DETAILED DESCRIPTION OF THE INVENTION

[0035] In accordance with the subject invention systems are provided for assaying proteomes as to a specific protein or group of related proteins and to determine the effects of agents on such proteins in a proteome. The systems include methods for identifying probes for use in the assay, the probes, the methods of reacting the probes with the proteomic mixture, and methods for analyzing the data from the assay for at least semi-quantitatively determining the target members of the proteome and the effect of the agents on the activity of a portion of the proteome. Kits are also provided of combinatorial libraries for screening proteomes to identify members having specific affinities and for screening proteomes, where the kits comprise different probes for different proteins or related groups of proteins. As part of the system for discovering probes for any related group of proteins, combinatorial libraries are employed having a common reactive functionality as part of a functional group and usually a common linker, linking the functional group to a ligand for which a receptor is available or a chemically reactive functionality reacting with a reciprocal functionality for adding a ligand. The results with an active proteome are repeated with an inactive proteome to determine the degree of activity of the total target protein as compared to active protein. Depending on the nature of the functional group, part of linker or part of the functional group may comprise the variable component.

[0036] The system uses probes specific for a specific or group of related proteins and combines one or a mixture of probes, depending on the specificity of the probes and the variety in the group or groups of related proteins to be assayed. The reaction mixture provides conditions under which the probes react substantially preferentially with active target proteins. By “active” is intended that the protein is in its native conformation and is able to interact with an entity that it normally interacts with, e.g. enzyme with substrate and cofactor, receptor with ligand, etc. Using the ligand, target conjugated probes are sequestered by means of the ligand and different protocols may be employed to determine the amount of the target proteins present in the medium as a group or individually. Optionally the sequestered proteins may be further assayed to identify the specific proteins to which the probes bound.

[0037] The combinatorial aspect of the present invention will be described first. There are provided combinatorial chemical libraries containing a plurality of activity-based probes (ABPs), where the individual members may be correspondingly tagged or labeled. The methods of the invention employ ABP-containing libraries for identifying bioactive compounds and for identifying target proteins in a mixture of proteins. It will be appreciated that the ABPs of the invention are class-selective and activity based. Therefore, the present invention allows for rapid target identification and isolation. (see U.S. Ser. No. 60/195,954, filed Apr. 10, 2000 and 60/212,891, filed Jun. 20, 2000, both of which are incorporated by reference in their entirety herein).

[0038] The combinatorial chemical libraries of the present invention are useful as screening tools for discovering new lead structures through evaluation of the compounds in the library across an array of biological assays, including the discovery of selective inhibition patterns across isozymes and related enzymes, where the enzymes share a common functionality at the active site, allelic proteins, binding to a family of ligands, etc. Thus, the library is useful as a tool for drug discovery, i.e., it is a means to discover novel lead compounds by screening the library against a variety of biological targets, and also as a tool for the development of structure-activity relationships in large families of related compounds. The combinatorial libraries after reacting with a proteome provide compositions of conjugates between members of the library and target proteins. Such compositions are useful for producing antibodies for sequestering the target proteins from the proteomic mixture, digestion and identification of the target protein using mass spectrometric analysis and data banks, as standards for measuring the amount of conjugate formed in other analyses with the same or different probes, and the like.

[0039] The inventive methods employ affinity-labeled or target protein directed protein reactive reagents, ABPs that allow for the selective detection and subsequent isolation of active proteins from complex mixtures. The isolated proteins are characteristic of the presence of a protein function, e.g., an enzymatic activity, protein complex formation, protein-nucleic acid interactions, etc., in those mixtures. Isolated proteins are optionally characterized by mass spectrometric (MS) techniques. In particular, the sequence of isolated proteins can be determined using tandem MS (MS ⁿ ) techniques, and by application of sequence database searching techniques, the protein from which a sequenced peptide originated can be identified. The ABPs also provide for differential labeling, e.g. isotopic or atomic (different atomic weight elements), of the isolated proteins, which facilitates quantitative determination by mass spectrometry of the relative amounts of active proteins in different samples.

[0040] A “combinatorial library” is a collection of compounds in which the compounds comprising the collection are composed of one or more types of sub-units. The library will have at least 2 members, rarely less than about 5 members, usually at least about 10 members, frequently will have about 50 members or more, usually fewer than about 1,000 members, more usually fewer than about 500 members. The sub-units may be selected from natural or unnatural moieties, including a variety of chemical moieties, such as synthetic compounds, naturally occurring compounds, e.g. amino acids, nucleotides, sugars, lipids, and carbohydrates, and synthetic analogs thereof, which are readily available commercially in a large variety of compounds. The compounds of the combinatorial library differ in one or more ways with respect to the number, order, type or types of or modifications made to one or more of the sub-units comprising the compounds. Alternatively, a combinatorial library may refer to a collection of “core molecular organization” which vary as to the conformation, size and charge distribution as a result of the presence of other moieties or differences in the way the core molecular organization is organized. The collection of compounds is generated in a systematic way. Any method of systematically generating a collection of sub-units differing from each other in one or more of the ways set forth above is a combinatorial library.

[0041] For understanding of the terms used in the subject application, a number of the generic terms is illustrated with examples coming within the genus.

[0042] A “chemical group” is an atom or assemblage of atoms and organic chemical groups include but are not limited to alkyl, alkenyl, alkynyl, alkoxy, aryl, alkylaryl, heterocycle including heteroaryl, amide, thioamide, ester, amine, ether, thioether, halo, imine, cyano, nitro, carboxy, keto, aldehydo, and combinations thereof.

[0043] “Alkyl” is intended to include aliphatically saturated linear or branched, hydrocarbon structures and combinations thereof “Lower alkyl” means alkyl groups of from 1 to 8 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, hexyl, octyl, and the like. Preferred alkyl groups are those of C20 or below, particularly C ₁₀ or below.

[0044] “Cycloalkyl” includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of lower cycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbornyl, decalin, and the like, and may be aliphatically saturated or unsaturated.

[0045] “Alkenyl” includes C2-C8 unsaturated hydrocarbons of a linear or branched configuration and combinations thereof. Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, 2,4-hexadienyl and the like.

[0046] “Alkynyl” includes C2-C8 hydrocarbons of a linear or branched configuration and combinations thereof containing at least one carbon-carbon triple bond. Examples of alkynyl groups include ethyne, propyne, butyne, pentyne, 3-methyl-1-butyne, 3,3-dimethyl-1-butyne and the like.

[0047] “Alkoxy” refers to groups of from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy and the like.

[0048] “Acylamino” refers to acylamino groups of from 1 to 8 carbon atoms of a straight, branched or cyclic configuration and combinations thereof. Examples include acetylamino, butyrylamino, cyclohexylanoylamino, and the like.

[0049] “Hydrocarbylamino” refers to a moiety consisting of hydrogen and carbon bonded to nitrogen and of from about 1 to 8 carbon atoms for each hydrocarbyl group, there being up to 4, usually 3, hydrocarbyl groups. By “hydrocarbyl is intended any molecule or core of a molecule composed solely of hydrogen and carbon.

[0050] “Halogen” includes F, Cl, Br, and I.

[0051] “Halophenyl” means phenyl substituted with 1-5 halogen atoms. Examples include pentachlorophenyl, pentafluorophenyl and 2,4,6-trichlorophenyl.

[0052] “Aryl” and “heteroaryl” mean a 5- or 6-membered aromatic or heteroaromatic ring containing 0-3 heteroatoms selected from O, N, or S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, or S; or a tricyclic 13- or 1 4-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, or S; each of which rings is optionally substituted with 1-3 lower alkyl, substituted alkyl, substituted alkynyl, ═O, —NO ₂ , halogen, hydroxy, alkoxy, OCH(COOH) ₂ , cyano, —NZZ, acylamino, phenyl, benzyl, phenoxy, benzyloxy, heteroaryl, or heteroaryloxy; each of said phenyl, benzyl, phenoxy, benzyloxy, heteroaryl, and heteroaryloxy is optionally substituted with 1-3 substituents selected from lower alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy, cyano, phenyl, benzyl, benzyloxy, carboxamido, heteroaryl, heteroaryloxy, —NO ₂ or —NZZ (wherein Z is independently H, lower alkyl or cycloalkyl, and -ZZ may be fused to form a cyclic ring with nitrogen).

[0053] The aromatic 6- to 14-membered carbocyclic rings include, e.g., benzene, naphthalene, indane, tetralin, and fluorene and the 5- to 10-membered aromatic heterocyclic rings include, e.g., imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole and pyrazole.

[0054] “Arylalkyl” means an alkyl residue attached to an aryl ring. Examples are benzyl, phenethyl and the like.

[0055] “Heteroarylalkyl” means an alkyl residue attached to a heteroaryl ring. Examples include, e.g., pyridinylmethyl, pyrimidinylethyl and the like.

[0056] “Heterocycloalkyl” means a cycloalkyl where one to two of the methylene (CH ₂ ) groups is replaced by a heteroatom such as O, NZ′ (wherein Zis H or alkyl), S or the like; with the proviso that except for nitrogen when two heteroatoms are present, they must be separated by at least one carbon atom. Examples of heterocycloalkyl include tetrahydrofuranyl, piperidine, dioxanyl and the like.

[0057] “Alkylcarbonyl” means —C(O)R′, wherein R′ is alkyl.

[0058] “Substituted” alkyl, alkenyl, alkynyl, cycloalkyl, or heterocycloalkyl means alkyl, alkenyl, alkynyl, cycloalkyl, or heterocycloalkyl wherein up to three H atoms on each C atom therein are replaced with halogen, hydroxy, loweralkoxy, carboxy, carboalkoxy, carboxamido, cyano, carbonyl, —NO ₂ , —NZZ; alkylthio, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, heteroaryloxy, or substituted phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, or heteroaryloxy.

[0059] “Aa” represents an amino acid, naturally occurring or synthetic, and is intended to include the racemates and all optical isomers thereof. The amino acid side chains of Aa include, e.g., methyl (alanine), hydroxymethyl (serine), phenylmethyl (phenylalanine), thiomethyl (cysteine), carboxyethyl (glutamic acid), etc. Primary and secondary amino acids are intended to include alanine, asparagine, N-β-trityl-asparagine, aspartic acid, aspartic acid-beta-t-butyl ester, arginine, N ^s -Mtr-arginine, cysteine, S-trityl-cysteine, glutamic acid, glutamic acid-γ-t-butyl ester, glutamine, N ⁶⁵ -trityl-glutamine, glycine, histidine, N ^im -trityl-histidine, isoleucine, leucine, lysine, N ^ε -Boc-lysine, methionine, phenylalanine, proline, serine, O-t-butyl-serine, threonine, tryptophan, N ⁱⁿ -Boc-tryptophan, tyrosine, valine, sarcosine, L-alanine, chloro-L-alanine, 2-aminoisobutyric acid, 2-(methylamino)isobutyric acid, D,L-3-aminoisobutyric acid, (R)-(−)-2 aminoisobutyric acid, (S)-(+)-2-aminoisobutyric acid, D-leucine, L-leucine, D-norvaline, L-norvaline, L-2-amino-4-pentenoic acid, D-isoleucine, L-isoleucine, D-norleucine, 2,3-diaminopropionic acid, L-norleucine, D,L-2-aminocaprylic acid, β-alanine, D,L-3-aminobutyric acid, 4-aminobutyric acid, 4-(methylamino)butyric acid, 5-aminovaleric acid, 5-aminocaproic acid, 7-aminoheptanoic acid, 8-aminocaprylic acid, 11-aminodecanoic acid, 12-aminododecanoic acid, carboxymethoxylamine, D-serine, D-homoserine, L-homoserine, D-allothreonine, L-allothreonine, D-threonine, L-threonine, D,L-4-amino-3-hydroxybutyric acid, D-,L-3-hydroxynorvaline, (3S,4S)-(−)-statine, 5-hydroxy-D,L-lysine, 1-amino-1-cyclopropanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cyclohexanecarboxylic acid, 5-amino-1,3-cyclohexadiene-1-carboxylic acid, 2-amino-2-norbomanecarboxylic acid, (S)-(−)-2-azetidinecarboxylic acid, cis-4-hydroxy-D-proline, cis-4-hydroxy-L-proline, trans-4-hydroxy-L-proline, 3,4-dehydro-D,L-proline, 3,4-dehydro-L-proline, D-pipecolinic acid, L-pipecolinic acid, nipecotic acid, isonipecotic acid, mimosine, 2,3-diaminopropionic acid, D,L-2,4-diaminobutyric acid, (S)-(+)-diaminobutyric acid, D-ornithine, L-ornithine, 2-methylornithine, N-ε-methyl-L-lysine, N-methyl-D-aspartic acid, D,L-2-methylglutamic acid, D,L-2-aminoadipic acid, D-2-aminoadipic acid, L-2-aminoadipic acid, (+/−)-3-aminoadipic acid, D-cysteine, D-penicillamine, L-penicillamine, D,L-homocysteine, S-methyl-L-cysteine, L-methionine, D-ethionine, L-ethionine, S-carboxymethyl-L-cysteine, (S)-(+)-2-phenylglycine, (R)-(−)-2-phenylglycine, N-phenylglycine, N-(4-hydroxyphenyl)glycine, D-phenylalanine, (S)-(−)indoline-2-carboxylic acid, α-methyl,D,L-phenylalanine, α-methyl-D,L-phenylalanine, D-homophenylalanine, L-homophenylalanine, D,L-2-fluorophenylglycine, D,L-2-fluorophenylalanine, D,L-3-fluorophenylalanine, D,L-4-fluorophenylalanine, D,L-4-chlorophenylalanine, L-4-chlorophenylalanine, 4-bromo-D,L-phenylalanine, 4-iodo-D-phenylalanine, 3,3′,5-triiodo-L-thyronine, (+)-3,3′,5-triiodo-L-thyronine, D-thyronine, L-thyronine, D,L-tyrosine, D-4-hydroxyphenylglycine, D-tyrosine, L-tyrosine, O-methyl-L-tyrosine, 3-fluoro-D,L-tyrosine, 3-iodo-L-tyrosine, 3-nitro-L-tyrosine, 3,5-diiodo-L-tyrosine, D,L-dopa, L-dopa, 2,4,5-trihydroxyphenyl-D,L-alanine, 3-amino-L-tyrosine, 4-amino-D-phenylalanine, 4-amino-L-phenylalnine, 4-amino-D,L-phenylalanine, 4-nitro-L-phenylalanine, 4-nitro-D,L-phenylalanine, 3,5-dinitro-L-tyrosine, D,L-α-methyltyrosine, L-α-methyltyrosine, (−)-3-(3,4-dihydroxyphenyl)-2-methyl-L-alanine, D,L-threo-3-phenylserine, trans-4-minomethyl)cyclohexane carboxylic acid, 4-(aminomethyl)benzoic acid, D,L-3-aminobutyric acid, 3-aminocyclohexane carboxylic acid, cis-2-amino-1-cyclohexane carboxylic acid, gamma-amino-beta-(p-chlorophenyl)butyric acid (Baclofen), D,L-3-aminophenylpropionic acid, 3-amino-3-(4-chlorophenyl)propionic acid, 3-amino-3-(2-nitrophenyl)propionic acid, and 3-amino-4,4,4-trifluorobutyric acid.

[0060] An “alkylaryl group” refers to an alkyl (as described above), covalently joined to an aryl group (as described above).

[0061] “Carbocyclic aryl groups” are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.

[0062] “Heterocyclic aryl groups” are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl,pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.

[0063] An “amide” refers to an —C(O)—NH—, where Z is either alkyl, aryl, alklyaryl or hydrogen.

[0064] A “thioamide” refers to —C(S)—NH—Z, where Z is either alkyl, aryl, alklyaryl or hydrogen.

[0065] An “ester” refers to an —C(O)—OZ′, where Z′ is either alkyl, aryl, or alklyaryl.

[0066] An “amine” refers to a —N(Z″)Z′″, where Z″ and Z′″, is independently either hydrogen, alkyl, aryl, or alklyaryl, provided that Z″ and Z′″ are not both hydrogen.

[0067] An “ether” refers to Z—O—Z, where Z is either alkyl, aryl, or alkylaryl.

[0068] A “thioether” refers to Z—S—Z, where Z is either alkyl, aryl, or alkylaryl.

[0069] A “cyclic molecule” is a molecule which has at least one chemical moiety which forms a ring. The ring may contain three atoms or more. The molecule may contain more than one cyclic moiety, the cyclic moieties may be the same or different.

[0070] A “linear molecule” does not contain a ring structure. However, the molecule may be straight or branched.

[0071] An “active site” of a protein refers to the specific area on the surface of a protein, e.g., an enzyme molecule or surface membrane receptor, to which a binding molecule, e.g. substrate or reciprocal ligand. is bound and results in a change in the ligand, e.g. substrate or complex formation with the protein as a result of ligand binding. For a receptor, the conformation may change, the protein may become susceptible to phosphorylation or dephosphorylation or other processing. For the most part, the active site will be the site(s) of an enzyme where the substrate and/or a cofactor bind, where the substrate and cofactor undergo a catalytic reaction, where two proteins form a complex, e.g. the site at which a G protein binds to a surface membrane receptor, two kringle structures bind, sites at which transcription factors bind to other proteins, sites at which proteins bind to specific nucleic acid sequences, etc. In the case of a membrane receptor, the active site will be the external site to a membrane, where the ligand binds and causes transduction of a signal.

[0072] The “activity-based probes” or “ABP”s of the invention are chemical reagents that are polyfunctional molecules for non-competitive or substantially irreversible binding to a target protein and inhibiting the action of the target protein. The ABPs will comprise at least a reactive functionality and a ligand and have an affinity for a related group of proteins, whereby the ABP will bind to the target protein and substantially inactivate the protein, and the ligand will permit detection and/or isolation.

[0073] In referring to affinity for an ABP to a target protein, one is concerned with the on-rate of the ABP with the target protein, since there is no off-rate, where the ABP covalently bonds to the target protein. One can determine relative on-rates between ABPs by having less than a stoichiometric amount of the target protein as compared to the total amount of a plurality of ABPs and then measuring the relative amounts of the conjugates for each of the ABPs. In this way one can obtain a measure of the relative activity of each of the ABPs toward the target protein, which for the purposes of this invention may be considered the affinity, if not the binding affinity, of the ABP for the target protein.

[0074] Exemplary protein targets described herein include enzymes, included in the groups oxidoreductases, hydrolases, ligases, isomerases, transferases, and lyases and include such enzymes or enzyme groups as serine hydrolases, metallo-hydrolases, dehydrogenases, e.g. alcohol and aldehyde dehydrogenases, and nucleotide triphosphate (NT)-dependent enzymes, although, the invention envisions ABPs which recognize any protein, e.g., enzyme, family. Other proteins include proteins that bind to each other or to nucleic acids, such as transcription factors, kringle structure containing proteins, nucleic acid binding proteins, G-protein binding receptors, cAMP binding proteins, etc. The structure of ABPs of the invention is described more fully below.

[0075] An “active protein” of the invention refers to a protein, e.g., enzyme, in its normal wild-type conformation, e.g. a catalytically active state, as opposed to an inactive state. The active state allows the protein, to function normally. An inactive state may be as a result of denaturation, inhibitor binding, either covalently or non-covalently, mutation, secondary processing, e.g. phosphorylation or dephosphorylation, etc. Functional states of proteins or enzymes as described herein may be distinct from the level of abundance of the same proteins or enzymes. An active site is an available wild-type conformation at a site that has biological activity, such as the catalytic site of an enzyme, a cofactor-binding site, the binding site of a receptor for its ligand, and the binding site for protein complexes, for example. In many instances, one is interested in knowing the level of availability of such sites. Targets of interest will be particularly enzymes, other proteins include receptors, transcription factors, G-proteins, and the like.

[0076] The subject systems are useful for, among other things, developing new drugs and identifying new drug targets. One embodiment of the subject invention is especially useful for rapidly generating and developing large numbers of drug candidate molecules. The invention is useful for systematically synthesizing a large number of molecules that may vary greatly in their chemical structure or composition, or that may vary in minor aspects of their chemical structure or composition. The invention is also useful for randomly generating a large number of drug candidates, and later optimizing those candidates that show the most medicinal promise. The combinatorial libraries of the present invention may also be screened for diagnostically useful compounds. By diagnostically useful is meant that the compound can be used to indicate the presence of a particular disease in a human or animal.

[0077] The combinatorial libraries of the present invention may be screened for pharmacologically active compounds, including analogs, that is compounds that can affect the biological status of a biological system, usually a cellular system. The biological system will depend on the use of a biological source that will include cells and/or viruses. By pharmacologically active is meant that a compound may effect the function of a protein, e.g, an enzyme, including physiological process such as signal transduction by a cellular receptor, initiation, cessation or modulation of an immune response, modulation of heart function, nervous system function, or any other organ or organ system. A pharmacologically active compound may also stimulate or inhibit the activity of a bacteria, virus, fungus, or other infectious agent. A pharmacologically active compound may modulate the effects of a disease, that is prevent or decrease the severity of or cure a disease such as cancer, diabetes, atherosclerosis, high blood pressure, Parkinson's disease and other disease states. Screening for pharmacological activity may be performed by assays as would be known in the art, depending on the function or activity to be assessed. Compounds which have been shown to be pharmacologically active compounds may be formulated for therapeutic administration by methods known in the art. Methods have been reported in the literature by which individual members of combinatorial libraries may be encoded by “tagging molecules” (“tags” or “labels”). See, for example, U.S. Pat. Nos. 5,721,099 and 6,001,579. Thus, a single molecular structure synthesized on a resin bead, for instance, is uniquely defined by a series of other, readily detectable molecules also bound to a bead. Individual beads are treated to release their library member, by a process which does not displace the tag, and following identification of this compound as an “active” in a biological screen, the tags are released and analyzed to deduce the identity of the “hit”. To allow for maximum diversity in a library it is critical that the chemistry used to introduce the tags is tolerated by a wide range of functionality. Thereby, introduction of the tagging molecule does not lead to undesired elaboration of the library structure, or alternatively, place limits upon the chemistry used to construct the library. Similarly, if the tag is removed prior to the library member, the conditions for removal of the tag does not destroy or react in some manner with the designed molecule.

[0078] The material upon which the combinatorial syntheses of the present invention are performed are referred to as solid supports, beads or resins. These terms are intended to include beads, pellets, disks, fibers, gels, or particles such as cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene and optionally grafted with polyethylene glycol and optionally functionalized with amino, hydroxy, carboxy, or halo groups, grafted co-poly beads, poly-acrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N,N′-bis-acryloyl ethylene diamine, glass particles coated with hydrophobic polymer, and the like, e.g., material having a rigid or semi-rigid surface; and soluble supports such as low molecular weight non-cross-linked polystyrene.

[0079] By “biological status” is intended to include mRNA profile, protein profile, total and/or active, spatial distribution profile of the proteins and mRNA, maturity of cells, population of surface membrane proteins, amount and spatial distribution of complexes, amount of ligands present, bound and unbound, lipid population, processing of proteins, such as glycosylation, methylation, acylation, phosphorylation, ubiquination, farnesylation, etc., those differences that distinguish cellular populations.

[0080] Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present invention is meant to include all such possible diastereomers, as well as, their racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0081] Activity-based probes (ABPs) are provided for specific reaction with the active site of one or more target proteins, where the target protein is a member of a class of proteins, particularly enzymes, for detection of the presence and quantitation of one or more active members. The ABPs have a common electrophile whose environment is changed in each of the ABPs to provide a different reactivity with different target proteins. The probes may be divided into four general regions: (1) a functional group (F) that specifically and covalently bonds to the active site of a protein; (2) a label or ligand (hereinafter collectively referred to as “ligand”) for sequestering and detecting the conjugate of the ABP and the active protein (X) 3) a linker L, between the F and the L; and 4) binding moiety or affinity label, that may be associated with or part of the linker region and/or the functional group (R). A linker is a bond or chemical group used to link one moiety to another, serving as a divalent bridge, where it provides a group between two other chemical moieties. Binding or affinity moiety refers to a chemical group, which may be a single atom, that is conjugated to the reactive functional group or associated with the linker, as a side chain or in the chain of the linker, and provides enhanced binding affinity for protein targets. A ligand refers to a molecule that can be used to detect and/or capture the ABP in combination with any other moieties that are bound strongly to the ligand so as to be retained in the process of the reaction of the functional group with the target active protein. The ABP may include a chemically reactive functionality, not found in proteins, that will react with a reciprocal functionality, e.g. vic.-diols with boronic acid, aldehydes and ketones, etc. These reactive functionalities may be used to bind to a ligand after reaction with the target protein. In some embodiments described herein, the ABP may be truncated and lack the ligand, but will always have a functional group, F, a linker, L and an R group (binding moiety), but no ligand, X (see FIG. 1 ).

[0082] The ABP will have an affinity for an active site, which may be specific for a particular active site or generally shared by a plurality of related proteins. The affinity may result from the functional group, the linker, or the binding moiety or combination thereof. For drug discovery, one may be interested in specificity for a single target, while for proteome analysis, one will usually be interested in binding to a plurality of targets that are related.

[0083] Exemplary Fs as used in an ABP of the invention include an alkylating agent, acylating agent, ketone, aldehyde, sulphonate or a phosphorylating agent. Examples of particular Fs include, but are not limited to fluorophosphonyl, fluorophosphoryl, fluorosulfonyl, alpha-haloketones or aldehydes or their ketals or acetals, respectively, alpha-haloacyls, nitriles, sulfonated alkyl or aryl thiols, iodoacetylamide group, maleimides, sulfonyl halides and esters, isocyanates, isothiocyanantes, tetrafluorophenyl esters, N-hydroxysuccinimidyl esters, acid halides, acid anhydrides, unsaturated carbonyls, alkynes, hydroxamates, alpha-halomethylhydroxamates, aziridines, epoxides, or arsenates and their oxides. Sulfonyl groups may include sulfonates, sulfates, sulfinates, sulfamates, etc., in effect, any reactive functionality having a sulfur group bonded to two oxygen atoms. Epoxides may include aliphatic, aralkyl, cycloaliphatic and spiro epoxides, the latter exemplified by fumagillin, which is specific for metalloproteases.

[0084] The ABPs of the subject invention may be illustrated by the following formula:

R*(F-L)-X

[0085] where the symbols are as defined previously and the asterisk intends that R may be included in F or L and X is bonded to L, more specifically: wherein:

[0086] X is a ligand present prior to formation of said product or added to a reactive functionality to provide said ligand, said ligand having the same chemical structure for each of said members of said library;

[0087] L is a bond or linking group, which is the same in each of the members of said library;

[0088] F is a functional group reactive at an active site of a protein member, which functional group comprises the same reactive functionality in each of the members of said library; and

[0089] R is a group of less than 1 kDal, that is different in each of the members of the library; the * intends that R is a part of F or L; and

[0090] wherein members of said library have different on rates with said protein member. For example, when X is biotin or any ligand, L is any linker of varied composition and length, F is a sulfonate and R is a pyridyl group, a distinct protein profile is observed as compared with the same ABP wherein the R group is methyl ( FIG. 2 ). Thus by varying R when bonded to a sulfonyl group, different binding profiles will be obtained, so that one can look for specificity, which may lead to designing a drug based on the structure of R or look for binding to related target proteins for proteome analysis.

[0091] Illustrative of the method is the use of a reactive functionality having a leaving group that is varied to provide the combinatorial library for identifying a chemical compound which affects the activity of a protein. The method includes contacting a combinatorial chemical library with a biological sample, where the library comprises a plurality of differing functional groups (F-R) reactive with an active protein, wherein, for example, a sulfonate ester can have R as any group, such as alkyl, heterocyclic, such as pyridyl, substituted pyridyl, imidazole, pyrrole, thiophene, furan, azole, oxazole, aziridine, etc., aryl, substituted aryl, amino acid or peptidyl, oligonucleotide or carbohydrate group; and detecting an effect on a biological activity in a biological sample (e.g., inhibition of cell proliferation or inhibition of an enzyme activity). One can take an ABP identified by screening the libraries as described herein and confirm the specificity of the ABP for the protein. For example, as exemplified in FIG. 3 , one can take an individual ABP identified from a library of sulfonates and identify through MALDI mapping, a target enzyme, as shown in the Experimental section, aldehyde dehydrogenase (ADH). An ABP lacking X, or a label (e.g., biotin) is utilized to confirm the inhibition of ADH activity by the specific ABP identified. To further characterize the bond between the sulfonate ABP and the ADH, for example, one can include modifying agents in an assay for detection of inhibition of ADH activity. For example, in FIG. 7 , addition of a variety of free thiols, including glutathione (GSH or glut), dithiothreitol (DTT), or a-mercaptoethanol (BME), did not quench the bonding of the ABP/ADH, and provides further information as to the bond between the ABP and newly identified ADH. Other protein effectors, thiols, metals, chelators (e.g., EDTA), ATP, calcium and the like can be added to reactions between an ABP and protein target to provide further characterization.

[0092] The ligand portion permits capture of the conjugate of the target protein and the probe. The ligand may be displaced from the capture reagent by addition of a displacing ligand, which may be free ligand or a derivative of the ligand, or by changing solvent (e.g., solvent type or pH) or temperature conditions or the linker may be cleaved chemically, enzymatically, thermally or photochemically to release the isolated materials (see discussion of the linker moiety, below).

[0093] Examples of ligands (including labels), X, include, but are not limited to, biotin, deiminobiotin, dethiobiotin, vicinal diols, such as 1,2-dihydroxyethane, 1,2-dihydroxycyclohexane, etc., digoxigenin, maltose, oligohistidine, glutathione, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, a peptide of polypeptide, a metal chelate, a saccharide, rhodamine or fluorescein, or any hapten to which an antibody can be generated.

[0094] Examples of ligands and their capture reagents include but are not limited to: dethiobiotin or structurally modified biotin-based reagents, including deiminobiotin, which bind to proteins of the avidin/streptavidin family, which may, for example, be used in the forms of strepavidin-Agarose, oligomeric-avidin-Agarose, or monomeric-avidin-Agarose; any 1,2-diol, such as 1,2-dihydroxyethane (HO—CH ₂ —CH ₂ —OH), and other 1,2-dihyroxyalkanes including those of cyclic alkanes, e.g., 1,2-dihydroxycyclohexane which bind to an alkyl or aryl boronic acid or boronic acid esters, such as phenyl-B(OH) ₂ or hexyl-B(OEthyl) ₂ which may be attached via the alkyl or aryl group to a solid support material, such as Agarose; maltose which binds to maltose binding protein (as well as any other sugar/sugar binding protein pair or more generally to any ligand/ligand binding protein pairs that has properties discussed above); a hapten, such as the dinitrophenyl group, for any antibody where the hapten binds to an anti-hapten antibody that recognizes the hapten, for example the dinitrophenyl group will bind to an anti-dinitrophenyl-lgG; a ligand which binds to a transition metal, for example, an oligomeric histidine will bind to Ni(II), the transition metal capture reagent may be used in the form of a resin bound chelated transition metal, such as nitrilotriacetic acid-chelated Ni(II) or iminodiacetic acid-chelated Ni(II); glutathione which binds to glutathione-S-transferase.

[0095] In general, any affinity label-capture reagent commonly used for affinity enrichment which meets the suitability criteria discussed above can be used in the method of the invention. Biotin and biotin-based affinity tags are particularly illustrated herein. Of particular interest are structurally modified biotins, such as deiminobiotin or dethiobiotin, which will elute from avidin or streptavidin (strept/avidin) columns with biotin or under solvent conditions compatible with ESI-MS analysis, such as dilute acids containing 10-20% organic solvent. It is expected that deiminobiotin tagged compounds will elute in solvents below about pH 4.

[0096] The linker group, while potentially can be a bond, is preferred to be other than a bond. The linker can be a cleavable linker that is cleaved, for example, by thermal, chemical or photochemical reaction. The choice of linker for the label and the functional group will be part of the synthetic strategy, since depending on the synthetic strategy, the linking group can result in a residual functionality on the product upon release from the support. It will usually be difficult, but feasible, to further modify the product after detachment from the bead. In designing the synthetic strategy, one can use a functionality to be retained in the product as the point of attachment for the linking group. Alternatively, when permitted by the nature of the product, one could use a cleavage or detachment method which removes the linking functionality, e.g., an arylthioether or silyl with a metal hydride or acid. Since in many cases, the synthetic strategy will be able to include a functionalized site for linking, the functionality can be taken advantage of in choosing the linking group. In some instances it may be desirable to have different functionalities at the site of linking the product to the support, which may necessitate using different modes of linking, which modes must accommodate either the same detachment method or different detachment methods which may be carried out concurrently or consecutively, e.g., irradiation with light and acid hydrolysis. The choice of linker, as with the choice of an R group, has been shown to alter the specificity of an ABP. For example, a linker for FP biotin, described in the Examples herein (see also FIG. 1 ), and a linker comprising PEG, have distinct specificities and provide distinct protein profiles (see FIG. 6 ). Thus, one of skill in the art can select the linker portion of the ABP in order to provide additional specificity of the ABP for a particular protein or protein class.

[0097] Photocleavable groups in the linker may include the 1-(2-nitrophenyl)ethyl group. Thermally labile linkers may include a double-stranded duplex formed from two complementary strands of nucleic acid, a strand of a nucleic acid with a complementary strand of a peptide nucleic acid, or two complementary peptide nucleic acid strands which will dissociate upon heating. Cleavable linkers also include those that have disulfide bonds, acid or base labile groups, including diarylmethyl or trimethylarylmethyl groups, silyl ethers, carbamates, oxyesters, thioesters, thionoesters and alpha-fluorinated amides and esters. Enzymatically cleavable linkers can contain protease-sensitive amides or esters, beta-lactamase-sensitive beta-lactam analogs and linkers that are nuclease-cleavable or glycosidase cleavable.

[0098] Linker groups include among others, ethers, polyethers, diamines, ether diamines, polyether diamines, amides, polyamides, polythioethers, disulfides, silyl ethers, alkyl or alkenyl chains (straight chain or branched and portions of which may be cyclic) aryl, diaryl or alkyl-aryl groups. While normally amino acids and oligopeptides are not preferred, when used they will normally employ amino acids of from 2-3 carbon atoms, i.e. glycine and alanine. Aryl groups in linkers can contain one or more heteroatoms (e.g., N, O or S atoms). Linkages also include substituted benzyl ethers, esters, acetals or ketals, diols, and the like (See, U.S. Pat. No. 5,789,172 for a list of useful functionalities and manner of cleavage, herein incorporated by reference). The linkers, when other than a bond, will have from about 1 to 60 atoms, usually 1 to 30 atoms, where the atoms include C, N, O, S, P, etc., particularly C, N and O, and will generally have from about 1 to 12 carbon atoms and from about 0 to 8, usually 0 to 6 heteroatoms. The atoms are exclusive of hydrogen in referring to the number of atoms in a group, unless indicated otherwise.

[0099] The linker and/or the ligand may be isotopically labeled, for example by substitution of one or more atoms in the linker with a stable isotope. For example, ¹ H can be substituted with ² H or ¹² C can be substituted with ¹³ C. Alternatively one may substitute one atom for another, such as H with F, use unsaturation to provide a different mass, or other known means. While ligands or linking groups may have different isotopic distributions, for the purposes of this invention they will be considered to be of the same chemical composition, where the atomic numbers of the atoms and their organization in the ligands or linking groups is the same. Therefore, in one aspect, the method of the invention provides for labeling of the ligand and/or linker to facilitate quantitative analysis by mass spectrometry of the amounts of proteins in different samples or in samples subjected to different conditions (e.g., in the presence and absence of a drug). Further, the label or linker can be non-radioisotopically labeled, e.g, with a fluorophore. In one aspect, the label produces an electromagnetic signal.

[0100] The process and compositions described in WO00/11208, which is specifically incorporated by reference herein, may be used with the suhject invention. In the application, one uses an affinity tagged, substantially chemically identical and differentially isotopically labeled probe, where the conjugates or fragments thereof are identified by mass spectrometry. The ratio of the different isotopic probes for each of the proteins with which the probes have reacted provides for the relative quantities of the individual proteins.

[0101] Linkers may be varied widely depending on their function, including alkyleneoxy and polyalkyleneoxy groups, where alkylene is of from 2-3 carbon atoms, methylene and polymethylene, polyamide, polyester, and the like, where individual monomers will generally be of from 1 to 6, more usually 1 to 4 carbon atoms. The oligomers will generally have from about 1 to 10, more usually 1 to 8 monomeric units. The monomeric units may be amino acids, both naturally occurring and synthetic, oligonucleotides, both naturally occurring and synthetic, condensation polymer monomeric units and combinations thereof. Alteration in the linker region has been shown to alter the specificity of the ABP for a target protein or class of proteins (e.g., enzymes).

[0102] Since the combinatorial production of probes is intended to find probes specific for target proteins and/or probes that provide information about the active site of a protein, these probes will usually be modified so as to be identifiable. Also, there can be a cleavable site between the target protein and at least a portion of the probe, where the identifiable moiety of the probe can be released from the target protein. Once the probe has been identified, for example, by mass spectrometry, fluorometry, electrochemically, etc., or combination thereof, the single probe may then be used with the same proteome mixture. At this stage, the protein target(s) can then be determined by conventional ways, using immunoassays, if available, sequencing, mass spectrometry and the like. The affinity label will then provide a basis for the design of a drug specific for the target protein.

[0103] With the ABP compounds described herein, screening assays such as FACS sorting and cell lawn assays may be used. When ligand, X, is detached prior to evaluation, its relationship to its solid support can be maintained, e.g., by location within the grid of a standard 96-well plate or by location of activity on a lawn of cells. Whether the compounds are tested attached to or detached from, the solid supports, the tags attached to the solid support associated with bioactivity may then be decoded to reveal the structural or synthetic history of the active compound (see for example, Ohlmeyer et al., Proc. Natl. Acad. Sci. USA, 90, 10922-10926, 1993). The usefulness of such libraries as screening tools is demonstrated by Burbaum et al., (Proc. Natl. Acad. Sci. USA, 92, 6027-6031, 1995), who describe the assaying of encoded combinatorial libraries for, e.g., carbonic anhydrase inhibition. Even when none of the compounds in a particular assay are found to be active for a given screen, such lack of activity often, however, provides useful structure-activity information.

[0104] In one embodiment, the invention employs two different compositions, intact ABPs and truncated ABPs lacking the ligand. See FIG. 1 . In most cases, the combination will employ combinatorial libraries that are being screened for bioactivity. For most intact ABPs containing ligands, it is difficult to obtain entry into an intact cell. Using electroporation, permeabilizing agents, or the like will change the status of the cell and could interfere with the assay. To use convenient ligands, such as biotin, one prepares two libraries, intact ABPs and truncated ABPs lacking the ligand. One then introduces the truncated ABPs under conditions that allow the truncated ABPs to enter the cell without significant disruption of the cell membrane. Any change in phenotype may be determined, such as apoptosis, proliferation, change in surface membrane proteins, inability to respond to ligands for cellular receptors, etc. If the change in phenotype is of interest, the cells may then be lysed and the lysate treated with the intact ABPs. In addition, one would use an inactivated lysate, to compare and select only those proteins that were conjugated in the active lysate and not conjugated in the inactivated lysate. The proteins to which the ABPs are conjugated can then be characterized in manners discussed in the specification and in the Experimental section. Alternatively, one may establish conditions for transport of the ABP into the cell without modifying the cell to change its response or choose lipophilic ligands or provide conditions where the ABPs will be entrained with another compound that provides permeability. In this way, one can determine conditions that will allow the screening of libraries where the ABPs will enter the cells without an undesirable change in the character of the cell. These approaches allow for the determination of ABPs that can serve as precursors in the design of drugs for specific binding to target proteins, while at the same time determining the effect on the phenotype of modifying the activity of the target protein. Of course, depending on the nature and size of the combinatorial library, one or more libraries may be used.

[0105] The above described method may also find application in determining whether the cellular environment affects the reaction of the ABP with a protein. One can use a radioactive label with the truncated ABP to identify whether the protein that reacted in the lysate also reacted in the cell. Alternatively, one may prepare monoclonal antibodies to the protein-ABP conjugates obtained in the lysate and use them to fish out the analogous protein from a cell that has been treated with the truncated ABP. By establishing that the truncated ABP bound to the same protein as the analogous ABP, one would establish that the specific affinity of the ABP provided the same intracellular activity or affinity as observed in the lysate.

[0106] Finally, one of skill in the art can identify the biological target in more detail by standard methods including SDS-PAGE or Western Blot analysis. As an illustrative example, the following protocol can be used to identify biological targets in a sample. After incubation of protein sample (0.5-2.5 mL, 0.5-1.0 mg/mL) with the ABPs, the sample is diluted to 2.5 mL in Tris or phosphate buffer and passed over a PD-10 size exclusion column to remove excess unreacted ABP. The protein is collected from the column in 3.5 mL of buffer, treated with SDS (final concentration of 0.5%), and heated for 10 min at 80° C. The sample is then diluted 2.5× and incubated with 100 μL of avidin agarose beads (Sigma) for 1-4 hours at room temp. The beads are then washed with several volumes of buffer to remove unbound protein and the ABP-labeled proteins are eluted with 100 μL of standard SDS-PAGE loading buffer by heating at 90° C. for 5 minutes. The eluted proteins are run on an SDS-PAGE gel and ABP-labeled proteins identified by staining and/or avidin blotting, excised from the gel, digested with trypsin, and the resulting peptide mixture characterized by MALDI and/or electrospray mass spectrometry. The mass spectrometric information is used in database searches to identify the ABP-labeled proteins.

[0107] Once one has established a probe, by combinatorial or other means, generally the probe may then be used to analyze a proteome for active protein(s). The probe may be specific for a single protein or more usually a related group of proteins. By related group of proteins is intended proteins that perform the same activity, as with enzymes that belong to the same group and catalyze the same reaction, e.g. hydrolysis, phosphorylation, oxidation, etc., and usually having one or more of the following characteristics: the same functionality at the active site; the same spatial orientation of functional groups that bind to the ligand; similar spatial structure and conformation; similar molecular weight; the same or similar cofactors or complexing proteins; and similar function. To enhance the distinction between active proteins and inactive proteins, special chemically reactive groups are employed.

[0108] A “chemically reactive group” is a moiety including a reactive functionality that does not react efficiently with the generally available functional groups of proteins, e.g. amino, hydroxy, carboxy, and thiol, but will react with a functionality present in a particular conformation on a surface. In some situations the reactive functionality will serve to distinguish between an active and an inactive protein. In other situations, the conformation of the chemically reactive group will bind to the specific conformation of the target protein(s), whereby with a slowly reactive functionality or one that requires activation, the predominant reaction will be at the active site. For example a photoactivatable group may be used such as a diazoketone, arylazide, psoralen, arylketone, arylmethylhalide, etc. any of which can bind non-selectively to the target protein, while the probe is bound to the active site. Olefins and acetylenes to which are attached electron withdrawing groups such as a sulfone, carbonyl, or nitro group may be used to couple to sulfhydryl groups.

[0109] A detectable label is a group that is detectable at low concentrations, usually less than micromolar, preferably less than nanomolar, that can be readily distinguished from other analogous molecules, due to differences in molecular weight, redox potential, electromagnetic properties, binding properties, and the like. The detectable label may be a hapten, such as biotin, or a fluorescer, or an oligonucleotide, capable of non-covalent binding to a complementary receptor other than the active protein; a mass tag comprising a stable isotope; a radioisotope; a metal chelate or other group having a heteroatom not usually found in biological samples; a fluorescent or chemiluminescent group preferably having a quantum yield greater than 0.1; an electroactive group having a lower oxidation or reduction potential than groups commonly present in proteins; a catalyst such as a coenzyme, organometallic catalyst, photosensitizer, or electron transfer agent; a group that affects catalytic activity such as an enzyme activator or inhibitor or a coenzyme.

[0110] Detectable labels may be detected directly by mass spectroscopy, detection of electromagnetic radiation, measurement of catalytic activity, potentiometric titration, cyclic voltametry, and the like. Alternatively labels may be detected by their ability to bind to a receptor thereby causing the conjugate to bind to the receptor. Binding of the conjugate to a receptor can be detected by any standard method such as ellipsometry, acoustic wave spectroscopy, surface plasmon resonance, evanescent wave spectroscopy, etc. when binding is to a surface, or by an immunoassay such as ELISA, FRET, SPA, RIA, in which the receptor may carry a label and an antibody to the active protein can be employed which may optionally carry a second label. Detectable labels may also be detected by use of separation methods such as HPLC, capillary or gel electrophoresis, chromatography, immunosorption, etc. In these methods the conjugate can be caused to bind to a member of an array of specific binding substances such as an array of antibodies where each member is an antibody for a specific active protein.

[0111] One aspect of the method of the invention is subjecting a portion of the sample to conditions that inactivate proteins in the sample. Significantly, information from studies with ABPs is preferable if one compares the level of protein conjugates in a portion of the sample that has been treated with inactivating conditions to a portion of the sample that contains active, wild-type or untreated proteins. Active wild-type proteins intend proteins with their natural conformation that are capable of carrying out binding and other functions, as appropriate. Other functions include enzymatic activity, ability to be modified, e.g. phosphorylation or dephosphorylation, acylation, etc., binding to cofactors or other proteins, functions that are necessary for biological activity. Differences between the protein profile in each of the active and the inactive portion are detected in order to identify active proteins, e.g. enzymes, in the sample. Inactivating conditions include chemical or physical means for inactivating, normally by denaturing the protein. For example, chemical means include denaturants such as organic solvents, harsh detergents, e.g. SDS, chaotropic.agents, e.g. urea, guanidinium chloride or isocyanate, etc., and other denaturing agents. Physical means include heat, freezing, electromagnetic radiation, shearing, drying, electrical discharge and the like. Inactivating agents that bind to the active site or an allosteric site affecting activity may bind covalently or non-covalently, with non-covalent binding being preferable. In a preferred embodiment, proteins in a sample are inactivated by heating, although other agents will be preferred if heating results in precipitation of the protein making it unavailable for reaction with an ABP.

[0112] Samples that can be analyzed by methods of the invention include biological samples, such as cell lysates, microsomal fractions, cell fractions, tissues, organelles, etc., and biological fluids including urine, sputum, saliva, blood, cerebrospinal fluid, tears, ejaculate, serum, pleural fluid, ascites fluid, stool, or a biopsy sample.

[0113] If the sample is impure (e.g., plasma, serum, stool, ejaculate, sputum, saliva, cerebrospinal fluid, or blood or a sample embedded in paraffin), it may be treated prior to employing a method of the invention, frequently to remove contaminants of the components of interest. Procedures include, for example, filtration, extraction, centrifugation, affinity sequestering, etc. Where the probes do not readily pass through a cellular membrane, intact or permeabilized, or where a lysate is desirable, the cells are treated with a reagent effective for lysing the cells contained in the fluids, tissues, or animal cell membranes of the sample, and for exposing the proteins contained therein and, as appropriate, partially separating the proteins from other aggregates or molecules such as microsomes, lipids, carbohydrates and nucleic acids in the sample. Methods for purifying or partially purifying proteins from a sample are well known in the art (e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989, herein incorporated by reference).

[0114] The samples may come from different sources and be used for different purposes. In many instances, the ABPs will be used to analyze a protein sample for active enzyme. This may include a relatively pure sample of the enzyme to determine the activity in relation to total protein of the sample. The sample may be a single cell or a mixture of cells, a neoplastic sample or other biopsy or tissue comprising a single cell type or a mixture of cell types, such as tissue from an organ, e.g. heart, lung, esophagus, kidney, brain, blood, etc., diseased tissue or healthy tissue, etc. The cells may be prokaryotic or eukaryotic, vertebrate or non-vertebrate, particularly mammalian and more particularly human. The cells or tissues, or lysates thereof may be prepared in a variety of ways, including fractionation, using chromatography, centrifugation, fluorescence activated cell sorting, dilution, dialysis, concentration, etc. The sample will usually be treated so as to preserve the activity of the target enzyme(s), so that the manner of treatment will be mild, ambient or lower temperatures will be used, particularly below 37° C., and other denaturing conditions will be avoided, such as organic solvents, or high salts.

[0115] Usually, a proteome will be analyzed. By a proteome is intended at least about 20% of total protein coming from a biological sample source, usually at least about 40%, more usually at least about 75%, and generally 90% or more, up to and including all of the protein obtainable from the source. Thus the proteome may be present in an intact cell, a lysate, a microsomal fraction, an organelle, a partially extracted lysate, biological fluid, and the like. The proteome will be a mixture of proteins, generally having at least about 20 different proteins, usually at least about 50 different proteins and in most cases 100 different proteins or more. In effect, the proteome is a complex mixture of proteins from a natural source and will usually involve having the potential of having 10, usually 20, or more proteins that are target proteins for the ABPs that are used to analyze the proteome profile. The sample will be representative of the target proteins of interest.

[0116] Generally, the sample will have at least about 0.05 mg of protein, usually at least about 1 mg of protein and may have 10 mg of protein or more, conveniently at a concentration in the range of about 0.1-10 mg/ml. The sample may be adjusted to the appropriate buffer concentration and pH, if desired. One or more ABPs may then be added, each at a concentration in the range of about 0.001 mM to 20 mM. After incubating the reaction, generally for a time for the reaction to go substantially to completion, generally for about 1-60 min, at a temperature in the range of about 20-40° C., the reaction may be quenched. The sample may now be assayed in different ways, depending upon the reagents to be used.

[0117] In one aspect of the invention, the method provides for quantitative measurement of specific active proteins in biological fluids, cells or tissues. Target identification can be applied to determine the global protein activity profiles in different cells and tissues. The same general strategy can be broadened to achieve the proteome-wide, qualitative and quantitative analysis of the state of activity of proteins, by employing ABPs or libraries of ABPs with differing specificity for reaction with proteins. The method and reagents of this invention can be used to identify active proteins of low abundance, active in complex mixtures and can be used to selectively analyze specific groups or classes of proteins, such as membrane or cell surface proteins, or proteins contained with organelles, sub-cellular fractions, or biochemical fractions such as immunoprecipitates. Further, these methods can be applied to analyze differences in expressed proteins in different cell states. For example, the methods and reagents herein can be employed in diagnostic assays for the detection of the presence or the absence of one or more active proteins indicative of a disease state, such as cancer, particularly profiles. The ABPs may be a single ABP that usually binds to at least 5, more usually at least about 10, different target proteins or may be a mixture of ABPs that bind to the same number or fewer proteins and may bind to related or unrelated proteins. Usually the mixture will have from about 2-20, more usually 2-15 different ABPs, where the profile will include a multiplicity of target proteins, encompassing individual or groups of related proteins. Usually, there will be the capability of binding to at least 10 different proteins, more usually at least about 15 different proteins and the number of proteins may be 20 or more, one at least one ABP will be capable of bonding to at least about 5 different target proteins.

[0118] The ABPs of the invention may be used to isolate and identify members of a class from the same or different species. With a neutral ABP (does not significantly discriminate between more than half of the members of the class of a single species, where the class has at least about 15 members, more usually at least about 20 members, usually being able to bind to at least 10 members or more), one can determine the available binding activity in a physiological sample of the members that bind, one can isolate new members, and one can inhibit the activity of members of the class, where such inhibition is of interest. In the case of affinity labels, one can determine the available activity in a protein composition of the target proteins, one can differentiate the activity between the target protein and other members of the class on the properties of the protein composition, e.g. cell(s) or lysate, one can obtain a protein activity profile for tissue, cells or lysate in response to various stimuli and one can screen compounds for their binding affinity to the target protein, e.g. drug screening. (It should be understood that enzymes are particularly exemplary of the target proteins and classes of enzymes will be the primary targets. To that extent, enzymes are paradigmatic of the class of target proteins and will be referred to in the future as exemplary and not limiting of the targets).

[0119] In this way the probes can be used in research, isolation and identification of proteins of a target class, diagnosis and developing therapies, and with combinatorial libraries designing target compounds having affinity for the target site of the target protein. For enzymes, because of their roles in regulation, cellular activity, response to external stimuli, and the like, there is a particular interest in being able to determine the enzyme activity in a composition, e.g. cell, as distinct from total enzyme, which could include enzyme that is not active, and how the enzyme activity varies in relation to external stimuli and/or changes in the status of the cell. For reacting with the active form of the enzyme or other protein, it is desirable that one employs a functionality that is at least relatively specific for the target enzyme genus. By relatively specific is intended that less than 20%, usually less than 10%, of the proteins other than the target enzymes in the genus will react with the functionality. Equally important is that the functionality optimally does not react, desirably less than 25%, at other than the wild-type active site, particularly with the inactive protein. Methods as those described in this application are employed to distinguish this non-active site labeling from activity-dependent labeling of the active site.

[0120] For many of the enzyme genera, functionalities are known that do not significantly react with enzymes of other genera, particularly non-enzymatic proteins and enzymes that have different reactive sites. It is also desirable that the functionality does not react with inactive target enzyme. Examples of inactive states include: 1) proenzymes, e.g. requiring cleavage of the protein; 2) enzymes bound by endogenous inhibitors (either covalent or non-covalent); 3) enzymes in an inactive conformation (e.g. enzymes that require the bindng of another protein, a conformational change, covalent modification by phosphorylation,/reduction/oxidation/methylation/acylation (e.g. formic or acetic acid) for conversion to an active state; 4) denatured enzymes; 5) mutant enzymes; 6) enzymes bound by either reversible or irreversible exogenous inhibitors; and 7) enzymes requiring a cofactor for activity. The enzymes of interest will usually have at least one of serine, threonine, cysteine, histidine, lysine, arginine, aspartate or glutamate as a member of the active site involved in the catalysis of the enzyme reaction. One or more of the functionalities of these amino acids may be the target of the ABP. The manner in which the inactive enzyme is inactivated is chosen to emphasize the differences in bonding of the ABP between the active and inactive state. However, if through the course of implementing the subject methodology, an exogenous inactivator is added to the protein sample and the effects of this treatment on the target protein activity profile of the sample relative to a control (absence of the exogenous inactivator in the sample) are determined, knowledge will be gained as to the form, quantity, and identity of the targeted protein (i.e. inactivated) by this inhibitor.

[0121] Enzymes typically fall within six main classes including oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. In a particular embodiment illustrated herein, an enzyme group of interest includes the class of hydrolases. One genus of the class is serine hydrolases, which includes sub-genera such as proteases, e.g.trypsins, chymotrypsins, esterases, such as acetylcholinesterases, thioesterases, amidases, such as FAAH, and acylpeptide hydrolases, lipases, transacylases, such as lecithin:cholesterol acyltransferase. Another sub-genus is cysteine hydrolases, such as caspases, cathepsins, and palmitoyl acyltransferases. Another sub-genus is metallohydrolases, including matrix metalloproteinases (“MMPs”), e.g. MMP 1-13, membrane type metalloproteinases, aminopeptidases, and ADAMalysins. In addition, are phosphatases, such as alkaline phosphatases, acid phosphatases, protein tyrosine phosphatases, and serine/threonine phosphatases. Further included are the GTPases and ATPases. Besides hydrolases are kinases, which include enzymes such as tyrosine kinases, e.g. src, abl, and lck, serine/threonine kinases, e.g. MAP kinases, MAPK kinases, CAM kinases, protein kinase C, and casein kinases. Also of interest are oxidoreductases, such as cytochrome P450s, amine oxidases, alcohol dehydrogenases, aldehyde dehydrogenases, such as ALDH1, ALDH2, ALDH3, desaturases, etc. Other proteins that are of interest include receptors, such as HLA antigens, hormone receptors, G-proteins coupled receptors, ion channels, transcription factors, protein inhibitors and the like.

[0122] The enzymes and/or the sites to which the ABPs bind may be identified in a variety of conventional ways, such as isolating the enzyme, e.g. using an affinity matrix, and characterizing it by mass spectrometry, isolating and sequencing the enzyme or proteolytically fragmenting the enzyme and determining the fractions as a profile for a specific enzyme, electrophoretic separation and Western blotting, or immunoassays employing labeled antibodies specific for the enzyme. The conditions under which the binding is determined will generally be mild conditions, conveniently ambient, using a buffer solution, where the buffer concentration will generally be in the range of about 50-200 mM and the concentration of each active enzyme will generally be about 0.01 pg (picograms)/ml to 0.1 mg (milligrams)/ml. After sufficient time for enzyme binding, non-specifically bound enzyme may be washed away. One may wish to use conditions of increasing stringency, by increasing salt concentration, organic solvent, temperature, etc., to determine levels of binding affinity. By comparison of the sequences at different levels of affinity, one may readily optimize the affinity sequence. In this way one or more libraries of affinity moieties are developed and can be used in conjunction with the other members of the ABP. By having a repertoire of affinity moieties, a specific affinity moiety can be selected that provides the least amount of background in a particular environment. For example, one affinity moiety may be preferred for a target enzyme in a particular background of other enzymes of the same genus.

[0123] The conjugates between ABPs and active proteins can be detected and analyzed by a number of different methods. For Western blotting analysis, conventional conditions are employed; quenching can be performed with conventional quenching media, e.g. 2×SDS-PAGE loading buffer (reducing), heated for 5-10 min at 80° C. and then run on an SDS-PAGE gel (8-14% acrylamide). After transferring the protein from the gel to a nitrocellulose paper by electroblotting, the blot is: 1) blocked for 15-60 min with 3% non-fat dry milk in TBS-Tween; 2) incubated with avidin-enzyme conjugate, e.g. horse radish peroxidase (where biotin has been employed as the ligand or other receptor-enzyme conjugate for a different ligand) for sufficient time for complex formation (1-2 hrs); 3) washed with TBS-Tween to remove non-specifically bound receptor-enzyme conjugate; 4) treated with an appropriate enzyme substrate for production of a detectable signal; and 5) detecting the site on the blot of the ABP bound to target. Quantification of differentially expressed enzyme activities among different protein samples is conducted by film densitometry using an Alphalmager 2000 (AlphaInnotech). Alternatively, one may analyze blots using a chemiluminescence detection system, such as the Lumi-Imager (Roche).

[0124] Other analytical techniques include binding of the conjugate to a surface by means of the ligand. Conjugated monoclonal antibodies conjugated with a label and specific for one or group of enzymes are added, with the antibodies binding to any target enzyme that is bound to the surface. The presence and amount of the enzyme may then be determined by the label, where the label may be a fluorescent label or an enzyme label, where the enzyme product provides a detectable signal, e.g. fluorescence. Other techniques include releasing the conjugate from the receptor, adding fluorescent receptor and using capillary electrophoresis to quantitate the enzyme.

[0125] One may also determine minimal or partial activity. One can do this by comparing the biotin (or other compound binding to a receptor) signals of protein activities found in crude samples to those produced by a fully biotinylated protein standard. For example, take a purified active serine hydrolase and conjugate it to completion with an appropriate ABP, so that there is no further enzyme activity. Then use this conjugated enzyme to generate a standard curve of signals on a gel blot that also contains crude proteomes conjugated with the same ABP. A protein activity in the crude proteome whose signal intensity matches the signal intensity of, for example 10 ng, of the standard enzyme would be considered to represent minimally 10 ng of the active enzyme in the proteome. By performing kinetics and probe concentration dependence assays, one can further determine the average partial activity, where the enzyme is only partially active.

[0126] For two samples in which the active proteins of a given family present in these samples are to be quantitatively compared, the following method can be used. A portion of each sample is treated so that the active proteins in the one portion are inactivated. Protein portions of the active and inactive samples are then treated with isotopic variants of the same ABP (e.g., one variant contains 5-10 hydrogens (light probe) and is applied to the inactive portions, the second variant has these 5-10 hydrogens substituted with deuteriums (heavy probe) and is applied to the active portions). After sufficient reaction time, the inactive and active portions of each sample are then separated from their respective ABPs (e.g., by gel filtration chromatography), combined to form a mixed sample, and this mixed sample is digested with a protease (e.g., trypsin) to create a mixture of peptides. These peptides are then treated with an affinity support to selectively isolate peptides covalently tagged with an ABP (e.g., avidin is the affinity support if the probe's tag is biotin). The isolated peptides are then optionally separated by a liquid chromatography step (e.g. HPLC) and characterized by mass spectrometry. ABP-tagged peptides representing active proteins are defined as those found in significantly greater excess (e.g., at least three-fold greater in mass ion abundance) bonded to the heavy probe than to the light probe. The molecular sequence of these peptides can be determined by Tandem Mass Spectrometry to provide the identity of the active proteins from which the ABP-labeled peptides are derived. This first procedure will thereby determine the members of a given protein family that are both present and active in the sample. Two protein portions of the active sample are then treated with the heavy and light probes and processed as described above. The levels of active protein activities will be quantitatively compared across the two samples by ratioing the mass ion abundances corresponding to heavy and light probe-bonded versions of individual peptides. Only those peptides that were determined in the first procedure to represent active proteins will be compared in this manner. To analyze simultaneously more than two samples, the same method may be followed, but an additional, unique isotopic variant of the activity-based probe will be required for each additional sample.

[0127] Of particular interest as ABPs are labeled fluorophosphonates, such as biotin-linked fluorophosphonates.

[0128] For the most part, the compounds come within the following formula:

X-L-R-(O) _m -P(O)(OT)-F

[0129] wherein:

[0130] F, P and O have their normal meaning of fluoro, phospho and oxy;

[0131] X is a ligand (including detectable label);

[0132] L is a linking group;

[0133] R is an aliphatic group of at least 2 carbon atoms, usually at least 4 carbon atoms and not more than about 16 carbon atoms, usually not more than about 12 carbon atoms, usually being straight chain alkylene or alkyleneoxy (wherein the alkylene groups are of from 2-3 carbon atoms), saturated or unsaturated, usually having not more than 2 sites of unsaturation;

[0134] mis 0 or 1; and

[0135] T is alkyl of from 1 to 6, usually 1 to 3 carbon atoms.

[0136] For the most part the ligand will be biotin or derivative thereof, e.g. deiminobiotin or dethiobiotin, and the detectable label may be a fluorescent compound, e.g. fluorescein, rhodamine, Texas red, etc.

[0137] Also of interest are compounds come within the following formula:

X-L-SO ₂ -R

[0138] wherein:

[0139] X is a ligand (including detectable label);

[0140] L is a linking group;

[0141] R is an aryl or heterocyclic group of from 5 to 12 carbon atoms having from 1-2 nitrogen atoms, which may be substituted or unsubstituted, where substituents may be halo, nitro, cyano, oxy, thio, amino, etc. In some instances, alkyl and substituted alkyl of from 1 to 20, usually 1-12 carbon atoms can find use.

[0142] Of particular interest are compounds where the linking group includes a dicarboxamido-α, α-alkylene, particularly where biotin (including derivatives thereof) is used which includes a carboxyl group naturally. The alkylene will generally be of about 2-6 carbon atoms, the length will be desirably related to not interfering with the binding of the ligand to its respective receptor and reaction of the sulfonate.

[0143] The ligand can be any ligand that does not interfere with the binding of the subject compounds to the serine hydrolases, relatively small, less than about lkdal, frequently less than about 500 Dal, has an appropriate receptor and is synthetically accessible. There are a number of popular ligands, such as biotin, dethiobiotin, deiminobiotin, digoxin, 2,4-dinitrophenyl, and derivatives thereof, fluorescein, etc. These ligands have strongly binding natural receptors, such as strept/avidin for biotin and dethio- or deiminobiotin, and antibodies for the remaining listed ligands. In some instances it will be desirable to release the serine hydrolase bonded to the inhibitor of this invention from the receptor. A useful pair is dethiobiotin or deiminobiotin, which can be replaced by biotin.

[0144] The subject compounds can be prepared using an α,ω (halo or pseudohalo)alkene, where the halo or pseudohalo group is displaced with a trialkylphosphite, followed by selective oxidation of the olefin to a carboxy or aldehyde. The activated carboxy, e.g. N-succinimidyl ester or carbodiimide anhydride, may be reacted with the ligand or detectable label bonded to a linking group terminating in an amino group to form an amide. The aldehyde may be bonded to an amine by forming an imine or Schiff's base or by reductive amination, forming an alkylated amine. Other than the ligand, the subject compounds will have fewer than 30 carbon atoms, usually fewer than 25 carbon atoms. There may be and preferably is 1 or more functionalities in the