METHOD FOR IDENTIFYING LIPIDBINDING PROTEIN AS TARGET MOLECULES FOR DRUG BINDING

Description

TECHNICAL FIELD

[0001] The present application is concerned with a high throughput method of identifying lipid binding proteins as drug binding targets.CROSS-REFERNCES

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 139,576, filed March 27, 2015.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format. Said ASCII copy, created on March 16, 2016, is named 48054-701.601_SL.txt and is 27,141 bytes in size.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0004] The invention disclosed herein was made, at least in part, with U.S. government support under Grant Nos. CA132630 and DA032541 by the National Institutes of Health. Accordingly, the U.S. Government has certain rights in this invention.BACKGROUND OF THE INVENTION

[0005] Small-molecule metabolites modulate various components of life. In some instances, their biological functions are mediated and regulated by interactions with proteins. In some cases, these metabolite-protein interactions include ligand-receptor, substrate-enzyme, and clientcarrier relationships, many of which represent key nodes in biochemical networks that regulate cell physiology and disease. Eukaryotic and prokaryotic cells harbor numerous structurally distinct metabolites. Among these natural products, lipids display a capacity to interact with and to affect the functions of proteins.

[0006] Kambe et al. ("Mapping the Protein Interaction Landscape for Fully Functionalized Small-Molecule Probes in Human Cells", J. Am. Chem. Soc., 2014, 136, 30, 10777-10782) describes the synthesis of an ~60-member fully functionalized probe library, prepared from Ugiazide condensation reactions to impart structural diversity and introduce diazirine and alkyne functionalities for target capture and enrichment, respectively.

[0007] Hulce et al. ("Proteome-wide Mapping of Cholesterol-Interacting Proteins in Mammalian Cells", Nature Methods, volume 10, pages259-264 (2013)) describes a chemoproteomic strategy that uses clickable, photoreactive sterol probes in combination with quantitative mass spectrometry to globally map cholesterol-protein interactions directly in living cells.

[0008] US 2011 / 0020837 A1 provides tools and methods for studying the interaction of proteins and lipids in vivo as well as in vitro. In particular, US 2011 / 0020837 A1 relates to a method for isolating or identifying a target protein interacting with a lipid in a cell.

[0009] US 2006 / 0257938 A1 describes method to screen for drugs that interfere with the binding between a specific lipid-binding protein and a selected ligand.

[0010] Dubinsky et al. ("Diazirine based photoaffinity labeling", Bioorg Med Chem., 2012, 20(2): 554-570) is a review which summarizes recent advances in the use of diazirines in photoaffinity labeling.

[0011] Xia et al. ("Photoactivatable Lipid Probes for Studying Biomembranes by Photoaffinity Labeling", Chem. Rev., 2013, 113, 10, 7880-7929) describes phoactivatable lipid probes for studying biomembranes.

[0012] Niphakis et al. ("A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells", Cell, 2015, 161(7): 1668-1680) describes a set of lipid-based chemical proteomic probes and their global interaction map in mammalian cells.SUMMARY OF THE INVENTION

[0013] The invention is set out in the appended claims.

[0014] Disclosed herein, in certain examples, is a method of identifying a lipid binding protein as a drug binding target, comprising: (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the lipid binding proteins from the set of lipid probe-protein complexes; (d) based on the value assigned in c), identifying a lipid binding protein as a drug binding target. In some examples, the sample comprises a first cell solution and a second cell solution. In some examples, the method further comprises contacting the first cell solution with a first lipid probe and contacting the second cell solution with a second lipid probe. In some examples, the first lipid probe and the second lipid probe are the same. In some examples, the second cell solution comprises a drug. In some examples, the second cell solution further comprises a buffer or a media. In some examples, the method further comprises 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. In some examples, the method further comprises contacting the first cell solution with a first set of lipid probes wherein each of the lipid probes comprises a lipid, a photoreactive group, and an affinity handle, and wherein each lipid probe is different within the set. In some examples, the method further comprises contacting the second cell solution with a second set of lipid probes wherein each of the lipid probes comprises a lipid, a photoreactive group, and an affinity handle, and wherein each lipid probe is different within the set. In some examples, the first set of lipid probes and the second set of lipid probes are the same. In some examples, the method further comprises treating the first cell solution and the second cell solution by a photoreactive means to generate a third group of lipid probe-protein complexes and a fourth group of lipid probe-protein complexes. In some examples, the lipid is a bioactive lipid. In some examples, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some examples, the lipid is a member of the fatty acyls group. In some examples, 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. In some examples, the lipid is a fatty acid. In some examples, the fatty acid comprises a saturated fatty acid, a monounsaturated fatty acid, or a polyunsaturated fatty acid. In some examples, the saturated fatty acid comprises propanoic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic acid, octacosanoic acid, nonacosanoic acid, triacontanoic acid, henatriacontanoic acid, dotriacontanoic acid, tritriacontanoic acid, tetratriacontanoic acid, pentatriacontanoic acid, or hexatriacontanoic acid. In some examples, the monounsaturated fatty acid comprises palmitoleic acid, vaccenic acid, oleic acid, eicosenoic acid, erucic acid, gadoleic acid, myristoleic acid, or nervonic acid. In some examples, the polyunsaturated fatty acid comprises omega-3 fatty acids, omega-6 fatty acids, omega-9 fatty acids, or conjugated fatty acids. In some examples, the polyunsaturated fatty acid comprises hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic acid, linoleic acid, gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid, tetracosapentaenoic acid, mead acid, rumenic acid, alpha-calendic acid, beta-calendic acid, jacaric acid, alpha-eleostearic acid, beta-eleostearic acid, catalpic acid, punicic acid, rumelenic acid, alpha-parinaric acid, beta-parinaric acid, bosseopentaenoic acid, pinolenic acid, or podocarpic acid. In some examples, the lipid is a sterol lipid. In some examples, the sterol lipid comprises sterols, steroids, secosteroids, or bile acids. In some examples, the sterol comprises cholesterol, ergosterol, C24-propyl sterols, or stanol. In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some examples, the photoreactive group is diazirine, or its derivatives thereof. In some examples, the photoreactive group further comprises a linker. In some examples, the affinity handle is a bioorthogonal affinity handle. In some examples, 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. In some examples, the affinity handle comprises an alkyne or an azide group. In some examples, the affinity handle is conjugated to the terminal carbon atom of the lipid probe. In some examples, the photoreactive group and the affinity handle are conjugated to the same site of the lipid probe. In some examples, the photoreactive group and the affinity handle are conjugated to different sites of the lipid probe. In some examples, the photoreactive means comprises ultraviolet light. In some examples, the affinity handle is further conjugated to an affinity ligand. In some examples, the affinity ligand comprises a chromophore, a labeling group, or a combination thereof. In some examples, the chromophore comprises fluorochrome, non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate. In some examples, the fluorophore comprises rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, thiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyren derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavin, acridine orange, acridine yellow, arylmethine derivatives, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphin, phtalocyanine, bilirubin 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 5(6)-FAM, 5-FAM, Fluorescein dT, 5-TAMRA-cadavarine, 2-aminoacridone, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA, TAMRA ™< (NHS Ester), TEX 615, ATTO ™< 488, ATTO ™< 532, ATTO ™< 550, ATTO ™< 565, ATTO ™< Rho101, ATTO ™< 590, ATTO ™< 633, ATTO ™< 647N, TYE ™< 563, TYE ™< 665, or TYE ™< 705. In some examples, the labeling group is biotin moiety, streptavidin moiety, bead, resin, a solid support, or a combination thereof. In some examples, the lipid probe comprises: a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls; a photoreactive linker; and an affinity handle. In some examples, 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. In some examples, the lipid probe has one of the following structures as illustrated in Table 1. In some examples, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some examples, 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. In some examples, the first cell solution further comprises a control. In some examples, the control is dimethyl sulfoxide (DMSO). In some examples, the proteomic analysis means comprises a mass spectroscopy method. In some examples, the value assigned to each of the lipid binding protein in step c) is the area-under-the curve from a plot of signal intensity as a function of mass-to-charge ratio. In some examples, the identifying in step d) 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. In some examples, the ratio of greater than 2 indicates that the lipid binding protein is a candidate for interacting with the drug. In some examples, the ratio of greater than 3 indicates that the lipid binding protein is a candidate for interacting with the drug. In some examples, the method further comprises contacting the second cell solution with an additional drug. In some examples, the method further comprises generating a drug-lipid binding protein profile. In some examples, the drug-lipid binding protein profile is a profile that indicates the array of lipid binding proteins that interact with the drug. In some examples, the method further comprises classifying the drug as a specific inhibitor or a pan inhibitor. In some examples, the cell is obtained from a tumor cell line. In some examples, the cell is obtained from a Neuro2a or A549 cell line. In some examples, the cell is obtained from a tumor sample. In some examples, the sample is a tissue sample. In some examples, the method is an in situ method.

[0015] Described herein is a method of mapping a ligand binding site on a lipid binding protein, comprising (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; and (c) based on step b), locating a ligand binding site on the lipid binding protein. In some examples, step b) further comprises treating the set of lipid probe-protein complexes with a protease to generate a set of protein fragments. In some examples, each protein fragment is attached to a lipid probe. In some examples, locating further comprises determining the sequence of the set of protein fragments. In some examples, the protein fragment correlates to a ligand binding site on a lipid binding protein. In some examples, the sample comprises a first cell solution and a second cell solution. In some examples, the second cell solution comprises a drug. In some examples, the second cell solution further comprises a buffer or a media. In some examples, the method further comprises 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. In some examples, the lipid is a bioactive lipid. In some examples, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some examples, the lipid is a member of the fatty acyls group. In some examples, 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. In some examples, the lipid is a sterol lipid. In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some examples, the affinity handle is a bioorthogonal affinity handle. In some examples, 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. In some examples, the lipid probe comprises a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls; a photoreactive linker; and an affinity handle. In some examples, 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. In some examples, the lipid probe has one of the following structures as illustrated in Table 1. In some examples, the lipid binding protein is a soluble protein or a membrane protein. In some examples, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some examples, 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. In some examples, the proteomic analysis means comprises a mass spectroscopy method. In some examples, the sample is a tissue sample.

[0016] Described herein, in certain examples, is a lipid probe-protein composition comprising a lipid probe and a lipid binding protein. In some examples, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some examples, the lipid binding protein is a protein encoded by a gene of Table 3, a protein encoded by a gene of Table 4, a protein encoded by a gene of Table 5, or a protein encoded by a gene of Table 6. In some examples, the lipid binding protein is nucleobindin-1 (NUCB1). In some examples, the lipid binding protein is a protein fragment. In some examples, the protein fragment is a protein fragment of Table 6. In some examples, the lipid is a bioactive lipid. In some examples, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some examples, the lipid is a member of the fatty acyls group. In some examples, 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. In some examples, the lipid is a fatty acid. In some examples, the lipid is a sterol lipid. In some examples, the sterol lipid comprises sterols, steroids, secosteroids, or bile acids. In some examples, the sterol comprises cholesterol, ergosterol, C24-propyl sterols, or stanol. In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some examples, the affinity handle is a bioorthogonal affinity handle. In some examples, 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. In some examples, the lipid probe comprises: a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls; a photoreactive linker; and an affinity handle. In some examples, 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. In some examples, the lipid probe has one of the following structures as illustrated in Table 1.

[0017] Described herein, in certain examples, is a lipid probe-protein composition produced by a process comprising contacting a sample with a lipid probe, and treating the sample comprising the lipid probe by a photoreactive means, wherein the treating time is from about 5 minutes to about 1 hour. In some examples, the treating time is about 10 minutes. In some examples, the lipid probe comprises a lipid, a photoreactive group, and an affinity handle. In some examples, the lipid is a bioactive lipid. In some examples, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some examples, the lipid is a member of the fatty acyls group. In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some examples, the affinity handle is a bioorthogonal affinity handle. In some examples, 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. In some examples, the lipid probe comprises a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls; a photoreactive linker; and an affinity handle. In some examples, 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. In some examples, the lipid probe has one of the following structures as illustrated in Table 1. In some examples, the protein from the lipid probe-protein composition is a lipid binding protein. In some examples, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some examples, the lipid binding protein is a protein encoded by a gene of Table 3, a protein encoded by a gene of Table 4, a protein encoded by a gene of Table 5, or a protein encoded by a gene of Table 6. In some examples, the lipid binding protein is nucleobindin-1 (NUCB1). In some examples, the method further comprises treating the lipid probe-protein composition with a protease. In some examples, the protein of the protease treated lipid probe-protein composition is a protein fragment. In some examples, the protein fragment is a protein fragment of Table 6. In some examples, the photoreactive means comprises ultraviolet light. In some examples, the sample is a cell solution sample. In some examples, the sample is a tissue sample.

[0018] Described herein, in certain examples, is a composition comprising an isolated sample wherein the isolated sample is an isolated cell or a tissue sample; and a lipid probe to be assayed for its ability to interact with a lipid binding protein expressed in the isolated sample. In some examples, the composition further comprises a drug as a test compound. In some examples, the lipid probe is assayed for its ability to interact with a lipid binding protein expressed in the sample in the presence of the drug.

[0019] Described herein, in certain examples, is an isolated treated cell comprising a lipid probe attached to a lipid binding protein. In some examples, the lipid probe is attached to the lipid binding protein through a covalent bond. In some examples, the isolated treated cell further comprises a set of lipid probes wherein each of the lipid probes is attached to a lipid binding protein. In some examples, each lipid probe within the set is different. In some examples, each lipid probe within the set is the same.

[0020] Described herein, in certain examples, is an isolated treated population of cells comprising a set of lipid probes attached to lipid binding proteins. In some examples, each of the lipid probes is attached to a lipid binding protein through a covalent bond. In some examples, each lipid probe within the set is different. In some examples, each lipid probe within the set is the same.

[0021] Described herein, in certain examples, is a lipid probe comprising a lipid, a photoreactive group, and an affinity handle wherein the lipid probe is constructed for detecting a drug-lipid binding protein interaction. In some examples, the lipid is a bioactive lipid. In some examples, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some examples, the lipid is a member of the fatty acyls group. In some examples, 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. In some examples, the lipid is a sterol lipid. In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some examples, the photoreactive group is diazirine, or its derivatives thereof. In some examples, the affinity handle is a bioorthogonal affinity handle. In some examples, 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. In some examples, the lipid probe comprises a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls; a photoreactive linker; and an affinity handle. In some examples, 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. In some examples, PG is attached to A. In some examples, AH is attached to A. In some examples, the lipid probe has one of the following structures as illustrated in Table 1. In some examples, the probe is a lipid binding protein ligand. In some examples, the probe is a competitive ligand for interaction with a lipid binding protein in the presence of a drug. In some examples, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some examples, 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. In some examples, the lipid binding protein is nucleobindin-1 (NUCB1).

[0022] Described herein, in certain examples, is an isolated and purified polypeptide comprising at least 90% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Table 6, wherein the isolated and purified polypeptide is at most 50 amino acids in length. In some examples, the isolated and purified polypeptide comprising at least 95% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Table 6, wherein the isolated and purified polypeptide is at most 50 amino acids in length. In some examples, the isolated and purified polypeptide comprising 100% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Table 6, wherein the isolated and purified polypeptide is at most 50 amino acids in length. In some examples, the isolated and purified polypeptide consisting 100% sequence identity to the full length of an amino acid sequence selected from Table 6, wherein the isolated and purified polypeptide is at most 50 amino acids in length.

[0023] Described herein, in certain examples, is a nucleic acid encoding a polypeptide comprising at least 90% sequence identity at least seven contiguous amino acids of an amino acid sequence selected from Table 6. In some examples, the nucleic acid encoding a polypeptide comprising at least 95% sequence identity at least seven contiguous amino acids of an amino acid sequence selected from Table 6. In some examples, the nucleic acid encoding a polypeptide comprising 100% sequence identity at least seven contiguous amino acids of an amino acid sequence selected from Table 6. In some examples, the nucleic acid encoding a polypeptide consisting 100% sequence identity to the full length of an amino acid sequence selected from Table 6.

[0024] Described herein, in certain examples, is a method of screening a drug for interaction with a lipid binding protein, comprising: (a) contacting a solution comprising a purified recombinant lipid binding protein and a drug with a lipid probe, wherein the lipid probe comprises a lipid and a fluorophore; and (b) detecting a change in fluorescence polarization relative to a control, wherein the change in fluorescence polarization indicates an interaction between the drug and the lipid binding protein. In some examples, the lipid is a bioactive lipid. In some examples, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some examples, the lipid is a member of the fatty acyls group. In some examples, 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. In some examples, the lipid is a fatty acid. In some examples, the lipid is a sterol lipid. In some examples, the sterol lipid comprises sterols, steroids, secosteroids, or bile acids. In some examples, the sterol comprises cholesterol, ergosterol, C24-propyl sterols, or stanol. In some examples, the lipid probe further comprises a photoreactive group. In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some examples, the photoreactive group is diazirine, or its derivatives thereof. In some examples, the photoreactive group further comprises a linker. In some examples, the fluorophore comprises rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, thiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyren derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavin, acridine orange, acridine yellow, arylmethine derivatives, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphin, phtalocyanine, bilirubin 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 5(6)-FAM, 5-FAM, Fluorescein dT, 5-TAMRA-cadavarine, 2-aminoacridone, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA, TAMRA ™< (NHS Ester), TEX 615, ATTO ™< 488, ATTO ™< 532, ATTO ™< 550, ATTO ™< 565, ATTO ™< Rho101, ATTO ™< 590, ATTO ™< 633, ATTO ™< 647N, TYE ™< 563, TYE ™< 665, or TYE ™< 705. In some examples, the fluorophore is conjugated to the hydrophilic portion of the lipid probe. In some examples, the photoreactive group and the fluorophore are conjugated to different sites of the lipid probe. In some examples, the lipid probe comprises an arachidonic acid and a fluorophore. In some examples, the lipid binding protein is a soluble protein or a membrane protein. In some examples, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some examples, 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. In some examples, the lipid binding protein is nucleobindin-1 (NUCB1). In some examples, the method further comprises conjugating the lipid probe to the isolated recombinant lipid binding protein through a photoreactive means. In some examples, the photoreactive means comprises ultraviolet light. In some examples, the method further comprises contacting the solution with an additional drug. In some examples, the method further comprises generating a drug-lipid binding protein profile. In some examples, the drug-lipid binding protein profile is a profile that indicates the array of lipid binding proteins that interact with the drug. In some examples, the method further comprises classifying the drug as a specific inhibitor or a pan inhibitor. In some examples, the method is a high throughput screening method.

[0025] Described herein, in certain examples, is a lipid probe comprising a lipid and a fluorophore wherein the lipid probe is constructed for detecting a drug-lipid binding protein interaction. In some examples, the lipid is a bioactive lipid. In some examples, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some examples, the lipid is a member of the fatty acyls group. In some examples, 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. In some examples, the lipid is a sterol lipid. In some examples, the lipid probe further comprises a photoreactive group. In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some examples, the photoreactive group is diazirine, or its derivatives thereof. In some examples, the fluorophore is conjugated to the hydrophilic portion of the lipid probe. In some examples, the lipid probe comprises arachidonic acid and a fluorophore. In some examples, the lipid probe is 2-(6-(Dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((5-((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenamido)pentyl)carbamoyl)benzoate. In some examples, the probe is a ligand for a lipid binding protein. In some examples, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some examples, 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. In some examples, the lipid binding protein is nucleobindin-1 (NUCB1).

[0026] Described herein, in certain examples, is a lipid probe comprising a lipid and a fluorophore wherein the lipid probe is constructed for detecting a drug-nucleobindin-1 (NUCB1) interaction. In some examples, the lipid is a bioactive lipid. In some examples, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some examples, the lipid probe comprises arachidonic acid and a fluorophore. In some examples, the lipid probe is 2-(6-(Dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((5-((5Z,82,11Z,14Z)-icosa-5,8,11,14-tetraenamido)pentyl)carbamoyl)benzoate.

[0027] Described herein, in certain examples, is a compound of Formula (II), or a pharmaceutically acceptable salt, or solvate thereof: wherein: R 1< is H, halogen, -CN, -OH, C 1 -C 4 alkoxy, C 1 -C 4 fluoroalkoxy, -NO 2 , -S-C 1 -C 4 alkyl, - S(=O)-C 1 -C 4 alkyl, or -S(=O) 2 -C 1 -C 4 alkyl; R 2< is H, halogen, C 1 -C 4 alkyl, C 3 -C 6 cycloalkyl, or substituted or unsubstituted phenyl, where if R 2< is substituted then R 2< is substituted with 1 or 2 R 5< ; each R 5< is independently selected from the group consisting of H, halogen, -CN, - NO 2 , -OH, -SR 6< , -S(=O)R 6< , -S(=O) 2 R 6< , -C(=O)R 6< , -CO 2 H, -CO 2 R 6< , -NH 2 , -NHR 6< , -N(R 6< ) 2 , -C(=O)NH 2 , -C(=O)NHR 6< , -C(=O)N(R 6< ) 2 , C 1 -C 4 alkyl, C 1 -C 4 fluoroalkyl, C 1 -C 4 alkoxy, C 1 -C 4 fluoroalkoxy, C 3 -C 6 cycloalkyl, C 2 -C 6 heterocycloalkyl, phenyl, and phenoxy; R 3< is H, or C 1 -C 4 alkyl; R 4< is hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, -NHNH 2 , -NH 2 , -NHR 6< , or -N(R 6< ) 2 ; each R 6< is independently selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 fluoroalkyl, C 3 -C 6 cycloalkyl, a substituted or unsubstituted phenyl, or substituted or unsubstituted benzyl; or two R 6< groups attached to the same N atom are taken together with the N atom to which they are attached to form a C 2 -C 6 heterocycloalkyl that is unsubstituted or substituted with C 1 -C 4 alkyl or C 3 -C 6 cycloalkyl.

[0028] In some examples, R 1< is H, Cl, -NO 2 , or -S(=O) 2 -CH 3 ; R 3< is H, or -CH 3 .

[0029] In some examples, R 4< is -NHNH 2 , -NH 2 , -NHR 6< , or -N(R 6< ) 2 .

[0030] In some examples, the compound has the following structure of Formula (III):

[0031] In some examples, each R 6< is independently selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 fluoroalkyl, C 3 -C 6 cycloalkyl, or benzyl; or two R 6< groups attached to the same N atom are taken together with the N atom to which they are attached to form a C 2 -C 6 heterocycloalkyl that is unsubstituted or substituted with C 1 -C 4 alkyl or C 3 -C 6 cycloalkyl, wherein the C 2 -C 6 heterocycloalkyl is pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, aziridinyl, or azetidinyl.

[0032] In some examples, the compound has one of the following structures as illustrated in Table 2. In some examples, the compound is a nucleobindin-1 (NUCB1) ligand.

[0033] Described herein, in certain examples, is a composition comprising a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, and an excipient. In some examples, the composition is formulated for administration to a mammal by intravenous administration, subcutaneous administration, oral administration, inhalation, nasal administration, dermal administration, or ophthalmic administration. In some examples, the composition is in the form of a tablet, a pill, a capsule, a liquid, a suspension, a gel, a dispersion, a solution, an emulsion, an ointment, or a lotion.

[0034] Described herein, in certain examples, is a method of identifying a lipid binding protein, comprising (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the proteins from the set of lipid probe-protein complexes; and (d) based on the value assigned in c), identifying a protein as a lipid binding protein. In some examples, the sample comprises a first cell solution and a second cell solution. In some examples, the method further comprises contacting the first cell solution with a first lipid probe and contacting the second cell solution with a second lipid probe. In some examples, the first lipid probe and the second lipid probe are the same. In some examples, the second cell solution comprises a buffer or a media. In some examples, the method further comprises treating the first cell solution by a photoreactive means. In some examples, the method further comprises contacting the first cell solution with a first set of lipid probes wherein each of the lipid probes comprises a lipid, a photoreactive group, and an affinity handle, and wherein each lipid probe is different within the set. In some examples, the method further comprises contacting the second cell solution with a second set of lipid probes wherein each of the lipid probes comprises a lipid, a photoreactive group, and an affinity handle, and wherein each lipid probe is different within the set. In some examples, the first set of lipid probes and the second set of lipid probes are the same. In some examples, the method further comprises treating the first cell solution by a photoreactive means. In some examples, the set of lipid probe-protein complexes further comprises a first group of lipid probe-protein complexes from the first cell solution and a second group of lipid probe-protein complexes from the second cell solution. In some examples, the lipid is a bioactive lipid. In some examples, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some examples, the lipid is a member of the fatty acyls group. In some examples, 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. In some examples, the lipid is a fatty acid. In some examples, the fatty acid comprises a saturated fatty acid, a monounsaturated fatty acid, or a polyunsaturated fatty acid. In some examples, the lipid is a sterol lipid. In some examples, the sterol lipid comprises sterols, steroids, secosteroids, or bile acids. In some examples, the sterol comprises cholesterol, ergosterol, C24-propyl sterols, or stanol. In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some examples, the photoreactive group is diazirine, or its derivatives thereof. In some examples, the photoreactive group further comprises a linker. In some examples, the affinity handle is a bioorthogonal affinity handle. In some examples, 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. In some examples, the affinity handle comprises an alkyne or an azide group. In some examples, the affinity handle is conjugated to the terminal carbon atom of the lipid probe. In some examples, the photoreactive group and the affinity handle are conjugated to the same site of the lipid probe. In some examples, the photoreactive group and the affinity handle are conjugated to different sites of the lipid probe. In some examples, the lipid probe comprises: a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls; a photoreactive linker; and an affinity handle. In some examples, 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. In some examples, the lipid probe has one of the following structures as illustrated in Table 1. In some examples, the protein is a soluble protein or a membrane protein. In some examples, the protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some examples, the 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. In some examples, the photoreactive means comprises ultraviolet light. In some examples, the affinity handle is further conjugated to an affinity ligand. In some examples, the affinity ligand comprises a chromophore, a labeling group, or a combination thereof. In some examples, the chromophore comprises fluorochrome, non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate. In some examples, the labeling group is biotin moiety, streptavidin moiety, bead, resin, a solid support, or a combination thereof. In some examples, the first cell solution further comprises a control. In some examples, the control is dimethyl sulfoxide (DMSO). In some examples, the value assigned to each of the protein in step c) is the area-under-the curve from a plot of signal intensity as a function of mass-to-charge ratio. In some examples, the identifying in step d) further comprises (i) locating a first value assigned to a protein from the first group of lipid probe-protein complex and a second value of the same protein from the second group of lipid probe-protein complex; and (ii) calculating a ratio between the two values. In some examples, the ratio of greater than 2 indicates that the protein is a candidate for interacting with the lipid probe. In some examples, the ratio of greater than 3 indicates that the protein is a candidate for interacting with the lipid probe. In some examples, the cell is obtained from a tumor cell line. In some examples, the cell is obtained from a Neuro2a or A549 cell line. In some examples, the cell is obtained from a tumor sample. In some examples, the sample is a tissue sample. In some examples, the method is an in situ method.

[0035] Described herein, in certain examples, is a method of identifying a fatty acyl binding protein, comprising: (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the proteins from the set of lipid probe-protein complexes; and (d) based on the value assigned in c), identifying a protein as a fatty acyl binding protein.

[0036] Described herein, in certain examples, is a method of identifying a fatty acyl binding protein as a drug binding target, comprising: (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the fatty acyl binding proteins from the set of lipid probe-protein complexes; and (d) based on the value assigned in c), identifying a fatty acyl binding protein as a drug binding target.

[0037] Described herein, in certain examples, is a method of generating a drug-lipid binding protein profile, comprising: (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the lipid binding proteins to generate a set of values; and (d) based on the set of values assigned in c), generate a drug-lipid binding protein profile. In some examples, the drug-lipid binding protein profile is a profile that indicates the array of lipid binding proteins that interacts with the drug.

[0038] Described herein, in certain examples, is a method of evaluating the selectivity of a drug for binding to a lipid binding protein, comprising: (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the lipid binding proteins from the first and second sets of lipid probe-protein complexes; and (d) based on the value assigned in c), classifying the drug as a specific inhibitor of a lipid binding protein or as a pan inhibitor.

[0039] Described herein, in certain examples, is a method of mapping a drug-induced metabolic change in a cell, comprising: (a) contacting a sample with a lipid binding protein ligand; (b) harvesting a set of lipids from a sample; (c) analyzing the set of metabolites by a proteomic analysis means; (d) based on step c), assigning a value to each of the metabolites from the set; and (e) based on step c), identifying one or more metabolites those expression levels have been changed relative to a control, wherein the changes in the one or more metabolite levels correlate to the drug-induced metabolic change in the cell. In some examples, the control is the expression levels of the one or more metabolites in the absence of the drug.

[0040] Described herein, in certain examples, is a method of monitoring the metabolic change of a cell with incubation of two or more drugs, comprising: (a) contacting a sample with a lipid binding protein ligand; (b) harvesting a set of lipids from a sample; (c) analyzing the set of metabolites by a proteomic analysis means; (d) based on step c), assigning a value to each of the metabolites from the set; and (e) based on step d), identifying one or more metabolites those expression levels have been changed relative to a control, wherein the changes in the one or more metabolite levels correlate to the metabolic change of the cell with incubation of two or more drugs. In some examples, the control is the expression levels of the one or more metabolites in the absence of the two or more drugs.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Various aspects of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the invention are utilized, and the accompanying drawings of which: Fig. 1 illustrates structural representations of lipid probes described herein. Fig. 2 illustrates a conceptual schematic of an exemplary computer server to be used for processing a method described herein. Fig. 3 illustrates chemical proteomic probes for mapping lipid-binding proteins in cells. (A) Structures of lipid probes featuring arachidonoyl (AEA-DA, AA-DA and A-DA), oleoyl (OEADA and O-DA), palmitoyl (PEA-DA) and stearoyl (S-DA) acyl chains, as well as photoreactive (diazirine) and alkyne groups. (B) AEA-DA and A-DA probes which show overlapping, but distinct protein interaction profiles in HEK293T cells. Cells were treated with each probe (20 µM) for 30 min in situ before photocrosslinking and analysis of probe-modified proteins as described in Figure 4. (C) Arachidonoyl probe labeling of membrane and soluble proteins depend on UV irradiation of cells. (D) Comparative labeling profiles of lipid probes (20 µM, 30 min) in HEK293T cells. Red and blue arrows mark representative proteins preferentially labeled by arachidonoyl and oleoyl / palmitoyl probes, respectively. See Figure 4C for profiles of A-DA, O-DA and S-DA. Fig. 4 illustrates chemical proteomic probes for mapping lipid-binding proteins in cells. (A) Experimental workflow for gel-based profiling of lipid-binding proteins in mammalian cells. Cells are incubated with lipid probes for 30 min prior to crosslinking with UV light (10 min, 4 °C) and subsequent cell lysis. Probe-labeled proteins are then conjugated a rhodamineazide (Rh-N3) reporter tag by copper-catalyzed azide-alkyne cycloaddition (CuAAC or "click") chemistry to allow for visualization of probe-labeled targets by SDS-PAGE and in-gel fluorescent scanning. (B) Membrane and soluble protein labeling profiles for the AEA-DA and AA-DA probes (20 µM) in HEK293T cells. Note that the AA-DA probe almost exclusively labels membrane proteins, whereas the AEA-DA probe interacts with both membrane and soluble proteins. (C) Comparative protein labeling profiles of the A-DA, O-DA and S-DA probes in HEK293T cells, revealing a greater degree of labeling by the A-DA versus O-DA or S-DA probes. Fig. 5 shows protein targets of lipid probes mapped by quantitative MS-based proteomics. (A) Depiction of experimental workflow for mapping lipid-binding proteins in cells by quantitative MS-based profiling. Light- and heavy amino acid-labeled SILAC cells were first treated with the indicated lipid probe (20 µM) and comparison condition in complete media supplemented with fetal bovine serum (10%). After 30 min, the media was removed and the cells were irradiated for 10 min at 4 °C. Heavy and light proteomes from the treated cells were mixed in equal proportions and the combined proteome was separated into membrane and soluble fractions by ultracentrifugation. Probe-labeled proteins were then conjugated to a biotin tag using CuAAC. Following streptavidin enrichment, probe targets were then digested on-bead and the resulting tryptic peptides were analyzed by multidimensional (strong cation exchange / reverse phase) liquid chromatography and tandem mass spectrometry. Targets were defined as proteins with ≥ 3 unique quantified peptides and SILAC ratios ≥ 3 in probe-versus-No UV experiments and ≤ 2 in probe-probe control experiments. (B) Heavy / light SILAC ratio plots for total proteins identified in experiments comparing the labeling profiles of the indicated lipid probe (20 µM) versus no-UV light (No UV; 20 µM probe without UV irradiation) or probe (both heavy and light cells treated with 20 µM probe) controls in Neuro2A cells. Dashed lines mark threshold ratio values (≥ 3-fold in No UV experiments and ≤ 2-fold in probe-probe experiments) for designation of lipid probe targets in Neuro2a cells. (C) Lipid probe targets that have been genetically linked to human diseases based on searches of the OMIM database. (D) Heat map showing the relative enrichment values for lipid probe targets by the A-DA probe compared to O-DA and S-DA probes, as well as compared to the A-DA probe itself with (A-DA) or without UV irradiation (No UV) as controls, in HEK293T cells. See also Table 3 (48054-701-101Table3.txt) for complete list of lipid probe targets and SILAC ratios from each indicated experiment. (E) Peptide MS1 chromatograms for representative AEA-DA-selective (HEATR3) and PEADA-selective (LSS) targets. Fig. 6 illustrates protein targets of lipid probes mapped by quantitative proteomics. (A) Heavy / light SILAC ratio plots for total proteins identified in experiments comparing the labeling profiles of lipid probes (20 µM) versus a 'No UV' control (20 µM probe without UV irradiation) or the equivalent probe (both heavy and light cells treated with 20 µM of the same probe) in HEK293T cells. Dashed lines mark threshold ratio values (≥ 3-fold in No UV experiments) for designation of lipid probe targets (also see Figure 5). (B) Venn diagram of shared and unique protein targets of AEA-DA and A-DA in HEK293T and Neuro2a cells. (C-F) Analysis of lipid probe targets based on (C) presence (membrane) or absence (soluble) of known / predicted transmembrane domains; (D) known / predicted subcellular distribution; (E) involvement in specific biological processes; and (F) protein class distribution. Categories were assigned based on UniProt annotations. (G) Diagram highlighting lipid probe targets (red) in major fatty acid metabolic pathways. SILAC ratios from probe-versus-No UV experiments are indicated in parentheses next to gene names (data shown are for the A-DA probe in HEK293T cells except for CPT1A, which was detected with the A-DA probe in Neuro2a cells). For instances where multiple isoforms of a given protein is enriched (i.e., ACSL and GPAT), the highest ratio across all isoforms is presented. (H) Heat map showing the relative protein enrichment values for the AEA-DA probe compared to OEA-DA and PEA-DA probes, as well as compared to the AEA probe itself with (AEA-DA) or without UV irradiation (No UV) as controls, in HEK293T cells. See Figure 5D for a similar analysis of the A-DA probe series and Table 3 for complete list of lipid probe targets. Fig. 7 shows experimental validation of representative lipid probe targets. (A) SILAC ratio plot for AEA-DA versus PEA-DA (20 µM) probe labeling in HEK293T cells highlighting targets selected for experimental validation. (B) Representative peptide MS1 chromatograms for selected targets showing relative labeling by AEA-DA and PEA-DA probes. (C) Lipid probe labeling of Myc-tagged recombinant proteins expressed by transient transfection in HEK293T cells. Top panels show in situ labeling profiles for the AEA-DA and PEA-DA probes with indicated targets (lane 1, mock-transfected cells; lanes 2 & 3, target-transfected cells; see Figure 10A for full gel profiles). Middle panels, anti-Myc blotting. Lower panels, anti-actin blotting as a loading control. (D) Upper panel shows in vitro competition profiles of AEA-DA probe labeling of NENF by hemin, protoporphyrin IX (Pp-IX), and the arachidonoyl lipids AA, AEA and 2-AG (1-100 µM) (experiments performed in NENF-transfected HEK293T lysates). Lower panel shows concentration-dependent inhibition of AEA-DA labeling of NENF by hemin (CI = 95% confidence interval) as determined from gel profiles. Data represent mean values ± SD from three independent experiments. (E) In vitro competition profiles of NUCB1 labeling by the AEA-DA (5.0 µM) probe using various lipids (20x) as competitors. Experiments were performed in lysates from NUCB1-transfected HEK293 T cells. (F) Calcium-dependent enhancement of NUCB1 labeling by the AEA-DA probe. Data represent mean values ± SEM; n = 3 / condition. ***P < 0.001 for untreated versus CaCl2 (100 µM)-treated samples. Fig. 8 illustrates experimental validation of representative lipid probe targets. (A) Full gel profiles of cropped images found in top panels of Figure 7A showing in situ probe labeling profiles for the indicated targets. (B) Full gel profiles of cropped images found in Figure 7D showing in vitro competition of NENF labeling by AEA-DA. Note that hemin competes NENF-labeling but in some instances does not compete with other probe-labeled targets. (C) Calcium-dependent enhancement of NUCB1 labeling by AEA-DA (experiments performed in lysates from NUCB 1-transfected HEK293T cells). In contrast, CaCl2 treatment did not affect the AEA-DA-labeling of most other proteins in HEK293T lysates. Note that EDTA-treated samples were supplemented with an equivalent amount of CuSO4 (relative to EDTA) prior to fluorophore conjugation in order to prevent EDTA from quenching the CuAAC reaction. Fig. 9 shows the lipid-interaction proteome is rich in drug targets. (A) Categorization of lipid probe targets based on distribution in DrugBank (left pie chart) and further analysis of non-DrugBank targets by protein classes considered ligandable (e.g., enzymes, receptors, transporters) or not (Others). (B) Scheme for in situ competitive profiling of ligands using lipid probes. Isotopically light and heavy cells are treated with vehicle (DMSO) or competitor ligand, respectively, along with a lipid probe for 30 min. Cells are then UV-irradiated, lysed, and light and heavy lysates combined, enriched, and digested for LC-MS / MS analysis. Ligand targets are designated as proteins that show light / heavy ratios of ≥ 3.0. (C) Chemical structures of the dual PTGS1 / 2-inhibitor (±)-flurbiprofen and PTGS2-selective inhibitor rofecoxib and representative peptide MS1 chromatograms for PTGS1 and PTGS2 in Neuro2a and A549 cells, respectively, showing that (±)-flurbiprofen (25 µM) competes A-DA (5 µM) labeling of both PTGS1 and PTGS2, whereas rofecoxib (25 µM) selectively competes PTGS2 labeling. (D) SILAC ratio plots for in situ competition experiments of A-DA (5 µM) labeling by (±)- flurbiprofen (25 µM) and rofecoxib (25 µM) validating target engagement and selectivity across PTGS isoforms and other lipid probe targets. Fig. 10 illustrates potential drug targets within the lipid-interaction proteome. (A) Categorization of lipid probe targets according to their classification as membrane or soluble proteins and by their presence or absence in DrugBank. (B, C) Representative peptide MS1 chromatograms for PTGS1 (A) and PTGS2 (B) enriched from A-DA-treated Neuro2a and A549 cells, respectively, with or without UV-irradiation, confirming PTGS1 and PTGS2 as UV-dependent A-DA targets. (D) Heavy / Light SILAC ratio plots of No UV (blue) and probe-versus-probe (red) experiments for A-DA (5 µM) in A549 cells showing several hundred UV-dependent targets (SILAC ratio ≥ 3.0), including PTGS2 (ratio = 3.2). See Table 3 (48054-701-101Table3.txt) for complete list of UV dependent targets. Fig. 11 illustrates in situ drug profiling with lipid probes. (A) Structures of compounds analyzed by competitive profiling with lipid probes. (B) Heatmap showing SILAC ratios for primary targets of drugs (25 µM, except for Ro 48-8071, which was assayed at 5 µM) from competitive profiling experiments performed in Neuro2a and A549 cells. All drugs were profiled in both cell lines, and target engagement for SOAT1, NAMPT, and ABCB1B is shown for Neuro2a cells with the AEA-DA probe (5 µM) and, for LSS, in A549 cells with the A-DA probe (5 µM) (also see Table 5,). (C) Box-whisker plots of protein SILAC ratios from in situ competition experiments showing on-(red) and off- (blue) targets (ratios ≥ 3.0) for tested drugs. (D) Heatmap of competed off-targets for tested drugs measured with the AEA-DA probe in Neuro2a cells. (E) Plot of SILAC ratios from AEA-DA competition experiments with tested drugs (25 µM) versus the lipid competitor AEA (200 µM). For simplicity, only the highest drug competition SILAC ratio is plotted for each target. (F) Categorization of drug-competed lipid probe targets based on their presence or absence in DrugBank and by protein class (also see Table 5). Fig. 12 shows in situ drug profiling with lipid probes. (A) SILAC ratio plots for in situ competition experiments performed with avasimibe (25 µM), FK-866 (25 µM), elacridar (25 µM), and Ro 48-8071 (5 and 50 µM) measured with the AEADA (5 µM) or A-DA (for Ro 48-8071) probe in the indicated cell lines. Data correspond to measurements plotted in Figure 11C. The known target of each drug is highlighted in red. (B) In situ competitive profiles for the lipid competitor AEA (50-200 µM) measured with the AEA-DA probe (10 µM) in Neuro2a cells. Red arrows highlight AEA-competed proteins. (C) In situ competition profiles of arachidonoyl probe (AEA-DA or A-DA; 5 µM) labeling of recombinant TMEM97 with AEA, FK-866, avasimibe, elacridar or Ro 48-8071 (25 µM each) in HEK293T cells transiently transfected with TMEM97. TMEM97 competition was observed for elacridar and Ro 48-8071, but not other competitors, matching the MS-based competition profiles of endogenous TMEM97 in Neuro2a and A549 cells (See Figure 11D and Table 5). (D) Concentration-dependent competition of AEA-DA (5 µM) probe labeling of ABCB1, TMEM97, and FECH by elacridar in Neuro2a cells. Fig. 13 shows modified lipid probes for high throughput screening (HTS) for the discovery of NUCB1 ligands. (A) Structure of Fl-AEA probe. (B) Incubation of the Fl-AEA probe (0.5 µM) with recombinant human NUCB1 (1.0 µM) produced a strong FluoPol signal that was significantly suppressed by the competitive lipid AA (20 µM; Z' = 0.54). (C) Concentration-dependent suppression of the NUCB 1-FluoPol signal by arachidonoyl lipids AEA, 2-AG and AA, but not palmitoyl lipids PEA, 2-palmitoyl glycerol (2-PG) or palmitic acid (PA). See Figure 14A for profiling of additional lipids. (D) Screen of 16,000 compounds identified small molecules that inhibited the NUCB1-FluoPol signal by 20% or greater (dotted black line). (E) Structure of confirmed HTS hit 1 and positions modified for medicinal chemistry optimization. See Figure 16B-Dfor summary of medicinal chemistry optimization of NUCB1 ligands. (F) Concentration-dependent blockade of AEA-DA (5 µM) labeling of purified, recombinant NUCB1 (0.25 µM) doped into HEK293T lysates (0.75 mg / mL) by HTS hit 1 (0.1-100 µM). (G, H) Structures and competitive profiling results (G) and IC50 curves and values (H) for NUCB1 ligands MJN228 and KML110 and the inactive control compound KML181. Data in B, C, and H represent mean values ± SD from at least three independent experiments. Fig. 14 shows optimization of NUCB1 ligands. (A) A panel of lipids, including sphingosine, cholesterol, prostaglandins (A 1 , B 1 and D 2 ) and prostamide species (PGD 2 -EA and PGF 2 α-EA), shows minimal blockade of Fl-AEA binding to recombinant NUCB1 as determined using a FluoPol assay. Error bars represent standard deviation (n = 5). (B) Gel profile of competition experiment performed with select HTS hits (A-M) (50 µM each) versus the AEA-DA probe (2.5 µM) in NUCB1-transfected HEK293T cell lysates, identifying HTS hit 1 (K) as a selective NUCB1 ligand. (C, D) Structures and competition gel profiles of amide and ester analogs HTS hit 1 (25 µM) (C) and analogs of optimized NUCB1 ligand MJN228 (11) (D). Competition assays were performed with 25 µM of each competitor against the AEA-DA probe (5 µM) in HEK293T lysates (0.75 mg / mL) doped with purified, recombinant human NUCB1 (0.25 µM). (E) Full gel profile for competition experiment performed with MJN228 (1.0-100 µM) versus the AEA-DA probe (5 µM) in HEK293T lysates (0.75 mg / mL) doped with purified, recombinant human NUCB1 (0.25 µM). Fig. 15 shows target engagement and lipid metabolism effects of NUCB1 ligands. (A) Representative peptide MS1 chromatograms showing blockade of AEA-DA probe labeling of endogenous NUCB1 in Neuro2a cells by MJN228 and KML110, but not KML181. (B) SILAC ratio plot for in situ competition experiment performed with MJN228 (10 µM) and the AEA-DA probe (5 µM). (C) LC-MS / MS identification of a MJN228-sensitive, AEA-DA-modified NUCB1 peptide (aa 53-68) in Neuro2a cells. (D) Untargeted metabolite profiling reveals that Neuro2a cells treated with MJN228 (10 µM) show elevated fatty acid amides (NAEs and NATs) compared to cells treated with DMSO or KML181 (10 µM) (P < 0.0001, n = 5 per condition). See also Table 7. (E) Targeted MRM measurements showing elevations in NAEs in Neuro2a cells treated with NUCB1 ligands MJN228 and KML110 (10 µM, 6 h), but not KML181, FK-866, or avasimibe. See Figure 16G for MRM measurements of NATs. (F) Left, Western blot showing knockdown of NUCB1 in shNUCB1 A549 cell lines compared to a control cell line (shGFP). Right, both shNUCB1 cells show significant elevations in NAEs compared to the control shGFP cell line. (G) NUCB1 ligands MJN228 and KML110 (5 and 25 µM), but not KML181 or FK-866 (25 µM each), suppress the conversion of exogenous AEA (20 µM, 30 min) to PGF 2 α-EA in PMAstimulated A549 cells. Rofecoxib (25 µM) also blocked PGF 2alpha -EA synthesis. For (E-G), data represent mean values ± SEM; n = 3-4 / group. *P < 0.05, **P < 0.01, ***P <0.001 for DMSO-treated (E) or shGFP cells (F) versus compound-treated (E) or shNUCB1 (F) cells. Fig. 16 shows cellular target engagement and lipid metabolism effects of NUCB1 ligands. (A) Concentration-dependent competition of AEA-DA (5 µM) probe labeling of NUCB1 and select off-targets (ABCB1B, TMEM97, CERS2, NUCB2) by MJN228 in Neuro2a cells. Data are normalized to the maximal competition value for each protein, which provides an estimate of fractional target engagement across the tested drug concentration range. (B) MS / MS spectrum of AEA-DA-modified NUCB1 peptide localizes probe crosslinking to His67. Purified, recombinant hNUCB1 was treated with DMSO or AEA-DA (10 µM), irradiated with UV light and subjected to proteolytic digestion, whereupon tryptic peptides were analyzed by LC-MS / MS and the resulting mass spectra were extracted using the ProLuCID algorithm designating a variable peptide modification of (+343.2511) for all residues. (C) Extracted ion chromatogram for m / z value of the NUCB1 AEA-DA-modified peptide described in (B) showing signals in AEA-DA-treated (red trace), but not DMSO-treated NUCB1. (D) AEA-hydrolysis assay using purified, recombinant humanized rat FAAH (Mileni et al., 2008) showing that MJN228, KML110, and KLM181 (50 or 100 µM) do not substantially inhibit FAAH activity. (E) AEA-hydrolysis assay in Neuro2a lysates showing minimal inhibition of FAAH activity by MJN228 and KML110 up to 250 µM. (F) Gel profiles of AEA-DA (20 µM) and A-DA (20 µM) probe labeling of HEK293T cells transiently transfected with hFAAH or empty vector. The clear differences in gel bands detected in FAAH-transfected cells suggest that the AEA-DA and A-DA probes are metabolized by FAAH. (G) Targeted MRM measurements showing NUCB1 ligands (MJN228 and KML110, 10 µM) and FAAH inhibitor PF-7845 (1 µM), but not inactive control compounds KML181, FK-866 or avasimibe (10 µM each), produce elevations in N-acyl taurines (NATs) in Neuro2a cells. (H) MJN228 and KML110 (10 µM) do not affect AA levels in Neuro2a cells, but produce a modest increase in 2-AG that is also observed to a greater degree with the selective FAAH inhibitor PF-7845 (1 µM). (I) Heatmap of drug targets in Neuro2a cells as measured by competitive profiling with the AEA-DA probe (5 µM). Note that MJN228 and KML110 both target NUCB1 whereas all other compounds do not, and, conversely, many of the additional targets of MJN228 and KML110 are shared by the other tested drugs. (J) Targeted MRM measurements of NAEs derived from A549 cells treated with MJN228 (10 µM), KML110 (10 µM) or PF-7845 (1 µM) for 6 h. (K) NUCB1 ligands MJN228 and KML110 (10 µM) do not produce elevations in NAEs when co-treated with PF-7845 (1 µM). (L) AEA oxygenation to PGE2-EA by purified, recombinant hPTGS2 is inhibited by celecoxib, but not MJN228 or KML110 (25 or 50 µM). (M) NUCB1 ligands MJN228 and KML110, but not KML181, suppress PGE2 (left) and TXB2 (right) synthesis from endogenous pools of AA in A549 cells pre-treated with the indicated compound (10 µM, 30 min) and then stimulated with PMA for 6 h. FK-866 selectively suppressed TXB2, but not PGE2 synthesis. Data are presented as means ± SEM; n = 3 / group. *P < 0.05, **P < 0.01, ***P < 0.001 for DMSO-treated versus compound-treated cells. Fig. 17 illustrates clickable photoreactive sterol probes. Panel A shows structures of cholesterol and three diastereomeric sterol chemoproteomic probes. Panel B shows three-dimensional structures of cholesterol and sterol probes as determined by x-ray crystallography; cholesterol structure derived from PDBID: 3GKI24. In A and B, from top to bottom; cholesterol, trans-sterol probe, epi-sterol probe, cis-sterol probe. Fig. 18 shows a gel-based profiling of sterol-binding proteins in HeLa cells. Panel A shows a scheme for treatment of live cells with sterol probes and competitive treatments. Panel B shows HeLa cells treated with 10 µM trans-sterol probe, with and without 365 UV irradiation before click chemistry and SDS-PAGE analysis. Panel C shows concentration-dependent labeling of live HeLa cells with each probe (cis, epi, trans) at 1, 5, 10, and 20 µM. Panel D shows competition of sterol probe labeling profiles (10 µM probes) with increasing cholesterol from 0, 10 (1×), 50 (5×), and 100 µM (10×). Fluorescence gel images are shown in grayscale. Fig. 19 illustrates MS-based profiling of sterol-binding proteins in HeLa cells. Panel A illustrates a scheme for enrichment and analysis of sterol probe labeling profiles in mammalian cells by biotin-streptavidin methods and SILAC MS analysis. Panels B-D show heavy / light ratio plots for total proteins identified in experiments that compared the labeling profiles of the trans-sterol probe versus no-UV light control (B; 20 µM trans probe / 20 µM trans probe with no UV), the PEA-DA probe (C; 20 µM trans probe / 20 µM PEA-DA probe), and 10× cholesterol competition (D; 10 µM trans probe / 10 µM trans probe + 100 µM cholesterol). Representative MS1 traces with calculated ratios for proteins that fall into Groups I-IV, as well as the MS1 traces for a non-specific background protein, are shown to the right of the global ratio plots. Ratios of > 20 are listed as 20. Panel E illustrates a Venn diagram showing the distribution of Group I-IV proteins for the trans-sterol probe labeling profile. Final F shows top-five pathways determined by searching Group I proteins on the KEGG database, and top-12 biological function networks determined by searching Group I proteins on the DAVID gene ontology server. Panel G shows trans-sterol probe labeling profile for the cholesterol biosynthetic pathway, with colors reflecting each enzyme's Group designation (black: not detected). Fig. 20 shows analysis of Group I proteins. Panel A shows breakdown of Group I proteins by biochemical functions. Panel B shows group I proteins known to be genetically associated with human disease based on the OMIM database; the 'cholesterol' group represents diseases from all other groups that are known to manifest via aberrant cholesterol homeostasis. Panel C shows fraction of Group I proteins that possess known or predicted transmembrane (TM) domains. TM proteins are further divided into single- versus multipass TM proteins. Panel D shows known or predicted subcellular localization of Group I proteins. Subcellular localization predictions were made by examining protein sequences by the PSORT II algorithm (http: / / psort.hgc.jp / form2.html). Panel E shows cholesterol regulation of Group I proteins at the mRNA level. 'SREBP bs' denotes the fraction of Group I proteins with SREBP transcription factor binding sites in the gene / promoter regions based on the Qiagen SABiosciences transcription factor database (http: / / www.sabiosciences.com / chipqpersearch.php?app=TFBS). '30 min' and '12 h' denote the fraction of Group I proteins with substantial (≥ two-fold) changes in mRNA levels after 100 µM cholesterol treatment for the indicated time. Panel F shows levels of enrichment (≥ 2-fold, 2-10 fold, ≥ 10-fold) of Groups I, II & III, and IV proteins in trans-sterol probe data sets compared to their abundance in unenriched membranes. DETAILED DESCRIPTION OF THE INVENTIONCertain Terminology

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[0043] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Hence "about 5 µL" means "about 5 µL" and also "5 µL." Generally, the term "about" includes an amount that would be expected to be within experimental error.

[0044] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Overview

[0045] Disclosed herein, in certain examples, are methods, compositions, probes, assays, kits, and services for identifying a lipid binding protein as a drug binding target, mapping a ligand binding site on a lipid binding protein, identifying a protein as a lipid binding protein, and generating drug profiles with the use of a lipid probe. Also disclosed herein are methods, assays, compositions, and probes for screening a drug for interaction with a lipid binding protein utilizing recombinant proteins and a lipid probe described herein.

[0046] In some instances, described herein is a method which comprises harvesting a sample incubated in the presence of a lipid probe and a drug, and utilizes a proteomic means for analysis to identify drug binding targets. In some examples, the method further comprises comparing the data with a control. In some instances, the control is a sample incubated in the presence of a lipid probe but in the absence of a drug.

[0047] In some examples, the drug is a small molecule, its fragment or derivatives thereof; a polypeptide; a nucleic acid molecule; or combinations thereof. In some instances, the drug is a small molecule, its fragments or derivatives thereof. In some cases, the drug is a test compound. In some cases, the test compound has a therapeutic effect. In other cases, the test compound does not have a therapeutic effect. In some instances, the drug is a polypeptide (e.g., an antibody or an antibody conjugate). In additional cases, the drug is a nucleic acid molecule (e.g., a naked nucleic acid molecule or a nucleic acid molecule conjugate). In some instances, the drug has a therapeutic effect. In other instances, the drug does not have a therapeutic effect.

[0048] In some examples, the sample is a cell sample. In some instances, the sample is a tissue sample. In some examples, the cell sample comprises a cell solution. In some instances, the cell sample comprises a first cell solution and a second cell solution. In some examples, the method further comprises contacting the first cell solution with a first lipid probe and contacting the second cell solution with a second lipid probe. In some instances, the first lipid probe and the second lipid probe are the same. In some cases, the first and second lipid probes are different. In some instants, the cell solution comprises a buffer. In some instances, the cell solution comprises a media. In some examples, the second cell solution further comprises a drug. In some instances, the second cell solution further comprises a drug and a media. In some examples, the second cell solution further comprises a drug and an enriched media. In some instances, the method further comprises 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.

[0049] In some instances, a first set of lipid probes are added to the first cell solution and a second set of lipid probes are added to the second cell solution. In some cases, each lipid probe is different within the set. In some instances, the first set of lipid probes is the same as the second set of lipid probes. In some cases, the method further comprises treating the first cell solution and the second cell solution by a photoreactive means to generate a third group of lipid probe-protein complexes and a fourth group of lipid probe-protein complexes.

[0050] In some instances, the photoreactive means comprises visible light or ultraviolet light. In some cases, after treatment by a photoreactive means, the sample is harvested and lysed and the lipid probe-protein complexes are collected for analysis by a proteomic analysis means. In some instances, the proteomic analysis means involve analysis by a mass spectroscopy, by gel electrophoresis, an antibody based detection method, and the like. In some instances, the lipid probe-protein complexes are further enriched on a bead, such as an affinity tagged bead (e.g. streptavidin-coupled beads), and then subjected to a mass spectroscopic analysis.

[0051] In some cases, a value is assigned to each of the lipid binding protein from the lipid probe-protein complexes after proteomic analysis. In some instances, the value is an area-under-the curve from a plot of signal intensity as a function of mass-to-charge ratio. In some examples, a first value is assigned to a lipid binding protein from the first group of lipid probe-protein complex of the first cell solution and a second value of the same lipid binding protein from the second group of lipid probe-protein complex of the second cell solution. In some instances, a ratio is then calculated between the two values, the first value and the second value, assigned to the same lipid binding protein. In some instances, a ratio of greater than 2 indicates that the lipid binding protein is a candidate for interacting with the drug. In some instances, the ratio is greater than 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10. In some cases, the ratio is at most 20. In some instances, the same lipid probe interacts with a number of lipid binding proteins in the presence of a drug. In some instances, the drug will modulate the interaction of a lipid probe with its lipid binding protein partners. In some instances, the spectrum of ratios for a lipid probe with its interacting protein partners in the presence of a drug indicates the specificity of the drug toward the protein. In some instances, the spectrum of ratio indicates whether the drug is a specific inhibitor to a protein or a pan inhibitor. In some instances, the method is an in-situ method.

[0052] In some examples, also described herein is a method of identifying a lipid binding protein as a drug binding target, comprising: (a) contacting a first sample with a first lipid probe and a second sample with a second lipid probe and a drug, wherein the first lipid probe and the second lipid probe are the same; (b) harvesting a first set of lipid probe-protein complexes from the first sample and a second set of lipid probe-protein complexes from the second sample; (c) analyzing the first and second sets of lipid probe-protein complexes by a proteomic analysis means; (d) based on step c), assigning a value to each of the lipid binding proteins from the first and second sets of lipid probe-protein complexes; and (e) based on the value assigned in d), identifying a lipid binding protein as a drug binding target.

[0053] In some examples, described herein is a method of identifying a lipid binding protein as a drug binding target, comprising: (a) harvesting a first set of lipid probe-protein complexes from a first cell solution and a second set of lipid probe-protein complexes from a second cell solution wherein the second cell solution comprises enriched media and a drug; (b) analyzing the first and second sets of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the lipid binding proteins from the first and second sets of lipid probe-protein complexes; and (d) based on the value assigned in c), identifying a lipid binding protein as a drug binding target.

[0054] In some examples, described herein is a method of identifying a fatty acyl binding protein as a drug binding target, comprising: (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the fatty acyl binding proteins from the set of lipid probe-protein complexes; and (d) based on the value assigned in c), identifying a fatty acyl binding protein as a drug binding target.

[0055] In some instances, described herein are methods which allow for identification of novel targets for drugs, such as for example new protein targets for drugs with previous known targets. In some instances, the methods described herein are used to identify and / or evaluate off-target effects of one or more drugs. In some instances, the methods described herein are used for off-target screening of therapeutic compounds.

[0056] In some examples, also described herein is a method of mapping a ligand binding site on a lipid binding protein, comprising (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; and (c) based on step b), locating a ligand binding site on the lipid binding protein.

[0057] In some examples, a method of mapping a ligand binding site on a lipid binding protein described herein comprises (a) contacting a first cell solution with a first lipid probe and a second cell solution with a second lipid probe and a drug, wherein the first lipid probe and the second lipid probe are the same; (b) harvesting a first set of lipid probe-protein complexes from the first sample and a second set of lipid probe-protein complexes from the second sample; (c) treating the first set of lipid probe-protein complexes and the second set of lipid probe-protein complexes by a protease; (d) harvesting the treated first set of lipid probe-protein complexes and the treated second set of lipid probe-protein complexes; (e) analyzing the first and second sets of treated lipid probe-protein complexes by a proteomic analysis means; (f) based on step e), assigning a value to each of the lipid binding proteins from the first and second sets of lipid probe-protein complexes; and (g) based on the value assigned in f), identifying a lipid binding protein as a drug binding target.

[0058] In some examples, a method of mapping a ligand binding site on a lipid binding protein described herein comprises (a) harvesting a first set of lipid probe-protein complexes from a first cell solution and a second set of lipid probe-protein complexes from a second cell solution wherein the second cell solution comprises enriched media and a drug; (b) treating the first set of lipid probe-protein complexes and the second set of lipid probe-protein complexes by a protease; (c) harvesting the treated first set of lipid probe-protein complexes and the treated second set of lipid probe-protein complexes; (d) analyzing the first and second sets of treated lipid probe-protein complexes by a proteomic analysis means; (e) based on step d), assigning a value to each of the lipid binding proteins from the first and second sets of lipid probe-protein complexes; and (f) based on the value assigned in e), identifying a lipid binding protein as a drug binding target.

[0059] In some examples, further described herein is a method of identifying a lipid binding protein, comprising: (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the proteins from the set of lipid probe-protein complexes; (d) based on the value assigned in c), identifying a protein as a lipid binding protein. In some instances, described is a method of identifying a fatty acyl binding protein, comprising: (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the proteins from the set of lipid probe-protein complexes; and (d) based on the value assigned in c), identifying a protein as a fatty acyl binding protein.

[0060] In some examples, additionally described herein are methods of generating a drug-lipid binding protein profile and evaluating the selectivity of a drug for binding to a lipid binding protein. In some instances, a method of generating a drug-lipid binding protein profile comprises (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the lipid binding proteins to generate a set of values; and (d) based on the set of values assigned in c), generate a drug-lipid binding protein profile. In some instances, a method of evaluating the selectivity of a drug for binding to a lipid binding protein, comprises (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; (c) based on step b), assigning a value to each of the lipid binding proteins from the first and second sets of lipid probe-protein complexes; and (d) based on the value assigned in c), classifying the drug as a specific inhibitor of a lipid binding protein or as a pan inhibitor.

[0061] In some examples, also disclosed herein are cells, populations of cells, compositions, and probes to a lipid probe described herein and a lipid binding protein.

[0062] In some examples, further disclosed herein are assay and kits that utilize one or more of the methods described herein.Lipid Probe

[0063] In some examples, the lipid probe comprises a lipid, a photoreactive group, and an affinity handle. Lipid is a broad family of small molecules capable of forming structures such as vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. In some examples, the lipid is a bioactive lipid. In some examples, the lipid is an amphipathic lipid. In some instances, the lipid is a hydrophobic lipid.

[0064] In some instances, the lipids described herein comprise fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some instances, the lipid is a lipid derived from a cell membrane.

[0065] In some examples, the lipid is a member of the fatty acyls group. In some examples, 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.

[0066] In some examples, the lipid is a fatty acid. In some instances, the fatty acid comprises straight chain fatty acids or branched fatty acids. In some instances, the fatty acid comprises hydroperoxy, hydroxy, oxo, epoxy, methoxy, halogenated, amino, cyano, nitro, thia, carbocyclic, heterocyclic fatty acid, or its derivatives thereof.

[0067] In some instances, the fatty acid or its derivative thereof comprises a C3-C36 fatty acid or derivative thereof. In some instances, the fatty acid or its derivative thereof comprises a C5-C30, C10-C25, or C15-C21 fatty acid or derivative thereof. In some instances, the fatty acid or its derivative comprises a C16, C17, C18, C19, or C20 fatty acid or derivative thereof.

[0068] In some cases, the fatty acid comprises a saturated fatty acid, a monounsaturated fatty acid, or a polyunsaturated fatty acid.

[0069] Exemplary saturated fatty acid includes, but is not limited to, propanoic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic acid, octacosanoic acid, nonacosanoic acid, triacontanoic acid, henatriacontanoic acid, dotriacontanoic acid, tritriacontanoic acid, tetratriacontanoic acid, pentatriacontanoic acid, or hexatriacontanoic acid.

[0070] Exemplary monounsaturated fatty acid includes, but is not limited to, palmitoleic acid, vaccenic acid, oleic acid, eicosenoic acid, erucic acid, gadoleic acid, myristoleic acid, or nervonic acid.

[0071] In some instances, the polyunsaturated fatty acid comprises omega-3 fatty acids, omega-6 fatty acids, omega-9 fatty acids, or conjugated fatty acids. Exemplary polyunsaturated fatty acid includes, but is not limited to, hexadecatrienoic acid, alpha-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic acid, linoleic acid, gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid, tetracosatetraenoic acid, tetracosapentaenoic acid, mead acid, rumenic acid, alpha-calendic acid, beta-calendic acid, jacaric acid, alpha-eleostearic acid, beta-eleostearic acid, catalpic acid, punicic acid, rumelenic acid, alpha-parinaric acid, beta-parinaric acid, bosseopentaenoic acid, pinolenic acid, or podocarpic acid.

[0072] In some instances, the lipid is an eicosanoid, such as prostaglandin, leukotriene, thromboxane, lipoxin, hydroxy / hydroperoxyeicosatrienoic acid, hydroxy / hydroperoxyeicosatetraenoic acid, hydroxy / hydroperoxyeicosapentaenoic acid, epoxyeicosatrienoic acid, hepoxilin, levuglandin, isoprostane, calvulone, or its derivatives thereof.

[0073] In some examples, the lipid is a sterol lipid. In some instances, the sterol lipid comprises sterols, steroids, secosteroids, or bile acids. In some instances, the sterol comprises cholesterol, ergosterol, C24-propyl sterols, or stanol. In some instances, the lipid is cholesterol.

[0074] In some examples, the lipid is a glycerolipid. Glycerolipids comprise of mono-, di-, and tri-substituted glycerols. In some instances, the glycerolipids comprise monoradylglycerols, diradylglycerols, triadylglycerols, glycosylmonoradylglycerols, or glycosyldiradylglycerols.

[0075] In some examples, the lipid is a glycerophospholipid. Glycerophospholipids or phospholipids serve as key components of the lipid bilayer. In some instances, the glycerophospholipid or phospholipids comprise glycerophosphocholines, glycerophosphoethanolamines, glycerophosphoserines, glycerophosphoglycerols, glycerophosphoglycerophosphate, glycerophosphoinositols, glycerophosphoinositol monophosphates, glycerophosphoinositol bisphosphates, glycerophosphoinositol trisphosphates, glycerophosphates, glyceropyrophosphates, glycerophosphoglycerophosphoglycerols, CDP-glycerols, glycosylglycerophospholipids, glycerophosphoinositolglycans, glycerophosphonocholines, glycerophosphonoethanolamines, di-glycerol tetraether phospholipids (caldarchaeols), glycerol-nonitol tetraether phospholipids, or oxidized glycerophospholipids.

[0076] In some examples, the lipid is a sphingolipid. Sphingolipids are a family of lipid molecules that share a common sphingoid base backbone which is synthesized de novo from serine and a long-chain fatty acyl CoA. In some instances, the sphingoid base is referred to as sphingosine. In some examples, sphingolipids are further subdivided into ceramides, phosphosphingolipids, phosphonosphingolipids, glycosphingolipids, or arsenosphingolipids. In some instances, the glycosphingolipid is a neutral glycosphingolipid, an acidic glycosphingolipid, a basic glycosphingolipid, or an amphoteric glycosphingolipid.

[0077] In some examples, the lipid is a prenol lipid. Prenol lipids are synthesized from isopentenyl diphosphate and dimethylallyl diphosphate. In some examples, the prenol lipids comprise isoprenoids, quinones and hydroquinones, or polyprenols.

[0078] In some examples, the lipid is a saccharolipid. Saccharolipids are lipid molecules which comprise of fatty acids covalently attached to sugar backbones. In some instances, the saccharolipid comprise an acylated glucosamine precursor of a Lipid A component of lipopolysaccharide.

[0079] In some examples, the lipid is a polyketide. Polyketides are a class of structurally diverse secondary metabolites which are further divided into type I polyketides (macrolides from multimodular megasyntheses), type II polyketides (aromatic molecules from iterative action of dissociated enzymes), and type III polyketides (small aromatic molecules from fungal species). In some instances, polyketides include geldanamycin, doxycycline, erythromycin, and aflatoxin B1.

[0080] In some examples, a lipid is further classified as a neutral lipid, a cationic lipid, or an anionic lipid. In some examples, a neutral lipid is any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0081] Cationic lipids, which carry a net positive charge at about physiological pH, include, but are not limited to, N,N-dioleoyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleoyloxy)propyl-N,N-N-triethylammonium chloride ("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); 1,2-Dioleoyloxy-3-trimethylaminopropane chloride salt ("DOTAP.C1"); 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl carboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine ("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"), N,N-dimethyl-2,3-dioleoyloxy)propylamine ("DODMA"), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide ("DMRIE").

[0082] Anionic lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol.

[0083] In some examples, a lipid probe disclosed herein comprises a) a lipid such as fatty acyl, glycerolipid, glycerophospholipid or phospholipid, sphingolipid, sterol lipid, prenol lipid, saccharolipid, or polyketide, b) a photoreactive group, and c) an affinity handle. In some instances, the lipid probe comprises a fatty acyl, a photoreactive group, and an affinity handle. In some instances, the lipid probe comprises a glycerolipid, a photoreactive group, and an affinity handle. In some examples, the lipid probe comprises a phospholipid, a photoreactive group, and an affinity handle. In some instances, the lipid probe comprises a sphingolipid, a photoreactive group, and an affinity handle. In some instances, the lipid probe comprises a sterol lipid, a photoreactive group, and an affinity handle.

[0084] In some examples, the lipid probe comprises a) a lipid selected from an arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls, b) a photoreactive group, and c) an affinity handle. In some examples, the lipid probe comprises an arachidonoyl fatty acyl, a photoreactive group, and an affinity handle. In some examples, the lipid probe comprises an arachidoyl fatty acyl, a photoreactive group, and an affinity handle. In some examples, the lipid probe comprises an oleoyl fatty acyl, a photoreactive group, and an affinity handle. In some examples, the lipid probe comprises a palmitoyl fatty acyl, a photoreactive group, and an affinity handle. In some examples, the lipid probe comprises a stearoyl fatty acyl, a photoreactive group, and an affinity handle.

[0085] As used herein, the term "lipid" includes any suitable naturally occurring lipid and its derivatives thereof. For example in some instances, the term "lipid" includes arachidonic acid, a polyunsaturated omega-6 fatty acid, and its derivatives such as for example, (5Z,8Z,112)-N-(2-hydroxyethyl)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienamide (AEA-DA), (5Z,8Z,11Z)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienoic acid (AA-DA), (5Z,8Z,11Z,14Z)-N-(2-(3-methyl-3H-diazirin-3-yl)ethyl)icosa-5,8,11,14-tetraen-19-ynamide (A-DA), (5Z,8Z,11Z)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienamide (ANH 2 -DA), potassium 2-((5Z,8Z,11Z)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienamido)ethane-1-sulfonate (AT-DA), or 1,3-dihydroxypropan-2-yl (5Z,8Z,11Z)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienoate (2-AG-DA).Photoreactive Group

[0086] In some instances, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones.

[0087] In some instances, the photoreactive group is diazirine, or its derivatives thereof.

[0088] In some instances, the photoreactive group is attached at the hydrophobic portion of the lipid. In some instances, the photoreactive group is attached to the hydrophilic terminus of the lipid. In some instances, the photoreactive group is attached to an internal carbon of the lipid. In some instances, the photoreactive group is attached to an internal main carbon backbone of the lipid. In some instances, the photoreactive group is attached to an internal side chain group of the lipid.

[0089] In some examples, the photoreactive group is attached to a fatty acyl, a glycerolipids, a glycerophospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a saccharolipid, or a polyketide. In some instances, the photoreactive group is attached to a fatty acyl (e.g. a fatty acid). In some instances, the photoreactive group is attached at C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C35, C35, or C36 position of the fatty acyl. In some instances, the photoreactive group is attached at C11, C12, C13, C14, C15, C16, C17, or C18 position.

[0090] In some instances, the photoreactive group further comprises a linker. In some instances, the linker bridges the lipid and the photoreactive group.

[0091] In some instances, the linker comprises a saturated, monosaturated, or polysaturated carbon chain group. In some instances, the linker is from about 1 to about 30 carbons long. In some instances, the linker is from about 2 to about 25, from about 3 to about 20, or from about 4 to about 14 carbons long.

[0092] As used herein, the term "photoreactive group" refers to a group capable of becoming covalently bound to another molecule upon irradiation by light. In some instances, the light is visible light. In some instances, the light is ultraviolet light. In some instances, the term "photoreactive means" refers to ultraviolet light. In some cases, upon irradiation such as by ultraviolet light, the photoreactive group further undergoes photolysis. As used herein, photolysis refers to the activation of the photoreactive group (e.g. diazirine) to generate a reactive species (e.g. carbene), which interact with a molecule in close proximity to form a covalent binding with the molecule.Affinity Handle

[0093] In some examples, the affinity handle is a bioorthogonal affinity ligand. In some examples, the affinity handle utilizes bioorthogonal chemistry. As used herein, bioorthogonal chemistry refers to any chemical reaction that occurs inside of a living system (e.g. a cell) without interfering with native biochemical processes.

[0094] In some instances, 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. In some instances, the affinity handle is an alkyne group, or an azide group.

[0095] In some instances, the affinity handle is an alkyne group. The term "alkyne group" as used in the context of an affinity handle refers to a group with a chemical formula of H-C≡C-R, HC 2 R, R 1 -C≡C-R 2 , or R 1 C 2 R 2 . In the context of the present chemical formula, R, R 1 , and R 2< are independently a lipid described herein, a lipid precursor, a lipid portion such that the alkyne group is at an internal site within the lipid, a linker, or a combination thereof. In some cases, the alkyne group is capable of being covalently linked in a chemical reaction with a molecule containing an azide. In some instances, the affinity handle is an azide group.

[0096] In some instances, the affinity handle (e.g. alkyne group or azide group) serve as nonnative and non-perturbed bioorthogonal chemical handles. In some instances, the affinity handle (e.g. alkyne group or azide group) is further derivatized through chemical reactions such as click chemistry. In some instances, the click chemistry is a copper(I)-catalyzed [3+2]-Huisgen 1,3-dipolar cyclo-addition of alkynes and azides leading to 1,2,3-triazoles. In other instances, the click chemistry is a copper free variant of the above reaction.

[0097] In some instances, the affinity handle is attached to the hydrophilic terminus of the lipid. In some instances, the affinity handle is attached to an internal site of the lipid. In some instances, the affinity handle is attached to an internal main carbon backbone of the lipid. In some instances, the affinity handle is attached to an internal side chain group of the lipid.

[0098] In some examples, the affinity handle is attached to a fatty acyl, a glycerolipids, a glycerophospholipid, a sphingolipid, a sterol lipid, a prenol lipid, a saccharolipid, or a polyketide. In some instances, the affinity handle is attached to a fatty acyl (e.g. a fatty acid). In some instances, the affinity handle is attached at C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C35, C35, or C36 position of the fatty acyl. In some instances, the affinity handle is attached to a terminal carbon atom.

[0099] In some instances, the affinity handle further comprises a linker. In some instances, the linker bridges the lipid and the affinity handle.

[0100] In some instances, the linker comprises a saturated, monosaturated, or polysaturated carbon chain group. In some instances, the linker is from about 1 to about 30 carbons long. In some instances, the linker is from about 2 to about 25, from about 3 to about 20, or from about 4 to about 14 carbons long.

[0101] In some instances, the photoreactive group and the affinity handle are conjugated to the same site on the lipid probe.

[0102] In some cases, the photoreactive group and the affinity handle are conjugated to different sites of the lipid probe.

[0103] In some instances, the affinity handle is further conjugated to an affinity ligand. In some examples, the affinity ligand comprises a chromophore, a labeling group, or a combination thereof. In some instances, the chromophore comprises fluorochrome, non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate.

[0104] In some instances, the fluorophore comprises rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, thiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyren derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavin, acridine orange, acridine yellow, arylmethine derivatives, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphin, phtalocyanine, bilirubin 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 5(6)-FAM, 5-FAM, Fluorescein dT, 5-TAMRA-cadavarine, 2-aminoacridone, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA, TAMRA ™< (NHS Ester), TEX 615, ATTO ™< 488, ATTO ™< 532, ATTO ™< 550, ATTO ™< 565, ATTO ™< Rho101, ATTO ™< 590, ATTO ™< 633, ATTO ™< 647N, TYE ™< 563, TYE ™< 665, or TYE ™< 705.

[0105] In some cases, the labeling group is a biotin moiety, a streptavidin moiety, bead, resin, a solid support, or a combination thereof. In some instances, the labeling group is a biotin moiety. In some instances, the biotin moiety further comprises a linker such as a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more residues in length. In some instances, as described elsewhere herein, the linker further comprises a cleavage site, such as a protease cleavage site. In some cases, the biotin moiety interacts with a streptavidin moiety. In some instances, the biotin moiety is further attached to a bead, such as a streptavidin-coupled bead. In some instances, the biotin moiety is further attached to a resin or a solid support, such as a streptavidin-coupled resin or a streptavidin-coupled solid support. In some instances, the solid support is a plate, a platform, a cover slide, a microfluidic channel, and the like.Lipid Probes of Formula I

[0106] In some examples, 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.

[0107] In some instances, PG is a photoreactive group described supra. In some examples, PG comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some instances, PG is diazirine, or its derivatives thereof. In some instances, PG is attached to A. In some instances, PG is attached to at a terminal site in A. In some instances, PG is attached to an internal site in A. In some instances, PG is attached to D.

[0108] In some cases, AH is an affinity handle described supra. In some cases, AH is a bioorthogonal affinity handle. In some cases, AH is a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group. In some instances, the affinity handle is an alkyne group, or an azide group. In some instances, AH is attached to A. In some instances, AH is attached to at a terminal site in A. In some instances, AH is attached to an internal site in A. In some cases, AH is attached to D.

[0109] In some instances, the lipid probe has one of the following structures as illustrated in Fig. 1. In some instances, the lipid probe is a lipid probe from Fig. 1. In some instances, the lipid probe is a lipid probe selected from Table 1. Table 1Lipid Probe Chemical Name 1.AEA-DA(5Z,8Z,11Z)-N-(2-hydroxyethyl)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienamide2.AA-DA(5Z,8Z,11Z)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienoic acid3.A-DA(5Z,8Z,11Z,14Z)-N-(2-(3-methyl-3H-diazirin-3-yl)ethyl)icosa-5,8,11,14-tetraen-19-ynamide4.ANH 2 -DA(5Z,8Z,11Z)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienamide5.AT-DApotassium 2-((5Z,8Z,11Z)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienamido)ethane-1-sulfonate6.2-AG-DA1,3-dihydroxypropan-2-yl (5Z,8Z,11Z)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienoate7.EEA-DAN-(2-hydroxyethyl)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradecanamide8.ENH 2 -DA14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradecanamide9.ET-DApotassium 2-(14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradecanamido)ethane-1-sulfonate10.2-EG-DA1,3-dihydroxypropan-2-yl 14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradecanoate11.EA-DA14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradecanoic acid12.OEA-DA(Z)-N-(2-hydroxyethyl)-12-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)dodec-9-enamide13.ONH 2 -DA(Z)-12-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)dodec-9-enamide14.OT-DApotassium (Z)-2-(12-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)dodec-9-enamido)ethane-1-sulfonate15.2-OG-DA1,3-dihydroxypropan-2-yl (2)-12-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)dodec-9-enoate16.OA-DA(Z)-12-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)dodec-9-enoic acid17.O-DA(Z)-N-(2-(3-methyl-3H-diazirin-3-yl)ethyl)octadec-9-en-17-ynamide18.PEA-DAN-(2-hydroxyethyl)-10-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)decanamide19.PNH 2 -DA10-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)decanamide20.PT-DApotassium 2-(10-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)decanamido)ethane-1-sulfonate21.2-PG-DA1-hydroxy-2-(hydroxymethyl)-13-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tridecan-4-one22.PA-DA10-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)decanoic acid23.S-DAN-(2-(3-Methyl-3H-diazirin-3-yl)ethyl)octadec-17-ynamide

[0110] In some instances, the lipid probe is selected from (5Z,8Z,11Z)-N-(2-hydroxyethyl)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienamide (AEA-DA), (5Z,8Z,11Z)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienoic acid (AA-DA), (5Z,8Z,11Z,14Z)-N-(2-(3-methyl-3H-diazirin-3-yl)ethyl)icosa-5,8,11,14-tetraen-19-ynamide (A-DA), (Z)-N-(2-hydroxyethyl)-12-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)dodec-9-enamide (OEA-DA), (Z)-N-(2-(3-methyl-3H-diazirin-3-yl)ethyl)octadec-9-en-17-ynamide (O-DA), N-(2-hydroxyethyl)-10-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)decanamide (PEA-DA), and N-(2-(3-Methyl-3H-diazirin-3-yl)ethyl)octadec-17-ynamide (S-DA). In some instances, the lipid probe is (5Z,8Z,112)-N-(2-hydroxyethyl)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienamide (AEA-DA). In some instances, the lipid probe is (5Z,8Z,11Z)-14-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)tetradeca-5,8,11-trienoic acid (AA-DA). In some instances, the lipid probe is (5Z,8Z,11Z,14Z)-N-(2-(3-methyl-3H-diazirin-3-yl)ethyl)icosa-5,8,11,14-tetraen-19-ynamide (A-DA). In some instances, the lipid probe is (Z)-N-(2-hydroxyethyl)-12-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)dodec-9-enamide (OEA-DA). In some instances, the lipid probe is (Z)-N-(2-(3-methyl-3H-diazirin-3-yl)ethyl)octadec-9-en-17-ynamide (O-DA). In some instances, the lipid probe is N-(2-hydroxyethyl)-10-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)decanamide (PEA-DA). In some instances, the lipid probe is N-(2-(3-Methyl-3H-diazirin-3-yl)ethyl)octadec-17-ynamide (S-DA).

[0111] As used herein in the context of lipid probe of Formula I, the term "alkyl" refers to a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbons. Exemplary saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Exemplary saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

[0112] The term "alkenyl" refers to an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Exemplary straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

[0113] Illustrative examples of cation include lithium (Li +< ), sodium (Na +< ), potassium (K +< ), cesium (Cs +< ), iron (Fe 2+< or Fe 3+< ), calcium (Ca 2+< ), vanadium (V 4+< ), zinc (Zn 2+< ), cadmium (Cd 2+< ), silver (Ag +< ), aluminum (Al 3+< ), and the like.Lipid Interacting Proteins

[0114] In some instances, the lipid binding protein is a soluble protein or a membrane protein. In some instances, the lipid binding protein is involved in one or more of a biological process such as protein transport, lipid metabolism, apoptosis, transcription, electron transport, mRNA processing, or host-virus interaction. In some instances, the lipid binding protein is associated with one or more of diseases such as cancer or one or more disorders or conditions such as immune, metabolic, developmental, reproductive, neurological, psychiatric, renal, cardiovascular, or hematological disorders or conditions. In some cases, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone.

[0115] An enzyme is a protein molecule that accelerates or catalyzes chemical reaction. Exemplary enzymes include, but are not limited to, AARS Alanine--tRNA ligase, cytoplasmic; ABCB1 multidrug resistant protein 1; ABHD10 Abhydrolase domain-containing protein 10, mitochondrial; ABHD12 Monoacylglycerol lipase ABHD12; ABHD16A Abhydrolase domain-containing protein 16A; ABHD6 Monoacylglycerol lipase ABHD6; ACAD9 Acyl-CoA dehydrogenase family member 9, mitochondrial; ACAD1 Long-chain specific acyl-CoA dehydrogenase, mitochondrial; ACAT2 Acetyl-CoA acetyltransferase, cytosolic; ACLY ATP-citrate synthase; ACOT2 Acyl-coenzyme A thioesterase 2, mitochondrial; ACP2 Lysosomal acid phosphatase; ACP6 Lysophosphatidic acid phosphatase type 6; ACSL1 Long-chain-fatty-acid-CoA ligase 1; ACSL3 Long-chain-fatty-acid--CoA ligase 3; ACSL4 Long-chain-fatty-acid--CoA ligase 4; ACSL6 Long-chain-fatty-acid--CoA ligase 6; ADK Adenosine kinase; ADPGK ADP-dependent glucokinase; ADSS Adenylosuccinate synthetase isozyme 2; AFG3L2 AFG3-like protein 2; AGK Acylglycerol kinase, mitochondrial; AGPAT1 1-acyl-sn-glycerol-3-phosphate acyltransferase alpha; Agpat2 1-acyl-sn-glycerol-3-phosphate acyltransferase beta; Agpat4 1-acyl-sn-glycerol-3-phosphate acyltransferase delta; AGPAT5 1-acyl-sn-glycerol-3-phosphate acyltransferase epsilon; AGPAT6 Glycerol-3-phosphate acyltransferase 4; Agpat9 Glycerol-3-phosphate acyltransferase 3; AGPS Alkyldihydroxyacetonephosphate synthase, peroxisomal; AHCY Adenosylhomocysteinase; AIFM1 Apoptosis-inducing factor 1, mitochondrial; AIFM2 Apoptosis-inducing factor 2; Akr1b8 Aldose reductase-related protein 2; Akr1c13 Aldo-keto reductase family 1 member C13; ALDH18A1 Delta-1-pyrroline-5-carboxylate synthase; ALDH1A2 Retinal dehydrogenase 2; ALDH1B1 Aldehyde dehydrogenase X, mitochondrial; ALDH2 Aldehyde dehydrogenase, mitochondrial; ALDH3A2 Fatty aldehyde dehydrogenase; Aldh3b1 Aldehyde dehydrogenase family 3 member B1; ALDH7A1 Alpha-aminoadipic semialdehyde dehydrogenase; ALG1 Chitobiosyldiphosphodolichol beta-mannosyltransferase; ALG10 Dol-P-Glc:Glc(2)Man(9)GlcNAc(2)-PP-Dol alpha-1,2-glucosyltransferase; ALG11 GDP-Man:Man(3)GlcNAc(2)-PP-Dol alpha-1,2-mannosyltransferase; ALG12 Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol alpha-1,6-mannosyltransferase; Alg2 Alpha-1,3 / 1,6-mannosyltransferase ALG2; ALG5 Dolichyl-phosphate beta-glucosyltransferase; ALG6 Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase; ALG9 Alpha-1,2-mannosyltransferase ALG9; AMFR E3 ubiquitin-protein ligase AMFR; ASAH1 acid ceramidase; ASNS Asparagine synthetase; ASPH Aspartyl / asparaginyl beta-hydroxylase; ATAD1 ATPase family AAA domain-containing protein 1; ATIC Bifunctional purine biosynthesis protein PURH; ATL2 Atlastin-2; ATP13A1 Probable cation-transporting ATPase 13A1; ATP1A1 Sodium / potassium-transporting ATPase subunit alpha; ATP1A3 Sodium / potassium-transporting ATPase subunit alpha; ATP2A2 Sarcoplasmic / endoplasmic reticulum calcium ATPase; ATP2B1 MCG13663, isoform CRA_a; ATP4A Potassiumtransporting ATPase alpha chain 1; ATP5B ATP synthase subunit beta, mitochondrial; B3GNT1 N-acetyllactosaminide beta-1,3-N-acetylglucosaminy; BDH1 D-beta-hydroxybutyrate dehydrogenase, mitochondrial; CAD CAD protein; CBS cystathionine beta-synthase; CLPP putative ATP-dependent Clp protease proteolytic subunit; COMT catechol O-methyltransferase; COPS5 COP9 signalosome complex subunit 5; COQ5 2-methoxy-6-polyprenyl-1,4-benzoquinol methylase; CPT1A Carnitine O-palmitoyltransferase 1, liver isoform; CPT2 Carnitine O-palmitoyltransferase 2, mitochondrial; CPVL Probable serine carboxypeptidase CPVL; CSNK1A1 casein kinase I isoform alpha; CTH cystathionine gamma-lyase; CTPS1 CTP synthase 1; CTSA Lysosomal protective protein; CTSB Cathepsin B; CTSD Cathepsin D; CYB5R3 NADH-cytochrome b5 reductase 3; Cyp20a1 Cytochrome P450 20A1; CYP51A1 Lanosterol 14-alpha demethylase; DAD1 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase; DCTPP1 dCTP pyrophosphatase 1; DCXR L-xylulose reductase; DDOST Dolichyl-diphosphooligosaccharide--protein glycosyltransferase; DDX17 Probable ATP-dependent RNA helicase DDX17; DCTN1 Dynactin subunit 1; DDX17 Probable ATP-dependent RNA helicase DDX17; DDX20 Probable ATP-dependent RNA helicase DDX20; DDX3X ATP-dependent RNA helicase DDX3X; DDX5 Probable ATP-dependent RNA helicase DDX5; DEGS1 Sphingolipid delta(4)-desaturase DES1; DHCR24 Delta(24)-sterol reductase; DHCR7 7-dehydrocholesterol reductase; DHFR Dihydrofolate reductase; DHODH Dihydroorotate dehydrogenase (quinone), mitochondrial; DHRS1 Dehydrogenase / reductase SDR family member 1; DHRS3 Short-chain dehydrogenase / reductase 3; DHRS7 Dehydrogenase / reductase SDR family member 7; DHRS7B Dehydrogenase / reductase SDR family member 7B; DHX15 Putative pre-mRNA-splicing factor ATP-dependent RN; DHX30 Putative ATP-dependent RNA helicase DHX30; DHX9 ATP-dependent RNA helicase A; DLD Dihydrolipoyl dehydrogenase, mitochondrial; DNM1L Dynamin-1-like protein; DNM2 Dynamin-2; DOLK Dolichol kinase; DOLPP1 Dolichyldiphosphatase 1; DPM1 Dolicholphosphate mannosyltransferase; Dpp7 Dipeptidyl peptidase 2; DPY19L1 Protein dpy-19 homolog 1; EBP 3-beta-hydroxysteroid-Delta(8),Delta(7)-isomerase; ECE1 Endothelinconverting enzyme 1; ECH1 Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial; ECHS1 Enoyl-CoA hydratase, mitochondrial; Eci1 Enoyl-CoA delta isomerase 1, mitochondrial; EIF3F Eukaryotic translation initiation factor 3 subunit; EIF4A1 Eukaryotic initiation factor 4A-I; EIF4A2 Eukaryotic initiation factor 4A-II; ELOVL2 Elongation of very long chain fatty acids protein; ENDOD1 Endonuclease domain-containing 1 protein; EPHX1 Epoxide hydrolase 1; EPHX2 Bifunctional epoxide hydrolase 2; ERMP1 Endoplasmic reticulum metallopeptidase 1; ERO1L ERO1-like protein alpha; Fads1 Fatty acid desaturase 1; FADS2 Fatty acid desaturase 2; FADS3 Fatty acid desaturase 3; FAH fumarylacetoacetase; Fkbp11 Peptidyl-prolyl cis-trans isomerase FKBP11; FKBP1A Peptidyl-prolyl cis-trans isomerase FKBP1A; Fkbp2 Peptidyl-prolyl cis-trans isomerase FKBP2; FKBP4 Peptidyl-prolyl cis-trans isomerase FKBP4; FKBP8 Peptidyl-prolyl cis-trans isomerase FKBP8; FNTB Protein farnesyltransferase subunit beta; G6PD Glucose-6-phosphate 1-dehydrogenase; GAA Lysosomal alpha-glucosidase; GALNT1 Polypeptide N-acetylgalactosaminyltransferase 1; GANAB Neutral alpha-glucosidase AB; GARS Glycine--tRNA ligase; GART Trifunctional purine biosynthetic protein adenosine;GBA Glucosylceramidase; GDPD1 Glycerophosphodiester phosphodiesterase domain-containing 1;GK Glycerol kinase; GLA Alpha-galactosidase A; GLB1 Beta-galactosidase; GLO1 Lactoylglutathione lyase; GLT8D1 Glycosyltransferase 8 domain-containing protein 1; GMPPB Mannose-1-phosphate guanyltransferase beta; GMPS GMP synthase; GNPAT Dihydroxyacetone phosphate acyltransferase; GPD2 Glycerol-3-phosphate dehydrogenase, mitochondrial; GPX8 Probable glutathione peroxidase 8; GSR Glutathione reductase, mitochondrial; GSTM1 Glutathione S-transferase Mu 1; GSTM2 Glutathione S-transferase Mu 2; GSTO1 Glutathione S-transferase omega-1; GSTP1 Glutathione S-transferase P; HACL1 2-hydroxyacyl-CoA lyase 1; HADH Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial;HADHA Trifunctional enzyme subunit alpha, mitochondrial; HADHB Trifunctional enzyme subunit beta, mitochondrial; HARS Histidine--tRNA ligase, cytoplasmic; HAT1 Histone acetyltransferase type B catalytic subunit;HK1 Hexokinase-1; HK2 Hexokinase-2; HM13 Minor histocompatibility antigen H13; HMGCS1 Hydroxymethylglutaryl-CoA synthase, cytoplasmic; HMOX2 Heme oxygenase 2; HPRT1 Hypoxanthine-guanine phosphoribosyltransferase; HSD17B10 3-hydroxyacyl-CoA dehydrogenase type-2; HSD17B11 Estradiol 17-beta-dehydrogenase 11; HSD17B12 Estradiol 17-beta-dehydrogenase 12; HSD17B4 Peroxisomal multifunctional enzyme type 2; HSD17B7 3-keto-steroid reductase; HSDL2 Hydroxysteroid dehydrogenase-like protein 2; HUWE1 E3 ubiquitin-protein ligase HUWE1; IARS Isoleucine--tRNA ligase, cytoplasmic; IARS2 Isoleucine--tRNA ligase, mitochondrial; ICMT Protein-S-isoprenylcysteine O-methyltransferase; IDH1 Isocitrate dehydrogenase; IDH2 Isocitrate dehydrogenase; IFI30 Gamma-interferon-inducible lysosomal thiol reductase;ILVBL Acetolactate synthase-like protein; IMPDH2 Inosine-5-monophosphate dehydrogenase 2; INPP5K Inositol polyphosphate 5-phosphatase K; KDM1B Lysine-specific histone demethylase 1B; KDSR 3-ketodihydrosphingosine reductase ;KDM1B Lysine-specific histone demethylase 1B; KDSR 3-ketodihydrosphingosine reductase; L2HGDH L-2-hydroxyglutarate dehydrogenase, mitochondrial; LARS Leucine--tRNA ligase, cytoplasmic; LCLAT1 Lysocardiolipin acyltransferase 1; LNPEP Leucyl-cystinyl aminopeptidase; LPCAT1 Lysophosphatidylcholine acyltransferase 1; LPCAT2 Lysophosphatidylcholine acyltransferase 2; LPCAT3 Lysophospholipid acyltransferase 5; LPGAT1 Acyl-CoA:lysophosphatidylglycerol acyltransferase; LSS Lanosterol synthase; LTA4H Leukotriene A-4 hydrolase; MAOA Amine oxidase; MARCH5 E3 ubiquitin-protein ligase MARCH5; MARCH6 E3 ubiquitin-protein ligase MARCH6; MBLAC2 Metallo-beta-lactamase domain-containing protein 2;MBOAT7 Lysophospholipid acyltransferase 7; MCCC2 Methylcrotonoyl-CoA carboxylase beta chain, mitochondrial; MCEE Methylmalonyl-CoA epimerase, mitochondrial; MCM2 DNA replication licensing factor MCM2; MCM4 DNA replication licensing factor MCM4; MCM6 DNA replication licensing factor MCM6; MCM7 DNA replication licensing factor MCM7; MEST Mesoderm-specific transcript protein; METAP1 Methionine aminopeptidase 1; METTL 7 A Methyltransferase-like protein 7A; MGEA5 Bifunctional protein NCOAT; MGST1 Microsomal glutathione S-transferase 1; MIF Macrophage migration inhibitory factor; MOGS Mannosyl-oligosaccharide glucosidase; MOV10 Putative helicase MOV-10; MPI Mannose-6-phosphate isomerase; MSMO1 Methylsterol monooxygenase 1; MT-CO1 Cytochrome c oxidase subunit 1; MT-ND1 NADH-ubiquinone oxidoreductase chain 1; MT-ND2 NADH-ubiquinone oxidoreductase chain 2; MT-ND5 NADH-ubiquinone oxidoreductase chain 5; MTAP S-methyl-5-thioadenosine phosphorylase; MTHFD1 C-1-tetrahydrofolate synthase, cytoplasmic; NAMPT Nicotinamide phosphoribosyltransferase; NDUFS1 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial;NDUFS2 NADH dehydrogenase; NDUFS3 NADH dehydrogenase; NDUFS7 NADH dehydrogenase; NLN Neurolysin, mitochondrial; NME2 Nucleoside diphosphate kinase B; NNT NAD(P) transhydrogenase, mitochondrial; NPEPPS Puromycin-sensitive aminopeptidase; NRD1 Nardilysin; NSDHL Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating;NSUN2 tRNA (cytosine(34)-C(5))-methyltransferase; NT5C3L Cytosolic 5-nucleotidase III-like protein; NT5DC2 5-nucleotidase domain-containing protein 2; NTMT1 N-terminal Xaa-Pro-Lys N-methyltransferase 1; NUDT1 7,8-dihydro-8-oxoguanine triphosphatase; NUDT9 ADP-ribose pyrophosphatase, mitochondrial; NXN Nucleoredoxin; OAT Ornithine aminotransferase, mitochondrial; OPA1 Dynamin-like 120 kDa protein, mitochondrial; OTUB1 Ubiquitin thioesterase OTUB1; P4HA1 Prolyl 4-hydroxylase subunit alpha-1; P4HA2 Prolyl 4-hydroxylase subunit alpha-2; P4HB Protein disulfide-isomerase; P4HTM Transmembrane prolyl 4-hydroxylase; PAICS Multifunctional protein ADE2; PANK4 Pantothenate kinase 4; PAOX Peroxisomal N(1)-acetyl-spermine / spermidine oxidase;PAPSS1 Bifunctional 3-phosphoadenosine 5-phosphosulfate; PARL Presenilins-associated rhomboid-like protein, mitochondrial; PCK2 Phosphoenolpyruvate carboxykinase; PCYOX1 Prenylcysteine oxidase 1; PCYOX1L Prenylcysteine oxidase-like; PCYT1A Choline-phosphate cytidylyltransferase A; PDIA6 Protein disulfide-isomerase A6; PDP1; Pdss1 Decaprenyl-diphosphate synthase subunit 1; PFKL 6-phosphofructokinase, liver type PFKP 6-phosphofructokinase type C PGAM5 Serine / threonine-protein phosphatase PGAM5, mitochondrial; PHGDH D-3-phosphoglycerate dehydrogenase; PI4KB Phosphatidylinositol 4-kinase beta; PIGK GPI-anchor transamidase; PIGM GPI mannosyltransferase 1; PISD Phosphatidylserine decarboxylase proenzyme; PITRM1 Presequence protease, mitochondrial; PKM Pyruvate kinase isozymes M1 / M2; Pla2g15 Group XV phospholipase A2; PLBD2 Putative phospholipase B-like 2; PLD3 Phospholipase D3; PLOD1 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1; PLOD3 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 3; PMPCA Mitochondrial-processing peptidase subunit alpha; PMPCB Mitochondrial-processing peptidase subunit beta; PNPLA2 Patatin-like phospholipase domain-containing protein;PNPLA6 Neuropathy target esterase; POLR2B DNA-directed RNA polymerase II subunit RPB2; POLRMT DNA-directed RNA polymerase, mitochondrial; PON2 Serum paraoxonase / arylesterase 2; POR NADPH--cytochrome P450 reductase; PPAPDC2 Presqualene diphosphate phosphatase; PPAT Amidophosphoribosyltransferase; PPM1L Protein phosphatase 1L; PPT1 Palmitoyl-protein thioesterase 1; PRCP Lysosomal Pro-X carboxypeptidase;PRDX1 Peroxiredoxin-1; PRDX3 Thioredoxin-dependent peroxide reductase, mitochondrial;PREP Prolyl endopeptidase;PREPL Prolyl endopeptidase-like; PRKDC DNA-dependent protein kinase catalytic subunit;PRMT1 Protein arginine N-methyltransferase 1; PSMA4 Proteasome subunit alpha type-4; PSMA6 Proteasome subunit alpha type-6;PSMB1 Proteasome subunit beta type-1; PSMB2 Proteasome subunit beta type-2; PSMB3 Proteasome subunit beta type-3; PSMB4 Proteasome subunit beta type-4; PSMBS Proteasome subunit beta type-5; PSMB6 Proteasome subunit beta type-6; PSMB7 Proteasome subunit beta type-7; PSMB8 Proteasome subunit beta type-8; PSMD14 26S proteasome non-ATPase regulatory subunit 14; PTDSS1 Phosphatidylserine synthase 1; PTDSS2 Phosphatidylserine synthase 2; PTGES2 Prostaglandin E synthase 2; PTGR2 Prostaglandin reductase 2; PTGS1 Prostaglandin G / H synthase 1; PTPLAD1 3-hydroxyacyl-CoA dehydratase 3; PTPLB 3-hydroxyacyl-CoA dehydratase 2; PTPN1 Tyrosine-protein phosphatase non-receptor type 1; PTRH2 Peptidyl-tRNA hydrolase 2, mitochondrial; QPCTL Glutaminyl-peptide cyclotransferase-like protein; RABGGTB Geranylgeranyl transferase type-2 subunit beta; RANBP2 E3 SUMO-protein ligase RanBP2; RARS Arginine--tRNA ligase, cytoplasmic;RDH10 Retinol dehydrogenase 10; RDH11 Retinol dehydrogenase 11; RDH13 Retinol dehydrogenase 13; RDH14 Retinol dehydrogenase 14; RETSAT All-trans-retinol 13,14-reductase; RHOT1 Mitochondrial Rho GTPase 1; RHOT2 Mitochondrial Rho GTPase 2; RNF14 E3 ubiquitin-protein ligase RNF14; RPN1 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase;RPN2 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase;RPS3 40S ribosomal protein S3; RPS6KA1 Ribosomal protein S6 kinase alpha-1; RPS6KA2 Ribosomal protein S6 kinase alpha-2; RPS6KA3 Ribosomal protein S6 kinase alpha-3; RRM2 Ribonucleoside-diphosphate reductase subunit M2; RUVBL1 RuvB-like 1; RUVBL2 RuvB-like 2; SACM1L Phosphatidylinositide phosphatase SAC1; SCCPDH Saccharopine dehydrogenase-like oxidoreductase; SCD Acyl-CoA desaturase; SCD2 Acyl-CoA desaturase 2; SCP2 Non-specific lipid-transfer protein; SCPEP1 Retinoid-inducible serine carboxypeptidase ;SDHA Succinate dehydrogenase; SDHB Succinate dehydrogenase; SEC11A Signal peptidase complex catalytic subunit SEC11A; SEC11C Signal peptidase complex catalytic subunit SEC11C; SGPL1 Sphingosine-1-phosphate lyase 1; SHMT1 Serine hydroxymethyltransferase, cytosolic; SHMT2 Serine hydroxymethyltransferase, mitochondrial; SLC27A2 Very long-chain acyl-CoA synthetase; SLC27A3 Long-chain fatty acid transport protein 3; SMPD4 Sphingomyelin phosphodiesterase 4; SNRNP200 U5 small nuclear ribonucleoprotein 200 kDa helicase;SOAT1 Sterol O-acyltransferase 1; SPCS2 Signal peptidase complex subunit 2; SPTLC1 Serine palmitoyltransferase 1; SPTLC2 Serine palmitoyltransferase 2; SQLE Squalene monooxygenase; SRM Spermidine synthase; SPTLC1 Serine palmitoyltransferase 1; SPTLC2 Serine palmitoyltransferase 2; SQLE Squalene monooxygenase; SRM Spermidine synthase; STT3A Dolichyl-diphosphooligosaccharide--protein glycosyltransferase;STT3B Dolichyl-diphosphooligosaccharide--protein glycosyltransferase;SYVN1 E3 ubiquitin-protein ligase synoviolin; TECR Trans-2,3-enoyl-CoA reductase; THOP1 Thimet oligopeptidase; TM7SF2 Delta(14)-sterol reductase; TMPPE Transmembrane protein with metallophosphoesterase; TMX3 Protein disulfide-isomerase TMX3; TOR1A Torsin-1A; TOR1B Torsin-1B; TPI1 Triosephosphate isomerase; TPP1 Tripeptidyl-peptidase 1; TRIM28 Transcription intermediary factor 1-beta; UGCG Ceramide glucosyltransferase; UGT8 2-hydroxyacylsphingosine 1-beta-galactosyltransferase;UQCRFS1 Cytochrome b-c1 complex subunit Rieske, mitochondrial;USP28 Ubiquitin carboxyl-terminal hydrolase 28; USP47 Ubiquitin carboxyl-terminal hydrolase 47; USP5 Ubiquitin carboxyl-terminal hydrolase 5; USP7 Ubiquitin carboxyl-terminal hydrolase 7; VAT1 Synaptic vesicle membrane protein VAT-1 homolog; VAT1L Synaptic vesicle membrane protein VAT-1 homolog-likeVCP Transitional endoplasmic reticulum ATPase; VKORC1L1 Vitamin K epoxide reductase complex subunit 1-like;VKORC1L1 Vitamin K epoxide reductase complex subunit 1-like; WHSC1 Probable histone-lysine N-methyltransferase NSD2; XPNPEP1 Xaa-Pro aminopeptidase 1; XRCC5 X-ray repair cross-complementing protein 5; XRCC6 X-ray repair cross-complementing protein 6; YKT6 Synaptobrevin homolog YKT6; YME1L1 ATP-dependent zinc metalloprotease YME1L1; ZADH2 Zinc-binding alcohol dehydrogenase domain-containing 2; and ZMPSTE24 CAAX prenyl protease 1 homolog.

[0116] In some examples, one or more lipid probes described herein interact or bind to one or more of the enzymes described above and elsewhere herein.

[0117] A transporter, also referred to as a transmembrane pump, transporter protein, escort protein, acid transport protein, cation transport protein, or anion transport protein, is a protein molecule that moves materials (e.g. ions, small molecules, macromolecules, proteins) within an organism, such as across a biological membrane. Exemplary transporters include, but are not limited to, AAAS Aladin; ABCB1 multidrug resistant protein 1; ABCB10 ATP-binding cassette sub-family B member 10, mitochondrial; ABCB7 ATP-binding cassette sub-family B member 7, mitochondrial; ABCD3 ATP-binding cassette sub-family D member 3; ACBD5 Acyl-CoA-binding domain-containing protein 5; ACTN4 alpha-actinin-4; AMFR E3 ubiquitin-protein ligase AMFR; AP3B1 AP-3 complex subunit beta-1; AP3M1 AP-3 complex subunit mu-1; APOL2 Apolipoprotein L2; APOO Apolipoprotein O; APOOL Apolipoprotein O-like; ARF1 ADP-ribosylation factor 1; ARF3 ADP-ribosylation factor 3; ARF4 ADP-ribosylation factor 4; ARF5 ADP-ribosylation factor 5; ARFGAP1 ADP-ribosylation factor GTPase-activating protein; ASNA1 ATPase ASNA1; ATP13A1 Probable cation-transporting ATPase 13A1; ATP1A1 Sodium / potassium-transporting ATPase subunit alpha; ATP1A3 Sodium / potassium-transporting ATPase subunit alpha; ATP2A2 Sarcoplasmic / endoplasmic reticulum calcium ATPase; ATP2B1 MCG13663, isoform CRA_a; ATP4A Potassium-transporting ATPase alpha chain 1; ATPSA1 ATP synthase subunit alpha, mitochondrial; ATP5B ATP synthase subunit beta, mitochondrial; ATP5C1 ATP synthase subunit gamma, mitochondrial; ATP5F1 ATP synthase subunit b, mitochondrial; ATP5H ATP synthase subunit d, mitochondrial; ATP5I ATP synthase subunit e, mitochondrial; ATP5J2 ATP synthase subunit f, mitochondrial; ATP5L ATP synthase subunit g, mitochondrial; ATP5O ATP synthase subunit O, mitochondrial; ATP6AP1 V-type proton ATPase subunit S1; ATP6V0A1 V-type proton ATPase 116 kDa subunit a isoform 1; ATP6V0A2 V-type proton ATPase 116 kDa subunit a isoform 2; ATP6V0C V-type proton ATPase 16 kDa proteolipid subunit; ATP6V1B2 V-type proton ATPase subunit B, brain isoform; BAG6 Large proline-rich protein BAG6; BCAP29 B-cell receptor-associated protein 29; BCAP31 B-cell receptor-associated protein 31; BNIP1 vesicle transport protein SEC20; CDIPT CDP-diacylglycerol--inositol 3-phosphatidyltransferase; CDKAL1 Threonylcarbamoyladenosine tRNA methylthiotransferase; CDS2 Phosphatidate cytidylyltransferase 2; CEPT1 Choline / ethanolaminephosphotransferase 1; CERS5 ceramide synthase 5; CERS6 ceramide synthase 6; CHDH choline dehydrogenase, mitochondrial; CHUK inhibitor of nuclear factor kappa-B kinase subunit; CKB creatine kinase B-type; COPB1 coatomer subunit beta; COPE coatomer subunit epsilon; CPT1A Carnitine O-palmitoyltransferase 1, liver isoform; CPT2 Carnitine O-palmitoyltransferase 2, mitochondrial; CSE1L exportin-2; CYB5A Cytochrome b5; CYB5B Cytochrome b5 type B; CYC1 Cytochrome c1, heme protein, mitochondrial; DCTN1 Dynactin subunit 1; DERL1 Derlin-1; DYNC1H1 Cytoplasmic dynein 1 heavy chain 1; ERGIC1 Endoplasmic reticulum-Golgi intermediate compartment; ERGIC2 Endoplasmic reticulum-Golgi intermediate compartment; ESYT1 Extended synaptotagmin-1; ESYT2 Extended synaptotagmin-2; ETFDH Electron transfer flavoprotein-ubiquinone oxidoreductase; FABP5 Fatty acid-binding protein, epidermal; FADS1 Fatty acid desaturase 1; FADS2 Fatty acid desaturase 2; FADS3 Fatty acid desaturase 3; FMR1 Fragile X mental retardation protein 1 homolog; GET4 Golgi to ER traffic protein 4 homolog; GOLIM4 Golgi integral membrane protein 4; GOLPH3 Golgi phosphoprotein 3; GOLT1B Vesicle transport protein GOT1B; GOPC Golgi-associated PDZ and coiled-coil motif-containing;GOSR1 Golgi SNAP receptor complex member 1; GPR89B Golgi pH regulator B; HIGD1A HIG1 domain family member 1A; HNRNPA1 Heterogeneous nuclear ribonucleoprotein A1; HNRNPA1L2 Heterogeneous nuclear ribonucleoprotein A1-like 2; IGF2BP1 Insulin-like growth factor 2 mRNA-binding protein; IGF2BP3 Insulin-like growth factor 2 mRNA-binding protein; IPO11 Importin-11; IPO4 Importin-4; IPO5 Importin-5; IPO7 Importin-7; IPO9 Importin-9; KDELR1 ER lumen protein retaining receptor 1; KDELR2 ER lumen protein retaining receptor 2; KDELR3 ER lumen protein retaining receptor 3; KHSRP Far upstream element-binding protein 2; KPNA2 Importin subunit alpha-2; KPNB1 Importin subunit beta-1; LAPTM4A Lysosomal-associated transmembrane protein 4A; LMAN1 Protein ERGIC-53 LMAN2 Vesicular integral-membrane protein VIP36; LRPPRC Leucine-rich PPR motif-containing protein, mitochondrial; MAGT1 Magnesium transporter protein 1; MCFD2 Multiple coagulation factor deficiency protein 2; MICU1 Calcium uptake protein 1, mitochondrial; MMGT1 Membrane magnesium transporter 1; MPC2 Mitochondrial pyruvate carrier 2; MT-ATP6 ATP synthase subunit a; MT-CO1 Cytochrome c oxidase subunit 1; MT-CO2 Cytochrome c oxidase subunit 2; MT-ND1 NADH-ubiquinone oxidoreductase chain 1; MT-ND2 NADH-ubiquinone oxidoreductase chain 2; MT-ND5 NADH-ubiquinone oxidoreductase chain 5; MTCH1 Mitochondrial carrier homolog 1; MTCH2 Mitochondrial carrier homolog 2; MTTP Microsomal triglyceride transfer protein large subunit; MTX1 Metaxin-1; MTX2 Metaxin-2; NASP Nuclear autoantigenic sperm protein; NCBP1 Nuclear cap-binding protein subunit 1; NDUFA10 NADH dehydrogenase; NDUFA11 NADH dehydrogenase; NDUFA13 NADH dehydrogenase; NDUFA4 NADH dehydrogenase; NDUFA8 NADH dehydrogenase; NDUFA9 NADH dehydrogenase; NDUFB10 NADH dehydrogenase; NDUFB6 NADH dehydrogenase; NDUFB8 NADH dehydrogenase; NDUFB9 NADH dehydrogenase; NDUFS1 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial; NDUFS2 NADH dehydrogenase; NDUFS3 NADH dehydrogenase; NDUFS7 NADH dehydrogenase; NUP133 Nuclear pore complex protein Nup133; NUP155 Nuclear pore complex protein Nup155; NUP160 Nuclear pore complex protein Nup160; NUP205 Nuclear pore complex protein Nup205; NUP210 Nuclear pore membrane glycoprotein 210; NUP37 Nucleoporin Nup37; NUP93 Nuclear pore complex protein Nup93; OSBPL8 Oxysterol-binding protein-related protein 8; PDCD6IP Programmed cell death 6-interacting protein; PEX14 Peroxisomal membrane protein PEX14; PITPNB Phosphatidylinositol transfer protein beta isoform;PLIN3 Perilipin-3; PRAF2 PRA1 family protein 2; PREB Prolactin regulatory element-binding protein; RAB10 Ras-related protein Rab-10; RAB11A Ras-related protein Rab-11A; RAB11B Ras-related protein Rab-11B; RAB18 Ras-related protein Rab-18; RAB1A Ras-related protein Rab-1A; RAB1B Ras-related protein Rab-1B; RAB2A Ras-related protein Rab-2A; RAB9A Ras-related protein Rab-9A; RANBP2 E3 SUMO-protein ligase RanBP2; RBP1 Retinol-binding protein 1; RFT1 Protein RFT1 homolog; RTN3 Reticulon-3; SAR1A GTP-binding protein SAR1a; SAR1B GTP-binding protein SAR1b; SCAMP1 Secretory carrier-associated membrane protein 1; SCAMP2 Secretory carrier-associated membrane protein 2; SCAMP3 Secretory carrier-associated membrane protein 3; SCP2 Non-specific lipid-transfer protein; SDHA Succinate dehydrogenase; SDHB Succinate dehydrogenase; SDHC Succinate dehydrogenase cytochrome b560 subunit, mitochondrial; SDHD Succinate dehydrogenase; SEC13 Protein SEC13 homolog; SEC22B Vesicle-trafficking protein SEC22b; SEC23A Protein transport protein Sec23A; SEC23B Protein transport protein Sec23B; SEC61A1 Protein transport protein Sec61 subunit alpha isoform; SEC61A2 Protein transport protein Sec61 subunit alpha isoform; SEC63 Translocation protein SEC63 homolog; SFXN1 Sideroflexin-1; SFXN4 Sideroflexin-4; SIGMAR1 Sigma non-opioid intracellular receptor 1; SIGMAR1 Sigma non-opioid intracellular receptor 1; SLC16A1 Monocarboxylate transporter 1; SLC1A5 Neutral amino acid transporter B(0); SLC25A1 Tricarboxylate transport protein, mitochondrial; SLC25A10 Mitochondrial dicarboxylate carrier; SLC25A11 Mitochondrial 2-oxoglutarate / malate carrier protein; SLC25A12 Calcium-binding mitochondrial carrier protein Aral; SLC25A13 Calcium-binding mitochondrial carrier protein Aral; SLC25A15 Mitochondrial ornithine transporter 1; SLC25A16 Graves disease carrier protein homolog; SLC25A17 Peroxisomal membrane protein PMP34; SLC25A19 Mitochondrial thiamine pyrophosphate carrier; SLC25A20 Mitochondrial carnitine / acylcarnitine carrier protein; SLC25A21 Mitochondrial 2-oxodicarboxylate carrier; SLC25A22 Mitochondrial glutamate carrier 1; SLC25A24 Calcium-binding mitochondrial carrier protein SCaM;SLC25A25 Calcium-binding mitochondrial carrier protein SCaM; SLC25A26 S-adenosylmethionine mitochondrial carrier protein; SLC25A3 Phosphate carrier protein, mitochondrial; SLC25A32 Mitochondrial folate transporter / carrier; SLC25A33 Solute carrier family 25 member 33; SLC25A4 ADP / ATP translocase 1; SLC25A44 Solute carrier family 25 member 44; SLC25A5 ADP / ATP translocase 2; SLC25A51 Solute carrier family 25 member 51; SLC25A6 ADP / ATP translocase 3; SLC29A3 Equilibrative nucleoside transporter 3; SLC2A3 Solute carrier family 2, facilitated glucose transporter;SLC30A6 Zinc transporter 6; SLC30A7 Zinc transporter 7; SLC33A1 Acetyl-coenzyme A transporter 1; SLC35B2 Adenosine 3-phospho 5-phosphosulfate transporter;SLC35E1 Solute carrier family 35 member E1; SLC38A2 Sodium-coupled neutral amino acid transporter 2; SLC39A7 Zinc transporter SLC39A7; SLC7A1 High affinity cationic amino acid transporter 1; SLC7A3 Cationic amino acid transporter 3; SNX1 Sorting nexin-1; SNX2 Sorting nexin-2; SNX27 Sorting nexin-27; SNX5 Sorting nexin-5; SNX6 Sorting nexin-6; SNX9 Sorting nexin-9; SPNS1 Protein spinster homolog 1; STX18 Syntaxin-18; Stx5 Syntaxin-5; SV2C Synaptic vesicle glycoprotein 2C; TIMM17A Mitochondrial import inner membrane translocase subunit; TIMM17B Mitochondrial import inner membrane translocase subunit; TIMM22 Mitochondrial import inner membrane translocase subunit; TIMM23 Mitochondrial import inner membrane translocase subunit; TIMM44 Mitochondrial import inner membrane translocase subunit; TIMM50 Mitochondrial import inner membrane translocase subunit; TMED 1 Transmembrane emp24 domain-containing protein 1; TMED10 Transmembrane emp24 domain-containing protein 10; TMED2 Transmembrane emp24 domain-containing protein 2; TMED3 Transmembrane emp24 domain-containing protein 3; TMED4 Transmembrane emp24 domain-containing protein 4; TMED5 Transmembrane emp24 domain-containing protein 5; TMED7 Transmembrane emp24 domain-containing protein 7; TMED9 Transmembrane emp24 domain-containing protein 9; TMEM38B Trimeric intracellular cation channel type B; TMEM48 Nucleoporin NDC1; Tmem66 Store-operated calcium entry-associated regulatory;TMX1 Thioredoxin-related transmembrane protein 1; TMX4 Thioredoxin-related transmembrane protein 4; TNPO1 Transportin-1; TNPO2 Transportin-2; TNPO3 Transportin-3; TOMM20 Mitochondrial import receptor subunit TOM20 homolog;TOMM22 Mitochondrial import receptor subunit TOM22 homolog;TOMM40 Mitochondrial import receptor subunit TOM40 homolog;TPR Nucleoprotein TPR; TRAM1 Translocating chain-associated membrane protein 1; TSPO Translocator protein; TTYH3 Protein tweety homolog 3; TUSC3 Tumor suppressor candidate 3; UNC119 Protein unc-119 homolog A; UNC119B Protein unc-119 homolog B; UQCR10 Cytochrome b-c1 complex subunit 9; UQCRC1 Cytochrome b-c1 complex subunit 1, mitochondrial; UQCRC2 Cytochrome b-c1 complex subunit 2, mitochondrial; UQCRFS1 Cytochrome b-c1 complex subunit Rieske, mitochondrial; UQCRQ Cytochrome b-c1 complex subunit 8; VCP Transitional endoplasmic reticulum ATPase; VDAC1 Voltage-dependent anion-selective channel protein; VDAC2 Voltage-dependent anion-selective channel protein; VDAC3 Voltage-dependent anion-selective channel protein; VPS35 Vacuolar protein sorting-associated protein 35; XPO1 Exportin-1; XPO5 Exportin-5; XPOT Exportin-T; YIF1A Protein YIF1A; and YKT6 Synaptobrevin homolog YKT6.

[0118] In some examples, one or more lipid probes described herein interact or bind to one or more of the transporters described above and elsewhere herein.

[0119] A receptor is a protein molecule that is embedded within the plasma membrane of a cell. In some instances, the receptor upon receiving a signal, such as a chemical signal, respond by producing a change in the cell. In some examples, a receptor is further divided into type 1 receptors or ionotropic receptors which are targeted by fast neurotransmitters such as acetylcholine (nicotinic) and GABA; type 2 receptors or G protein-coupled receptors (GPCRs); type 3 receptors or kinase linked and related receptors such as tyrosine kinase receptor and enzyme-linked receptor; and type 4 receptors or nuclear receptors. Exemplary receptors include, but are not limited to, ITGB1 Integrin beta-1, KDELR1 ER lumen protein retaining receptor 1, KDELR2 ER lumen protein retaining receptor 2, KDELR3 ER lumen protein retaining receptor 3, LBR Lamin-B receptor, LMBR1 Limb region 1 protein homolog, NGFR Tumor necrosis factor receptor superfamily member, NR3C1 Glucocorticoid receptor, PGRMC1 Membrane-associated progesterone receptor component, PGRMC2 Membrane-associated progesterone receptor component, PHB2 Prohibitin-2, SCARB1 Scavenger receptor class B member 1, SCARB2 Lysosome membrane protein 2, SIGMAR1 Sigma non-opioid intracellular receptor 1, SLC1A5 Neutral amino acid transporter B(0), SRPRB Signal recognition particle receptor subunit beta, and TFRC Transferrin receptor protein 1.

[0120] In some examples, one or more lipid probes described herein interact or bind to one or more of the receptors described above and elsewhere herein.

[0121] An adaptor is an accessory protein molecule which mediates specific protein-protein interactions that drive the formation of protein complexes. In some instances, an adaptor protein lacks intrinsic enzymatic activity. Exemplary adaptor proteins include, but are not limited to, adaptor protein complex 1, adaptor protein complex 2, Casitas B-lineage lymphoma (Cbl) adaptor protein, Lck-interacting molecule (LIME), non-catalytic region of tyrosine kinase (Nck) adaptor protein, BAIAP2 Brain-specific angiogenesis inhibitor 1-associated; MPP6 MAGUK p55 subfamily member 6; SPTAN1 spectrin alpha chain, non-erythrocytic 1; YWHAB 14-3-3 protein beta / alpha; YWHAE 14-3-3 protein epsilon; YWHAG 14-3-3 protein gamma; YWHAH 14-3-3 protein eta; YWHAQ 14-3-3 protein theta; and YWHAZ 14-3-3 protein zeta / delta.

[0122] In some examples, one or more lipid probes described herein interact or bind to one or more of the adaptors described above and elsewhere herein.

[0123] A channel protein is a pore-forming membrane protein which allows the transport of molecules such as ions and small molecules across a cell membrane. In some instances, a channel protein is a ligand-gated, voltage-gated, or mechanically-gated channel protein. In some instances, a channel protein allows for passive diffusion of molecules such as ions and small molecules. Exemplary channel proteins include, but are not limited to, VDAC1 Voltage-dependent anion-selective channel protein, VDAC2 Voltage-dependent anion-selective channel protein, and VDAC3 Voltage-dependent anion-selective channel protein.

[0124] In some examples, one or more lipid probes described herein interact or bind to one or more of the channel proteins described above and elsewhere herein.

[0125] A chaperone is a protein molecule that assists in folding or unfolding of protein molecules and / or assembly or disassembly of macromolecular structures. Exemplary chaperones include, but are not limited to, ABCE1 ATP-binding cassette sub-family E member 1; AHSA1 Activator of 90 kDa heat shock protein ATPase homolog 1; ANP32B acidic leucine-rich nuclear phosphoprotein 32 family; BAG6 Large proline-rich protein BAG6; BCS1L mitochondrial chaperone BCS1; CALR calreticulin; CANX calnexin; CCT2 T-complex protein 1 subunit beta; CCT3 T-complex protein 1 subunit gamma; CCT4 T-complex protein 1 subunit delta; CCT5 T-complex protein 1 subunit epsilon; CCT6A T-complex protein 1 subunit zeta; CCT7 T-complex protein 1 subunit eta; CD74 H-2 class II histocompatibility antigen gamma chai; CDC37 Hsp90 co-chaperone Cdc37; CLGN calmegin; DNAJA1 DnaJ homolog subfamily A member 1; DNAJC1 DnaJ homolog subfamily C member 1; DNAJC11 DnaJ homolog subfamily C member 11; HSP90AA1 Heat shock protein HSP 90-alpha; HSP90AB1 Heat shock protein HSP 90-beta; HSP90B1 Endoplasmin; HSPA1B Heat shock 70 kDa protein 1A / 1B; HSPA2 Heat shock-related 70 kDa protein 2; HSPA8 Heat shock cognate 71 kDa protein; HSPA9 Stress-70 protein, mitochondrial; HSPD1 60 kDa heat shock protein, mitochondrial; HYOU1 Hypoxia up-regulated protein 1; NDUFAF2 Mimitin, mitochondrial ; SCO1 Protein SCO1 homolog, mitochondrial ;SCO2 Protein SCO2 homolog, mitochondrial; ST13 Hsc70-interacting protein; TBCD Tubulin-specific chaperone D; TCP1 T-complex protein 1 subunit alpha; TIMMDC1 Translocase of inner mitochondrial membrane domain; and TMEM126B Transmembrane protein 126B.

[0126] In some examples, one or more lipid probes described herein interact or bind to one or more of the chaperones described above and elsewhere herein.

[0127] In some examples, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone described supra. In other examples, the lipid binding protein is a protein disclosed in Tables 3-6 (which are incorporated in files 48054-701-101Table3.txt, 48054-701-101 Table4.txt, 48054-701-101 Table5.txt, and 48054-701-101Table6.txt). Sometimes, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone described in Tables 3-6.

[0128] In some examples, one or more lipid probes interact or bind to one or more of the lipid binding proteins disclosed in Tables 3-6. In some instances, one or more lipid probes interact or bind to one or more of the enzyme, transporter, receptor, adaptor, channel protein, or chaperone described in Tables 3-6.Samples, Analytical Techniques, and Instrumentation

[0129] In certain examples, one or more of the methods disclosed herein comprise a sample. In some examples, the sample is a cell sample or a tissue sample. In some instances, the sample is a cell sample. In some examples, the sample for use with the methods described herein is obtained from cells of an animal. In some instances, the animal cell includes a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal. In some instances, the mammalian cell is a primate, ape, equine, bovine, porcine, canine, feline, or rodent. In some instances, the mammal is a primate, ape, dog, cat, rabbit, ferret, or the like. In some cases, the rodent is a mouse, rat, hamster, gerbil, hamster, chinchilla, or guinea pig. In some examples, the bird cell is from a canary, parakeet or parrots. In some examples, the reptile cell is from a turtles, lizard or snake. In some cases, the fish cell is from a tropical fish. In some cases, the fish cell is from a zebrafish (e.g. Danio rerio). In some cases, the worm cell is from a nematode (e.g. C. elegans). In some cases, the amphibian cell is from a frog. In some examples, the arthropod cell is from a tarantula or hermit crab.

[0130] In some examples, the sample for use with the methods described herein is obtained from a mammalian cell. In some instances, the mammalian cell is an epithelial cell, connective tissue cell, hormone secreting cell, a nerve cell, a skeletal muscle cell, a blood cell, or an immune system cell.

[0131] Exemplary mammalian cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells , 293 H cells, HEK 293 cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F ™< cells, Flp-In ™< T-REx ™< 293 cell line, Flp-In ™< -293 cell line, Flp-In ™< -3T3 cell line, Flp-In ™< -BHK cell line, Flp-In ™< -CHO cell line, Flp-In ™< -CV-1 cell line, Flp-In ™< -Jurkat cell line, FreeStyle ™< 293-F cells, FreeStyle ™< CHO-S cells, GripTite ™< 293 MSR cell line, GS-CHO cell line, HepaRG ™< cells, T-REx ™< Jurkat cell line, Per.C6 cells, T-REx ™< -293 cell line, T-REx ™< -CHO cell line, T-REx ™< -HeLa cell line, NC-HIMT cell line, and PC12 cell line.

[0132] In some instances, the sample for use with the methods described herein is obtained from cells of a tumor cell line. In some instances, the sample is obtained from cells of a solid tumor cell line. In some instances, the solid tumor cell line is a sarcoma cell line. In some instances, the solid tumor cell line is a carcinoma cell line. In some examples, the sarcoma cell line is obtained from a cell line of alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma of soft tissue, dedifferentiated liposarcoma, desmoid, desmoplastic small round cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoid tumor, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, infantile fibrosarcoma, inflammatory myofibroblastic tumor, Kaposi sarcoma, leiomyosarcoma of bone, liposarcoma, liposarcoma of bone, malignant fibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH) of bone, malignant mesenchymoma, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, myxofibrosarcoma, myxoid liposarcoma, myxoinflammatory fibroblastic sarcoma, neoplasms with perivascular epitheioid cell differentiation, osteosarcoma, parosteal osteosarcoma, neoplasm with perivascular epitheioid cell differentiation, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, PNET / extraskeletal Ewing tumor, rhabdomyosarcoma, round cell liposarcoma, small cell osteosarcoma, solitary fibrous tumor, synovial sarcoma, or telangiectatic osteosarcoma.

[0133] In some examples, the carcinoma cell line is obtained from a cell line of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0134] In some instances, the sample is obtained from cells of a hematologic malignant cell line. In some instances, the hematologic malignant cell line is a T-cell cell line. In some instances, B-cell cell line. In some instances, the hematologic malignant cell line is obtained from a T-cell cell line of: peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia / lymphoma (ATLL), blastic NK-cell lymphoma, enteropathy-type T-cell lymphoma, hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, nasal NK / T-cell lymphomas, or treatment-related T-cell lymphomas.

[0135] In some instances, the hematologic malignant cell line is obtained from a B-cell cell line of: acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), high-risk chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk small lymphocytic lymphoma (SLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[0136] In some examples, the sample for use with the methods described herein is obtained from a tumor cell line. Exemplary tumor cell line includes, but is not limited to, 600MPE, AU565, BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF-7, MDA-MB-231, SkBr3, T-47D, HeLa, DU145, PC3, LNCaP, A549, H1299, NCI-H460, A2780, SKOV-3 / Luc, Neuro2a, RKO, RKO-AS45-1, HT-29, SW1417, SW948, DLD-1, SW480, Capan-1, MC / 9, B72.3, B25.2, B6.2, B38.1, DMS 153, SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs 817.T, LMH, LMH / 2A, SNU-398, PLHC-1, HepG2 / SF, OCI-Ly1, OCI-Ly2, OCI-Ly3, OCI-Ly4, OCI-Ly6, OCI-Ly7, OCI-Ly10, OCI-Ly18, OCI-Ly19, U2932, DB, HBL-1, RIVA, SUDHL2, TMD8, MEC1, MEC2, 8E5, CCRF-CEM, MOLT-3, TALL-104, AML-193, THP-1, BDCM, HL-60, Jurkat, RPMI 8226, MOLT-4, RS4, K-562, KASUMI-1, Daudi, GA-10, Raji, JeKo-1, NK-92, and Mino.

[0137] In some examples, the sample for use in the methods is from any tissue or fluid from an individual. Samples include, but are not limited to, tissue (e.g. connective tissue, muscle tissue, nervous tissue, or epithelial tissue), whole blood, dissociated bone marrow, bone marrow aspirate, pleural fluid, peritoneal fluid, central spinal fluid, abdominal fluid, pancreatic fluid, cerebrospinal fluid, brain fluid, ascites, pericardial fluid, urine, saliva, bronchial lavage, sweat, tears, ear flow, sputum, hydrocele fluid, semen, vaginal flow, milk, amniotic fluid, and secretions of respiratory, intestinal or genitourinary tract. In some examples, the sample is a tissue sample, such as a sample obtained from a biopsy or a tumor tissue sample. In some examples, the sample is a blood serum sample. In some examples, the sample is a blood cell sample containing one or more peripheral blood mononuclear cells (PBMCs). In some examples, the sample contains one or more circulating tumor cells (CTCs). In some examples, the sample contains one or more disseminated tumor cells (DTC, e.g., in a bone marrow aspirate sample).

[0138] In some examples, the samples are obtained from the individual by any suitable means of obtaining the sample using well-known and routine clinical methods. Procedures for obtaining tissue samples from an individual are well known. For example, procedures for drawing and processing tissue sample such as from a needle aspiration biopsy is well-known and is employed to obtain a sample for use in the methods provided. Typically, for collection of such a tissue sample, a thin hollow needle is inserted into a mass such as a tumor mass for sampling of cells that, after being stained, will be examined under a microscope.Sample Preparation and Analysis

[0139] In some examples, the sample is a sample solution. In some instances, the sample solution comprises a solution such as a buffer (e.g. phosphate buffered saline) or a media. In some instances, the sample solution is a cell solution.

[0140] In some examples, the sample (e.g. cells or a cell solution) is incubated with a lipid probe for analysis of protein lipid probe interactions. In some instances, the sample is further compared with a control. In some instances, a difference is observed in a set of lipid probe protein interactions between the sample and the control. In some instances, the difference correlates to a set of lipid binding proteins that interacts with a lipid probe.

[0141] In some instances, the sample (e.g. cells or a cell solution) is incubated with a drug for analysis of protein lipid probe interactions and lipid binding protein-drug interactions. In some cases, the sample is further compared with a control. In some instances, a difference is observed in a set of lipid probe protein interactions between the sample and the control. In some instances, the difference correlates to a set of lipid binding proteins that interacts with a drug. In some instances, one lipid binding protein is observed to interact with a drug. In some instances, multiple lipid binding proteins are observed to interact with a drug. In some aspects, a drug is a specific inhibitor of a lipid binding protein. In some instances, a drug is a pan-inhibitor of a lipid binding protein. In some instances, the drug is a competitor of a lipid probe for interaction with a lipid binding protein.

[0142] In some examples, the sample (e.g. cells or a cell solution) is further labeled for analysis of protein lipid probe interactions and lipid binding protein-drug interactions. In some instances, the sample (e.g. cells or a cell solution) is labeled with an enriched media. In some cases, the sample (e.g. cells or a cell solution) is labeled with isotope-labeled amino acids, such as 13< C or 15< N-labeled amino acids. In some cases, the labeled sample is further compared with a non-labeled sample to detect differences in protein lipid probe interactions between the two samples. In some instances, this difference is a difference of a protein and its interaction with a lipid probe in the labeled sample versus the non-labeled sample. In some instances, the difference is an increase, decrease or a lack of protein lipid probe interaction in the two samples. In some instances, the isotope-labeled method is termed SILAC, stable isotope labeling using amino acids in cell culture.

[0143] In some instances, the sample is divided into a first cell solution and a second cell solution. In some instances, the first cell solution is contacted with a first lipid probe and a second cell solution is contacted with a second lipid probe. In some examples, the first lipid probe and the second lipid probe are the same. In some instances, the second cell solution comprises an enriched media and a drug. In some examples, enriched cell culture media is a stable isotope labeled media. In some instances, the enriched cell culture media contains 13< C labeled amino acids, 15< N labeled amino acids, or a combination thereof. In some examples, the first cell solution comprises unlabeled media, such as a fetal bovine serum based or serum-free media.

[0144] In some instances, upon incubation with a lipid probe and / or a drug, the first cell solution and / or second cell solution are then treated by visible light or ultraviolet light to crosslink the lipid probe with its binding partner from the cell solutions. In some instances, both cell solutions are treated by a photoreactive means (e.g. ultraviolet light). In some instances, only one of the cell solutions is treated by a photoreactive means.

[0145] In some instances, when a drug is added to the second cell solution for determining a lipid binding protein as a drug binding target, both cell solutions are treated by a photoreactive means (e.g. ultraviolet light). In some cases, a drug competes with a lipid probe for interaction with a lipid binding protein. In some instances, lipid probe and protein interaction from both the first cell solution and second cell solution are analyzed and the difference (e.g. the lack or decreased lipid probe and protein interaction due to a drug competition effect in the second cell solution) in lipid probe and protein interaction between the two cell solutions allow for identification of the lipid binding proteins as drug targets. In some instances, a control (e.g. dimethyl sulfoxide (DMSO)) is added to the first cell solution or the second cell solution.

[0146] In some examples, only the first cell solution is treated by a photoreactive means (e.g. ultraviolet light). In some instances, both cell solutions are then further processed to identify proteins as lipid binding proteins. In some instances, the difference between the two cell solutions, e.g. the lack or decreased lipid probe and protein interaction due to an absence of photolysis in the second cell population, allows for identification of proteins as lipid binding proteins.

[0147] In some instances, after treatment by a photoreactive means, the cell solutions are then treated by conventional means to harvest the lipid probe-protein complexes. In some instances, the lipid probe-protein complexes are harvested involving centrifugation; lysis such as by vortex, homogenization, or freeze-thaw step; filtration step; and enrichment steps such as concentration, chemical extraction, or affinity based extraction. In some instances, the cell lysate generated from the cell solutions is further separated into a solution fraction and a membrane fraction and lipid probe-protein complexes are harvested from the two fractions.

[0148] In some examples, the lipid probe-protein complex is further conjugated to a chromophore, such as a fluorophore. In some instances, the lipid probe-protein complex is separated and visualized utilizing an electrophoresis system, such as through a gel electrophoresis, or a capillary electrophoresis. Exemplary gel electrophoresis includes agarose based gels, polyacrylamide based gels, or starch based gels. In some instances, the lipid probe-protein is subjected to a native electrophoresis condition. In some instances, the lipid probe-protein is subjected to a denaturing electrophoresis condition.

[0149] In some instances, the lipid probe-protein is further fragmentized to generate protein fragments. In some instances, fragmentation is generated through mechanical stress, pressure, or chemical means. In some instances, the protein from the lipid probe-protein complexes is fragmented by a chemical means. In some examples, the chemical means is a protease. Exemplary proteases include, but are not limited to, serine proteases such as chymotrypsin A, penicillin G acylase precursor, dipeptidase E, DmpA aminopeptidase, subtilisin, prolyl oligopeptidase, D-Ala-D-Ala peptidase C, signal peptidase I, cytomegalovirus assemblin, Lon-A peptidase, peptidase Clp, Escherichia coli phage K1F endosialidase, CIMCD self-cleaving protein, nucleoporin 145, lactoferrin, murein tetrapeptidase LD-carboxypeptidase, or rhomboid-1; threonine proteases such as ornithine acetyltransferase; cysteine proteases such as TEV protease, amidophosphoribosyltransferase precursor, gamma-glutamyl hydrolase (Rattus norvegicus), hedgehog protein, DmpA aminopeptidase, papain, bromelain, cathepsin K, calpain, caspase-1, separase, adenain, pyroglutamyl-peptidase I, sortase A, hepatitis C virus peptidase 2, sindbis virus-type nsP2 peptidase, dipeptidyl-peptidase VI, or DeSI-1 peptidase; aspartate proteases such as beta-secretase 1 (BACE1), beta-secretase 2 (BACE2), cathepsin D, cathepsin E, chymosin, napsin-A, nepenthesin, pepsin, plasmepsin, presenilin, or renin; glutamic acid proteases such as AfuGprA; and metalloproteases such as peptidase_M48.

[0150] In some instances, the fragmentation is a random fragmentation. In some instances, the fragmentation generates specific lengths of protein fragments, or the shearing occurs at particular sequence of amino acid regions.

[0151] In some instances, the protein fragments are further analyzed by a proteomic method such as by liquid chromatography (LC) (e.g. high performance liquid chromatography), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or nuclear magnetic resonance imaging (NMR).

[0152] In some examples, the LC method is any suitable LC methods well known in the art, for separation of a sample into its individual parts. This separation occurs based on the interaction of the sample with the mobile and stationary phases. Since there are many stationary / mobile phase combinations that are employed when separating a mixture, there are several different types of chromatography that are classified based on the physical states of those phases. In some examples, the LC is further classified as normal-phase chromatography, reverse-phase chromatography, size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, flash chromatography, chiral chromatography, and aqueous normal-phase chromatography.

[0153] In some examples, the LC method is a high performance liquid chromatography (HPLC) method. In some examples, the HPLC method is further categorized as normal-phase chromatography, reverse-phase chromatography, size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, chiral chromatography, and aqueous normal-phase chromatography.

[0154] In some examples, the HPLC method of the present disclosure is performed by any standard techniques well known in the art. Exemplary HPLC methods include hydrophilic interaction liquid chromatography (HILIC), electrostatic repulsion-hydrophilic interaction liquid chromatography (ERLIC) and reverse phase liquid chromatography (RPLC).

[0155] In some examples, the LC is coupled to a mass spectroscopy as a LC-MS method. In some examples, the LC-MS method includes ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS), ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS / MS), reverse phase liquid chromatography-mass spectrometry (RPLC-MS), hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS), hydrophilic interaction liquid chromatography-triple quadrupole tandem mass spectrometry (HILIC-QQQ), electrostatic repulsion-hydrophilic interaction liquid chromatography-mass spectrometry (ERLIC-MS), liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), multidimensional liquid chromatography coupled with tandem mass spectrometry (LC / LC-MS / MS). In some instances, the LC-MS method is LC / LC-MS / MS. In some examples, the LC-MS methods of the present disclosure are performed by standard techniques well known in the art.

[0156] In some examples, the GC is coupled to a mass spectroscopy as a GC-MS method. In some examples, the GC-MS method includes two-dimensional gas chromatography time-of-flight mass spectrometry (GC-GC-TOFMS), gas chromatography time-of-flight mass spectrometry (GC-QTOF-MS) and gas chromatography-tandem mass spectrometry (GC-MS / MS).

[0157] In some examples, CE is coupled to a mass spectroscopy as a CE-MS method. In some examples, the CE-MS method includes capillary electrophoresis- negative electrospray ionization-mass spectrometry (CE-ESI-MS), capillary electrophoresis-negative electrospray ionization-quadrupole time of flight-mass spectrometry (CE-ESI-QTOF-MS) and capillary electrophoresis-quadrupole time of flight-mass spectrometry (CE-QTOF-MS).

[0158] In some examples, the nuclear magnetic resonance (NMR) method is any suitable method well known in the art for the detection of one or more lipid binding proteins or protein fragments disclosed herein. In some examples, the NMR method includes one dimensional (1D) NMR methods, two dimensional (2D) NMR methods, solid state NMR methods and NMR chromatography. Exemplary 1D NMR methods include 1< Hydrogen, 13< Carbon, 15< Nitrogen, 17< Oxygen, 19< Fluorine, 31< Phosphorus, 39< Potassium, 23< Sodium, 33< Sulfur, 87< Strontium, 27< Aluminium, 43< Calcium, 35< Chlorine, 37< Chlorine, 63< Copper, 65< Copper, 57< Iron, 25< Magnesium, 199< Mercury or 67< Zinc NMR method, distortionless enhancement by polarization transfer (DEPT) method, attached proton test (APT) method and 1D-incredible natural abundance double quantum transition experiment (INADEQUATE) method. Exemplary 2D NMR methods include correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), 2D-INADEQUATE, 2D-adequate double quantum transfer experiment (ADEQUATE), nuclear overhauser effect spectroscopy (NOSEY), rotating-frame NOE spectroscopy (ROESY), heteronuclear multiple-quantum correlation spectroscopy (HMQC), heteronuclear single quantum coherence spectroscopy (HSQC), short range coupling and long range coupling methods. Exemplary solid state NMR method include solid state 13< Carbon NMR, high resolution magic angle spinning (HR-MAS) and cross polarization magic angle spinning (CP-MAS) NMR methods. Exemplary NMR techniques include diffusion ordered spectroscopy (DOSY), DOSY-TOCSY and DOSY-HSQC.

[0159] In some examples, the protein fragments are analyzed by method as described in Weerapana et al., "Quantitative reactivity profiling predicts functional cysteines in proteomes," Nature, 468:790-795 (2010).

[0160] In some examples, a value is assigned to each of the protein from the lipid probe-protein complex. In some instances, the value is the area-under-the curve from a plot of signal intensity as a function of mass-to-charge ratio. In some examples, a first value is assigned to the protein obtained from the first cell solution and a second value is assigned to the same protein obtained from the second cell solution. In some instances, a ratio is calculated between the two values. In some instances, a ratio of greater than 2 indicates that the protein is a candidate for interacting with a drug or that the protein is a lipid binding protein. In some instances, the ratio is greater than 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, the ratio is at most 20.

[0161] In some instances, the ratio is calculated based on averaged values. In some instances, the averaged value is an average of at least two, three, or four values of the protein from each cell solution, or that the protein is observed at least two, three, or four times in each cell solution and a value is assigned to each observed time. In some instances, the ratio further has a standard deviation of less than 12, 10, or 8.

[0162] In some instances, a value is not an averaged value. In some instances, the ratio is calculated based on value of a protein observed only once in a cell population. In some instances, the ratio is assigned with a value of 20.

[0163] In some examples, in the context of identifying a lipid binding protein as a drug binding target, a first ratio is obtained from two cell solutions in which both cell solutions have been treated by photoreactive means and the second cell solution is incubated with a drug. In some instances, the first ratio is further compared to a second ratio in which both cell solutions have been treated by photoreactive means in the absence of a drug. In some instances, the first ratio is greater than 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some instances, the second ratio is greater than 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some instances, if the first ratio is greater than 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and the second ratio is from about 0.5 to about 2, the two ratios indicate that a protein is a drug binding target.

[0164] In some examples, in the context of identifying a protein as a lipid binding protein, a first ratio is obtained from two cell solutions in which both cell solutions have been treated by photoreactive means. In some instances, the first ratio is further compared to a second ratio in which one of the cell solutions (e.g. the first cell solution) is treated by a photoreactive means. In some instances, the first ratio is greater than 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some instances, the second ratio is greater than 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some instances, if the first ratio is from about 0.5 to about 2, and the second ratio is greater than 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, the two ratios indicate that a protein is a lipid binding protein.Method of mapping ligand binding site on a lipid binding protein

[0165] In some instances, also disclosed herein is a method of mapping the ligand binding site on a lipid binding protein. In some examples, the method comprises (a) harvesting a set of lipid probe-protein complexes from a sample wherein the lipid probe comprises a lipid, a photoreactive group, and an affinity handle; (b) analyzing the set of lipid probe-protein complexes by a proteomic analysis means; and (c) based on step b), locating a ligand binding site on the lipid binding protein.

[0166] In some examples, the method further comprises treating the set of lipid probe-protein complexes with a protease to generate a set of protein fragments. The protease is a serine protease, a threonine protease, a cysteine protease, an aspartate protease, a glutamic acid protease, or a metalloprotease. In some instances, the protease is a serine protease. In some instances, the protease is trypsin. In some instances, lipid probe-protein complex is further attached to a labeling group such as a biotin moiety. In some instances, the labeling group such as a biotin moiety further comprises a linker. In some instances, the linker is a peptide. In some instances, the peptide linker is about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in length. In some instances, the peptide linker contains a cleavage site. A non-limiting list of cleavage sites includes Tobacco Etch Virus (TEV), thrombin (Thr), enterokinase (EKT), activated Factor X (Xa), or human Rhinovirus 3C protease (3C / PreScission). In some instances, the peptide linker contains a TEV protease cleavage site. In some instances, the TEV protease cleavage site comprises the following sequence Gly-Gln-Phe-Tyr-Leu-Asn-Glu. In some instances, the biotin moiety is further coupled to a bead (e.g. a streptavidin-coupled bead).

[0167] In some instances, the protein from the lipid probe-protein complex attached to the bead (via a biotin moiety comprising a linker and attached to a streptavidin-coupled bead) is digested with trypsin, and the immobilized peptide or protein fragment is further separated and collected. In some instances, the collected peptide or protein fragment is then digested by a protease (e.g. TEV protease), and the treated protein fragment is then separated, and collected for analysis. In some instances, the analysis is a proteomic analysis as described above and elsewhere herein. In some instances, the sequence of the protein fragment is further determined. In some instances, the protein fragment correlates to a drug binding site on the lipid binding protein.

[0168] In some examples, the sequence of the protein fragment correlates to a sequence as illustrated in Table 6. In some instances, the sequence as shown in Table 6 correlates to a site on the full length protein as a drug binding site. In some instances, the sequence as shown in Table 6 correlates to a drug binding site. In some instances, polypeptides comprising one or more of the sequences as shown in Table 6 serve as probes for drug screening.

[0169] In some examples, the lipid probe comprises a lipid, a photoreactive group, and an affinity handle. In some examples, the lipid is a bioactive lipid. In some instances, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some cases, the lipid is a member of the fatty acyls group. In some cases, 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. In some instances, the lipid is a sterol lipid.

[0170] In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some examples, the photoreactive group is a diazirine or its derivatives thereof.

[0171] In some examples, the affinity handle is a bioorthogonal affinity handle. In some instances, 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. In some examples, the affinity handle comprises an alkyne group or an azide group.

[0172] In some examples, the lipid probe comprises: a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls; a photoreactive linker; and an affinity handle.

[0173] In some examples, 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.

[0174] In some examples, the lipid probe has one of the following structures as exemplified in Figure 1 and in Table 1. In some examples, the lipid binding protein is a soluble protein or a membrane protein. In some instances, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some instances, 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.

[0175] In some instances, the sample comprises a cell sample or a tissue sample. In some instances, the sample is a cell sample. In some instances, the cell sample comprises a first cell solution and a second cell solution. In some cases, the second cell solution comprises an enriched media and a drug. In some cases, the enriched media is a stable isotope labeled media. In some instances, the first cell solution and the second cell solution are further treating 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.Compositions, process of producing a lipid probe-protein composition, and lipid probes

[0176] In some examples, disclosed herein include compositions of the lipid probe-protein complex, and compositions that comprises a lipid probe-protein complex and a sample.

[0177] In some examples, disclosed herein is a lipid probe-protein composition which comprises a lipid probe and a lipid binding protein. In some examples, also described herein is a lipid probe-protein composition produced by a process which comprises contacting a sample with a lipid probe, and treating the sample which comprises the lipid probe by a photoreactive means, wherein the treating time is from about 5 minutes to about 1 hour. In some instances, the treating time is from about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 30, 40, 50, or 60 minutes. In some instances, the treating time is about 10 minutes. In some instances, the treating temperature is at about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 37°C, or 40°C. In some instances, the treating temperature is at about 4°C.

[0178] In some instances, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some examples, the lipid binding protein is a protein encoded by a gene of Table 3, a protein encoded by a gene of Table 4, a protein encoded by a gene of Table 5, or a protein encoded by a gene of Table 6. In some instances, the lipid binding protein is nucleobindin-1 (NUCB1). In some cases, the lipid binding protein is a protein fragment. In some cases, the protein fragment is a protein fragment of Table 6.

[0179] In some examples, the lipid is a bioactive lipid. In some instances, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some instances, the lipid is a member of the fatty acyls group. In some instances, 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. In some cases, the lipid is a fatty acid. In some cases, the lipid is a sterol lipid. In some instances, the sterol lipid comprises sterols, steroids, secosteroids, or bile acids. In some instances, the sterol comprises cholesterol, ergosterol, C24-propyl sterols, or stanol.

[0180] In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some examples, the affinity handle is a bioorthogonal affinity handle. In some instances, 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.

[0181] In some instances, the lipid probe comprises: a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls; a photoreactive linker; and an affinity handle.

[0182] In some examples, 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.

[0183] In some instances, the lipid probe has one of the following structures as exemplified in Figure 1 and in Table 1.

[0184] In some examples, disclosed herein is a composition which comprises an isolated sample wherein the isolated sample is an isolated cell or a tissue sample; and a lipid probe to be assayed for its ability to interact with a lipid binding protein expressed in the isolated sample. In some instances, the composition further comprises a drug as a test compound. In some cases, the lipid probe is assayed for its ability to interact with a lipid binding protein expressed in the sample in the presence of the drug.

[0185] In some examples, also described herein is an isolated treated cell which comprises a lipid probe attached to a lipid binding protein. In some examples, the lipid probe is attached to the lipid binding protein through a covalent bond. In some instances, the isolated treated cell further comprises a set of lipid probes wherein each of the lipid probes is attached to a lipid binding protein. In some cases, each lipid probe within the set is different. In other cases, each lipid probe within the set is the same.

[0186] In some examples, further described herein is an isolated treated population of cells which comprises a set of lipid probes attached to lipid binding proteins. In some instances, each of the lipid probes is attached to a lipid binding protein through a covalent bond. In some cases, each lipid probe within the set is different. In other cases, each lipid probe within the set is the same.

[0187] In some examples, disclosed herein is a lipid probe which comprises a lipid, a photoreactive group, and an affinity handle wherein the lipid probe is constructed for detecting a drug-lipid binding protein interaction. In some instances, the lipid is a bioactive lipid. In some cases, the lipid comprises fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, or polyketides. In some instances, the lipid is a member of the fatty acyls group. In some cases, 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. In other instances, the lipid is a sterol lipid. In some examples, the photoreactive group comprises azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some cases, the photoreactive group is diazirine, or its derivatives thereof. In some instances, the affinity handle is a bioorthogonal affinity handle. In some cases, 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.

[0188] In some examples, the lipid probe comprises: a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls; a photoreactive linker; and an affinity handle.

[0189] In some examples, 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.

[0190] In some instances, PG is attached to A. In some cases, AH is attached to A. In some cases, the lipid probe has one of the following structures as exemplified in Fig. 1 and Table 1. In some cases, the probe is a lipid binding protein ligand. In some instances, the probe is a competitive ligand for interaction with a lipid binding protein in the presence of a drug. In some cases, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some cases, 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. In some cases, the lipid binding protein is nucleobindin-1 (NUCB1).Lipid binding protein fragments as ligand interaction sites

[0191] In some examples, described herein includes an isolated and purified polypeptide that is derived from a lipid binding protein and serves as a ligand interaction site. In some instances, the isolated and purified polypeptide comprises at least 90% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Table 6, wherein the isolated and purified polypeptide is at most 50 amino acids in length. In some instances, the isolated and purified polypeptide comprises at least 95% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Table 6, wherein the isolated and purified polypeptide is at most 50 amino acids in length. In some instances, the isolated and purified polypeptide comprises 100% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Table 6, wherein the isolated and purified polypeptide is at most 50 amino acids in length. In some instances, the isolated and purified polypeptide consists of 100% sequence identity to the full length of an amino acid sequence selected from Table 6, wherein the isolated and purified polypeptide is at most 50 amino acids in length.

[0192] In some instances after the generation of a polypeptide, the polypeptide is subjected to one or more rounds of purification steps to remove impurities. In some instances, the purification step is a chromatographic step utilizing separation methods such as affinity-based, size-exclusion based, ion-exchange based, or the like. In some cases, the polypeptide is at most 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities. In some cases, the polypeptide is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities.

[0193] In some examples, described herein includes nucleic acid encoding a polypeptide that is derived from a lipid binding protein and serves as a ligand interaction site. In some examples, described herein includes nucleic acid encoding a polypeptide comprising at least 90% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Table 6. In some cases, the nucleic acid encoding a polypeptide comprising at least 95% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Table 6. In some cases, the nucleic acid encoding a polypeptide comprising 100% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Table 6. In some cases, the nucleic acid encoding a polypeptide consisting 100% sequence identity to the full length of an amino acid sequence selected from Table 6.

[0194] In some instances, the nucleic acid is subjected to one or more rounds of purification steps to remove impurities. In some instances, the purification step is a chromatographic step utilizing separation methods such as affinity-based, size-exclusion based, ion-exchange based, or the like. In some cases, the nucleic acid is at most 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities. In some cases, the nucleic acid is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities.

[0195] As used herein, a polypeptide includes natural amino acids, unnatural amino acids, or a combination thereof. In some instances, an amino acid residue refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. The term amino acid, as used herein, includes, without limitation, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

[0196] The term "α-amino acid" refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the α-carbon.

[0197] The term "β-amino acid" refers to a molecule containing both an amino group and a carboxyl group in a β configuration.

[0198] "Naturally occurring amino acid" refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.

[0199] The following table shows a summary of the properties of natural amino acids: Amino Acid3-Letter Code1-Letter CodeSide-chain PolaritySide-chain charge (pH 7.4)Hydropathy IndexAlanineAlaAnonpolarneutral1.8ArginineArgRpolarpositive-4.5AsparagineAsnNpolarneutral-3.5Aspartic acidAspDpolarnegative-3.5CysteineCysCpolarneutral2.5Glutamic acidGluEpolarnegative-3.5GlutamineGlnQpolarneutral-3.5GlycineGlyGnonpolarneutral-0.4HistidineHisHpolarpositive(10%)-3.2neutral(90%)IsoleucineIleInonpolarneutral4.5LeucineLeuLnonpolarneutral3.8LysineLysKpolarpositive-3.9MethionineMetMnonpolarneutral1.9PhenylalaninePheFnonpolarneutral2.8ProlineProPnonpolarneutral-1.6SerineSerSpolarneutral-0.8ThreonineThrTpolarneutral-0.7TryptophanTrpWnonpolarneutral-0.9TyrosineTyrYpolarneutral-1.3ValineValVnonpolarneutral4.2

[0200] "Hydrophobic amino acids" include small hydrophobic amino acids and large hydrophobic amino acids. "Small hydrophobic amino acid" are glycine, alanine, proline, and analogs thereof. "Large hydrophobic amino acids" are valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof. "Polar amino acids" are serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof. "Charged amino acids" are lysine, arginine, histidine, aspartate, glutamate, and analogs thereof.

[0201] The term "amino acid analog" refers to a molecule which is structurally similar to an amino acid and which is substituted for an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogs include, without limitation, β-amino acids and amino acids where the amino or carboxy group is substituted by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester).

[0202] The term "non-natural amino acid" refers to an amino acid which is not one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.

[0203] In some instances, amino acid analogs include β-amino acid analogs. Examples of β-amino acid analogs include, but are not limited to, the following: cyclic β-amino acid analogs; β-alanine; (R)-β-phenylalanine; (R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (R)-3-amino-4-(1-naphthyl)-butyric acid; (R)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (R)-3-amino-4-(2-chlorophenyl)-butyric acid; (R)-3-amino-4-(2-cyanophenyl)-butyric acid; (R)-3-amino-4-(2-fluorophenyl)-butyric acid; (R)-3-amino-4-(2-furyl)-butyric acid; (R)-3-amino-4-(2-methylphenyl)-butyric acid; (R)-3-amino-4-(2-naphthyl)-butyric acid; (R)-3-amino-4-(2-thienyl)-butyric acid; (R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(3,4-dichlorophenyl)butyric acid; (R)-3-amino-4-(3,4-difluorophenyl)butyric acid; (R)-3-amino-4-(3-benzothienyl)-butyric acid; (R)-3-amino-4-(3-chlorophenyl)-butyric acid; (R)-3-amino-4-(3-cyanophenyl)-butyric acid; (R)-3-amino-4-(3-fluorophenyl)-butyric acid; (R)-3-amino-4-(3-methylphenyl)-butyric acid; (R)-3-amino-4-(3-pyridyl)-butyric acid; (R)-3-amino-4-(3-thienyl)-butyric acid; (R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(4-bromophenyl)-butyric acid; (R)-3-amino-4-(4-chlorophenyl)-butyric acid; (R)-3-amino-4-(4-cyanophenyl)-butyric acid; (R)-3-amino-4-(4-fluorophenyl)-butyric acid; (R)-3-amino-4-(4-iodophenyl)-butyric acid; (R)-3-amino-4-(4-methylphenyl)-butyric acid; (R)-3-amino-4-(4-nitrophenyl)-butyric acid; (R)-3-amino-4-(4-pyridyl)-butyric acid; (R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-pentafluoro-phenylbutyric acid; (R)-3-amino-5-hexenoic acid; (R)-3-amino-5-hexynoic acid; (R)-3-amino-5-phenylpentanoic acid; (R)-3-amino-6-phenyl-5-hexenoic acid; (S)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (S)-3-amino-4-(1-naphthyl)-butyric acid; (S)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (S)-3-amino-4-(2-chlorophenyl)-butyric acid; (S)-3-amino-4-(2-cyanophenyl)-butyric acid; (S)-3-amino-4-(2-fluorophenyl)-butyric acid; (S)-3-amino-4-(2-furyl)-butyric acid; (S)-3-amino-4-(2-methylphenyl)-butyric acid; (S)-3-amino-4-(2-naphthyl)-butyric acid; (S)-3-amino-4-(2-thienyl)-butyric acid; (S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(3,4-dichlorophenyl)butyric acid; (S)-3-amino-4-(3,4-difluorophenyl)butyric acid; (S)-3-amino-4-(3-benzothienyl)-butyric acid; (S)-3-amino-4-(3-chlorophenyl)-butyric acid; (S)-3-amino-4-(3-cyanophenyl)-butyric acid; (S)-3-amino-4-(3-fluorophenyl)-butyric acid; (S)-3-amino-4-(3-methylphenyl)-butyric acid; (S)-3-amino-4-(3-pyridyl)-butyric acid; (S)-3-amino-4-(3-thienyl)-butyric acid; (S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(4-bromophenyl)-butyric acid; (S)-3-amino-4-(4-chlorophenyl) butyric acid; (S)-3-amino-4-(4-cyanophenyl)-butyric acid; (S)-3-amino-4-(4-fluorophenyl) butyric acid; (S)-3-amino-4-(4-iodophenyl)-butyric acid; (S)-3-amino-4-(4-methylphenyl)-butyric acid; (S)-3-amino-4-(4-nitrophenyl)-butyric acid; (S)-3-amino-4-(4-pyridyl)-butyric acid; (S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-pentafluoro-phenylbutyric acid; (S)-3-amino-5-hexenoic acid; (S)-3-amino-5-hexynoic acid; (S)-3-amino-5-phenylpentanoic acid; (S)-3-amino-6-phenyl-5-hexenoic acid; 1,2,5,6-tetrahydropyridine-3-carboxylic acid; 1,2,5,6-tetrahydropyridine-4-carboxylic acid; 3-amino-3-(2-chlorophenyl)-propionic acid; 3-amino-3-(2-thienyl)-propionic acid; 3-amino-3-(3-bromophenyl)-propionic acid; 3-amino-3-(4-chlorophenyl)-propionic acid; 3-amino-3-(4-methoxyphenyl)-propionic acid; 3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid; D-β-phenylalanine; β-leucine; L-β-homoalanine; L-β-homoaspartic acid γ-benzyl ester; L-β-homoglutamic acid δ-benzyl ester; L-β-homoisoleucine; L-β-homoleucine; L-β-homomethionine; L-β-homophenylalanine; L-β-homoproline; L-β-homotryptophan; L-β-homovaline; L-Nω-benzyloxycarbonyl-β-homolysine; Nω-L-β-homoarginine; O-benzyl-L-β-homohydroxyproline; O-benzyl-L-β-homoserine; O-benzyl-L-β-homothreonine; O-benzyl-L-β-homotyrosine; γ-trityl-L-β-homoasparagine; (R)-β-phenylalanine; L-β-homoaspartic acid γ-t-butyl ester; L-β-homoglutamic acid δ-t-butyl ester; L-Nω-β-homolysine; Nδ-trityl-L-β-homoglutamine; Nω-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-β-homoarginine; O-t-butyl-L-β-homohydroxy-proline; O-t-butyl-L-β-homoserine; O-t-butyl-L-β-homothreonine; O-t-butyl-L-β-homotyrosine; 2-aminocyclopentane carboxylic acid; and 2-aminocyclohexane carboxylic acid.

[0204] In some instances, amino acid analogs include analogs of alanine, valine, glycine or leucine. Examples of amino acid analogs of alanine, valine, glycine, and leucine include, but are not limited to, the following: α-methoxyglycine; α-allyl-L-alanine; α-aminoisobutyric acid; α-methyl-leucine; β-(1-naphthyl)-D-alanine; β-(1-naphthyl)-L-alanine; β-(2-naphthyl)-D-alanine; β-(2-naphthyl)-L-alanine; β-(2-pyridyl)-D-alanine; β-(2-pyridyl)-L-alanine; β-(2-thienyl)-D-alanine; β-(2-thienyl)-L-alanine; β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine; β-(3-pyridyl)-D-alanine; β-(3-pyridyl)-L-alanine; β-(4-pyridyl)-D-alanine; β-(4-pyridyl)-L-alanine; β-chloro-L-alanine; β-cyano-L-alanin; β-cyclohexyl-D-alanine; β-cyclohexyl-L-alanine; β-cyclopenten-1-yl-alanine; β-cyclopentyl-alanine; β-cyclopropyl-L-Ala-OH.dicyclohexylammonium salt; β-t-butyl-D-alanine; β-t-butyl-L-alanine; γ-aminobutyric acid; L-α,β-diaminopropionic acid; 2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine; 2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine; 3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine; 4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH.dicyclohexylammonium salt; 4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine; 4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoic acid; cyclopentyl-D-Gly-OH.dicyclohexylammonium salt; cyclopentyl-Gly-OH.dicyclohexylammonium salt; D-α,β-diaminopropionic acid; D-α-aminobutyric acid; D-α-t-butylglycine; D-(2-thienyl)glycine; D-(3-thienyl)glycine; D-2-aminocaproic acid; D-2-indanylglycine; D-allylglycine-dicyclohexylammonium salt; D-cyclohexylglycine; D-norvaline; D-phenylglycine; β-aminobutyric acid; β-aminoisobutyric acid; (2-bromophenyl)glycine; (2-methoxyphenyl)glycine; (2-methylphenyl)glycine; (2-thiazoyl)glycine; (2-thienyl)glycine; 2-amino-3-(dimethylamino)-propionic acid; L-α,β-diaminopropionic acid; L-α-aminobutyric acid; L-α-t-butylglycine; L-(3-thienyl)glycine; L-2-amino-3-(dimethylamino)-propionic acid; L-2-aminocaproic acid dicyclohexyl-ammonium salt; L-2-indanylglycine; L-allylglycine.dicyclohexyl ammonium salt; L-cyclohexylglycine; L-phenylglycine; L-propargylglycine; L-norvaline; N-α-aminomethyl-L-alanine; D-α,γ-diaminobutyric acid; L-α,γ-diaminobutyric acid; β-cyclopropyl-L-alanine; (N-β-(2,4-dinitrophenyl))-L-α,β-diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,β-diaminopropionic acid; (N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,β-diaminopropionic acid; (N-β-4-methyltrityl)-L-α,β-diaminopropionic acid; (N-β-allyloxycarbonyl)-L-α,β-diaminopropionic acid; (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,γ-diaminobutyric acid; (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyric acid; (N-γ-4-methyltrityl)-D-α,γ-diaminobutyric acid; (N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid; (N-γ-allyloxycarbonyl)-L-α,γ-diaminobutyric acid; D-α,γ-diaminobutyric acid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine; D-homocyclohexylalanine; L-1-pyrenylalanine; L-2-aminocaproic acid; L-allylglycine; L-homocyclohexylalanine; and N-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.

[0205] In some instances, amino acid analogs include analogs of arginine or lysine. Examples of amino acid analogs of arginine and lysine include, but are not limited to, the following: citrulline; L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me) 2 -OH; Lys(N 3 )-OH; Nδ-benzyloxycarbonyl-L-ornithine; Nω-nitro-D-arginine; Nω-nitro-L-arginine; α-methyl-ornithine; 2,6-diaminoheptanedioic acid; L-ornithine; (Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-ornithine; (N6-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-ornithine; (Nδ-4-methyltrityl)-D-ornithine; (Nδ-4-methyltrityl)-L-ornithine; D-ornithine; L-ornithine; Arg(Me)(Pbf)-OH; Arg(Me) 2 -OH (asymmetrical); Arg(Me)2-OH (symmetrical); Lys(ivDde)-OH; Lys(Me)2-OH.HCl; Lys(Me3)-OH chloride; Nω-nitro-D-arginine; and Nω-nitro-L-arginine.

[0206] In some instances, amino acid analogs include analogs of aspartic or glutamic acids. Examples of amino acid analogs of aspartic and glutamic acids include, but are not limited to, the following: α-methyl-D-aspartic acid; α-methyl-glutamic acid; α-methyl-L-aspartic acid; γ-methylene-glutamic acid; (N-γ-ethyl)-L-glutamine; [N-α-(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelic acid; L-α-aminosuberic acid; D-2-aminoadipic acid; D-α-aminosuberic acid; α-aminopimelic acid; iminodiacetic acid; L-2-aminoadipic acid; threo-β-methyl-aspartic acid; γ-carboxy-D-glutamic acid γ,γ-di-t-butyl ester; γ-carboxy-L-glutamic acid γ,γ-di-t-butyl ester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and pyroglutamic acid.

[0207] In some instances, amino acid analogs include analogs of cysteine and methionine. Examples of amino acid analogs of cysteine and methionine include, but are not limited to, Cys(farnesyl)-OH, Cys(farnesyl)-OMe, α-methyl-methionine, Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH, 2-amino-4-(ethylthio)butyric acid, buthionine, buthioninesulfoximine, ethionine, methionine methylsulfonium chloride, selenomethionine, cysteic acid, [2-(4-pyridyl)ethyl]-DL-penicillamine, [2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine, 4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine, 4-methylbenzyl-L-penicillamine, benzyl-D-cysteine, benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine, carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine, t-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine, cystathionine, homocystine, L-homocystine, (2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine, Cys(StBu)-OH, and acetamidomethyl-D-penicillamine.

[0208] In some instances, amino acid analogs include analogs of phenylalanine and tyrosine. Examples of amino acid analogs of phenylalanine and tyrosine include β-methyl-phenylalanine, β-hydroxyphenylalanine, α-methyl-3-methoxy-DL-phenylalanine, α-methyl-D-phenylalanine, α-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine, 2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine, 2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine, 2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine, 2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine, 2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine, 2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine, 2-nitro-L-phenylalanine, 2;4;5-trihydroxy-phenylalanine, 3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine, 3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine, 3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine, 3,5,3'-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine, 3-(trifluoromethyl)-D-phenylalanine, 3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine, 3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine, 3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine, 3-chloro-L-tyrosine, 3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine, 3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine, 3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine, 3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine, 3-nitro-L-phenylalanine, 3-nitro-L-tyrosine, 4-(trifluoromethyl)-D-phenylalanine, 4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine, 4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine, 4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine, 4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine, 4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine, 4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine, thyroxine, 3,3-diphenylalanine, thyronine, ethyl-tyrosine, and methyl-tyrosine.

[0209] In some instances, amino acid analogs include analogs of proline. Examples of amino acid analogs of proline include, but are not limited to, 3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline.

[0210] In some instances, amino acid analogs include analogs of serine and threonine. Examples of amino acid analogs of serine and threonine include, but are not limited to, 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic acid, 2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid, and α-methylserine.

[0211] In some instances, amino acid analogs include analogs of tryptophan. Examples of amino acid analogs of tryptophan include, but are not limited to, the following: α-methyl-tryptophan; β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan; 5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan; 7-methyl-tryptophan; D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid; 7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.

[0212] In some instances, amino acid analogs are racemic. In some instances, the D isomer of the amino acid analog is used. In some cases, the L isomer of the amino acid analog is used. In some instances, the amino acid analog comprises chiral centers that are in the R or S configuration. Sometimes, the amino group(s) of a β-amino acid analog is substituted with a protecting group, e.g., tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like. Sometimes, the carboxylic acid functional group of a β-amino acid analog is protected, e.g., as its ester derivative. In some cases, the salt of the amino acid analog is used.

[0213] In some examples, nucleic acid molecules refer to at least two nucleotides covalently linked together. In some instances, a nucleic acid described herein contains phosphodiester bonds, although in some cases, as outlined below (for example in the construction of primers and probes such as label probes), nucleic acid analogs are included that have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid (also referred to herein as "PNA") backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996)). Other analog nucleic acids include those with bicyclic structures including locked nucleic acids (also referred to herein as "LNA"), Koshkin et al., J. Am. Chem. Soc. 120.13252 3 (1998); positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169 176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. "Locked nucleic acids" are also included within the definition of nucleic acid analogs. LNAs are a class of nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom with the 4'-C atom. In some instances, these modifications of the ribose-phosphate backbone are done to increase the stability and half-life of such molecules in physiological environments. For example, PNA:DNA and LNA-DNA hybrids exhibit higher stability and thus are used in some examples. The target nucleic acids are single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. Depending on the application, the nucleic acids are DNA (including, e.g., genomic DNA, mitochondrial DNA, and cDNA), RNA (including, e.g., mRNA and rRNA) or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.High throughput method of screening utilizing recombinant proteins

[0214] In some instances, disclosed herein are methods of screening a drug utilizing recombinant lipid binding proteins. In some examples, the method comprises (a) contacting a solution comprising a purified recombinant lipid binding protein and a drug with a lipid probe, wherein the lipid probe comprises a lipid and a fluorophore; and (b) detecting a change in fluorescence polarization relative to a control, wherein the change in fluorescence polarization indicates an interaction between the drug and the lipid binding protein. In some instances, the method is a high throughput method.

[0215] In some instances, the change in fluorescence polarization is an increase in fluorescence polarization relative to a control. In some instances, the change in fluorescence polarization is a decrease in fluorescence polarization relative to a control. In some instances, the decrease in fluorescence polarization correlates to an interaction between the drug and the lipid binding protein. As used in this context, the control is the fluorescence polarization of a lipid probe in the presence of a recombinant protein in the absence of a drug.

[0216] In some instances, the lipid probe comprises a bioactive lipid. In some instances, the lipid probe comprises a fatty acyl, glycerolipid, glycerophospholipid, sphingolipid, sterol lipid, prenol lipid, saccharolipid, or polyketide. In some instances, the lipid probe comprises a lipid from a member of the fatty acyls group. In some instances, 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. In some instances, the lipid is a fatty acid. In some instances, the lipid probe comprises a sterol lipid. In some instances, the sterol lipid comprises sterols, steroids, secosteroids, or bile acids. In some cases, the sterol comprises cholesterol, ergosterol, C24-propyl sterols, or stanol.

[0217] In some instances, the lipid probe further comprises a photoreactive group, such as azides, benzophenone, diazo compounds, diazirines, diazonium salts, or diaryl ketones. In some instances, the photoreactive group further comprises a linker.

[0218] In some instances, the lipid probe comprises a fluorophore such as rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, thiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyren derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavin, acridine orange, acridine yellow, arylmethine derivatives, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphin, phtalocyanine, bilirubin 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 5(6)-FAM, 5-FAM, Fluorescein dT, 5-TAMRA-cadavarine, 2-aminoacridone, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA, TAMRA ™< (NHS Ester), TEX 615, ATTO ™< 488, ATTO ™< 532, ATTO ™< 550, ATTO ™< 565, ATTO ™< Rho101, ATTO ™< 590, ATTO ™< 633, ATTO ™< 647N, TYE ™< 563, TYE ™< 665, or TYE ™< 705.

[0219] In some instances, the fluorophore is conjugated to the hydrophilic portion of the lipid probe. In some instances, the photoreactive group and the fluorophore are conjugated to different sites of the lipid probe.

[0220] In some instances, the lipid probe comprises a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls and a fluorophore. In some instances, the lipid probe comprises an arachidonoyl fatty acyl and a fluorophore. In some instances, the lipid probe comprises an arachidoyl fatty acyl and a fluorophore. In some instances, the lipid probe comprises an oleoyl fatty acyl and a fluorophore. In some instances, the lipid probe comprises a palmitoyl fatty acyl and a fluorophore. In some instances, the lipid probe comprises a stearoyl fatty acyl and a fluorophore.

[0221] In some instances, the lipid probe comprises a lipid probe such as those described in Table 1 in which the lipid probe is conjugated to a fluorophore.

[0222] In some instances, the lipid probe comprises arachidonic acid and a fluorophore. In some instances, the lipid probe is 2-(6-(Dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((5-((52,8Z2,11Z,14Z)-1cosa-5,8,11,14-tetraenamido)pentyl)carbamoyl)benzoate.

[0223] In some examples, a recombinant protein is constructed, expressed, and purified using methods well known in the art. In some instances, the vector is any suitable vectors derived from either a eukaryotic or prokaryotic sources. In some instances, vectors include bacteria (e.g. E. coli), insects, yeast (e.g. Pichia pastoris), or mammalian source.

[0224] In some instances, bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2. In some instances the vector is pET21 from E.coli. Insect vector may include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2.

[0225] In some instances, yeast vectors include Gateway ®< pDEST ™< 14 vector, Gateway ®< pDEST ™< 15 vector, Gateway ®< pDEST ™< 17 vector, Gateway ®< pDEST ™< 24 vector, Gateway ®< pYES-DEST52 vector, pBAD-DEST49 Gateway ®< destination vector, pAO815 Pichia vector, pFLD1 Pichi pastoris vector, pGAPZA,B, & C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B, & C yeast vector, or pYES3 / CT yeast vector.

[0226] In some instances, mammalian vectors include transient expression vectors or stable expression vectors. In some cases, mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. In some instances, mammalian stable expression vector include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0227] In some instances, any suitable protein expression and purification methodologies are applicable for expression and purification of a recombinant protein described herein. In some instances, protein purification includes a purification tag, such as a HIS(6)-Tag, HA-tag, Myc-tag, V5-tag, FLAG-tag, maltose binding protein (MBP) tag, and the like. In some instances, the purification utilizes affinity chromatographic methods, extraction methods, precipitation methods, and the like.

[0228] In some instances, the lipid binding protein is an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some cases, 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. In some instances, the lipid binding protein is nucleobindin-1 (NUCB1). NUCB1 is a small calcium-binding EF-hand protein and participate in Golgi calcium homeostatis and Ca 2+< regulated signal transduction events. In some instances, NUCB1 serves as a repressor of the S1P-mediated ATF6 activation. TF6 is a bZIP transcription factor that plays a major role in UPR transcriptional induction via binding to the ER stress response elements in the promoter of target genes. In some instances, the NUCB1 gene is an endoplasmic reticulum (ER) stress-indicible gene.Lipid probe for use in a high throughput screening method utilizing recombinant proteins

[0229] In some instances, also described herein is a lipid probe which comprises a lipid and a fluorophore wherein the lipid probe is constructed for detecting a drug-lipid binding protein interaction. As described above, the lipid probe comprises a lipid such as a bioactive lipid (e.g. a fatty acyl). In some instances, the lipid probe further comprises a photoreactive group. In some instances, the lipid probe comprises a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls and a fluorophore. In some instances, the lipid probe comprises an arachidonoyl fatty acyl and a fluorophore. In some instances, the lipid probe comprises an arachidoyl fatty acyl and a fluorophore. In some instances, the lipid probe comprises an oleoyl fatty acyl and a fluorophore. In some instances, the lipid probe comprises a palmitoyl fatty acyl and a fluorophore. In some instances, the lipid probe comprises a stearoyl fatty acyl and a fluorophore.

[0230] In some instances, the lipid probe comprises a lipid probe such as those described in Table 1 in which the lipid probe is conjugated to a fluorophore.

[0231] In some instances, the lipid probe comprises arachidonic acid and a fluorophore. In some instances, the lipid probe is 2-(6-(Dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((5-((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenamido)pentyl)carbamoyl)benzoate.

[0232] In some instances, the lipid probe is a ligand for a lipid binding protein. In some instances, the lipid probe is a ligand to an enzyme, a transporter, a receptor, an adaptor, a channel protein, or a chaperone. In some instances, the lipid probe is a ligand to 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. In some instances, the lipid probe is a ligand to nucleobindin-1 (NUCB1).

[0233] In some instances, also described herein is a lipid probe which comprises a lipid and a fluorophore wherein the lipid probe is constructed for detecting a drug-nucleobindin-1 (NUCB1) interaction. In some instances, the lipid probe comprises arachidonic acid and a fluorophore. In some instances, the lipid probe is 2-(6-(Dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((5-((52,8Z2,11Z,14Z)-1cosa-5,8,11,14-tetraenamido)pentyl)carbamoyl)benzoate.NUCB1 Ligand

[0234] In some examples, disclosed herein is a NUCB 1 ligand. In some instances, the NUCB1 ligand is a compound of Formula (II), or a pharmaceutically acceptable salt, or solvate thereof: wherein: R 1< is H, halogen, -CN, -OH, C 1 -C 4 alkoxy, C 1 -C 4 fluoroalkoxy, -NO 2 , -S-C 1 -C 4 alkyl, - S(=O)-C 1 -C 4 alkyl, or -S(=O) 2 -C 1 -C 4 alkyl; R 2< is H, halogen, C 1 -C 4 alkyl, C 3 -C 6 cycloalkyl, or substituted or unsubstituted phenyl, where if R 2< is substituted then R 2< is substituted with 1 or 2 R 5< each R 5< is independently selected from the group consisting of H, halogen, -CN, - NO 2 , -OH, -SR 6< , -S(=O)R 6< , -S(=O) 2 R 6< , -C(=O)R 6< , -CO 2 H, -CO 2 R 6< , -NH 2 , -NHR 6< , -N(R 6< ) 2 , -C(=O)NH 2 , -C(=O)NHR 6< , -C(=O)N(R 6< ) 2 , C 1 -C 4 alkyl, C 1 -C 4 fluoroalkyl, C 1 -C 4 alkoxy, C 1 -C 4 fluoroalkoxy, C 3 -C 6 cycloalkyl, C 2 -C 6 heterocycloalkyl, phenyl, and phenoxy; R 3< is H, or C 1 -C 4 alkyl; R 4< is hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, -NHNH 2 , -NH 2 , -NHR 6< , or -N(R 6< ) 2 ; each R 6< is independently selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 fluoroalkyl, C 3 -C 6 cycloalkyl, a substituted or unsubstituted phenyl, or substituted or unsubstituted benzyl; or two R 6< groups attached to the same N atom are taken together with the N atom to which they are attached to form a C 2 -C 6 heterocycloalkyl that is unsubstituted or substituted with C 1 -C 4 alkyl or C 3 -C 6 cycloalkyl.

[0235] In some instances, R 1< is H, Cl, -NO 2 , or -S(=O) 2 -CH 3 ; R 3< is H, or -CH 3 .

[0236] In some instances, R 4< is -NHNH 2 , -NH 2 , -NHR 6< , or -N(R 6< ) 2 .

[0237] In some instances, the compound has the following structure of Formula (III):

[0238] In some instances, each R 6< is independently selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 fluoroalkyl, C 3 -C 6 cycloalkyl, or benzyl; or two R 6< groups attached to the same N atom are taken together with the N atom to which they are attached to form a C 2 -C 6 heterocycloalkyl that is unsubstituted or substituted with C 1 -C 4 alkyl or C 3 -C 6 cycloalkyl, wherein the C 2 -C 6 heterocycloalkyl is pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, aziridinyl, or azetidinyl.

[0239] In some examples, the NUCB1 ligand has a structure selected from Table 2.

[0240] In some instances, further disclosed herein is a composition which comprises a compound such as a NUCB1 ligand or a pharmaceutically acceptable salt or solvate thereof, and an excipient.

[0241] In some instances, any suitable excipient is used. A summary of pharmaceutical compositions described herein may be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).

[0242] In some instances, the composition further comprises of carriers, stabilizers, diluents, dispersing agents, suspending agents, and / or thickening agents.

[0243] A "carrier" or "carrier materials" include any commonly used excipients in pharmaceutics and should be selected on the basis of compatibility with compounds disclosed herein, such as, compounds of ibrutinib, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. "Pharmaceutically compatible carrier materials" include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).

[0244] "Dispersing agents," and / or "viscosity modulating agents" include materials that control the diffusion and homogeneity of a drug through liquid media or a granulation method or blend method. In some examples, these agents also facilitate the effectiveness of a coating or eroding matrix. Exemplary diffusion facilitators / dispersing agents include, e.g., hydrophilic polymers, electrolytes, Tween ®< 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone ®< ), and the carbohydrate-based dispersing agents such as, for example, hydroxypropyl celluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M), carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinyl pyrrolidone / vinyl acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g., Pluronics F68 ®< , F88 ®< , and F108 ®< , which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic 908 ®< , also known as Poloxamine 908 ®< , which is a tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany, N.J.)), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone / vinyl acetate copolymer (S-630), polyethylene glycol, e.g., the polyethylene glycol has a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates, chitosans and combinations thereof. Plasticizers such as cellulose or triethyl cellulose are also used as dispersing agents. Dispersing agents particularly useful in liposomal dispersions and self-emulsifying dispersions are dimyristoyl phosphatidyl choline, natural phosphatidyl choline from eggs, natural phosphatidyl glycerol from eggs, cholesterol and isopropyl myristate.

[0245] "Suspending agents" include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

[0246] Thickening agent or viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.

[0247] In some instances, the composition further comprises one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[0248] In some instances, the composition also includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0249] In some instances, the composition is formulated for administration to a mammal by intravenous administration, subcutaneous administration, oral administration, inhalation, nasal administration, dermal administration, or ophthalmic administration.

[0250] In some instances, the composition is in the form of a tablet, a pill, a capsule, a liquid, a suspension, a gel, a dispersion, a solution, an emulsion, an ointment, or a lotion. In some instances, the compositions described herein are formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by an individual to be treated, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. In some examples, the compositions are formulated into capsules. In some examples, the compositions are formulated into solutions (for example, for intravenous (IV) administration). In some instances, the dosage form further comprises acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof.

[0251] As used in the context of Formula II and Formula III, the terms below, as used herein, have the following meanings, unless indicated otherwise:

[0252] "Alkyl" refers to a straight or branched hydrocarbon chain radical, having from one to twenty carbon atoms, and which is attached to the rest of the molecule by a single bond. An alkyl comprising up to 10 carbon atoms is referred to as a C 1 -C 10 alkyl, likewise, for example, an alkyl comprising up to 6 carbon atoms is a C 1 -C 6 alkyl. Alkyls (and other moieties defined herein) comprising other numbers of carbon atoms are represented similarly. Alkyl groups include, but are not limited to, C 1 -C 10 alkyl, C 1 -C 9 alkyl, C 1 -C 8 alkyl, C 1 -C 7 alkyl, C 1 -C 6 alkyl, C 1 -C 5 alkyl, C 1 -C 4 alkyl, C 1 -C 3 alkyl, C 1 -C 2 alkyl, C 2 -C 8 alkyl, C 3 -C 8 alkyl and C 4 -C 8 alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl, i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethyl-propyl, and the like. In some examples, the alkyl is methyl, ethyl, s-butyl, or 1-ethyl-propyl. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted as described below. "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group. In some examples, the alkylene is -CH 2 -, -CH 2 CH 2 -, or - CH 2 CH 2 CH 2 -. In some examples, the alkylene is -CH 2 -. In some examples, the alkylene is - CH 2 CH 2 -. In some examples, the alkylene is -CH 2 CH 2 CH 2 -.

[0253] "Alkoxy" refers to a radical of the formula -OR where R is an alkyl radical as defined. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted as described below. Representative alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy. In some examples, the alkoxy is methoxy. In some examples, the alkoxy is ethoxy.

[0254] "Cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. Cycloalkyls may be saturated, or partially unsaturated. Cycloalkyls may be fused with an aromatic ring (in which case the cycloalkyl is bonded through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having from 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloakyls having from three to ten carbon atoms, from three to eight carbon atoms, from three to six carbon atoms, or from three to five carbon atoms. Monocyclic cyclcoalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some examples, the monocyclic cyclcoalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, and 3,4-dihydronaphthalen-1(2H)-one. Unless otherwise stated specifically in the specification, a cycloalkyl group may be optionally substituted.

[0255] "Heterocycloalkyl" or "heterocyclyl" or "heterocyclic ring" refers to a stable 3- to 14-membered non-aromatic ring radical comprising 2 to 13 carbon atoms and from one to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless stated otherwise specifically in the specification, the heterocycloalkyl radical may be a monocyclic, or bicyclic ring system, which may include fused (when fused with an aryl or a heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized. The nitrogen atom may be optionally quaternized. The heterocycloalkyl radical is partially or fully saturated. Examples of such heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl. In some examples the heterocycloalkyl is morpholinyl, thiomorpholinyl, piperidinyl, or pyrrolidinyl. In some examples the heterocycloalkyl is morpholinyl. The term heterocycloalkyl also includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. Unless otherwise noted, heterocycloalkyls have from 2 to 10 carbons in the ring. In some examples, heterocycloalkyls have from 2 to 8 carbons in the ring. In some examples, heterocycloalkyls have from 2 to 8 carbons in the ring and 1 or 2 N atoms. It is understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring). Unless stated otherwise specifically in the specification, a heterocycloalkyl group may be optionally substituted.

[0256] The term "optionally substituted" or "substituted" means that the referenced group may be substituted with one or more additional group(s) individually and independently selected from alkyl, haloalkyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, -OH, alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxide, aryl sulfoxide, alkylsulfone, arylsulfone, -CN, alkyne, C 1 -C 6 alkylalkyne, halogen, acyl, acyloxy, -CO 2 H, -CO 2 alkyl, nitro, and amino, including mono- and di-substituted amino groups (e.g. -NH 2 , -NHR, -N(R) 2 ), and the protected derivatives thereof. In some examples, optional substituents are independently selected from alkyl, alkoxy, haloalkyl, cycloalkyl, halogen, -CN, -NH 2 , -NH(CH 3 ), -N(CH 3 ) 2 , -OH, -CO 2 H, and -CO 2 alkyl. In some examples, optional substituents are independently selected from fluoro, chloro, bromo, iodo, - CH 3 , -CH 2 CH 3 , -CF 3 , -OCH 3 , and -OCF 3 . In some examples, substituted groups are substituted with one or two of the preceding groups. In some examples, an optional substituent on an aliphatic carbon atom (acyclic or cyclic, saturated or unsaturated carbon atoms, excluding aromatic carbon atoms) includes oxo (=O).Kits and Articles of Manufacture

[0257] Disclosed herein, in certain examples, are kits and articles of manufacture for use with one or more methods described herein. In some examples, described herein is a kit for identifying a lipid binding protein as a drug binding target. In some instances, also described herein is a kit for mapping binding sites on a lipid binding protein. In some cases, described herein is a kit for identifying lipid binding proteins. In some examples, also described herein is a kit for a high throughput screening of a drug for interaction with a lipid binding protein.

[0258] In some examples, such kit includes lipid probes such as the lipid probes described herein, test compounds such as drugs and / or controls, and reagents suitable for carrying out one or more of the methods described herein. In some instances, the kit further comprises samples, such as a cell sample, and suitable solutions such as buffers or media. In some examples, the kit further comprises recombinant proteins for use in one or more of the methods described herien. In some examples, additional components of the kit comprises a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, plates, syringes, and test tubes. In one example, the containers are formed from a variety of materials such as glass or plastic.

[0259] The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, bags, containers, and any packaging material suitable for a selected formulation and intended mode of use.

[0260] For example, the container(s) include lipid probes, test compounds, and one or more reagents for use in a method disclosed herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.

[0261] A kit typically includes labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0262] In one example, a label is on or associated with the container. In one example, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one example, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.Services

[0263] The methods provided herein may also be performed as a service. In some instances, a service provider obtain from the customer a plurality of drug candidates for analysis with one or more of the lipid probes for screening. In some examples, the service provider screens the drug candidates using one or more of the methods described herein, and then provide the results to the customer. In some instances, the service provider provides the appropriate reagents to the customer for analysis utilizing one or more of the lipid probes and one or more of the methods described herein. In some cases, the customer performs one or more of the methods described herein and then provide the results to the service provider for analysis. In some examples, the service provider then analyzes the results and provides the results to the costumer. In some cases, the customer further analyze the results by interacting with software installed locally (at the customer's location) or remotely (e.g., on a server reachable through a network). Exemplary customers include pharmaceutical companies, clinical laboratories, physicians, patients, and the like. In some instances, a customer is any suitable customer or party with a need or desire to use the methods, systems, compositions, and kits described herein.Digital Processing Device

[0264] In some examples, the methods described herein include a digital processing device, or use of the same. In further examples, the digital processing device includes one or more hardware central processing units (CPU) that carry out the device's functions. In still further examples, the digital processing device further comprises an operating system configured to perform executable instructions. In some examples, the digital processing device is optionally connected to a computer network. In further examples, the digital processing device is optionally connected to the Internet such that it accesses the World Wide Web. In still further examples, the digital processing device is optionally connected to a cloud computing infrastructure. In other examples, the digital processing device is optionally connected to an intranet. In other examples, the digital processing device is optionally connected to a data storage device.

[0265] In accordance with the description herein, suitable digital processing devices include, but are not limited to, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Suitable tablet computers include those with booklet, slate, or convertible configurations.

[0266] In some examples, the digital processing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device's hardware and provides services for execution of applications. Suitable server operating systems include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD ®< , Linux, Apple ®< Mac OS X Server ®< , Oracle ®< Solaris ®< , Windows Server ®< , and Novell ®< NetWare ®< . Suitable personal computer operating systems include, by way of non-limiting examples, Microsoft ®< Windows ®< , Apple ®< Mac OS X ®< , UNIX ®< , and UNIX-like operating systems such as GNU / Linux ®< . In some examples, the operating system is provided by cloud computing. Suitable mobile smart phone operating systems include, by way of non-limiting examples, Nokia ®< Symbian ®< OS, Apple ®< iOS ®< , Research In Motion ®< BlackBerry OS ®< , Google ®< Android ®< , Microsoft ®< Windows Phone ®< OS, Microsoft ®< Windows Mobile ®< OS, Linux ®< , and Palm ®< WebOS ®< . Suitable media streaming device operating systems include, by way of non-limiting examples, Apple TV ®< , Roku ®< , Boxee ®< , Google TV ®< , Google Chromecast ®< , Amazon Fire ®< , and Samsung ®< HomeSync ®< . Suitable video game console operating systems include, by way of non-limiting examples, Sony ®< PS3 ®< , Sony ®< PS4 ®< , Microsoft ®< Xbox 360 ®< , Microsoft Xbox One, Nintendo ®< Wii ®< , Nintendo ®< Wii U ®< , and Ouya ®< .

[0267] In some examples, the device includes a storage and / or memory device. The storage and / or memory device is one or more physical apparatuses used to store data or programs on a temporary or permanent basis. In some examples, the device is volatile memory and requires power to maintain stored information. In some examples, the device is non-volatile memory and retains stored information when the digital processing device is not powered. In further examples, the non-volatile memory comprises flash memory. In some examples, the non-volatile memory comprises dynamic random-access memory (DRAM). In some examples, the non-volatile memory comprises ferroelectric random access memory (FRAM). In some examples, the non-volatile memory comprises phase-change random access memory (PRAM). In other examples, the device is a storage device including, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, magnetic disk drives, magnetic tapes drives, optical disk drives, and cloud computing based storage. In further examples, the storage and / or memory device is a combination of devices such as those disclosed herein.

[0268] In some examples, the digital processing device includes a display to send visual information to a user. In some examples, the display includes a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic light emitting diode (OLED) display, a plasma display, a video projector, or a combination thereof.

[0269] In some examples, the digital processing device includes an input device to receive information from a user. In some examples, the input device is a keyboard. In some examples, the input device is a pointing device including, by way of non-limiting examples, a mouse, trackball, track pad, joystick, game controller, or stylus. In some examples, the input device is a touch screen or a multi-touch screen. In other examples, the input device is a microphone to capture voice or other sound input. In other examples, the input device is a video camera or other sensor to capture motion or visual input. In further examples, the input device is a Kinect ™< , Leap Motion ™< , or the like. In still further examples, the input device is a combination of devices such as those disclosed herein.

[0270] In some examples, the systems and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked digital processing device. In further examples, a computer readable storage medium is a tangible component of a digital processing device. In still further examples, a computer readable storage medium is optionally removable from a digital processing device. In some examples, a computer readable storage medium includes, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, cloud computing systems and services, and the like. In some cases, the program and instructions are permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the media.

[0271] In some examples, the systems and methods disclosed herein include at least one computer program, or use of the same. A computer program includes a sequence of instructions, executable in the digital processing device's CPU, written to perform a specified task. In some examples, computer readable instructions are implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), data structures, and the like, that perform particular tasks or implement particular abstract data types.

[0272] In some examples, the functionality of the computer readable instructions are combined or distributed as desired in various environments. In some examples, a computer program comprises one sequence of instructions. In some examples, a computer program comprises a plurality of sequences of instructions. In some examples, a computer program is provided from one location. In other examples, a computer program is provided from a plurality of locations. In various examples, a computer program includes one or more software modules. In various examples, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.

[0273] In some examples, a computer program includes a web application. A web application, in various examples, utilizes one or more software frameworks and one or more database systems. In some examples, a web application is created upon a software framework such as Microsoft ®< .NET or Ruby on Rails (RoR). In some examples, a web application utilizes one or more database systems including, by way of non-limiting examples, relational, non-relational, object oriented, associative, and XML database systems. In further examples, suitable relational database systems include, by way of non-limiting examples, Microsoft ®< SQL Server, mySQL ™< , and Oracle ®< . A web application, in various examples, is written in one or more versions of one or more languages. In some examples, a web application is written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some examples, a web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or eXtensible Markup Language (XML). In some examples, a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some examples, a web application is written to some extent in a client-side scripting language such as Asynchronous Javascript and XML (AJAX), Flash ®< Actionscript, Javascript, or Silverlight ®< . In some examples, a web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion ®< , Perl, Java ™< , JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python ™< , Ruby, Tcl, Smalltalk, WebDNA ®< , or Groovy. In some examples, a web application is written to some extent in a database query language such as Structured Query Language (SQL). In some examples, a web application integrates enterprise server products such as IBM ®< Lotus Domino ®< . In some examples, a web application includes a media player element. In various further examples, a media player element utilizes one or more of many suitable multimedia technologies including, by way of non-limiting examples, Adobe ®< Flash ®< , HTML 5, Apple ®< QuickTime ®< , Microsoft ®< Silverlight ®< , Java ™< , and Unity ®< .

[0274] In some examples, a computer program includes a mobile application provided to a mobile digital processing device. In some examples, the mobile application is provided to a mobile digital processing device at the time it is manufactured. In other examples, the mobile application is provided to a mobile digital processing device via the computer network described herein.

[0275] In view of the disclosure provided herein, a mobile application is created by techniques using hardware, languages, and development environments. Suitable programming languages include, by way of non-limiting examples, C, C++, C#, Objective-C, Java ™< , Javascript, Pascal, Object Pascal, Python ™< , Ruby, VB.NET, WML, and XHTML / HTML with or without CSS, or combinations thereof.

[0276] Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non-limiting examples, AirplaySDK, alcheMo, Appcelerator ®< , Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available without cost including, by way of non-limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone and iPad (iOS) SDK, Android ™< SDK, BlackBerry ®< SDK, BREW SDK, Palm ®< OS SDK, Symbian SDK, webOS SDK, and Windows ®< Mobile SDK.

[0277] In some examples, commercial forums for distribution of mobile applications include, by way of non-limiting examples, Apple ®< App Store, Android ™< Market, BlackBerry ®< App World, App Store for Palm devices, App Catalog for webOS, Windows ®< Marketplace for Mobile, Ovi Store for Nokia ®< devices, Samsung ®< Apps, and Nintendo ®< DSi Shop.

[0278] In some examples, a computer program includes a standalone application, which is a program that is run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in. In some instances, standalone applications are compiled. A compiler is a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, Java ™< , Lisp, Python ™< , Visual Basic, and VB .NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some examples, a computer program includes one or more executable complied applications.

[0279] In some examples, the computer program includes a web browser plug-in. In computing, a plug-in is one or more software components that add specific functionality to a larger software application. Makers of software applications support plug-ins to enable third-party developers to create abilities which extend an application, to support easily adding new features, and to reduce the size of an application. When supported, plug-ins enable customizing the functionality of a software application. For example, plug-ins are commonly used in web browsers to play video, generate interactivity, scan for viruses, and display particular file types. In some instances, web browser plug-ins include Adobe ®< Flash ®< Player, Microsoft ®< Silverlight ®< , and Apple ®< QuickTime ®< . In some examples, the toolbar comprises one or more web browser extensions, add-ins, or add-ons. In some examples, the toolbar comprises one or more explorer bars, tool bands, or desk bands.

[0280] In view of the disclosure provided herein, plug-in frameworks are available that enable development of plug-ins in various programming languages, including, by way of non-limiting examples, C++, Delphi, Java ™< , PHP, Python ™< , and VB .NET, or combinations thereof.

[0281] Web browsers (also called Internet browsers) are software applications, designed for use with network-connected digital processing devices, for retrieving, presenting, and traversing information resources on the World Wide Web. Suitable web browsers include, by way of non-limiting examples, Microsoft ®< Internet Explorer ®< , Mozilla ®< Firefox ®< , Google ®< Chrome, Apple ®< Safari ®< , Opera Software ®< Opera ®< , and KDE Konqueror. In some examples, the web browser is a mobile web browser. Mobile web browsers (also called mircrobrowsers, mini-browsers, and wireless browsers) are designed for use on mobile digital processing devices including, by way of non-limiting examples, handheld computers, tablet computers, netbook computers, subnotebook computers, smartphones, music players, personal digital assistants (PDAs), and handheld video game systems. Suitable mobile web browsers include, by way of non-limiting examples, Google ®< Android ®< browser, RIM BlackBerry ®< Browser, Apple ®< Safari ®< , Palm ®< Blazer, Palm ®< WebOS ®< Browser, Mozilla ®< Firefox ®< for mobile, Microsoft ®< Internet Explorer ®< Mobile, Amazon ®< Kindle ®< Basic Web, Nokia ®< Browser, Opera Software ®< Opera ®< Mobile, and Sony ®< PSP ™< browser.

[0282] In some examples, the systems and methods disclosed herein include software, server, and / or database modules, or use of the same. In view of the disclosure provided herein, software modules are created and implemented in a multitude of ways. In various examples, a software module comprises a file, a section of code, a programming object, a programming structure, or combinations thereof. In further various examples, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, or combinations thereof. In various examples, the one or more software modules comprise, by way of non-limiting examples, a web application, a mobile application, and a standalone application. In some examples, software modules are in one computer program or application. In other examples, software modules are in more than one computer program or application. In some examples, software modules are hosted on one machine. In other examples, software modules are hosted on more than one machine. In further examples, software modules are hosted on cloud computing platforms. In some examples, software modules are hosted on one or more machines in one location. In other examples, software modules are hosted on one or more machines in more than one location.

[0283] In some examples, the methods and systems disclosed herein include one or more databases, or use of the same. In view of the disclosure provided herein, databases are suitable for storage and retrieval of analytical information described elsewhere herein. In various examples, suitable databases include, by way of non-limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entity-relationship model databases, associative databases, and XML databases. In some examples, a database is internet-based. In further examples, a database is web-based. In still further examples, a database is cloud computing-based. In other examples, a database is based on one or more local computer storage devices.Server

[0284] In some examples, the methods provided herein are processed on a server or a computer server (Fig. 2). In some examples, the server 401 includes a central processing unit (CPU, also "processor") 405 which is a single core processor, a multi core processor, or plurality of processors for parallel processing. In some examples, a processor used as part of a control assembly is a microprocessor. In some examples, the server 401 also includes memory 410 (e.g. random access memory, read-only memory, flash memory); electronic storage unit 415 (e.g. hard disk); communications interface 420 (e.g. network adaptor) for communicating with one or more other systems; and peripheral devices 425 which includes cache, other memory, data storage, and / or electronic display adaptors. The memory 410, storage unit 415, interface 420, and peripheral devices 425 are in communication with the processor 405 through a communications bus (solid lines), such as a motherboard. In some examples, the storage unit 415 is a data storage unit for storing data. The server 401 is operatively coupled to a computer network ("network") 430 with the aid of the communications interface 420. In some examples, a processor with the aid of additional hardware is also operatively coupled to a network. In some examples, the network 430 is the Internet, an intranet and / or an extranet, an intranet and / or extranet that is in communication with the Internet, a telecommunication or data network. In some examples, the network 430 with the aid of the server 401, implements a peer-to-peer network, which enables devices coupled to the server 401 to behave as a client or a server. In some examples, the server is capable of transmitting and receiving computer-readable instructions (e.g., device / system operation protocols or parameters) or data (e.g., sensor measurements, raw data obtained from detecting metabolites, analysis of raw data obtained from detecting metabolites, interpretation of raw data obtained from detecting metabolites, etc.) via electronic signals transported through the network 430. Moreover, in some examples, a network is used, for example, to transmit or receive data across an international border.

[0285] In some examples, the server 401 is in communication with one or more output devices 435 such as a display or printer, and / or with one or more input devices 440 such as, for example, a keyboard, mouse, or joystick. In some examples, the display is a touch screen display, in which case it functions as both a display device and an input device. In some examples, different and / or additional input devices are present such an enunciator, a speaker, or a microphone. In some examples, the server uses any one of a variety of operating systems, such as for example, any one of several versions of Windows ®< , or of MacOS ®< , or of Unix ®< , or of Linux ®< .

[0286] In some examples, the storage unit 415 stores files or data associated with the operation of a device, systems or methods described herein.

[0287] In some examples, the server communicates with one or more remote computer systems through the network 430. In some examples, the one or more remote computer systems include, for example, personal computers, laptops, tablets, telephones, smart phones, or personal digital assistants.

[0288] In some examples, a control assembly includes a single server 401. In other situations, the system includes multiple servers in communication with one another through an intranet, extranet and / or the Internet.

[0289] In some examples, the server 401 is adapted to store device operation parameters, protocols, methods described herein, and other information of potential relevance. In some examples, such information is stored on the storage unit 415 or the server 401 and such data are transmitted through a network.EXAMPLES

[0290] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.Example I Materials

[0291] Internal standards for LC / MS analysis (AEA-d 4 , PEA-d 4 , AA-ds, 2-AG-ds, PGE 2 -d 9 , PGD 2 -d 9 , TXB 2 -d 4 , PGE 2 -EA-d 4 ) were purchased from Cayman Chemical Company. Lipid standards and competitors, including (±)-flurbiprofen, rofecoxib, avasimibe, FK-866 and Ro 48-8071 were purchased from Cayman Chemical Company and used without further purification. Lipid probes and NUCB1 ligands were synthesized according to methods outlined below.Cell culture

[0292] HEK293T and Neuro2a cells were maintained in high-glucose DMEM supplemented with 10% (v / v) fetal bovine serum (FBS), penicillin (100 U / mL), streptomycin (100 µg / mL) and Lglutamine (2 mM). A549 cells were grown in RPMI-1640 (Mediatech) supplemented as above. All cell lines were grown at 37 °C in a humidified 5% CO2 atmosphere. For SILAC experiments, each cell line was passaged at least six times in either SILAC DMEM or SILAC RPMI-1640 (Thermo), which lack L-lysine and L-arginine, and supplemented with 10% (v / v) dialyzed FBS (Gemini), PSQ (as above), and either [ 13< C 6 , 15< N 2 ]- L-lysine and [ 13< C 6 , 15< N 4 ]- Larginine (Sigma) (100 µg / mL each) or L-lysine•HCl and L-arginine•HCl (Sigma) (100 µg / mL each). Heavy and light cells were maintained in parallel and cell aliquots were frozen after six passages in SILAC media and stored in liquid N 2 until needed. When thawed, cells were passaged at least three times before use in experiments.Live cell labeling with clickable photoaffinity probes

[0293] At ~80-90% confluency, cells were trypsinized and counted using an automated cell counter (Bio-Rad). For gel-based analysis, cells were plated at a density of 2.5 x 10 6< cells / 6 cm plate and grown for 18-24 h before labeling. The indicated photoaffinity probe and, if applicable, competitors or vehicle were dissolved in fresh media (1.5 mL) in a glass vial and warmed to 37 °C in a water bath. For competition experiments, serum-free media was used, whereas for probe-probe comparisons, standard growth media containing 10% (v / v) FBS was used. The media from each 6 cm plate was then aspirated and the cells were washed with DPBS (2 x 3.0 mL) before adding media containing probes and / or competitors. In some instances, media was either poured directly from the glass vials onto the cells or transferred using a glass Pasteur pipette. Cells were incubated at 37 °C for 30 min before the media was removed and the cells were either directly exposed to 365 nm light for 10 min at 4 °C (Stratagene, UV StratalinkerTM 1800 Crosslinker) or incubated at 4 °C for 10 min in ambient light (No UV control experiments).

[0294] For MS-based experiments, cell labeling was performed in a similar manner as above. Modifications to this protocol included using isotopically light and heavy SILAC cells and increasing the cell count to increase protein yield. Specifically, SILAC cells were plated at a density of 4 x 10 6< cells / 10 cm plate and grown to near complete confluency prior to labeling. Additionally, probe and, if applicable, vehicles or competitors were dissolved together in 4.0 mL of fresh, serum-free SILAC media in glass vials. Isotopically light cells were labeled with the arachidinoyl probe (AEA-DA or A-DA) and irradiated with UV for 10 min at 4 °C. "Heavy" cells were subjected to variable conditions as specified in each experiment, including treatments with alternative lipid probes (OEA-DA, PEA-DA, O-DA or S-DA) or competitors.Proteome preparation for gel- and MS-based analyses

[0295] Cells were then harvested by scraping in cold DPBS (1.5 mL) and cell pellets isolated by centrifugation (1,400 x g, 3 min) and then washed by addition of cold DPBS (1.0 mL), vortexing and re-centrifugation (1,400 x g, 3 min). To the cell pellets was added cold DPBS (100-500 µL) and the cells were lysed by probe sonication using a Branson Sonifier probe sonicator (10 pulses, 50% duty cycle, output setting = 3). In some instances, PMA-stimulated SILAC A549 cells were lysed in DPBS containing indomethacin (10 µM) to inhibit PTGS2 prior to mixing heavy and light proteomes in order to prevent non-UV dependent covalent adduction of this enzyme. When analyzing soluble and membrane components separately, cell lysates were then centrifuged (100,000 x g, 45 min) to provide the soluble (supernatant) and membrane (pellet) fractions. For SILAC experiments, isotopically "heavy" and "light" cell lysates were mixed in equal proportions (2.0 mg of protein each) prior to fractionation.

[0296] For gel-based analysis, after sonicating the membrane pellets in cold DPBS, protein concentrations of each fraction were determined using the BCA protein assay (Bio-Rad) and a microplate reader (Tecan, Infinite F500).Gel-based analysis of crosslinked proteins

[0297] Proteomes were diluted to 1.0 mg / mL (total protein concentration) and 50 µL of each proteome was transferred to separate wells in a 96-well plate. To each proteome sample was added 6 µL of a freshly prepared "click" reagent mixture containing TBTA (3 µL / sample, 1.7 mM in 4:1 DMSO:t-BuOH), CuSO 4 (1.0 µL / sample, 50 mM in H 2 O), TCEP (1.0 µL / sample, 50 mM in DPBS and Rh-N 3 (1.0 µL / sample, 1.25 mM in DMSO). Upon addition of the "click" mixture, each reaction was mixed by pipetting up-and-down several times and then allowed to react at room temperature. After 1 h, each reaction was quenched with 4X SDS loading buffer (17 µL), and proteins were resolved using SDS-PAGE (10% acrylamide 13 gel) and analyzed by in-gel fluorescent scanning on a Hitachi FMBIO-II flatbed fluorescence scanner.MS-based analysis of crosslinked proteins

[0298] Isotopically heavy and light proteomes from derived from probe labeled cells were mixed in equal proportions (1.0 mg each) and diluted to a final volume of 1.0 mL with DPBS. A mixture of TBTA (60 µL / sample, 1.7 mM in 4:1 DMSO:t-BuOH), CuSO 4 (20 µL / sample, 50 mM in H2O), TCEP (20 µL / sample, 50 mM in DPBS and Biotin-N 3 (10 µL / sample, 10 mM in DMSO) was added and the proteome was vortexed and placed on a rotator at room temperature. After 1 h, cold MeOH (2 mL), CHCl 3 (0.5 mL) and DPBS (1 mL) were added sequentially and the cloudy mixture was vortexed and then centrifuged (5,000 x g, 15 min, 4 °C). The organic and aqueous layers were aspirated leaving a protein disc which had formed between phases. The protein disc was washed with cold 1:1 MeOH:CHCl 3 (3 x 1 mL) while intact and then probe sonicated in cold 4:1 MeOH:CHCl 3 (2.5 mL). Insoluble proteins were pelleted via centrifugation (5,000 x g, 15 min, 4 °C) and the supernatant was removed. The remaining pellet was redissolved in a freshly prepared solution of urea (500 µL, 6 M in DPBS). When analyzing membrane proteomes, 10% (w / v) SDS (20 µL) was added and the solution was warmed to 37 °C to facilitate the dissolution of poorly soluble proteins. TCEP (50 µL, 100 mM in DBPS, pH 7) was next added, and after the proteome solution was incubated for 30 min at 37 °C, iodoacetamide (70 µL, 400 mM in DPBS) was added. After incubating for an additional 30 min at room temperature, 10% (w / v) SDS (120 µL) was added followed by DPBS (5.5 mL) and then pre-washed streptavidin beads (100 µL, 1:1, Pierce). Proteomes were rotated with the beads for 1.5 h at room temperature and the beads were pelleted by centrifugation (1,000 x g, 2 min) and sequentially washed with 0.25% SDS (3 x 10 mL), DPBS (3 x 10 mL) and ddH 2 O (3 x 10 mL). The beads were transferred to a Protein LoBind tube (Eppendorf) and enriched proteins were trypsinized by addition of urea (200 µL, 2.0 M in DPBS), CaCl 2 (2.0 µL, 100 mM in H 2 O) and sequence grade porcine trypsin (Promega). After digesting overnight at 37 °C, the supernatant was transferred to a clean Protein LoBind tube, acidified with HCO2H (16 µL) and stored at -20 °C until the analyzed by LC / LC-MS / MS.Proteomic analysis by mass spectrometry and data analysis

[0299] Proteomic samples were analyzed using a Thermo Orbitrap Velos mass spectrometer according to Weerapana et al., "Quantitative reactivity profiling predicts functional cysteines in proteomes," Nature 468:790-795 (2010). Peptides from on-bead tryptic digests were pressure loaded onto a 250 µm (inner diameter) fused silica capillary column packed with 4 cm C18 resin (5 µm, Phenomenex). Peptides were then eluted onto a 100 µm (inner diameter) fused silica capillary column packed with 3 cm strong cation exchange (SCX) resin followed by 10 cm C18 resin. Chromatographically separation of the peptide mixture was achieved using a 5-step multidimensional LC-MS (MudPIT) according to Washburn et al., "Large-scale analysis of the yeast proteome by multidimensional protein identification technology," Nat Biotechnol 19:242-247 (2001) in which Washburn et al. teaches of 0%, 25%, 50%, 80% and 100% salt bumps of NH 4 OAc (500 mM) salt bumps followed by an increasing gradient of CH 3 CN (0.1% HCO 2 H) in H 2 O. Peptides were analyzed by a Thermo Orbitrap Velos mass spectrometer set in data-dependent acquisition mode where two MS1 microscans [400-1800 mass to charge ratio (m / z)] were followed by 30 data-dependent fragmentation (MS2) scans. Dynamic exclusion (repeat count of 1, exclusion duration of 20 s) and monoisotopic precursor selection were enabled, whereas all other parameters were left at default values. MS2 spectra were extracted from raw data files using RAW Xtractor and searched using the ProLuCID algorithm against mouse and human, reverse-concatenated non-redundant (gene-centric) FASTA databases that were assembled from the Uniprot database (www.uniprot.org). The precursor-ion mass tolerance was set to 50 ppm and the fragment-ion mass tolerance was the default assignment of 0. Searches allowed for oxidation of methionine (+15.9949 m / z) as a variable modification and specified static modification of cysteine residues (+57.0215 m / z; iodoacetamide alkylation). Datasets were independently searched with light and heavy parameter files where light searches applied default masses to each amino acid and heavy searches specified static modifications on lysine (+8.0142 m / z) and arginine (+10.0082 m / z). Matched MS2 spectra from ProLuCID searches were assembled by protein and filtered using DTASelect (version 2.0.47) which allowed only half-tryptic or fully-tryptic peptides for identification and quantification. Peptides were restricted to a specified false positive rate of 1%. Redundant peptide identifications, if common between multiple proteins, were allowed, as database entries were limited to a single consensus splice variant. SILAC ratios were determined using in-house software (CIMAGE) (Weerapana et al., 2010)]. Briefly, MS1 ion chromatograms (±10 ppm) from "light" and "heavy" target peptide masses (m / z) were generated using a retention time window (±10 min) centered at the time the peptide ion was selected for MS / MS fragmentation, and subsequently identified. The ratio of "light" and "heavy" peptide peak areas are then calculated. To ensure the correct peak-pair is used for quantification, CIMAGE applies a co-elution correlation score filter (R2 ≥ 0.8) for "heavy" and "light" peptide peaks to exclude target peptides with bad co-elution profiles. Furthermore, an "envelope correlation score" filter is applied to ensure the experimentally observed high-resolution MS1 spectrum matches (R2 > 0.8) the predicted isotopic distribution. Peptides detected as singletons, where only the heavy or light isotopically labeled peptide was detected and sequenced, but which passed all other filters described above, were given a standard ratio of 20, which is the maximum SILAC ratio reported here.Proteomics data filtering

[0300] SILAC results for identification of UV-dependent probe targets and comparison of structurally related lipid probes in HEK293T and Neuro2a represents data combined from 2-3 separate biological replicates. The soluble and membrane fractions from each biological replicate were analyzed separately to improve protein coverage. Median peptide SILAC ratios were then filtered to assure each protein ratio was derived from three or more unique and quantified peptides and that the combined quantified peptide ratios possessed a standard deviation of less than 10. SILAC ratios complying with these criteria were then averaged with ratios acquired from replicates and the alternate fraction (membrane or soluble) to provide a final value which is reported in Table 3 (48054-701-101Table3.txt). If no replicate values were detected, the SILAC ratio from this single occurrence was included only if each target was also quantified in probe-versus-probe experiments according to the above criteria. Data from instances where a target was identified in a single replicate was included due to the analysis of probe-versus-No UV data where a >90% confirmation was found in the UV-dependence (SILAC ratio ≥ 3.0) of targets identified across multiple replicates. In some instances, a subpopulation of these singly quantified targets was also identified in both cell lines (Neuro2a and HEK293T). See Table 6 for a list of individual peptide sequences.

[0301] UV-dependent lipid probe targets were defined as proteins that complied with the following criteria: 1) the protein was identified and quantified (according to the above criteria) in both probe-versus-probe and probe-versus-No UV datasets; 2) the protein exhibited a mean SILAC ratio of ≥ 3.0 in probe-versus-No UV experiments; 3) the protein exhibited a mean SILAC ratio of < 2.0 and > 0.5 in probe-versus-probe. Only UV-dependent targets are shown in Table 3. For competition experiments, SILAC results were derived from 1-3 experiments. Only proteins that qualified as UV-dependent targets according to the above criteria were included in the analysis (see Table 4 and 5; 48054-701-101 Table4.txt, 48054-701-101 Table5.txt). Furthermore, SILAC ratios for each competition experiment were only included if they were derived from two or more unique and quantified peptides.Lipid probe target meta-analysis

[0302] Analyses of the final lipid probe target list as found in Table 3 and Figure 5 and 6 were conducted using the ID Mapping feature of UniProt Database (http: / / www.uniprot.org / uploadlists / ) with the exception of the OMIM analysis which was performed using the DAVID Bioinformatics Database (http: / / david.abcc.ncifcrf.gov / tools.jsp). Membrane proteins were defined as proteins possessing known or predicted transmembrane domains (UniProt analysis), and the remaining targets were considered soluble. Statistical analysis of membrane and soluble targets for each probe were determined by combining No UV datasets from HEK293T (n = 2) and Neuro2a (n = 2) cell lines for AEA-DA and A-DA probes, and calculating the percent of total probe-enriched targets (SILAC ratio ≥ 3.0) that possess or lack transmembrane domains (i.e. membrane or soluble, respectively). Data are presented as the mean percentage of total probe targets ± SEM; n = 4 / group. **P < 0.01 for AEA-DA versus A-DA probe targets.Transient protein expression in HEK293T cells

[0303] Full-length cDNAs encoding for the each protein of interest were subcloned into pcDNA3.1 myc-His A (Invitrogen) or used directly for transfections if available in a eukaryotic expression vector. HEK293T cells were grown to ~70% confluence under standard growth conditions before adding the appropriate cDNA [control cells ("mock") received an equal amount of empty pcDNA3.1 myc-His A vector] and FuGENE 6 or X-tremeGENE HP (Roche) transfection reagent according to the manufacturer's instructions. Cells were incubated for 36-48 hours before labeling in situ or harvesting for in vitro experiments.Recombinant NUCB1 expression and purification

[0304] Human NUCB1 lacking the N-terminal signal sequence (M1-A26) was subcloned into pET45b(+) vector (Novagen) using the following PCR primers for amplification: 5'-CAT CAA TTG TGT GCC CCT GGA GAG AG-3' and 5'-AGC AAG CTT TAC AGA TGT TGG GGC ACC-3'. Following transformation of Escherichia coli strain BL21(DE3) with hNucb1ΔSSpET45b(+), a single colony was isolated and grown in Terrific Broth containing carbenicillin (100 mg / L) at 37 °C to an OD 600 of 0.8. Protein expression was induced with IPTG (1.0 mM) and the cells were incubated at 20 °C with shaking overnight. After centrifugation, cells pellets were resuspended in cold buffer containing HEPES (0.05M), NaCl (0.03 M), imidazole (5.0 mM), DNase (0.1 mg / mL), MgCl2 (1.0 mM), CaCl 2 (1.0 mM) and EDTA-free Complete Protease Inhibitor Cocktail and lysed using a microfluidizer. Cell lysates were centrifuged (8,000 x g, 30 min, 4 °C) and the supernatant was stirred with Talon cobalt affinity resin (0.4 mL / g cells) (Clontech) for 1 h at 4 °C. The suspension was applied to a column and the retained resin was washed with HEPES buffer containing 500 mM NaCl until the eluent was clean of DNA and protein (A280 / 260) and then with HEPES buffer containing 10 mM imidazole (5 mL / 1 mL resin). Protein was then slowly eluted with HEPES buffer containing 50 mM imidazole in 1.0 mL fractions and the combined fractions containing hNUCB1 were passed through a Sephadex G-25M column to remove imidazole and concentrated in an Amicon centrifugal filter device (Millipore). These conditions produced HNUCB1 at approximately 10 mg / L of culture; however, several lower molecular weight fragments were also present in this preparation. hNUCB1 was further purified by size exclusion chromatography.FluoPol assay optimization

[0305] FluoPol conditions were optimized by screening various concentrations of Fl-AEA, hNUCB1, CaCl 2 and Pluronic F-127 against positive and negative controls. Arachidonic acid (20 µM final) was used as a positive control, and DMSO as a negative. Additionally, protein-free controls were used to normalize for background signal. Optimal conditions were selected based on maximal probe competition, and were found to be: 1.0 µM hNucb1, 0.5 µM Fl-AEA, 20 µM CaCl 2 , and 0.01% Pluronic F-127, using DPBS as a buffer system. The Z'-factor for these conditions was determined to be 0.54, indicating a high signal to noise assay.High throughput screen

[0306] A 10 µL solution of hNUCB1 (1 µM), Fl-AEA (0.5 µM), CaCl 2 (20 µM), and Pluronic F-127 (0.01%) was added to a Greiner 384-well plate. Additionally, in some wells protein was omitted to serve as a blank control for background signal. To the protein containing wells 16,000 compounds (50 nL in DMSO, 2 mM) from the Maybridge Hitfinder library were added by a pin-tool robot. Additionally, on each plate to some wells arachidonic acid (20 µM final) or DMSO were added as positive and negative controls to allow normalization between plates. The plates were allowed to incubate for 5 minutes at room temperature, then fluorescence polarization was measured by measuring fluorescence intensity through polarized 595 nm filters. Hits were selected based on maximal probe competition.In vitro gel-based competition lipid probes

[0307] Cell lysates (1.0 mg / mL), the indicated lipid probe (2.5 or 5 µM) and, for recombinant NUCB1 competition experiments, purified hNUCB1 (250 nM) and CaCl 2 (100 µM) were mixed on ice. DMSO or competitors were then added to the proteome mixture (50 µM) in 96-well plates and each sample was incubated for 15 min at 37 °C. The samples were then irradiated with UV light and subjected to previously described click chemistry conditions. As before, each reaction was quenched with 4X SDS loading buffer (17 µL), and proteins were resolved using SDS-PAGE (10% acrylamide gel) and analyzed by in-gel fluorescent scanning on a Hitachi FMBIO-II flatbed fluorescence scanner. NUCB1 competition was quantitatively assessed by measuring the fluorescent intensity of the corresponding gel band using ImageJ 1.48v and normalizing this value by a non-competed gel band to control for loading differences. Because residual, non-competitive NUCB1 labeling were observed at the highest concentration of the tested NUCB1 ligands, the IC50's were determined by calculating the percent of maximal competition (100 µM, MJN228) rather than percent of the background signal in lanes without recombinant NUCB1 added.Mapping probe-modified peptides

[0308] Mapping probe-modified peptides with purified NUCB1: To a solution of recombinant hNUCB1 (50 µL, 5.0 mg / mL) in DPBS and CaCl 2 (100 µM) was added either DMSO or AEADA (50 µM final concentration). After incubating at 37 °C for 15 min, the mixture was irradiated with UV light for 10 min at 4 °C. Standard reduction and alkylation steps were omitted because of the absence of cysteine residues in hNUCB1. Thus, the probe crosslinked hNUCB1 solution was diluted with urea (150 µL, 8.0 M in DPBS) and trypsinized by addition of sequence grade porcine trypsin (5.0 µg, Promega) and incubation overnight at 37 °C. The samples were then acidified with HCO 2 H (16 µL) and a 10 µL aliquot was pressure loaded onto a 100 µm (inner diameter) fused silica capillary column with a 5 µm tip containing 10 cm C18 resin (5 µm, Phenomenex). Peptides were eluted from the column using a 180 min gradient from 0% to 50% Buffer B (Buffer A: 5% acetonitrile, 95% water, 0.1% formic acid; Buffer B: 80% acetonitrile, 20% water, 0.1% formic acid) and analyzed on an LTQ-Orbitrap mass spectrometer (ThermoFisher).

[0309] Mapping probe-modified peptides in cells: Experiments were performed as described above using the AEA-DA probe (100 µM) in 3.0 mL of DMEM (serum-free) and Neuro2A cells without (twelve replicates; ten biological; two technical). For the ligand competition study, cells were treated with the AEA-DA probe (50 µM) and DMSO or MJN228 (50 µM) treatment. Cells were harvested and processed as described above. Whole cell lysates (500 µL at 1.5 mg / mL) were processed for MS analysis using the previously described isoTOPABPP protocol (Speers and Cravatt, 2005). In brief, for global mapping of AEA-DA-modified peptides, proteomes were split into two fractions, which were subjected to click chemistry conditions with either light or heavy isotopically labeled TEV-tags. For the MJN288 competition study, proteomes were subjected to click chemistry conditions with either light (DMSO) or heavy (MJN228-treated) TEV-tags. Light and heavy-tagged proteomes in each experiment were then combined, and, following enrichment of probe-labeled targets using streptavidin beads, proteins were digested on-bead with trypsin and remaining immobilized peptides released with a subsequent TEV protease digestion. The resulting probe-modified peptides were collected for MS analysis, which was performed as described above with differences in the salt bumps applied in the chromatographic gradients which were 0%, 30%, 60%, 90% and 100% NH4OAc (500 mM). The protein identification searches of the MS data were performed using the same software packages described above with the following changes applied to identify the peptides modified with AEA-DA and the cleaved TEV tag. All amino acids were considered as possible residues for modification. To facilitate the computational searches, sets of up to 3 amino acids were searched using ProLuCID and filtered with DTASelect as described above. The mass of the modification used to search for probe-modified peptides was +727.4745 m / z for the probe plus the light TEV-tag and +733.4831 m / z for the heavy counterpart. The isoTOP ratios for probe labeled peptides were quantified using the in-house software CIMAGE (Weerapana et al., 2010). For global mapping experiments, AEA-DA-modified peptides were expected to show a ratio of "heavy" and "light" signals of ~1.0 (0.5 < ratio < 2.0) and were required to have been detected in at least two of the twelve replicate experiments. Proteins containing probe-modified peptides were further categorized as having: i) one principal probe-modified peptide (where only a single probe-modified peptide was identified for the protein or the protein possessed a probe-modified peptide that was detected in at least 75% of the experiments and no other probe-modified peptide that was detected in more than 33% of the experiments); ii) two probe-modified peptides; or iii) three or more probe-modified peptides.Untargeted metabolomics

[0310] Discovery metabolite profiling was performed as described by Saghatelian et al., "Assignment of endogenous substrates to enzymes by global metabolite profiling," Biochemistry 43:14332-14339 (2004) with several modifications. Neuro2a cells were plated at a density of 4 x 10 6< cells / 10 cm plate and grown for 18 h. In glass vials, solutions of vehicle or test compounds were prepared in serum- and dye-free DMEM and warmed to 37 °C in a water bath. The media from the cells was removed and cells were washed with DPBS (2 x 5.0 mL) before adding freshly prepared media solutions containing vehicle or compound. After incubating for 6 h at 37 °C and subsequent removal of the media, the cells were harvested by scrapping in cold DPBS (3 mL) and pelleted by centrifugation in glass vials. The pellets were washed once with cold DPBS (3 mL) and then cold CH 3 Cl:MeOH (2:1) (3 mL) was added. After vortexing, the vials were centrifuged (3,000 x g, 10 min, 4 °C) and the bottom (organic) layer was transferred to a new glass vial and concentrated under a stream of N 2 . Samples were stored at -80 °C until the day of analysis when they were reconstituted in Mobile Phase A (100 µL, see below).

[0311] Lipid extracts were analyzed by LC / MS in both positive and negative modes using an Agilent 6520 QTOF instrument and LC separation performed on a Kinetex reversed phase C18 column (50 x 4.6 mm, 2.6 µm particle size, 100Å). Mobile phase A was composed of 5:1:4 isopropanol: methanol: water and Mobile phase B was composed of 99:1 isopropanol: water. Both mobile phases also contained 0.1% AcOH and NH 4 OAc (5 mM) to facilitate ionization and LC s...

Claims

1. A high throughput method of identifying lipid binding proteins as drug binding targets, comprising: a) harvesting a set of 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, wherein the proteomic analysis means comprises a mass spectroscopy method; c) based on step b), determining i) first and second values for each of the lipid binding proteins from the set of lipid probe-protein complexes, wherein the first value is determined for a lipid binding protein from a first group of lipid probe-protein complexes of the first cell solution and the second value is determined for the same lipid binding protein from a second group of lipid probe-protein complexes of the second cell solution, and the first and second values are the area-under-the curve from a plot of signal intensity as a function of mass-to-charge ratio, and calculating a ratio between the two values assigned to the same lipid binding protein; 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 ratio 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 that interact with the drug; wherein the lipid probe is a lipid probe of Formula (I): wherein: A is C16-C20alkyl or C16-C20alkenyl; D is -OH, -NH2, -NHR7, or -OCH(3-n)R8; R7 is C1-C4alkyl, (C1-C5alkyl)OH, or (C1-C5)SO3M; R8 is (CH2OH)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.

2. The method of claim 1, wherein the lipid probe comprises a lipid selected from arachidonoyl, arachidoyl, oleoyl, palmitoyl, or stearoyl fatty acyls.

3. The method of either claim 1 or claim 2, 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.

4. The method of any preceding claim, wherein the photoreactive group comprises azides, benzophenones, diazo compounds, diazirines, diazonium salts, or diaryl ketones, and optionally a linker.

5. The method of any preceding claim, wherein the affinity handle is a bioorthogonal affinity handle, optionally conjugated to an affinity ligand.

6. The method of any preceding claim, 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 an isocyanate group.

7. The method of claim 5, wherein the affinity ligand comprises a chromophore, a labeling group, or a combination thereof.

8. The method of any preceding claim, wherein the photoreactive group and the affinity handle are conjugated to the same site of the lipid probe.

9. The method of any one of claims 1 to 7, wherein the photoreactive group and the affinity handle are conjugated to different sites of the lipid probe.