Lipid probes and uses thereof

Patent metadata

Field	Value
Publication number	US-10168342-B2
Application number	US-201615080767-A
Country	US
Kind code	B2
Filing date	Mar 25, 2016
Priority date	Mar 27, 2015
Publication date	Jan 1, 2019
Grant date	Jan 1, 2019

Abstract

Disclosed herein are methods, compositions, probes, assays and kits for identifying a lipid binding protein as a drug binding target. Also disclosed herein are methods, compositions, and probes for mapping a ligand binding site on a lipid binding protein, identification of lipid binding proteins, generating drug-lipid binding protein profiles, high throughput drug screening, and identification of drugs as potential lipid binding protein ligands.

First claim

What is claimed is:

1. A high-throughput method of identifying lipid binding proteins as drug binding targets, comprising:
a) harvesting a set of competitive lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle, and wherein the sample comprises a first cell solution and a second cell solution, and the second cell solution comprises a drug;
b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means;
c) based on step b), determining i) a value for each of the lipid binding proteins from the set of lipid probe-protein complexes, and ii) a ligand binding site on each of the lipid binding proteins from the set of lipid probe-protein complexes; and
d) based on the value from c), generating a drug-lipid binding protein profile, wherein the drug-lipid binding protein profile is a profile that indicates an array of lipid binding proteins and respective ligand binding sites that interact with the drug, and wherein the drug-lipid binding protein profile is generated by the high-throughput method.

2. The method of claim 1 , further comprising contacting the first cell solution with a first lipid probe and contacting the second cell solution with a second lipid probe.

3. The method of claim 2 , wherein the first lipid probe and the second lipid probe are the same.

4. The method of claim 1 , wherein the second cell solution comprises a buffer or a media.

5. The method of claim 1 , further comprising treating the first cell solution and the second cell solution by a photoreactive means to generate a first group of lipid probe-protein complexes and a second group of lipid probe-protein complexes, wherein the first group and the second group of lipid probe-protein complexes comprise the set of lipid probe-protein complexes.

6. The method of claim 1 , wherein the lipid is a bioactive lipid.

7. The method of claim 1 , wherein the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccarolipids, or polyketides.

8. The method of claim 7 , wherein the fatty acyls comprise fatty acids, octadecanoids, eicosanoids, docosanoids, fatty alcohols, fatty aldehydes, fatty esters, fatty amides, fatty nitriles, fatty ethers, or fatty acyl glycosides.

9. The method of claim 7 , wherein the sterol lipid comprises sterols, steroids, secosteroids, or bile acids.

10. The method of claim 1 , wherein the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones; and optionally a linker.

11. The method of claim 1 , wherein the affinity handle is a bioorthogonal affinity handle, optionally conjugated to an affinity ligand.

12. The method of claim 1 , wherein the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group.

13. The method of claim 1 , wherein the photoreactive group and the affinity handle are conjugated to the same site of the lipid probe, or the photoreactive group and the affinity handle are conjugated to different sites of the lipid probe.

14. The method of claim 11 , wherein the affinity ligand comprises a chromophore, a labeling group, or a combination thereof.

15. The method of claim 14 , wherein the chromophore comprises fluorochrome, non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate.

16. The method of claim 14 , wherein the labeling group is biotin moiety, streptavidin moiety, bead, resin, a solid support, or a combination thereof.

17. The method of claim 1 , wherein the lipid probe comprises:
a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls;
a photoreactive linker; and
an affinity handle.

18. The method of claim 1 , wherein the lipid probe is a lipid probe of Formula (I):

wherein:
A is C 16 -C 20 alkyl or C 16 -C 20 alkenyl;
D is —OH, —NH 2 , —NHR 7 , or —OR 8 ;
R 7 is C 1 -C 4 alkyl, (C 1 -C 5 alkyl)OH, or (C 1 -C 5 )SO 3 M;
R 8 is (CH 2 OH)n;
M is monovalent or divalent cation;
n is 1, 2, or 3;

PG is a photoreactive group; and
AH is an affinity handle;
wherein PG is attached to A or D and AH is attached to A or D.

19. The method of claim 1 , wherein the lipid probe has one of the structures as illustrated in Table 1.

20. The method of claim 1 , wherein the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone.

21. The method of claim 1 , wherein the lipid binding protein is a protein encoded by a gene of Table 3, a protein encoded by a gene of Table 4, or a protein encoded by a gene of Table 5.

22. The method of claim 1 , wherein the proteomic analysis means comprises a mass spectroscopy method.

23. The method of claim 1 , wherein the value for each of the lipid binding proteins in step c) is the area-under-the curve from a plot of signal intensity as a function of mass-to-charge ratio.

24. The method of claim 5 , wherein the generating in step d) of claim 1 further comprises
i) locating a first value assigned to a lipid binding protein from the first group of lipid probe-protein complex and a second value of the same lipid binding protein from the second group of lipid probe-protein complex; and
ii) calculating a ratio between the two values assigned to the same lipid binding protein.

25. The method of claim 24 , wherein the ratio of greater than 2 indicates that the lipid binding protein is a candidate for interacting with the drug.

26. The method of claim 1 , further comprising classifying the drug as a specific inhibitor or a pan inhibitor.

Assignees

• Scripps Research Inst

Inventors

• Cravatt Benjamin F
• Niphakis Micah J
• Lum Kenneth
• Correia Bruno
• Cognetta Armand
• Hulce Jonathan

Classifications

• G01N2500/04

  Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A) · CPC title

• G01N33/92 Primary

  involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)} · CPC title

• G01N2570/00

  Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes · CPC title

• G01N33/6842

  Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins · CPC title