Science , , J Am Chem Soc , , Molecular Biosystems , 4, Protein Science , 16, Structure , 15, ACS Chem Biol , 2, Acta Cryst D , 63, ChemBioChem , 7, Langmuir , 22, J Photochem Photobiol B , 85, Curr Opin Struct Biol , 16, Acta Cryst F , 61, Protein Science , 13, Eur J Biochem , , Structure , 11, Acta Cryst D , 59, Curr Opin Struct Biol , 12, ChemBioChem , 2, Acta Cryst D , 56, Nature , , Nature Structural Biology , 5, Biochemistry , 36, Cell , 91, Nature , , Nature Structural Biology , 3, Proteins , 21, Crystallographic analysis of the VL domain confirms the structural model.
Journal of Chromatography A , , Gene , , Book Section. In Biomineralization, edited by E. In Encyclopedia of Nanoscience and Nanotechnology, edited by J. Schwarz, Contescu, C. Zuletzt aktualisiert: Direkt zum Inhalt. AG Essen Prof. The unchanged diacyl moiety permits insertion and retention of Pea-PIP n s in a lipid bilayer to facilitate recruitment of PtdlnsP n -specific binding proteins to a membrane surface environment. These new hybrid lipids can serve as direct enzymatic substrates that can be delivered into a cell in order to measure direct turnover.
This is a great improvement over measuring competitive displacement with a surrogate. Accordingly, Pea-PIP n s of the present invention have potential for the production of unique, reporter-based high throughput screens or assays for in vitro biochemical activity and for monitoring real time in situ biochemical activity in living cells.
The following figures form part of the present specification and are included to further demonstrate certain aspects of the disclosed embodiments of the invention. The embodiments of the invention may be better understood by reference to one or more of the figures in combination with the detailed description of specific embodiments of the invention presented herein. Illustrates at c an exemplary hybrid lipid of the present invention that possess a phosphatidylethanolamine PE, or Pea head group at the 1-position and a Ptdlns 4,5 P 2 head group at the 4-position in accordance with one embodiment of the present invention.
Illustrates exemplary synthesis of differentially functionalized 2,3-diacylthreitol backbones in accordance with an embodiment of the present invention. Illustrates exemplary backbone phosphorylation and synthesis of Pea-PIP 2 derivatives Illustrates exemplary Pea-PIP n reporter groups according to an embodiment of the present invention. Reaction of the free Pea amino group of with four N-hydroxysuccinimidyl NHS esters afforded the corresponding biotinylated derivative 4 a , the fluorescent N- 7-nitrobenzoxa-1,3-diazolyl NBD and 6-carboxyfluorescein derivatives 4 - b and 4 - c , and the spin-labeled 3-carboxy-2,2,5,5-tetramethylpyrrolidinyloxy PROXYL derivative 4 - d.
Illustrates the synthesis of exemplary short PEG linkers. Illustrates the synthesis of an exemplary protected threitol backbone with PEG linkers. In particular embodiments, a reporter group, e.
The reporter would thus be targeted to the lipid-water interface at a site distant from the key PtdlnsPn head group for example, Ptdlns 4,5 P 2 recognition features of the binding protein. In various embodiments, the diacyl moiety permits insertion and retention of Pea-PIP n s in a lipid bilayer to facilitate recruitment of PtdlnsPn head group— for example, Ptdlns 4,5 P 2 — specific binding proteins to a membrane surface environment.
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The method for producing the hybrid phospholipids of the present invention involves diethyl D-tartrate as the chiral precursor for the extended glycerol backbone of the target hybrid lipid. The corresponding acetal is reducted with lithium aluminum hydride and protected with 1 equivalent of PMB-Cl to give the monobenzyl ether. Coupling with a carbonylbenloxy Cbz -protected aminoethoxy phosphoramidite yields, after oxidation, the protected phosphatidyl analogue ready for addition of the acyl chains and the selected PIP n head group.
After acetal hydrolysis, the 2,3-dipalmitoyl derivative is prepared. As shown for exemplary molecules in FIG. Any PIP n head group may be prepared and used according to the invention. PIP n head groups to be utilized in this invention may be commercially obtained or prepared by standard methods known in the art. According to this embodiment, the Pea-diacylthreitol synthetic module is prepared and coupled with any selected phosphoinositide head group. In another embodiment of the present invention, a linker may be utilized between the PIP n headgroup and a reporter.
In a particular embodiment, the length of the linker between the PIP n headgroup and the reporter moiety at the primary amine is lengthened. In a particular embodiment, the extension for the linker comprises an oligo-polyethylene glycol linker. Other embodiments include additional or alternative linkers. For example, one such alternative linker is a diamino linker that will yield a phosphoramidate final product rather than a phosphate linkage. Another alternative linker uses both phospho- and non-phospho linked spacers.
In certain embodiments, this can be accomplished by replacing the Pea group at C- 4 , such as with a simple ester, amide or other linkage that allows a pendant functionality to be incorporated. Another alternative linker is to use a phosphatidylserine or other carboxylic acid instead of Pea to permit further functionalization at the end distal to the PIP n recognition element Various methods for producing alternative linkers are well known in the art.
Pea extension of the invention may also include aminoalcohols as linkers, for example 3-aminopropanol, 4-aminobutanol, and others. In certain embodiments heteroatom-containing derivatives such as 1-aminohydroxy-3,6,9-trixaundecane or similar aminoalcohols with water soluble spacer chains may be used. These can include branched aminoalcohols including, but not limited to, for example, 2-aminomethylaminopropanol which has multiple reactive amino termini for addition of two or more biochemical probes.
One of ordinary skill in the art will know methods of Pea extension beyond those listed above. Methods of modifying the length or degree of double bonds in an acyl chain are well known in the art. In certain embodiments, the carbons will have a number of double bonds, for example from 0 to 6. In alternative embodiments, one or both acyl chains can be replaced with an ether chain of the same length, degree of unsaturation or terminal functionalization. Replacement of one or both acyl chains can be accomplished by any standard method known in the art.
According to other embodiments, the phosphate groups of this invention can be chemically modified to increase stability or resistance to chemical or enzymatic hydrolysis. In certain embodiments, the phosphate groups will be on the inositol, the phosphodiester or the PE phosphodiester. Other methods of chemically modifying the phosphate groups of this invention are known in the art.
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In certain embodiments, a Pea-PIP n of the present invention has a triester analog at P-1 to allow for an additional site for derivation. See, e. Gu and G. In yet another embodiment, the Pea-PIP n carbon threitol backbone can include four, five, six or more carbons. In particular embodiments, such carbon backbone may be branched. In a certain embodiment, such carbon threitol backbone is 2,3-diacylthreitol or erythritol. Attached to the Pea amino group, the reporter would thus be targeted to the lipid-water interface at a site distant from the specific features of the PtdlnsP n head group necessary for interaction with lipid recognition proteins or other chemicals or compounds that specifically interact with the PtdlnsP n at a membrane surface.
In various embodiments, the diacyl moiety permits insertion and retention of Pea-PIP n s in a lipid bilayer to facilitate recruitment of PtdlnsP n -specific binding proteins to a membrane surface environment. An individual Pea-PIPn may have one or more reporters which may be of the same or different types. In various embodiments, the Pea-PIPns will be transported into cells, with or without a reporter. All stereoisomers, which include enantiomers or diasteromers, for any component of the Pea-PIP n molecules can be employed in any embodiment of this invention.
Carbohydrate Chemistry, Biology and Medical Applications
Such modifications are well known in the art. Certain embodiments will include Pea-PIP n s bound to a surface, for example for use in a biochemical assay. In particular embodiments, such surface will include a plate, a bead or nitrocellulose. In a particular embodiment, such surfaces are selected from the group consisting of, but not limited to, a chemically activated glass, plastic or other surface; activated agarose, polystyrene or any other type of bead and nitrocellulose.
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In other embodiments, the Pea-PIP n s are attached to a metal surface, such as gold. In particular embodiments, attachment to gold is accomplished by introducing to the Pea-PIP n a pendant alkyl thiol moiety that is capable of attaching to a gold surface. Methods for making such pendant alkyl thiol derivatives are well known in the art. In alternative embodiments, the Pea-PIP n s will be incorporated in a liposome.
Such liposome incorporated Pea-PIP n s may include a reporter. Certain embodiments allow for the use of Pea-PIP n s for assays. The compositions and methods of their use can be used in any assay that currently uses a modified Pea-PIP. These may be in a high-throughput format. Alternative embodiments include in vitro enzyme assays, lipid kinase or phosphatase activity, cell-based assays and agonist or antagonist assays.
Such assays include, but are not limited to, in vitro enzyme assays, in vitro agonist or antagonist assays or cell-based assays. In some embodiments of the invention, labeled Pea-PIP n s can also be linked or bound to plates, beads or other surfaces which may, in particular embodiments, be coated with a means for binding Pea-PIP n thereto. Labels of use in the present invention may be activated by any method known in the art in order to effect attachment to a Pea-PIP n of the present invention.
Methods of use in the attachment of a label include, but are not limited to, the activation of an ester, carbonyldiimidazole activation and use of any Michael acceptor, such as acrylates, acrylamide, maleimides, vinylsulfone, a,b-unsaturated ketones, esters, aldhydes, amides, and the like. An example of such a means of linking or binding a Pea-PIP n s is streptavidin. Another example is NHS activation.
In other embodiments, the Pea-PIPn is bound to nitrocellulose.
In particular embodiments, the reporter is biotin or a fluorescent label. One embodiment of the present invention provides a method of screening for phosphoinositide-specific binding proteins in a membrane surface environment. Such compounds include, but are not limited to, for example, agonists and antagonists for protein kinases and phosphoinositide kinases and for phosphoinositide and inositol phosphate binding proteins that are regulated by PIP n s or IP n s and may serve as downstream effectors in signaling pathways important for therapeutic interventions. In particular embodiments, lipid phosphatases or phospholipases are identified.
In alternative embodiments, HTS assays include cell-based assays using intracellular PIP n s introduced by the shuffling system and can use primary cells, immortalized cells, cancer cells, cells transformed with plasmids encoding key enzymes or other proteins, and the like. The assays could also use in vitro cell extracts or partially purified or homogeneous proteins. In other embodiments the Pea-PIP n s are introduced into cells. Such introduced Pea-PIP n s may be labeled, or tagged, with one or more reporters. In a particular embodiment, fluorescent acyl-modified Pea-PIP n s are shuttled into cells where they exhibit subsequent appropriate membrane localization.
In certain embodiments, assays according to the present invention are performed in living cells. Particular embodiments of the invention provide compositions and methods for visualizing the location of labeled phosphoinositides within a cell. A method for facilitating uptake of a Pea-PIP n s into a cell comprises contacting the cell with a composition of matter comprising a Pea-PIP n and a shuttle, or other method of introducing the Pea-PIP n into the cell. Compounds suitable for shuttling Pea-PIP n s into a cell can include, but are not limited to, polyamines.
Such polyamines can include, for example, aminoglycosidic aminocyclitols e. The polybasic polypeptide may also contain unnatural or non-protein amino acids, N-acylglycine groups, and any of a known group of amide group replacements known as peptide bond isosteres. In particular embodiments, assays performed in living cells are monitored by high-content screening methods or confocal microscopy. Such in vitro assays allow for minimal disruption of the normal cellular environment. The disclosed compositions and methods of their use can be used for the discovery of new pharmaceutical agents and targets related to PtdlnsP n s compounds.
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The skilled artisan will realize that the chemical modifications listed above are exemplary only and that many variations may be used, depending on the particular type of Pea-PIP, to be synthesized. For the purposes of the present invention, the following terms shall have the following meanings:. In various embodiments of the invention, such attachment may be either covalent or non-covalent In certain embodiments of the invention, the labels have physical characteristics that facilitate the identification of the label.
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