CHEMOPROTEOMIC APPROACH FOR DISCOVERING COVALENT LIGANDS FOR DIVERSE PROTEIN TARGETS

Abstract

Despite its power in identifying highly potent ligands for select protein targets, conventional medicinal chemistry is limited by its low throughput and lack of proteomic selectivity information. We seek to develop a chemoproteomic approach for discovering covalent ligands for protein targets in an unbiased, high-throughput manner. Tripartite probe compounds composed of a heterocyclic core, an electrophilic warhead and an alkyne tag have been designed and synthesized for covalently labeling and identifying targets in cells. We have developed a novel condensation reaction to prepare 2-chloromethylquinoline (2-CMQ), a novel electrophilic heterocycle. These chloromethylquinolines potently and covalently bind to a number of cellular protein targets including Prostaglandin E Synthase 2 (PTGES2), a critical regulator of cell proliferation, apoptosis, angiogenesis, inflammation, and immune surveillance.

Claims

1. A compound having formula I or formula II: ##STR00017## wherein: Rf.sub.1 is an alkyne-containing moiety or azido-containing moiety; X.sub.1 is NR or O or X.sub.1 is absent with LK being directly bonded to Pc where R is H or C.sub.1-6 alkyl; LK.sub.1, LK.sub.2 are each independently a hydrocarbon-containing linking group; PC.sub.1 is aryl or heteroaryl; PC.sub.2 is an alkyl, aryl, heteroaryl, cycloalkyl, or heteroatom-containing ring system; and Lm is a leaving group-containing moiety or an electrophilic moiety

2. The compound of claim 1 wherein PC.sub.1 is C.sub.6-12 aryl, or C.sub.5-12 heteroaryl.

3. The compound of claim 1 wherein PC.sub.2 is: ##STR00018## where hydrogens are replaced with Lm and Lk or Rf.sub.1.

4. The compound of claim 1 wherein LK.sub.1 or LK.sub.2 is C.sub.1-12 alkyl, C.sub.6-12 aryl, or C.sub.5-12 heteroaryl.

5. The compound of claim 1 wherein Lm is Cl, CHCH.sub.2, or NCS.

6. The compound of claim 1 wherein: Rf.sub.1 is ##STR00019## n is 1-6; and R.sub.1 is H or C.sub.1-6 alkyl.

7. The compound of claim 1 wherein: Rf.sub.1 is N.sub.3 or ##STR00020## and n is 1-6.

8. The compound of claim 1 wherein: Lm is halo or CH(X.sub.2)R.sub.1; X.sub.2 is a leaving group; and R.sub.1 is H or C.sub.1-6 alkyl.

9. The compound of claim 1 having formula III: ##STR00021## wherein: R.sub.2 is H, halo, C.sub.1-6 alkyl, nitro, or cyano; R.sub.3 is halo, C.sub.1-6 alkyl, nitro, cyano or aryl; and p is 0, 1, 2, 3, or 4.

10. The compound of claim 9 having formula IV: ##STR00022##

11. The compound of claim 10 having formula V: ##STR00023## wherein X.sub.2 is a leaving group or an electrophilic functional group.

12. The compound of claim 1 having formula VI: ##STR00024## wherein R.sub.2 is halo, C.sub.1-6 alkyl, nitro, or cyano; R.sub.3 is C.sub.1-12 alkyl, C.sub.6-12 aryl, C.sub.5-12 heteroaryl, C.sub.2-24 alkyl ether groups, C.sub.12-24 aryl ether groups, or C.sub.12-24 aryl alkyl ether groups; and p is 0, 1, 2, or 3.

13. The compound of claim 12 wherein: Rf.sub.1 is ##STR00025## n is 1-6; and R.sub.5 is H or C.sub.1-6 alkyl.

14. The compound of claim 1 wherein: Rf.sub.1 is N.sub.3 or ##STR00026## and n is 1-6.

15. The compound of claim 1 having formula VII: ##STR00027## wherein R.sub.5, R.sub.6, R.sub.7 is halo, C.sub.1-6 alkyl, nitro, or cyano.

16. The compound of claim 15 having formula VIII: ##STR00028## wherein: n is 1-6; and R.sub.5 is H or C.sub.1-6 alkyl.

17. The compound of claim 1 having formula IX: ##STR00029## R.sub.5, R.sub.6, R.sub.7 is halo, C.sub.1-6 alkyl, nitro, or cyano.

18. The compound of claim 17 having formula X: ##STR00030## wherein: n is 1-6; and R.sub.5 is H or C.sub.1-6 alkyl.

19. A method of finding covalent ligands comprising: contacting a protein-containing sample with any of the compounds of claim 1 to form a modified protein; and contacting the modified protein with a probe compound that includes an alkyne-containing moiety Rf.sub.1 or azido-containing moiety Rf.sub.2, with the proviso that when Rf.sub.1 is an alkyne-containing moiety, Rf.sub.2 is an azido-containing moiety or when Rf.sub.2 is an alkyne-containing moiety, Rf.sub.1 is an azido-containing moiety wherein Rf.sub.1 reacts with Rf.sub.2 to form a ring.

20. The method of claim 19 wherein the probe compound includes a fluorophore.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1A-B. A novel approach for proteome-wide discovery of covalent ligands is shown. FIG. 1A shows an analogy between tri-partite small-molecule probes and fishing gears. FIG. 1B shows a schematic representation of the procedure for proteome-wide discovery of covalent ligands using tri-partite probes to fish out protein targets from proteomes.

[0018] FIGS. 2A-B. A novel one-step condensation reaction yielding a panel of diverse 2-chloromethylquinoline (2-CMQ) probes is shown. FIG. 2A shows a condensation reaction developed from simple acetamides to access 2-CMQs, a class of novel electrophilic heterocyclic compounds. FIG. 2B shows a panel of synthesized 2-CMQ probes.

[0019] FIGS. 3A-C. Proteome labeling by 2-chloromethylquinoline probes in situ is shown. FIG. 3A shows in-gel fluorescence images of proteome labeled by eight 2-CMQ probes. HEK293H cells were treated with the probe at indicated concentrations, lysed, clicked with TAMRA-azide, and resolved by SDS-PAGE before being fluorescence imaged. Bands A-F denote the major targets that are labeled by compound 16. FIG. 3B shows that coomassie staining of the same gel shows largely even loading across the lanes.

[0020] FIG. 4. A pulse-chase experiment revealed apparent affinity of 2-CMQ to its targets in situ. Cells were pretreated with varying concentrations of compound 17 that contains no alkyne tag for 1 hr followed by incubation of 3 M of probe 3 for 1 hr. Cells were then lysed, clicked with TAMRA-azide, and resolved by SDS-PAGE before being fluorescence imaged.

[0021] FIGS. 5A-B. Targets of 2-CMQs were identified using affinity pull-down followed by LC-MS/MS analysis. FIG. 5A shows target proteins enriched and visualized by coomassie staining. Cells were treated with probe 15, clicked to biotin-azo-azide, and enriched on streptavidin resin. The target proteins were then eluted with sodium dithionite, resolved by SDS-PAGE, and stained with coomassie blue. Slabs were cut off the gel for trypsinization and LC-MS/MS analysis. FIG. 5B shows three proteins that were identified from the gel slabs by LC-MS/MS.

[0022] FIG. 6. Confirmation of PTGES2 as the target responsible for band D and identification of the probe modification site. Transient transfection of FLAG-tagged wild-type PTGES2 soluble fragment led to enhanced fluorescent band at 37 kDa. When a PTGES2 variant in which Cys110 was mutated to Ala was used instead, no fluorescence increase was observed. The FLAG western blot revealed comparable levels of PTGES2 proteins across the transfection conditions.

[0023] FIG. 7. Recombinant PTGES2 was labeled by probe 15 near saturation at low micromolar concentrations. 6His-tagged PTGES2 was expressed and affinity-purified from E. coli. The protein was incubated with probe 15 at 37 C. for 1 hr, followed by click reaction with TAMRA-azide and fluorescence imaging.

[0024] FIG. 8. Recombinant PTGES2 was occupied by compound 17 at low micromolar concentrations. 6His-tagged PTGES2 was expressed and affinity-purified from E. coli. The protein was first treated with various concentrations of compound 17 at 37 C. for 1 hr, then incubated with 3 M of probe 15 at 37 C. for 1 hr, followed by click reaction with TAMRA-azide and fluorescence imaging.

DETAILED DESCRIPTION

[0025] Reference will now be made in detail to presently preferred compositions, embodiments and methods of the present invention, which constitute the best modes of practicing the invention presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the invention and/or as a representative basis for teaching one skilled in the art to variously employ the present invention.

[0026] Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and/or use are to be understood as modified by the word about in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary: all R groups (e.g. R.sub.i where i is an integer) include alkyl, lower alkyl, C.sub.1-6 alkyl, C.sub.6-10 aryl, or C.sub.6-10 heteroaryl; single letters (e.g., n or o) are 1, 2, 3, 4, or 5; percent, parts of, and ratio values are by weight; the term polymer includes oligomer, copolymer, terpolymer, and the like; molecular weights provided for any polymers refers to weight average molecular weight unless otherwise indicated; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.

[0027] It is also to be understood that this invention is not limited to the specific embodiments and methods described below, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way.

[0028] It must also be noted that, as used in the specification and the appended claims, the singular form a, an, and the comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components.

[0029] The term comprising is synonymous with including, having, containing, or characterized by. These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.

[0030] The phrase consisting of excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0031] The phrase consisting essentially of limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

[0032] With respect to the terms comprising, consisting of, and consisting essentially of, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

[0033] As used herein alkyl refers to C.sub.1-20 inclusive, linear (i.e., straight-chain), branched, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. Lower alkyl refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C.sub.1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. Higher alkyl refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkyl group can be optionally substituted (i.e., a substituted alkyl) with another atom or functional group such as alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, mercapto, and the like.

[0034] As used herein alkenyl means a substituted or unsubstituted monovalent unsaturated hydrocarbon group that has at least one carbon to carbon double bond. The alkenyl can be linear or branched. In a refinement, the alkenyl can have 1, 2 or 3, carbon-carbon double bonds. In a refinement, the alkenyl groups have from 2 to 10 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and the like. The term alkenylene refers a divalent alkenyl group.

[0035] As used herein alkynyl means a monovalent unsaturated hydrocarbon group which may be linear or branched and which has a carbon-carbon triple bond. In a refinement, such alkynyl groups have from 2 to 10 carbon atoms. Representative alkynyl groups include, by way of example, ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like. The term alkynylene means a divalent alkynyl group. Therefore, the term alkyne-containing moiety includes C.sub.2-10 alkynyl groups or C.sub.2-10 alkynylene groups. In context of the present invention, alkyne-containing moiety preferably have one carbon-carbon triple bond.

[0036] As used herein aryl means a monovalent aromatic hydrocarbon having a single ring (i.e., phenyl) or fused rings (i.e., naphthalene). In a refinement, such aryl groups include from 6 to 12 carbon ring atoms. In another refinement, such aryl groups include 6 to 10 carbon ring atoms. Representative aryl groups include, by way of example, phenyl biphenyl, naphthyl, anthranyl, and naphthalene-1-yl, naphthalene-2-yl, and the like. The term arylene means a divalent aryl group. Each aryl can be substituted with a functional group such as alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, mercapto, and the like.

[0037] As used herein heteroaryl means a monovalent aromatic group having a single ring or two fused rings and containing in the ring at least one heteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygen or sulfur. In a refinement, heteroaryl groups typically contain from 5 to 10 total ring atoms. In a refinement, heteroaryl groups have from 6 to 16 total ring atoms. In a refinement, the heteroaryl is a C.sub.5-12 heteroaryl. Examples of heteroaryl include, but are not limited to, monovalent species of pyrrole, imidazole, thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, indole, benzofuran, benzothiophene, benzimidazole, benzthiazole, quinoline, isoquinoline, quinazoline, quinoxaline and the like, where the point of attachment is at any available carbon or nitrogen ring atom. Additional examples heteroaryl groups include, but are not limited to, furanyl, thienyl, and pridinyl group. The term heteroarylene means a divalent heteroaryl group. Each heteroaryl can be substituted with a functional group such as alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, mercapto, and the like.

[0038] As used herein, polycyclic heteroaryl means heteroaryl groups having at least 2 total rings (e.g., fused rings). In a refinement, polycyclic heteroaryl groups have from 9 to 18 total ring atoms. In another refinement, polycyclic heteroaryl groups have from 9 to 15 total ring atoms. In some refinements, the polycyclic heteroaryl groups are C.sub.6-16 polycyclic heteroaryl groups.

[0039] As used herein alkyl aryl means a substituted or unsubstituted functional group that includes an aromatic ring (e.g., phenyl, biphenyl, naphthyl, etc) with an attached C.sub.1-8 alkyl group.

[0040] As used herein alkyl heteroaryl means a substituted or unsubstituted heteroaryl group (typically, C.sub.4-15) with an attached C.sub.1-8 alkyl group.

[0041] As used herein heteroatom-containing ring system means a substituted or unsubstituted functional group that includes aromatic or aliphatic ring systems containing 1 to 3 heteroatoms such as nitrogen, sulfur, and oxygen. In a refinement, the heteroatom-containing ring system has from 5 to 10 total ring atoms. In a refinement, heteroaryl groups have from 6 to 16 total ring atoms. In a refinement, the heteroatom-containing ring system is a C.sub.5-12 heteroatom-containing ring system.

[0042] As used herein leaving group means a functional group or atom which can be displaced by another functional group or atom in a substitution reaction (e.g., a nucleophilic substitution reaction). Examples of leaving groups include, but are not limited to, chloro, bromo and iodo groups; sulfonic ester groups (e.g., tosylate, mesylate, brosylate, nosylate, etc.); and acyloxy groups (e.g., trifluoroacetoxy, etc).

[0043] Throughout this application, when a group is described as substituted, examples of substituents include, but are not limited to, nitro, cyano, halo (e.g., F, Cl, Br, I), hydroxyl, ester, carboxylate, and the like.

[0044] In an embodiment of the present invention, a compound that is useful for identifying covalent ligands is provided. The compound of this embodiment is described by formula I or formula II:

##STR00002##

wherein:

[0045] Rf.sub.1 is an alkyne-containing moiety (e.g., an ethynyl-containing moiety) or azido-containing moiety;

[0046] X.sub.1 is NR or O or X.sub.1 is absent with LK being directly bonded to Pc where R is H or C.sub.1-6 alkyl;

[0047] LK.sub.1, LK.sub.2 are each independently a hydrocarbon-containing linking group;

[0048] PC.sub.1 is aryl or heteroaryl;

[0049] PC.sub.2 is an alkyl, aryl, heteroaryl, cycloalkyl, or heteroatom-containing ring system; and

[0050] Lm is a leaving group-containing moiety or an electrophilic moiety.

[0051] In a refinement of the present embodiment, PC.sub.1 and/or PC2 are each independently a C.sub.6-12 aryl, or C.sub.5-12 heteroaryl. In another refinement, PC.sub.1 and/or PC2 are each independently a polycyclic heteroaryl (e.g., a C.sub.7-12 polycyclic heteroaryl). In another refinement of the present embodiment, PC.sub.1 and/or PC2 are each independently:

##STR00003##

where hydrogens are replaced with Lm, Lk.sub.1, Lk.sub.2, and/or Rf.sub.1.

[0052] In some variations, LK.sub.1 or LK.sub.2 are each independently a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl. In a refinement, LK.sub.1 or LK.sub.2 are each independently a substituted or unsubstituted C.sub.1-12 alkyl, substituted or unsubstituted C.sub.6-12 aryl, or substituted or unsubstituted C.sub.5-12 heteroaryl.

[0053] In most variations, Lm is Cl, CHCH.sub.2, or NCS. In a refinement, Rf.sub.1 is:

##STR00004##

where n is 1-6 and R.sub.1 is H or a C.sub.1-6 alkyl. In another variation, Rf.sub.1 is N.sub.3 or

##STR00005##

and n is 1-6. Lm can be halo or CH(X.sub.2)R.sub.1 where X.sub.2 is a leaving group and R.sub.1 is H or C.sub.1-6 alkyl.

[0054] In a variation of the present embodiment, the compound that is useful for identifying covalent ligands is described by formula III:

##STR00006##

wherein:

[0055] R.sub.2 is H, halo, C.sub.1-6 alkyl, nitro, or cyano;

[0056] R.sub.3 is halo, C.sub.1-6 alkyl, nitro, cyano or aryl; and

[0057] p is 0, 1, 2, 3, or 4.

[0058] In another variation of the present embodiment, the compound that is useful for identifying covalent ligands is described by formula IV:

##STR00007##

[0059] In another variation of the compound that is useful for identifying covalent ligands, is described by formula V:

##STR00008##

[0060] wherein X.sub.2 is a leaving group or an electrophilic functional group.

[0061] In another variation of the compound, that is useful for identifying covalent ligands, is described by formula VI:

##STR00009##

[0062] wherein R.sub.2 is halo, C.sub.1-6 alkyl, nitro, or cyano;

[0063] R.sub.3 is C.sub.1-12 alkyl, C.sub.6-12 aryl, C.sub.5-12 heteroaryl, C.sub.2-24 alkyl ether groups, C.sub.12-24 aryl ether groups, or C.sub.12-24 aryl alkyl ether groups; and

[0064] p is 0, 1, 2, or 3.

[0065] In a refinement, Rf.sub.1 is

##STR00010##

n is 1-6, and R.sub.5 is H or C.sub.1-6 alkyl. In another refinement of the present embodiment, Rf.sub.1 is N.sub.3 or

##STR00011##

and n is 1-6.

[0066] In a variation of the present embodiment, the compound that is useful for identifying covalent ligands is described by formula VII:

##STR00012##

wherein R.sub.5, R.sub.6, R.sub.7 are each independently halo, C.sub.1-6 alkyl, nitro, or cyano.

[0067] In a variation of the present embodiment, the compound is described by formula VIII:

##STR00013##

[0068] wherein:

[0069] n is 1-6; and

[0070] R.sub.5 is H or C.sub.1-6 alkyl.

[0071] In another variation of the compound, that is useful for identifying covalent ligands, is described by formula IX:

##STR00014##

[0072] wherein R.sub.5, R.sub.6, R.sub.7 are each independently halo, C.sub.1-6 alkyl, nitro, or cyano.

[0073] In a variation of the present embodiment, the compound that is useful for identifying covalent ligands is described by formula X:

##STR00015##

wherein:

[0074] n is 1-6; and

[0075] R.sub.5 is H or C.sub.1-6 alkyl.

[0076] In another embodiment of the present invention, a method of finding covalent ligands is provided. The method comprises contacting a protein-containing sample with any of the above-mentioned compounds to form a modified protein; and contacting the modified protein with a probe compound that includes an alkyne-containing moiety Rf.sub.1 or azido-containing moiety Rf.sub.2, with the proviso that when Rf.sub.1 is an alkyne-containing moiety, Rf.sub.2 is an azido-containing moiety or when Rf.sub.2 is an alkyne-containing moiety, Rf.sub.1 is an azido-containing moiety wherein Rf.sub.1 reacts with Rf.sub.2 to form a ring:

##STR00016##

In a refinement of the method of finding covalent ligands, the compound includes a fluorophore.

[0077] The following examples illustrate the various embodiments of the present invention. Those skilled in the art will recognize many variations that are within the spirit of the present invention and scope of the claims.

[0078] Quinolines and their derivatives are one of the most important classes of heterocyclic compounds. According to the DrugBank, 47 FDA-approved drugs contain a quinoline core. These quinoline-containing drugs exhibit a broad range of medicinal activities including antimalarial, antibacterial, antihypertensive, anti-HIV, antitubercular, anticancer, and antiasthmatic activity. The quinoline thus represents a privileged pharmacophore in medicine. 2-Chloromethylquinolines (2-CMQ) contain an electrophile of chloromethyl group attached at the 2 position of the quinoline ring. Herein, we developed a novel condensation reaction to synthesize a panel of tripartite 2-CMQs that contain a quinoline core, an electrophilic warhead and an alkyne reporter. These 2-CMQs were evaluated for their covalent interactions with the whole proteome. The alkyne tag facilitated the use of copper-assisted azide-alkyne cycloaddition (CuAAC) for in-gel fluorescence analysis and mass-spectrometry-based identification of protein targets (FIG. 1).

[0079] A limited number of methods were reported for the preparation of 2-CMQ in the past. For example, 2-CMQs can be synthesized from quinaldines by converting them to their N-oxides, followed by the reaction with sulfonyl chlorides. A second method is the radical-mediated chlorination reaction of 2-methylquinolines with N-chlorosuccinimide. However, this method suffers from the formation of multiple side products including dichlorinated, trichlorinated, and other undesired side products. Chlorination of 2-methylquinolines using chlorine as the halogen source has benefits of low cost and high yield, but chlorine is an irritating toxic gas. Although 2-CMQ can be prepared from 2-quinolinemethanols by an alternative chlorination approach, the starting materials 2-quinolinemethanols are usually not commercially available. Chlorination of 2-methylquinolines into 2-(chloromethyl)quinolines in the tetrabutylammonium iodide and 1,2-dichloroethane system, via in situ generated ICl, has been developed recently. Unfortunately, all of the above methods are either incompatible with alkyne functional group or require multi-step synthesis to produce the tripartite quinolines (FIG. 1A). A more efficient method is needed to generate probes of diverse functionality and substitution pattern for interacting with various targets in the proteome.

[0080] To fill in the gap, we have developed a one-step condensation method for the preparation of 2-chloromethylquinolines. In this approach, PCl.sub.5 is used to condense two molecules of arylacetamide, a readily available material with tremendous choices of structural diversity, to construct the quinoline core, and meantime installs both chloromethyl electrophile and alkyne tag. By changing the substitution position and pattern, we can attach the alkyne tag to various positions in the N-arylacetamide and alter geometries in the probes. In a similar manner, a non-alkyne substituent can also be introduced to broaden the diversity and functionality in the probes.

[0081] We first examined the proteome reactivity of a panel of 2-CMQs (FIG. 2B). HEK293H cells were treated with each probe at 0.1 or 1.0 M for 1 hour before being lysed and clicked with TAMRA-azide using CuAAC. The samples were resolved by SDS-PAGE and then scanned for in-gel fluorescence (FIG. 3, lanes selected). 2-CMQs potently labeled a few distinct bands in the proteome. For example, 1 M of probe 16 labeled six prominent bands of sizes between 20 and 60 kDa (FIG. 3A). The different probes have subtly different labeling patterns indicating different spectrum of protein targets. These data demonstrate that the proteome reactivity of 2-CMQs can be finely adjusted by modulating the sterics of the quinoline ring system, thereby providing a highly tunable electrophile for chemoproteomic studies.

[0082] We then chose probe 6 and probe 15 as exemplary probes for further investigation of their proteomic targets. These two probes produced different labeling patterns as band C was labeled by probe 15 but not probe 6 (FIG. 4). To determine the binding saturability and affinity, we prepared two compounds 6c and 15c (FIG. 4) that contain a methoxy group instead of the propargoxy group using the same condensation reaction. The pulse-chase experiment involved pretreating cells with various concentrations of a competitor for 1 hr followed by incubation of 3 M of a probe for 1 hr. Cells were then lysed, clicked with TAMRA-azide, and resolved by SDS-PAGE before being fluorescence imaged. The competition assay showed that majority of the band labelings were wiped off by competitor compounds, supporting that the labeling is saturable. IC.sub.50 values derived from the dose-response curve signify apparent binding affinity of the competitor to the target protein in situ. Our results revealed IC.sub.50 values of 1 M for binding of the 2-CMQs to targets A-D, a good starting point considering that no chemical optimization was done yet.

[0083] We then set out to identify the target proteins that were covalently labeled by the 2-CMQ probes. HEK293H cells were incubated with mock or 10 M of 15c for 1 hr before further incubation with 3 M of probe 15 for 1 more hr at 37 C. The cells were then lysed, conjugated with biotin-azo-azide via CuAAC, and pulled down using streptavidin agarose beads. Addition of sodium dithionite cleaved the target proteins off the beads, which were resolved by SDS-PAGAE and visualized by coomassie staining (FIG. 5). The sections corresponding to the region containing bands A-D were cut off from the gel and subjected to MS analysis. This led to the identification of glutathione S-transferase omega-1, heme oxygenase 2, and prostaglandin E synthase 2 as target proteins B, C and D, respectively.

[0084] Transient transfection of FLAG-tagged wild-type PTGES2 soluble fragment led to enhanced fluorescent band at 37 kDa, confirming PTGES2 as the target protein responsible for band D (FIG. 6). When a PTGES2 variant in which Cys110 was mutated to Ala was transfected instead, no fluorescence increase was observed supporting that Cys110 is the site of probe modification (FIG. 6). To test if pure PTGES2 could also be labeled by 2-CMQ, 6His-tagged PTGES2 was expressed and affinity-purified from E. coli. The recombinant PTGES2 was labeled by low micromolar of probe 15 to saturated levels, exhibiting similar behaviors to the protein in HEK293H cells (FIG. 7). In addition, pretreatment of the recombinant PTGES2 with increasing concentrations of compound 17 blocked the protein from subsequent labeling by probe 15, suggesting that 2-CMQ2 binding to PTGES2 is close to saturation at 1 M (FIG. 8).

[0085] In conclusion, we have developed a novel condensation reaction to prepare 2-chloromethylquinoline, a new electrophile for chemoproteomic studies. These chloromethylquinolines covalently and potently bind to a number of cellular protein targets including Prostaglandin E Synthase 2, a critical regulator of cell proliferation, apoptosis, angiogenesis, inflammation, and immune surveillance. The novel PTGES2 inhibitors that we discovered using chemoproteomics have the potential to serve as novel therapies for the treatment of human diseases such as inflammation. Beyond this case study, our methodology can be extended to other electrophiles and scaffolds for discovering covalent ligands for diverse targets in the whole proteome.

[0086] While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation, and it is understood that various changes may be made without departing from the spirit and scope of the invention. Additionally, the features of various implementing embodiments may be combined to form further embodiments of the invention.