# Patent

## Title
Methods for the identification of phosphatidylinositol kinase interacting molecules and for the purification of phosphatidylinositol kinase proteins

## Abstract

The present invention relates to immobilization compounds of formula (I), immobilization products and preparations thereof as well as methods and uses for the identification of phosphatidylinositol kinase interacting compounds or for the purification or identification of phosphatidylinositol kinase proteins.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national stage filing under 35 U.S.C. §371 of international application PCT/EP2010/002987, filed May 14, 2010, which claims benefit of the filing date of European Application No. 09006786.9, filed May 20, 2009.
The present invention relates to immobilization compounds, immobilization products and preparations thereof as well as methods and uses for the identification of phosphatidylinositol kinase interacting compounds or for the purification or identification of phosphatidylinositol kinase proteins.
Phosphatidylinositol, a component of eukaryotic cell membranes, is unique among phospholipids in that its head group can be phosphorylated at multiple free hydroxyls. Several phosphorylated derivatives of phosphatidylinositol, collectively termed phosphoinositides, have been identified in eukaryotic cells. Phosphoinositides are involved in the regulation of diverse cellular processes, including proliferation, survival, cytoskeletal organization, vesicle trafficking, glucose transport, and platelet function. The enzymes that phosphorylate phosphatidylinositol and its derivatives are termed phosphatidylinositol kinases or phosphoinositide kinases (Fruman et al., 1998. Annual Rev. Biochem. 67:481-507).
Phosphoinositide 3-kinases (also called Phosphatidylinositol 3-kinases, PI3Ks) represent a a superfamily of signaling lipid kinases that catalyse the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2 or phosphatidylinositol (PtdIns) at the 3′-OH group, giving rise to the second messengers phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) or phosphatidylinositol-3-phosphate (PtdIns(3)P). PtdIns(3,4,5)P3 can be converted into PtdIns(3,4)P2 by SH2-containing inositol phosphatase (SHIP), or can be dephosphorylated by phosphatase and tensin homologue (PTEN) phosphatase to regenerate PtdIns(4,5)P2. The 3′-phosphorylated phosphoinositides, PtdIns(3,4,5)P3, PtdIns(3,4)P2 PtdIns(4,5)P2, PtdIns(5)P and PtdIns(3)P, recruit and activate various signalling proteins (PtdInsbinding proteins; PtdIns-BPs) through direct lipid-protein interactions. Some PI3Ks also display protein kinase activity (Fruman et al., 1998, Annu. Rev. Biochem. 67:481-507).
Different types of PI3K have been identified and grouped into three classes according to their primary and secondary structures, mode of regulation and substrate specificity. Class I PI3K has been the most extensively studied so far, and includes heterodimeric proteins that consist of a catalytic and a regulatory adaptor subunit, the nature of which determines a further subdivision into class IA and IB PI3K. Class II PI3K uses PtdIns as in vivo substrate, yielding phosphatidylinositol-3-phosphate (PtdIns(3)P). Some evidence has been presented that class II enzymes, similarly to class I can be activated by external stimuli via receptor tyrosine kinases (RTKs), cytokine receptors and integrins, suggesting roles in cancer, wound healing and insulin signaling. By contrast, the class III PI3K, represented by a single species (hVps34) in humans, has relatively high activity even in resting cells. The class IA—PI3Kα, β and δ (PIK3CA, PIK3CB and PIK3CD)—consists of a SH2-domain-containing regulatory subunit (p85; five distinct isoforms of which have been identified) that forms a complex with one of three catalytic subunits, p110α, p110β or p110δ. PI3Kγ, the only member of class IB (PIK3CG), associates with either of two regulatory subunits, p101 and p84, that control its activation and subcellular location (Bader et al., 2005, Nat. Rev. Cancer 5 (12):921-9).
Phosphatidylinositol 4-kinases catalyse the production of phosphatidylinositol 4-phosphate (PtdIns 4-phosphate, PtdIns4P) from phosphatidylinositol, the first step in the formation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3, two lipid products whose functions as regulatory molecules are best understood. Four distinct phosphatidylinositol 4-kinases have been identified in mammalian cells (PI4KIIα, PI4KIIβ, PI4KIIIα (synonym PIK4CA), and PI4KIIIβ (synonym PIK4CB)) (Balla and Balla, 2006. Trends in Cell Biology 16(7):351-361).
Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphorylating phosphatidylinositol 4-phosphate. As a precursor for second messengers generated by phospholipase C isoforms and class I PI3Ks, PIP2 is indispensable for cellular signaling by membrane receptors. Three isoforms of PIP5k with alternative splice variants have been cloned and characterized (PIP5K2A, PIP5K2B and PIP5K2C) so far (Weernink et al., 2004. Europ. J. Pharmacol. 500, 87-99).
The in vitro investigation of phosphatidylinositol kinase activity is typically performed using radioactively labelled ATP and the transfer of phosphor-groups into phospholipid substrates incorporated in unilamellar lipid vesicle (ULVs) followed by thin layer chromatography (TLC) analysis of reaction products. These assays are sensitive and specific but require vesicle preparation which can be challenging for large-scale production necessary for high-throughput screening to identify phosphatidylinositol kinase inhibitors. In addition, typically these assays require the availability of purified or recombinant phosphatidylinositol kinases.
For example, PI3K phosphatidylinositol kinase activity can be measured using purified or recombinant enzyme in a solution-based assay with phospholipid vesicles. The reaction is terminated by the addition of acidified organic solvents and subsequent phase separation by extraction or thin layer chromatography analysis (Carpenter et al., 1990, J. Biol. Chem. 265, 19704-19711). Another PI3K assay described in the art is based on the phosphate transfer from radiolabeled ATP to phosphatidylinositol immobilized on plates. This assay type uses recombinant PI3Kγ enzyme and can be performed in a high-throughput mode as a vesicle free assay format (Fuchikami et al., 2002, J. Biomol. Screening 7, 441-450). A high-throughput liposome PI3K assay with an automated lipid extraction process was described that allows to quantitatively measure inhibitor activity (Lingaraj et al., 2008. J. Biomol. Screening 13(9):906-11). For the phosphatidylinositol-4-phosphate 5-kinases (PIP5K) a vesicle assay was reported that uses radioactive ATP and recombinant PIP5Ks followed by thin layer chromatography or HPLC analysis of the reaction products (Tolias et al, 1998. J. Biol. Chem. 273, 18040-18046).
Another, although not in all instances necessary prerequisite for the identification of selective kinase inhibitors is a method that allows to determine the target selectivity of these molecules. For example, it can be intended to provide molecules that bind to and inhibit a particular drug target but do not interact with a closely related target, inhibition of which could lead to unwanted side effects. Conventionally large panels of individual enzyme assays are used to assess the inhibitory effect of a compound for protein kinases (Bain et al., 2007. Biochemical Journal 408(3):297-315) and lipid kinases (Garcia-Martinez et al., 2009. Biochemical Journal 421(1):29-42, PMID: 19402821).
More recently, kinases or kinase domains displayed on bacteriophages have been employed to assess the ability of a given compound to interact with a large set of kinases (Karaman et al., 2008. Nature Biotechnology 26, 127-132). In addition, chemical proteomics methods have been described which allow the profiling of kinase inhibitors against the proteome (WO 2006/134056; WO2008/015013; Bantscheff et al., 2007. Nature Biotechnology 25, 1035-1044; Patricelly et al., 2007. Biochemistry 46, 350-358; Gharbi et al., 2007. Biochem. J. 404, 15-21).
In view of the above, there is a need for providing effective tools and methods for the identification and selectivity profiling of phosphatidylinositol kinase interacting compounds as well as for the purification of phosphatidylinositol kinases.
The present invention relates inter alia to an immobilization compound of formula (I)




or a salt thereof, wherein

    R is C1-4 alkyl optionally substituted with one or more fluoro (preferably unsubstituted C1-4 alkyl; more preferably, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; even more preferably, isopropyl, or tert-butyl); and
    n is 1, 2, or 3 (preferably, 1, or 2; more preferably, 1).


“C1-4 alkyl” means an alkyl chain having 1-4 carbon atoms, i.e. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. Each hydrogen of a C1-4 alkyl carbon may be replaced by a substituent as further specified.
Preferred immobilization compounds of formula (I) are selected from the group consisting of




or a mixture of both.

The immobilization compounds of the present invention can be prepared by methods well known in the art. Exemplary analogous routes for the synthesis are described in, for example, in WO-A 2008/025821.
A general route for the synthesis of immobilization compounds of the present invention is shown in Example 1.
The invention further relates to a method for the preparation of an immobilization product, wherein at least one immobilization compound according to the invention is immobilized on a solid support. Such immobilization products obtainable according to the method of the invention are e.g. useful in the methods of the invention for the identification of kinase interacting compounds or in diagnostic methods for the diagnosis of inflammatory diseases, proliferative diseases and metabolic diseases.
According to the method of the invention, at least one immobilization compound of the invention is immobilized on a solid support. Throughout the invention, the term “solid support” relates to every undissolved support being able to immobilize a small molecule ligand on its surface.
According to the invention, the term “at least one immobilization compound” means either that at least one immobilization compound of the same type is immobilized on the solid support or that one or more different immobilization compounds (each of them either in singular or plural) may be immobilized on the solid support. Preferably, one or two different immobilization compounds are immobilized on the solid support, more preferably the preferred immobilization compounds of formula (I) of the present invention selected from the group consisting of




are immobilized.

The solid support may be selected from the group consisting of agarose, modified agarose, sepharose beads (e.g. NHS-activated sepharose), latex, cellulose, and ferro- or ferrimagnetic particles.
In case that the solid support is a material comprising various entities, e.g. in case that the solid support comprises several beads or particles, it is envisaged within the present invention that, if different immobilization compounds are immobilized, on each single entity, e.g. each bead or particle, one or more different immobilization compounds are immobilized. Therefore, in case that two immobilization compounds are used, it is envisaged within the present invention that on each single entity one or two different immobilization compounds are immobilized. If no measures are taken that on one entity only one different immobilization compound is immobilized, it is very likely that on each entity all different immobilization compounds will be present.
The immobilization compound or compounds of the invention may be coupled to the solid support either covalently or non-covalently. Non-covalent binding includes binding via biotin affinity ligands binding to steptavidin matrices.
Preferably, the immobilization compound or compounds are covalently coupled to the solid support.
Methods for immobilizing compounds on solid supports are known in the art and further exemplified in Example 1.
In general, before the coupling, the matrixes can contain active groups such as NHS, Carbodimide etc. to enable the coupling reaction with the immobilization compound. The immobilization compound can be coupled to the solid support by direct coupling (e.g. using functional groups such as amino-, sulfhydryl-, carboxyl-, hydroxyl-, aldehyde-, and ketone groups) and by indirect coupling (e.g. via biotin, biotin being covalently attached to the immobilization product of the invention and non-covalent binding of biotin to streptavidin which is bound directly to the solid support).
The linkage to the solid support material may involve cleavable and non-cleavable linkers. The cleavage may be achieved by enzymatic cleavage or treatment with suitable chemical methods.
Therefore, according to a preferred embodiment of the invention, the immobilization product results from a covalent direct or linker mediated attachment of the at least one immobilization compound of the invention to the solid support.
The linker may be a C1-10 alkylene group, which is optionally interrupted or terminated by one or more atoms or functional groups selected from the group consisting of S, O, NH, C(O)O, C(O), and C(O)NH and wherein the linker is optionally substituted with one or more substituents independently selected from the group consisting of halogen, OH, NH2, C(O)H, C(O)NH2, SO3H, NO2, and CN.
The term “C1-10 alkylene” means an alkylene chain having 1-10 carbon atoms, e.g. methylene, ethylene, —CH═CH—, —C≡C—, n-propylene and the like, wherein each hydrogen of a carbon atom may be replaced by a substituent.
The term “interrupted” means that the one or more atoms or functional groups are inserted between two carbon atoms of the alkylene chain or—when “terminated”—at the end of said chain.
The invention further relates to an immobilization product, obtainable by the method of the invention.
Furthermore, the present invention relates to an immobilization product, comprising the immobilization compound of the invention immobilized on a solid support, in particular wherein the solid support is selected from the group consisting of agarose, modified agarose, sepharose beads (e.g. NHS-activated sepharose), latex, cellulose, and ferro- or ferrimagnetic particles.
Therefore, an immobilization product which is obtainable by the method of the invention is or comprises an immobilization compound of the present invention immobilized on a solid support. This immobilization product will be referred to in the following as the immobilization product of the invention and is used in the methods of the present invention.
In a preferred embodiment, the immobilization compound or immobilization product of the invention may further be labeled.
By “labeled” is meant that the respective substance is either directly or indirectly labeled with a molecule which provides a detection signal, e.g. radioisotope, fluorescent tag, chemiluminescent tag, a peptide or specific binding molecules. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin. The label can directly or indirectly provide a detectable signal. The tag can also be a peptide which can be used, for example, in an enzyme fragment complementation assay (e.g. beta-galactosidase enzyme fragment complementation; Zaman et al., 2006. Assay Drug Dev. Technol. 4(4):411-420). The labeled compounds would be useful not only in imaging techniques but also in assays, both in vitro and in vivo, for identifying kinase interacting compounds by inhibition of binding of the labeled compound, for example in kinase assays that contain such labeled compounds.
Radioisotopes are commonly used in biological applications for the detection of a variety of biomolecules and have proven to be useful in binding assays. Several examples of probes have been designed to incorporate 3H (also written as T for tritium) because it can replace hydrogen in a probe without altering its structure (Fenteany et al., 1995. Science 268:726-731). An “isotopically” or “radio-labeled” compound is a compound of the invention where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). Suitable radionuclides that may be incorporated in compounds of the present invention include but are not limited to 2H (also written D for Deuterium), 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 18F, 35S, 36Cl, 82Br, 75Br, 76Br, 77Br, 123I, 124I, 125I and 131I.
Guidance for the selection and methods for the attachment of fluorescent tags (e.g. fluorescein, rhodamine, dansyl, NBD (nitrobenz-2-oxa-1,3-diazole), BODIPY (dipyrromethene boron difluoride), and cyanine (Cy)-dyes) to small molecule ligands are generally known in the art (Vedvik et al., 2004. Assay Drug Dev. Technol. 2(2): 193-203; Zhang et al., 2005. Analytical Biochemistry 343(1):76-83). The application of fluorescent probes (fluorophores) in assays for high throughput screening (HTS) of protein kinases was described (Zaman et al., 2003. Comb. Chem. High Throughput Screen 6(4): 313-320). The change of the fluorescent properties after binding of the fluorescent probe to the target kinase can be determined by measuring for example fluorescence polarization (Kashem et al., 2007. J. Biomol. Screening 12(1):70-83), fluorescence resonance energy transfer (FRET; Zhang et al., 2005. Analytical Biochemistry 343(1):76-83) or fluorescence lifetime (Moger et al., 2006. J. Biomol. Screening 11(7): 765-772). In addition, the ALPHAScreen technology can be used where the excitation of a donor bead at 680 nm produces singlet oxygen which can diffuse to an acceptor bead undergoing a chemiluminescent reaction (Glickman et al., 2002. J. Biomol. Screen. 7(1):3-10).
One possible use of the immobilization products of the invention is in the context of the identification of compounds interacting with phosphatidylinositol kinases. Therefore, the present invention also relates to such methods and uses.
In a first aspect of the methods of the invention, the invention therefore relates to a method for the identification of a phosphatidylinositol kinase interacting compound, comprising the steps of

    
    
        a) providing a protein preparation containing a variety of phosphatidylinositol kinases,
        b) contacting the protein preparation with the immobilization product of the invention under conditions allowing the formation of one or more different complexes between one of the phosphatidylinositol kinases and the immobilization product,
        c) incubating the one or more different complexes with a given compound, and
        d) determining whether the compound is able to separate the phosphatidylinositol kinase from the immobilization product.
    
    


In a second aspect, the present invention relates into a method for the identification of a phosphatidylinositol kinase interacting compound, comprising the steps of

    
    
        a) providing a protein preparation containing a variety of phosphatidylinositol kinases,
        b) contacting the protein preparation with the immobilization product of the invention and with a given compound under conditions allowing the formation of one or more different complexes between one of the phosphatidylinositol kinases and the immobilization product, and
        c) detecting the complex or the complexes formed in step b).
    
    


In a third aspect, the present invention relates to a method for the identification of a phosphatidylinositol kinase interacting compound, comprising the steps of:

    
    
        a) providing two aliquots of a protein preparation containing a variety of phosphatidylinositol kinases,
        b) contacting one aliquot with the immobilization product of the invention under conditions allowing the formation of one or more different complexes between one of the phosphatidylinositol kinases and the immobilization product,
        c) contacting the other aliquot with the immobilization product of the invention and with a given compound under conditions allowing the formation of one or more different complexes between one of the phosphatidylinositol kinases and the immobilization product, and
        d) determining the amount of the complex or the complexes formed in steps b) and c).
    
    


In a fourth aspect, the invention relates to a method for the identification of a phosphatidyl-inositol kinase interacting compound, comprising the steps of:

    
    
        a) providing two aliquots of a cell preparation comprising each at least one cell containing a variety of phosphatidylinositol kinases,
        b) incubating one aliquot with a given compound,
        c) harvesting the cells of each aliquot,
        d) lysing the cells in order to obtain protein preparations,
        e) contacting the protein preparations with the immobilization product of the invention under conditions allowing the formation of one or more different complexes between one of the phosphatidylinositol kinases and the immobilization product, and
        f) determining the amount of the complex or the complexes formed in each aliquot in step e).
    
    


In the context of the present invention, it has been found that the immobilization products of the present invention are suitable for the identification of compounds interacting with phosphatidylinositol kinases.
The immobilization products of the present invention bind to a variety of kinases, especially phosphatidylinositol kinases. Especially, they bind to kinases listed in tables 4, 5 and 6 shown in the examples.
For example, the following kinases were identified in example 2 (Table 4):

    PIK3Ca, PIK3Cb, PIK4Ca, PIP5K2C, PIK3Cg, P1K3Cd.


In addition, for example, the following kinases were identified in example 3 (Table 5):

    PIP5K2A, PIK4C2B, PIK3C3.


In addition, for example, the following kinases were identified in example 4 (Table 6):

    PIP5K2B, PIK3C2b.


Consequently, in the methods of the present invention, these immobilization products can be used to identify compounds binding to at least one kinase out of said variety of phosphatidylinositol kinases.
According to the present invention, the expression “phosphatidylinositol kinase” means enzymes that phosphorylate phosphatidylinositol or its phosphorylated derivatives.
According to the present invention, the term “variety” means one or more different types of the enzyme class of interest, in the present case phosphatidylinositol kinases.
Examples of phosphatidylinositol kinases are:

    Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3Ca),
    Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3Cb);
    Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3Cg);
    Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3Cd);
    Phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing beta (PIK3C2b);
    Phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3; VPS34 homolog);
    Phosphatidylinositol 4-kinase alpha (PIK4Ca);
    Phosphatidylinositol 4-kinase type 2-beta (PIK4C2B);
    Phosphatidylinositol-4-phosphate 5-kinase type-2 alpha (PIP5K2A);
    Phosphatidylinositol-4-phosphate 5-kinase type-2 beta (PIP5K2B);
    Phosphatidylinositol-4-phosphate 5-kinase type-2 gamma (PIP5K2C).


According to the present invention, the expression “phosphatidylinositol kinase” relates to both human and other proteins of this family. The expression especially includes functionally active derivatives thereof, or functionally active fragments thereof, or a homologues thereof, or variants encoded by a nucleic acid that hybridizes to the nucleic acid encoding said protein under low stringency conditions. Preferably, these low stringency conditions include hybridization in a buffer comprising 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% BSA, 100 ug/ml denatured salmon sperm DNA, and 10% (wt/vol) dextran sulfate for 18-20 hours at 40° C., washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS for 1-5 hours at 55° C., and washing in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4) 5 mM EDTA, and 0.1% SDS for 1.5 hours at 60° C.
Moreover, according to the present invention, the expression “phosphatidylinositol kinase” includes mutant forms said kinases. For example, the PIK3CA gene encoding the catalytic subunit p110α is frequently mutated in human solid tumours. Cancer-specific mutations are clustered in the helical and the kinase domains of p110α with amino acid residues E542, E545 and H1047 as prominent mutational hotspots (Bader et al., 2005. Nature Reviews Cancer 5, 921-929),
In some aspects of the invention, first a protein preparation containing said phosphatidylinositol kinases or kinase is provided. The methods of the present invention can be performed with any protein preparation as a starting material, as long as the respective kinase is solubilized in the preparation. Examples include a liquid mixture of several proteins, a cell lysate, a partial cell lysate which contains not all proteins present in the original cell or a combination of several cell lysates. The term “protein preparation” also includes dissolved purified protein.
In another aspect of the invention, aliquots of a cell preparation are provided as the starting material. In the context of the present invention, the term “cell preparation” refers to any preparation containing at least one cell with the desired properties. Suitable cell preparation are described below.
The presence of the phosphatidylinositol kinases in a protein preparation of interest can be detected on Western blots probed with antibodies that are specifically directed against said kinase. Alternatively, also mass spectrometry (MS) could be used to detect the kinases (see below).
Cell lysates or partial cell lysates can be obtained by isolating cell organelles (e.g. nucleus, mitochondria, ribosomes, golgi etc.) first and then preparing protein preparations derived from these organelles. Methods for the isolation of cell organelles are known in the art (Chapter 4.2 Purification of Organelles from Mammalian Cells in “Current Protocols in Protein Science”, Editors: John. E. Coligan, Ben M. Dunn, Hidde L. Ploegh, David W. Speicher, Paul T. Wingfield; Wiley, ISBN: 0-471-14098-8).
In addition, protein preparations can be prepared by fractionation of cell extracts thereby enriching specific types of proteins such as cytoplasmic or membrane proteins (Chapter 4.3 Subcellular Fractionation of Tissue Culture Cells in “Current Protocols in Protein Science”, Editors: John. E. Coligan, Ben M. Dunn, Hidde L. Ploegh, David W. Speicher, Paul T. Wingfield; Wiley, ISBN: 0-471-14098-8).
Furthermore protein preparations from body fluids can be used (e.g. blood, cerebrospinal fluid, peritoneal fluid and urine).
For example whole embryo lysates derived from defined development stages or adult stages of model organisms such as C. elegans can be used. In addition, whole organs such as heart dissected from mice can be the source of protein preparations. These organs can also be perfused in vitro in order to obtain a protein preparation.
Furthermore, the protein preparation may be a preparation containing the kinase or the kinases which has been recombinantely produced. Methods for the production of recombinant proteins in prokaryotic and eukaryotic cells are widely established (Chapter 5 Production of Recombinant Proteins in “Current Protocols in Protein Science”, Editors: John. E. Coligan, Ben M. Dunn, Hidde L. Ploegh, David W. Speicher, Paul T. Wingfield; Wiley, 1995, ISBN: 0-471-14098-8).
In a preferred embodiment of the methods of the invention, the provision of a protein preparation includes the steps of harvesting at least one cell containing the phosphatidylinositol kinase or the kinases and lysing the cell.
Suitable cells for this purpose as well as for the cell preparations used as the starting material in one aspect of the present invention are e.g. those cells or tissues where the kinases are expressed. In any given cell or tissue only a subset of the kinome may be expressed. Therefore it may be necessary to generate multiple protein preparations from a variety of cell types and tissues to cover the kinome, especially for selectivity profiling of kinase inhibitors. As established cell lines may not reflect the physiological expression pattern of kinases, primary animal or human cells may be used, for example cells isolated from blood samples.
Therefore, in a preferred embodiment, cells isolated from peripheral blood represent a suitable biological material. Procedures for the preparation and culture of human lymphocytes and lymphocyte subpopulations obtained from peripheral blood (PBLs) are widely known (W. E Biddison, Chapter 2.2 “Preparation and culture of human lymphocytes” in Current Protocols in Cell Biology, 1998, John Wiley & Sons, Inc.). For example, density gradient centrifugation is a method for the separation of lymphocytes from other blood cell populations (e.g. erythrocytes and granulocytes). Human lymphocyte subpopulations can be isolated via their specific cell surface receptors which can be recognized by monoclonal antibodies. The physical separation method involves coupling of these antibody reagents to magnetic beads which allow the enrichment of cells that are bound by these antibodies (positive selection).
As an alternative to primary human cells cultured cell lines (e.g. MOLT-4 cells, Jurkat, Ramos or HeLa cells) can be used.
In a preferred embodiment, the cell is part of a cell culture system and methods for the harvest of a cell out of a cell culture system are known in the art (literature supra).
The choice of the cell will mainly depend on the expression of the phosphatidylinositol kinases, since it has to be ensured that the protein is principally present in the cell of choice. In order to determine whether a given cell is a suitable starting system for the methods of the invention, methods like Westernblot, PCR-based nucleic acids detection methods, Northernblots and DNA-microarray methods (“DNA chips”) might be suitable in order to determine whether a given protein of interest is present in the cell.
The choice of the cell may also be influenced by the purpose of the study. If the in vivo efficacy for a given drug needs to be analyzed then cells or tissues may be selected in which the desired therapeutic effect occurs (e.g. B-cells). By contrast, for the elucidation of protein targets mediating unwanted side effects the cell or tissue may be analysed in which the side effect is observed (e.g. cardiomyocytes, vascular smooth muscle or epithelium cells).
Furthermore, it is envisaged within the present invention that the cell containing the phosphatidylinositol kinases or the kinase may be obtained from an organism, e.g. by biopsy. Corresponding methods are known in the art. For example, a biopsy is a diagnostic procedure used to obtain a small amount of tissue, which can then be examined microscopically or with biochemical methods. Biopsies are important to diagnose, classify and stage a disease, but also to evaluate and monitor drug treatment.
It is encompassed within the present invention that by the harvest of the at least one cell, the lysis is performed simultaneously. However, it is equally preferred that the cell is first harvested and then separately lysed.
Methods for the lysis of cells are known in the art (Karwa and Mitra: Sample preparation for the extraction, isolation, and purification of Nuclei Acids; chapter 8 in “Sample Preparation Techniques in Analytical Chemistry”, Wiley 2003, Editor: Somenath Mitra, print ISBN: 0471328456; online ISBN: 0471457817). Lysis of different cell types and tissues can be achieved by homogenizers (e.g. Potter-homogenizer), ultrasonic desintegrators, enzymatic lysis, detergents (e.g. NP-40, Triton X-100, CHAPS, SDS), osmotic shock, repeated freezing and thawing, or a combination of these methods.
According to the methods of the invention, the protein preparation containing one or more phosphatidylinositol kinases is contacted with the immobilization product under conditions allowing the formation of a complex between the said kinase and the immobilization product of the invention.
In the present invention, the term “a complex between a phosphatidylinositol kinase and the immobilization product” denotes a complex where the immobilization product interacts with a phosphatidylinositol kinase, e.g. by covalent or, most preferred, by non-covalent binding.
In the context of the present invention, compounds are identified which interfere with the formation of a complex between the immobilization product and a phosphatidylinositol kinase present in a cell or protein preparation. In case that only one phosphatidylinositol kinase is to be detected or present, the formation of one complex is observed and tested. In case that several kinases are to be detected or present, the formation of several, different complexes is observed and tested.
The skilled person will know which conditions can be applied in order to enable the formation of said complex.
In the context of the present invention, the term “under conditions allowing the formation of the complex” includes all conditions under which such formation, preferably such binding is possible. This includes the possibility of having the solid support on an immobilized phase and pouring the lysate onto it. In another preferred embodiment, it is also included that the solid support is in a particulate form and mixed with the cell lysate. Such conditions are known to the person skilled in the art.
In the context of non-covalent binding, the binding between the immobilization product and the kinase is, e.g., via salt bridges, hydrogen bonds, hydrophobic interactions or a combination thereof.
In a preferred embodiment, the steps of the formation of said complex are performed under essentially physiological conditions. The physical state of proteins within cells is described in Petty, 1998 (Howard R. Petty, Chapter 1, Unit 1.5 in: Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, and Kenneth M. Yamada (eds.) Current Protocols in Cell Biology Copyright © 2003 John Wiley & Sons, Inc. All rights reserved. DOI: 10.1002/0471143030.cb0101s00Online Posting Date: May, 2001Print Publication Date: October, 1998).
The contacting under essentially physiological conditions has the advantage that the interactions between the ligand, the cell preparation (i.e. the phosphatidylinositol kinase to be characterized) and optionally the compound reflect as much as possible the natural conditions. “Essentially physiological conditions” are inter alia those conditions which are present in the original, unprocessed sample material. They include the physiological protein concentration, pH, salt concentration, buffer capacity and post-translational modifications of the proteins involved. The term “essentially physiological conditions” does not require conditions identical to those in the original living organism, wherefrom the sample is derived, but essentially cell-like conditions or conditions close to cellular conditions. The person skilled in the art will, of course, realize that certain constraints may arise due to the experimental set-up which will eventually lead to less cell-like conditions. For example, the eventually necessary disruption of cell walls or cell membranes when taking and processing a sample from a living organism may require conditions which are not identical to the physiological conditions found in the organism. Suitable variations of physiological conditions for practicing the methods of the invention will be apparent to those skilled in the art and are encompassed by the term “essentially physiological conditions” as used herein. In summary, it is to be understood that the term “essentially physiological conditions” relates to conditions close to physiological conditions, as e.g. found in natural cells, but does not necessarily require that these conditions are identical.
For example, “essentially physiological conditions” may comprise 50-200 mM NaCl or KCl, pH 6.5-8.5, 20-37° C., and 0.001-10 mM divalent cation (e.g. Mg++, Ca++,); more preferably about 150 m NaCl or KCl, pH7.2 to 7.6, 5 mM divalent cation and often include 0.01-1.0 percent non-specific protein (e.g. BSA). A non-ionic detergent (Tween, NP-40, Triton-X100) can often be present, usually at about 0.001 to 2%, typically 0.05-0.2% (volume/volume). For general guidance, the following buffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HCl, pH5-8, with optional addition of divalent cation(s) and/or metal chelators and/or non-ionic detergents.
Preferably, “essentially physiological conditions” mean a pH of from 6.5 to 7.5, preferably from 7.0 to 7.5, and/or a buffer concentration of from 10 to 50 mM, preferably from 25 to 50 mM, and/or a concentration of monovalent salts (e.g. Na or K) of from 120 to 170 mM, preferably 150 mM. Divalent salts (e.g. Mg or Ca) may further be present at a concentration of from 1 to 5 mM, preferably 1 to 2 mM, wherein more preferably the buffer is selected from the group consisting of Tris-HCl or HEPES.
The skilled person will appreciate that between the individual steps of the methods of the invention, washing steps may be necessary. Such washing is part of the knowledge of the person skilled in the art. The washing serves to remove non-bound components of the cell lysate from the solid support. Nonspecific (e.g. simple ionic) binding interactions can be minimized by adding low levels of detergent or by moderate adjustments to salt concentrations in the wash buffer.
According to the identification methods of the invention, the read-out system is either the detection or determination of a phosphatidylinositol kinase (first aspect of the invention), the detection of the complex between a phosphatidylinositol kinase and the immobilization product (second aspect of the invention), or the determination of the amount of the complex between a phosphatidylinositol kinase and the immobilization product (second, third and fourth aspect of the invention).
In the method according to the first aspect of the invention, the detection or determination of the amount of separated phosphatidylinositol kinase is preferably indicative for the fact that the compound is able to separate the phosphatidylinositol kinase from the immobilization product. This capacity indicates that the respective compound interacts, preferably binds to the phosphatidylinositol kinase, which is indicative for its therapeutic potential.
In one embodiment of the method according to the second aspect of the invention, the complex formed during the method of the invention is detected. The fact that such complex is formed preferably indicates that the compound does not completely inhibit the formation of the complex. On the other hand, if no complex is formed, the compound is presumably a strong interactor with the phosphatidylinositol kinase, which is indicative for its therapeutic potential.
According to the methods of the second, third and fourth aspect of the invention the amount of the complex formed during the method is determined. In general, the less complex in the presence of the respective compound is formed, the stronger the respective compound interacts with the phosphatidylinositol kinase, which is indicative for its therapeutic potential.
The detection of the complex formed according to the second aspect of the invention can be performed by using labeled antibodies directed against the phosphatidylinositol kinase and a suitable readout system.
According to a preferred embodiment of the second aspect of the invention, the complex between one phosphatidylinositol kinase and the immobilization product is detected by determining its amount.
In the course of the second, third and fourth aspect of the invention, it is preferred that the phosphatidylinositol kinase are separated from the immobilization product in order to determine the amount of said complex.
According to invention, separating means every action which destroys the interactions between the immobilization compound and the phosphatidylinositol kinase. This includes in a preferred embodiment the elution of the phosphatidylinositol kinase from the immobilization compound.
The elution can be achieved by using non-specific reagents as described in detail below (ionic strength, pH value, detergents). In addition, it can be tested whether a compound of interest can specifically elute the phosphatidylinositol kinase from the immobilization compound. Such phosphatidylinositol kinase interacting compounds are described further in the following sections.
Such non-specific methods for destroying the interaction are principally known in the art and depend on the nature of the ligand enzyme interaction. Principally, change of ionic strength, the pH value, the temperature or incubation with detergents are suitable methods to dissociate the target enzymes from the immobilized compound. The application of an elution buffer can dissociate binding partners by extremes of pH value (high or low pH; e.g. lowering pH by using 0.1 M citrate, pH2-3), change of ionic strength (e.g. high salt concentration using NaI, KI, MgCl2, or KCl), polarity reducing agents which disrupt hydrophobic interactions (e.g. dioxane or ethylene glycol), or denaturing agents (chaotropic salts or detergents such as Sodium-docedyl-sulfate, SDS; Review: Subramanian A., 2002, Immunoaffinty chromatography).
In some cases, the solid support has preferably to be separated from the released material. The individual methods for this depend on the nature of the solid support and are known in the art. If the support material is contained within a column the released material can be collected as column flowthrough. In case the support material is mixed with the lysate components (so called batch procedure) an additional separation step such as gentle centrifugation may be necessary and the released material is collected as supernatant. Alternatively magnetic beads can be used as solid support so that the beads can be eliminated from the sample by using a magnetic device.
In step d) of the method according to the first aspect of the invention, it is determined if the phosphatidylinositol kinase has been separated from the immobilization product of the invention. This may include the detection of the phosphatidylinositol kinase or the determination of the amount of the phosphatidylinositol kinase.
Consequently, at least in preferred embodiments of all identification methods of the invention, methods for the detection of a separated phosphatidylinositol kinase or for the determination of their amount are used. Such methods are known in the art and include physico-chemical methods such as protein sequencing (e.g. Edmann degradation), analysis by mass spectrometry methods or immunodetection methods employing antibodies directed against the kinase.
Throughout the invention, if an antibody is used in order to detect a phosphatidylinositol kinase or in order to determine its amount (e.g. via ELISA), the skilled person will understand that, if a specific phosphatidylinositol kinase is to be detected or if the amount of a phosphatidylinositol kinase is to be determined, a specific antibody may be used (Sasaki et al., 2000, Nature 406, 897-902; Deora et al., 1998, J. Biol. Chem. 273, 29923-29928). As indicated above, such antibodies are known in the art. Furthermore, the skilled person is aware of methods for producing the same.
Preferably, a phosphatidylinositol kinase is detected or the amount of a phosphatidyl-inositol kinase is determined by mass spectrometry or immunodetection methods.
The identification of proteins with mass spectrometric analysis (mass spectrometry) is known in the art (Shevchenko et al., 1996, Analytical Chemistry 68: 850-858; Mann et al., 2001, Analysis of proteins and proteomes by mass spectrometry, Annual Review of Biochemistry 70, 437-473) and is further illustrated in the example section.
Preferably, the mass spectrometry analysis is performed in a quantitative manner, for example by using iTRAQ technology (isobaric tags for relative and absolute quantification) or cICAT (cleavable isotope-coded affinity tags) (Wu et al., 2006. J. Proteome Res. 5, 651-658).
According to a further preferred embodiment of the present invention, the characterization by mass spectrometry (MS) is performed by the identification of proteotypic peptides of the kinase. The idea is that the phosphatidylinositol kinase is digested with proteases and the resulting peptides are determined by MS. As a result, peptide frequencies for peptides from the same source protein differ by a great degree, the most frequently observed peptides that “typically” contribute to the identification of this protein being termed “proteotypic peptide”. Therefore, a proteotypic peptide as used in the present invention is an experimentally well observable peptide that uniquely identifies a specific protein or protein isoform.
According to a preferred embodiment, the characterization is performed by comparing the proteotypic peptides obtained in the course of practicing the methods of the invention with known proteotypic peptides. Since, when using fragments prepared by protease digestion for the identification of a protein in MS, usually the same proteotypic peptides are observed for a given phosphatidylinositol kinase, it is possible to compare the proteotypic peptides obtained for a given sample with the proteotypic peptides already known for phosphatidylinositol kinases and thereby identifying the phosphatidylinositol kinase being present in the sample.
As an alternative to mass spectrometry analysis, the eluted phosphatidylinositol kinase (including coeluted binding partners such as regulatory subunits), can be detected or its amount can be determined by using a specific antibody directed against the phosphatidylinositol kinase.
Furthermore, in another preferred embodiment, once the identity of the coeluted binding partner (e.g. regulatory subunit) has been established by mass spectrometry analysis, each binding partner can be detected with specific antibodies directed against this protein.
Suitable antibody-based assays include but are not limited to Western blots, ELISA assays, sandwich ELISA assays and antibody arrays or a combination thereof. The establishment of such assays is known in the art (Chapter 11, Immunology, pages 11-1 to 11-30 in: Short Protocols in Molecular Biology. Fourth Edition, Edited by F. M. Ausubel et al., Wiley, New York, 1999).
These assays can not only be configured in a way to detect and quantify a phosphatidylinositol kinase interacting protein of interest (e.g. a catalytic or regulatory subunit of a kinase complex), but also to analyse posttranslational modification patterns such as phosphorylation or ubiquitin modification.
Furthermore, the identification methods of the invention involve the use of compounds which are tested for their ability to be a phosphatidylinositol kinase interacting compound.
Principally, according to the present invention, such a compound can be every molecule which is able to interact with the phosphatidylinositol kinase, eg. by inhibiting its binding to the immobilization product of the invention. Preferably, the compound has an effect on the phosphatidylinositol kinase, e.g. a stimulatory or inhibitory effect.
Preferably, said compound is selected from the group consisting of synthetic or naturally occurring chemical compounds or organic synthetic drugs, more preferably small molecule organic drugs or natural small molecule compounds. Preferably, said compound is identified starting from a library containing such compounds. Then, in the course of the present invention, such a library is screened.
Such small molecules are preferably not proteins or nucleic acids. Preferably, small molecules exhibit a molecular weight of less than 1000 Da, more preferred less than 750 Da, most preferred less than 500 Da.
A “library” according to the present invention relates to a (mostly large) collection of (numerous) different chemical entities that are provided in a sorted manner that enables both a fast functional analysis (screening) of the different individual entities, and at the same time provide for a rapid identification of the individual entities that form the library. Examples are collections of tubes or wells or spots on surfaces that contain chemical compounds that can be added into reactions with one or more defined potentially interacting partners in a high-throughput fashion. After the identification of a desired “positive” interaction of both partners, the respective compound can be rapidly identified due to the library construction. Libraries of synthetic and natural origins can either be purchased or designed by the skilled artisan.
Examples of the construction of libraries are provided in, for example, Breinbauer R, Manger M, Scheck M, Waldmann H. Natural product guided compound library development. Curr. Med. Chem. 2002; 9(23):2129-2145, wherein natural products are described that are biologically validated starting points for the design of combinatorial libraries, as they have a proven record of biological relevance. This special role of natural products in medicinal chemistry and chemical biology can be interpreted in the light of new insights about the domain architecture of proteins gained by structural biology and bioinformatics. In order to fulfill the specific requirements of the individual binding pocket within a domain family it may be necessary to optimise the natural product structure by chemical variation. Solid-phase chemistry is said to become an efficient tool for this optimisation process, and recent advances in this field are highlighted in this review article. The current drug discovery processes in many pharmaceutical companies require large and growing collections of high quality lead structures for use in high throughput screening assays. Collections of small molecules with diverse structures and “drug-like” properties have, in the past, been acquired by several means: by archive of previous internal lead optimisation efforts, by purchase from compound vendors, and by union of separate collections following company mergers. Although high throughput/combinatorial chemistry is described as being an important component in the process of new lead generation, the selection of library designs for synthesis and the subsequent design of library members has evolved to a new level of challenge and importance. The potential benefits of screening multiple small molecule compound library designs against multiple biological targets offers substantial opportunity to discover new lead structures.
In a preferred embodiment of the second and third aspect of the invention, the phosphatidylinositol kinase containing protein preparation is first incubated with the compound and then with the immobilization product. However, the simultaneous incubation of the compound and the immobilization product of the invention (coincubation) with the phosphatidylinositol kinase containing protein preparation is equally preferred (competitive binding assay).
In case that the incubation with the compound is first, the phosphatidylinositol kinase is preferably first incubated with the compound for 10 to 60 minutes, more preferred 30 to 45 minutes at a temperature of 4° C. to 37° C., more preferred 4° C. to 25° C., most preferred 4° C. Preferably compounds are used at concentrations ranging from 1 nM to 100 μM, preferably from 10 nM to 10 μM. The second step, contacting with the immobilized ligand, is preferably performed for 10 to 60 minutes at 4° C.
In case of simultaneous incubation, the phosphatidylinositol kinase is preferably simultaneously incubated with the compound and the immobilization product of the invention for 30 to 120 minutes, more preferred 60 to 120 minutes at a temperature of 4° C. to 37° C., more preferred 4° C. to 25° C., most preferred 4° C. Preferably compounds are used at concentrations ranging from 1 nM to 100 μM, preferably from 10 nM to 10 μM.
Furthermore, steps a) to c) of the second aspect of the invention may be performed with several protein preparations in order to test different compounds. This embodiment is especially interesting in the context of medium or high throughput screenings (see below).
In a preferred embodiment of the method of the invention according to the third or fourth aspect, the amount of the complex formed in step c) is compared to the amount formed in step b)
In a preferred embodiment of the method of the invention according to the third or fourth aspect, a reduced amount of the complex formed in step c) in comparison to step b) indicates that a phosphatidylinositol kinase is a target of the compound. This results from the fact that in step c) of this method of the invention, the compound competes with the immobilized compound for the binding of the kinase. If less kinase is present in the aliquot incubated with the compound, this means preferably that the compound has competed with the inhibitor for the interaction with the enzyme and is, therefore, a direct target of the protein and vice versa.
Preferably, the identification methods of the invention are performed as a medium or high throughput screening.
The interaction compound identified according to the present invention may be further characterized by determining whether it has an effect on the phosphatidylinositol kinase, for example on its kinase activity (Carpenter et al., 1990, J. Biol. Chem. 265, 19704-19711).
The compounds identified according to the present invention may further be optimized (lead optimisation). This subsequent optimisation of such compounds is often accelerated because of the structure-activity relationship (SAR) information encoded in these lead generation libraries. Lead optimisation is often facilitated due to the ready applicability of high-throughput chemistry (HTC) methods for follow-up synthesis. An example for lead optimization of PI3Kγ inhibitors was reported (Pomel et al., 2006. J. Med. Chem. 49(13): 3857-3871).
The invention further relates to a method for the preparation of a pharmaceutical composition comprising the steps of

    
    
        a) identifying a phosphatidylinositol kinase interacting compound as described above, and
        b) formulating the interacting compound to a pharmaceutical composition.
    
    


Methods for the formulation of identified compounds are known in the art. Furthermore, it is known in the art how to administer such pharmaceutical compositions.
The obtained pharmaceutical composition can be used for the prevention or treatment of diseases where the respective phosphatidylinositol kinase plays a role, e.g. for the prevention or treatment of cancer (Wymann and Schneiter, 2008. Nature Reviews Molecular cell Biology 9, 162-176). For example, phosphatidylinositol kinase inhibitors may be useful for the treatment of inflammatory diseases, cancer or metabolic diseases.
The invention further relates to a method for the purification of a phosphatidylinositol kinase, comprising the steps of

    
    
        a) providing a protein preparation containing said kinase,
        b) contacting the protein preparation with the immobilization product of the invention under conditions allowing the formation of a complex between the phosphatidylinositol kinase and the immobilization product, and
        c) separating the phosphatidylinositol kinase from the immobilization product.
    
    


As mentioned above, it has been surprisingly found that the compound of the invention and therefore also the immobilization product of the invention is a ligand which recognizes the kinases mentioned above. This enables efficient purification methods for said kinases.
Preferred kinases to be purified include:

    PIK3Ca, PIK3Cb, PIK4Ca, PIP5K2C, PIK3Cg, PIK3Cd, PIP5K2A, PIK4C2B, PIK3C3, PIP5K2B, PIK3C2b.


With respect to the phosphatidylinositol kinases, the protein preparation containing the phosphatidylinositol kinases, the conditions for contacting with the immobilization product of the invention, the immobilization product of the invention, the complex between the phosphatidylinositol kinases and the immobilization product of the invention, the separation of the phosphatidylinositol kinases from the immobilization product of the invention, and the detection of the phosphatidylinositol kinases or the determination of its amount, the embodiments as defined above for the identification methods of the invention also apply to the purification method of the invention.
In a preferred embodiment, the purification method of the invention further comprises after step c) the identification of proteins being capable of binding to said phosphatidylinositol kinases. This is especially interesting when the formation of the complex is performed under essentially physiological conditions, because it is then possible to preserve the natural condition of the enzyme which includes the existence of binding partners, enzyme subunits or post-translational modifications, which can then be identified with the help of mass spectrometry (MS).
Consequently, in a preferred embodiment, the purification method of the invention further comprises after step c) the determination whether the phosphatidylinositol kinase is further posttranslationally modified, e.g. by ubiquitin modification.
The binding proteins or the posttranslational modifications can be determined as explained above for the detection of phosphatidylinositol kinases or the determination of the amount of phosphatidylinositol kinases. Preferably, said methods include mass spectrometry of immunodetection methods as described above.
The invention further relates to a method for determining the presence of one or more kinases in a sample, comprising the steps of:

    
    
        a) providing a protein preparation expected to contain said one or more phosphatidylinositol kinases,
        b) contacting the protein preparation with the immobilization product of the invention under conditions allowing the formation of a complex between one of the phosphatidylinositol kinases and the immobilization product, and
        c) detecting whether one or more phosphatidylinositol kinases have formed a complex with the immobilization product.
    
    


In a preferred embodiment of the invention, said detecting in step c) is performed by separating said one or more phosphatidylinositol kinases from the immobilization product and further identification of said one or more phosphatidylinositol kinases.
Said identification may be performed by mass spectrometry or immunodetection methods as described above.
Preferably, also in the context of this method of the invention the phosphatidylinositol kinase is PIK3Ca, PIK3Cb, PIK4Ca, PIP5K2C, PIK3Cg, PIK3Cd, PIP5K2A, PIK4C2B, PIK3C3, PIP5K2B, PIK3C2b.
According to an especially preferred embodiment of this method of the invention, the kinase contains at least one mutation.
With respect to said one or more phosphatidylinositol kinases, the protein preparation containing said phosphatidylinositol kinases, the conditions for contacting with the immobilization product of the invention, the immobilization product of the invention, the complex between said phosphatidylinositol kinase and the immobilization product of the invention, the separation of phosphatidylinositol kinases from the immobilization product of the invention, and the detection of kinases or the determination of its amount, the embodiments as defined above for the identification methods of the invention also apply to the purification method of the invention.
The invention further relates to the use of the immobilization compound or the immobilization product of the invention for the identification of a phosphatidylinositol kinase interacting compound and for the purification of a phosphatidylinositol kinase. The embodiments as defined above also apply to the uses of the invention.
The invention further relates to a kit comprising the compound or the immobilization product of the invention. Such a kit is especially useful for performing the methods of the invention. Further components of the kit may be antibodies for the detection of kinase proteins, for example antibodies specific for phosphoinositide kinases. Such antibodies and their use are known in the art and they are commercially available (Sasaki et al., 2000, Nature 406, 897-902; Deora et al., 1998, J. Biol. Chem. 273, 29923-29928). Furthermore, the kit may contain further auxiliary components like buffers, means for the detection of antibodies, and positive controls. Such components are known in the art.
The invention is further illustrated by the following figures and examples, which are not considered as being limiting for the scope of protection conferred by the claims of the present application. In case where in the following examples the term “affinity matrix” is used, this term refers to an immobilization product as defined in the present application.



SHORT DESCRIPTION OF THE FIGURES
FIG. 1: Methods used in the synthesis of immobilization compounds as described in example 1.
FIG. 2: Structure of 5-(2-(3-(2-aminoethyl)ureido)-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-isopropylpyridine-3-sulfonamide (CZC00031207).
FIG. 3: Structure of 5-(2-(3-(2-aminoethyl)ureido)-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-tert-butylpyridine-3-sulfonamide (CZC00025236).
FIG. 4: Kinobeads experiment with the immobilized compound CZC31207 for mass spectrometry analysis of captured proteins.
A protein gel after staining with Coomassie brilliant blue is shown. The experiment was performed as described in example 2 with a mix of HeLa and placenta cell lysates. Proteins bound to the affinity matrix were eluted with SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis. The indicated gel areas were cut out as gel slices, proteins were treated with trypsin and ITRAQ-labeled peptides were analysed by mass spectrometry. Left lane (P28737B): cell lysate treated with 10 μM free compound CZC31207; middle lane: protein molecular weight marker; right lane (P28738B): DMSO control.
FIG. 5: Kinobeads experiment with the immobilized compound CZC31207 for mass spectrometry analysis of captured proteins.
A protein gel after staining with Coomassie brilliant blue is shown. The experiment was performed as described in example 3 with a mix of Jurkat and Ramos cell lysates. Proteins bound to the affinity matrix were eluted with SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis. The indicated gel areas were cut out as gel slices, proteins were treated with trypsin and ITRAQ-labeled peptides were analysed by mass spectrometry. Left lane (P28733B): cell lysate treated with 10 μM free compound CZC31207; middle lane: protein molecular weight marker; right lane (P28734B): DMSO control.
FIG. 6: Selectivity profiling experiment for test compound CZC00024513 using CZC00025236 as a capture compound.
A protein gel after staining with Coomassie brilliant blue is shown. The experiment was performed as described in example 4 with a mix of HeLa and placenta cell lysates. Proteins bound to the affinity matrix were eluted with SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis. The indicated gel areas were cut out as gel slices, proteins were treated with trypsin and ITRAQ-labeled peptides were analysed by mass spectrometry.
First lane (P28835B): cell lysate treated with 10 μM of test compound CZC00024513;
Second lane (P28836B): cell lysate treated with 1 μM of test compound CZC00024513;
Third lane (P28837B): cell lysate treated with 0.1 μM of test compound CZC00024513;
Fourth lane (P28838B): cell lysate treated 0.5% DMSO.
M: protein molecular weight marker.
FIG. 7: Dose response curve for PI3Kalpha (PIK3CA; IC50=1.55 μM)
FIG. 8: Dose response curve for PI3 Kbeta (PIK3CB; IC50=0.31 μM)
FIG. 9: Dose response curve for PI3 Kgamma (PIK3CG; IC50=0.06 μM)
FIG. 10: Dose response curve for PI3 Kdelta (PIK3CD; IC50=0.62 μM)
FIG. 11: Amino acid sequence of human PIK3CA (IPI0031386.2). Peptides identified by mass spectrometry are underlined (HeLa Placenta experiment, P28738B)
FIG. 12: Amino acid sequence of human PIK3CB (IPI00031388.1). Peptides identified by mass spectrometry are underlined (HeLa Placenta experiment, P28738B)
FIG. 13: Amino acid sequence of human PIK4Ca (IPI00070943.3). Peptides identified by mass spectrometry are underlined (HeLa Placenta experiment, P28738B)
FIG. 14: Amino acid sequence of human PIP5K2C (IPI00152303.7). Peptides identified by mass spectrometry are underlined (HeLa Placenta experiment, P28738B)
FIG. 15: Amino acid sequence of human PIK3Cg (IPI00292690.1). Peptides identified by mass spectrometry are underlined (HeLa Placenta experiment, P28738B)
FIG. 16: Amino acid sequence of human PIK3Cd (IPI00298410.2). Peptides identified by mass spectrometry are underlined (HeLa Placenta experiment, P28738B)



EXAMPLES
Example 1
Preparation of the Affinity Matrix
This example describes the synthesis of compounds and methods for their immobilization on a solid support yielding the affinity matrix used in the following examples for the capturing of kinases from cell lysates.
Analytical Methods
NMR spectra were obtained on a Brucker dpx400.
LCMS was carried out on an Agilent 1100 using a Gemini C18, 3×30 mm, 3 microns column. Column flow was 1.2 mL/min. and solvents used were water and acetonitrile (0.1% formic acid) with an injection volume of 3 or 10 ul. Wavelengths were 254 and 210 nm.







TABLE 1







Chromatography









Time (min)
Water
Acetonitrile












0
95
5


3
5
95


4.5
5
95


4.6
95
5








5.00
STOP









Synthesis of Compounds

1-(5-Bromo-pyridin-2-yl)-3-carboethoxy-thiourea




To a solution of 2-amino-5-bromopyridine (200.0 g, 1.156 mol) in DCM (2.0 L) cooled to 5° C. was added ethoxycarbonyl isothiocyanate (134.9 mL, 1.156 mol) dropwise over 15 min. The reaction mixture was then allowed to warm to room temperature (20° C.) and stirred for 16 h. Evaporation in vacuo gave a yellow solid which was collected by filtration, thoroughly washed with petrol (3×500 mL) and air-dried to afford the title compound (351.5 g, quantitative). No further purification was required.
1H NMR (d6-DMSO)  12.22 (br s, 1H), 11.75 (br s, 1H), 8.66 (br s, 1H), 8.57 (d, 1H), 8.16 (dd, 1H), 4.26 (q, 2H), 1.28 (t, 3H).
LCMS, (M+H+) 304/406, Rt=2.84 min.
6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine (A)




To a suspension of hydroxylamine hydrochloride (409.2 g, 5.888 mol) in EtOH/MeOH (1:1, 2.5 L) was added N,N-diisopropylethylamine (606.1 mL, 3.480 mol), the mixture was stirred at room temperature (20° C.) for 1 h. 1-(6-Bromo-pyridin-2-yl)-3-carboethoxy-thiourea (352.8 g, 1.160 mol) was then added and the mixture slowly heated to reflux (Note: bleach scrubber required to quench H2S evolved). After 2 h at reflux the mixture was allowed to cool and filtered to collect the precipitated solid. The collected solid was washed successively with water (1.0 L), EtOH/MeOH (1:1, 1.0 L) and diethyl ether (500 mL) then air-dried to afford the title compound as a white solid (169.2 g, 69%). No further purification was required.
1H NMR (d6-DMSO)  8.94 (d, 1H), 7.58 (dd, 1H), 7.36 (d, 1H), 6.16 (br s, 2H).
LCMS (M+H+) 213/214, Rt=1.45 min.
Method 1 Synthesis of Sulfonamides
5-bromo-N-tert-butylpyridine-3-sulfonamide




To a solution of 5-bromopyridine-3-sulfonyl chloride (5 g, 17 mmol) in pyridine (10 mL) at 0° C. was added tert-butylamine (3.6 mL, 2 equiv., 34 mmol). The reaction mixture was allowed to warm to room temperature and then heated to 40° C. for 14 h. After this time the crude reaction mixture was again cooled to 0° C. and diluted with dilute HCl (0.05M, 40 mL). The reaction was stirred at 0° C. for 30 min and the resulting precipitate collected by filtration. The solid was washed with water and dried to afford the title compound as a yellow solid (2.12 g, 7.3 mmol, 42%). No further purification was required.
LCMS (method A) (M+H+) 292/294, Rt=2.41 min
5-bromo-N-isopropylpyridine-3-sulfonamide




Prepared according to Method 1 using isopropylamine.
LCMS (method A) (M+H+) 281/283, Rt=2.28 min
Method 2 Suzuki Coupling
5-(2-Amino-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-pyridine-3-sulfonic acid tert-butylamide




5-bromo-N-tert-butylpyridine-3-sulfonamide (375 mg, 0.986 mmol), bis(pinacolato) diboron (276 mg, 1.085 mmol), potassium acetate (290 mg, 2.96 mmol), [1,1′bis(diphenylphosphino)ferrocene]dichloro-palladium (II) complex with CH2Cl2 (40 mg, 0.049 mmol) and dioxane (3 ml) were heated to 120° C. for 60 minutes in the microwave. After this time aryl bromide (A) (147 mg, 0.69 mmol), Na2CO3 (2M aqueous solution, 2 mL), EtOH (0.4 ml) and a further portion of [1,1′bis(diphenylphosphino)ferrocene]dichloro-palladium (II) complex with CH2Cl2 was added and reaction mixture heated further for 60 minutes at 120° C. in the microwave. After this time the solvents were removed in vacuo and the brown residue redissolved in 2M HCl (30 mL), the aqueous phase was washed with ethyl acetate (3×20 mL) and then neutralized with concentrated NaOH to pH 7.0. The aqueous phase was then extracted with ethyl acetate (3×20 mL), the organic extracts were combined, dried over sodium sulfate, filtered and the solvent removed in vacuo to afford the desired product as a brown solid (441 mg, 54%).
LCMS, (M+H+) 347, RT=1.89 min.
5-(2-amino-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-isopropylpyridine-3-sulfonamide




Prepared according to method 2
LCMS, (M+H+) 333, Rt=1.86 min
Method 3 Synthesis of Ureas
tert-butyl 2-(3-(6-(5-(N-tert-butylsulfamoyl)pyridin-3-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)ureido)ethylcarbamate




5-(2-Amino-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-pyridine-3-sulfonic acid tert-butylamide (50 mg, 0.14 mmol) was suspended in tetrahydrofuran:pyridine (30 mL, 5:1) and cooled to 0° C. Triphosgene (41 mg, 0.14 mmol) was added in one portion and the reaction mixture heated at 35° C. for 2 hours. After this time the solvent was decanted and the resultant semi-solid dissolved in DMF:pyridine (1 mL, 10:1), N-Boc-ethylenediamine (50 mg, 0.32 mmol) was added in one portion and the reaction mixture heated for 18 hours at 65° C. The desired product was isolated as a white solid, directly from the reaction mixture, by preparative HPLC (35 mg)
LCMS, (M+H+) 533, RT=1.66 min.
tert-butyl 2-(3-(6-(5-(N-isopropylsulfamoyl)pyridin-3-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)ureido)ethylcarbamate




Prepared according to method 3. LCMS, (M+H+) 519, Rt=1.59 min.
Method 4 Boc Deprotection
5-(2-(3-(2-aminoethyl)ureido)-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-tert-butylpyridine-3-sulfonamide




tert-butyl 2-(3-(6-(5-(N-tert-butylsulfamoyl)pyridin-3-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)ureido)ethylcarbamate (35 mg) was suspended in HCl (4M in dioxane, 2 mL) and DCM (2 mL) and the reaction mixture stirred overnight at room temperature. After this time the reaction mixture was pippetted slowly onto diethyl ether at −78° C. and maintained at this temperature for 20 minutes. The resultant white solid was filtered, washed with further diethyl ether and dried under vacuum to afford the title compound (21 mg).
1H NMR δ (d6-DMSO) δ 10.27 (s, 1H), 9.42 (d, 1H), 9.23 (d, 1H), 9.01 (d, 1H), 8.59 (t, 1H), 8.37 (t, 1H), 8.10 (dd, 1H), 7.99-7.92 (m, 2H), 7.86 (s, 1H), 7.83 (d, 1H), 7.35 (s, 1H), 7.23 (s, 1H), 7.10 (s, 1H), 3.50 (q, 2H), 3.0-2.96 (m, 2H), 1.16 (s, 9H).
LCMS, (M+H+) 433, Rt=1.60 min.
5-(2-(3-(2-aminoethyl)ureido)-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-isopropylpyridine-3-sulfonamide




Prepared according to method 4
1H NMR δ (d6-DMSO) δ 10.30 (s, 1H), 9.43 (d, 1H), 9.25 (d, 1H), 8.99 (d, 1H), 8.56 (t, 1H), 8.36 (t, 1H), 8.12 (dd, 1H), 7.96-7.93 (m, 3H), 7.82 (d, 1H), 3.50 (q, 2H), 3.41 (septet, 1H), 2.98 (q, 2H), 0.99 (d, 6H). LCMS, (M+H+) 419, Rt=1.50 min.







TABLE 2





Abbreviations
















Boc
tert-butoxycarbonyl


DCM
Dichloromethane


DMSO
dimethylsulfoxide


MeOH
Methanol


EtOH
Ethanol


iPr2NEt
Diisopropylethylamine


NH2OH•HCl
hydroxylaminehydrochloride


Pd(dppf)(Cl)2
[1,1′bis(diphenylphosphino)ferrocene] dichloro-palladium (II)


DMF
N,N-Dimethylformamide


THF
tetrahydrofuran


s
singlet


d
Doublet


dd
Doubledoublet


br
Broad


mL
millilitres


L
litre


t
Triplet


m
Multiplet


Rt
Retention time









Immobilization of Compounds on Beads (Affinity Matrix)

NHS-activated Sepharose 4 Fast Flow (Amersham Biosciences, 17-0906-01) was equilibrated with anhydrous DMSO (Dimethylsulfoxid, Fluka, 41648, H20<=0.005%). 1 ml of settled beads was placed in a 15 ml Falcon tube, compound stock solution (usually 100 mM in DMF or DMSO) was added (final concentration 0.2-2 μmol/ml beads) as well as 15 μl of triethylamine (Sigma, T-0886, 99% pure). Beads were incubated at room temperature in darkness on an end-over-end shaker (Roto Shake Genie, Scientific Industries Inc.) for 16-20 hours. Coupling efficiency is determined by HPLC. Non-reacted NHS-groups were blocked by incubation with aminoethanol at room temperature on the end-over-end shaker over night. Beads were washed with 10 ml of DMSO and were stored in isopropanol at −20° C. These beads were used as the affinity matrix in the following examples. Control beads (no compound immobilized) were generated by blocking the NHS-groups by incubation with aminoethanol as described above.
Example 2
Kinobeads Experiment Using Immobilized Compound CZC31207 and a Mix of HeLa and Placenta Cell Lysates
This example demonstrates the use of an immobilized compound (structure shown in FIG. 2, CZC31207) for the capturing and identification of phosphatidylinositol kinases from cell lysate in a competition binding assay. To the first aliquot of cell lysate 10 μM of the free compound (CZC31207) was added and allowed to bind to proteins in the lysate. Then the affinity matrix with the immobilized compound (Example 1) was added to capture proteins that were not interacting with the previously added free compound. Beads were separated from the lysate and bead bound proteins were eluted in SDS sample buffer and subsequently separated by SDS-Polyacrylamide gel electrophoresis (FIG. 4). Suitable gel bands were cut out and subjected to in-gel proteolytic digestion with trypsin. The second lysate aliquot was processed identically, however no free compound was added (DMSO solvent control). Peptides originating from samples 1 and 2 were labeled with iTRAQ reagents (iTRAQ 115 and iTRAQ 117) and the combined samples were analyzed with a nano-flow liquid chromatography system coupled online to a tandem mass spectrometer (LC-MS/MS) experiment followed by iTRAQ reporter ion quantification in the MS/MS spectra (Ross et al., 2004. Mol. Cell. Proteomics 3(12):1154-1169). Further experimental protocols can be found in WO2006/134056 and a previous publication (Bantscheff et al., 2007. Nature Biotechnology 25, 1035-1044).
The identified kinases are shown in Table 4 including the percent competition values for the sample to which 10 μM free compound had been added. In total 18 different kinases were identified and competed by different degrees. For illustration, the identified peptides for PIK3Ca, PIK3Cb, PIK4Ca, PIP5K2C, PIK3Cg and PIK3Cd are shown in FIGS. 11 to 16. Sequence identifiers are defined by the International Protein Index (IPI) (Kersey et al., 2004. Proteomics 4(7): 1985-1988).
1. Cell Culture
In this example a mix of HeLa and placenta cell lysates was used (Bantscheff et al., 2007. Nature Biotechnology 25, 1035-1044). HeLa cells (American Type Culture Collection-No CCL-2) were either obtained from an external supplier (CIL SA, Mons, Belgium) or grown in one litre Spinner flasks (Integra Biosciences, #182101) in suspension in RPMI 1640 medium (Invitrogen, #21875-034) supplemented with 10% Fetal Bovine Serum (Invitrogen, #10270-106). Cells were harvested by centrifugation, washed once with 1×PBS buffer (Invitrogen, #14190-094) and cell pellets were frozen in liquid nitrogen and subsequently stored at −80° C.
2. Preparation of Cell Lysates
Cells were homogenized in a Potter S homogenizer in lysis buffer: 50 mM Tris-HCl, 0.8% NP40, 5% glycerol, 150 mM NaCl, 1.5 mM MgCl2, 25 mM NaF, 1 mM sodium vanadate, 1 mM DTT, pH 7.5. One complete EDTA-free tablet (protease inhibitor cocktail, Roche Diagnostics, 1 873 580) per 25 ml buffer was added. The material was dounced 20 times using a mechanized POTTER S, transferred to 50 ml falcon tubes, incubated for 30 minutes rotating at 4° C. and spun down for 10 minutes at 20,000×g at 4° C. (10,000 rpm in Sorvall SLA600, precooled). The supernatant was transferred to an ultracentrifuge (UZ)-polycarbonate tube (Beckmann, 355654) and spun for 1 hour at 145.000×g at 4° C. (40.000 rpm in Ti50.2, precooled). The supernatant was transferred again to a fresh 50 ml falcon tube, the protein concentration was determined by a Bradford assay (BioRad) and samples containing 50 mg of protein per aliquot were prepared. The samples were immediately used for experiments or frozen in liquid nitrogen and stored frozen at −80° C.
3. Capturing of Kinases from Cell Lysate
Sepharose-beads with the immobilized compound (100 μl beads per pull-down experiment) were equilibrated in lysis buffer and incubated with a cell lysate sample containing 50 mg of protein on an end-over-end shaker (Roto Shake Genie, Scientific Industries Inc.) for 2 hours at 4° C. Beads were collected, transferred to Mobicol-columns (MoBiTech 10055) and washed with 10 ml lysis buffer containing 0.4% NP40 detergent, followed by 5 ml lysis buffer containing 0.2% detergent. To elute bound proteins, 60 μl 2×SDS sample buffer was added to the column. The column was incubated for 30 minutes at 50° C. and the eluate was transferred to a siliconized microfuge tube by centrifugation. Proteins were then alkylated with 108 mM iodoacetamid. Proteins were then separated by SDS-Polyacrylamide electrophoresis (SDS-PAGE).
4. Protein Identification by Mass Spectrometry
4.1 Protein Digestion Prior to Mass Spectrometric Analysis
Gel-separated proteins were digested in-gel essentially following a previously described procedure (Shevchenko et al., 1996, Anal. Chem. 68:850-858). Briefly, gel-separated proteins were excised from the gel using a clean scalpel, destained twice using 100 μl 5 mM triethylammonium bicarbonate buffer (TEAB; Sigma T7408) and 40% ethanol in water and dehydrated with absolute ethanol. Proteins were subsequently digested in-gel with porcine trypsin (Promega) at a protease concentration of 10 ng/μl in 5 mM TEAB. Digestion was allowed to proceed for 4 hours at 37° C. and the reaction was subsequently stopped using 5 μl 5% formic acid.
4.2 Sample Preparation Prior to Analysis by Mass Spectrometry
Gel plugs were extracted twice with 20 μl 1% formic acid and three times with increasing concentrations of acetonitrile. Extracts were subsequently pooled with acidified digest supernatants and dried in a vacuum centrifuge.
4.3 iTRAQ Labeling of Peptide Extracts
The peptide extracts of samples treated with 10 μM of free compound (CZC31326) and the solvent control (0.5% DMSO) were treated with different variants of the isobaric tagging reagent (iTRAQ Reagents Multiplex Kit, part number 4352135, Applied Biosystems, Foster City, Calif., USA). The iTRAQ reagents are a set of multiplexed, amine-specific, stable isotope reagents that can label peptides on amino groups in up to four different biological samples enabling simultaneous identification and quantitation of peptides. The iTRAQ reagents were used according to instructions provided by the manufacturer. The samples were resuspended in 10 μl 50 mM TEAB solution, pH 8.5 and 10 μl ethanol were added. The iTRAQ reagent was dissolved in 120 μl ethanol and 10 μl of reagent solution were added to the sample. The labeling reaction was performed at room temperature for one hour on a horizontal shaker and stopped by adding 5 μl of 100 mM TEAB and 100 mM glycine in water. The two labeled sampled were then combined, dried in a vacuum centrifuge and resuspended in 10 μl of 0.1% formic acid in water.
4.4 Mass Spectrometric Data Acquisition
Peptide samples were injected into a nano LC system (CapLC, Waters or nano-LC 1D+, Eksigent) which was directly coupled either to a quadrupole TOF (QTOF Ultima, QTOF Micro, Waters), ion trap (LTQ) or Orbitrap mass spectrometer. Peptides were separated on the LC system using a gradient of aqueous and organic solvents (see below). Solvent A was 0.1% formic acid and solvent B was 70% acetonitrile in 0.1% formic acid.







TABLE 3







Peptides elution off the LC system









Time (min)
% solvent A
% solvent B












0
95
5


8.0
95
5


15
85
15


64.5
60
40


84.5
38
62


87
5
95


91
250
95


91.5
2095
5









4.5 Protein Identification

The peptide mass and fragmentation data generated in the LC-MS/MS experiments were used to query a protein data base consisting of an in-house curated version of the International Protein Index (IPI) protein sequence database combined with a decoy version of this database (Elias and Gygi, 2007, Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nature Methods 4, 207-214). Proteins were identified by correlating the measured peptide mass and fragmentation data with data computed from the entries in the database using the software tool Mascot (Matrix Science; Perkins et al., 1999. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551-3567). Search criteria varied depending on which mass spectrometer was used for the analysis. Protein acceptance thresholds were adjusted to achieve a false discovery rate of below 1% as suggested by hit rates on the decoy data base (Elias and Gygi, 2007, Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nature Methods 4, 207-214).
4.6 Protein Quantitation
Relative protein quantitation was performed using peak areas of iTRAQ reporter ion signals essentially as described in an earlier publication (Bantscheff et al., 2007. Nature Biotechnology 25, 1035-1044).







TABLE 4







Identified kinases with compound CZC31207 from mixed HeLa 


and placenta cell lysates











Representative
Kinase
Kinase
Quantified
Compe-


Sequence
Name
Group
Spectra
tition %














IPI00003479.3
Erk2
CMGC
6
−2.9


IPI00021331.1
NEK2
Other
5
40.4


IPI00022633.3
TNK1
TK
8
−1.9


IPI00031386.2
PIK3Ca
Lipid Kinase
7
68.6


IPI00031388.1
PIK3Cb
Lipid Kinase
67
85.6


IPI00070943.3
PIK4Ca
Lipid Kinase
477
39.9


IPI00152303.7
PIP5K2C
Lipid Kinase
57
78.3


IPI00169392.5
CaMK2g
CAMK
8
15.2


IPI00180781.3
MLKL
TKL
23
34.2


IPI00292690.1
PIK3Cg
Lipid Kinase
28
80


IPI00296337.2
DNAPK
Atypical
44
35.3


IPI00298410.2
PIK3Cd
Lipid Kinase
57
83.6


IPI00298612.1
BCKDK
Atypical
187
8


IPI00298940.3
AurA
Other
4
12.8


IPI00303550.2
JNK2
CMGC
5
24.6


IPI00418221.3
MAP3K6
STE
3
10.3


IPI00513678.1
FRAP
Atypical
33
25.9


IPI00787127.1
MAP3K1
STE
12
13.3









Example 3
Kinobeads Experiment Using Immobilized Compound CZC31207 and a Mix of Jurkat and Ramos Cell Lysates
This example demonstrates the use of an immobilized compound (structure shown in FIG. 2, CZC31207) for the capturing and identification of kinases from amix of Jurkat and Ramos cell lysates in a competition binding assay. To the first aliquot of cell lysate 10 μM of the free compound CZC31207 was added and allowed to bind to proteins in the lysate. Then the affinity matrix with the immobilized compound was added to capture proteins that were not interacting with the previously added free compound. Beads were separated from the lysate and bead bound proteins were eluted in SDS sample buffer and subsequently separated by SDS-Polyacrylamide gel electrophoresis (FIG. 5). Suitable gel bands were cut out and subjected to in-gel proteolytic digestion with trypsin. The second lysate aliquot was processed identically, however no free compound was added (DMSO solvent control). Peptides originating from samples 1 and 2 were labeled with iTRAQ reagents (iTRAQ 114 and iTRAQ 116) and the combined samples were analyzed with a nano-flow liquid chromatography system coupled online to a tandem mass spectrometer (LC-MS/MS) experiment followed by iTRAQ reporter ion quantification in the MS/MS spectra as described in Example 2.
Jurkat cells (ATCC number T1B-152) and Ramos cells (ATCC number CRL-1596) were either obtained from an external supplier (CIL SA, Mons, Belgium) or grown in one litre Spinner flasks (Integra Biosciences, #182101) in suspension in RPMI 1640 medium (Invitrogen, #21875-034) supplemented with 10% Fetal Bovine Serum (Invitrogen, #10270-106) at a density between 0.2×106 and 1.0×106 cells/ml. Cells were harvested by centrifugation, washed once with 1×PBS buffer (Invitrogen, #14190-094) and cell pellets were frozen in liquid nitrogen and subsequently stored at −80° C.
The identified kinases are shown in Table 5 including the percent competition values for the sample to which 10 μM free compound had been added. In total of 20 different kinases were identified and competed by different degrees.







TABLE 5







Identified kinases with compound CZC31207 from a mix of 


Jurkat and Ramos cell lysates











Representative
Kinase
Kinase
Quantified
Compe-


Sequence
Name
Group
Spectra
tition %














IPI00009334.4
PKD2
CAMK
12
37.9


IPI00009688.1
PIP5K2A
Lipid Kinase
8
68.5


IPI00011488.4
MST1
STE
4
24.5


IPI00021331.1
NEK2
Other
21
17


IPI00023529.1
CDK6
CMGC
4
−1.6


IPI00026689.4
CDC2
CMGC
4
34.1


IPI00031388.1
PIK3Cb
Lipid Kinase
48
94.8


IPI00070943.3
PIK4Ca
Lipid Kinase
111
28.5


IPI00152303.7
PIP5K2C
Lipid Kinase
23
75.5


IPI00169392.5
CaMK2g
CAMK
58
−3.1


IPI00291068.3
PIK4C2B
Lipid Kinase
3
20.3


IPI00292690.1
PIK3Cg
Lipid Kinase
273
91.5


IPI00296337.2
DNAPK
Atypical
126
38.2


IPI00298410.2
PIK3Cd
Lipid Kinase
160
91.3


IPI00298612.1
BCKDK
Atypical
62
−0.1


IPI00299755.2
PIK3C3
Lipid Kinase
9
52.4


IPI00337426.1
BIKE
Other
90
26.1


IPI00513678.1
FRAP
Atypical
5
−87.1


IPI00787127.1
MAP3K1
STE
68
14.1


IPI00828081.1
CaMK2d
CAMK
21
−1.3









Example 4
Kinobeads Selectivity Profiling
This example illustrates the use of a competition binding assay in cell lysate to establish the kinase selectivity profile of the test compound CZC24513. This compound was added at defined concentrations (10 μM, 1 μM and 0.1 μM CZC24513) to a mix of HeLa and placenta cell lysates thereby allowing the test compound to bind to the target proteins in the lysate. Then the lysate was contacted with the immobilized compound CZC25236 to capture remaining free target proteins. The proteins bound to the immobilized compound were eluted with detergent-containing buffer, separated on a SDS-polyacrylamide gel and analyzed by mass spectrometry as described in example 2.
The peptide extracts corresponding to samples treated with different concentrations of the test compound (10 μM, 1 μM and 0.1 μM CZC24513) and the solvent control (0.5% DMSO) were treated with different variants of the isobaric tagging reagent (iTRAQ Reagents Multiplex Kit, part number 4352135, Applied Biosystems, Foster City, Calif., USA). The iTRAQ reagents are a set of multiplexed, amine-specific, stable isotope reagents that can label peptides in up to four different biological samples enabling simultaneous identification and quantitation of peptides. The iTRAQ reagents were used according to instructions provided by the manufacturer.
The test compound CZC24513 was used at three different concentrations in the cell lysate and the IC50 values were normalized to the DMSO control. For selected kinases the IC50 values were plotted against the concentration of CZC24513 and curve fitting was performed using the Xlfit program (ID Business Solutions Ltd.) as previously described. (Bantscheff et al., 2007. Nature Biotechnology 25, 1035-1044). The IC50 value corresponds to the test compound concentration at which the relative intensity of the MS signal for a kinase is 50% compared to the solvent (DMSO) control. Examples of dose response curves for individual kinases are shown in FIGS. 7 to 10.







TABLE 6







Selectivity profiling of test compound CZC24513











Representative
Kinase
Kinase
Quantified
IC50


Sequence
Name
Group
Spectra
(μM)














IPI00003479.3
Erk2
CMGC
5
10


IPI00009688.1
PIP5K2A
Lipid Kinase
11
0.61


IPI00022633.3
TNK1
TK
9
10


IPI00024006.3
PIK3R4
Other
2
6.01


IPI00031386.2
PIK3Ca
Lipid Kinase
10
1.55


IPI00031388.1
PIK3Cb
Lipid Kinase
104
0.31


IPI00070943.3
PIK4Ca
Lipid Kinase
593
2.26


IPI00152303.7
PIP5K2C
Lipid Kinase
78
0.75


IPI00216470.1
PIP5K2B
Lipid Kinase
1
0.27


IPI00292056.4
PIK3C2b
Lipid Kinase
46
0.99


IPI00292690.1
PIK3Cg
Lipid Kinase
18
0.1


IPI00296337.2
DNAPK
Atypical
148
3.04


IPI00298410.2
PIK3Cd
Lipid Kinase
50
0.62


IPI00337426.1
BIKE
Other
30
10


IPI00479760.6
AAK1
Other
7
4.31


IPI00513678.1
FRAP
Atypical
22
10


IPI00787127.1
MAP3K1
STE
5
10
















TABLE 7







Preparation of 5x-DP buffer












Final conc. in 1 x
Add for 1l 5 x 


Substance:
Stock solution
lysis buffer
lysis buffer





Tris/HCl pH 7.5
1M
 50 mM
250 ml


Glycerol
87%
5%
288 ml


MgCl2
1M
 1.5 mM
 7.5 ml


NaCl
5M
150 mM
150 ml


Na3VO4
100 mM
 1 mM
 50 m1









The 5×-DP buffer was filtered through a 0.22 μm filter and stored in 40 ml-aliquots at −80° C. Stock solutions were obtained from the following suppliers: 1.0 M Tris/HCl pH 7.5 (Sigma, T-2663), 87% Glycerol (Merck, catalogue number 04091.2500); 1.0 M MgCl2 (Sigma, M-1028); 5.0 M NaCl (Sigma, S-5150).

## Claims

1. An immobilization compound of formula (I)



or a salt thereof, wherein
R is C1-4 alkyl optionally substituted with one or more fluoro; and
n is 1, 2, or 3.

2. The immobilization compound of claim 1, selected from the group consisting of

3. A method for the preparation of an immobilization product, said method comprising immobilizing the immobilization compound of claim 1 to a solid support by a covalent direct or linker mediated attachment.

4. The method of claim 3, wherein the solid support is selected from the group consisting of agarose, modified agarose, sepharose beads latex, cellulose, and ferro- or ferrimagnetic particles.

5. An immobilization product, comprising the immobilization compound of claim 1 immobilized on a solid support.

6. A method for the identification of a phosphatidylinositol kinase interacting compound, comprising the steps of
a) providing a protein preparation containing a variety of phosphatidylinositol kinases,
b) contacting the protein preparation with the immobilization product of claim 5 and with a given compound under conditions allowing the formation of one or more different complexes between one of the phosphatidylinositol kinases and the immobilization product, and
c) detecting the complex or the complexes formed in step b).

7. A method for the identification of a phosphatidylinositol kinase interacting compound, comprising the steps of:
a) providing two aliquots of a protein preparation containing a variety of phosphatidylinositol kinases,
b) contacting one aliquot with the immobilization product of claim 5 under conditions allowing the formation of one or more different complexes between one of the phosphatidylinositol kinases and the immobilization product,
c) contacting the other aliquot with the immobilization product of claim 5 and with a given compound under conditions allowing the formation of one or more different complexes between one of the phosphatidylinositol kinases and the immobilization product, and
d) determining the amount of the complex or the complexes formed in steps b) and c).

8. A method for the identification of a phosphatidylinositol kinase interacting compound, comprising the steps of:
a) providing two aliquots of a cell preparation comprising each at least one cell containing a variety of phosphatidylinositol kinases,
b) incubating one aliquot with a given compound,
c) harvesting the cells of each aliquot,
d) lysing the cells in order to obtain protein preparations,
e) contacting the protein preparations with the immobilization product of claim 5 under conditions allowing the formation of one or more different complexes between one of the phosphatidylinositol kinases and the immobilization product, and
f) determining the amount of the complex or the complexes formed in each aliquot in step e).

9. The method of claim 7, wherein a reduced amount of the complex formed in the aliquot incubated with the compound in comparison to the aliquot not incubated with the compound indicates that said phosphatidylinositol kinase interacts with the compound.

10. The method of claim 7, wherein the amount of the complex is determined by separating the phosphatidylinositol kinase from the immobilization product and subsequent detection of the separated phosphatidylinositol kinase or subsequent determination of the amount of the separated phosphatidylinositol kinase.

11. The method of claim 7, wherein said given compound is selected from the group consisting of synthetic compounds, or organic synthetic drugs and natural small molecule compounds.

12. The method of claim 7, wherein the given compound is a phosphatidylinositol kinase inhibitor.

13. The method of claim 7, wherein the provision of a protein preparation includes the steps of harvesting at least one cell containing phosphatidylinositol kinases and lysing the cell.

14. The method of claim 7, wherein the steps of the formation of the complex are performed under essentially physiological conditions.