Hybrid phosphoinositide phospholipids: compositions and uses

• US 20050148042A1
• Filed: 03/31/2003
• Published: 07/07/2005
• Est. Priority Date: 03/29/2002
• Status: Abandoned Application

• Current Assignee: Angie M. Branch, Colin G. Ferguson, Glenn Prestwich, Lee R. Crosby, Paul O. Neilsen, Piotr W. Rzepecki
• Original Assignee: Angie M. Branch, Colin G. Ferguson, Glenn Prestwich, Lee R. Crosby, Paul O. Neilsen, Piotr W. Rzepecki
• Inventors: Neilsen, Paul O., Branch, Angie M., Ferguson, Colin G., Crosby, Lee R., Rzepecki, Piotr W., Prestwich, Glenn
• Application Number:
  US10/510,197
• Publication Number:
  US 20050148042A1
• Time in Patent Office: Days
• US Class Current: 435/15
• CPC Class Codes: C07F 9/117 with cycloaliphatic alcohols
• CPC Class Codes: C07F 9/65324 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/65517 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743

Abstract

The methods and compositions disclosed herein concern the synthesis of a novel class of “two-headed” phospholipid-phosphoinositide hybrids possessing a carbon backbone, such as 2,3-diacylthreitol, erythritol or a synthetic module. The second phospholipid head group allows introduction of a biochemical or chemical moiety in a position orthogonal in space to those occupied by the phosphoinositide head group and the two acyl chains. The diacyl moieties allow for the incorporation of Pea-PIP₂ into a lipid bilayer, while the Ptdlns(4,5)P₂ moiety in the aqueous layer is specifically recognized by lipid binding proteins. In alternative embodiments of the invention, reporters, for example biotin, fluorophores and/or spin labels, are attached to the free amino group of the head groups of such molecules to specifically target the reporters to the lipid-water interface.

Claims

1. A functionalized phosphoinositide polyphosphate comprising a carbon backbone, a phosphatidylethanolamine head group at carbon position 1, and a PtdlnsP_n head group at carbon position 4.

2. A functionalized phosphoinositide polyphosphate of claim 1, wherein said carbon backbone is selected from the group consisting of 2,3-diacylthreitol, erythritol and a synthetic module.

3. A functionalized phosphoinositide polyphosphate of claim 2, wherein said synthetic module is prepared and coupled with a PtdlnsP_n head group.

4. A functionalized phosphoinositide polyphosphate of claim 3, wherein said PtdlnsP_n head group is selected from the group consisting of (PI, PI(3)_p, PI(4)P, PI(5)P, PI(4,5)P₂, PI(3,4)P₂, PI(3,5)P₂, PI(3,4,5)P₃).

5. A functionalized phosphoinositide polyphosphate of claim 1, further comprising a reporter group.

6. A functionalized phosphoinositide polyphosphate of claim 5, wherein the reporter is selected from the group consisting of a flourophore, a spin label, biotin, a radio label, a chemiluminescent label, a photophore, a chromophore, a nanogold particle and mixtures thereof.

7. A functonalized phosphoinositide polyphosphate of claim 6, wherein the flourophore is selected from the group consisting of acrylodan, AMCA, BODIPY, Cascade-Blue, CINERF, dansyl, dialkylaminocoumarin, eosin, erythrosine, fluorescein, hydroxycoumarin, NBD, Oregon green, PyMPO, pyrene, rhodamine, Rhodol Green, TMR, Texas Red, and X-Rhodamine.

8. A functionalized phosphoinositide polyphosphate of claim 5, wherein the reporter is covalently attached to a free amino group on the phosphatidylethanolamine head group.

9. A functionalized phosphoinositide polyphosphate of claim 8, wherein the reporter is attached to a free amino group by a linker.

10. A functionalized phosphoinositide polyphosphate of claim 9, wherein said linker comprises an oligo-polyethylene glycol linker.

11. A functonalized phosphoinositide polyphosphate of claim 9, wherein said linker may include an additional or alternative linker.

12. A functionalized phosphoinositide polyphosphate of claim 11, wherein said additional or alternative linker is selected from the group consisting of a diamino linker, a linker utilizing both phospho- and non-phospho linked spacers, a phosphatidylserine linker and a carboxylic acid linker.

13. A functionalized phosphoinositide polyphosphate of claim 11, wherein said linker includes aminoalcohols, heteroatom-containing derivatives or branched aminoalcohols.

14. A functionalized phosphoinositide polyphosphate of claim 13, further comprising a diamino linker.

15. A functionalized phosphoinositide polyphosphate of claim 1 selected from the group consisting of Pea-PI, Pea-PI(3)P, Pea-PI(4)P, Pea-PI(5)P, Pea-PI(3,4)P₂, Pea-PI(3,5)P₂, Pea-PI(4,5)P₂ and Pea-PI(3, 4,5)P₃.

16. A functionalized phosphoinositide polyphosphate of claim 1, wherein the carbon backbone comprises 2 acyl chains having from 2 to 26 carbons.

17. A functionalized phosphoinositide polyphosphate of claim 1, wherein the carbon backbone comprises an ether chain in the place of one or both acyl chains.

18. A functionalized phosphoinositide polyphosphate of claim 1, further comprising one or more phosphate groups that have been chemically modified to stabilize the compound against chemical or enzymatic hydrolysis.

19. A functionalized phosphoinositide polyphosphate of claim 1, further comprising a polymerizable group.

20. A method of screening for phosphoinositide-specific binding proteins comprising (a) contacting a functionalized phosphoinositide polyphosphate comprising a carbon backbone, a phosphatidylethanolamine head group at carbon position 1, and a PtdlnsP_n head group at carbon position 4 with a putative phosphoinositide-specific binding protein-containing composition;

and (b) measuring binding

21. A method of claim 20, wherein said functionalized phosphoinositide polyphosphate is attached to a surface.

22. A method of claim 21, wherein said surface is selected from the group consisting of plates, beads, liposomes, nitrocellulose and metals.

23. A method of claim 21, wherein said attaching a functionalized phosphoinositide polyphosphate is selected from the group consisting of streptavidin and NHS activation.

24. A method of claim 20, wherein said functionalized phosphoinositide polyphosphate is selected from the group consisting of Pea-PI, Pea-PI(3)P, Pea-PI(4)P, Pea-PI(5)P, Pea-PI(3,4)P₂, Pea-PI(3,5)P₂, Pea-PI(4,5)P₂ and Pea-PI(3, 4,5)P₃.

25. A method of claim 20, wherein said functionalized phosphoinositide polyphosphate further comprises a reporter.

26. A method of claim 25, wherein said reporter is selected from the group consisting of a flourophore, a spin label, biotin, a radio label, a chemiluminescent label, a photophore, a chromophore, a nanogold particle and mixtures thereof.

27. A method of claim 20, wherein said method comprises an assay selected from the group consisting of an in vitro enzyme assay, an in vitro agonist assay, an in vitro antagonist assay, a cell-based assay, a lipid kinase activity assay, a protein kinase activity assay, a lipid phosphatase activity assay, a protein phosphatase activity assay, a phospholipase assay and a phosphatase activity assay.

28. A method of claim 20, wherein said phosphoinositide-specific binding protein is selected from the group consisting of protein kinases, phosphoinositide kinases, phosphoinositide binding proteins, inositol phosphate binding proteins, lipid phosphatases and phospholipases.

32. A method of claim 28, wherein said method comprises a cell-based assay.

29. A method of identifying compositions that affect phosphoinositide recognition or signaling at a cell membrane comprising (a) contacting a functionalized phosphoinositide polyphosphate comprising a carbon backbone, a phosphatidylethanolamine head group at carbon position 1, and a PtdlnsP_n head group at carbon position 4 with a composition that putatively affects phosphoinositide recognition or signaling;

and (b) measuring recognition and signaling.

30. A method of claim 29, wherein said composition that putatively affects phosphoinositide recognition or signaling comprises a compound selected from the group consisting of a chemical, a natural product, and a synthetic compound.

31. A method of claim 29, wherein said composition that putatively affects phosphoinositide recognition or signaling comprises a compound selected from the group consisting of lipid phosphatases and phospholipases.

33. A functionalized phosphoinositide polyphosphate comprising Pea-PI, Pea-PI(3)P, Pea-PI (4)P, Pea-PI(5)P, Pea-PI (3, 4)P₂, Pea-PI (3,5)P₂, Pea-PI(4,5)P₂ or Pea-PI(3,4,5)P₃.