide, polypeptide or protein synthesis, and the use of crosslinkers and their proteinaceous molecules or their Including, but not limited to, the use of other methods that impose conformational constraints on analogs.

  Derivatives of nucleic acid sequences that can be used according to the methods of the invention can also be obtained by single or multiple nucleotide substitutions, deletions and / or additions, including fusions with other nucleic acid molecules. Derivatives of nucleic acid molecules used in the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in co-suppression, and fusions of nucleic acid molecules. Nucleic acid sequence derivatives include degenerate variants.

  It should be understood that a “variant” of sphingosine kinase or SKAM1 means a variant molecule that exhibits at least a portion of the functional activity of the sphingosine kinase or SKAM1 form. The variation can take any form and can be natural or non-natural. A mutant molecule is a molecule that exhibits a modified functional activity.

  "Homolog" means that the molecule is derived from a species that is different from the species to be treated according to the methods of the invention.

  It should be understood that a “mimetic” is a molecule that exhibits any one or more of the functional activities of the molecule of interest, the functional equivalent of which may be from any source, For example, it may be chemically synthesized or identified through a screening process, such as natural product screening. For example, chemical or functional equivalents can be designed and / or identified using well-known techniques such as combinatorial chemistry or high-throughput screening of recombinant libraries or by natural product screening. These molecules can also be used to screen for any modulatory agent useful in the methods of the invention.

  For example, a library containing small organic molecules can be screened. The organic molecules used therein have many specific parent groups substituted. General synthetic schemes may follow published methods (eg, Bunin et al. (1994) Proc. Natl. Acad. Sci. USA, 91: 4708-4712; DeWitt 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 of the selected tube subpopulations on the array. The selection of the tube subpopulation is performed so that all possible permutations of the different substituents used to create the library are generated. One suitable permutation strategy is outlined in US Pat. No. 5,763,263.

  There is much interest today in using random organic molecular combinatorial libraries to search for biologically active compounds (see, eg, US Pat. No. 5,763,263). Ligands discovered by screening this type of library can be useful to mimic or block natural ligands or to interfere 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 sphingosine kinase SKAM1 agonists or antagonists. Sphingosine kinase and / or SKAM1 or related portions thereof can be used in accordance with the present invention in combinatorial libraries formed by various solid phase or solution phase synthesis methods (eg, US Pat. No. 5,763,263 and citations thereof). See literature). By using techniques such as those disclosed in US Pat. No. 5,753,187, hundreds of new chemical and / or biological compounds can be screened in a flow operation within a few weeks. Of the large number of compounds identified, only those exhibiting the appropriate biological activity are further analyzed.

  For high-throughput library screening methods, selected biological agents, such as biomolecules, macromolecular complexes or oligomeric or small molecule library compounds capable of specifically interacting with cells, are among the well-known methods as described above. Screens using combinatorial library devices that are readily selected by those skilled in the art. In such a method, each member of the library is screened for its ability to interact specifically with the selected factor. In carrying out this method, biological agents are loaded into compound-containing tubes and allowed to interact with individual library compounds in each tube. The interaction is designed to produce a detectable signal that can be used to observe the presence of the desired interaction. Preferably, the biological agent is prepared as an aqueous solution and additional conditions are used depending on the desired interaction. Detection can be performed, for example, by any well-known function-based or non-function-based method for the detection of substances.

  An “analog” of sphingosine kinase, SKAM1 or agonistic or antagonistic agents contemplated in the present invention includes side chain modifications, incorporation of unnatural amino acids and / or derivatives during peptide, polypeptide or protein synthesis, and This includes, but is not limited to, other methods of imposing conformational constraints on crosslinkers and their analogs. The particular form that can arise from such modifications is believed to depend on whether the molecule of interest is proteinaceous or non-proteinaceous. The nature and / or suitability of a particular modification can be routinely determined by those skilled in the art.

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

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

  The carboxyl group can be modified by activation of carbodiimide through O-acylisourea formation followed by subsequent derivatization, eg, derivatization to the corresponding amide.

  The sulfhydryl group may be carboxymethylated with iodoacetic acid or iodoacetamide; performic acid oxidation 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 chloromercury benzoate, 4-chloromercury phenylsufonic acid, phenylmercury chloride, 2-chloromercury-4-nitrophenol and other mercury agents; methods such as carbamoylation with cyanate at alkaline pH Can be modified.

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 halogenated sulfenyl. On the other hand, tyrosine residues can be altered to form 3-nitrotyrosine derivatives by nitration with tetranitromethane.

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

  Examples of incorporating unnatural amino acids and derivatives 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 D-isomers of amino acids. A list of unnatural amino acids contemplated in the present invention is shown in Table 1.

(Table 1)

Crosslinking agents include, for example, homobivalent crosslinking agents such as divalent imide esters having a (CH ₂ ) _n spacer group (n = 1 to n = 6), glutaraldehyde, N-hydroxysuccinimide esters, and usually It can be used to stabilize the three-dimensional conformation using a heterobifunctional reagent that contains an amino-reactive moiety, such as N-hydroxysuccinimide and another group-specific reactive moiety.

  It should be understood that the methods of the invention can be practiced in the context of a cell source in either an in vitro or in vivo context.

  Since sphingosine kinase is a molecule that is central to the function of intracellular signal transduction pathways, the methods of the present invention provide a means of modulating cellular activity that is controlled or regulated by sphingosine kinase signaling. For example, sphingosine kinase signaling pathways up-regulate cellular activity such as inflammation, cell transformation, apoptosis, cell proliferation, production of inflammatory mediators such as cytokines, chemokines, eNOS, and up-regulation of adhesion molecule expression. It is known to control the resulting cellular activity. The upregulation can be induced by a number of stimuli including, for example, inflammatory cytokines such as tumor necrosis factor alpha and interleukin 1, endotoxin, oxidized or modified lipids, radiation, or tissue damage. In this context, reference to “modulation of cellular activity” refers to any one or more of the activities that a cell can exert according to sphingosine kinase signaling, eg, chemokine production, cytokine production, nitric oxide. Reference to up-regulation, down-regulation or otherwise modification of one or more of, but not limited to, the synthesis of, adhesion molecule expression, and production of other inflammatory modulators. Although the preferred method is to down-regulate undesirable cellular activity by down-regulating the activity of sphingosine kinase, the present invention nevertheless includes the up-regulation of cellular activity that may be desired in a particular environment. Should be understood.

  Accordingly, yet another aspect of the present invention is to provide cells in an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, homolog or mimetic thereof, and sphingosine kinase. A method of modulating cellular activity comprising the step of contacting, wherein induction of or otherwise agonizing said association up-regulates cellular activity, and inhibition or otherwise antagonizing down-regulates cellular activity.

  Preferably, the invention comprises contacting the cell with an effective amount of the agent for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, homolog or mimetic thereof, and sphingosine kinase 1. Induction of the association or otherwise agonizing up-regulates cellular activity, and inhibition of the association or otherwise antagonizing down-regulates cellular activity.

  In a most preferred embodiment, the present invention provides that an effective amount of a drug is administered to a cell for a time and under conditions sufficient to modulate the interaction of a sphingosine kinase with an amino acid region corresponding to residues 51-132 of SEQ ID NO: 1. Modulation of human cell activity, comprising inducing contact or otherwise agonizing up-regulates human cell activity and inhibiting or otherwise antagonizing down-regulating human cell activity Regarding the method.

  Most preferably, said modulation is an upregulation of cellular activity achieved by inducing or agonizing the interaction of sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1.

  Most preferably, the agent is the peptide itself comprising the sequence of SKAM1 or SEQ ID NO: 6.

  A further aspect of the invention relates to the use of the invention in connection with the treatment and / or prevention of disease states. While the present invention is not limited to any one theory or mode of action, for a wide range of cellular functional activities that are regulated through the sphingosine kinase signaling pathway, the control of sphingosine kinase function is associated with normal and disease states. It is an essential element for all aspects of both physiological processes. Thus, the methods of the present invention provide a valuable tool for the modulation of abnormal or otherwise undesirable cellular functional activity, controlled through the sphingosine kinase signaling pathway.

  Accordingly, yet another aspect of the present invention is to provide an effective amount of an agent in a mammal for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, homolog or mimetic thereof, and sphingosine kinase. Inducing or otherwise agonizing up-regulation of cellular activity, and inhibiting or otherwise down-regulating cellular activity of abnormal, undesirable, Or relates to methods for the treatment and / or prevention of conditions in mammals that are otherwise characterized by inappropriate cellular activity.

  Yet another aspect of the present invention is to administer an effective amount of an agent to a mammal for a time and under conditions sufficient to modulate the interaction of SKAM1, or a functional derivative, homolog or mimetic thereof, and sphingosine kinase. Inducing or otherwise agonizing up-regulation of sphingosine kinase activity; otherwise inhibiting or otherwise down-regulating sphingosine kinase activity; Or relates to a method for the treatment and / or prevention of a condition in a mammal characterized by an otherwise inappropriate functional activity of sphingosine kinase.

  Preferably, the interaction occurs with the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1.

  In a most preferred embodiment, the present invention provides an effective amount of an agent in humans for a time and under conditions sufficient to modulate the interaction of the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1 with sphingosine kinase. Inducing or otherwise agonizing the interaction, and inhibiting or otherwise antagonizing down-regulating cellular activity, including the step of administering Or a method for the treatment and / or prevention of conditions in humans characterized by otherwise inappropriate cellular activity.

  In another most preferred embodiment, the present invention provides an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of the sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1. Inducing or otherwise upregulating the functional activity of sphingosine kinase, and inhibiting or otherwise antagonizing down the functional activity of sphingosine kinase. It relates to a method for the treatment and / or prophylaxis of conditions in humans characterized by the functional activity of sphingosine kinase that regulates, abnormal, undesirable or otherwise inappropriate.

  Most preferably, the modulation is upregulation and the agent is a peptide comprising the sequence of SKAM1 or SEQ ID NO: 6, or a functional derivative, homolog or mimetic thereof.

  Reference to "abnormal, undesirable or otherwise inappropriate" cellular activity refers to excessive cellular activity, inappropriate cellular activity or insufficient in that it is physiologically normal but undesirable It should be understood that this is a reference to cell activity. This definition applies in a similar manner to "abnormal, undesirable or otherwise inappropriate" sphingosine kinase activity. For example, as long as the cells are neoplastic, it is desirable that cell proliferation and anti-apoptotic promotion be down-regulated. Similarly, diseases characterized by inflammation, such as rheumatoid arthritis, atherosclerosis, asthma, autoimmune diseases, and inflammatory bowel disease are known to involve cellular activities that lead to the synthesis and secretion of inflammatory mediators, such as adhesion molecules. is there. In such situations, it is also desirable to down-regulate such activity. In other situations, it may be desirable to agonize or otherwise induce the activation of sphingosine kinase to stimulate cell proliferation, eg, to promote angiogenesis.

  As used herein, the term “mammal” includes humans, primates, livestock animals (eg, sheep, pigs, cows, horses, donkeys), laboratory animals (eg, mice, rabbits, rats, guinea pigs). ), Pet animals (eg, dogs, cats) and captive wild animals (eg, foxes, kangaroos, deer). Preferably, the mammal is a human or laboratory animal. Even more preferably, the mammal is a human.

  “Effective amount” means an amount necessary to at least partially obtain the desired response or to delay the onset of, or inhibit the progression of, or to stop both onset or progression of the particular condition being treated To do. This amount depends on the health and physiological status of the individual being treated, the taxonomic classification of the individual being treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors Change. This amount is relatively broad and it is believed that these can be determined through routine testing.

  References herein to “treatment” and “prevention” should be taken in their broadest sense. 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. Accordingly, treatment and prevention includes amelioration of symptoms of a particular condition or suppression or otherwise reduction of the risk of developing a particular condition. The term “prevention” can be taken to reduce the severity or onset of a particular condition. “Treatment” can also reduce the severity of an already existing condition.

  The invention further provides the mammal with cytotoxic agents or radiation therapy in therapeutic combinations, such as other agents, drugs or treatments that may be useful in connection with the treatment of the subject's condition, such as the treatment of cancer. It is intended to administer the drug with.

  Administration of the modulating agent in the form of a pharmaceutical composition can be performed by any convenient means. A modulatory agent in a pharmaceutical composition is intended to exhibit therapeutic activity when administered in an amount that depends on the particular case. The variability depends, for example, on the human or animal selected and the regulatory agent. A wide range of doses may be applicable. Depending on the patient, for example, from about 0.1 mg / kg to about 1 mg of regulatory agent per kg body weight can be administered per day. The usage can be adapted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, weekly, monthly, or at other suitable time intervals, and the dose may be reduced in proportion to the urgency of the situation.

  The modulatory agent can be in any convenient manner, such as the oral route, intravenous route (if water soluble), intraperitoneal route, intramuscular route, subcutaneous route, intradermal route, or suppository route, or It can be administered by implantation (eg, using sustained release molecules). The modulatory agent is administered in the form of a pharmaceutically acceptable non-toxic salt, such as an acid addition salt or a complex with a metal complex, such as zinc, iron, etc. (these are considered salts for purposes of this application). obtain. Examples of such acid addition salts are hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbine Acid salts, tartrate salts, and the like. When the active ingredient is administered in the form of a tablet, the tablet may include 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, nasopharynx, intravenous, intraperitoneal, subcutaneous, intracranial, intradermal, intramuscular, intraocular, intrathecal, intracerebral, intranasal, infusion, oral, rectal , Via intravenous drip, patches, and transplants.

  According to these methods, an agent defined according to the present invention can be co-administered with one or more other compounds or molecules. “Simultaneous administration” means administering simultaneously as the same formulation or as two different formulations through the same route or different routes, or sequentially administered by the same route or different routes. For example, a subject agent can be administered with an agonistic agent to enhance its effect. “Sequential” administration means that there is 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 is as defined hereinabove in the manufacture of a medicament for the treatment of a condition in a mammal characterized by abnormal, undesirable or otherwise inappropriate cellular activity. The agent modulates the interaction of sphingosine kinase, induction of the interaction or otherwise agonizing upregulates cellular activity, inhibition of the interaction or otherwise antagonism Nize is intended for use, down-regulating cellular activity.

  Yet another aspect of the present invention relates to the manufacture of a medicament for the treatment of a condition in a mammal characterized by abnormal, undesirable or otherwise inappropriate functional activity of sphingosine kinase. Use of an agent as defined above, said agent modulating the interaction of SKAM1, or a functional derivative, homologue or mimetic thereof with sphingosine kinase, inducing or otherwise agonizing said interaction Is intended for use in up-regulating the functional activity of sphingosine kinase and inhibiting the interaction or otherwise antagonizing down-regulates the functional activity of sphingosine kinase.

  Preferably, the interaction is an interaction with the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1.

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

  In yet another further aspect, the present invention contemplates a pharmaceutical composition comprising a modulatory agent 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 injectable solutions or sterile powders for the quick preparation of dispersions, or suitable for cream form or topical application Other forms may be possible. They must be stable under the conditions of manufacture and storage and must be preserved against the contamination 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. Inhibition of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by using agents that delay absorption in the composition, for example, aluminum monostearate and gelatin.

  Sterile injectable solutions are prepared by adding the required amount of the active compound to a suitable solvent containing the various other ingredients listed above as required and sterilizing it by filtration. Generally, dispersions are prepared by adding the various sterilized active ingredients to a sterile medium that contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is the reduced-pressure and lyophilization techniques in which the active ingredient and any additional desired ingredient powders are produced from their pre-filter sterilized solutions.

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

  Tablets, troches, pills, capsules and the like may also contain the ingredients listed below: binders such as gum, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as Corn starch, potato starch, alginic acid and the like; lubricants such as sodium stearate; and sweeteners such as sucrose, lactose, or saccharin, or flavoring agents such as peppermint, winter green oil, or cherry flavor. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the above types of materials. Various other materials can be added 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 to prepare any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound can be added to sustained-release and sustained-release formulations.

  The pharmaceutical composition can also include a vector that carries a genetic molecule, eg, a nucleic acid molecule capable of transfecting a target cell and encoding a preparative agent. The vector can be, for example, a viral vector.

  Yet another aspect of the present invention relates to an agent as defined herein above for use in the method of the present invention.

  A still further aspect of the invention includes a region corresponding to residues 51-132 of SEQ ID NO: 1 or a region exhibiting at least 40% similarity thereto, interacting with sphingosine kinase and the unique property of sphingosine kinase SKAM1 mimics that can up-regulate catalytic activity.

  Preferably, the percent similarity is 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99%, or more.

  It should be understood that the present invention extends to nucleic acid molecules that encode the above mimetics.

  The invention is further illustrated by reference to the following non-limiting examples.

Example 1
Activation of sphingosine kinase through interaction with SKAM1 Materials and Methods Materials Glutathione and agarose powders were purchased from Sigma Chemical Co (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium (DMEM), HEPES buffer, fetal bovine serum, penicillin and streptomycin were purchased from CSL Biosciences (Parkville, Australia). Protease inhibitors (Complete ™) were purchased from Roche Diagnostics GMbH (Mannheim, Germany), Benchmark ™ colored protein ladders were purchased from Invitrogen (San Diego, CA), and nitrocellulose membranes were purchased from Schleicher and Schuell ( Keene, USA), isopropyl β-D-thiogalactoside (IPTG) was purchased from Promega (USA). Labtek chamber slides were purchased from Nalge Nunc International (Naperville, IL). FITC-conjugated sheep anti-rabbit and anti-mouse IgG were purchased from AMRAD (Melbourne, Australia). Alexa (594) -conjugated anti-rabbit and anti-mouse IgG were purchased from Molecular Probes (Eugene, Oregon USA). The fluorescent staining mounting medium was purchased from DAKO Corporation (CA, USA), and the protein assay staining solution was purchased from Bio-Rad Laboratories (CA, USA). D-erythro-sphingosine was purchased from Biomol Research Laboratories Inc (Plymouth, PA) and [γ- ³² P] ATP was purchased from Perkin Elmer (Melbourne, Australia).

Yeast two-hybrid screening Yeast two-hybrid screening was performed using Matchmaker Gal4 two-hybrid system 3 (Clontech) according to the manufacturer's instructions. Full-length hSK1 cDNA (Genbank accession number AF200328) was cloned in frame with the Gal4 DNA binding domain into pGBKT7 (Clontech). The bait construct was then transformed with the bait construct along with pACT2 (Clontech) containing a human leukocyte cDNA library. A total of 1 × 10 ⁶ independent clones were screened and 10 positive positive clones were obtained.

であった。PCR産物をEcoRIで切断し、pCMV（HA）哺乳動物発現ベクター（Clontech）にインフレームでクローニングした。次いでSKAM1CおよびSKAM1Yを、プライマー

を用いてpCMV(HA)-SKAM1AからPCRにより生成した。次いでこれらの生成物をpCMV(HA)プラスミドにクローニングした。得られたSKAM1 cDNAは両方向から配列決定し、それらの完全性を確認した。 Preparation of mammalian expression construct of SKAM1 Partial sequences of SKAM1 isolated by yeast two-hybrid screening were analyzed through NCBI database, and primers for PCR amplification of various SKAM1 isoforms (Genbank accession number AK001534) were designed. SKAM1A and SKAM1B were PCR amplified from placental cDNA, and SKAM1D was PCR amplified from total mononuclear cell cDNA. Primer is

Met. The PCR product was cut with EcoRI and cloned in frame into the pCMV (HA) mammalian expression vector (Clontech). SKAM1C and SKAM1Y are then used as primers

Was generated from pCMV (HA) -SKAM1A by PCR using PCR. These products were then cloned into the pCMV (HA) plasmid. The resulting SKAM1 cDNA was sequenced from both directions to confirm their integrity.

Cell culture and transfection Human embryonic kidney cells (HEK-293T) were cultured in 10 cm culture dishes with 10% FCS, 2 mM glutamine, 0.2% (w / v) sodium bicarbonate, 1.2 mg / ml penicillin and 1.6 mg / ml. Plated in DMEM supplemented with streptomycin. After 24 hours, cells were transfected with the mammalian expression construct using the calcium phosphate precipitation method according to Sambrook et al (1989). The next day, HEK-293T transfected cells were harvested by scraping in 10 ml cold PBS and then centrifuged at 1500 rpm, 4 ° C. for 5 minutes. Cells transfected with either pCMV- (HA) -SKAM1 and / or pcDNA3.SK1 (FLAG) (Pitson et al., 2000a, supra) by sonication (2 watts, 4 ° C. for 30 seconds) Contains 50 mM Tris / HCl (pH 7.4), 10% glycerol, 0.05% Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, 2 mM Na ₃ VO ₄ , 10 mM NaF, 1 mM EDTA and Complete ™ protease inhibitor Dissolved in extraction buffer. Cells transfected with pcDNA3.SK2 (FLAG) (Roberts et al., 2004, Anal. Biochem. 331: 122-129) have been reported that Triton X-100 inhibits SK2 activity (Liu et al., 2000, supra) and thus collected in the same manner except that an extraction buffer containing no Triton X-100 was used. For analysis of SKAM1-hSK2 interaction by co-immunoprecipitation, cells were lysed in 1 ml of the above extraction buffer containing 0.5% Triton X-100 by passing 5 times through a 26 gauge needle. Lysates were usually flash frozen in liquid nitrogen and stored at -80 ° C until further analysis.

Immunoprecipitation and Western blot analysis
Lysates expressing hSK1 (FLAG) or hSK2 (FLAG) alone and / or with the HA-SKAM1 isoform were centrifuged at 13,000 g for 10 minutes at 4 ° C. to remove insolubles. The antibody was added to the lysate and incubated at 4 ° C. with stirring for 3 hours. The immune complexes are then captured by incubating with 50 μl of a 50% slurry of Protein A Sepharose (Amersham Pharmacia Biotech) for 3 hours at 4 ° C., washed with cold extraction buffer, and subjected to SDS-PAGE to allow the protein to be nitrocellulose. Transferred to membrane. hSK1 was quantified using either a monoclonal M2 anti-FLAG antibody (Sigma) or a polyclonal chicken or rabbit anti-SK1 antibody raised against recombinant hSK1 produced in E. coli (Pitson et al ., 2000a, supra). SKAM1 was determined using either anti-HA antibody (12CA5; Sigma) or anti-SKAM1 antibody. Immune complexes were detected with HRP-conjugated anti-mouse (Pierce), anti-rabbit (Pierce) or anti-chicken IgG (IMVS, Adelaide, Australia) using an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech) .

Preparation of GST-SKAM1 isoform
pCMV (HA) -SKAM1A, B, C, D and Y were cut with EcoRI, then cloned in frame into the pGEX4T2 vector and transformed into E. coli JM109 by electroporation. Scrap the colonies and mix with either 30 ml L-broth or super broth (35 g / L tryptone, 20 g / L yeast extract, 5 g / L NaCl, 20 g / L glucose and 100 μg / ml ampicillin, pH 7.5) Cultured overnight. The overnight culture was diluted 1:30 with either 300 ml L-broth or super broth and incubated with shaking at 37 ° C. until the OD ₆₀₀ reached approximately 0.8. The expression of GST-SKAM1 protein in these cultures was then induced with 1 mM IPTG for 1 hour at 37 ° C. The culture was then centrifuged at 7500 rpm, 4 ° C. for 15 minutes, the supernatant was discarded, and the bacterial cell pellet was snap frozen under liquid nitrogen and stored at −80 ° C. The frozen bacterial pellet was thawed and resuspended in 20 ml PBS containing Complete ™ protease inhibitor and incubated on ice for 30 minutes. The cell suspension was then sonicated 3 times at 5 Watts for 30 seconds and then incubated on ice for 15 minutes in the presence of 1% Triton X-100. The cell lysate was then centrifuged at 20,000 rpm, 4 ° C. for 30 minutes. The GST-SKAM1 isoform was isolated from the washed lysate by adding 200 μl of a 50% slurry of glutathione sepharose (Amersham Pharmacia Biotech) and incubating with stirring at 4 ° C. for 3 hours. Glutathione sepharose containing bound GST-SKAM1 was collected by centrifugation (3000 rpm, 4 ° C., 5 minutes) and washed 3 times with cold PBS. Next, GST-SKAM1 protein was eluted by adding 250 μl of 20 mM glutathione and stirring at 4 ° C. for 30 minutes. The eluted GST-SKAM1D protein was collected, dialyzed in PBS, and analyzed by SDS-PAGE and Coomassie staining (Sigma Chemical Co). Protein concentration was determined in solution with Coomassie Brilliant Blue reagent (BioRad) or on Coomassie stained gels based on the intensity of the band against the BSA standard.

SK enzyme activity assay The activity of sphingosine kinase was determined using D-erythro-sphingosine and [γ- ³² P] ATP as substrates as previously described (Pitson et al., 2000a, supra). The assay for hSK1 was performed using sphingosine solubilized with Triton X-100, and the assay for hSK2 (Roberts et al., 2004, supra) showed that sphingosine was supplied as a complex with BSA. Except in the same manner. One unit (U) of activity was defined as the activity of forming 1 pmol S1P per minute with 1 mg protein. Substrate dynamics analysis of SK was performed by fixing ATP at 1 mM and changing sphingosine concentration (1-50 μM), and by fixing sphingosine (Sph) at 100 μM and changing ATP concentration (5-500 μM) . Kinetic data were analyzed with the nonlinear regression program Hyper 1.1s using the Michaelis-Menten velocity equation.

Immunodepletion of endogenous SKAM1
Prepare lysates from total mononuclear cells (TMNC), HEK293-T cells and NIH3T3 and centrifuge for 15 minutes at 13,000 rpm at 4 ° C to remove insoluble material. It used for PAGE. The membrane was blocked on a rocking platform in PBS containing 5% SMP and 0.1% Tween-20 for 30 minutes at room temperature. While blocking the membrane, a 1: 10000 dilution of anti-SKAM1 antibody was incubated on the rocking platform for 1 hour at room temperature with either glutathione-sepharose-conjugated GST-SKAM1D or glutathione-sepharose-conjugated GST. Glutathione-Sepharose beads were centrifuged at 2000 rpm and 4 ° C. for 5 minutes. The control supernatant and immunodepleted antiserum SKAM1 were then used for Western blot analysis.

Cell component fraction For cell component fractionation, cells were collected and lysed the next day by sonication in 200 μl extraction buffer without Triton X-100. Total cell lysate was collected and centrifuged at 600 rpm at 4 ° C. for 10 minutes to remove nuclei. Subsequently, the post nuclear supernatant was ultracentrifuged at 55,000 rpm and 4 ° C. for 1 hour. The cytosolic fraction (supernatant) was collected. The pellet was resuspended in extraction buffer containing 0.8% Triton X-100, sonicated at 3 Hz for 30 seconds and incubated on ice for 30 minutes. The Triton insoluble matter was then separated by centrifugation at 13,000 rpm and 4 ° C. for 10 minutes. The Triton soluble membrane fraction was collected and the pellet, the Trion insoluble cytoskeletal fraction, was resuspended again in extraction buffer containing 0.8% Triton X-100 and sonicated at 3 Hz for 30 seconds. All fractions were subjected to 15% and 12% polyacrylamide gels followed by Western blot using anti-HA and anti-FLAG antibodies as previously described.

Immunofluorescence examination Mouse fetal fibroblasts (NIH3T3) and breast cancer cell line (MFC-7) were plated on DMEM on 6 cm culture dishes. After 24 hours, using pCMV (HA) -SKAM1A, 1B, 1C and 1D alone or with pcDNA3 vector encoding GFP-SK1 (Pitson et al., 2003, supra) using Lipofectamine 2000 according to the manufacturer's instructions NIH3T3 cells were transfected. The following day Labtek slides were coated with 200 μl of 50 ng / ml fibronectin and incubated at 37 ° C. for 30 minutes. Meanwhile, transfected NIH3T3 and untransfected MCF-7 cells were washed with PBS, resuspended in 5 ml DMEM, counted and then diluted with an appropriate amount of DMEM. Fibronectin was removed from 8-well labtek slides and 12,500 cells were seeded per well and incubated overnight at 37 ° C. For immunofluorescence analysis, the medium was removed, cells were washed with 200 μl PBS per well, and fixed with 200 μl 4% paraformaldehyde for 10 minutes. Cells were washed twice with 200 μl PBS (PBST) containing 0.1% Triton X-100. Another 200 μl PBST was added and left for 10 minutes to permeabilize the cells. PBST was removed and incubated for 1 hour at room temperature with 100 μl of anti-HA antibody diluted 1: 4000 and / or 1: 1000 or 1: 4000 diluted anti-SKAM1 antibody in PBST containing 3% BSA. The antibody was removed and the cells were washed 3 times with 200 μl PBST. Cells were incubated with 100 μl of anti-mouse Alexa594 diluted 1: 500 in PBST containing 3% BSA and 1: 1000 diluted anti-rabbit FITC for 1 hour at room temperature in the dark. The secondary antibody was removed and the cells were washed three times with 200 μl PBST and then with 200 μl PBS. Slides were drip dried and fluorescent staining mounting medium (Dako) and cover slip applied.

result
Identification of SKAM1 as an hSK1 interacting protein
In order to identify proteins that interact with hSK1, yeast two-hybrid screening was performed using a human leukocyte cDNA library with full-length hSK1 as a bait. This screen identified 10 putative hSK1-interacting proteins, including a protein called sphingosine kinase activation molecule 1 (SKAM1). A database search revealed four different transcripts. These appear to arise by alternative splicing and encode four different naturally occurring SKAM1 protein isoforms (A, B, C and D) (FIG. 1). A partial cDNA of SKAM1 isolated through yeast two-hybrid screening encoded a protein identical to the C-terminal region of the SKAM1D isoform (FIG. 1A). The reason is that it has 8 amino acids at the C-terminal end that are unique to this isoform.

Sequence analysis showed that SKAM1 is a protein present in many organisms ranging from humans to worms. At present, the structure and function of this protein have not yet been elucidated. Furthermore, no domain can be found in SKAM1 that can be identified by comparison with other known proteins. The results of the first domain survey using the SMART database (Schultz et al., 1998, Proc. Natl. Acad. Sci. USA 95: 5857-5864) revealed that SKAM1A may have an AAA domain . AAA ( A TPases a ssociated with diverse cellular a ctivities) proteins are involved in ATP-dependent protein folding, protein complex formation, protein transport through membrane fusion, and protein degradation (Ogura & Wilkinson, 2001, Genes Cells 6: 575-597). However, more detailed sequence analysis shows that although SKAM1A shows a certain sequence similarity to the AAA domain, the Walker A (GXXXXGK (T / S)) motif and Walker are essential for ATP binding and classification into AAA domain families. Clarified that it does not have the B ((D / E) XX) motif. In addition, SKAM1A also does not have other highly conserved amino acid sequences characteristic of the AAA family, such as the second homology region (SRH) or sensor-1 motif and proline-rich region (Karata et al., 1999, J. Biol. Chem. 274: 26225-26232.

Cloning and expression of SKAM1 isoform
To confirm the interaction and effects of various SKAM1 isoforms on hSK1, mammalian expression constructs were made for four naturally occurring SKAM1 isoforms (A, B, C and D). Based on the partial cDNA of SKAM1 isolated by Y2H screening for proteins that interact with hSK1, an artificial shortened SKAM1 was also prepared (SKAM1Y). This involves PCR amplification of the SKAM1 isoform from TMNC and placental cDNA and cloning the resulting product into the vector pCMV (HA) for expression in mammalian cells with an N-terminal hemagglutinin (HA) tag. Went by. Four naturally occurring SKAM1 isoforms (AD) and artificially shortened SKAM1Y were then overexpressed in mammalian cells. Transfect HEK-293T cells with pCMV (HA) -SKAM1A, B, C, D and Y, collect and lyse cells 24 hours after transfection, Immunoblots through their HA epitope tags for the presence of overexpressed proteins. All HA-tagged SKAM1 proteins were detected at molecular weights of 28 kDa (SKAM1A), 24 kDa (SKAM1B), 19 kDa (SKAM1C), 15 kDa (SKAM1D) and 10 kDa (SKAM1Y) as expected by immunoblotting with anti-HA antibody (Figure 1B).

Confirmation of SKAM1-hSK1 interaction
The interaction between hSK1 and SKAM1 isoforms was assessed by co-immunoprecipitation studies. Immunize SKAM1 protein by immunoprecipitation with anti-HA monoclonal antibody lysates from HEK-293T cells expressing SKAM1A, B, C, D and Y isoforms with HA tag and hSK1 with FLAG epitope tag Allowed to settle. The presence of hSK1 in the immune complex was then analyzed by immunoblot analysis using chicken anti-SK1 antibody (Pitson et al., 2003, supra). The expected 45 kDa hSK1 was detected in all SKAM1-hSK1 cotransfection lysates (FIG. 2). This indicated that there was an interaction between hSK1 and the four naturally occurring SKAM1A, B, C, D isoforms and artificially shortened SKAM1Y.

  To further confirm the interaction between SKAM1 isoform and hSK1, reverse immunoprecipitation was performed using anti-FLAG antibody and the immune complex was analyzed by Western blot using anti-HA antibody or anti-SKAM1 antibody. All four naturally occurring SKAM1 isoforms effectively co-immunoprecipitated with hSK1 (FIG. 3). The artificially shortened SKAM1Y isoform could not co-immunoprecipitate with hSK1 in this manner, suggesting that its binding ability is low (FIG. 3). Endogenous SKAM1 from lysates from cells that overexpress only hSK1 was also immunoprecipitated using anti-SKAM1 antibody and attempted to detect hSK1 using chicken anti-SK1 antibody. A 45 kDa protein was detected from the immunoprecipitate of lysates of hSK1 overexpressing cells (FIG. 4), indicating an interaction between overexpressed hSK1 and endogenous SKAM1.

A recent study (Pitson et al., 2003, supra) showed that one mechanism of hSK1 activation involves its phosphorylation at Ser225. In order to determine whether the SKAM1-hSK1 interaction depends on this phosphorylation, an analysis was performed to determine whether ^SKAM1D can interact with non-phosphorylated hSK1 (hSK1 ^S225A ). The results (FIG. 5) demonstrated that SKAM1D and hSK1 ^S225A actually co-immunoprecipitate, indicating that hSK1 phosphorylation of Ser225 is not required for this interaction.

Overexpression of SKAM1 isoforms that increase the activity of endogenous human SK1 To study the effect of overexpressed SKAM1 isoforms on endogenous SK activity, HEK-293T cells were transformed into pCMV (HA) -SKAM1A, B, C , D and Y constructs were transfected. After 24 hours, cells were harvested and total cell lysates were analyzed for SK enzyme activity. The results (FIG. 6) demonstrate that overexpression of all naturally occurring SKAM1 isoforms increases baseline SK activity by approximately 2-3 fold. Similarly, the expression of an artificially shortened SKAM1Y isoform also increased the SK activity of the cells by a factor of two.

Direct interaction between human SK1 and SKAM1 in cells that mediates increased cellular SK activity
We analyzed whether SK activity increased by SKAM1 overexpression was the result of a direct interaction between SKAM1 and hSK1. Thus, SKAM1A was previously shown to act as a dominant negative to block hSK1 activation (Pitson et al., 2000b, supra) in cells that also express the catalytically inactive form of hSK1 (hSK1 ^G82D ). And the ability of overexpression of D to enhance SK activity was tested. SKAM1A and D and hSK1 ^G82D are first detected by immunoprecipitation of SKAM from these lysates with anti-HA antibody ^followed by detection of hSK1 ^G82D in the immune complex by Western blot using anti-hSK1 antibody. The interaction between was confirmed (Figure 7). Testing of endogenous SK activity in these cells showed that when ^SKAM1 was co-expressed with hSK1 ^G82D , the ability of SKAM1A and D to enhance endogenous SK activity was lost (FIG. 7). This suggests that SKAM1 has a direct effect on hSK1 activity in cells.

Preparation of GST-SKAM1 protein isoform
To test the direct effect of SKAM1 on hSK1 in vitro and to generate polyclonal anti-SKAM1 antibodies, the SKAM1 isoform was generated as a purified recombinant GST fusion protein. SKAM1A, B, C, D and Y DNA was cloned into the pGEX4T2 vector and recombinant GST-SKAM1 protein was produced in E. coli. The protein produced was then analyzed by SDS-PAGE and Coomassie staining. Proteins with a molecular weight of 54 kDa (GST-SKAM1A), 50 kDa (GST-SKAM1B), 45 kDa (GST-SKAM1C), 41 kDa (GST-SKAM1D) and 35 kDa (GST-SKAM1Y), 26 kDa (GST) were visualized (FIG. 8). Other proteins present in GST-SKAM1A, D and Y extracts are considered to be degradation products. The yield of GST-SKAM1 protein from this preparation was 364 μg (SKAM1A), 850 μg (SKAM1B), 870 μg (SKAM1C), 500 μg (SKAM1D) and 1500 μg (SKAM1Y) per 300 ml bacterial culture.

In vitro effect of SKAM1 on hSK1
Since hSK1 is a labile enzyme (Pitson et al., 2000a, supra), first, overexpression of SKAM1 enhances SK activity in cells by increasing SK stability in cells or cell extracts I hypothesized that it might be. Therefore, recombinant hSK1 (rec hSK1) and GST-SKAM1D were incubated together at 45 ° C. for 240 minutes and the effect of SKAM1 on the in vitro stability of hSK1 was measured by measuring the remaining SK activity at various time points. Tested. The results (Figure 9) showed that SKAM1D actually has a certain effect on the in vitro stability of rec-hSK1 and increases its half-life at 45 ° C from 7 minutes to 15 minutes. . However, the presence of SKAM1D markedly enhanced rec-hSK1 activity, more than twice as much as the GST alone control. This effect was confirmed by repeated analysis (Fig. 10), even though this was due to an unexpected SK activity of SKAM1D (Fig. 10), and the effect of SKAM1D on changes in the stability of rec-SK1 in this enzyme assay It was proved not to be caused by This is because rec-hSK1 was stable and showed a linear reaction rate under the present assay conditions (FIG. 11).

  To test whether this effect of GST-SKAM1D on rec-hSK1 is dose-dependent, the activity of rec-hSK1 was measured using increasing concentrations of GST-SKAM1D. The results showed that rec-hSK1 activity increased with increasing concentrations of GST-SKAM1D to at least a 10-fold molar excess of SKAM1D (FIG. 12). The maximum activity of rec-hSK1 was observed with a 20-fold molar excess of GST-SKAM1D.

Effect of GST-SKAM1D on human SK1 substrate dynamics
Since SKAM1D directly increases rec-hSK1 activity, we evaluated the effect of SKAM1D on hSK1 substrate kinetics. The effect of GST-SKAM1D on rec-hSK1 activity in the presence of increasing concentrations of ATP (FIG. 13) or sphingosine (FIG. 14) was measured and the data was analyzed using the Michaelis-Menten rate equation. The results (Table 2) show that the K _m value of rec-hSK1 for both ATP and sphingosine is not altered by the presence of GST-SKAM1D. However, in contrast, the K- _cat value (catalytic rate constant or number of revolutions) of rec-hSK1 was increased approximately 4-fold by GST-SKAM1D. This suggests that SKAM1 increases the rate of the enzymatic reaction and does not increase the binding affinity of hSK1 to the substrate.

Effects of other SKAM1 isoforms on recombinant human SK1 activity
GST-SKAM1 protein was incubated with rec-hSK1 in a 20-fold molar excess and the mixture was subjected to an in vitro SK activity assay. In the presence of these proteins, rec-hSK1 activity was increased approximately 4-fold by GST-SKAM1A and B and 2-3-fold by GST-SKAM1C, D and Y (FIG. 15). Furthermore, the effect of GST-SKAM1 isoform on hSK1 was very similar to that observed from overexpression of SKAM1 isoform in HEK-293T cells on endogenous hSK1 activity (FIG. 6).

SKAM1 expression does not alter TNFα-mediated SK activation Previous findings in this study have demonstrated that SKAM1 overexpression enhances cellular SK activity, so ERK1 / 2 promotes hSK1 in Ser225 The effect of phosphorylation mediated TNFα on intracellular SK activation (Pitson et al., 2003, supra) was tested. HEK293T cells overexpressing SKAM1A and SKAM1D (maximum and minimal naturally occurring SKAM isoforms) were treated with TNFα for 10 minutes, collected, and the SK activity of the cells was measured. As previously observed, overexpression of SKAM1 increased baseline SK activity approximately 2-fold compared to control cells (FIG. 16). Treatment of these SKAM1A or D overexpressing cells with TNFα further increases SK activity in the same manner as observed in control cells, so that SKAM1 does not affect phosphorylation-mediated SK activation It has been shown.

Interaction and activation of hSK2 by SKAM1
Since SKAM1 interacted with hSK1 and had a direct effect on its activity, its potential effect on the other human SK isoform, hSK2, was tested. The interaction of SKAM1A and D (maximum and minimal naturally occurring SKAM1 isoforms) with hSK2 was assessed by co-immunoprecipitation. Lysates from HEK-293T cells co-expressing FLAG-tagged hSK2 with either HA-SKAM1A or D were co-immunoprecipitated with anti-FLAG antibody. The presence of SKAM1 protein in the immune complex was then analyzed by immunoblotting using anti-HA antibody. 28 kDa (SKAM1A) and 15 kDa (SKAM1D) proteins were detected in the co-immunoprecipitated lysate (FIG. 17), indicating that there was an interaction between hSK2 and SKAM1A and D.

  Now that SKAM1 has been demonstrated to interact with hSK2, we next investigated whether GST-SKAM1 also has an effect on hSK2 activity in vitro. GST-SKAM1A, B, C, D and Y isoforms were incubated with rec-hSK2 in vitro and the resulting SK activity was measured. Similar to the results observed in rec-hSK1, all SKAM1 isoforms enhanced rec-hSK2 activity. hSK2 activity was increased approximately 5-fold by GST-SKAM1A, 4-fold by GST-SKAM1B, and about 2-3-fold by GST-SKAM1C, D and Y (FIG. 18).

Generation and characterization of polyclonal anti-SKAM1 antibodies To generate anti-SKAM1 polyclonal antibodies, New Zealand white rabbits were inoculated with purified recombinant GST-SKAM1D protein. SKAM1D is the smallest naturally occurring SKAM1 isoform, and since nearly all of its polypeptide sequence is present in all other SKAM1 isoforms, it was selected as the antigen. In other words, SKAM1D was considered to be the SKAM1 isoform with the highest possibility of producing antibodies that detect all SKAM1 proteins. The activity and specificity of the anti-SKAM1 antibody was tested with a final crude antiserum that detects SKAM1D overexpressed in HEK293T cells. SKAM1D with an estimated molecular weight of 15 kDa was detected by serial dilutions of anti-SKAM1 antiserum (FIG. 19A). The antiserum was very specific at high dilutions and showed only one band corresponding to SKAM1D. However, although some non-specific bands were seen on the blots at low dilutions, approximately 27 kDa and 23 kDa proteins representing endogenous SKAM1A and B isoforms were also observed (FIG. 19A).

  Since the anti-SKAM1 antibody can effectively detect overexpressed SKAM1D (the protein used to make the antibody), an investigation was made as to whether this antibody could detect other SKAM1 isoforms. Lysates from HEK293T cells expressing HA-SKAM1A, B, C, D and Y were immunoblotted with both anti-SKAM1 and anti-HA antibodies (FIGS. 19B, C). All SKAM1 isoforms were specifically detected by anti-SKAM1 antibodies at 28 kDa (SKAM1A), 24 kDa (SKAM1B), 19 kDa (SKAM1C), 15 kDa (SKAM1D) and 10 kDa (SKAM1Y). The difference in protein intensity in the anti-SKAM1 blot represents the difference in protein expression level in the lysate as shown by immunoblotting with anti-HA antibody (FIG. 19C).

  To assess the ability of anti-SKAM1 antibodies to immunoprecipitate overexpressed SKAM1 isoforms, lysates from HEK293T cells expressing HA-SKAM1A, B, C, D and Y are immunoprecipitated with anti-SKAM1 polyclonal antibodies It was. The immunoprecipitated SKAM1 protein was then immunoblotted with anti-SKAM1 antibody. FIG. 19D shows that all naturally occurring SKAM1 isoforms can be effectively immunoprecipitated. Artificial SKAM1Y immunoprecipitated with low efficiency.

  Since the anti-SKAM1 antibody was confirmed to be able to detect overexpressed SKAM1 through both Western blot and immunofluorescence, the ability of this antibody to detect endogenous SKAM1 in different cell lines was tested. It was impossible to accurately detect endogenous SKAM1 in HEK-293T cells. This is most likely due to the fact that these cells express SKAM1 only at low levels. Therefore, various other cell lines were tested for the presence of endogenous SKAM1 isoforms using this anti-SKAM1 antibody. Total mononuclear cells (TMNC), HEK293T, NIH3T3, MCF-7, human lymphocytes, leukemia cell line (HL60), human umbilical vein endothelial cells (HUVEC), human monocytes, mouse bone marrow, human neutrophils and mouse fetal liver Cells were subjected to SDS-PAGE and immunoblotted with 1: 5000 dilution of anti-SKAM1. A mouse cell line was employed because of high sequence conservation (99.6% amino acid identity) between human and mouse SKAM1. An approximately 23 kDa protein was detected in TMNC, human lymphocytes and MCF-7 cells and mouse fetal liver (FIG. 20). Endogenous SKAM1B appears to be this protein because it is 1 kDa smaller than SKAM1B overexpressed due to the HA epitope. In the lysates of HEK293T and NIH3T3 cells, there was a weak detection of two proteins of approximately 27 kDa and 23 kDa that would represent endogenous SKAM1A and B (FIG. 20A).

  To demonstrate that this protein is endogenous SKAM1, immunodepletion studies were performed using various lysates suspected of the presence of endogenous SKAM1, namely TMNC, HEK-293 and NIH3T3. Lysates from these cell lines were subjected to Western blot using antisera pretreated with either GST-SKAM1D conjugated to glutathione-Sepharose or GST alone. The results showed that the strength of the putative endogenous SKAM1 protein was greatly reduced after immune removal of anti-SKAM1 antiserum by GST-SKAM1D (FIG. 20B). This strongly suggests that the anti-SKAM1 antibody can detect endogenous SKAM1.

  The signal of endogenous SKAM1 in these lysates, especially HEK-293T and NIH3T3 lysates, was very small. Therefore, to enhance these signals, TMNC, HEK-293T and 3T3 cell lysates were immunoprecipitated with anti-SKAM1 antibody and then immunoblotted with anti-SKAM1 antibody. Two proteins of approximately 27 kDa and 23 kDa molecular weight, which would represent endogenous SKAM1A and B, were immunoprecipitated from the lysates of these three cells (FIG. 20C).

Intracellular localization of SKAM1
To test the intracellular localization of the SKAM1 protein, HA-tagged SKAM1A, B, C and D were overexpressed in NIH3T3 cells and subjected to immunofluorescence analysis using anti-HA antibody and anti-SKAM1 antibody. Both antibodies showed similar staining patterns and all SKAM1 isoforms were present throughout the cell, but most of this protein was present in the cytosol and not in the nucleus or part of the nucleolus (Figure 21). However, when SKAM1 proteins were very highly expressed in NIH3T3 cells, a partial localization of these proteins was observed in the endosome-like perinuclear structure (FIG. 22).

Intracellular co-localization of SKAM1 and hSK1
To test whether SKAM1 and hSK1 colocalize in the cells, the localization of both proteins was tested by immunofluorescence. NIH3T3 was transfected with GFP-tagged SK1 alone and with an HA-tagged SKAM1 isoform and then subjected to anti-HA immunofluorescence staining. When hSK1 was expressed alone, an extensive cytosolic staining pattern was seen as previously reported (Pitson et al., 2003, supra) (FIG. 23). As already described, the various SKAM1 isoforms also exhibit predominantly cytosolic diffusion, with some localized in a different endosomal-like perinuclear region (FIG. 23). When GFP-hSK1 was co-expressed with various SKAM1 isoforms, the localization of hSK1 shifted slightly, and hSK1 and a portion of SKAM1 co-localized in the perinuclear structure not yet identified (FIG. 23).

  Those skilled in the art should understand that the invention described herein may accept variations and modifications other than those specifically described herein. It should be understood that the invention encompasses all such variations and modifications. The present invention also contemplates all or all of the steps, features, compositions and compounds mentioned or shown herein, individually or collectively, and any and all of any two or more of these steps or features. Of combinations.

(Table 2) Overview of hSK1 substrate dynamics in the presence of SKAM1D

Bibliographic information

FIG. 2 is a schematic diagram for sequence alignment and expression of SKAM1 isoforms. (A) Alignment of predicted amino acid sequences of naturally occurring SKAM1 (A-SEQ ID NO: 1, B-SEQ ID NO: 2, C-SEQ ID NO: 3, and D-SEQ ID NO: 4) isoforms . Y2H (SEQ ID NO: 5) was the first protein that interacted with SK1 isolated in the yeast two-hybrid screen. SKAM1Y (SEQ ID NO: 6) represents the smallest SKAM1 region presumed to interact with SK1. Shaded identical and conserved amino acid substitutions. (B) Schematic diagram of the conserved region of SKAM1 isoform, (C) Expression of SKAM1 isoform added with HA epitope tag in HEK293T. Expression of SKAM1 isoforms, SKAM1A (28 kDa), SKAM1B (24 kDa), SKAM1C (19 kDa), SKAM1D (15 kDa) and SKAM1Y (10 kDa) was detected by Western blot using anti-HA monoclonals. It is an image about the interaction between SKAM1 isoform and hSK1 by immunoprecipitation using an anti-HA antibody. The interaction between hSK1 and SKAM1A, B, C, D and Y was assessed by co-immunoprecipitation. Lysates from cells co-transfected with HA-SKAM1A, B, C, D and Y along with hSK1 (FLAG) were immunoprecipitated with anti-HA antibody and then both chicken anti-SK1 antibody and anti-HA monoclonal antibody were used Analyzed by immunoblotting. It is an image about the interaction between SKAM1 isoform and hSK1 by immunoprecipitation using an anti-FLAG antibody. The interaction between hSK1 and SKAM1A, B, C, D and Y was assessed by co-immunoprecipitation. Lysates of cells co-transfected with hSK1 (FLAG) and HA-SKAM1A, B, C, D and Y were immunoprecipitated with anti-FLAG antibody and then with anti-SKAM1 antibody or anti-HA monoclonal antibody and anti-FLAG antibody Analyzed by immunoblotting. 28 kDa, 24 kDa, 19 kDa, and 15 kDa proteins representing SKAM1 isoforms were detected, indicating that SKAM1A, B, C, and D interact with hSK1. Image of the interaction between endogenous SKAM1 and overexpressed hSK1. The interaction between overexpressed hSK1 and endogenous SKAM1 was assessed by immunoprecipitation analysis. Lysates from cells transfected with either an empty vector or a plasmid encoding SKAM1A, SKAM1D, or hSK1 (FLAG) are immunoprecipitated with anti-SKAM1 antibody and then immunoblotted with chicken anti-SK1 antibody did. It is an image about the interaction between hSK1 ^S225A and SKAM1D. The interaction between hSK1 ^S225A and SKAM1D was evaluated by co-immunoprecipitation. ^Lysates from cells co-transfected with HA-SKAM1D and hSK1 ^S225A were immunoprecipitated using anti-HA antibodies. Immunoblots using anti-HA and anti-FLAG antibodies showed transgene expression. The immunoprecipitated complex was then immunoblotted using chicken anti-SK1 antibody and anti-HA monoclonal antibody. Figure 2 is a graph for the effect of SKAM1 isoform on endogenous SK activity. HEK-293T cells are transfected with SKAM1A, B, C, D and Y isoforms or empty pCMV (HA) expression vector (control) and total cell lysates are prepared 24 hours after transfection to increase SK activity It was measured. Data are the mean (± S.D.) Of 5 individual experiments performed in duplicate. FIG. 6 is an image of the effect of overexpression of hSK1 (hSK1 ^G82D ), which is inactive as a catalyst for SKAM1-mediated activation of endogenous SK activity. HEK293T cells were transfected with empty pCMV (HA) expression vector (control), SKAM1A and D isoforms alone or with hSK1 ^G82D . (A) Lysates from HEK293T cells co-expressing HA-SKAM1A and D together with hSK1 ^G82D were immunoprecipitated using anti-HA antibody. The immune complex was then immunoblotted using chicken anti-SK1 antibody and anti-HA monoclonal antibody. IgG is the light chain of the anti-HA antibody used in immunoprecipitation. (B) SK activity was measured in the cytosolic fraction. Data are the mean (± standard error) of two individual experiments and each experiment was performed in duplicate. It is an image about preparation of GST-SKAM1 protein. Coomassie stained gels show 54 kDa, 50 kDa, 45 kDa, 41 kDa and 35 kDa expression corresponding to GST-SKAM1A, B, C, D, Y and GST. Figure 2 is a graph of the effect of SKAM1 on hSK1 in vitro stability. GST-SKAM1D or GST was incubated with rec-hSK1 at 45 ° C. for 4 hours. (A) SK activity assays were performed at various designated times. (B) The half-life in the presence of hSK1 alone or in the presence of GST-SKAM1D was determined by plotting the data on a logarithmic scale. It is a graph about the effect of GST-SKAM1D with respect to rec-hSK1 activity. GST-SKAM1D or GST was incubated with rec-hSK1 and then subjected to an in vitro SK activity assay. It is a graph about the stability of hSK1 in a SK assay. Recombinant hSK1 was subjected to in vitro SK activity assay and aliquots were removed over time to determine S1P. It is a graph about the titration of GST-SKAM1D with respect to rec-hSK1 activity. Increasing concentrations of GST-SKAM1D or GST were incubated with rec-hSK1 and subjected to in vitro SK activity assay. The graph shows that SK1 activity increases in a dose response manner and reaches maximum activity with a 20-fold molar excess of GST-SKAM1D. It is a graph about the enzyme dynamics of ATP concentration with respect to hSK1. GST-SKAM1D and GST were added to rec-hSK1 at a concentration such that the molar ratio of hSK1 / GST-SKAM1 was 20, and SK activity was assayed using increasing concentrations of ATP. (A) Substrate dynamics of rec-hSK1 using ATP in a concentration range of 0-500 μM. (B) Line Weber-Burk plot of substrate dynamics. It is a graph about the enzyme dynamics of the sphingosine concentration with respect to hSK1. GST-SKAM1D and GST were added to rec-hSK1 at a concentration such that the molar ratio of hSK1 / GST-SKAM1 was 20, and SK activity was assayed using increasing concentrations of sphingosine. (A) Substrate dynamics of rec-hSK1 using sphingosine at a concentration of 0-50 μM. (B) Line Weber-Burk plot of substrate dynamics. Figure 2 is a graph for the effect of GST-SKAM1 isoform on hSK1 activity. GST-SKAM1A, B, C, D and Y at a concentration of 20 hSK1 / GST-SKAM1 molar ratio were incubated with rec-hSK1 and then subjected to in vitro SK activity assay. The graph shows that SK1 activity was increased approximately 4-fold by GST-SKAM1A or B and approximately 2-fold by GST-SKAM1C, D or Y compared to control GST. FIG. 6 is a graph for the effect of SKAM1 overexpression on hSK1 activation. HEK-293T cells were transfected with an empty vector or a plasmid encoding SKAM1A and D. Cells were serum starved for 5 hours 24 hours after transfection and then treated with TNFα (1 μg / ml) for 10 minutes to prepare total cell lysates and measure SK activity. Data are the mean (± SEM) of two individual experiments performed in duplicate. It is an image about the interaction between SKAM1 and hSK2. The interaction between hSK2 and SKAM1 isoforms A and D was assessed by coimmunoprecipitation. Lysates of cells co-transfected with HA-SKAM1A and D together with hSK2 (FLAG) were immunoprecipitated with anti-HA antibody. The immunoprecipitated complex was immunoblotted with both anti-HA monoclonal antibody and anti-FLAG antibody. IgG-H and IgG-L are the heavy and light chains of anti-HA antibodies, respectively. Figure 2 is a graph of the effect of GST-SKAM1 isoform on rec-hSK2 activity. GST-SKAM1A, B, C, D and Y at a concentration of 15 hSK2 / GST-SKAM1 molar ratio were incubated with rec-hSK2 and then subjected to in vitro SK activity assay. It is an image about the characteristic of an anti- SKAM1 antibody. (A) Titration of anti-SKAM1 antiserum using HEK293T cells overexpressing SKAM1D. A typical Western blot shows that 1: 1000 and 1: 10000 anti-SKAM1 antiserum dilutions detected 15 kDa SKAM1D in crude lysates of HEK293T cells. (B) Detection of HA-SKAM1 isoform using anti-SKAM1 antibody. A typical Western blot using both anti-SKAM1 and anti-HA antibodies shows HA-SKAM1 isoforms containing A, B, C, D, and Y with estimated molecular weights of 28 kDa, 24 kDa, 19 kDa, 15 kDa, and 10 kDa, respectively. Expression is shown. (C) Immunoprecipitation of HA-SKAM1 isoform using anti-SKAM1 antibody. Lysates from cells transfected with SKAM1A, B, C, D and Y were immunoprecipitated with anti-SKAM1 antibody and then immunoblotted with anti-SKAM1 antibody. It is an image about the detection of endogenous SKAM using an anti-SKAM1 antibody. (A) Lysates from various human and mouse cell lines were immunoblotted with anti-SKAM1 antibody. Approximately 27 kDa and 23 kDa proteins suspected to be endogenous SKAM1A and B were detected in lysates of TMNC, human lymphocytes, MCF-7, HEK-293T, and NIH3T3 cells. The SKAM1 positive control lane is a lane that pools all lysates from overexpressed HA-SKAM1A, B, C, and D isoforms. HUVEC are human umbilical vein endothelial cells. (B) Immunodepletion of endogenous SKAM1. A typical blot shows that the protein intensity representing endogenous SKAM1 is greatly reduced after incubation of the blot with anti-SKAM1 antibody immunodepleted by glutathione-sepharose-conjugated GST-SKAM1D, but anti-SKAM1 antibody is glutathione-sepharose-bound This is not the case for controls that were immunodepleted with GST alone. The SKAM1 positive control lane is a lane where all lysates from overexpressed HA-SKAM1A, B, C and D isoforms are pooled together. (C) Immunoprecipitation of endogenous SKAM1 using anti-SKAM1 antibody. Lysates from TMNC, HEK-293T and NIH3T3 were immunoprecipitated and two proteins of approximately 27 kDa and 23 kDa corresponding to endogenous SKAM1A and B were obtained by immunoblotting with anti-SKAM1 antibody. The SKAM1 positive control lane is a lane that pools all lysates of overexpressed HA-SKAM1A, B, C, and D isoforms. The IP control lane is a lane in which the lysate was incubated under the same conditions except for the anti-SKAM1 antibody. It is an image about the immunofluorescence test of HA-SKAM1 isoform. NIH3T3 cells were transfected with HA-SKAM1A, B, C and D, fixed and stained with both anti-SKAM1 and anti-HA antibodies. It is an image about the immunofluorescence test by the anti- SKAM1 antibody of the HA-SKAM1 isoform expressed at a high level. HA-SKAM1A, B, C and D were overexpressed in NIH3T3 cells, fixed and stained with anti-SKAM1 antibody. It is an image about the colocalization of SKAM1 isoform and hSK1. GFP-hSK1 was transiently expressed in NIH3T3 cells alone or with an HA-tagged SKAM1 isoform. Cells were plated on slides, fixed, permeabilized and then immunostained with anti-HA antibody followed by Alexa594 labeled secondary antibody.

Claims (43)

  A method of modulating sphingosine kinase-mediated signaling, wherein the time and conditions are sufficient to modulate the interaction of SKAM1, or a functional derivative, variant, homolog or mimetic thereof, and sphingosine kinase A step of contacting the sphingosine kinase with an effective amount of an agent, wherein induction or otherwise agonizing upregulates the catalytic activity of the sphingosine kinase, and inhibiting or otherwise antagonizing the A method of down-regulating catalytic activity.   A method for modulating sphingosine kinase-mediated cellular activity, wherein the drug is effective in a time and under conditions sufficient to modulate the interaction of SKAM1 or a functional derivative, homolog or mimetic thereof with sphingosine kinase Inducing or otherwise agonizing up-regulates the cellular activity, and inhibiting or otherwise antagonizing down-regulating the cellular activity.   The method according to claim 1 or 2, wherein SKAM1 is SKAM1A, SKAM1B, SKAM1C, SKAM1D, or abbreviated SKAM1Y.   The method according to any one of claims 1 to 3, wherein the sphingosine kinase is human sphingosine kinase.   5. The method of claim 4, wherein the human sphingosine kinase is sphingosine kinase 1.   5. The method according to claim 4, wherein the human sphingosine kinase is sphingosine kinase 2.   The modulation is an upregulation of sphingosine kinase catalytic activity level, wherein the upregulation is a nucleic acid molecule encoding SKAM1A, SKAM1B, SKAM1C, SKAM1D, or a truncated SKAM1Y, or a functional equivalent, derivative, or homologue thereof, or The method according to claim 1 or 2, achieved by introducing into the cell a SKAM1A, SKAM1B, SKAM1C, SKAM1D, or truncated SKAM1Y expression product, or a functional derivative, homologue, analog, equivalent or mimetic thereof. Method.   The regulation is an upregulation of sphingosine kinase activity level, wherein the upregulation is achieved by contacting the cell with a proteinaceous or non-proteinaceous molecule that functions as an agonist of the sphingosine kinase / SKAM1 interaction. Or the method of 2.   The regulation is a down-regulation of sphingosine kinase activity level, wherein the down-regulation is achieved by contacting the cell with a proteinaceous or non-proteinaceous molecule that functions as an antagonist of the sphingosine kinase / SKAM1 interaction. Or the method of 2.   10. The method of claim 9, wherein the antagonist is a SKAM1 competitor.   10. The method of claim 9, wherein the antagonist is against the amino acid region defined by residues 51-132 of SEQ ID NOs: 1-4.   12. The method according to claim 9 or 11, wherein the antagonist is an antibody against SKAM1.   10. The method of claim 9, wherein the antagonist is an antisense nucleic acid molecule, siRNA, or a nucleic acid molecule suitable for inducing co-suppression, wherein the molecule is against SKAM1.   A method for the treatment and / or prevention of a condition in a mammal characterized by abnormal, undesirable or otherwise inappropriate sphingosine kinase-mediated cellular activity comprising SKAM1, or a functional derivative thereof Administering an effective amount of the agent to the mammal for a time and under conditions sufficient to modulate the interaction of the sphingosine kinase with a homolog or mimetic, wherein induction of the association or otherwise agonizing comprises A method wherein the cellular activity is upregulated and inhibition of the association or otherwise antagonizing downregulates the cellular activity.   A method for the treatment and / or prevention of a condition in a mammal characterized by abnormal, undesirable or otherwise inappropriate sphingosine kinase functional activity, comprising SKAM1, or a functional derivative, homologue thereof Or administering an effective amount of the agent to the mammal for a time and under conditions sufficient to modulate the interaction of the mimetic with the sphingosine kinase, wherein the induction of the association or else the agonizing comprises the sphingosine kinase Wherein the inhibition or otherwise antagonization down-regulates the functional activity of sphingosine kinase.   16. The method of claim 14 or 15, wherein the condition is a neoplastic condition or other undesirable cell proliferation and the sphingosine kinase-SKAM1 interaction is downregulated.   16. The method of claim 14 or 15, wherein the condition is an inflammatory condition and the sphingosine kinase-SKAM1 interaction is downregulated.   15. The method of claim 14, wherein the undesirable cellular activity is secretion of inflammatory mediators or expression of adhesion molecules and the sphingosine kinase / SKAM interaction is downregulated.   18. The method of claim 17, wherein the inflammatory condition is associated with rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease, or inflammatory bowel disease.   The modulation is an upregulation of sphingosine kinase catalytic activity level, wherein the upregulation is a nucleic acid molecule encoding SKAM1A, SKAM1B, SKAM1C, SKAM1D, or a truncated SKAM1Y, or a functional equivalent, derivative, or homologue thereof, or The method according to claim 14 or 15, which is achieved by introducing an expression product of SKAM1A, SKAM1B, SKAM1C, SKAM1D, or a truncated SKAM1Y, or a functional derivative, homolog, analog, equivalent or mimetic thereof, into a cell. Method.   15. The modulation is upregulation of sphingosine kinase activity level, wherein the upregulation is achieved by contacting the cell with a proteinaceous or non-proteinaceous molecule that functions as an agonist of the sphingosine kinase / SKAM1 interaction. Or the method of 15.   15. The regulation is downregulation of sphingosine kinase activity levels, wherein the downregulation is achieved by contacting the cell with a proteinaceous or non-proteinaceous molecule that functions as an antagonist of the sphingosine kinase / SKAM1 interaction. The method of any one of -19.   24. The method of claim 22, wherein the antagonist is a SKAM1 competitor.   24. The method of claim 22, wherein the antagonist is to the amino acid region defined by residues 51-132 of SEQ ID NOs: 1-4.   25. The method of claim 22 or 24, wherein the antagonist is an antibody against SKAM1.   23. The method of claim 22, wherein the antagonist is an antisense nucleic acid molecule, siRNA, or a nucleic acid molecule suitable for inducing co-suppression, said molecule being against SKAM1.   27. The method according to any one of claims 1 to 26, wherein the mammal is a human.   Use of a drug in the manufacture of a medicament for the treatment of a condition in a mammal characterized by abnormal, undesirable or otherwise inappropriate cellular activity, wherein the drug interacts with SKAM1 and sphingosine kinase Use, wherein the induction of the interaction or otherwise agonizing upregulates the cellular activity, and the inhibition of the interaction or otherwise antagonizing downregulates the cellular activity.   Use of a drug in the manufacture of a medicament for the treatment of a condition in a mammal characterized by an abnormal, undesirable or otherwise inappropriate sphingosine kinase functional activity, said drug comprising SKAM1 and sphingosine kinase Inhibition of the interaction or otherwise antagonizing down-regulates the functional activity of sphingosine kinase Use.   30. Use according to claim 28 or 29, wherein the condition is a neoplastic condition or other undesirable cell proliferation and the sphingosine kinase / SKAM1 interaction is downregulated.   30. Use according to claim 28 or 29, wherein the condition is an inflammatory condition and the sphingosine kinase / SKAM1 interaction is downregulated.   29. Use according to claim 28, wherein the undesirable cellular activity is secretion of inflammatory mediators or expression of adhesion molecules and the sphingosine kinase / SKAM1 interaction is downregulated.   32. The use of claim 31, wherein the inflammatory condition is associated with rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease, or inflammatory bowel disease.   The modulation is an upregulation of sphingosine kinase catalytic activity level, wherein the upregulation is a nucleic acid molecule encoding SKAM1A, SKAM1B, SKAM1C, SKAM1D, or a truncated SKAM1Y, or a functional equivalent, derivative, or homologue thereof, or The method according to claim 28 or 29, which is achieved by introducing an expression product of SKAM1A, SKAM1B, SKAM1C, SKAM1D, or a truncated SKAM1Y, or a functional derivative, homologue, analog, equivalent or mimetic thereof, into a cell. use.   28. The modulation is upregulation of sphingosine kinase activity level, wherein the upregulation is achieved by contacting the cell with a proteinaceous or non-proteinaceous molecule that functions as an agonist of the sphingosine kinase / SKAM1 interaction. Or use of 29 description.   28. The regulation is downregulation of sphingosine kinase activity level, wherein the downregulation is achieved by contacting the cell with a proteinaceous or non-proteinaceous molecule that functions as an antagonist of the sphingosine kinase / SKAM1 interaction. Use according to any one of .about.33.   40. Use according to claim 36, wherein the antagonist is a SKAM1 competitor.   37. Use according to claim 36, wherein the antagonist is against the amino acid region defined by residues 51-132 of SEQ ID NOs: 1-4.   39. Use according to claim 36 or 38, wherein the antagonist is an antibody against SKAM1.   37. Use according to claim 36, wherein the antagonist is an antisense nucleic acid molecule, siRNA or a nucleic acid molecule suitable for inducing co-suppression, said molecule being against SKAM1.   41. Use according to any one of claims 28 to 40, wherein the mammal is a human.   An agent that modulates the interaction of SKAM1 and sphingosine kinase when used according to the method of any one of claims 1-31, together with one or more pharmaceutically acceptable carriers and / or diluents A pharmaceutical composition comprising.   An agent that modulates the interaction of SKAM1 and sphingosine kinase when used according to the method of any one of claims 1-27.

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